chapter 0 - intro - title - toc - summary
Post on 11-Feb-2017
221 Views
Preview:
TRANSCRIPT
Research Collection
Doctoral Thesis
Medium-chain-length poly(R-3-hydroxyalkanoates): frombiosynthesis towards medical applications
Author(s): Furrer, Patrick Christian
Publication Date: 2008
Permanent Link: https://doi.org/10.3929/ethz-a-005705895
Rights / License: In Copyright - Non-Commercial Use Permitted
This page was generated automatically upon download from the ETH Zurich Research Collection. For moreinformation please consult the Terms of use.
ETH Library
Diss. ETH No. 17654
Medium-chain-length poly([R]-3-hydroxyalkanoates):
from biosynthesis towards medical applications
A dissertation submitted to
ETH ZURICH
for the degree of
Doctor of Sciences
presented by
Patrick Christian Furrer
Dipl. Chem. University of Basel
born January 19, 1976
citizen of Attinghausen, UR
accepted on the recommendation of
Prof. Dr. S. Panke, examiner
Prof. Dr. T. Egli, co-examiner
Dr. M. Zinn, co-examiner
2008
2
For my parents
3
Table of contents
Summary 6
Zusammenfassung 8
Chapter 1: Poly([R]-3-hydroxyalkanoates): biosynthesis,
recovery, and their potential for biomedical appl ications 11
1.1 Introduction 12
1.2 Biosynthesis 13
1.3 Fermentative production 18
1.4 Material properties 20
1.5 Recovery methods 22
1.5.1 Chemical digestion of non PHA-biomass 23
1.5.2 Standard method: solvent extraction and
precipitation of PHA in non-solvents 26
1.5.3 Special approaches for the recovery of PHA 28
1.6 Purification of PHA 29
1.6.1 Sources and characterization of contaminations 30
1.6.2 Methods to purify extracted PHA 32
1.7 Potential applications of PHA in medicine and pharmacy 34
1.7.1 PHA as drug carrier 34
1.7.2 PHA as scaffold material in tissue engineering 35
1.8 Conclusions and outlook 37
4
Chapter 2: Quantitative analysis of bacterial mediu m-chain-length
poly([R]-3-hydroxyalkanoates) by gas chromatograp hy 39
2.1 Introduction 40
2.2 Materials and methods 42
2.3 Results and discussion 45
2.4 Conclusions 54
2.5 Acknowledgement 55
2.6 Supplementary information 56
Chapter 3: Biosynthesis of medium-chain-length
poly([R]-3-hydroxyalkanoate): endotoxin reduction
by phosphorus limitation 61
3.1 Introduction 62
3.2 Materials and methods 64
3.3 Results and discussion 67
3.4 Conclusions 77
3.5 Acknowledgements 78
Chapter 4: Efficient recovery of low endotoxin medium-chain-le ngth
poly([R]-3-hydroxyalkanoate) from bacterial bioma ss 79
4.1 Introduction 80
4.2 Materials and methods 82
4.3 Results and discussion 86
4.4 Conclusions 94
4.5 Acknowledgement 95
5
Chapter 5: Biocompatibility of untreated and chemically modifi ed
medium-chain-length poly(3-hydroxyalkanoates) 97
5.1 Introduction 98
5.2 Materials and methods 100
5.3 Results and discussion 104
5.3.1 Physical and chemical properties of PHAs 104
5.3.2 Cytotoxicity according to the eluate test 105
5.3.3 Cell culturing on PHA coatings 107
5.4 Conclusions 112
Chapter 6: General conclusions 115
References 119
Curriculum vitae 141
6
Summary
Poly(3-hydroxyalkanoate) (PHA) is a class of biodegradable polyesters that is
accumulated by a large number of bacteria as carbon storage compound under
nutrient limited growth conditions. PHA with side-chains of medium-chain-length
(mcl-PHA) represents a promising class of polymers because of its broad spectrum
of physical properties which are determined by the monomeric composition. Mcl-PHA
has a wide variety of potential applications in medicine and pharmacy due to its
versatility and biodegradability. However, a major breakthrough of mcl-PHA in the
biomedical field has not taken place yet. An important reason for this is that
biosynthetic products must fulfil strict requirements to be approved for medical
applications. In particular biosynthesis with Gram-negative bacteria is delicate,
because endotoxins from the cell membrane can contaminate the product.
In the first part of this doctoral thesis a method for the quantitative analysis of
mcl-PHA was developed, to optimise the fermentative production and the
downstream processing (DSP) of mcl-PHA (chapter 2). Standard methods for the
quantification of short-chain-length PHA proved to be inappropriate for the analysis of
mcl-PHA. It could be shown that the transesterification catalyzed by protic acids was
not quantitative for mcl-PHA under typical conditions. This was due to slow reaction
kinetics and the formation of side-products in case of mcl-PHA with functionalised
side-chains. An improved method using boron trifluoride as catalyst was developed
to quantitatively methanolyse different mcl-PHAs (recovery > 94%) and to analyse
the resulting methyl esters by gas chromatography.
The fermentative production and the DSP of mcl-PHA were investigated in the
second part of this doctoral thesis with the objective to generate mcl-PHA of high
purity and minimal endotoxic contamination. Phosphorus limited culturing conditions
were applied to Pseudomonas putida GPo1 in the chemostat to reduce the endotoxic
activity of lipopolysaccharides which act as endotoxins (chapter 3). Indeed, the
combination of phosphorus, carbon and nitrogen limitation drastically changed the
cell composition. The content of phospholipids and the endotoxic activity of dried
biomass were decreased by up to 90% under phosphorus limitation compared to
conditions with excess phosphorus. Hence, the endotoxic contamination of mcl-PHA
caused by the fermentation process could be drastically decreased.
7
To obtain mcl-PHA of high purity, the recovery from biomass was optimised
(chapter 4). Classical non-solvent precipitation was replaced by applying
temperature-controlled extraction and precipitation. N-hexane was found to be a
suitable solvent to recover poly(3-hydroxyoctanoate) and copolymers thereof with
this technique. A purity of >97% (w/w) and endotoxicities around 15 EU g-1 PHA were
obtained. Additional re-dissolution in 2-propanol combined with temperature-
controlled precipitation resulted in an enhanced purity of close to 100% (w/w) and a
minimal endotoxicity of 2 EU g-1 PHA.
In the third part of this doctoral thesis the biocompatibility of different mcl-PHAs
was studied with fibroblasts (chapter 5). Cytotoxicity experiments with aqueous
eluates of mcl-PHA as well as cell-culturing experiments on mcl-PHA coatings
showed that these polymers caused no toxic effect, when an effective purification
procedure, such as repeated dissolution and precipitation, was applied. Cell
adhesion was in fact limited on the non-polar surfaces of mcl-PHA, but chemical
derivatisation of the polymer surface clearly improved its hydrophilicity and cell
adhesion. However, the functionality of fibroblasts cultured on mcl-PHA surfaces
seemed to be limited as a reduced collagen production was measured.
The results achieved in this work show that an optimised DSP is of immense
importance to efficiently recover mcl-PHA of high purity which can be used for
biomedical applications. Furthermore, the activity of endotoxins can significantly be
reduced during biosynthesis of mcl-PHA which facilitates the DSP considerably. For
most biomedical applications chemical modification of hydrophobic mcl-PHA is
required to assure favourable interactions with cells.
8
Zusammenfassung
Poly(3-hydroxyalkanoat) (PHA) ist eine Klasse von biologisch abbaubaren
Polyestern, die von einer Vielzahl von Bakterien unter Nährstoff-limitierten
Wachstumsbedingungen als Kohlenstoffspeicher akkumuliert wird. PHA mit
Seitenketten von mittlerer Kettenlänge (mcl-PHA) repräsentiert eine vielver-
sprechende Klasse von Polymeren wegen ihres breiten Spektrums an physikalischen
Eigenschaften, welche durch die monomerische Zusammensetzung bestimmt
werden. Mcl-PHA besitzt eine grosse Vielfalt von möglichen Anwendungen im
medizinischen und pharmazeutischen Bereich aufgrund ihrer Vielseitigkeit und
biologischen Abbaubarkeit. Allerdings fehlt ein entscheidender Durchbruch von mcl-
PHA im biomedizinischen Bereich immer noch. Ein wichtiger Grund dafür ist, dass
biosynthetische Produkte strenge Anforderungen erfüllen müssen, um für
medizinische Anwendungen zugelassen zu werden. Speziell die Biosynthese mit
Gram-negativen Bakterien ist kritisch, weil Endotoxine der Zellmembrane das
Produkt verunreinigen können.
Im ersten Teil dieser Doktorarbeit wurde eine Methode zur quantitativen Analyse
von mcl-PHA entwickelt, um die fermentative Produktion und die Aufarbeitung von
mcl-PHA zu optimieren (Kapitel 2). Standardmethoden für die Quantifizierung von
PHAs mit kurzen Seitenketten erwiesen sich als ungeeignet für die Analyse von mcl-
PHAs. Es konnte gezeigt werden, dass die durch protische Säuren katalysierte
Umesterung nicht quantitativ ablief für mcl-PHAs unter typischen Bedingungen.
Grund dafür war die langsame Reaktionskinetik und die Bildung von Nebenprodukten
im Fall von mcl-PHAs mit funktionellen Seitenketten. Eine verbesserte Methode mit
Bortrifluorid als Katalysator wurde entwickelt um verschiedene mcl-PHAs quantitativ
zu methanolysieren (Wiederfindung >94%) und um die resultierenden Methylester
gaschromatografisch zu analysieren.
Die fermentative Produktion und die Aufarbeitung von mcl-PHA wurden im
zweiten Teil dieser Doktorarbeit untersucht mit dem Ziel mcl-PHA von hoher Reinheit
und minimaler endotoxischer Kontamination herzustellen. Phosphorlimitierte
Wachstumsbedingungen wurden angewendet für Pseudomonas putida Gpo1 im
Chemostat, um die endotoxische Aktivität der Lipopolysaccharide, welche als
Endotoxine wirken, herabzusetzen (Kapitel 3). Tatsächlich wurde die
Zellzusammensetzung durch kombinierte Phosphor-, Kohlenstoff- und Stickstoff-
9
limititation drastisch verändert. Der Phospholipidgehalt und die endotoxische Aktivität
der trockenen Biomasse wurden um bis zu 90% herabgesetzt unter
Phosphorlimitation verglichen mit Bedingungen mit Phosphor im Überschuss. Somit
konnte die Kontamination von mcl-PHA mit Endotoxinen, verursacht durch den
Fermentationsprozess, drastisch gesenkt werden.
Um mcl-PHA von hoher Reinheit zu erhalten, wurde der Aufreinigungsprozess
aus der Biomasse optimiert (Kapitel 4). Die klassische Fällung des Polymers mit Hilfe
eines Nicht-Lösungsmittels wurde ersetzt durch die Anwendung der temperatur-
kontrollierten Extraktion und Fällung. N-Hexan erwies sich als geeignetes Lösungs-
mittel, um Poly(3-Hydroxyoktanoat) und dessen Copolymere mit dieser Technik
aufzureinigen. Eine Reinheit von >97% (w/w) und eine endotoxische Aktivität von
ungefähr 15 EU g-1 PHA wurden damit erreicht. Zusätzliches Lösen in 2-Propanol
kombiniert mit temperaturkontrollierter Fällung resultierten in einer erhöhten Reinheit
von nahezu 100% (w/w) und einer minimalen endotoxischen Aktivität von 2 EU g-1
PHA.
Im dritten Teil dieser Doktorarbeit wurde die Biokompatibilität von verschiedenen
mcl-PHAs anhand von Fibroblasten untersucht (Kapitel 5). Zytotoxizitätstests mit
wässrigen Eluaten der mcl-PHAs und Zellkulturexperimente auf mcl-PHA Schichten
zeigten, dass diese Polymere keinen toxischen Effekt verursachten, wenn eine
effektive Aufreinigungsprozedur wie das wiederholte Lösen und Fällen angewendet
wurden. Die Zelladhäsion auf unpolaren mcl-PHA Oberflächen war limitiert, doch
durch chemische Derivatisierung der Polymeroberfläche konnte die Hydrophilie und
die Zelladhäsion deutlich verbessert werden. Allerdings schien die Funktionalität von
Fibroblasten, welche auf mcl-PHA Oberflächen kultiviert wurden, eingeschränkt zu
sein, da eine reduzierte Produktion von Kollagen gemessen wurde.
Die Resultate dieser Arbeit zeigen, dass ein optimiertes Aufreinigungsverfahren
von immenser Wichtigkeit ist, um mcl-PHA effizient und mit hoher Reinheit zu
erhalten, welches für biomedizinische Zwecke eingesetzt werden kann. Ausserdem
kann die Aktivität der Endotoxine während der Biosynthese von mcl-PHA signifikant
reduziert werden, was das Aufreinigungsverfahren wesentlich vereinfacht. Für die
meisten biomedizinischen Anwendungen wird eine chemische Modifikation des
hydrophoben mcl-PHAs benötigt, um günstige Interaktionen mit Zellen zu
gewährleisten.
10
11
Chapter 1
Poly([R]-3-hydroxyalkanoates):
Biosynthesis, recovery, and their potential
for biomedical applications
Patrick Furrer, Sven Panke and Manfred Zinn. 2007. Accepted for
publication in the Handbook of Natural-based Polymers for Biomedical
Applications.
Introduction PHA 12
1.1 Introduction
New biomaterials of the third generation are needed for particular medical
applications such as vascular implants, heart valves and cardiovascular fabrics
(Hench and Polak 2002; Hubbell 1995; Ueda and Tabata 2003). They are designed
to stimulate specific cellular responses at the molecular level and to combine the
concepts of bioactive and resorbable materials. They are supposed to help the body
heal itself by prompting cells to repair their own tissues. Today’s polymeric and
biodegradable systems used in medicine are mainly based on poly(lactic acid) (PLA),
on poly(glycolic acid) (PGA), and on their co-polymers (Gomes and Reis 2004).
Other biodegradable polymers have been proposed, but could not enter the market
yet, due to lacking FDA approval (Gomes and Reis 2004).
One of the candidates is poly([R]-hydroxyalkanoate) (PHA), a class of
biodegradable and biocompatible polyesters with many potential applications in the
medical field, such as heart valve scaffolds (Sodian et al. 2000a; Sodian et al. 2002),
pulmonary conduits (Stock et al. 2000), sutures, screws, bone plates, repair patches,
stents, bone marrow scaffolds, and many others over the last years as recently
reviewed by Chen and Wu (Chen and Wu 2005). PHA is composed of 3, 4, or rarely
5-hydroxy fatty acid monomers, which form linear polyesters. The general structure
of poly([R]-hydroxyalkanoate) is shown in Fig. 1.1. PHAs can be separated into 3
classes according to the size of comprising monomers: short-chain-length PHAs (scl-
PHA) with monomers of 3-5 carbon atoms, medium-chain-length PHAs (mcl-PHA)
with monomers of 6-14 carbon atoms and long-chain-length PHAs (lcl-PHAs) with
monomers of more than 14 carbon atoms. PHA is produced as reserve material by
many archae and eubacteria in aerobic and anaerobic habitats. PHA accumulation
occurs when microorganisms experience a metabolic stress, such as limitation by an
essential nutrient in excess of a suitable carbon source (Lageveen 1986, Lee 1996b,
Schlegel and Gottschalk 1962, Ward et al. 1977, Zinn and Hany 2005).
O
O
n
R
m
m=1-3
Fig. 1.1: General structure of poly([R]-hydroxyalkanoate).
13 Chapter 1
The purity of PHA is a crucial factor for sophisticated applications. It is
determined by the fermentation process and the downstream processing. The
production strain and the selective recovery procedure are essential factors for
obtaining PHA of high quality. Although there has been a continuous progress in
downstream processing of PHA for bulk applications (more than 50 patents have
been filed in the past 40 years) further improvements for high-quality PHAs and in
particular for mcl-PHAs are to be expected.
The restricted availability of PHAs has been limiting research significantly. Poly(3-
hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)
are the only PHA-polymers that are currently commercially available (e.g. from
Metabolix and Biomer). At present, polymers based on poly(4-hydroxybutyrate)
(P4HB) are developed for medical use and one of them has been approved by FDA
recently (Rizk 2005). In 2008, Telles, a joint venture between Metabolix and Archer
Daniels Midland, plans to start the production of PHA under the name MirelTM at an
annual rate of 50 kt.
PHAs offer a wide range of physical properties due to their chemical diversity and
due to chemical modifications of functional groups following biosynthesis.
1.2 Biosynthesis
PHAs are a class of polyesters produced as reserve materials by many archae and
eubacteria (Gram-negative and Gram-positive, see Table 1.1) in aerobic and
anaerobic habitats. Up to date, more than 300 microorganisms are known to
synthesize PHA (Suriyamongkol et al. 2007). PHA accumulation is generally
triggered when the microorganisms experience a metabolic stress, such as limitation
by an essential nutrient (e.g., nitrogen, phosphorus or oxygen) and a concomitant
excess of a suitable carbon source (Anderson and Dawes 1990). PHA is stored
intracellularly in form of granules as a carbon and energy storage compound. The
PHA granules are surrounded by a phospholipid monolayer and several proteins like
the PHA polymerase and depolymerase and structural proteins called phasins are
part of the granule surface (see Fig. 1.2). Upon carbon starvation or a change of the
environmental pH (Ruth et al. 2007), intracellular PHA depolymerases, which are
attached to the granule, release 3-hydroxyalkanoic acids.
Introduction PHA 14
Table 1.1: Important PHA-producing genera, substrates and resulting PHAs.
Genus Gram stain
Substrates PHA Ref.
Alcaligenes - Fatty acids PHB Doi et al. 1992, Fukui and Doi 1998
Azospirillum - Fatty acids, saccharides
PHB Itzigsohn et al. 1995
Bacillus + Fatty acids (C2-C6), saccharides, lactones
PHB, PHBV, PHBHx, P4HB
Caballero et al. 1995, Chen et al. 1991, McCool et al. 1996, Tajima et al. 2003, Valappil et al. 2007
Beijerinckia - Glucose PHB Jendrossek et al. 2007
Brevundimonas - Saccharides PHB Silva et al. 2007
Burkholderia - Saccharides PHB, PH4PE Rodrigues et al. 2000
Caulobacter - Saccharides PHB Qi and Rehm 2001
Chromobacterium - 1,3-propandiol Scl- and mcl-PHA
Kimura et al. 2002
Clostridium + Acetate PHB Emeruwa and Hawirko 1973
Corynebacterium + Saccharides PHBV Haywood et al. 1991
Cupriavidus (former Wautersia)
- Saccharides, fatty acids
PHB, PHBV, P4HB
Kimura et al. 1999, Volova et al. 2006, Yan et al. 2003
Haloferax - Saccharides PHB, PHBV Koller et al. 2007, Lillo and Rodriguezvalera 1990
Methylobacterium - Alcohols, fatty acids
PHB, PHBV Yezza et al. 2006
Nocardia + Fatty acids, saccharides
PHB, PHBV Alvarez et al. 1997
Pseudomonas - Fatty acids, saccharides
PHHp - PHDd Kessler and Palleroni 2000
Rhizobium - Saccharides PHBV Lakshman et al. 2004
Rhodobacter - Fatty acids PHB, PHBV Kranz et al. 1997
Rhodococcus + Saccharides, fatty acids
PHBV, PHPi Füchtenbusch et al. 1998, Füchtenbusch and Steinbüchel 1999
Sphingopyxis - Saccharides PHBV Godoy et al. 2003
Staphylococcus + Saccharides PHB Szewczyk and Mikucki 1989
Streptomyces + Saccharides PHB Manna et al. 1999
PHB: Poly(3-hydroxybutyrate), PHBV: Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PHPi: Poly(3-hydroxypivalate), P4HB: Poly(4-hydroxybutyrate), PHBHx: Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), P3HB-4HB: Poly(3-hydroxybutyrate-co-4-hydroxybutyrate), PHHp: Poly(3-hydroxyheptenoate), PHDd: Poly(3-hydroxydodecanoate), PH4PE: Poly(3-hydroxypent-4-enoate)
15 Chapter 1
Fig. 1.2: Structure of PHA- granules accumulated by bacteria (Zinn et al. 2001).
PHAs have the general chemical structure depicted in Fig.1.1. The high
stereoselectivity of the producing enzyme machinery guarantees complete
stereospecificity (all chiral carbon atoms in the backbone are in the R configuration),
which is essential for the biodegradability and biocompatibility of PHA (Bachmann
and Seebach 1999; Hocking et al. 1996). The type of bacterium and the growth
conditions determine the chemical composition of PHAs and the molecular weight,
which typically ranges from 2x105 to 3x106 Da (Byrom 1987).
Research has focused on the substrate specificity of the PHA polymerase (Rehm
2003). Four major classes of PHA polymerases have been proposed with respect to
their primary structures, their substrate specificities and their subunit composition
(Rehm 2007) (see Table 1.2). It was found that the substrate specificity of the PHA
polymerase and the supply of cells with a particular substrate control the monomeric
composition of the resulting PHA. In general, different pathways for the biosynthesis
of PHA are possible (see Fig. 1.3). Acetyl-CoA activated precursors of PHA are
either synthesized in the course of anabolism through de novo fatty acid synthesis or
stem in the course of catabolism from the β-oxidation of fatty acids that are supplied
to cells.
Introduction PHA 16
Fig. 1.3: Metabolic routes of PHA biosynthesis. PhaA, β-ketothiolase; PhaB, NADPH-dependent acetoacetyl-CoA reductase; PhaC, PHA synthase; PhaG, 3-hydroxyacyl-ACP-CoA transferase; PhaJ, (R)-enoyl-CoA hydratase; FabD, malonyl-CoA-ACP transacylase; FabG, 3-ketoacyl-ACP reductase.
Table 1.2: The four classes of polyester synthases (Rehm 2003).
HA-CoA: coenzyme A thioester of [R]-hydroxy fatty acids of variable length
17 Chapter 1
To date, more than 150 different hydroxyalkanoic acids are known as PHA
constituents (Rehm 2007), but only few of the corresponding PHAs have been
produced in large quantities and well characterized (Kessler and Witholt 1998;
Witholt and Kessler 1999). As a consequence, little is known about the chemical and
mechanical properties of the larger number of these polymers. To date, PHA
monomers with straight, branched, saturated and unsaturated side chains have been
identified (see Fig. 1.4) (Steinbüchel and Valentin 1995). Of special interest are
functionalized groups in the side chain that allow further chemical modification, e.g.
halogens, carboxy, hydroxy, epoxy, phenoxy, cyanophenoxy, nitrophenoxy,
thiophenoxy, and methylester groups. The size of the monomer and its functional
group considerably influence properties like the bioplastic’s melting point, the glass
transition temperature, or the crystallinity, and therefore determine its final
application.
HO OH
n
HO
R
OH
O O
HO OH
O
HO OH
O
HO
X
OH
n O
HO OH
n O
m
HO OH
O
O
n
Halogen and cyano substituentsX=F, Br, Cl, CN
Epoxy substituent
Linear and branched alkyl substituentsR=alkyl substituent (C1-C12)
Terminal double bond
Cyclohexane substituent Aromatic substituent
Substituent with internal double bonds
OH
O
HOOH
OHO
4-hydroxy fatty acid 5-hydroxy fatty acid
R
R
Fig. 1.4: Monomers found in poly([R]-3,4 and 5-hydroxyalkanoates).
Introduction PHA 18
1.3 Fermentative production
Most PHA-producing bacteria start to accumulate PHA when their cell growth is
impaired by the limitation of an essential nutrient (e.g., N, P, Mg, K, O or S) in the
presence of excess carbon source. It is therefore important to use a suitable
fermentation strategy to enhance the production of PHA. The PHA content is thereby
the most important parameter for an efficient and economic downstream processing.
For fed-batch cultures a two step process is usually employed. In the first phase
the cells are grown to a desired cell concentration and in the second phase PHA
biosynthesis is triggered by a nutrient limitation. For continuous cultures, cell growth
and PHA accumulation are controlled by the ratio of the carbon source to the limiting
nutrient as well as by the dilution rate.
PHB has been efficiently produced with Alcaligenes latus in a two-step fed-batch
fermentation. Nitrogen limitation was chosen as the best strategy as it allowed the
greatest enhancement of PHB production. A cell concentration of 111.7 g L-1 and a
PHB content of 88 w% were reached resulting in the productivity of 4.94 g PHB L-1 h-1
(Wang and Lee 1997a). With Cupriavidus necator (former Wautersia eutropha and
Ralstonia eutropha) a PHB content of 76 w% was obtained at a cell concentration of
164 g L-1 resulting in a productivity of 2.42 g L-1 h-1 under nitrogen limitation (Kim et
al. 1994b).
PHBV has been successfully produced with C. necator. The two-step fed-batch
strategy was applied using nitrogen or phosphorus limitation in the second step. The
mole fraction of HV in the co-polymer could be controlled by changing the ratio of
glucose to propionic acid in the feed. PHBV contents of up to 75 w% and cell
concentrations of up to 158 g L-1 were obtained under nitrogen limitation (Kim et al.
1994a). However, with an increasing HV mole fraction, the PHA content decreased.
P3HB4HB could be produced in fed-batch cultures under nitrogen limitation. Cell
concentration of 34-49 g L-1 and PHA contents of 39-50 w% were reached with 4HB
mole fractions of 1.6-25.2 mol% (Kim et al. 2005).
For the efficient production of scl-PHAs, recombinant E. coli and C. necator have
been intensively investigated. In contrast to natural PHA-producing bacteria, PHA
accumulation by recombinant E. coli was not triggered by nutrient limitation. In a fed-
batch fermentation PHB was produced with recombinant E. coli reaching a cell
19 Chapter 1
concentration of 204 g L-1 with a PHB content of 77 w% and a productivity of
3.2 g L-1 h-1 (Wang and Lee 1997b). A higher PHB productivity of 4.63 g L-1 h-1 was
obtained with recombinant E. coli harboring an optimally designed plasmid containing
the PHA biosynthesis genes from A. latus (Choi et al. 1998). PHBV with 11 mol% HV
could be produced with a concentration of 159 g L-1 and a productivity of
2.88 g L-1 h-1 with recombinant E. coli (Choi and Lee 1999b). Recombinant C. necator
was investigated for the production of PHB and PHBV but the final cell
concentrations and PHA contents were only slightly increased compared to the
parent strain (Lee et al. 1996; Park et al. 1995; Valentin and Steinbuchel 1995).
Although efforts have been made to use recombinant E. coli and recombinant
Pseudomonas for the production of mcl-PHA, few fermentation processes were
based on recombinant strains (Prieto et al. 1999).The production of mcl-PHA was
extensively investigated with Pseudomonas species (Diniz et al. 2004; Eggink et al.
1992; Hartmann et al. 2006; Hoffmann and Rehm 2004; Hori et al. 1994; Huijberts
and Eggink 1996; Kim et al. 2000). Various alkanes, alkanoic acids as well as
glucose, fructose and glycerol were used as substrates for the production of mcl-PHA
(Huijberts et al. 1992; Timm and Steinbüchel 1990). Especially with structurally
related carbon sources, such as alkanes and aliphatic acids efficient mcl-PHA
synthesis occurs (Lageveen et al. 1988). But these carbon sources are poorly water
miscible or/and toxic to bacteria at rather low concentration (Sun et al. 2007). Hence
the concentration of these carbon sources has to be well controlled.
For fed-batch cultures the two-step fermentation strategy has often been applied.
Using octanoic acid as substrate, PHA contents up to 75 w% at a cell concentration
of 55 g L-1 and a productivity of 0.63 g L-1 h-1 were obtained with Pseudomonas
putida GPo1 under nitrogen limitation (Kim 2002). With Pseudomonas IPT 046 cell
concentrations of up to 50 g L-1 with a PHA content of 63 w% and a productivity of
0.8 g L-1 h-1 were reached under phosphate limitation using a mixture of glucose and
fructose as carbon source (Diniz et al. 2004). When oleic acid was used as substrate
for the cultivation of Pseudomonas putida KT2442, a cell concentration of 141 g L-1
with a PHA content of 51 w% and a productivity of 1.91 g L-1 h-1 were obtained under
phosphate limitation (Lee et al. 2000b).
Continuous fermentation has been optimized for the efficient production of mcl-
PHA (Hartmann et al. 2006; Huijberts and Eggink 1996; Prieto et al. 1999). It has
Introduction PHA 20
been shown that the PHA content decreased with increased specific growth rate.
Thus, a compromise between PHA content and productivity is required in a single-
stage continuous process (Sun et al. 2007). A two-stage continuous process was
developed to overcome this limitation (Jung et al. 2001). Cell densities of 18 g L-1
with a PHA content of 63 w% and an overall volumetric productivity of 1.06 g L-1 h-1
were obtained.
1.4 Material properties
Interestingly, the material properties of PHA are similar within one class as shown in
Table 1.3. They are strongly dependent on the monomeric composition, in particular
on the monomers’ side-chain and on the distance between the ester linkages in the
backbone. In general, PHA is water insoluble, biodegradable, and biocompatible. It
can be degraded at a moderate rate (3–9 months) by many microorganisms into
carbon dioxide and water using their own secreted PHA depolymerases (Jendrossek
2001). Its primary breakdown products, 3-hydroxyacids, are naturally found in
animals and humans.
Table 1.3: Physical properties of short-chain-length (scl) and medium-chain-length (mcl) PHAs.
Scl-PHA Mcl-PHA
Crystallinity [%] 40-80 20-40
Glass transition temperature [°C] 2 -36
Melting point [°C] 80-180 30-80
Elongation to break [%] 6-10 300-450
Density [g cm-3] 1.25 1.05
Scl-PHAs are typically crystalline thermoplasts. The homopolymer PHB is a
relatively stiff and brittle bioplastic, which can be processed by melt extrusion. PHA
co-polymers composed of primarily HB with a fraction of longer chain monomers,
such as HV, HHx or HO, are more flexible and less brittle thermoplasts. Their
structure is shown in Fig. 1.5. They can be used in melt-extrusion processes to form
a wide variety of products including containers, bottles, razors, and materials for food
21 Chapter 1
packaging. PHB and PHBV was used as water-proof film on the back of diaper
sheets (Martini et al. 1989). PHBV is more flexible, more impact resistant and was
marketed under the trade name Biopol™ by ICI/Zeneca and later on by Monsanto till
1995. Co-polymers consisting of 3-hydroxybutyrate and a 3-hydroxyalkanoic acid
with at least 6 carbon atoms, for instance PHBHx was developed by Procter and
Gamble and commercialized under the name Nodax. In analogy to PHBV, PHBHx is
less crystalline than PHB and more flexible.
O
O
O
n
O
O
n
poly(3-hydroxybutyrate-co-3-hydroxyvalerate)
poly(4-hydroxybutyrate)
O
m
O
O
O
npoly(3-hydroxybutyrate-co-3-hydroxyhexanoate)
O
m
Fig. 1.5: Chemical structures of commercially important scl-PHAs: Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) and poly(4-hydroxybutyrate) (P4HB).
Common mcl-PHAs are less crystalline and more elastomeric. Their properties
largely depend on their side-chains. For instance, the glass transition temperature of
a mcl-PHA containing linear chains up to seven carbon atoms and aromatic groups
varies between -39 °C and -6 °C with an increasing content of aromatic side-chains
(Hartmann et al. 2004). The physical properties of mcl-PHAs containing functional
groups in the side-chains can be adapted through chemical modification. Thereby,
the field of possible applications is considerably extended. Through the insertion of
hydroxy groups in the side-chains of mcl-PHA, the polymer’s polarity and solubility in
polar solvents could be drastically changed (Eroglu et al. 2005; Lee et al. 2000a;
Renard et al. 2005). Epoxidation and crosslinking of mcl-PHAs with unsaturated side-
chains increased the polymer’s tensile strength and Young’s modulus by a factor of 4
and 39, respectively (Ashby et al. 2000). Polyhedral oligomeric silsesquioxane
(POSS) were linked to the side-chains of unsaturated mcl-PHAs thereby increasing
the crystallinity and rising the melting point up to 120 °C (Hany et al. 2005).
Introduction PHA 22
Very little is known about lcl-PHA. It has been shown that the ability to polymerize
long-chain-length PHAs is limited to Pseudomonas putida KTOY06 (Liu and Chen
2007) and probably this is valid for most PHA accumulating bacteria.
1.5 Recovery methods
Recovery processes can be divided into two categories: solvent extraction and
chemical digestion. In the former one PHA is extracted from biomass by using an
organic solvent like methylene chloride. In the latter case the rest biomass is
digested by applying chemicals like sodium hypochlorite or hydrolytic enzymes.
Through cross-flow filtration or centrifugation PHA is separated then from cell debris.
The main problem of chemical digestion is severe degradation of PHA, resulting in a
reduction of the molecular weight.
To certify polymers for medical applications, very demanding specifications have
to be fulfilled. Considerable amounts of impurities like proteins, surfactants and
endotoxins are not tolerated by regulatory agencies. The presence of bacterial
endotoxins in medical polymers is one of the biggest concerns of suppliers (Williams
et al. 1999). Up to now only few methods for depyrogenation of PHA were described
in literature, although this aspect is very important for medical applications. More
details are given in section 1.6.
Fig. 1.6: PHA recovery through chemical digestion (A) and solvent extraction (B).
23 Chapter 1
1.5.1 Chemical digestion of non PHA-biomass
Digestion of non-PHA biomass is performed in aqueous environment without or with
only small amounts of organic solvents (Fig. 1.6A). The suspended cells are usually
lysed to release the PHA granules, which form an aqueous suspension. For cell lysis
various techniques used in biotechnology can be applied as shown in Table 1.4. The
granule envelope protects the PHA to a certain degree from chemical degradation.
All the residual biomass constituents are rendered water soluble by chemical
modification. This can be done by using chemical agents like peroxides,
hypochlorites and ozone or by using hydrolytic enzymes. After cell disruption and
digestion of residual biomass, PHA granules can be separated from cell debris by
conventional methods like centrifugation or cross-flow filtration. While PHB granules
have a density of about 1.2 g cm-1, mcl-PHA granules possess a density close to that
of water (Preusting et al. 1993) and therefore do not settle in aqueous suspensions
(Marchessault et al. 1995). Hence, ultracentrifugation or cross-flow filtration has to be
applied (deKoning et al. 1997; Yasotha et al. 2006). Although the PHA granules are
not physically dissolved in H2O, they form a stable suspension (latex). Once the PHA
granules are isolated further purification steps usually follow. In the following we will
focus the discussion to the first step, the degradation of residual biomass, because
this is crucial for the further processing.
Introduction PHA 24
Table 1.4: Methods to lyse bacterial cells for the recovery of PHA.
Physical
methods
Ref. Chemical
methods
Ref.
Bead milling
Tamer et al. 1998a,
Tamer et al. 1998b
Reducing or oxidizing
agents
Ramsay et al. 1992,
Tamer et al 1998a
Homogenization Ling et al 1997b,
Tamer et al. 1998a
Detergents
de Koning et al. 1997
Ultrasonication
Hwang et al. 2006 Lytic enzymes Kim et al. 1996
Thermal shock
Cumming et al.
1994
Solvents (osmotic
shock)
de Koning and Witholt
1997, Escalona et al.
1996
Supercritical fluid
treatment
Castor and Hong
2003, Hejazi et al.
2003
pH-shock Tamer et al. 1998b
Cell debris digestion with hypochlorite (Berger et al. 1989; Hahn et al. 1994;
Hahn et al. 1995; Lee and Choi 1998; Ling et al. 1997b; Middelberg et al. 1995;
Tamer et al. 1998a; Taniguchi et al. 2003) was one of the earliest methods for
degrading residual biomass. Most cell constituents are oxidized by hypochlorite and
thus become water soluble. However, PHA was shown to become severely degraded
with a decrease in molecular weight Mw from e.g. 1’200 kDa to 600 kDa under
optimized conditions (Berger et al. 1989; Hahn et al. 1994; Hahn et al. 1995; Ramsay
1990). In addition, it is difficult to eliminate residues of sodium hypochlorite
completely in the subsequent steps. Hypochlorite digestion could be improved by
combining it with surfactants or organic solvents and resulted in PHA of higher purity
with less polymer degradation (Bordoloi et al. 2003; Hahn et al. 1994; Ramsay 1990).
Alternative oxidizing agents like hydrogen peroxide or ozone were investigated to
oxidize residual biomass. Peroxides were especially useful to degrade nucleic acids
and to decrease the viscosity of the lysate (Greer 1994; Greer 1997; Liddell and
Locke 1997). In combination with enzymes and surfactants, the efficiency of rest
25 Chapter 1
biomass degradation with peroxides could be increased (George and Liddell 1997).
Treatment of PHA containing biomass or partially purified PHA with ozone yielded an
enhanced level of purity in combination with other purification steps. The resulting
polymer was basically odor-free (Horowitz and Brennan 1999). Furthermore,
coloration of PHA was drastically reduced with ozone or peroxide. It has not clearly
been described in literature if PHA was degraded by hydrogen peroxide or ozone.
But a certain degradation has to be expected due to the strong reactivity of these
agents.
Simple alkaline digestion of suspended biomass provided PHB with a purity of
85 % or higher, but a minor degradation of PHA has also been observed (Choi and
Lee 1999a). Combination with chelates and surfactants improved cell disruption and
solubilisation of the rest biomass, and therefore PHA of higher purity was obtained.
The use of enzymes is an alternative to harsh chemical agents. Cell fragments
can be degraded more selectively and the degradation of PHA is negligible. Enzyme
cocktails consisting of various hydrolytic enzymes like proteinases, nucleases,
phospholipases, lysozymes and others in combination with surfactants and chelate
formers have been applied (Byrom 1987; George and Liddell 1997; Holmes and Lim
1990; Kim et al. 1996; Ramsay et al. 1992; Yamamoto et al. 1995). Combined with
heat treatment, PHA purities of up to 95% could be achieved (deKoning and Witholt
1997). Heat accelerated the degradation of residual biomass, while surfactants and
chelates improved the solubility of all components. Mcl-PHA with a purity of 93 %
was obtained by enzymatic digestion combined with ultrafiltration (Yasotha et al.
2006).
Generally the isolation of the PHA-granules is much easier with bacteria that
have a high PHA content (>60 % in dry mass). Only one or two steps are necessary
to achieve a purity of around 90 %. If the PHA content is below 60%, the separation
process is more complicated. For this case a combined method with enzymes and
reducing agents like sodium dithionite was effective (Schumann and Müller 2003).
Cell components like proteins, nucleic acids and polysaccharides were decomposed
without drastically changing PHA properties.
To avoid the problem of a dramatic increase in viscosity caused by the liberation
of DNA, a nuclease-encoding gene was integrated into the genome of PHA
Introduction PHA 26
producing bacteria (Boynton et al. 1999). Thereby the amount of enzymes or
chemicals necessary for the digestion of the rest biomass was reduced. The lysate
viscosity was significantly reduced without affecting the PHA production or the strain
stability.
Major disadvantages of the enzymatic digestion are the high costs of hydrolytic
enzymes and the additional purification steps necessary to reach a high degree of
purity. Moreover surfactants can hardly be separated from PHA.
The purification of PHA granules would be much more efficient if they were
secreted into the medium. Separation from the cells could be done by centrifugation
without digesting the residual biomass. The production of extracellular PHA has been
proposed (Sabirova et al. 2006). However, this seems to be irrational as excreted
PHA granules would hardly be accessible to the bacteria for a later use of PHA.
1.5.2 Standard method: solvent extraction and prec ipitation of PHA in non-
solvents
As discussed before, the alternative method to chemical or enzymatic digestion of
the residual biomass is the selective extraction of PHA from biomass with an organic
solvent. Most recovery procedures based on the extraction with organic solvents
follow the steps shown in Fig.1.6B. Bacterial cells are collected by centrifugation or
filtration and dried to remove water that inhibits an effective extraction. Pre-treating
dry biomass with methanol can be effective to remove some of the lipids and coloring
impurities (Gorenflo et al. 2001; Jiang et al. 2006). PHA is extracted from the dried
biomass with an organic solvent under stirring and in some cases heating. The
resulting suspension is filtered or centrifuged to remove particulate cell debris and
subsequently PHA is either precipitated with a non-solvent, or obtained by
evaporation of the solvent. In some cases the crude PHA thus obtained is washed
with a non-solvent. Repeated extraction, precipitation and washing steps result in a
higher purity.
Solvent extractions normally use large amounts of solvents, typically 5-20 times
of the dry weight of biomass and similar amounts of non-solvents for precipitation.
Solvent recycling is energy consuming, especially for solvents with high boiling
points. The high viscosity of even diluted PHA solutions (e.g. 5% w/v) impedes
27 Chapter 1
extensive solvent savings and limits economical optimization. Soxhlet extraction was
used to work with reduced volumes of solvent (Valappil et al. 2007).
The solubility of PHA is strongly dependent on the polymer composition, the
molecular weight and on the temperature and pressure (Terada and Marchessault
1999). The extraction of scl-PHA was usually performed with chlorinated solvents like
methylene chloride, chloroform, 1,2-dichloroethane, 1,1,2-trichloroethane and
1,1,2,2-tetrachloroethane (Baptist 1962b; Barham 1982; Holmes et al. 1980;
Numazawa et al. 1987; Ramsay et al. 1994; Schmidt et al. 1986; Stageman 1985;
Vanlautem and Gilain 1982; Vanlautem and Gilain 1987) because most chlorinated
solvents are capable of dissolving PHB at a relatively high concentration, but only
little of the rest biomass is dissolved as well. Preferentially the extraction was carried
out at elevated temperatures to increase the solubility and to reduce the viscosity of
the polymer solution and the extraction time. To reach high temperatures without
evaporation of the solvent, pressurization has been applied (Kurdikar et al. 1998).
PHA degradation at high extraction temperatures has been observed, especially
when water was present in solution. At temperatures above 200 °C degradation
occurs also in the absence of water by a non-radical random chain scission reaction
(cis-elimination) (Lee et al. 2001). Recently, it has been shown that even at moderate
temperatures thermal degradation occurs via E1cB mechanism if carboxylate groups
are present (Kawalec et al. 2007). To precipitate the dissolved polymer, different non-
solvents like methanol, ethanol, water, or ether have been used (Baptist 1962a;
Holmes 1980; Kessler and Witholt 1998; Numazawa et al. 1987; Stageman 1985).
Thereby, ether could only be applied for scl-PHA as it dissolves mcl-PHAs. PHB with
a purity of 99% could be obtained with precipitation in water (Hrabak 1992). Mixtures
of chlorinated solvents with a non-solvent were applied to extract PHB from biomass
at high temperatures and to precipitate the polymer by cooling to room temperature
(Schmidt et al. 1986). Azeotrope-building chlorinated solvents (e.g., 1,1,2-
trichloroethane) were used for simultaneous azeotropic distillation and PHA
extraction from an aqueous suspension of microorganisms (Vanlautem and Gilain
1987). Thereby water was removed from the cell suspension as a minimum boiling
azeotrope. The remaining chlorinated solvent was used to extract PHA. Hence a
preceding drying of the cell suspension could be avoided.
Introduction PHA 28
In an effort to prevent the disadvantages of halogenated solvents, which are
known to pose health risks and environmental problems, alternative solvents like
cyclic carbonic acid esters (Lafferty 1977), methyl lactate (Metzner et al. 1997), ethyl
lactate (Metzner et al. 1997; Sela and Metzner 1996), acetic acid (Rapthel et al.
1993a; Rapthel et al. 1993b; Runkel et al. 1993a), acetic acid anhydride (Runkel et
al. 1993b; Schmidt et al. 1985), n-methylpyrrolidone (Schumann and Mueller 2001),
tetrahydrofuran (Matsushita et al. 1995) and mixtures of non-halogenated solvents
(Noda and Schechtman 1997) were investigated for the extraction of PHB. Co-
polymers with PHB were also extracted by non-chlorinated solvents like acetone
(Gorenflo et al. 2001), ethyl acetate (Chen et al. 2001), butyl acetate (Walsem et al.
2004), methyl isobutylketone (Walsem et al. 2004) and cyclo-hexanone (Walsem et
al. 2004).
Mcl-PHA has the advantage to be soluble in a broader spectrum of solvents than
PHB. Even at room temperature typical mcl-PHAs are soluble in acetone, THF or
diethyl ether. A patent from Firmenich S.A. describes an extraction method with non-
halogenated solvents at room temperature tailored for polyhydroxyoctanoate
(Ohleyer 1993). More recently solvents like acetone were used for extracting mcl-
PHA from biomass (Jiang et al. 2006).
A combined extraction-filtration method has been described using a circular
filtration system (Horowitz 2000), where the aqueous slurry was diafiltrated and an
organic solvent like acetone was continuously added. In the beginning the bacterial
cells were rejected, but at a certain acetone to water ratio of about 9:1 (w/w) the cells
were lysed, mcl-PHA was dissolved and passed the filtration membrane.
The recycling of solvent and non-solvent mixtures is time and energy
consuming. The utilisation of non-solvents can be avoided by temperature induced
extraction and precipitation, described in chapter 4.
1.5.3 Special approaches for the recovery of PHA
Supercritical fluid extraction (SFE) with carbon dioxide has been proposed for the
recovery of PHA (Williams et al. 1999). After extraction, carbon dioxide evaporates
immediately and a drying step is not required. However, published data are
conflicting. Pure PHB is soluble up to 8.01 g L-1 in supercritical CO2 at a temperature
29 Chapter 1
of 348 K and a pressure of 355 bar (Khosravi-Darani et al. 2003). In contrast to these
results, Seidel and Hampson showed that lipids, pigments and ubiquinones were
extracted from biomass containing PHB or PHO without dissolving the polymer under
similar conditions (Hampson and Ashby 1999; Seidel et al. 1991). In these reports
PHA was extracted from the purified biomass with an organic solvent, e.g. chloroform
(Hampson and Ashby 1999; Seidel et al. 1991). Consequently, it remains uncertain
whether SFE can be efficiently used to extract PHA from biomass.
A process for recovering PHA from biological material by comminution and air
classification has been patented (Noda 1995; Noda 1998). In principle no chemicals
or solvent is needed for this process. The dried biomass was defatted and grinded so
that the diameter of most particles was below 100 µm. An air stream was used to
suspend the particles and classify them according to their weight or size. At
appropriate stream velocities the particles were separated into a coarse and a fine
fraction. The fine fraction was then subjected to further purification steps to reach a
PHA purity of 80% or higher. So far, it has not been described whether this process
is successfully applied for the recovery of PHA at a larger scale.
Furthermore dissolved-air flotation was recently used to separate mcl-PHA
granules from the fermentation broth (van Hee et al. 2006). Flotation is potentially
cheap and is common in wastewater treatment. Flotation separates particles
according to their affinity to the air/liquid interface which is generated by bubbling air
(or another gas) through a liquid phase (e.g. water). Hydrophobic particles are
transported with the air-bubbles to the surface, whereas more hydrophilic ones
remain in the aqueous phase. The cells were pre-treated with enzymes to release the
PHA granules. Selective aggregation and flotation of mcl-PHA granules could be
triggered by adjusting the pH at around 3.5. A purity of 86% was obtained after three
consecutive batch flotation steps.
1.6 Purification of PHA
For applications in the medical field, PHA of high purity is needed. In particular
biologically active contaminants like proteins and lipopolysaccharides (LPS) have to
be reduced to a very low level as they could induce immunoreactions. The U.S. Food
Introduction PHA 30
and Drug Administration (FDA) requires the endotoxin content of medical devices not
to exceed 20 U.S. Pharmacopeia (USP) endotoxin units (EU) per device, except for
those devices that are in contact with the cerebrospinal fluid. In this case the content
must not exceed 2.15 USP EU per device. LPS act as endotoxins whereby minute
quantities can have severe effects in contact with blood and trigger immunoreactions.
Particularly for PHA derived from fermentation of Gram-negative bacteria,
contamination with endotoxins is a serious problem, as LPS are part of the outer
membrane. During cell lysis and product recovery, LPS are liberated from the outer
membrane and contaminate PHA. Therefore, PHA for use in medical devices has to
be carefully purified from such endotoxins.
Standard techniques used for the purification of PHA are re-dissolution and
precipitation, washing with a non-solvent, purification by chromatography, treatments
with chemical agents and filtration. The purity of PHA can also be increased by
washing the biomass before extracting PHA or by aqueous digestion to remove
major impurities as described before.
1.6.1 Sources and characterization of contamination s
The spectrum of possible PHA-contaminants from residual biomass is broad. But
basically, the extraction solvent determines which impurities will be carried over. For
instance, proteins and DNA have been frequently detected when PHA was recovered
by aqueous chemical digestion. Solvent extraction with non-polar solvents or with
solvents of average polarity is more susceptible to co-extract lipids and coloring
substances. Notably polar organic solvents like acetone and 2-propanol co-extract
chromophores and give the polymer a yellow to brownish color (see Fig. 1.7).
Lipopolysaccharides have been detected with aqueous digestion as well as with
solvent extraction. They are soluble in water, but their amphiphilic character renders
them to some extent soluble in non-polar solvents.
Besides lipids, proteins and lipopolysaccharides also antifoam agents from
fermentations, surfactants and hydrolytic enzymes from the purification procedure
are contaminants of the polymer. The most common impurities found in PHA are
summarized in Table 1.5. In summary the nature of contaminants is determined by
the biosynthesis as well as by the downstream processing.
31 Chapter 1
Table 1.5: Common contaminants found in PHA.
Contaminant Amount in
PHA
Ref. Recovery
method
Microorganism PHA
Lipids 0.1-3 mol%a Sevastianov et al.
2003
Ethanol-KOH
wash +
chloroform-
ethanol
extraction
R. eutropha
B5786
PHB,
PHBV
Proteins 0-9 w% Ling et al. 1997a,
Ling et al. 1997b
Homogenization
+ centrifugation
E. coli
PHB
n.d Seidel et al. 1991 Extraction M. rhodasianum PHB
n.d Williams et al.
1999
Aq. digestion n.d. PHO
DNA 0.03-2.2 w%
Ling et al. 1997b Homogenization
+ centrifugation
E. coli PHB
0.6-2.2 w% Ling et al. 1997a Homogenization
+ centrifugation
E. coli PHB
LPS >120 EU g-1 Williams et al.
1999
N.d. N.d. PHB
1-104 EU g-1 Lee et al. 1999 NaOH-digestion E. coli PHB
Antifoam
agents
< 1 w% Unpubl. results Chloroform-
ethanol
extraction
P. putida GPo1 PHO
SDS N.d. de Koning and
Witholt 1997
SDS
solubilisation +
enzym.
treatment
P. putida GPo1 PHO
UV absorbers N.d. Jiang et al. 2006 Acetone
extraction
P. putida
KT2440
mcl-
PHA
N.d. not defined, a) relative to the PHA monomers
Introduction PHA 32
Fig. 1.7: Coloring impurities co-extracted from biomass. PHO extracted with A) hexane, B) methylene chloride and C) acetone. The solvent was evaporated after filtration.
1.6.2 Methods to purify extracted PHA
Repeated dissolution and precipitation was commonly applied to reach a purity of
close to 100%. The efficiency of this method is dependent on several parameters like
the concentration of the polymer solution and the temperature but usually large
amount of non-solvents were used. Jiang et al. (Jiang et al. 2006) showed that the
concentration of UV absorbing molecules is considerably decreased by dissolution of
mcl-PHA in acetone and precipitation in cold methanol as shown in Fig. 1.8. Scl-PHA
was repeatedly dissolved in chloroform and precipitated in ethanol to remove
biologically active substances. Traces of fatty acids from C6 to C18 were eliminated
and the hemocompatibility increased (Sevastianov et al. 2003). The authors propose
that these fatty acids originated from lipopolysaccharides.
Fig. 1.8: UV spectra of crude extracted and purified mcl-PHA in chloroform. 1: acetone extract precipitated in methanol, 2-4: additional cycles of dissolution and precipitation (1-3 times). The absorption bands at 275 and 241 nm were attributed to aromatic amino acids and nucleic acids (Jiang et al. 2006).
33 Chapter 1
Temperature controlled dissolution and precipitation in 2-propanol was used to
purify certain mcl-PHA like PHO. A purity of nearly 100 % and an endotoxicity of
below 10 EU g-1 polymer was obtained with this method as shown in chapter 4.
To reduce the endotoxin content further to acceptable values, e.g. < 10 EU g-1 of
PHA, a treatment with an oxidizing agent such as hydrogen peroxide or benzoyl
peroxide was used successfully (Williams et al. 2001). Destruction of LPS by
applying basic conditions was also successful (Lee et al. 1999; Sevastianov et al.
2003). The concentration of base and the treatment time (see Fig. 1.9) are crucial for
an ideal detoxification. Otherwise, the destruction of LPS is incomplete or PHA is
depolymerised and its molecular weight reduced as previously mentioned.
Fig. 1.9: Endotoxic activity of PHB recovered by 1.2N NaOH digestion at 30 °C for various durations (Lee et al. 1999).
Further methods can be used for purification such as washing of PHA with a non-
solvent, purification by chromatography, filtration and treatment with endotoxin
removing agents, however, accurate data about their efficacy are not available. For
the protein purification in aqueous solutions, e.g., cationic endotoxin removal agents
have been shown to be very successful (Zhang et al. 2005).
Introduction PHA 34
1.7 Potential applications of PHA in medicine and p harmacy
PHA has the potential to become an important compound for medical applications
(Williams et al. 1999; Zinn et al. 2001). Biocompatibility and slow biodegradability are
thereby essential properties. The changing PHA composition also allows favorable
mechanical properties as shown in Table 1.3. In vitro cell experiments and in vivo
studies have focused on PHB, PHBV, P4HB, PHBHx and PHO. An overview of the
potential applications of PHA is given in Table 1.6. In the following we discuss the
applications in the field of drug delivery and tissue engineering. The discussion is
restricted to these fields, because the most promising research was done in these
areas.
Table 1.6: Potential applications of PHA in medicine and pharmacy.
Type of application Products Type of PHA
Wound management Sutures, skin substitutes, nerve cuffs, surgical meshed, staples, swabs
Scl, mcl
Vascular system Heart valves, cardiovascular fabrics, pericardial patches, vascular grafts
Mcl
Urology Urological stents Scl, mcl
Orthopaedy Scaffolds for cartilage engineering, spinal cages, bone graft substitutes, meniscus regeneration, internal fixation devices (e.g. screws)
Scl, (mcl)
Dental Barrier material for guided tissue regeneration in periodontosis
Scl, mcl
Computer assisted tomo-graphy and ultrasound imaging
Micro- and nanospheres for anticancer therapy
Scl, mcl
Drug delivery Chemoembolizing agents, micro- and nanospheres for anticancer therapy
Scl, mcl
1.7.1 PHA as drug carrier
PHAs became candidate material as drug carriers in the early 1990s due to their
inherent biocompatibility (Pouton and Akhtar 1996). Microspheres of PHB loaded
with rifampicin were investigated for their use as chemoembolizing agent (agent for
the selective occlusion of blood vessels) (Kassab et al. 1999; Kassab et al. 1997).
35 Chapter 1
The drug release of all microspheres was very rapid with almost 90% of the drug
released within 24h. The drug release rate could be controlled by the drug loading
and the particle size. A similar behavior was described by Sendil and coworkers for
PHBV supplemented with tetracycline (Sendil et al. 1999). PHBV was further
investigated as an antibiotic-loaded carrier to treat implant-related and chronic
osteomyelitis (Yagmurlu et al. 1999). The antibiotic sulbactamcefoperazone was
integrated into PHBV rods and implanted into a rabbit tibia that was artificially
infected by S. aureus. The infection subsided after 15 days and was nearly
completely healed after 30 days.
In search of an efficient transdermal drug delivery system a PHO-based system
with a polyamidoamine dendrimer was examined. Tamsulosin was used as the
model drug. The dendrimer was found to act as weak permeability enhancer. By
adding the dendrimer, the dendrimer-containing PHA matrix achieved the clinically
required amount of tamsulosin permeating through the skin model (Wang et al.
2003).
1.7.2 PHA as scaffold material in tissue engineerin g
PHBV was chosen as a temporary substrate for growing retinal pigment epithelium
cells as an organized monolayer before their subretinal transplantation. The surface
of the PHBV film was hydrophilized by oxygen plasma treatment to increase the
attachment of D407 cells to the polymer surface. The cells grew to confluency as an
organized monolayer. Hence, PHBV films can be used as temporary substrates for
subretinal transplantation to replace diseased or damaged retinal pigment epithelium
(Tezcaner et al. 2003).
An interesting approach is the implantation of biodegradable supporting scaffolds
that are seeded with tissue-engineered cells. This approach was exemplified by
Sodian and co-workers (Sodian et al. 2000c) who used PHO and P4HB for the
fabrication of a tri-leaflet heart valve scaffold. A porous surface was achieved with the
salt leaching technique resulting in pore size between 80 and 200 µm. The scaffold
was seeded with vascular cells from ovine carotid artery and subsequently tested in a
pulsatile flow bioreactor. The cells formed a confluent layer on the leaflets.
Introduction PHA 36
In another study, the native pulmonary leaflets were resected with the use of
cardiopulmonary bypass, and segments of pulmonary artery were replaced by
autologous cell-seeded heart valve constructs. All animals survived the procedure
without receiving any anticoagulation therapy. The tissue engineered constructs were
covered with tissue and no thrombus formation was observed. It was concluded that
tissue engineered heart valve scaffolds fabricated from PHO can be used for
implantation in the pulmonary position with an appropriate function for 120 days in
lambs (Sodian et al. 2000a).
The same group demonstrated that PHO and P4HB have thermoprocessible
advantages over PGA who has better property for ovine vascular cell growth (Sodian
et al. 2000b; Sodian et al. 2000c).
Vascular smooth muscle cells and endothelial cells from ovine carotid arteries
were seeded on P4HB scaffolds to study autologous tissue engineered blood vessels
in the descending aorta of juvenile sheep. Up to 3 months after implantation, grafts
were fully patent, without any signs of dilatation, occlusion or intimal thickening. A
confluent luminal endothelial cell layer was observed. In contrast, after 6 months graft
displayed significant dilatation and partial thrombus formation, most likely caused by
an insufficient elastic fiber synthesis (Opitz et al. 2004).
PHBHx was found to be a suitable biomaterial for osteoblast attachment,
proliferation and differentiation from bone marrow cells. The cells on PHBHx
scaffolds presented typical osteoblast phenotypes: round cell shape, high alkaline
phosphotase (ALP) activity, strong calcium deposition, and fibrillar collagen
synthesis. After incubation for 10 days, cells grown on PHBHx scaffolds were
approximately 40% more than that on PHB scaffolds and 60% more than that on PLA
scaffolds. ALP activity of the cells grown on PHBHx scaffolds was up to about 65
U g-1 scaffolds, 50% higher than that of PHB and PLA, respectively (Wang et al.
2004).
Similarly, it was observed that chondrocytes isolated from rabbit articular
cartilage proliferated better on PHB scaffolds blended with PHBHx than on pure PHB
scaffolds. Chondrocytes proliferated on the PHB-PHBHx scaffold and preserved their
phenotype up to 28 days (Deng et al. 2003).
37 Chapter 1
1.8 Conclusions and Outlook
Polyhydroxyalkanoates with a wide range of physical properties are accessible
through biosynthesis in bacteria. It has been shown that they have a potential in
several medical applications. Unfortunately, inappropriate downstream processing of
the polymer resulted in contamination of PHAs by bacterial cell compounds and
therefore may have affected first studies in a negative way. Improved purification
methods have been described in the last years which were successful in reducing
pyrogenic contaminations. Recently, a type of PHA, P4HB, obtained the approval of
the U.S. Federal Drug Administration for application as a suture material. It is to be
expected that more PHAs will follow because material properties of PHAs can
already be tailored for particular applications during biosynthesis or later on by
chemical and physical modifications.
Introduction PHA 38
39
Chapter 2
Quantitative analysis of bacterial medium-
chain-length poly(R-3-hydroxyalkanoates) by
gas chromatography
Patrick Furrer, Roland Hany, Daniel Rentsch, Andreas Grubelnik, Katinka Ruth,
Sven Panke, Manfred Zinn. 2007. Journal of Chromatography A, 1143, 199-206.
Quantitative analysis of mcl-PHA 40
2.1 Introduction
Polyhydroxyalkanoates (PHAs) are a class of biodegradable and biocompatible
polyesters with many potential applications in the medical field (Chen et al. 2005). In
particular, the scientific and industrial research on elastomeric PHA with medium-
chain-length side chains (mcl-PHA, containing 3-hydroxyalkanoate monomers with
chain lengths from C6-C14) has been intensified in the last years (Chen et al. 2005;
Sodian et al. 2000; Stock 2000; Williams et al. 1999; Zinn et al. 2001).
PHAs are produced by microorganisms under unbalanced growth conditions
(Anderson and Dawes 1990). In order to control and optimize the conditions of
fermentation and down-stream processing, the quantitative analysis of PHA in
biomass and of purified polymer is of great importance. Different methods have been
applied to determine the content of PHA in biomass and to analyze the monomeric
composition of PHA-copolymers. After the discovery of poly(3-hydroxybutyrate)
(PHB), a short-chain-length PHA (scl-PHA), Lemoigne saponified the extracted PHB,
and determined the amount of PHB by gravimetry after applying a complicated
treatment (Lemoigne 1926). Slepecky and Law converted PHB with concentrated
sulphuric acid to crotonic acid and estimated the content via its UV absorption at 235
nm (Slepecky and Law 1960). Recently, Fourier transform infrared spectroscopy
(FTIR) has been applied to determine the content of PHA in cell suspensions (Jarute
et al. 2004; Randriamahefa et al. 2003). However, all these methods lack the
specificity to discriminate between different monomers and hence they can not be
used to determine the monomeric composition of PHA copolymers. 1H and 13C NMR experiments were applied to scl- and mcl-PHA without chemical
derivatisation of the polymer (Doi et al. 1995; Doi et al. 1986; Jan et al. 1996). For
quantitative measurements, NMR is limited to purified PHA. For scl-PHA, 1H NMR
measurements are sufficient to elucidate their composition, whereas for mcl-PHA
time-consuming 13C-NMR measurements are necessary. The need for faster
analytical methods for the determination of PHA in biomass directly forced the
development of chromatographic methods such as high performance liquid
chromatography (HPLC) or gas chromatography (GC). This requires a quantitative
depolymerisation of the polymer, usually combined with a derivatisation. Ion-
exchange HPLC with conductivity detection was applied for the analysis of digested
41 Chapter 2
poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in activated sludge
(Hesselmann et al. 1999). However, the use of GC is more common due to the
simple detection by flame ionisation (FID). The first derivatisation methods for GC
were developed for PHB (Braunegg et al. 1978; Riis and Mai 1988). Sulphuric acid in
methanol and later hydrochloric acid in n-propanol were used as reagent for the
transesterification, forming methyl and propyl esters, respectively. In contrast to the
alkaline hydrolysis (Wallen and Rohwedder 1974), which led to a mixture of 3-
hydroxyalkanoic acid methyl esters and 2-alkenoic acid methyl esters, the acidic
transesterification resulted in the ideal case in only one methyl ester per component.
In addition, it was considerably faster because extraction and transesterification
proceeded in one step. Certain modifications have been introduced for the acidic
transesterification method of PHA in biomass, like an extended reaction time for the
analysis of PHB (Huijberts et al. 1994; Jan et al. 1995), and an increased
concentration of sulphuric acid for scl-PHA (Oehmen et al. 2005) and mcl-PHA
(Lageveen et al. 1988; Lee and Choi 1995). For mcl-PHA, sulphuric acid in methanol
was used (Huijberts et al. 1994; Lageveen et al. 1988; Lee and Choi 1995). However,
sulphuric acid is of limited use as a general catalyst for transesterification reactions,
due to further decomposition of the 3-hydroxy esters (Oehmen et al. 2005; Riis and
Mai 1988), e.g. by acid catalyzed elimination.
So far, the transesterification of mcl-PHA has not been studied in detail. In this
work, we show that the methods developed for scl-PHA can not be adapted to mcl-
PHA without reservation. This is because the reaction kinetics differ significantly
between scl- and mcl-PHAs and side products occur for certain side-chain
functionalized mcl-PHAs, e.g. olefinic mcl-PHAs. Both effects lead to a significant
underestimation of the effective PHA content and to an incorrect copolymer
composition. The aim of this work was to develop a novel method for the accurate
quantification of mcl-PHA by GC for purified polymer as well as for mcl-PHA in
biomass directly. We found that the Lewis acid boron trifluoride (BF3) in methanol is
an effective transesterification reagent without significant formation of side-products.
We attribute this mainly due to the avoidance of water and protic acids. BF3 in
methanol is well known for the derivatisation of fatty acids (Lopez-Lopez et al. 2001;
Moffat et al. 1991; Rotzsche 1991; Shantha et al. 1993). The transesterification with
BF3-methanol was quantitative under optimized conditions for all mcl-PHAs analyzed
Quantitative analysis of mcl-PHA 42
in this study, and the copolymer composition could be confirmed by NMR
measurements.
2.2 Materials and methods
PHA derivatisation. The recovery and the composition of different purified PHAs
and of PHAs in biomass directly was determined by GC after their depolymerisation
and derivatisation to methyl or propyl esters. Each sample was derivatised twice for
independent analysis. The propanolysis according to Riis and Mai was applied to a
set of scl- and mcl-PHA as described by Riis and Mai (Riis and Mai 1988).
Further, this method was adapted for mcl-PHA to improve the recovery of propyl
esters. The following conditions were used: About 10 mg of polymer was transferred
into a 10 mL Pyrex tube. One mL of methylene chloride containing 10 mg mL-1 of
2-ethyl-2-hydroxybutyric acid as internal standard was added and the polymer was
let to dissolve at room temperature for one hour. One mL of a 20/80 (v/v) mixture of
HCl (37%) and n-propanol was added, the tube was tightly sealed, and the mixture
was vigorously shaken. The tube was placed in an oven at 80 °C for 16 h.
Subsequently, it was cooled to room temperature and 2 mL of demineralized water
was added. The mixture was vigorously shaken and after phase separation, the
aqueous (upper) phase was removed. The organic phase was dried over Na2SO4,
neutralized by adding Na2CO3, and filtered through a 1.0 µm nylon filter. In contrast
to the original method (Riis and Mai 1988), we chose a lower reaction temperature
but an extended reaction time. Furthermore, methylene chloride was used as solvent
instead of 1,2-dichloroethane and 2-ethyl-2-hydroxybutyric acid was used as internal
standard instead of benzoic acid. Additionally, the organic phase was dried and
neutralized to remove residual water and acid.
A similar transesterification procedure was developed using boron trifluoride
(BF3) as catalyst. In contrast to the transesterification catalyzed by hydrochloric acid,
the transesterification catalyzed by BF3 did not work with n-propanol. Therefore
methanol was used instead, forming methyl esters. About 10 mg of polymer was
transferred into a 10 mL Pyrex tube. One mL of methylene chloride containing 10
mg mL-1 of 2-ethyl-2-hydroxybutyric acid as internal standard was added. The
polymer was let to dissolve at room temperature for one hour. One mL of BF3 (1.3 M)
43 Chapter 2
in methanol was added, the tube was tightly sealed and the mixture was vigorously
shaken. The tube was placed in an oven and kept at 80 °C for 20 h. The subsequent
extraction and drying procedure was similar to the procedure described above,
except that the sample was extracted twice with 1 mL of a saturated NaCl solution for
a complete removal of the catalyst. Method optimization was carried out with 50 mg
of polymer and corresponding volumes of solvent and reagent to decrease the
measurement uncertainty. The measurement uncertainty was calculated from the
standard deviation of the transesterification (n=8) combined with uncertainties
inherent to the analytical system. Thereby, the mass of derivatised PHA was
important. The transesterification of larger masses (50 mg instead of 10 mg)
decreased the measurement uncertainty from ± 7.2% to ± 4.0%.
For the direct analysis of PHA, 50 mg biomass was derivatised in the same way
as described for 10 mg of purified PHA. For comparison, 5 g of biomass was
extracted twice with 100 mL methylene chloride for 24 h. The PHA solution was
filtrated, concentrated to 20% (v/v) and the polymer was precipitated by adding 200
mL of ethanol at 0 °C. The polymer was dried under vacuum at 40 °C for two days.
Analytical methods. For gas chromatographic analysis, one µL of the ester solution
was analyzed on a GC (GC 8575 Mega 2, Fisons Instruments, Rodano, Italy)
equipped with a Supelco SPB-35 (30 m x 0.32 mm) column with a film thickness of
0.25 µm (Supelco, Bellefonte, USA) at a split ratio of 1:10 and an initial temperature
of 80 °C. The temperature was raised with a rate of 10 °C min-1 to 240 °C. For
quantification purposes by FID, known amounts of pure 3-hydroxybutyric, -valeric, -
hexanoic, -octanoic, -nonanoic and -undecanoic acid were derivatised and measured
to calculate their response factors. 3-Hydroxyalkenoic acids were not available
commercially and their response factors were assumed to be similar to those of their
corresponding saturated alkanoic acids. The response factor of 3-hydroxy-6-
heptenoic acid was calculated by linear interpolation. 2-Ethyl-2-hydroxybutyric acid
(Sigma-Aldrich, Buchs, Switzerland) was used as internal standard because of its
chemical similarity to the 3-hydroxyalkanoic acids and because its elution time is
different to the 3-hydroxyalkanoic acids measured. Tetradecane (Sigma-Aldrich,
Buchs, Switzerland) was used as second internal standard. 3-Hydroxyalkanoic acids
were purchased from Larodan (Malmö, Sweden).
Quantitative analysis of mcl-PHA 44
NMR experiments in solution were performed on a Bruker AV-400 spectrometer
at 297 K using a 5 mm broad-band probe. For PHBV samples, typically 5 mg polymer
were dissolved in 0.7 mL CDCl3, for purified mcl-PHA samples, 30 mg per 0.7 mL
CDCl3 were used. NMR samples of whole cells were prepared by stirring 50 mg
biomass in 1 mL CDCl3 for 5 h followed by filtration. Chemical shifts are given in ppm
relative to the remaining signals of chloroform as internal reference (1H NMR: 7.26
ppm; 13C NMR: 77.0 ppm). Proton (1H) NMR spectra were recorded at 400.13 MHz
with a 9.6 µs 90° pulse length, 4460 Hz spectral width, 64 k data points, and 24
scans with a relaxation delay of 20 s were accumulated. Carbon (13C) NMR spectra
at 100.61 MHz with 1H WALTZ decoupling were recorded for the mcl-PHA samples
with the following parameters: 3.2 µs 45° pulse length, 26250 Hz spectral width, 64 k
data points, 5000 scans, relaxation delay 10 s, and decoupling field 2.5 kHz. 13C
NMR chemical shifts of PHA monomer units are compiled in the Supplementary
Information.
Propyl and methyl esters and side-products were analyzed by HPLC on a C18
Nucleosil column (2 x 250 mm, 3 µm, 100 Å, Macherey-Nagel Inc., Easton, USA).
Separation was achieved using a linear gradient from 10% to 80% acetonitrile in 20
min. 0.1% acetic acid in double distilled water was used as mobile phase. The flow
rate was 0.2 mL min-1 with injection volumes of 7.5 µL. Sample compounds were
identified by mass spectroscopy with an APCI ion source and an ion trap mass
detector (esquire HCT, Bruker Daltonics, Bremen, Germany).
To describe the monomeric composition of PHAs, the notation Cn:x was used,
where n is the number of carbon atoms and x indicates the position of the double
bond.
PHA biosynthesis. The following PHAs were produced by cultivation of Cupriavidus
necator for scl-PHA and with Pseudomonas putida GPo1 for mcl-PHA as described
elsewhere (Hartmann et al. 2006; Zinn et al. 2003) (see Table 2.1): Poly(3-
hydroxybutyrate) (PHB), from a carbon feed of 100% butyric acid; poly(3-
hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), from a carbon feed of 80% (mole
mole-1) butyric acid and 20% (mole mole-1) valeric acid (PHBV-a), and 100% valeric
acid (PHBV-b); poly(3-hydroxyoctanoate-co-3-hydroxyhexanoate) (PHO), from a
carbon feed of 100% octanoic acid; poly(3-hydroxy-10-undecenoate-co-3-hydroxy-8-
45 Chapter 2
nonenoate-co-3-hydroxyoctanoate-co-3-hydroxy-6-heptenoate-co-3-hydroxy-
hexanoate) (PHOUE), from a carbon feed of 46% (mole mole-1) octanoic acid and
54% (mole mole-1) 10-undecenoic acid; poly(3-hydroxy-10-undecenoate-co-3-
hydroxy-8-nonenoate-co-3-hydroxy-6-heptenoate) (PHUE), from a carbon feed of
100% 10-undecenoic acid; and poly(3-hydroxyundecanoate-co-3-hydroxynonanoate-
co-3-hydroxyheptanoate-co-3-hydroxypentanoate) (PHUA), from a carbon feed of
100% undecanoic acid. Polymers were purified by repeated extraction and
precipitation with methylene chloride and ethanol, respectively.
2.3 Results and discussion
The propanolysis method developed by Riis and Mai (Riis and Mai 1988) was applied
to various scl- and mcl-PHA. The data obtained with GC-FID were compared with the
results from NMR as shown in Table 2.1.
The monomeric composition of PHBV copolymers was determined from
quantitative 1H NMR spectra. The signals of the methyl groups of HB units at 1.28
ppm and of HV units at 0.90 ppm are well separated, and the ratio between these
intensities was used to calculate the polymer composition. The overall intensities of
the methine HB- and HV- resonances at 5.26 and 5.16 ppm such as that of the
backbone methylene protons resonating at 2.54 ppm were used to cross-check the
individual HB and HV fractions.
The copolymer composition of mcl-PHAs was also investigated with NMR
spectroscopy. The combined fractions of (C6:0 + C8:0) units and (C7:6 + C9:8 +
C11:10) units were readily obtained from 1H NMR spectra by integration of the
overlapping methyl group signals at 0.87 ppm and the olefinic proton signals at 5.78
ppm. For standardization, the intensities of the backbone –CH protons resonating at
5.17 ppm was set to 100 (mol%). The signals of the different saturated and
unsaturated PHA units overlap in the 1H NMR spectrum, but the resonances are
separated in the corresponding 13C NMR spectrum (Supplementary Information
Table 2.3). In these spectra, the intensity of the backbone -CH carbons resonating at
69.8 – 71.3 was then set to 100 (mol%). This resulted in relative intensities of 99 -
103 for the overlapping carbonyl carbons at 168.8 – 169.8 ppm and of 96 - 101 for
the backbone methylene carbons at 39.1 ppm. From this good agreement between
Quantitative analysis of mcl-PHA 46
the intensities for backbone carbons C1, C2 and C3 we concluded that a relaxation
delay of 10 s between individual pulses was sufficient to yield quantitative 13C NMR
data, and the intensities of resolved carbon signals of different monomer units were
used to determine the individual fractions. For example for sample PHOUE,
integration of the resonances at 13.9, 22.5 and 31.5 ppm for C8:0 yielded an intensity
of (32.9 ± 1.7). Similarly, fractions of C6:0 = (7.1 ± 0.8), C7:6 = (12.7 ± 0.2), C9:8 =
(38.2 ± 0.3) and C11:10 = (9.1 ± 0.4) were determined. The sum of C6:0 and C8:0
units is 40 mol%, close to the 41.3 mol% determined from the 1H NMR spectrum.
Also for the sum of unsaturated monomer units, the agreement between the 13C
NMR (60 mol%) and 1H NMR (58.7 mol%) data is satisfactory. NMR results were
converted to % w/w for direct comparison with GC data.
The original propanolysis method was quantitative for the transesterification of
PHB and PHBV containing a low amount of valerate. Nearly 100% of the polymer
was recovered as propyl ester. However, for PHBV with a high amount of valerate
and for PHAs with longer side-chains the reaction was not quantitative and the
recovery of propyl esters fell below 50% for PHU. The reaction time was not sufficient
for a complete reaction of mcl-PHA and side-products were observed for PHA with
unsaturated side-chains.
Therefore we tried to improve the HCl-propanol method for PHO and PHUE, two
common mcl-PHAs (Bear et al. 1999; Dufresne et al. 2001). Experiments with these
two co-polymers revealed that the maximum recovery of 97% for PHO, and 55% for
PHUE was reached after 16 h of propanolysis at 80 °C. Increasing the reaction
temperature or reaction time decreased the recovery of PHUE due to the formation of
side-products. Already after 16 h side-products were observed for PHUE in the GC-
chromatogram as shown in Fig. 2.1a. In contrast to PHUE the recovery of PHO
reached a plateau after 16 h and no considerable side-products were observed. The
improved HCl-propanol transesterification method (16 h at 80 °C, [HCl] = 1.2 M) was
quantitative for scl- and mcl-PHAs with saturated side-chains. Table 2.1 shows that
the recovery had significantly decreased for PHA containing unsaturated side-chains,
due to the formation of side-products (see Fig. 2.1a). Thus HCl-propanol is an
effective reagent for the transesterification of all PHAs with saturated side-chains
under these conditions.
47 Chapter 2
Table 2.1: Monomeric composition and recovery (% w/w) of different scl- and mcl-PHA analyzed with different GC-transesterification methods and with 1H NMR for scl–PHA and 13C-NMR for mcl-PHA, respectively. For GC-measurements with 10 mg of PHA a measurement uncertainty of ± 7.2% was determined.
PHAa Analysis C4:0 C5:0 C6:0 C7:0 C8:0 C9:0 C11:0 C7:6 C9:8 C11:10 Total
NMR 100
BF3-MeOH 45 45
HCl-PrOH1 98 98
PHB
HCl-PrOH2 100 100
NMR 81 19
BF3-MeOH 34 12 46
HCl-PrOH1 79 18 97
PHBV-a
HCl-PrOH2 78 19 97
NMR 35 65
BF3-MeOH 17 48 65
HCl-PrOH1 33 65 98
PHBV-b
HCl-PrOH2 29 59 88
NMR 14 86
BF3-MeOH 11 90 101
HCl-PrOH1 12 85 97
PHO
HCl-PrOH2 9 64 73
NMR 6 32 11 40 11
BF3-MeOH 4 31 10 39 15 99
HCl-PrOH1 5 30 3 21 6 65
PHOUE
HCl-PrOH2 4 25 3 18 5 55
NMR 23 60 17
BF3-MeOH 22 59 17 98
HCl-PrOH1 6 41 8 55
PHUE
HCl-PrOH2 8 30 7 45
NMR 1 33 50 16
BF3-MeOH 2 34 49 15 100
HCl-PrOH1 2 32 51 14 99
PHUA
HCl-PrOH2 2 22 33 9 66 aFermentation conditions see materials and methods. 1Transesterification method with HCl-propanol adapted for mcl-PHA: 16h, 80 °C, [HCl]=1.2 M. 2Transesterification method with HCl-propanol developed by Riis and Mai for PHB.
Quantitative analysis of mcl-PHA 48
Fig. 2.1: GC-FID-chromatograms of PHUE transesterified with the adapted HCl-propanol method of this work (a) and with BF3-methanol (b).
We suspected the C7:6-monomer to excessively undergo side-reactions during
propanolysis and to form the side-products observed by GC. To confirm this
hypothesis and to elucidate the structure of the side-products, 3-hydroxy-6-heptenoic
acid produced by fermentation and separated by column chromatography (Ren et al.
2005) was propanolysed for 2 days with HCl-propanol at 100 °C to quantitatively
convert it into side-products. LC-MS measurements showed that these products had
identical retention times and mass spectra as the dominant side-products from PHUE
obtained by transesterification with HCl-propanol. Two products with a molecular
mass of 186 u were detected with LC-MS, both possessing the same fragmentation
pattern. NMR experiments revealed that the two products were (2-S, 5-R) and (2-R,
5-R) diastereomers of (5-methyl-tetrahydrofuran-2-yl)-acetic acid propyl ester as
shown in the supplementary information, Fig. 2.8.
These side-products were generated by the intramolecular cyclisation of propyl
3-hydroxy-6-heptenoate. We propose that a secondary cation is formed, catalysed by
protic acids and that the furane ring is built by an intramolecular nucleophilic attack,
as shown in Fig. 2.2A. The chiral centre in position 5 is in R configuration due to
biosynthesis whereas the chiral centre in position 2 is generated during cyclisation.
The ratio between the (2-S, 5-R) and (2-R, 5-R) diastereomers is 69:31 according to 1H NMR. The chirality in position 5 induces asymmetry in the new chiral centre in
position 2. The formation of pyrane analogs was not observed, since the formation of
an intermediate primary cation is less favoured. Likewise, the formation of 7- and 9-
0 5 1 0 1 5
C 1 1 :1 0
C 9 :8
C 7 :6Rel
ativ
e in
tens
ity [a
.u.]
T im e [m in ]
a
b
S id e p ro d u c tsC 7 :6
C 9 :8
C 1 1 :1 0
In te rn a l s ta n d a rd
49 Chapter 2
membered rings from propyl 3-hydroxy-8-nonenoate and propyl 3-hydroxy-10-
undecenoate was not observed. The addition of H2O to the double bond led to further
side-products. This reaction most likely proceeds again through the secondary
cation. Subsequently, H2O acts as nucleophile to generate a secondary alcohol as
shown in Fig. 2.2B. According to the NMR data for PHU, 32% and 53% of the C9:8-
and the C11:10-propyl esters were lost by this and other side-reactions, whereas
74% of the C7:6-monomer was lost mainly through cyclisation.
O
O
nHO
O
O
O
O
O
H
H+,∆T
-H+
n-propanol
A)
O
O
n
H+, ∆Tm
O
O
n
m
H
O
O
n
m
H
OH
H2O, ∆T
-H+
B)
Fig. 2.2: Side-reactions of PHUE during transesterification with HCl-propanol. A) Cyclisation of propyl 3-hydroxy-6-heptenoate. B) Water addition to unsaturated side-chains (m = 1-3).
Improved results for the transesterification of mcl-PHAs were obtained with
BF3-methanol as derivatisation reagent. PHO and PHUE were used to optimize the
conditions of this reaction. Best results were obtained for a reaction time of 20 h at 80
°C with a BF3-concentration of 0.65 M. Similar to the transesterification with HCl-
propanol, PHO was quantitatively methanolysed with BF3-methanol without forming
side-products. After 8 h at 100 °C and 20 h at 80 °C the maximal recovery was
reached as shown in Fig. 2.3. Further reaction time did not reduce the recovery
substantially. showed a similar reactivity, but the conversion to side-products was
Quantitative analysis of mcl-PHA 50
observed when the reaction time was further elongated, as shown in Fig. 2.4. At the
maximum recovery at 80 °C only negligible peaks of side-products were observed in
the chromatogram and about 98% of the polymer could be recovered as methyl
esters.
The influence of the boron trifluoride concentration in the reaction mixture was
studied for PHO and PHUE derivatised at 80 °C for 20 h. The optimum boron
trifluoride concentration was 0.65 M for both polymers. Increasing this concentration
to 0.87 M reduced the recovery of PHO from 100 to 53% (w/w) and the recovery of
PHUE from 98 to 61% (w/w) (Fig. 2.5 and Fig. 2.6). Similarly, a decrease in the
concentration of BF3 to 0.16 M reduced the recovery of PHO to 34% (w/w) and the
recovery of PHUE to 24% (w/w).
Low recoveries can also be caused by steps following the derivatisation of PHA.
Jan et al. (Jan et al. 1995) described the loss of short 3-hydroxyalkanoic acid methyl
esters during aqueous extraction, leading to incorrect results. To exclude this
phenomenon for mcl-PHA, the aqueous phases of the extraction from PHO and
PHUE were dried and analyzed, but no methyl esters could be detected. Hence,
3-hydroxyhexanoic methyl ester and the corresponding monomers with longer chains
did not migrate to the aqueous phase.
To study the possible influence of the polymer concentration on the recovery of
the BF3-transesterification, the concentration of PHO was varied between 0.1 - 21.4
mg mL-1. The recovered ratio for C8:0 to C6:0 was 8.9 (w/w) in all cases. However,
the total recovery decreased slightly for small concentrations. For concentrations
above ~3 mg mL-1, the recovery was always greater than 97% (w/w), and was
approximately (93.5 ± 1)% (w/w) in the concentration range 0.1 – 1.5 mg mL-1. This is
attributed to adsorptive effects of glass surfaces and of the GC injection system.
Similar trends were observed for the BF3-transesterification of PHU. This
concentration was varied between 0.1 – 19.7 mg mL-1. For example for C7:6, the
mean value of recovery was 20% (w/w), with maximum deviations of +3.4% (w/w) for
a concentration of 19.7 mg mL-1, and -3.5% (w/w) for 0.2 mg mL-1.
51 Chapter 2
0 25 500
50
100R
ecov
ery
[% w
/w]
Derivatisation time [h]
abcd
e
f
Fig. 2.3: BF3-methanol transesterification of PHO at 80 °C (�) and 100 °C (�), [BF3] = 0.65 M. a) sum of monomers at 80 °C, b) sum of monomers at 100 °C, c) 3-hydroxyoctanoate at 80 °C, d) 3-hydroxyoctanoate at 100 °C, e) 3-hydroxyhexanoate at 100 °C, f) 3-hydroxyhexanoate at 80 °C. A measurement uncertainty of ± 4% was determined for 50 mg of PHA.
0 25 500
50
100
c
a
b
hgfe
Rec
over
y [%
w/w
]
Derivatisation time [h]
d
Fig. 2.4: BF3-methanol transesterification of PHUE at 80 °C (�) and 100 °C (�), [BF3]=0.65 M. a) sum of monomers at 80 °C, b) sum of monomers at 100 °C, c) 3-hydroxy-8-nonenoate at 80 °C, d) 3-hydroxy-8-nonenoate at 100 °C, e) 3-hydroxy-6-heptenoate at 80 °C, f) 3-hydroxy-10-undecenoate at 80 °C, g) 3-hydroxy-10-undecenoate at 100 °C, h) 3-hydroxy-6-heptenoate at 100 °C. A measurement uncertainty of ± 4% was determined for 50 mg of PHA.
Quantitative analysis of mcl-PHA 52
0.1 0.5 0.90
50
100
ab
c
Rec
over
y [%
w/w
]
Concentration of BF3 [M]
Fig. 2.5: BF3-methanol transesterification of PHO at 80 °C for 20 h with variable concentrations of BF3. a) sum of monomers, b) 3-hydroxyoctanoate, c) 3-hydroxyhexanoate. A measurement uncertainty of ± 4% was determined for 50 mg of PHA.
0.1 0.5 0.90
50
100
Rec
over
y [%
w/w
]
Concentration of BF3 [M]
a
b
cd
Fig. 2.6: BF3-methanol transesterification of PHUE at 80 °C for 20 h with variable
concentrations of BF3. a) sum of monomers, b) 3-hydroxy-8-nonenoate, c) 3-hydroxy-10-undecenoate d) 3-hydroxy-6-heptenoate. A measurement uncertainty of ± 4% was determined for 50 mg of PHA.
53 Chapter 2
The transesterification with BF3-methanol was quantitative for all mcl-PHAs
and the data were in good agreement with the results obtained by NMR as shown in
Table 2.1. In contrast to mcl-PHAs, scl-PHAs were only recovered to 45-65% (w/w).
This loss is due to the partitioning of short-chain methyl esters between the organic
and the aqueous phase during extraction as described previously (Huijberts et al.
1994; Jan et al. 1995). In this work, NaCl-saturated water (Niskanen et al. 1978) was
used instead of pure water, but this could not inhibit an extensive loss of short-chain
methyl esters in our experiments. Hence, methanolysis should not be applied to scl-
PHA when an aqueous extraction is necessary to remove reagents. In case of PHUE
negligible amounts of side-products were observed. Compared with the HCl-
propanolysis, more than three times the amount of C7:6-monomer, and increased
amounts of C9:8 and C11:10 monomers were detected. The recovery improved from
55 to 98% (w/w).
The quantitative determination of PHA in biomass is of particular importance.
Therefore, the transesterification method with BF3-methanol was applied to biomass
with different contents of PHOUE. Equal amounts (50 mg) of these biomass samples
were methanolysed to study if other biomass components interfere with the methyl
esters from PHA. A small number of weak additional peaks was observed, but they
did not superpose the peaks of interest. The monomeric composition of PHOUE
remained nearly constant for samples 1-4 as shown in Table 2.2. For instance, the
relative amount of C8:0 in the polymer varied between 43.4% and 42.1% (w/w). Only
in sample 5, larger deviations were observed.
To determine the PHA content alternatively, large samples of biomass (5 g) were
extracted twice with methylene chloride and the polymer was precipitated with
ethanol. The mass of PHOUE was determined by gravimetry after drying under
vacuum. PHA contents between 3.4% and 16.8% (w/w) and between 2.9% and
15.9% (w/w) were determined by gravimetry and GC, as shown in Table 2.2. The
correlation with the gravimetric determination showed a relative difference between
0% and 5.5% except at the lowest PHA concentration where the difference was
14.7% (Table 2.2). The extraction recovery was higher than the one determined by
GC. This is reasonable since non-polar cell components like membrane lipids can be
co-extracted and lead to an overestimation of the PHA content determined by
gravimetry. Especially at low PHA contents this effect became marked. Further, we
Quantitative analysis of mcl-PHA 54
observed that biomass with low contents of PHA comprises higher amounts of non-
polar extractable impurities.
Table 2.2: Comparison between the quantitative analysis of mcl-PHA in biomass with GC after methanolysisa and the gravimetric determination after extractionb. Five biomass samplesc with different contents of PHOUE were analyzed to determine the relative monomeric composition (% w/w) and the PHA content (% wPHA/wbiomass).
Sample C6:0
GC
C8:0
GC
C7:6
GC
C9:8
GC
C11:10
GC
PHA
content
GC
PHA
content
gravimetry
Relative
difference
PHA
content
1 5.0 43.4 10.1 32.1 9.4 15.9 16.8 5.4
2 5.0 43.3 10.0 32.5 9.2 12.0 12.7 5.5
3 5.1 42.9 10.2 32.6 9.2 9.8 9.8 0.0
4 5.3 42.1 10.5 33.3 8.8 5.7 5.7 0.0
5 3.5 41.4 10.3 34.5 10.3 2.9 3.4 14.7
a) 50 mg of biomass was methanolysed with BF3-methanol (at 80 °C for 20 h). The measurement uncertainty is ≥ 7%. b) 5 g of biomass was extracted twice with methylene chloride, the solution was concentrated and precipitated with an excess of ethanol. The measurement uncertainty is around 5%. c) Sample 1 and 5 were produced by continuous fermentation under different conditions. Samples 2-4 were obtained by mixing sample 1 and 5 with varying ratios.
2.4 Conclusions
GC analysis is a common and efficient tool to quantify bacterial PHA. This study
however emphasised the need for novel derivatisation methods for the analysis of
mcl-PHA. A common transesterification method (Riis and Mai 1988) using HCl-
propanol was shown to be inadequate for the quantitative analysis of mcl-PHA due to
a serious underestimation of the PHA content. Although the adaptation of this
method was successful for mcl-PHA with saturated side-chains, another
derivatisation method was needed for mcl-PHA with unsaturated side-chains, to
avoid an excessive formation of side-products. The main side-products of PHUE
55 Chapter 2
derivatised with HCl-propanol were identified as the diastereomers of propyl 5-
methyltetrahydrofuran-2-acetate formed by cyclisation of propyl 3-hydroxy-6-
heptenoate, and as the products of water addition at the double bond. Other protic
catalysts like sulphuric acid will produce the same side-products and hence generate
incorrect results.
The formation of these side-products was avoided by using the Lewis-acid BF3 in
a water-free solvent. Transesterification with BF3-methanol was applied successfully
to all mcl-PHA investigated in this study. This novel method is well suited for the
detection of mcl-PHA in biomass where other analytical methods fail. In comparison
with the adapted HCl-propanol transesterification, only a few non-interfering
additional peaks from other lipids of biomass appeared in the spectrum, which
simplifies the analysis of data considerably.
NMR spectroscopy has been widely used to investigate various aspects of PHA,
including copolymer compositions, linkage sequences or metabolic pathway studies.
However, the measurement time for a single quantitative 13C spectrum was
approximately 16 hours; therefore, NMR spectroscopy is not suitable for the routine
determination of PHA copolymer composition. We also stirred whole cells in CDCl3
for 5 hours and measured NMR spectra directly after filtration. In these spectra,
several unidentified signals appeared that overlapped with PHA resonances. This
means that the dissolution of PHA from biomass is not selective and that also the
time-consuming extraction and purification steps can not be avoided for NMR
analysis of PHA. This is in contrast to the GC method with BF3-methanol presented in
this work that was used to quantify PHA in biomass directly. This method is
convenient to derivatise large amounts of samples in parallel and to measure them in
a short time, since one GC-measurement requires only about 20 minutes.
2.5 Acknowledgment
We gratefully acknowledge the financial support by the Empa R&D fund. We also
thank Thomas Ramsauer for assisting with polymer extractions, Elisabeth Michel for
supporting GC analyses, and Matthias Nagel for chemical advices.
Quantitative analysis of mcl-PHA 56
2.6 Supplementary information
Table 2.3: 13C NMR chemical shift assignments of the incorporated monomer units 3-hydroxypentanoate (C5:0), 3-hydroxyhexanoate (C6:0), 3-hydroxyoctanoate (C8:0), 3-hydroxy-6-heptenoate (C7:6), 3-hydroxy-8-nonenoate (C9:8), and 3-hydroxy-10-undecenoate (C11:10) in mcl-PHA.
δδδδ(13C) relative to δδδδ(CDCl3) = 77.0 ppm
Position C5:0 C6:0 C7:0 C8:0 C9:0 C11:0 C7:6 C9:8 C 11:10
1 168.8 – 169.8
2 38.7 39.1
3 69.8 – 71.3
4 26.7 35.9 33.5 33.6 33.8 33.8 32.9 33.8 33.8
5 9.3 18.3 27.1 24.7 25.0 25.1 29.2 24.5 25.0
6 13.8 22.4 31.5 29.0 29.5* 137.2 28.5 28.9*
7 13.9 22.5 31.7 29.4* 115.3 33.5 29.2*
8 13.9 22.5 29.2* 138.5 28.8*
9 14.0 31.8 114.6 33.7
10 22.6 139.0
11 14.1 114.2
* Positions not assigned
OO
O
8
176
2
3 4
510
9
Fig. 2.7: Numbering of carbon atoms used for the signal assignments of compound 1.
57 Chapter 2
Table 2.4: Chemical shifts of the furanoyl isomers.
Position a) δδδδ(1H) / ppm δδδδ (13C) / ppm
1a 1b 1a 1b
1 1.21 1.23 21.2 21.4
2 4.11 3.97 74.9 75.7
3 1.49/2.05 1.48/1.99 33.6 32.7
4 1.61/2.16 1.63/2.06 32.1 31.2
5 4.40 4.22 74.8 75.4
6 2.43/2.61 2.46/2.64 41.1 41.3
7 171.4 171.4
8 4.05 4.05 66.0 66.0
9 1.65 1.65 22.0 22.0
10 0.94 0.94 10.3 10.3
a) For atom numbers, see Fig. 2.7.
Quantitative analysis of mcl-PHA 58
Table 2.5: 1H, 13C-HMBC correlations used for NMR shift assignments and resolved 1H,
1H
coupling constants.
Compound HMBC correlations (w = weak) 1 J(H,H) / Hz
1a H-(1) → C-(2, 3); H-(3) → C-(1, 4); H-
(4) → C-(3, 6); H-(5) → C-(2, 3(w),
7(w)); H-(6) → C-(4, 5, 7); H-(8) → C-
(7, 9, 10); H-(9) → C-(8, 10); H-(10)
→ C-(8, 9)
J(1,2) = 6.1; J(5,6a) = 6.6; J(5,6b)
= 6.8; J(6a,6b) = 14.9; J(8,9) = 6.8;
J(9,10) = 7.5
1b H-(1) → C-(2, 3); H-(3) → C-(1,
4(w)); H-(4) → C-(3(w), 5(w)); H-(5)
→ C-(7(w)); H-(6) → C-(4, 5, 7); H-
(8) → C-(7, 9, 10); H-(9) → C-(8, 10);
H-(10) → C-(8, 9)
J(1,2) = 6.1; J(5,6a) = 6.7; J(5,6b)
= 6.8; J(6a,6b) = 15.1; J(8,9) = 6.8;
J(9,10) = 7.5
(S)
O(R) O
O
H3C
(R)
O(R) O
O
H3C
H
H
1a
1b
Fig. 2.8: Chemical structures with assignments of the absolute configuration of the mixture of isomers (1a and 1b) of (5-methyl-tetrahydro-furan-2-yl)-acetic acid propyl ester.
The signals at 4.40 and 4.22 ppm in the 1H NMR spectrum of the mixture of
isomers (1a and 1b, respectively) do not interfere with other resonances. Relative
amounts of 69% (1a) and 31% (1b) were determined from the integration of these
signals. The relative configurations were established with a series of 1D NOESY
spectra. When the pair of doublets at 1.2 ppm was selected, only H-(5) of 1a showed
the proximity to the corresponding methyl group, together with H-(2) for both
diastereomers at 4.11 and 3.97 ppm, respectively (see Fig. 2.9). Selective excitation
59 Chapter 2
at 4.40 ppm (H-(5) of 1a) showed an NOE to H-(1), whereas the analogous
experiment performed with H-(5) of 1b did not show an effect on the methyl group of
this isomer (spectra not shown).
4.14.24.34.4 ppm
a)
b)
4.14.24.34.4 ppm
a)
b)
Fig. 2.9: Sections of 1H NMR spectra of the 1a/1b mixture of diastereomers: a) 1D-NOESY experiment with selection of H-(1) at 1.2 ppm and b) normal 1H NMR spectrum.
Experimental
The 1H and 13C NMR spectra were recorded at 400.13 (100.61) MHz on a Bruker
Avance-400 NMR spectrometer. The 1D 1H and 13C NMR spectra and the 1H,1H and 1H,13C 2D correlation experiments were performed at 297 K using a 5 mm broadband
inverse probe with z-gradient (100% gradient strength of 10 G cm–1) and 90° pulse
lengths of 6.7 µs (1H) and 14.9 µs (13C). The chemical shifts were referenced
internally using the resonance signals of CDCl3 at 7.26 (1H) and at 77.0 (13C) ppm.
The gradient selected HSQC (Davis et al. 1992) (HMBC; (Bax and Marion 1988))
experiments were performed with selection of 1H,13C coupling constants of 145 (10)
Hz, gradient strengths of 80 : 20.1 : 11: –5 (50 : 30 : 40.1), applying a carbon
decoupling field of 3.1 kHz for the HSQC experiments (GARP decoupling; (Shaka et
al. 1985)). Before processing the data matrices of 1024 x 512 were zero filled to 1024
x 1024. The HSQC-TOCSY (Palmer et al. 1992) spectra were recorded with the
selection of 1J (1H,13C) = 145 Hz and a 27 µs 90° pulse length for the TOCSY transfer
with a total mixing time of 110 ms, applying the above mentioned carbon decoupling
Quantitative analysis of mcl-PHA 60
conditions, data matrices and processing conditions. The 1H,1H connectivity’s and the
relative configuration of the isomers were established by DQF-COSY and by 1D
NOESY experiments (mixing time of 1s, 50 ms selective 180° inversion pulse with
Gaussian shape) (Stott et al. 1997).
61
Chapter 3
Biosynthesis of medium-chain-length
poly(3-hydroxyalkanoate): endotoxin reduction by
phosphorus limitation
Biosynthesis of mcl-PHA under P-limitation 62
3.1 Introduction
PHAs with side chains of medium-chain-length (mcl-PHA, monomers with 6-14 car-
bon atoms) possess a wide range of physical and chemical properties due to their
manifold monomeric compositions. They are synthesized by microorganisms under
appropriate growth conditions, such as nitrogen limitation (Witholt and Kessler 1999).
The production of mcl-PHAs is well established with Gram-negative bacteria but its
contamination with lipopolysaccharide (LPS) from the outer membrane restrains the
application in the medical field (Valappil et al. 2007; Williams et al. 1999). LPS, also
referred to as endotoxin, represents one of the microbial molecular signals responsi-
ble for the activation of the innate immune system (Trent et al. 2006). It is recognised
by receptor TLR4 (toll-like receptor 4) that is present on many cell types like macro-
phages and dendritic cells. Even nanogram amounts of LPS cause fever, septic
shock and other immune responses when injected intravenously into humans.
Hence, its concentration in medical products has to be very low, e.g. the endotoxic
activity has to be smaller than 20 endotoxic units (EU) per medical device (U.S. De-
partment of health and human services 1997).
To obtain mcl-PHA of medical purity, several purification steps are necessary for
its recovery from biomass and special procedures have to be applied for endotoxin
removal (Lee et al. 1999; Williams et al. 2001). This complicates the downstream
processing and increases the production costs. Preventing the synthesis of LPS or
reducing their endotoxic activity during the production of mcl-PHA would drastically
simplify the recovery procedure. However, LPS are essential for the survival of
Gram-negative bacteria as they augment the rigidity of the cell wall and protect bac-
teria for instance from bile salts, gut enzymes and antibiotics (Holst 1995). Among
Gram-negative bacteria, only mutants of Neisseria meningitidis were viable without
LPS, presumably due to the presence of capsular polysaccharide (Bos and Tom-
massen 2005; Dixon and Darveau 2005; Raetz et al. 2007).
Indeed, the biological activity of LPS and in particular its endotoxic activity is
largely dependent on the structure of lipid A, the lipophilic part of LPS (Dixon and
Darveau 2005; Trent et al. 2006). Reducing the degree of acylation or phosphoryla-
tion drastically decreases the biological activity of LPS as shown by Rietschel et al.
(Fig. 3.1, (Rietschel et al. 1994)). Certain bacteria adapt the structure of lipid A to the
environmental growth conditions. Yersinia pestis for instance reduces the degree of
63 Chapter 3
acylation of lipid A during invasion of mammals to avoid the host’s immune defence
(Knirel et al. 2005). Furthermore, it has been observed that some pathogenic organ-
isms lack one or both phosphate groups of lipid A (Trent et al. 2006). But so far, it
has been unknown whether non-pathogenic bacteria can be stimulated by environ-
mental conditions to reduce the degree of phosphorylation of lipid A.
Fig. 3.1: Lipid A – structure activity relationships. Shown are independent chemical changes of the lipid A structure of Escherichia coli and the factor by which the modified structure is less biologically active than the reference lipid A (Rietschel et al. 1994).
In this work we investigated whether the endotoxic activity of crude PHA can be
lowered by phosphate-limited growth conditions such that the phosphorylation of lipid
A is reduced. This is of great importance because the endotoxic activity of lipid A is
decreased by a factor of 100 or more when one phosphate group is removed
(Rietschel et al. 1994). To realize such a scenario, we used carbon and nitrogen lim-
ited conditions ((C,N)-limitation) as starting point for the cultivation of Pseudomonas
putida GPo1 to induce PHA accumulation (Hartmann et al. 2006). Next, the phos-
phate concentration in the mineral medium was reduced stepwise to reach phospho-
rus limitation (P-limitation) as part of a triple (C,N,P)-limitation. Drastic changes in the
cell composition were observed with P-limitation.
Modification Reduction factor
1a 102
1b 102
1a + 1b Non toxic
2 107
3a 102
3a + 3b 107
4 102
5 101
Biosynthesis of mcl-PHA under P-limitation 64
3.2 Materials and methods
Biosynthesis of mcl-PHA
Polyhydroxyoctanoate (PHO) consisting of 87% (w/w) 3-hydroxyoctanoate and 13%
(w/w) 3-hydroxyhexanoate was produced by balance-controlled continuous cultiva-
tion of P. putida GPo1 (ATCC 29347) in a 3 L bioreactor (KLF 2000, Bioengineering,
Wald, Switzerland) with a working volume of 2.6 L and a dilution rate (D) of 0.125 h-1.
The preculture was prepared as described elsewhere (Durner et al. 2000; Hartmann
et al. 2006). An aliquot of 200 mL of a shake-flask culture was used to inoculate the
bioreactor containing 2.4 L of fermentation medium.
For continuous cultivation, the following medium was used (per liter): 0.80 g of
EDTA-Na2·2H2O, 3.78 g of (NH4)2SO4, 1.00 g of MgSO4·7H2O, 0.28 g of
FeSO4·7H2O, 0.03 g of CaCl2·2H2O and 1.05 g of MOPS was added as pH buffer.
The carbon to phosphorus ratio (C/P) in the mineral medium was adjusted by the ad-
dition of KH2PO4 to the medium (e.g. 1.17 g of KH2PO4 for C/P = 30 g g-1), whereas
the carbon source octanoic acid was pumped separately to the culture broth (result-
ing in a carbon feed concentration of 8.0 g C L-1). As a consequence the carbon to
nitrogen ratio (C/N) in the medium feed was fixed to 10 g g-1. For fermentations with
nitrogen excess the concentration of (NH4)2SO4 was increased to 11.34 g L-1 result-
ing in a C/N of 3.3 g g-1. Furthermore, 1 L of the medium was supplemented with 2
mL of trace element stock solution that contained per liter: 12.22 g of MnCl2·4H2O,
1.27 g of CoCl2·6H2O, 2.00 g of CuCl2·2H2O, 7.50 g of ZnSO4·7H2O, and 0.5 g of
Na2MoO4·2H2O in 1M HCl. The medium was sterilized by filtration. Octanoic acid
(95 % w/w) was pumped directly into the culture vessel by using a dosimat (Metrohm,
Herisau, Switzerland). The dissolved oxygen concentration was maintained between
30 and 50% relative to the maximum oxygen concentration in the aerated culture
medium before inoculation. Poly(propylene glycol) (Mw = 4000) was added to oc-
tanoic acid to 4% (v/v) to avoid excessive foam formation. For each phosphate con-
centration, at least five volume exchanges were allowed to reach steady state. Two
measurements were made within 24 h.
The fermentation broth was concentrated by centrifugation at 4’500 x g for 20
min. The paste was lyophilized for 48 h, ground, and the dry biomass was stored in a
closed container at -25 °C. The supernatant was filtrated (PES filter, 0.22 µm) and
stored at -25 °C.
65 Chapter 3
Analytical methods
PHA determination. The PHA content in biomass was determined in duplicate by
GC after propanolysis of biomass as described in Chapter 2.
Endotoxin detection. The endotoxic activity in aqueous solutions was determined
with the PyroGene recombinant factor C assay (Lonza Bioscience, Visp, Switzer-
land). For the endotoxic activity of biomass, 1 g of dried biomass was washed twice
with 50 mL of 0.9% NaCl by vortexing and centrifugation to remove free LPS. The
pellet was freeze dried. Twenty mg washed biomass was suspended in 10 mL buffer
(2.3 g SDS, 11.5 mL glycerol, 12.4 mL 0.5 M Tris-HCl and 76.1 mL H2O adjusted to
pH = 6.8 with HCl), heated up to 95 °C for 10 min to lyse the cells and then shaken at
150 rpm at room for 24 h. The suspension was filtrated (PES filter, 0.22 µm) and di-
luted in three steps (1:100, 1:100 and 1:500) to a total dilution of 1:5x106. One hun-
dred µl of this solution were used to measure the endotoxic activity with the recombi-
nant factor C assay. For the endotoxic activity of PHA, 200 mg of washed biomass
was extracted with 5 mL of methylene chloride under stirring at room temperature for
20 h. The suspension was centrifuged, filtrated (Nylon filter, 0.45 µm) and 3.5 mL of
the resulting polymer solution was poured into a small Petri dish (d=4 cm) that had
been depyrogenized previously at 200 °C for 6 h. The solvent was slowly evaporated
(3 days) to form PHA films with a homogenous surface. Endotoxins were extracted
from the surface with 3 mL of endotoxin-free water at room temperature for 24 h. An
aliquot of 100 µl of this solution was then used to measure the endotoxic activity.
The recombinant factor C of the Pyrogene assay is activated by endotoxin-
binding, and the active moiety created then acts to cleave a synthetic substrate,
which results in the generation of a fluorogenic compound. After one hour of incuba-
tion, the fluorescence at 440 nm can be measured by exciting at 380 nm and the en-
dotoxic activity is determined relative to an endotoxin standard. One endotoxic unit
(EU) corresponds to about 0.1 ng of standard endotoxin.
Biomass concentration. Cells were collected on pre-weighed polycarbonate filters
(0.2 µm, Nucleopore, Sterico AG, Dietikon, Switzerland). The filters were first washed
with 10 mM MgCl2, dried overnight at 105 °C and weighed. An aliquot of 3 mL of cell
suspension was filtered through the filter and the filter was dried again at 105 °C
overnight. The mass difference was used to calculate the concentration of dry bio-
mass in the culture.
Biosynthesis of mcl-PHA under P-limitation 66
Optical density. The cell suspension was diluted 1:100 with deionised water and the
optical density was determined at 450 nm.
Analyses of substrate concentrations in the culture supernatant. Ammonium
was measured by using a photometric ammonium test (Spectroquant, Merck Darm-
stadt, Germany). The measurement range was linear between 0.01 and 3 mg N L-1
(present as ammonium). If necessary, the samples were diluted with deionised water.
Octanoic acid was measured by gas chromatography after derivatisation to propyl
esters. An aliquot of 5 mL of methylene chloride containing 1 mg mL-1 of 2-ethyl-2-
hydroxybutyric acid as internal standard was added to 2 mL of supernatant in a 20
mL Pyrex tube. Five mL of a 20/80 (v/v) mixture of HCl (37%) and n-propanol was
added, the tube was tightly sealed, and the mixture was vigorously shaken. The tube
was placed in an oven at 80 °C for 16 h. The subsequent steps were similar to the
derivatisation of PHA as described above.
Phosphate was determined according to the ascorbic acid molybdane blue
method (Chen et al. 1956). An aliquot of 10 mL of supernatant was mixed with 5 mL
of freshly prepared reagent (containing sulphuric acid, ammonium molybdate and
ascorbic acid) and supplemented with deionised water to 50 mL. The mixture was
shaken and the absorbance at 800 nm was measured after 15 minutes of reaction at
room temperature. The measurement range was linear between 0.1 and 100 mg P
L-1 (present as phosphate), for higher concentrations dilution was necessary.
Analyses of biomass. Total phosphorus was determined as follows: About 300 mg
of dried biomass and 3 mL of HNO3 (65%) were added to a 20 mL cone. An aliquot of
2 mL of distilled water and 1 mL of H2O2 (30%) were added to a 30 mL vessel and
the cone was placed therein. The sample was digested in a microwave digester
(Milestone, MLS 1200 Mega, Sorisole, Italy) for 15 minutes with a stepwise increase
from 100 to 500 W. After cooling, the clear solution was filled up with distilled water to
a volume of 10 mL. The solution was diluted between 1:10 and 1:100 with 2% HNO3
and the emission was measured with ICP-OES (Perkin Elmer, Optima 3000,
Schwerzenbach, Switzerland) at 213.6 and 214.9 nm. Standard solutions were pre-
pared from a 1.00 mg P L-1 standard solution (Alfa Aesar, Karlsruhe, Germany) for
external calibration.
Phospholipids were extracted with chloroform-methanol according to method of
Folch (Folch et al. 1957). Quantitative analysis was done by a colorimetric method
based on the formation of a complex between phospholipids and ammonium
67 Chapter 3
ferrothiocyanate (Stewart 1980). Phospholipids extracted from 200 mg of dried bio-
mass were dissolved in 2 mL of chloroform. To an aliquot of 0.1 mL, 1.9 ml of chloro-
form and 2 mL of thiocyanate reagent was added. The mixture was vortexed for 1
min and centrifuged for 10 min at 2’000 x g. The lower layer was removed and the
light absorbance of this solution was measured at 488 nm. 1,2-Dipalmitoyl-sn-
glycero-3-phosphoethanolamine was used for calibration. The linear range was be-
tween 1.5 and 30 mg L-1.
3.3 Results and discussion
General observations
The adaptation of P. putida GPo1 to P-limitation was generally slow and a drastic de-
crease of the phosphorus concentration in the mineral medium feed (e.g. from 200 to
100 mg P L-1) was not tolerated by the cells. Excessive foam generation as well as
biofilm formation were observed that soon led to instable process conditions. Even
with small changes of the phosphorus concentration, wall growth could not be pre-
vented completely. C/P ratios higher than 90 g g-1 lead to instable cultures. One pos-
sible reason is that the specific growth rate of the cells was decreased by very low
phosphorus concentrations, so that octanoic acid could not be consumed completely,
resulting in a substrate inhibition finally leading to a wash-out of the culture. Under
phosphorus limitation, the cell shape became more rod-like and the brown colour of
the fermentation broth brightened.
Nutrient limitations
The accumulation of PHA in bacteria can be triggered by nutrient limitations such as
nitrogen limitation. Carbon limitation is commonly applied to prevent substrate inhibi-
tion. For the production of mcl-PHA with P. putida GPo1 the combined (C, N)-
limitation strategy has successfully been applied (Durner et al. 2000; Hartmann et al.
2006; Ruth et al. 2007).
Nutrient limitation has often been described using the kinetic model of Monod
(Monod 1949), although this model neglects bacterial adaptations to prolonged nutri-
ent limitation (Ferenci 1999). The concentration of the limiting nutrient (s) in the cul-
ture broth can be calculated with Monod’s equation if the specific growth rate (µ), the
maximum specific growth rate (µm) and the half saturation coefficient (Ks) are known:
Biosynthesis of mcl-PHA under P-limitation 68
[1]
For chemostat cultures at steady state the specific growth rate is equal to the di-
lution rate and consequently the concentration of the limiting nutrient is constant.
However, the Monod model is only valid for single nutrient limitation. A simple kinetic
model for dual and triple-limited fermentations does not exist. Hence a simple ap-
proximation was used to define kinetic nutrient limitation. Here limitation was defined
as the state where the residual concentration of a nutrient becomes virtually constant
at a low level. A further decrease of the nutrient concentration in the medium does
not affect the residual concentration significantly.
The measured concentration of a nutrient is in fact compromised by method of
measurement. During the time between sample drawing and centrifugation, the bac-
teria continue to consume the residual nutrients in the medium. The measured nutri-
ent concentrations are consequently lower than at the time point of sampling. Ex-
trapolating from the observed nutrient consumption rates, a decrease in phosphorus
concentration of about 0.7 mg L-1 has to be considered for a concentration of about 7
g biomass L-1 for a delay of 2 minutes between sampling and centrifugation. Simi-
larly, a reduction of 34 mg/L has to be taken into account for carbon and 3.4 mg/L for
nitrogen for a period of 2 minutes.
The limiting (measured) concentrations of nutrients obtained were 0.3 mg P L-1, 3
mg N L-1 and 10 mg C L-1. The values for phosphorus and nitrogen appeared reason-
able as similar values, 0.2 mg P/L and 2.2 mg N/L could be calculated with Ks values
from E. coli and P. putida PGA1 (17 µM for phosphate and 460 µM for ammonium,
respectively) using the Monod equation (Annuar et al. 2007; Owens and Legan
1987). According to the experiments, phosphorus limitation started at a C/P = 65
g g-1. Further, it was observed that nitrogen became non-limiting at a C/P = 75 g g-1
and octanoic acid at a C/P = 85 g g-1. According to these boundaries four regimes of
nutrient limitation could be distinguished:
- Dual (C,N)-limited regime: C/P = 30-65 g g-1 (I)
- Triple (C,N,P)-limited regime: C/P = 65-75 g g-1 (II)
- Dual (C,P)-limited regime: C/P = 75-85 g g-1 (III)
- Single P-limited regime: C/P = 85-95 g g-1 (IV)
s=KS
m−
69 Chapter 3
0.1
1
10
100
1000
1
10
100
1000
10000
20 30 40 50 60 70 80 90 1000.1
1
10
100
Con
cent
ratio
n of
nitr
ogen
[mg
L-1]
B
Con
cent
ratio
n of
car
bon
[mg
L-1]A
I II III IV
C/P [g g-1]
Con
cent
ratio
n of
phos
phor
us [m
g L-1
]
Fig. 3.2: Residual concentration of nutrients in the supernatant and their original concentration in the medium of fermentation 1. A) Free carbon from octanoic acid in medium (�), free carbon from octanoic acid in supernatant (�), free nitrogen (present as NH4
+) in medium (�), free nitrogen (present as NH4+) in supernatant
(�). B) Free phosphorus (present as phosphate) in medium (�), free phosphorus (present as phosphate) in supernatant (�), bound phosphorus in supernatant (�). Limitation regimes: I) (C,N)-limited, II) (C,N,P)-limited, III) (C,P)-limited, IV) (P)-limited.
Interestingly up to 6 mg P L-1 were found in extracellular substances under P-
limiting conditions (Fig. 3.2B). Likewise, in the N-limited regime (C/P = 30-70 g g-1) a
substantial amount of nitrogen was found as extracellular protein (140-200 mg pro-
tein L-1, corresponding to ~24-34 mg N L-1). Hence, the quantity of extracellular sub-
stances containing nitrogenous compounds was non-negligible. More detailed analy-
ses to identify the compounds were not carried out.
Biosynthesis of mcl-PHA under P-limitation 70
Biomass production and PHA accumulation
The optical density (OD) and the cell dry weight (CDW) increased towards higher C/P
and reached a maximum at C/P = 80 g g-1 (Fig. 3.3A). This increase was mainly due
to enhanced PHA accumulation as the concentration of residual biomass remained
nearly constant until C/P = 70 g g-1 and only slightly decreased towards higher C/P.
The PHA content of dried biomass increased with P-limitation (C/P > 60 g g-1) from
10 w% to 25 w% from regime I to regime III (Fig. 3.3B).
0
5
10
0
10
20
30
20 30 40 50 60 70 80 90 1000
10
20
Dry
wei
ghts
[g L
-1]
B
Opt
ical
den
sity
(45
0nm
) [-
]A
PH
A c
onte
nt [%
w/w
]
C/P [g g-1]
I II III IV
Fig. 3.3: A) Optical density (OD) measured at 450 nm (�), cell dry weight (CDW) (�), residual biomass weight (�), and PHA weight (�) and B) PHA content in dried biomass (�) of fermentation 1. Limitation regimes: I) (C,N)-limited, II) (C,N,P)-limited, III) (C,P)-limited, IV) (P)-limited.
Phosphorus and phospholipid in biomass
The phosphorus content in residual biomass and the content of phospholipids de-
creased with P-limitation (Fig. 3.4). Phosphorus in residual biomass was reduced un-
der P-limited conditions to virtually 50% of the maximum value, whereas the content
71 Chapter 3
in phospholipids was decreased to 10% of its maximum value. We therefore could
confirm that membrane phospholipids play a key role in the phosphorus pool of P.
putida GPo1, as has been observed for P. diminuta, P. aeruginosa and P. fluores-
cens (Minnikin and Abdolrah.H 1974; Minnikin et al. 1974; Yuasa 2002). For these
species, most of the membrane phospholipids were substituted under strong
P-limitation by nitrogen or sulphur containing lipids (Minnikin and Abdolrah.H 1974;
Minnikin et al. 1974; Yuasa 2002). Interestingly, under conditions of excess phos-
phate (C/P = 30 and 40 g g-1) the phospholipid content was also decreased relative
to the observed maximum at C/P of 50 g g-1. This may be due to a shift in the phos-
pholipid composition with excess phosphate eventually causing an underestimation.
20 30 40 50 60 70 80 90 1000
2
4
0
2
4
Con
tent
of p
hosp
holip
ids
in
resi
dual
bio
mas
s [%
w/w
]
C/P [g g-1]
Con
tent
of p
hosp
horu
s in
re
sidu
al b
iom
ass
[% w
/w]
I II III IV
Fig. 3.4: Content of phosphorus (�) and phospholipids (�) in residual biomass of fermentation 1. Limitation regimes: I) (C,N)-limited, II) (C,N,P)-limited, III) (C,P)-limited, IV) (P)-limited.
Biosynthesis of mcl-PHA under P-limitation 72
Endotoxins in biomass and extracted PHA
The endotoxic activity of biomass was considerably reduced with P-limitation, as
shown in Fig. 3.5A. Because the measurement of endotoxic activities suffered from a
large uncertainty, the fermentation experiment was repeated twice for independent
measurements. Large variations were observed between the three independent ex-
periments, but the same trend was clearly found for each experiment – the endotoxic
activity dropped between 12- and 20-fold. One reason for these variations might be
LPS that were released from the cell membrane, partially adhering to biomass during
centrifugation. It has been observed that special fermentation conditions trigger the
release of endotoxins (Svensson et al. 2005). Biomass was washed twice during
work-up to remove free LPS, but this procedure might not have been quantitative.
For fermentation 1 (F1) the endotoxic activity of biomass dropped from 1.46 x 106
(C/P = 30 g g-1) to 1.22 x 105 EU mg-1 biomass (C/P = 90 g g-1). Fermentation 2 (F2)
and 3 (F3) showed endotoxic activities of 2.35 x 106 and 3.55 x 106 EU mg-1 biomass
(C/P = 40 g g-1) and 1.04 x 105 and 2.62 x 105 EU mg-1 biomass (C/P = 90 g g-1), re-
spectively. An analogous trend was measured for PHA extracted from biomass al-
though here the effect was more pronounced. Methylene chloride was chosen as a
standard solvent because it is suitable for the extraction of all mcl-PHAs and is often
used for this purpose. Again the endotoxic activities of PHA from three independent
fermentation experiments varied considerably, but showed the same trend (Fig.
3.5B). In particular for the regime III and IV PHA showed a very low endotoxic activity
of 18, 66 and 28 EU g-1 PHA for F1, F2, and F3, respectively. In general, the en-
dotoxic activity of PHA was considerably reduced, e.g. by a factor of about 300 from
C/P = 30 g g-1 to CP = 90 g g-1 for F1, whereas the endotoxic activity of biomass was
reduced only by a factor of 12 for F1.
The reason for the decreased endotoxic activity of biomass and of PHA produced
under phosphorus limitation can be explained either by structural changes of lipid A,
most apparently from biphosphorylated lipid A to the mono- or non-phosphorylated
form (compare Fig. 3.1), or by a reduction of the concentration of LPS in the outer
membrane. Although the latter is rather unlikely because of the necessity for mem-
brane stabilisation and protection, we have so far been unsuccessful in confirming
the structural change of lipid A by separation of lipid A from other cell components
with thin layer chromatography and analysis by mass spectrometry. Thus, more de-
tailed investigations are required for clarification.
73 Chapter 3
100000
1000000
20 30 40 50 60 70 80 90 10010
100
1000
10000
End
otox
icity
[EU
mg-1
bio
mas
s]
End
otox
icity
[EU
g-1 P
HA
]
C/P [g g-1]
A
B
I II III IV
Fig. 3.5: A) Endotoxic activity of dried biomass B) Endotoxic activity of PHA extracted from dried biomass with methylene chloride. Results were obtained from three independent fermentation experiments. Fermentation 1 (�), fermentation 2 (�) and fermentation 3 (�). Limitation regimes: I) (C,N)-limited, II) (C,N,P)-limited, III) (C,P)-limited, IV) (P)-limited.
The consequences of P-limitation seem to be similar for phospholipids and LPS.
In contrast to phospholipids, the reduction of the endotoxic activity started already
when the concentration of free phosphorus was low (0.3-5 mg P L-1) but not limiting
and continued with P-limitation.
Yield coefficients
Carbon and nitrogen yield coefficients for residual (PHA-free) biomass were hardly
affected by P-limitation (Table 3.1). The carbon yield coefficient varied between 0.69
and 0.81 g g-1 and the nitrogen yield coefficient between 7.60 and 8.27 g g-1 for F1.
The phosphorus yield coefficient rose from 30 to 62 g g-1 for a change of C/P from 30
Biosynthesis of mcl-PHA under P-limitation 74
to 90 g g-1 for F1. F2 and F3 showed similar results. The carbon yield coefficients for
F2 and F3 varied only slightly as well as the nitrogen yield coefficients. The phospho-
rus yield coefficients increased by about 100% for a change of C/P from 40 to 90
g g-1 (F2 and F3).
By using the modified stoichiometric approach (Egli 1991), dual and triple-limited
growth regimes can be estimated. According to this approach growth limiting nutri-
ents are completely converted into biomass under nutrient limitation. The ratio of
these nutrient concentrations in the medium feed (Cf, Nf, and Pf, respectively) equals
the inverse ratio of the corresponding growth yield coefficients (YX/C, YX/N, and YX/P,
respectively). The growth yield coefficients for total biomass can be replaced in this
calculation by growth yield coefficients for residual biomass since this leads to the
same result. Under triple (C,N,P)-limitation the following equations must hold:
[2]
[3]
The yield ratios of F1 are shown in Table 3.1. According to the stoichiometric ap-
proach, triple limitation had to be found between C/P = 40 and 70 g g-1. For F2 and
F3 triple limitation was reached from C/P = 50 to 70 g g-1. This estimation was in
clear contrast to the previous determination of nutrient-limited growth regimes based
on the kinetic approach, where the triple-limited regime was determined from C/P 65
to 75 g g-1. The stoichiometric P-limitation was reflected in the decreasing phospho-
rus content in biomass (Fig. 3.4). P-limitation according to the kinetic approach went
together with a considerable increase of the PHA content in biomass. This was simi-
lar to N-limitation, where higher C/N ratios enhance PHA accumulation (Durner et al.
2000).
Hence, the stoichiometric approach indicated physiological adaptations to re-
duced concentrations of phosphorus. Effective limitation in the sense of very low re-
sidual phosphorus concentrations in the supernatant and enhanced PHA accumula-
tion was only determined with the kinetic approach.
Cf
P f
≈YX /P
C,N,P−lim.
YX /CC,N,P−lim.
Nf
Pf
≈YX /P
C,N,P−lim.
YX /NC,N,P−lim.
75 Chapter 3
Table 3.1: Yield factors for residual biomass and yield ratios for the determination of dual
and triple-limited growth regimes of fermentation 1. Dual limitation is reached
when the yield ratio equals the ratio of the corresponding nutrients in the feed
medium (C/P or N/P). For triple limitation both yield ratios have to fulfil this
condition.
Regime C/P N/P Y X/C YX/P YX/N YX/P/YX/C YX/P/YX/N
[g g-1] [g g-1] [g g-1] [g g-1] [g g-1] [g2 g-2] [g2 g-2]
I 30 3.0 0.79 30.1 7.93 38.0 3.8
I 40 4.0 0.77 31.9 7.73 41.2 4.1
I 50 5.0 0.76 39.2 7.60 51.6 5.1
I 60 6.0 0.81 48.8 8.11 60.3 6.0
II 70 7.0 0.80 57.6 7.99 72.1 7.2
III 80 8.0 0.76 62.1 8.07 81.7 7.7
IV 90 9.0 0.69 62.0 8.27 90.5 7.5
YX/Y: growth yield factor for residual biomass, YX/Y/YX/Z: yield ratio.
Cell physiological aspects with different nutrient limitations
Nitrogen limitation was applied in the previous experiments combined with C- and or
P-limitation because it was considered to be essential for PHA accumulation. To see
if the N-limitation played a crucial role for the PHA accumulation and for the en-
dotoxic activity, the concentration of nitrogen in the mineral medium was increased
threefold (resulting in a C/N = 3.3 g g-1) to ensure excess nitrogen. Two steady
states, C/P = 60 and C/P= 80 g g-1, were analysed and compared with the results ob-
tained with C/N = 10 g g-1 (see Table 3.2).
Excess nitrogen (C/N = 3.3 g g-1) induced an enhanced production of biomass of
8.3 g L-1 and an increased consumption of phosphorus causing P-limitation for C/P =
60 g g-1. PHA accumulation in dried biomass was similar to the one under N-limited
conditions. The endotoxic activity of PHA and biomass, however, was about 2.5
Biosynthesis of mcl-PHA under P-limitation 76
times higher under nitrogen in excess than under nitrogen limitation. Interestingly, the
content of phospholipids was reduced by more than 50% with excess nitrogen.
Excess carbon (30 mg L-1) and excess nitrogen (1’740 mg L-1) was measured in
the supernatant for C/P = 80 g g-1 and C/N = 3.3 g g-1 and hence this steady state
was only P-limited. The cell dry weight was reduced, the phospholipid content
strongly reduced and the PHA content was increased by about 50%. In comparison,
for a C/P = 80 g g-1 and a C/N = 3.3 g g-1, significant amounts of free nitrogen (48 mg
L-1) could be measured in the supernatant and hence this growth state was (C,P)- but
not N-limited. Concomitantly PHA accumulation was more efficient at a C/N = 10
g g-1. Significant differences were observed for the endotoxic activity of biomass and
PHA which was about 8- respectively 126-times smaller with C/N = 10 g g-1 com-
pared to the fermentation with excess nitrogen (C/N = 30 g g-1).
Concluding, increasing C/P from 60 to 80 g g-1 did raise the endotoxic activity
with C/N = 3.3 g g-1 to some extent. Thus, PHA accumulation was higher and the ac-
tivity of endotoxins was lower with a high carbon to nitrogen ratio. Hence, a low N/P
ratio (6 and 8 g g-1) was favourable over high N/P values (18 and 24 g g-1) for P-
limited conditions.
These effects can be explained by the reduced flexibility of cells with additional
nitrogen limitation, compared to a state with nitrogen in large excess, forcing the bac-
teria to efficiently use phosphorus and to accumulate mcl-PHA from carbon feed. For
instance the synthesis of proteins and membrane lipids lacking phosphorus (e.g. or-
nithine-based lipids) will be facilitated with excess nitrogen compared to N-limited
conditions. One could argue that both fermentations with C/P = 80 g g-1 were non-
nitrogen limited and should give similar results. But it seems that a large nitrogen ex-
cess has an inhibiting effect on the cell growth at this C/P ratio.
Some wall growth leading to biofilm formation was observed with P-limitation in-
dependent from N-limitation. It has been observed for P. putida in a laminar flow cell
reactor that biofilm accumulation rate was highest at C/P =100 g g-1 (Rochex and Le-
beault 2007). This confirms our observation that biofilm formation increased with a
higher C/P. Calcium crosslinking with extracellular polysaccharides is supposed to be
important for the matrix of biofilms and can be inhibited by increased precipitation of
calcium phosphate (Turakhia and Characklis 1989). But this is only possible with ex-
cess phosphate and hence biofilm formation seems to be an intrinsic problem of P-
limitation.
77 Chapter 3
Table 3.2: Comparison of different nutrient limitations with C/N = 10 and C/N = 3.3 g g-1.
Limited
nutrients
C/P C/N OD CDW CDW-
PHA
PHA
content
Phospholipid
content
Endotoxins
biomass
Endo-
toxins
PHA
[g g-1] [g g-1] [-] [g L-1] [g L-1] [w%] [w%] EU mg-1
biomass
EU g-1
PHA
C, N 60 10 25.6 7.3 6.5 10.7 4.4 4.2 x 105 828
C, P 60 3.3 26.6 8.3 7.3 11.5 2.0 10.9 x 105 2208
C, P 80 10 31.7 8.2 6.1 25.6 1.0 2.2 x 105 21
P 80 3.3 26.9 7.5 6.2 16.5 1.0 17.6 x 105 2661
OD: optical density measured at 450nm, CDW: cell dry weight, CDW-PHA: residual biomass.
3.4 Conclusions
P. putida GPo1 cultivated continuously under P-limitation combined with carbon and
nitrogen limitation drastically changed its cell composition. The amount of phospho-
lipids in dried biomass was decreased by about 90% under P-limitation compared to
a state without P-limitation. In parallel, the endotoxic activity of biomass was reduced
to 10% of its initial value. Furthermore, P-limitation combined with N-limitation con-
siderably increased the PHA accumulation. With combined (C,P)-limitation and ex-
cess nitrogen the PHA accumulation was significantly lower and the endotoxic activ-
ity was notably increased. This clearly demonstrated that combined (C,N,P)-limitation
leads to a better performance of the culture than (C,P)-limitation with nitrogen ex-
cess.
Consequently, by selecting appropriate fermentation conditions, the contamina-
tion of PHA with endotoxins can be significantly reduced. However, P-limitation can-
not be extended arbitrarily as the cell growth rate becomes restricted. Further, P-
limitation of P. putida GPo1 is prone to biofilm formation, which can limit the stability
and reproducibility of fermentation experiments and should be avoided.
Biosynthesis of mcl-PHA under P-limitation 78
Overall, cultivation under P-limitation could be a promising technique for fermen-
tations with Gram-negative bacteria to reduce the product contamination by endoto-
xins.
3.5 Acknowledgments
We gratefully acknowledge the financial support by the Empa R&D fund. We also
thank Ernst Pletscher and Dominik Noger for assisting with fermentations and for ad-
vices and Kathrin Grieder for ICP-OES measurements.
79
Chapter 4
Efficient recovery of low endotoxin medium-chain-
length poly([R]-3-hydroxyalkanoate) from bacterial
biomass
Patrick Furrer, Sven Panke, and Manfred Zinn. 2007. Journal of Microbiological
Methods 69, 206-213.
Efficient recovery of mcl-PHA 80
4.1 Introduction
New biomaterials, in particular degradable ones are needed for various medical
applications (Hubbell 1995; Ueda and Tabata 2003). Today’s biomaterials of the third
generation (Hench and Polak 2002) are designed to stimulate specific responses at
the molecular level and to combine the concepts of bioactive and resorbable
materials. Today’s polymeric and biodegradable systems used in medicine are
mainly based on poly(lactic acid) (PLA), on poly(glycolic acid) (PGA), and on their co-
polymers (Gomes and Reis 2004). Other biodegradable polymers have been
proposed, but could not enter the market yet, due to lacking FDA approval (Gomes
and Reis 2004).
One of the candidates is polyhydroxyalkanoate (PHA), a class of biodegradable
and biocompatible polyesters with many potential applications in the medical field,
such as heart valve scaffolds (Sodian et al. 2000a; Sodian et al. 2002), pulmonary
conduits (Stock et al. 2000), sutures, screws, bone plates, repair patches, stents,
bone marrow scaffolds and many others over the last years as recently reviewed by
Chen and Wu (Chen and Wu 2005). In particular the scientific and industrial research
on elastomeric PHA with medium-chain-length side-chains (mcl-PHA, containing 3-
hydroxyalkanoate monomers from C6-C14) has been intensified in the last years
(Chen et al. 2005; Sodian et al. 2002; Sodian et al. 2000b; Stock et al. 2000; Williams
et al. 1999; Zinn et al. 2001).
Mcl-PHA is synthesized by fluorescent pseudomonads belonging to the rRNA
homology group I (Huisman et al. 1989) and serves as an intracellular carbon and
energy storage compound. Its biosynthesis can be triggered by appropriate growth
conditions such as phosphorus and nitrogen limitation (Witholt and Kessler 1999).
Mcl-PHA is stored in distinct granules that are coated by amphiphilic phospholipids
and proteins (Pötter and Steinbüchel 2004).
Along with cell growth, all of the Gram-negative production strains produce
lipopolysaccharides (LPS) as an integral part of the outer membrane (Petsch and
Anspach 2000). LPS are pyrogenic and their concentration in a medical product is
strictly regulated. The US-American Food and Drug Administration has given clear
81 Chapter 4
regulations on the maximum endotoxin content of medical devices, namely 20 EU
per medical device (U.S. Department of health and human services 1997).
There are currently two well-established approaches for recovering PHA: solvent
extraction and aqueous digestion of non-PHA materials. The former one uses an
organic solvent to extract PHA from dried biomass, whereas the latter one involves
chemical agents or enzymes to lyse bacteria and thus liberating the PHA granules
forming a latex (Choi and Lee 1999; de Koning et al. 1997; de Koning and Witholt
1997; Ling et al. 1997; Taniguchi et al. 2003; Yu and Chen 2006). To purify and
depyrogenize PHA in latex form, oxidizing agents like hydrogen peroxide and sodium
hypochlorite as well as sodium hydroxide were applied (Lee et al. 1999; Sevastianov
et al. 2003; Williams et al. 1999). However, oxidizing agents may result in polymer
degradation as was reported for the treatment with sodium hypochlorite (Taniguchi et
al. 2003) and usually do require additional, expensive processes like
ultracentrifugation and microfiltration (de Koning and Witholt 1997; Marchessault et
al. 1995). In general, the PHA material obtained by this method is of low purity
(Kessler et al. 2001) and therefore not suitable for medical applications.
Various organic solvents can be used to extract mcl-PHA from dried biomass
(Chen et al. 2001; Kessler et al. 2001; Williams et al. 1999). The lyophilized biomass
is commonly extracted with a chlorinated solvent and the resulting PHA solution is
filtered (see Fig. 4.1A). In order to separate PHA from co-extracted impurities (e.g.
lipids and proteins), the polymer solution is usually mixed with a non-solvent like
methanol which induces precipitation of the polymer, leaving most contaminants in
solution. The extraction conditions and further processing steps strongly affect the
recovery and purity of the resulting PHA. Although polymer purities above 90% (w/w)
are easily obtained by a solvent extraction followed by a non-solvent precipitation,
the amount of endotoxins can still reach high levels (Sevastianov et al. 2003).
Typically values of more than 100 EU g-1 of polymer were found in a commercially
available PHA, namely poly(3-hydroxybutyrate) (Williams et al. 1999).
In this study, we investigated an alternative method for the purification of mcl-
PHA to obtain a polymer of high purity and low endotoxicity, namely by the heat
driven extraction with a solvent and the selective precipitation induced by
temperature reduction (see Fig. 4.1B). Special attention was paid to minimize the
contamination with endotoxins.
Efficient recovery of mcl-PHA 82
Fig. 4.1: Schematic of PHA recovery: A) extraction and non-solvent precipitation, B) extraction and temperature-controlled precipitation.
4.2 Materials and Methods
Biosynthesis. Polyhydroxyoctanoate (PHO) consisting of 87% (w/w)
3-hydroxyoctanoate and 13% (w/w) 3-hydroxyhexanoate was produced by
continuous cultivation of Pseudomonas putida GPo1 (Durner et al. 2000) in a 16 L
bioreactor (Bioengineering, Wald, Switzerland). The dilution rate was set to 0.1 h-1,
the ammonium content of the minimal medium was 800 mg N L-1 and the carbon feed
rate was adjusted to obtain a carbon to nitrogen ratio of 12 g g-1. The fermentation
broth was then concentrated with a continuous centrifuge (Cepa Z61H, Lahr,
Germany) at 17’500 x g and a flow rate of 50 L h-1. The paste was lyophilized for
48 h, ground, and the dry biomass was stored in a closed container at -25 °C until
extraction.
Analysis: Gel permeation chromatography (GPC). Molecular weights (number-
average (Mn) and weight-average (Mw)) were determined by gel permeation
chromatography (TDA 302, Viscotek, Waghausel-Kirrlach, Germany) equipped with a
viscometer, RI- and light-scattering detector. The system was calibrated by using 2
polystyrene standards (Polycal, Viscotek) with known molecular weights and intrinsic
viscosities (standard 1: Mw=115 kDa, Mn=112 kDa, IV=0.519, standard 2: Mw=250
kDa, Mn=100 kDa, IV=0.843). Forty mg of PHO-sample was dissolved in 10 mL of
THF over night, filtered through a 1.0 µm nylon filter, and aliquots of 100 µL were
83 Chapter 4
chromatographed with pure THF as solvent phase through three GPC columns
(Mixed bed, Viscotek) at 35 °C and a flow rate of 1 mL min-1.
Gas chromatography (GC). The purity of PHO and its composition with respect to
alkanoate monomers were determined by gas chromatography. About 50 mg of
polymer was transferred into a 20 mL Pyrex tube. Five mL of methylene chloride
containing 10.0 mg mL-1 of 2-ethyl-2-hydroxybutyric acid as internal standard and 5
mL of a 20/80 (v/v) mixture of HCl (37%) and n-propanol were added for the
propanolysis at 80 °C for 16 h. Subsequently, the sample was cooled on ice and 8
mL of demineralized water was added. The mixture was vigorously shaken, the
phases were separated, and the aqueous phase was removed. The organic phase
was dried over Na2SO4, neutralized by adding Na2CO3, and filtered through a 1.0 µm
nylon filter. One µL of this sample was injected into the GC (GC 8575 Mega 2, Fisons
Instruments, Rodano, Italy) onto a Supelcowax-10: 30 m x 0.32 mm column with a
film thickness of 0.5 µm (Supelco, Bellefonte, USA) at a split ratio 1:10 and an initial
temperature 120 °C, which was raised at 10° min-1 to 280 °C. Detection was
performed by FID detection.
Limulus amebocyte lysate (LAL)-test. The endotoxicity was measured using a
chromogenic LAL endpoint assay (QCL-1000, Cambrex, New Jersey, USA). About
300 mg of PHO were placed in a 15 mL pyrogen-free centrifuge tube (Corning, New
York, USA) and heated up to 80 °C for 24 h to obtain a smooth polymer surface.
Afterwards 6 mL of pyrogen-free water (Cambrex) was added. The tubes were
placed in a rotary shaker and incubated for 24 h at 37 °C under constant shaking at
180 rpm to extract the endotoxins from the polymer. The aqueous endotoxin-solution
was directly used for the LAL-test or diluted 1:10 or 1:100 if the concentration of
endotoxins was high. Each analysis was carried out at least twice.
Experimental setup. For the recovery of PHO, we applied the solvent extraction
method with precipitation by a non-solvent (standard extraction, Fig. 4.1A), and
temperature-controlled precipitation, which does not require a non-solvent (Fig.
4.1B).
Standard extraction. Five gram of dry biomass containing about 30% (w/w) PHO
were extracted with 75 mL of different organic solvents - methylene chloride
(CH2Cl2), ethyl acetate, acetone, n-hexane, tetrahydrofurane (THF) and 2-propanol of
purum grade or higher (Fluka, Buchs, Switzerland) - for 24 h at room temperature
Efficient recovery of mcl-PHA 84
under intense stirring. The biomass to solvent ratio of 1:15 (g mL-1) had been
identified as optimal in a preliminary test (data not shown) and was applied for all
extractions. The suspension was filtered through a regenerated cellulose membrane
filter (1.0 µm, Schleicher & Schuell Microscience GmbH, Dassel, Germany) with a
pressure filtration unit (200 mL stainless steel, Sartorius AG, Goettingen, Germany)
by applying an overpressure of typically 3 bars to obtain a clear PHO solution. The
polymer solution was either evaporated or concentrated by rotary evaporation to 1/5
of the initial volume and then precipitated at -20 °C by adding 10 times the volume of
methanol. Finally the polymer was dried under vacuum at 40 °C for 48 h. The
resulting polymers had molecular weights of about 200 kDa (Mw) and 100 kDa (Mn)
and hence a polydispersity of 2.0.
Temperature dependent solubility. About 1 g of pure PHO was dissolved in 100
mL organic solvent at 40 °C under intense stirring. The solution was cooled to 0 °C in
an ice bath under gentle stirring. After 4 hours the solution was decanted. The
precipitate was dried and the solvent was evaporated from the solution to determine
the ratio between precipitated and dissolved PHO.
Temperature-controlled extraction and precipitation . Five gram of dried biomass
was extracted with 75 mL of n-hexane at different temperatures under intense stirring
for 24 h. The suspension was filtered at the same temperature as the extraction took
place. In order to study the influence of temperature on the solubility of PHO in n-
hexane, the polymer solution was kept at a temperature between +20 °C and -10 °C
under gentle stirring for 24 h. The precipitated polymer was decanted and dried
under vacuum at 40 °C for 48 h.
Re-dissolution and precipitation. For further purification, dried PHO (extracted with
n-hexane at 50 °C and precipitated at 0 °C) was re-dissolved in 2-propanol (2%
(w/v)) at 45 °C under intense stirring for 8 h. PHO was precipitated by cooling the
solution to a temperature between 30 °C and 0 °C under gentle stirring for 24 h.
Finally the polymer was dried under vacuum at 40 °C for 48 h.
Calculation. The purity and the recoveries of PHO were calculated according to the
following equations:
85 Chapter 4
100Em
xOH-3m
EP ⋅=∑ [% w/w] [1]
100Pm
xOH-3m
PP ⋅=∑ [% w/w] [2]
100Bm
xOH-3m
PHOX ⋅=∑ [% w/w] [3]
where PE is the purity of the extract, PP the purity of the precipitate, XPHO the PHO
content in the dried biomass, mE is the mass of the extract, mP the mass of the
precipitate, mB the mass of the biomass, and m3-OH x are the masses of the individual
dehydrated 3-hydroxy acids (as present in the polymer) determined with GC as
described above.
100PHOBm
EPEm
ER ⋅⋅
⋅=
X [% w/w] [4]
100EPEmPPPm
PR ⋅⋅
⋅= [% w/w] [5]
The recovery is either calculated for the extract relative to the total mass of PHO
in the biomass (RE), or for the precipitate relative to the mass of extracted PHO (RP).
All measurements were carried out in duplicates. The bars in Fig. 4.2, Fig. 4.3
and Fig. 4.5 represent the uncertainty of measurement for the purity and the recovery
(±5% and ±6%, respectively) and the maximum standard deviation for the
endotoxicity (±10%). The uncertainty of measurement was calculated based on
additional experiments not described in this paper.
Efficient recovery of mcl-PHA 86
4.3 Results and discussion
Standard extraction and precipitation. Biomass was extracted according to the
conventional technique (Fig. 4.1A) to find solvents suitable to extract PHO selectively
with a good recovery. As shown in Table 4.1, methylene chloride was the most
efficient solvent at room temperature. 2-propanol and n-hexane were rather poor
PHO extracting solvents. However, ethyl acetate, tetrahydrofurane, and acetone had
recovery rates around 80% and provided PHO of purities > 80%. Especially ethyl
acetate and acetone extracts contained less than 10% impurities. Furthermore n-
hexane, methylene chloride, ethyl acetate, and acetone resulted in PHO of low
endotoxicity.
Table 4.1: Recovery (RE), purity (PE or PP) and endotoxicity of PHO extracted from dried biomass with different solvents.
Solvent evaporated Precipitated in MeOH
Solvent R E
[% w/w]
PE
[% w/w]
Endotoxicity
[EU g -1]
RE
[% w/w]
PP
[% w/w]
Endotoxicity
[EU g -1]
n-hexane 53 93 320 49 100 102
CH2Cl2 86 78 43 83 100 10
Ethyl
acetate
80 92 16 78 99 10
THF 80 84 589 77 100 10
Acetone 83 92 75 77 100 36
2-propanol 23 66 115 12 84 30
The subsequent precipitation of PHO with methanol drastically increased the
purity of the polymer. Purities close to 100% could be reached (Table 4.1). At the
same time, only small amounts of PHO, usually less than 5%, were lost.
87 Chapter 4
Temperature-controlled precipitation. To identify solvents capable of dissolving
PHO in a temperature dependent manner (Fig. 4.1B), PHO solutions of 1% (w/v) in
organic solvent were prepared. The concentration of 1% (w/v) was chosen because it
is typical for the concentration reached after biomass extraction with an organic
solvent at a ratio 1:15 (w/v). The polymer solutions were cooled down to 0 °C and
sol- and gel-phase were separated by decantation.
There were only two solvents with sufficiently different solubilities at 40 oC and
0 oC: 2-propanol and n-hexane (Table 4.2). Nearly all dissolved polymer could be
recovered from 2-propanol whereas in n-hexane about 26% of the polymer remained
in solution. The other solvents showed no polymer precipitation when the
temperature was adjusted to 0 °C, consequently they are not suitable for a
temperature induced precipitation of PHO. Eventually polymer precipitation would
have taken place at significantly lower temperatures but lower purities had to be
expected due to co-precipitation of impurities present in the solution.
Of the two solvents with a suitable temperature-dependent behavior, 2-propanol
is much less suitable for extracting PHO since it co-extracted a lot of cellular
compounds and thus resulted in PHA of low purity (Table 4.1). In contrast, n-hexane
selectively extracted PHO from biomass and was therefore suitable for a combined
extraction / temperature-controlled precipitation.
Table 4.2: Fractions of precipitated PHO after cooling the polymer solutions to 0 °C.
Solvent Precipitated polymer
[% w/w]
n-hexane 74
CH2Cl2 0
Ethyl acetate 0
THF 0
Acetone 0
2-propanol 97
Efficient recovery of mcl-PHA 88
Temperature optimization of PHO extraction. After having identified n-hexane as
the solvent with the most promising properties, we optimized the extraction step with
respect to temperature. PHO was extracted with n-hexane at temperatures between
20 °C and n-hexane’s boiling point at 69 °C. The recovery increased considerably
with rising extraction temperature. A plateau was reached at 50 °C (Fig. 4.20). The
purity of the resulting PHO slightly decreased with increasing temperature, but still
remained close to 90% at the boiling point. In contrast, the endotoxicity increased
dramatically with the extraction temperature from 10 to >1’000 EU g-1 polymer (Fig.
4.2). Hence the co-extraction of LPS from biomass is much more pronounced at
higher extraction temperatures. The molecular weight of PHO increased moderately
with increasing temperature, indicating that high molecular weight PHO dissolved
better above 20 °C than below (Table 4.3). Consequently, the extraction with n-
hexane was very efficient above 50 °C, but due to the increased contamination with
endotoxins, an extraction temperature below 50 °C was more reasonable.
In subsequent experiments, dry biomass was extracted with n-hexane at 50 °C
for 24 h. This temperature was chosen in order to clearly document a potential
endotoxin reduction effect of the precipitation.
0
25
50
75
100
10 20 30 40 50 60 70Extraction temperature [ °C]
Pur
ity a
nd re
cove
ry [%
w/w
]
1
10
100
1000
10000
End
otox
icity
[EU
g-1
pol
ymer
]
Recovery
PurityEndotoxicity
Fig. 4.2: Recovery, purity and endotoxicity for PHO extracted with n-hexane at different temperatures.
89 Chapter 4
Table 4.3: Weight averaged molecular weight (Mw), number averaged molecular weight (Mn) and polydispersity (D) of PHO extracted from biomass with n-hexane at different temperatures.
Extraction
temperature
[°C]
Mw
[kDa]
Mn
[kDa]
D
[-]
20 190 96 1.97
30 202 104 1.95
40 193 98 1.97
50 209 113 1.86
60 221 114 1.95
69 212 111 1.92
Temperature optimization of PHO precipitation. To investigate the optimum
precipitation temperature of PHO from n-hexane, the polymer solution was cooled
after filtration to temperatures between +20 and -10 °C. First precipitations were
observed at 20 °C. The amount of precipitated polymer increased further with
decreasing temperature and may have reached a maximum between 5 and 0 °C.
Surprisingly, precipitation became less efficient at a temperature below 0 °C as
shown in Fig. 4.3. Apparently, PHO formed a dispersion with n-hexane at
temperatures below 0 °C and precipitated very slowly. About 80% of the extracted
PHO could be precipitated at best. However, an improved recovery could be
obtained when the polymer solution was concentrated previously, e.g. by solvent
evaporation.
The purity of the resulting polymer was close to 100% for precipitation
temperatures down to 5 °C, and declined for lower temperatures (Fig. 4.3). This
indicates that non-polar contaminants co-precipitated significantly below 0 °C. The
endotoxicity increased at low temperatures (Fig. 4.3) in parallel with the observed
precipitation of other impurities.
Efficient recovery of mcl-PHA 90
0
25
50
75
100
-20 -10 0 10 20 30
Precipitation temperature [ °C]
Pur
ity a
nd re
cove
ry [%
w/w
]
1
10
100
1000
End
otox
icity
[EU
g-1
pol
ymer
]
RecoveryPurityEndotoxicity
Fig. 4.3: Recovery, purity and endotoxicity for PHO extracted with n-hexane at 50 °C, precipitated at different temperatures (20 °C: endotoxicity not determined).
Gel permeation chromatography (GPC) showed that particularly the high
molecular weight fractions of the polymer precipitated at higher temperatures and
that the low molecular weight fractions remained in solution as shown in Fig. 4.4.
This led to a decrease of the polydispersity (Mw/Mn) from 1.86 to 1.39 at 10 °C (Table
4.4). This is important since decreasing the amount of low molecular weight species
prevents a rapid and extensive release of mono- or oligomers in medical (implants)
and pharmaceutical (drug carriers) applications. For some polymers in medical
applications it has been reported that the release of low molecular weight
components led to a drastic decrease of the pH near the implant surface (Agrawal
and Athanasiou 1997) or often caused inflammations (Eckelt et al. 2004).
Since the goal was to produce low endotoxin containing PHA at a high yield, it
was concluded that the optimal precipitation temperature was 0 °C taking into
account that the polydispersity was somewhat larger (D = 1.56).
91 Chapter 4
Fig. 4.4: Normalized molecular weight distribution of PHO extracted with n-hexane at 50 °C, precipitated at different temperatures.
Table 4.4 : Weight averaged molecular weight (Mw), number averaged molecular weight (Mn) and polydispersity (D) of PHO extracted with n-hexane at 50 °C, precipitated at different temperatures (starting material: Mw=209 kDa, Mn=113 kDa, D=1.86).
Precipitation
temperature*
[°C]
Mw
[kDa]
Mn
[kDa]
D
[-]
10 363 262 1.39
5 233 157 1.48
0 228 146 1.56
-5 204 121 1.70
-10 217 125 1.73
*Molecular weight analysis could not be carried out for the sample at 20 °C because of
insufficient polymer recovery.
2-propanol re-dissolution and precipitation. In order to further improve the purity
of PHO above 97%, we investigated the re-dissolution of the purified polymer in
2-propanol followed by another temperature-controlled precipitation, because
2-propanol was very efficient in co-extracting cellular impurities (see above). Thus we
Efficient recovery of mcl-PHA 92
proposed that the sequential usage of n-hexane and 2-propanol for temperature-
controlled precipitation has a beneficial effect on the purity of PHA since impurities
may have a different affinity to these solvents. PHO obtained from n-hexane
extraction at 50 °C and precipitated at 0 °C was re-dissolved in 2-propanol at 45 °C
to a concentration of 20 g L-1. The solution was then cooled to temperatures between
30 and 0 °C. The PHO recovery increased with decreasing temperatures as shown in
Fig. 4.5. At 25 °C already 80% of the polymer had precipitated. At 0 °C nearly 100%
of the PHO was recovered. The purity was close to 100% for all temperatures. The
endotoxicity was lowered to less than 10 EU g-1 polymer at all temperatures, except
at 0 °C (Fig. 4.5). A temperature of 10 °C resulted in the lowest endotoxicity of PHO
with 2 EU g-1 polymer. GPC measurements again showed a fractionation effect
according to the molecular weight (Fig. 4.6 and Table 4.5). High molecular weight
fractions precipitated at elevated temperatures, whereby the low molecular weight
fractions remained in solution. The polydispersity was decreased from 1.50 to a
minimum of 1.26.
0
25
50
75
100
-5 0 5 10 15 20 25 30 35
Precipitation temperature [ °C]
Pur
ity a
nd re
cove
ry [%
w/w
]
1
10
100
End
otox
icity
[EU
g-1
pol
ymer
]
Recovery
Purity
Endotoxicity
Fig. 4.5: Recovery, purity and endotoxicity for PHO re-dissolved in 2-propanol at 40 °C and precipitated at different temperatures.
93 Chapter 4
Fig. 4.6: Normalized molecular weight distribution of PHO re-dissolved in 2-propanol and precipitated at different temperatures.
Table 4.5: Weight average molecular weight (Mw), number average molecular weight (Mn) and polydispersity (D) of PHO re-dissolved in 2-propanol and precipitated at different temperatures (starting material: Mw=228 kDa, Mn=146 kDa, D=1.56).
Precipitation
temperature
[°C]
Mw
[kDa]
Mn
[kDa]
D
[-]
30 321 255 1.26
25 260 199 1.31
20 244 184 1.33
15 234 159 1.47
10 231 156 1.48
5 239 159 1.50
0 227 156 1.46
Efficient recovery of mcl-PHA 94
4.4 Conclusions
Temperature-controlled extraction and precipitation can be effectively used to
increase the purity and reduce the endotoxin content of microbial PHO samples. For
this reason, it represents a valuable alternative to the classical approach where the
PHA-solution is mixed with an excess of non-solvent for precipitating mcl-PHA. Our
method yields in a polymer of high quality, is very simple and omits the
disadvantages of a non-solvent precipitation (e.g. separation and recycling of
solvents and non-solvents).
N-hexane was useful for a quantitative extraction of PHO at a temperature
around 50 °C. By decreasing the extraction temperature to 40 °C or below, the
recovery decreased moderately but the PHA contained much less endotoxins.
Precipitation induced by cooling led to PHO of high purity and low endotoxicity,
leaving most impurities in solution. However, a certain loss of polymer can not be
prevented except through a pre-concentration of the polymer solution. Especially the
low molecular weight fractions of PHO hardly precipitated.
The temperature dependent capacity to dissolve PHO made 2–propanol a
promising candidate for PHO extraction from biomass, but it dissolved other cell
components as well, which made it less suitable for the first extraction step.
However, its increased polarity relative to n-hexane led us to reason that it might be
an excellent solvent to re-dissolve PHO and selectively remove endotoxins. Indeed,
endotoxin concentrations of lower than 10 EU g-1 of polymer could be reached.
Additionally, the polymer polydispersity and the molecular weight can be controlled
by a temperature-controlled precipitation in order to achieve a more homogenous
degradation.
The solubility of various mcl-PHA in organic solvents can differ notably and
therefore the presented method might not be applicable to all mcl-PHAs without
adjustments. Recently, the method could be applied to a co-polymer produced from
octanoic and 10-undecenoic acid, where the relative composition was 50:50 w%
(Hartmann et al. 2006).
Concluding, we present a simple and very efficient method to produce PHO that
complies with the endotoxin requirements of the FDA for biomedical applications
95 Chapter 4
such as implants and drug delivery systems (U.S. Department of health and human
services 1997).
4.5 Acknowledgement
We gratefully acknowledge the financial support by Empa. We also thank Ernst
Pletscher and René Hartmann for their help with fermentations, Thomas Ramsauer
for assisting with polymer extractions and Manfred Schmid and Elisabeth Michel for
supporting GPC and GC analyses.
Efficient recovery of mcl-PHA 96
97
Chapter 5
Biocompatibility of untreated and chemically
modified medium-chain-length
poly(3-hydroxyalkanoates)
The results of this chapter were obtained in collaboration with:
Katharina Maniura and Philipp Spohn – cytotoxicity experiments
Sarah Cartmell – cell culturing on PHA coatings
Sarah Rathbone – cell culturing on PHA coatings
Patrick Furrer has carried out the following parts: PHA biosynthesis, PHA
extraction and purification, chemical derivatisation of PHA, solvent casting and
interpretation of cell culturing experiments.
Biocompatibility of mcl-PHA 98
5.1 Introduction
During the last two decades, significant advances have been made in the
development of biocompatible and biodegradable materials for biomedical
applications. The development of new biocompatible materials includes
considerations that go beyond the absence of toxic effects: the stimulation of specific
responses of tissue cells at the molecular level is the aim of so-called third
generation biomaterials (Hench and Polak 2002).
The chemical composition and surface structure of biomaterials are mainly
responsible for the interaction with biological systems. In the presence of serum, it is
thought that attachment of cells is mediated through adsorbed proteins and the
underlying surface chemistry controls protein adsorption. Through a protein layer, cell
adhesion and spreading can be prevented or promoted (Mrksich 2000). Physical and
chemical techniques can be used to modify the surface properties and hence to
control protein adsorption and cell adhesion. For instance plasma treatment (Qu et
al. 2005; Tezcaner et al. 2003), UV radiation (Shangguan et al. 2006; Viktor N.
Vasilets et al. 2004), collagen immobilisation (Tesema et al. 2004) and chemical
derivatisation with hydroxide (Rouxhet et al. 1998) have been used. Consequently,
the wettability or hydrophilicity of surfaces, as indicated by the water contact angle, is
of paramount importance for their interaction with cells. For osteoblasts, good
proliferation was obtained for fibronectin-coated surfaces with a water contact angle
of 50-90 ° (Kennedy et al. 2006).
Poly(3-hydroxyalkanoate) (PHA), a class of biodegradable polyesters, has the
potential to become an important biomaterial for medical applications (Williams et al.
1999; Zinn et al. 2001). Due to lower crystallinity and increased elasticity, mcl-PHA
offer physical properties that were quite different from scl-PHAs and give new
possibilities for medical applications such as scaffolds for tissue engineering and as
carrier for drug delivery. Functional groups in the side-chains of mcl-PHA allow to
regulate its properties. In addition, chemical modification of functionalized PHAs
enables further tailoring of the chemo-physical properties to suit particular
applications (Hazer and Steinbuchel 2007). For instance, the side-chain alkenes of
mcl-PHA were quantitatively transformed into hydroxyl groups with borane
99 Chapter 5
tetrahydrofurane (Renard et al. 2005). The hydrophilicity of the polymer increased
and the modified PHA was soluble in polar solvents like ethanol.
In vitro and in vivo studies have so far focused on short-chain-length PHA (scl-
PHA) with monomers of 3-5 carbon atoms and rather few studies were carried out
with medium-chain-length PHA (mcl-PHA) with monomers of 6-14 carbon atoms
(Chen and Wu 2005; Sodian et al. 2000a; Sodian et al. 2000b; Sodian et al. 2000c).
The combination of polyhedral oligomeric silsesquioxane (POSS) molecules and
polymers holds great promises for the future of biomedical devices, especially for
cardiovascular interfaces (Kannan et al. 2005). It has been shown that POSS
nanocomposites, unlike certain carbon nanotubes (Cui et al. 2005) are
cytocompatible and hence suitable for tissue engineering (Kannan et al. 2005;
Punshon et al. 2005). Further, It has been postulated that pendant nanocages
containing silicon atoms form foci with increased surface free energy thus promoting
the formation of an endothelial cell layer (endothelialization) and repelling coagulant
proteins (Kannan et al. 2005; Silver et al. 1999). The combination of POSS to mcl-
PHA would be of particular interest because the cell adhesion and proliferation on a
mcl-PHA matrix could be drastically enhanced.
In this work different mcl-PHAs with saturated and unsaturated side-chains were
produced and purified until the contamination with biological residues, in particular
with endotoxins was very low. The biocompatibility of these PHAs was studied.
Further, chemical modification was applied to hydrophobic mcl-PHAs with
unsaturated side-chains to increase their hydrophilicity and to improve cell adhesion
and proliferation. This was done by introducing carbonyl and carboxyl groups via
reaction with ozone or by covalently binding POSS to the unsaturated side-chains.
Biocompatibility of mcl-PHA 100
5.2 Materials and Methods
Biosynthesis. Mcl-PHAs were produced by continuous cultivation of Pseudomonas
putida GPo1 (ATCC 29347) as previously described (Ruth et al. 2007). Undecanoic,
10-undecenoic acid or a mixure of octanoic and 10-undecenoic acid were used as
carbon source for the production of poly(3-hydroxyundecanoate) (PHUA), poly(3-
hydroxy-10-undecenoate) (PHUE) and poly(3-hydroxyoctanoate-co-3-hydroxy-10-
undecenoate) (PHOUE). A 16 L laboratory bioreactor (Bioengineering, Wald,
Switzerland) with a working volume of 10 L was used. The dilution rate was set to
0.1 h-1, the ammonium content of the minimal medium was 800 mg N L-1 and the
carbon feed rate was adjusted to obtain a carbon to nitrogen ratio of 12 g g-1. The
fermentation broth was then concentrated with a continuous centrifuge (Cepa Z61H,
Lahr, Germany) at 17’500 x g and a flow rate of 50 L h-1. The paste was lyophilized
for 48 h, ground, and the dry biomass was stored in a closed container at -25 °C until
PHA extraction.
PHA extraction . PHUA, PHUE and PHOUE were extracted from dried biomass with
methylene chloride under stirring at room temperature for 24 h. After filtration, the
polymer solution was concentrated to one fifth of its original volume, cooled to 0 °C
and a tenfold excess of ethanol was added to induce precipitation. The polymer was
separated from the liquid phase by decantation and dried under vacuum. For
purification, the polymers were re-dissolved in methylene chloride, cooled to 0 °C and
a 10 fold excess of ethanol was added for precipitation. The solvent was decanted
and the PHA dried under vacuum.
Chemical derivatisation. PHOUE was linked with polyhedral oligomeric
silsesquioxane (POSS) via a free radical addition reaction as described elsewhere
(Hany et al. 2005). 1.9 g PHOUE, 14.0 g mercaptopropyl-isobutyl-POSS (POSS-SH)
and 0.27 g azobisisobutyronitrile (AIBN) were dissolved in 100 mL toluene and
heated to 75 °C for 18 h under nitrogen atmosphere. The solution was cooled to 0 °C
and 1 L of methanol was added for precipitation. To remove unreacted POSS-SH,
the polymer was dissolved in 200 mL methylene chloride and 1.5 L methanol was
added at room temperature for the precipitation of PHOUE-POSS. This procedure
was repeated four times for a complete removal of POSS-SH. The precipitate was
dried under vacuum.
101 Chapter 5
PHUE coatings in Petri dishes were treated with ozone generated with an
ozoniser (Certizon 200, Sander, Uetze-Eltze, Germany) supplied with pure oxygen.
The treatment time was set to 15 min for complete derivatisation of the double bonds
on the polymer surface based on contact angle measurements.
Solvent casting. Small (d=4 cm) and large (d=19.5 cm) glass Petri dishes were
coated with PHA for biocompatibility experiments after they had been depyrogenised
by heating to 200 °C for 6 h. To this end, the polymer was dissolved in
dichloromethane at a concentration of 2.5% w/v. The solution was filtrated (Nylon
filter, 0.45 µm) and cast into depyrogenised Petri dishes. The polymer solution was
slowly allowed to evaporate for 3 days at room temperature under an atmosphere
nearly saturated with dichloromethane to avoid the formation of bubbles. After that,
the polymer coatings were further dried under vacuum for 18 h at 40 °C. The
resulting polymer coating had a thickness of about 100 µm. Petri dishes with PHA
coatings were sterilised by x-ray previous to cell culturing.
PHA coating purification. For the cell culturing on PHA coatings, PHOUE-POSS
and PHUE treated with ozone (PHUE-O3) were purified to remove impurities of the
chemical derivatisation from the surface: PHOUE-POSS coatings were extracted with
3 mL of pure water for 3 h and with 3 mL of ethanol for 3 h and dried under vacuum.
PHUE-O3 was only dried under vacuum for 18 h to remove volatile impurities.
Endotoxin measurement. The endotoxicity was measured using a chromogenic
limulus amebocyte lysate (LAL) endpoint assay (QCL-1000, Cambrex, New Jersey,
USA). About 100 mg PHA was dissolved in 3 mL methylene chloride and solvent cast
into a depyrogenised glass Petri dish (d=4 cm). The solvent was slowly evaporated
under a nearly saturated atmosphere as described above the polymer coatings were
further dried under vacuum at 40 °C for 18 h. Three mL of pyrogen-free water
(Cambrex) was added to the Petri dish and incubated at 37 °C for 24 h to extract the
endotoxins from the polymer. The aqueous endotoxin-solution was directly used for
the LAL-test. Each analysis was carried out at least twice.
Water contact angle. The water contact angle was determined using a Krüss G10
contact angle measuring system (Hamburg, Germany) and pure water (Chromasolv,
Sigma-Aldrich). Three slides were coated for each polymer and two measurements
with droplets of 30 µL were made on each slide.
Biocompatibility of mcl-PHA 102
Gel permeation chromatography (GPC). Molecular weights (number-average Mn
and weight-average Mw) were determined by gel permeation chromatography (TDA
302, Viscotek, Waghausel-Kirrlach, Germany) equipped with a viscometer, RI- and
light-scattering detector. The system was calibrated by using 2 polystyrene standards
(Polycal, Viscotek) with known molecular weights and known intrinsic viscosities
(standard 1: Mw = 115 kDa, Mn = 112 kDa, IV=0.519, standard 2: Mw = 250 kDa, Mn =
100 kDa, IV=0.843). Then, 40 mg of PHA was dissolved in 10 mL of THF over night,
filtered through a 1 µm nylon filter, and aliquots of 100 µL were chromatographed
with pure THF as solvent phase through three GPC columns (Mixed bed, Viscotek) at
35 °C and a flow rate of 1 mL min-1.
Gas chromatography. Quantitative analysis of PHA has been performed by
methanolysis catalyzed by BF3 as described in chapter 2.
Cytotoxicity according to ISO10993-5
To test if a material releases biologically active substances to an aqueous
environment, the material was incubated for 3 days in water. Fibroblasts of type 3T3
were cultured with these extracts on 96 well plates. After 5 days the cell cultures
were analysed to determine the influence of the extract.
Extraction of water soluble PHA contaminations. The polymer coatings prepared
by solvent casting in Petri dishes (d=19.5 cm) had a thickness of about 10 µm and a
surface of 300 cm2. An aliquot of 25 mL of water containing 1% penicillin-
streptomycin-neomycin (PSN) antibiotics (Invitrogen, Basel, Switzerland) was added
to the Petri dish to extract substances from the PHA coating and incubated for 72 h
at 37 °C. The pH was initially adjusted to 7.4 with NaHCO3. The aqueous extract was
diluted with water and supplemented with 800 µL of Dulbecco’s minimal essential
medium (DMEM, Invitrogen), 300 µL NaHCO3 7.5%, 400 µL of foetal bovine serum
(FBS, Invitrogen) and 80 µL of PSN antibiotics to obtain solutions containing 0, 10,
20, 50 and 70 % of the aqueous extract.
Cell culturing. Each well was incubated with 200 µL of cell suspension containing
about 7500 3T3 cells per mL for 24 h. The medium was gently removed and 200 µL
of the diluted extract was added. After 5 days of incubation at 37 °C and a gas
atmosphere containing 5% CO2, DNA from permeabilised cells was measured with
103 Chapter 5
the Hoechst 33258 DNA stain (Invitrogen) and total protein was determined with the
bicinchonin acid protein assay kit (Pierce, Nr. 23225, Rockford Illinois) according to
the manufacturers protocols. To measure the metabolic activity, the mitochondrial
activity was determined by the MTT assay (Mosmann 1983) and the activity of
lysosymes was measured with the neutral red assay (Borenfreund et al. 1985).
Cell culturing on PHA coatings
Cell culture. Mouse connective tissue fibroblasts were bought (cell line L929 –
ECACC, 85011425, lot 04H008, Sigma, Poole, UK) and cultured in 88% DMEM high
glucose (Biosera L0106, East Sussex, UK), supplemented with 10% FCS (504002,
batch S1900-Biosera), 1% antibiotics (streptomycin and penicillin L00100, Biosera),
1% L-glutaraldehyde (G7513, Sigma-Aldrich), and incubated at 37 °C with a gas
atmosphere containing 5% CO2 in T175 culture flasks (Starstedt T175 vented red cap
83.1813.002, Leicester, UK). The cells were passaged 2 times and seeded at a
density of 100’000 cells/well into the small PHA-coated Petri dishes and into the wells
of Costar polystyrene (PS) 6-well plates (Corning, No. 3516, Massachusetts, USA)
and incubated to the required time point (7 days and 14 days). The medium was
changed every 4 days throughout the incubation period.
The cell viability was measured with the live/dead assay kit (Invitrogen,
L3224). To quantitate double-stranded DNA in solution, the PicoGreen® assay was
used (Invitrogen, P7589). Cells adhered to PHA and PS and cells in suspension were
measured separately. The manufacturers’ protocols were used for these two assays.
The hydroxyproline content was considered as an indicator for the collagen
production. It was determined as described elsewhere (Kivirikko et al. 1967).
Probability values of less than 0.05 (P<0.05) were considered to be significantly
different (2-tailed student T-test, n=5).
Biocompatibility of mcl-PHA 104
5.3 Results and discussion
5.3.1 Physical and chemical properties of PHAs
The purity of all PHAs was >95 w% according to GC measurements (Table 5.1). The
endotoxic activity of the polymer coatings was decreased below the detection limit
and was below 1 endotoxic unit per gram (EU g-1). The molecular weights were
similar for PHUA, PHUE, PHOUE and PHUE-O3 (Mw ≅ 200 kDa) and only for
PHOUE-POSS clearly increased, which was due to the linked POSS groups. The
water contact angle of untreated PHAs (PHUA, PHUE and PHOUE) was between
104° and 109°, whereas the POSS-derivatised PHOUE and the ozone-treated PHUE
showed lower values of 95° and 73° respectively. The monomeric composition of all
PHAs prior to chemical derivatisation is given in Table 5.2.
Table 5.1: Extraction, purification and derivatisation techniques applied for the production of different mcl-PHAs and their physical properties.
PHAs Extraction Purification Derivati-
sation
Contact
angle
Mw Mn Purity Endotoxicity
solvent-
non solvent
solvent-
non solvent
[°] [kDa] [kDa] [w%] [EU g-1]
PHUA CH2Cl2-EtOH CH2Cl2-EtOH 2x - 109 168 91 >95 <1
PHUE CH2Cl2-EtOH CH2Cl2-EtOH 2x - 104 231 115 >95 <1
PHOUE CH2Cl2-EtOH CH2Cl2-EtOH 2x - 108 255 151 >95 <1
PHOUE-
POSS
CH2Cl2-EtOH CH2Cl2-EtOH 2x,
CH2Cl2-EtOH2 4x
POSS-
SH
95 910 420 >95 <1
PHUE-
O3
CH2Cl2-EtOH CH2Cl2-EtOH 2x Ozone 73 231 115 >95 <1
2 Purification after chemical derivatisation
105 Chapter 5
Table 5.2 : Monomeric composition of mcl-PHAs determined by GC after transesterification.
PHAs Monomeric composition [mole%]
C5:0 a C6:0 C7:0 C8:0 C9:0 C11:0 C7:6 C9:8 C11:10
PHUA 2.8 33.8 49.0 14.4
PHUE 24.8 62.9 12.3
PHOUE 5.1 32.5 11.7 38.1 12.6
PHOUE-POSS 5.1 32.5 11.7 38.1 12.6
PHUE-O3 24.8 62.9 12.3
a) The notation Cn:x was used to denote the 3-hydroxy acids, where n is the number of
carbon atoms and x indicates the position of the double bond (0: no double bond present).
5.3.2 Cytotoxicity according to the eluate test
A critical point for the application of PHA (and other polymers) in the medical field is
the effect of water soluble substances contained in the polymer. To proof that no
toxic substances can diffuse from the polymer matrix to an aqueous environment the
cytotoxicity test according to ISO 10993-5 is commonly applied. Accordingly, PHA
coatings were extracted with water for 3 days and 3T3 fibroblasts were incubated
with diluted extracts for 5 days and finally the proliferation was determined. For pure
PHAs without chemical derivatisation a toxic effect in the aqueous eluate could
hardly be seen (Fig. 5.1A - C) in contrast to chemically derivatised PHAs where cell
proliferation was strongly reduced Fig. 5.1D - E).
Biocompatibility of mcl-PHA 106
0
50
100
0
50
100
0
50
100
0
50
100
0 20 40 60 800
50
100
Extract concentration [% v/v]
Rel
ativ
e co
ncen
trat
ion
[%]
Rel
ativ
e co
ncen
trat
ion
[%]
Rel
ativ
e co
ncen
trat
ion
[%]
Rel
ativ
e co
ncen
trat
ion
[%]
Rel
ativ
e co
ncen
trat
ion
[%]
A
B
C
D
E
Fig. 5.1: Cytotoxic activity of aqueous extracts from PHUA (A), PHUE (B), PHOUE (C), PHOUE-POSS (D) and PHUE-O3 (E). The toxic activity was determined with 3T3-fibroblasts by � DNA, � MTT, � protein and � neutral red assays.
107 Chapter 5
For PHUA, and PHUE no toxic effect could be seen with the DNA, MTT, protein
and neutral red assay (Fig. 5.1A - B). Their values varied between 90 and 115%
relative to the control, but there was no increase in the effect with increasing
concentration. Hence, these polymers appear to be free of biologically active
impurities that could be solubilised in an aqueous environment. For PHOUE a
cytotoxic effect was measured at the highest extract concentration where the values
for DNA, MTT and protein were reduced to about 80% of the control (Fig. 5.1C).
Apparently, PHOUE contained small amounts of biologically active impurities.
For PHOUE-POSS the cell proliferation and activity was decreased after 5 days
to about 60% (Fig. 5.1D). This toxic effect was attributed to side-products formed
during chemical derivatisation. Although the derivatised PHA was thoroughly purified
by dissolution-precipitation until no impurities of low molecular weight could be
detected by GPC, high toxicity was still measured.
Even more drastically the relative values of DNA, MTT, protein and neutral red
decreased for PHUE-O3. They reached around 20% at the highest extract
concentration (Fig. 5.1E). It has been reported that formaldehyde and small amounts
of formic acid are generated during oxidation of alkenes with ozone (Dubowski et al.
2004; Eliason et al. 2003). Adsorption of these products on the polymer surface, or
trapping in the polymer combined with slow diffusion can explain the toxic effect we
measured. Further purification of PHOUE-POSS and PHUE-O3 was required after
chemical derivatisation and was applied before cell culturing on PHA coatings.
5.3.3 Cell culturing on PHA coatings
Mouse connective tissue fibroblasts were seeded directly on the PHA coatings to
analyse their interaction with the polymer surface. Polystyrene (optimised for cell
attachment) was used as control material.
Unmodified PHAs: PHUA, PHUE and PHOUE
Proliferation had occurred during the 7 days according to the Picogreen assay. About
90%, 230% and 60% more cells were found on the PHUA, PHUE and PHOUE
coating, respectively in comparison to the control (Fig. 5.2). Most cells were attached
to the mcl-PHA surface and only few cells (~ 2%) were suspended in the medium.
Biocompatibility of mcl-PHA 108
Consequently, the loss of cells during medium change was negligible. This
observation was similar for all mcl-PHA coatings.
0
600000
1200000
Control PHUA PHUA coating Control PHUE PHUE coating Control PHOUE PHOUE coating
Cel
l num
ber
per
wel
l
Fig. 5.2: Cell numbers of adhered cells on PHUA, PHUE and PHOUE coatings and corresponding controls (control PHUA, control PHUE and control PHOUE) after 7 days, determined with the Picogreen assay.
The results of the live/dead assay for PHUA and PHUE showed that very few
viable cells were present on these PHA coatings in comparison to the control (Fig.
5.3a-c). This is in contrast to the measurements made with the Picogreen assay. The
cell film on the PHA coating was very confluent before the staining procedure.
However, before the live/dead staining was carried out, the cell density was not
evenly distributed on the PHUE coating but found in patches. The surface properties
of the PHA coating may have prevented the cells from adhering strongly, where they
became dislodged and washed away during washing steps of the live/dead assay.
Still, cell adhesion was strong enough to prevent a significant loss of cells during
routine medium change as shown with the Picogreen assay.
Contrarily, the live/dead assay for PHOUE showed that this coating had a high
density of viable cells, comparable to the cell density of the control (Fig. 5.3d and e).
This indicates that the surface properties were suitable for providing strong cell
attachment.
109 Chapter 5
Fig. 5.3: Fluorescence microscope images of live/dead-stained mouse connective tissue fibroblasts on PHUA after 7 days. (a) PHUA coating, (b) PS control for PHUA and PHUE, (c) PHUE coating, (d) PHOUE coating and (e) PS control for PHOUE. Viable cells were stained green.
The results of the hydroxyproline assay, which is used as an indicator for the
collagen production, showed that there was a higher concentration of hydroxyproline
in the PS control than in the coatings of PHUA, PHUE and PHOUE as exemplified for
PHUA in Fig. 5.4. The hydroxyproline concentration was reduced to 35% for PHUA
and PHUE and to 50% for PHOUE compared to the reference. However, for the
PHUE coating there was a considerable error in the hydroxyproline concentration.
This assay specifically measures the amount of hydroxyproline produced by
a b
c
d e
Biocompatibility of mcl-PHA 110
functional cells in the form of collagen. Hydroxyproline makes up for up to 13% of
collagen. However, the cells on PHA coatings seemed to be significantly less
functional than the control cells. Possibly, degradation products of PHA had effected
gene expression of certain extracellular matrix genes, such as switching off the gene
that produces hydroxyproline.
The results of these assays suggest that the PHUA, PHUE and PHOUE coatings
had no toxic effect and were supporting some cell growth with limited collagen
production. The surface properties of PHUA and PHUE may not suitable to allow
strong cell adherence. Only for PHOUE good cell attachment was observed. The
reason for improved cell adhesion was not clear. It could not be attributed to the
unsaturated side-chains because the cell adhesion was weak for PHUE, which
contained more unsaturated side-chains. Possibly the surface of PHOUE had an
increased roughness, thereby easing cell adhesion.
0
5
10
15
Control PHUA PHUA coating Control PHOUE-POSS PHOUE-POSS coating
Hyd
roxy
prol
ine
conc
entr
atio
n (
µg m
L-1
)
Fig. 5.4: Hydroxyproline assay for cells on PHUA and PHOUE-POSS coatings and corresponding PS controls (control PHUA and control PHOUE-POSS) after 14 days. P = 9.45 x10-10 and 0.029, respectively.
Chemically modified mcl-PHAs: PHOUE-POSS and PHOUE- O3
Similarly to PHOUE, a high cell number was observed with the Picogreen assay for
the PHOUE-POSS coating. In contrast to this, the cell number on the PHOUE-O3
coating was nearly identical to the one of the control and hence smaller compared to
all other PHA coatings.
111 Chapter 5
For PHOUE-POSS many viable cells were observed with the live/dead assay
(Fig. 5.5a). The density was comparable to the control. Hence, the cell adhesion was
strong, similar to that of PHOUE. In analogy, the live/dead assay showed a high cell
density for PHUE-O3 (Fig. 5.5c). Consequently, strong cell adhesion was obtained by
treating PHUE with ozone.
Fig. 5.5: Fluorescence microscope images of live/dead-stained mouse connective tissue fibroblasts on PHOUE-POSS (a), PHUE-O3 (c) and corresponding PS controls (b and d) after 7 days.
The results of the hydroxyproline assay for PHOUE-POSS showed that the
control had again a significantly higher hydroxyproline concentration than the coating
(Fig. 5.4). Hence, the functionality of fibroblasts on untreated and modified PHA
coatings seemed to be reduced. Surprisingly, the POSS derivatisation of PHOUE did
not improve cell adhesion, proliferation and collagen production considerably. For
PHUE-O3, the results of the hydroxyproline assay showed a reduced hydroxyproline
concentration, but they were not very significant because the error of the control was
big (data not shown).
a b
c d
Biocompatibility of mcl-PHA 112
The ozone derivatisation drastically decreased the water contact angle of the
surface and increased the cell adhesion compared to the untreated PHUE. In parallel
to this the cell proliferation was clearly decreased. The PHUE-O3 coating showed
similar properties to PS optimized for cell culturing, if the results from the
hydroxyproline were not considered.
According to our results, the PHAs can be divided into two groups according to
their suitability for cell attachment, proliferation and function:
PHOUE, PHOUE-POSS and PHUE-O3: These biomaterials provided good cell
attachment regarding the live/dead staining. The cell proliferation was equal or better
on these coatings than on PS. The collagen production was reduced with these
PHAs.
PHUA and PHUE: Neither coating provided very good cell attachment (live/dead
stain) and most of the cells became dislodged during the staining procedure. Both
coatings supported proliferation resulting in cell numbers higher than on the PS
control. The cells produced lower amounts of collagen on both coatings in
comparison to controls.
5.4 Conclusions
Cytotoxicity experiments as well as cell-culturing experiments on PHA coatings
showed that biosynthesised mcl-PHA with different chemical compositions are suited
for medical applications, when an effective purification procedure such as repeated
dissolution-precipitation was applied. Cell adhesion was limited on the non-polar
surfaces of mcl-PHA and hence a derivatisation of the PHA surface was required to
improve its hydrophilicity and to optimise the cell attachment. For long term
applications the type of chemical modification may be of crucial importance. Surface-
modified PHAs like PHUA-O3 could loose the polar groups during degradation and
consequently the surface would become more hydrophobic. Homogeneous PHAs on
the other hand will not change their surface polarity drastically during degradation.
Possibly, the cleavage of ester bonds during degradation will increase the surface
polarity through the formation of additional carboxyl and hydroxyl groups. Hence,
long time studies are needed to elucidate these questions. For applications where
113 Chapter 5
cell attachment has to be avoided, mcl-PHA with saturated side-groups could be well
suited.
Our measurements indicated that the collagen production of fibroblasts was
reduced on all PHA coatings, compared to the cells on PS. Hence the functionality of
fibroblasts on PHA coatings appeared to be reduced. The reason for this observation
was indistinct and further measurements with another assay are needed to confirm
this result. Furthermore the collagen type should be determined to identify eventual
changes.
Biocompatibility of mcl-PHA 114
115
Chapter 6
General conclusions
General conclusions 116
The use of polymeric materials for the administration of pharmaceutical and as bio-
medical devices has increased dramatically over the last ten years (Shikanov et al.
2005). Important biomedical applications of biodegradable polymers are in the areas
of drug delivery systems and in the form of implants and devices for fracture repairs,
surgical dressings, artificial heart valves, vascular grafts, and organ regeneration. It
has been concluded that PHA has the potential to become an important compound
for biomedical applications (Valappil et al. 2007; Williams et al. 1999; Zinn et al.
2001) and the results of this thesis further confirm this conclusion.
The aim of this work was to adapt the properties of mcl-PHA for medical applica-
tions by optimising its fermentative production and downstream processing (chapter
3 and 4). PHO was used as a model for the class of mcl-PHA. To analyze and opti-
mize the production of mcl-PHA, a new method for the quantitative analysis by gas
chromatography (GC) was developed (chapter 2). Finally, the biocompatibility of mcl-
PHA films was investigated in vitro using mouse fibroblasts and the polymers were
chemically modified to enhance their properties (chapter 5).
Quantitative analysis of PHA
Nuclear magnetic resonance spectroscopy (NMR) is a common tool to analyse and
quantify bacterial PHA. But NMR analyses are restricted to pure PHA and are very
time-consuming, whereas GC measurements are cheap and need less time. Estab-
lished methods for the transesterification of PHA in biomass with protic acids were
shown to be inadequate for the quantitative analysis of mcl-PHA due to an excessive
formation of side-products. This problem could be solved by using the Lewis-acid BF3
in water-free methanol. However, the development of a fast method that can be used
to derivatise and analyse PHA-containing biomass in a short time (e.g. 1 h) remains
a challenge. For PHB-containing biomass the derivatisation time was drastically de-
creased to about 4 minutes by using microwave heating (Betancourt et al. 2007).
Presumably, microwave heating can likewise be used for the accelerated transesteri-
fication of mcl-PHA. Alternatively, other derivatisation reactions like the reductive de-
polymerisation by LiAlH4 could be applied to mcl-PHA. Ideally, the PHA concentration
in a fermenter is measured continuously in situ, but a reliable method has not been
developed yet.
117 Chapter 6
PHA biosynthesis – possibilities to reduce the endo toxic contamination
P-limitation was successfully applied in this work to the fermentative production of
mcl-PHA to reduce the endotoxic contamination. The confirmation of the structural
change of lipid A was unsuccessful and it would be interesting to investigate it. How-
ever, P-limitation could not be extended arbitrarily in the chemostat as the cell growth
rate became restricted. Possibly, P-limitation would be more efficient in a two-stage
fed batch or two-stage continuous fermentation with P-limitation in the second stage.
In this case the endotoxic activity would only decrease if the structure of Lipid A is
changed during P-limitation. Recent experiments with Francisella novicida showed
that certain microorganisms own specific phosphatases for lipid A (Wang et al. 2004;
Wang et al. 2006). If similar phosphatases were also present in P. putida, the phos-
phorylation of lipid A could be modified during the adaptation of cells to altered cultur-
ing conditions. Otherwise, the genes coding for these phosphatases (LpxE and LpxF)
could be overexpressed in PHA producing bacteria like P. putida to detoxify the lipid
A, independently from culturing conditions.
Another alternative to avoid the contamination with LPS is the production of PHA
with Gram-positive bacteria. PHAs were produced with the genera Bacillus and
Streptomyces (Valappil et al. 2007). However, the ability to synthesize mcl-PHA with
Gram-positive bacteria appears to be rather limited (Valappil et al. 2007).
PHA recovery
PHA extraction with organic solvents and precipitation with non-solvents results in a
polymer of high quality, but this standard method is rather complicated and produces
large amounts of mixtures of solvent and non-solvent. Temperature controlled extrac-
tion and precipitation (TCP) was found to be a good alternative to the classical ap-
proach. It could be used to extract and precipitate PHO and co-polymers of PHO effi-
ciently. The recovery of PHA is basically complicated by the fact that PHA is stored
intracellularly. It would be simplified a lot if the PHA granules were secreted to the
medium. The cells could be separated from the supernatant by centrifugation and the
PHA granules would be recovered with the supernatant in high purity. However, the
transport of huge PHA granules through the membrane of intact cells appears to be
unrealistic. Recently, the production of extracellular PHA with Alcanivorax borkumen-
sis SK2 has been claimed (Sabirova et al. 2006), but it was not shown that extracel-
General conclusions 118
lular PHA did not originate from lysed cells. Hence, serious doubts remain and further
research is needed.
Towards biomedical applications
Cytotoxicity experiments with aqueous extracts from mcl-PHA as well as cell-
culturing experiments on films of mcl-PHA confirmed that these polymers were bio-
compatible when mcl-PHA of high purity was used. Cell adhesion could be improved
by chemical treatments of the non-polar PHA surface. However, the functionality of
mouse connective tissue fibroblasts was limited. The reason for this observation re-
mained obscure and further investigation is needed.
An important but restraining aspect is the processing of mcl-PHA. For most mcl-
PHA processing is difficult due to their elastomeric and sticky properties. Hence, ap-
propriate processing techniques have to be developed for a successful commercial
application. Again, chemical derivatisation may help to increase the crystallinity of
PHA, thereby facilitating its processing.
The results obtained in this doctoral thesis show, that optimized fermentation and
downstream processing generate mcl-PHA of high quality with very low endotoxic
contamination that can be used in the biomedical field. The broad range of different
mcl-PHAs combined with chemical modification allows adjusting the chemical and
physical properties to the target application. Thus, a basis for successful biomedical
applications of mcl-PHA was established with this work.
References 119
References
Agrawal, C. M., Athanasiou K. A. 1997. Technique to control pH in vicinity of biodegrading
PLA-PGA implants. J. Biomed. Mater. Res. 38: 105-114.
Alvarez, H. M., Kalscheuer R., Steinbüchel A. 1997. Accumulation of storage lipids in
species of Rhodococcus and Nocardia and effect of inhibitors and polyethylene glycol. Fett-
Lipid 99: 239-246.
Anderson, A. J., Dawes E. A. 1990. Occurence, metabolism, metabolic role, and industrial
uses of bacterial polyhydroxyalkanoates. Microbiol. Rev. 54: 450-472.
Annuar, M. S. M., Tan I. K. P., Ibrahim S., Ramacha ndran K. B. 2007. Production of
medium-chain-length poly(3-hydroxyalkanoates) from crude fatty acids mixture by
Pseudomonas putida. Food Bioprod. Process. 85: 104-119.
Ashby, R. D., Foglia T. A., Solaiman D. K. Y., Liu C. K., Nunez A., Eggink G. 2000.
Viscoelastic properties of linseed oil-based medium chain length poly(hydroxyalkanoate)
films: effects of epoxidation and curing. Int. J. Biol. Macromol. 27: 355-361.
Bachmann, B. M., Seebach D. 1999. Investigation of the enzymatic cleavage of
diastereomeric oligo(3-hydroxybutanoates) containing two to eight HB units. A model for the
stereoselectivity of PHB depolymerase from Alcaligenes faecalis T-1. Macromolecules 32:
1777-1784.
Baptist, J. N. W. R. Grace & Co., 1962a. Process for preparing PHB. US patent 3036959.
Baptist, J. N. W:R. Grace & co., 1962b. Process for preparing PHB. US patent 3044942.
Barham, P. J. Imperial Chemical Industries Ltd., UK, 1982. Extraction of poly(ß-
hydroxybutyric acid). EP patent 58480.
Bax, A., Marion D. 1988. Improved resolution and sensitivity In H-1-detected heteronuclear
multiple bond correlation spectroscopy. J. Magn. Reson. 78: 186-191.
Bear, M. M., Mallarde D., Langlois V., Randriamahef a S., Bouvet O., Guérin P. 1999.
Natural and artificial functionalized biopolyesters. II. Medium-chain length poly-
hydroxyoctanoates from Pseudomonas strains. J. Environ. Polym. Degrad. 7: 179-184.
Berger, E., Ramsay B. A., Ramsay J. A., Chaverie C. , Braunegg G. 1989. PHB recovery
by hypochlorite digestion of non-PHB biomass. Biotechnol. Tech. 3: 227-232.
References 120
Betancourt, A., Yezza A., Halasz A., Van Tra H., Ha wari J. 2007. Rapid microwave
assisted esterification method for the analysis of poly-3-hydroxybutyrate in Alcaligenes latus
by gas chromatography. J. Chromatogr. A 1154: 473-476.
Bordoloi, M., Borah B., Thakur P. S., Nigam J. N.; Council of scientific & industrial
research, India. 2003. Process for the isolation of polyhydroxybutyrate from Bacillus
mycoides RLJ B-017. US patent 2003027293.
Borenfreund, E. and Puerner, J. A. 1985. Toxicity determined in vitro by morphological
alterations and neutral red absorption. Toxicol. Lett. 24, 119-124.
Bos, M. P., Tommassen J. 2005. Viability of a capsule- and lipopolysaccharide-deficient
mutant of Neisseria meningitidis. Infect. Immun. 73: 6194-6197.
Boynton, Z. L., Koon J. J., Brennan E. M., Clouart J. D., Horowitz D. M., Gerngross T.
U., Huisman G. W. 1999. Reduction of cell lysate viscosity during processing of poly(3-
hydroxyalkanoates) by chromosomal integration of the staphylococcal nuclease gene in
Pseudomonas putida. Appl. Environ. Microb. 65: 1524-1529.
Braunegg, G., Lefebvre G., Genser K. F. 1998. Polyhydroxyalkanoates, biopolyesters from
renewable resources: Physiological and engineering aspects. J. Biotechnol. 65: 127-161.
Braunegg, G., Sonnleitner B., Lafferty R. M. 1978. A rapid gas chromatographic method
for the determination of poly-3-hydroxybutyric acid in microbial biomass. Eur. J. Appl.
Microbiol. Biotechnol. 6: 29-37.
Byrom, D. 1987. Polymer synthesis by microorganisms: technology and economies. Trends
Biotechnol. 5: 246-250.
Caballero, K. P., Karel S. F., Register R. A. 1995. Biosynthesis and characterization of
hydroxybutyrate-hydroxycaproate copolymers. Int. J. Biol. Macromol. 17: 86-92.
Castor, T. P., Hong G. T.; Aphios Corporation, USA, 2003. Method of fractionation of
biologically derived materials using critical fluids. US patent 6569640.
Chen, G. Q., Konig K. H., Lafferty R. M. 1991. Occurrence of poly-D(-)-3-
hydroxyalkanoates in the genus Bacillus. FEMS Microbiol. Lett. 84: 173-176.
Chen, G. Q., Wu Q. 2005. The application of polyhydroxyalkanoates as tissue engineering
materials. Biomaterials 26: 6565-6578.
Chen, G. Q., Wu Q., Wang Y. W., Zheng Z. 2005. Application of microbial polyesters-
polyhydroxyalkanoates as tissue engineering materials. Asbm6: Advanced Biomaterials Vi. p
437-440.
121 References
Chen, G. Q., Zhang G., Park S. J., Lee S. Y. 2001. Industrial scale production of poly(3-
hydroxybutyrate-co-3-hydroxyhexanoate). Appl. Microbiol. Biotechnol. 57: 50-55.
Chen, P. S., Toribara T. Y., Warner H. 1956. Microdetermination of phosphorus. Anal.
Chem. 28: 1756-1758.
Choi, J. I., Lee S. Y. 1999a. Efficient and economical recovery of poly(3-hydroxybutyrate)
from recombinant Escherichia coli by simple digestion with chemicals. Biotechnol. Bioeng.
62: 546-553.
Choi, J. I., Lee S. Y. 1999b. High-level production of poly(3-hydroxybutyrate-co-3-
hydroxyvalerate) by fed-batch culture of recombinant Escherichia coli. Appl. Environ. Microb.
65: 4363-4368.
Choi, J. I., Lee S. Y., Han K. 1998. Cloning of the Alcaligenes latus polyhydroxyalkanoate
biosynthesis genes and use of these genes for enhanced production of poly(3-
hydroxybutyrate) in Escherichia coli. Appl. Environ. Microb. 64: 4897-4903.
Cui, D., Tian F., Ozkan C. S., Wang M., Gao H. 2005. Effect of single wall carbon
nanotubes on human HEK293 cells. Toxicol. Lett. 155: 73-85.
Cumming, R. H., Rees P., Watson J. S.; Zeneca Ltd., UK, 1994. Process for the separation
of solid materials from microorganisms. WO patent 9411491.
Davis, A. L., Keeler J., Laue E. D., Moskau D. 1992. Experiments for recording pure-
absorption heteronuclear correlation spectra using pulsed field gradients. J. Magn. Reson.
98: 207-216.
de Koning, G. J. M., Witholt B. 1997. A process for the recovery of poly(hydroxyalkanoates)
from Pseudomonads. 1. Solubilization. Bioproc. Eng. 17: 7-13.
de Koning, G. J. M., Kellerhals M., vanMeurs C., Wi tholt B. 1997. A process for the
recovery of poly(hydroxyalkanoates) from Pseudomonads. 2. Process development and
economic evaluation. Bioproc. Eng. 17: 15-21.
Deng, Y., Lin X. S., Zheng Z., Deng J. G., Chen J. C., Ma H., Chen G. Q. 2003.
Poly(hydroxybutyrate-co-hydroxyhexanoate) promoted production of extracellular matrix of
articular cartilage chondrocytes in vitro. Biomaterials 24: 4273-4281.
Diniz, S. C., Taciro M. K., Gomez J. G. C., Pradell a J. G. D. 2004. High-cell-density
cultivation of Pseudomonas putida IPT 046 and medium-chain-length polyhydroxyalkanoate
production from sugarcane carbohydrates. Appl. Biochem. Biotechnol. 119: 51-69.
Dixon, D. R., Darveau R. P. 2005. Lipopolysaccharide heterogeneity: Innate host responses
to bacterial modification of lipid A structure. J. Dent. Res. 84: 584-595.
References 122
Doi, Y., Kawaguchi Y., Koyama N., Nakamura S., Hira mitsu M., Yoshida Y., Kimura H.
1992. Synthesis and degradation of polyhydroxyalkanoates in Alcaligenes eutrophus. FEMS
Microbiol. Rev. 103: 103-108.
Doi, Y., Kitamura S., Abe H. 1995. Microbial synthesis and characterization of poly(3-
hydroxybutyrate-co-3-hydroxyhexanoate). Macromolecules 28: 4822-4828.
Doi, Y., Kunioka M., Nakamura Y., Soga K. 1986. Nuclear magnetic resonance studies on
poly(ß-hydroxybutyrate) and a copolyester of ß-hydroxybutyrate and ß-hydroxyvalerate
isolated from Alcaligenes eutrophus H16. Macromolecules 19: 2860-2864.
Dubowski, Y., Vieceli J., Tobias D. J., Gomez A., L in A., Nizkorodov S. A., McIntire T.
M., Finlayson-Pitts B. J. 2004. Interaction of gas-phase ozone at 296 K with unsaturated
self-assembled monolayers: A new look at an old system. J. Phys. Chem. A 108: 10473-
10485.
Dufresne, A., Reche L., Marchessault R. H., Lacroix M. 2001. Gamma-ray crosslinking of
poly (3-hydroxyoctanoate-co-undecenoate). Int. J. Biol. Macromol. 29: 73-82.
Durner, R., Witholt B., Egli T. 2000. Accumulation of poly[(R)-3-hydroxyalkanoates] in
Pseudomonas oleovorans during growth with octanoate in continuous culture at different
dilution rates. Appl. Environ. Microbiol. 66: 3408-3414.
Eckelt, J., Haase T., Loske S., Wolf B. A. 2004. Large scale fractionation of macro-
molecules. Macromol. Mater. Eng. 289: 393-399.
Eggink, G., de Waard P., Huijberts G. N. M. 1992. The role of fatty acid biosynthesis and
degradation in the supply of substrates for poly(3-hydroxyalkanoate) formation in
Pseudomonas putida. FEMS Microb. Rev. 103: 159-164.
Egli, T. 1991. On multiple-nutrient-limited growth of microorganisms, with special reference
to dual limitation by carbon and nitrogen substrates. Antonie van Leeuwenhoek 60: 225-234.
Eliason, T. L., Aloisio S., Donaldson D. J., Cziczo D. J., Vaida V. 2003. Processing of
unsaturated organic acid films and aerosols by ozone. Atmos. Environ. 37: 2207-2219.
Emeruwa, A. C., Hawirko R. Z. 1973. Poly-ß-hydroxybutyrate metabolism during growth
and sporulation of Clostridium botulinum. J. Bacteriol. 116: 989-993.
Eroglu, M. S., Hazer B., Ozturk T., Caykara T. 2005. Hydroxylation of pendant vinyl groups
of poly(3-hydroxy undec-10-enoate) in high yield. J. Appl. Polym. Sci. 97: 2132-2139.
Escalona, A. M., Varela F. R., Gomis A. M.; Repsol Quimica S.A. Madrid, 1996. Procedure
for extraction of polyhydroxyalkanoates from halophilic bacteria which contain them. US
patent 5536419.
123 References
Ferenci, T. 1999. 'Growth of bacterial cultures' 50 years on: towards an uncertainty principle
instead of constants in bacterial growth kinetics. Res. Microbiol. 150: 431-438.
Folch, J., Lees M., Stanley G. H. S. 1957. A simple method for the isolation and purification
of total lipids from animal tissues. J. Biol. Chem. 226: 497-509.
Füchtenbusch, B., Fabritius D., Waltermann M., Stei nbüchel A. 1998. Biosynthesis of
novel copolyesters containing 3-hydroxypivalic acid by Rhodococcus ruber NCIMB 40126
and related bacteria. FEMS Microbiol. Lett. 159: 85-92.
Füchtenbusch, B., Steinbüchel A. 1999. Biosynthesis of polyhydroxyalkanoates from low-
rank coal liquefaction products by Pseudomonas oleovorans and Rhodococcus ruber. Appl.
Microbiol. Biotechnol. 52: 91-95.
Fukui, T., Doi Y. 1998. Efficient production of polyhydroxyalkanoates from plant oils by
Alcaligenes eutrophus and its recombinant strain. Appl. Microbiol. Biot. 49: 333-336.
George, N., Liddell J. M.; Monsanto Company, USA; George, Neil; Liddell, John
Macdonald. 1997. Separating polyester particles from fermentation broth. WO patent
9722654.
Godoy, F., Vancanneyt M., Martinez M., Steinbüchel A., Swings J., Rehm B. H. A. 2003.
Sphingopyxis chilensis sp. nov., a chlorophenol-degrading bacterium that accumulates
polyhydroxyalkanoate, and transfer of Sphingomonas alaskensis to Sphingopyxis alaskensis
comb. nov. Int. J. Sys. Evol. Micr. 53: 473-477.
Gomes, M. E., Reis R. L. 2004. Biodegradable polymers and composites in biomedical
applications: from catgut to tissue engineering - Part 1 - Available systems and their
properties. Int. Mater. Rev. 49: 261-273.
Gorenflo, V., Schmack G., Vogel R., Steinbüchel A. 2001. Development of a process for
the biotechnological large-scale production of 4-hydroxyvalerate-containing polyesters and
characterization of their physical and mechanical properties. Biomacromolecules 2: 45-57.
Greer, W.; Zeneca Ltd. 1994. Peroxide degradation of DNA for viscosity reduction. WO
patent 9410289.
Greer, W.; Zeneca Ltd. 1997. Peroxide degradation of DNA for viscosity reduction. US
patent 5627276.
Hahn, S. K., Chang Y. K., Kim B. S., Chang H. N. 1994. Optimization of microbial poly(3-
hydroxybutyrate) recovery using dispersions of sodium hypochlorite solution and chloroform.
Biotechnol. Bioeng. 44: 256-261.
References 124
Hahn, S. K., Chang Y. K., Lee S. Y. 1995. Recovery and characterization of poly(3-
hydroxybutyric acid) synthesized in Alcaligenes eutrophus and recombinant Escherichia coli.
Appl. Environ. Microbiol. 61: 34-39.
Hampson, J. W., Ashby R. D. 1999. Extraction of lipid-grown bacterial cells by supercritical
fluid and organic solvent to obtain pure medium chain-length polyhydroxyalkanoates. J. Am.
Oil Chem. Soc. 76: 1371-1374.
Hany, R., Hartmann R., Bohlen C., Brandenberger S., Kawada J., Lowe C., Zinn M.,
Witholt B., Marchessault R. H. 2005. Chemical synthesis and characterization of POSS-
functionalized poly [3-hydroxyalkanoates]. Polymer 46: 5025-5031.
Hartmann, R., Hany R., Geiger T., Egli T., Witholt B., Zinn M. 2004. Tailored biosynthesis
of olefinic medium-chain-length poly [(R)-3-hydroxyalkanoates] in Pseudomonas putida
GPo1 with improved thermal properties. Macromolecules 37: 6780-6785.
Hartmann, R., Hany R., Pletscher E., Ritter A., Wit holt B., Zinn M. 2006. Tailor-made
olefinic medium-chain-length poly[(R)-3-hydroxyalkanoates] by Pseudomonas putida GPo1:
Batch versus chemostat production. Biotechnol. Bioeng. 93: 737-746.
Haywood, G. W., Anderson A. J., Williams D. R., Daw es E. A., Ewing D. F. 1991.
Accumulation of a poly(hydroxyalkanoate) copolymer containing primarily 3-hydroxyvalerate
from simple carbohydrate substrates by Rhodococcus sp. ncimb 40126. Int. J. Biol.
Macromol. 13: 83-88.
Hazer, B., Steinbüchel A. 2007. Increased diversification of polyhydroxyalkanoates by
modification reactions for industrial and medical applications. Appl. Microbiol. Biotechnol. 74:
1-12.
Hejazi, P., Vasheghani-Farahani E., Yamini Y. 2003. Supercritical fluid disruption of
Ralstonia eutropha for poly(ß-hydroxybutyrate) recovery. Biotechnol. Progr. 19: 1519-1523.
Hench, L. L., Polak J. M. 2002. Third-generation biomedical materials. Science 295: 1014-
1018.
Hesselmann, R. P. X., Fleischmann T., Hany R., Zehn der A. J. B. 1999. Determination of
polyhydroxyalkanoates in activated sludge by ion chromatographic and enzymatic methods.
J. Microbiol. Methods 35: 111-119.
Hocking, P. J., Marchessault R. H., Timmins M. R., Lenz R. W., Fuller R. C. 1996.
Enzymatic degradation of single crystals of bacterial and synthetic poly(ß-hydroxybutyrate).
Macromolecules 29: 2472-2478.
125 References
Hoffmann, N., Rehm B. H. A. 2004. Regulation of polyhydroxyalkanoate biosynthesis in
Pseudomonas putida and Pseudomonas aeruginosa. FEMS Microbiol. Lett. 237: 1-7.
Holmes, P. A., Wright L. F., Alderson B., Senior P. J.; Imperial Chemical Industries Ltd.,
UK. 1980. A process for the extraction of poly(3-hydroxybutyric acid) from microbial cells. EP
patent 0015123.
Holmes, P. A., Lim G. B.; Imperial Chemical Industries PLC, 1990. Separation process. US
patent 4910145.
Holst, O. 1995. Endotoxin antagonists: possible candidates for the treatment of Gram-
negative sepsis. Angew. Chem. Int. Ed. Engl. 34: 2000-2002.
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.
Horowitz, D.; Metabolix, Inc., USA, 2000. Methods for purifying polyhydroxyalkanoates. WO
patent 2000068409.
Horowitz, D. M., Brennan E. M.; Metabolix, Inc., USA, 1999. Methods for separation and
purification of polyhydroxyalkanoates from biomass using ozone treatment. WO patent
9951760.
Hrabak, O. 1992. Industrial production of Poly-ß-hydroxybutyrate. FEMS Microbiol. Rev.
103: 251-255.
Hubbell, J. A. 1995. Biomaterials in tissue engineering. Nat. Biotechnol. 13: 565-576.
Huijberts, G. N. M., Eggink G. 1996. Production of poly(3-hydroxyalkanoates) by
Pseudomonas putida KT2442 in continuous cultures. Appl. Microbiol. Biotechnol. 46: 233-
239.
Huijberts, G. N. M., Eggink G., de Waard P., Huisma n G. W., 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.
Huijberts, G. N. M., Vanderwal H., Wilkinson C., Eg gink G. 1994. Gas-chromatographic
analysis of poly(3-hydroxyalkanoates) in bacteria. Biotechnol. Tech. 8: 187-192.
Huisman, G. W., 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.
References 126
Hwang, K. J., You S. F., Don T. M. 2006. Disruption kinetics of bacterial cells during
purification of poly-ß-hydroxyalkanoate using ultrasonication. J. Chin. Inst. Chem. Eng 37:
209-216.
Itzigsohn, R., Yarden O., Okon Y. 1995. Polyhydroxyalkanoate analysis in Azospirillum
brasilense. Can. J. Microbiol. 41: 73-76.
Jan, S., Roblot C., Courtois J., Courtois B., Barbo tin J. N., Seguin J. P. 1996. H-1 NMR
spectroscopic determination of poly(3-hydroxybutyrate) extracted from microbial biomass.
Enzyme Microb. Technol. 18: 195-201.
Jan, S., Roblot C., Goethals G., Courtois J., Court ois B., Saucedo J. E. N., Seguin J. P.,
Barbotin J. N. 1995. Study of parameters affecting poly(3-hydroxybutyrate) quantification by
gas chromatography. Anal. Biochem. 225: 258-263.
Jarute, G., Kainz A., Schroll G., Baena J. R., Lend l B. 2004. On-line determination of the
intracellular poly(β-hydroxybutyric acid) content in transformed Escherichia coli and glucose
during PHB production using stopped-flow attenuated total reflection FT-IR Spectrometry.
Anal.Chem. 76: 6353-6358.
Jendrossek, D. 2001. Microbial degradation of polyesters. Adv. Biochem. Eng. Biotechnol.:
Springer-Verlag, Berlin Heidelberg. p 293-325.
Jendrossek, D., Selchow O., Hoppert M. 2007. Poly(3-hydroxybutyrate) granules at the
early stages of formation are localized close to the cytoplasmic membrane in Caryophanon
latum. Appl. Environ. Microbiol. 73: 586-593.
Jiang, X., Ramsay J. A., Ramsay B. A. 2006. Acetone extraction of mcl-PHA from
Pseudomonas putida KT2440. J. Microbiol. Meth. 67: 212-219.
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.
Kannan, R. Y., Salacinski H. J., Butler P. E., Seif alian A. M. 2005. Polyhedral oligomeric
silsesquioxane nanocomposites: The next generation material for biomedical applications.
Accounts Chem. Res. 38: 879-884.
Kassab, A. C., Piskin E., Bilgic S., Denkbas E. B., Xu K. 1999. Embolization with
polyhydroxybutyrate (PHB) microspheres: In-vivo studies. J. Bioact. Compat. Polym. 14: 291-
303.
127 References
Kassab, A. C., Xu K., Denkbas E. B., Dou Y., Zhao S ., Piskin E. 1997. Rifampicin carrying
polyhydroxybutyrate microspheres as a potential chemoembolization agent. J. Biomater. Sci.
Polym. Ed. 8: 947-961.
Kawalec, M., Adamus G., Kurcok P., Kowalczuk K., Fo ltran I., Focarete M. L., Scandola
M. 2007. Carboxylate induced degradation of PHB. Biomacromolecules 8: 1053-1058.
Kennedy, S. B., Washburn N. R., Simon C. G., Amis E . J. 2006. Combinatorial screen of
the effect of surface energy on fibronectin-mediated osteoblast adhesion, spreading and
proliferation. Biomaterials 27: 3817-3824.
Kessler, B., Palleroni N. J. 2000. Taxonomic implications of synthesis of poly-ß-
hydroxybutyrate and other poly-ß-hydroxyalkanoates by aerobic Pseudomonads. Int. J. Syst.
Evol. Micr. 50: 711-713.
Kessler, B., Westhuis R., Witholt B., Eggink G. 2001. Production of microbial polyesters:
fermentation and downstream processes. Babel W, Steinbüchel A, editors. Berlin: Springer.
159-182 p.
Kessler, B., Witholt B. 1998. Synthesis, recovery and possible application of medium-chain-
length polyhydroxyalkanoates: A short overview. Macromol. Symp. 130: 245-260.
Khosravi-Darani, K., Vasheghani-Farahani E., Yamini Y., Bahramifar N. 2003. Solubility
of poly(ß-hydroxybutyrate) in supercritical carbon dioxide. J. Chem. Eng. Data 48: 860-863.
Kim, B. S. 2002. Production of medium chain length polyhydroxyalkanoates by fed-batch
culture of Pseudomonas oleovorans. Biotechnol. Lett. 24: 125-130.
Kim, B. S., Lee S. C., Lee S. Y., Chang H. N., Chan g Y. K., Woo S. I. 1994a. Production of
poly(3-hydroxybutyric-co-3-hydroxyvaleric acid) by fed-batch culture of Alcaligenes
eutrophus with substrate control using online glucose analyzer. Enzyme Microb. Tech. 16:
556-561.
Kim, B. S., Lee S. C., Lee S. Y., Chang H. N., Chan g Y. K., Woo S. I. 1994b. Production of
poly(3-hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus with glucose-
concentration control. Biotechnol. Bioeng. 43: 892-898.
Kim, D. Y., Kim Y. B., Rhee Y. H. 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, J. S., Lee B. H., Kim B. S. 2005. Production of poly(3-hydroxybutyrate-co-4-
hydroxybutyrate) by Ralstonia eutropha. Biochem. Eng. J. 23: 169-174.
References 128
Kim, K., Sonn Y., Lee M., Jung S.; Cheil Synthetics Inc., S. Korea. 1996. Preparation
process of poly-3-hydroxybutyrate. KR patent 9609065.
Kimura, H., Ohura T., Takeishi M., Nakamura S., Doi Y. 1999. Effective microbial
production of poly(4-hydroxybutyrate) homopolymer by Ralstonia eutropha H16. Polym. Int.
48: 1073-1079.
Kimura, H., Yamamoto T., Iwakura K. 2002. Biosynthesis of polyhydroxyalkanoates from
1,3-propanediol by Chromobacterium sp. Polym. J. 34: 659-665.
Kivirikko, K. I ., Laitinen O ., Prockop D.J . 1967. Modifications of specific assay for
hydroxyproline in urine. Anal. Biochem. 19: 249.
Knirel, Y. A., Lindner B., Vinogradov E. V., Kochar ova N. A., Senchenkova S. N.,
Shaikhutdinova R. Z., Dentovskaya S. V., Fursova N. K., Bakhteeva I. V., Titareva G. M.,
Balakhonov S. V., Holst O., Gremyakova T. A., Pier G. B., Anisimov A. P. 2005.
Temperature-dependent variations and intraspecies diversity of the structure of the
lipopolysaccharide of Yersinia pestis. Biochemistry 44: 1731-1743.
Koller, M., Hesse P., Bona R., Kutschera C., Atlic A., Braunegg G. 2007. Potential of
various archae- and eubacterial strains as industrial polyhydroxyalkanoate producers from
whey. Macromol. Biosci. 7: 218-226.
Kranz, R. G., Gabbert K. K., Locke T. A., Madigan M . T. 1997. Polyhydroxyalkanoate
production in Rhodobacter capsulatus: genes, mutants, expression, and physiology. Appl.
Environ. Microb. 63: 3003-3009.
Kurdikar, D. L., Strauser F. E., Solodar A. J., Pas ter M. D.; Monsanto Co., USA. 1998.
High temperature PHA extraction using PHA-poor solvents. WO patent 9846783.
Lafferty, R. M., Agroferm A.G., Switzerland, 1977. Use of cyclic carbonic acid esters as
solvent for poly(ß-hydroxybutyric acid). US patent 4140741.
Lageveen, R. 1986. Oxidation of aliphatic compounds by Pseudomonas oleovorans. PhD
thesis, Rijksuniversiteit Groningen.
Lageveen, R. G., Huisman G. W., Preusting H., Ketel aar 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.
References 129
Lakshman, K., Rastogi N. K., Shamala T. R. 2004. Simultaneous and comparative
assessment of parent and mutant strain of Rhizobium meliloti for nutrient limitation and
enhanced polyhydroxyalkanoate (PHA) production using optimization studies. Process
Biochem. 39: 1977-1983.
Lee, E. Y., Choi C. Y. 1995. Gas chromatography mass spectrometric analysis and its
application to a screening procedure for novel bacterial polyhydroxyalkanoic acids containing
long chain saturated and unsaturated monomers. J. Ferment. Bioeng. 80: 408-414.
Lee, I. Y., Kim G. J., Choi D. K., Yeon B. K., Park Y. H. 1996. Improvement of
hydroxyvalerate fraction in poly(ß-hydroxybutyrate-co-ß-hydroxyvalerate) by a mutant strain
of Alcaligenes eutrophus. J. Ferment. Bioeng. 81: 255-258.
Lee, M. Y., Lee T. S., Park W. H. 2001. Effect of side chains on the thermal degradation of
poly(3-hydroxyalkanoates). Macromol. Chem. Phys. 202: 1257-1261.
Lee, M. Y., Park W. H., Lenz R. W. 2000a. Hydrophilic bacterial polyesters modified with
pendant hydroxyl groups. Polymer 41: 1703-1709.
Lee, S. Y. 1996a. Plastic bacteria? Progress and prospects for polyhydroxyalkanoate
production in bacteria. Trends Biotechnol. 14: 431-438.
Lee, S. Y. 1996b. Bacterial polyhydroxyalkanoates. Biotechnol. Bioeng. 49: 1-14.
Lee, S. Y., Choi J. I. 1998. Effect of fermentation performance on the economics of poly(3-
hydroxybutyrate) production by Alcaligenes latus. Polym. Degrad. Stabil. 59: 387-393.
Lee, S. Y., Choi J. I., Han K., Song J. Y. 1999a. Removal of endotoxin during purification of
poly(3-hydroxybutyrate) from Gram-negative bacteria. Appl. Env. Microb. 65: 2762-2764.
Lee, S. Y., Wong H. H., Choi J. I., Lee S. H., Lee S. C., Han C. S. 2000b. Production of
medium-chain-length polyhydroxyalkanoates by high-cell-density cultivation of Pseudomonas
putida under phosphorus limitation. Biotechnol. Bioeng. 68: 466-470.
Lemoigne, M. 1926. Produits de deshydration et de polymerisation de l'acide β-oxybutyric.
Bull. Soc. Chem. Biol. 8: 770-782.
Liddell, J. M., Locke T. J.; Zeneca Limited, 1997. Production of plastics materials from
microorganisms. US patent 5691174.
Lillo, J. G., Rodriguezvalera F. 1990. Effects of culture conditions on poly(ß-hydroxybutyric
acid) production by Haloferax mediterranei. Appl. Environ. Microb. 56: 2517-2521.
Ling, Y., Williams D. R. G., Thomas C. J., Middelbe rg A. P. J. 1997a. Recovery of poly-3-
hydroxybutyrate from recombinant Escherichia coli by homogenization and centrifugation.
Biotechnol. Tech. 11: 409-412.
References 130
Ling, Y., Wong H. H., Thomas C. J., Williams D. R. G., Middelberg A. P. J. 1997b. Pilot-
scale extraction of PHB from recombinant Escherichia coli by homogenization and
centrifugation. Bioseparation 7: 9-15.
Liu, W., Chen G. Q. 2007. Production and characterization of medium-chain-length
polyhydroxyalkanoate with high 3-hydroxytetradecanoate monomer content by fadB and
fadA knockout mutant of Pseudomonas putida KT2442. Appl. Microbiol. Biot.
Lopez-Lopez, A., Castellote-Bargallo A. I., Lopez-S abater M. C. 2001. Comparison of two
direct methods for the determination of fatty acids in human milk. Chromatographia 54: 743-
747.
Manna, A., Banerjee R., Paul A. K. 1999. Accumulation of poly (3-hydroxybutyric acid) by
some soil Streptomyces. Curr. Microbiol. 39: 153-158.
Marchessault, R. H., Morin F. G., Wong S., Saracova n I. 1995. Artificial granule
suspensions of long side-chain poly(3-hydroxyalkanoate). Can. J. Microbiol. 41: 138-142.
Martini, F., Perazzo L., Vietto P.; W. R. Grace & Co.-Conn., 1989. Sheets materials of HB
polymers. US patent 4826493.
Matsushita, H., Yoshida S., Tawara T.; Mitsubishi Gas Chemical Co, Japan. 1995.
Extraction of poly(3-hydroxybutyric acid) from microorganisms. JP patent 07079788.
McCool, G. J., Fernandez T., Li N., Cannon M. C. 1996. Polyhydroxyalkanoate inclusion-
body growth and proliferation in Bacillus megaterium. FEMS Microbiol. Lett. 138: 41-48.
Metzner, K., Sela M., Schaffer J.; Buna Sow Leuna Olefinverbund GmbH, Germany, 1997.
Agents for extracting polyhydroxyalkanoic acids. WO patent 9708931.
Middelberg, A. P. J., Lee S. Y., Martin J., William s D. R. G., Chang H. N. 1995. Size
analysis of poly(3-hydroxybutyric acid) granules produced in recombinant Escherichia coli.
Biotechnol. Lett. 17: 205-210.
Minnikin, D. E., Abdolrah.H. 1974. Replacement of phosphatidylethanolamine and acidic
phospholipids by an ornithine-amide lipid and a minor phosphorus-free lipid in Pseudomonas
fluorescens Ncmb 129. FEBS Letters 43: 257-260.
Minnikin, D. E., Abdolrah.H, Baddiley J. 1974. Replacement of acidic phospholipids by
acidic glycolipids in Pseudomonas diminuta. Nature 249: 268-269.
Moffat, C. F., McGill A. S., Anderson R. S. 1991. The Production of artefacts during
preparation of fatty acid methyl esters from fish oils, food products and pathological samples.
HRC J. High Resolut. Chromatogr. 14: 322-326.
Monod, J. 1949. The growth of bacterial cultures. Annu. Rev. Microbiol. 3: 371-394.
131 References
Mosmann, T. 1983. Rapid colorimetric assay for cellular growth and survival: application to
proliferation and cytotoxicity assays. J. Immunol. Meth. 65, 55-63.
Mrksich, M. 2000. A surface chemistry approach to studying cell adhesion. Chem. Soc. Rev.
29: 267-273.
Niskanen, A., Kiutamo T., Raisanen S., Raevuori M. 1978. Determination of fatty acid
compositions of Bacillus cereus and related bacteria - rapid gas chromatographic method
using a glass capillary column. Appl. Environ. Microbiol. 35: 453-455.
Noda, I.; Procter and Gamble Co., USA, 1995. Process for recovering
polyhydroxyalkanoates using air classification. WO patent 9533064.
Noda, I.; The Procter & Gamble Company, USA, 1998. Process for recovering
polyhydroxyalkanoates using air classification. US patent 5849854.
Noda, I., Schechtman L. A.; Procter and Gamble Company, USA. 1997. Solvent extraction
of polyhydroxyalkanoates from biomass. WO patent 9707230.
Numazawa, R., Miyamori T., Sakimae A., Onishi H.; Mitsubishi Rayon Co., Ltd., Japan.
1987. Separation and purification of poly-ß-hydroxybutyric acid from cell extracts. JP patent
62205787.
Oehmen, A., Keller-Lehmann B., Zeng R. J., Yuan Z. G., Keller E. 2005. Optimisation of
poly-ß-hydroxyalkanoate analysis using gas chromatography for enhanced biological
phosphorus removal systems. J. Chromatogr. A 1070: 131-136.
Ohleyer, E.; Firmenich S. A., Switzerland, 1993. Extraction of poly-(ß)-hydroxyoctanoate
from microbial biomass. WO patent 9311656.
Opitz, F., Schenke-Layland K., Cohnert T. U., Starc her B., Halbhuber K. J., Martin D. P.,
Stock U. A. 2004. Tissue engineering of aortic tissue: dire consequence of suboptimal
elastic fiber synthesis in vivo. Cardiovasc. Res. 63: 719-730.
Owens, J. D., Legan J. D. 1987. Determination of the Monod substrate saturation constant
for microbial growth. FEMS Microbiol. Rev. 46: 419-432.
Palmer, A. G., Cavanagh J., Byrd R. A., Rance M. 1992. Sensitivity improvement in 3-
dimensional heteronuclear correlation NMR-Spectroscopy. J. Magn. Reson. 96: 416-424.
Park, J. S., Park H. C., Huh T. L., Lee Y. H. 1995. Production of poly(ß-hydroxybutyrate) by
Alcaligenes eutrophus transformants harboring cloned Phbcab genes. Biotechnol. Lett. 17:
735-740.
Petsch, D., Anspach F. B. 2000. Endotoxin removal from protein solutions. J. Biotechnol.
76: 97-119.
References 132
Pötter, M., Steinbüchel A. 2004. Poly(3-hydroxybutyrate) granule-associated proteins:
Impacts on poly(3-hydroxybutyrate) synthesis and degradation. Biomacromolecules 6: 552-
560.
Pouton, C. W., Akhtar S. 1996. Biosynthetic polyhydroxyalkanoates and their potential in
drug delivery. Adv. Drug. Deliver. Rev. 18: 133-162.
Preusting, H., Kingma J., Huisman G., Steinbüchel A ., Witholt B. 1993. Formation of
polyester blends by a recombinant strain of Pseudomonas oleovorans: different poly(3-
hydroxyalaknoates) are stored in separate granules. J. Environ. Polym. Degrad. 1: 11-21.
Prieto, M. A., Kellerhals M. B., Bozzato G. B., Rad novic D., Witholt B., Kessler B. 1999.
Engineering of stable recombinant bacteria for production of chiral medium-chain-length
poly-3-hydroxyalkanoates. Appl. Environ. Microb. 65: 3265-3271.
Punshon, G., Vara D. S., Sales K. M., Kidane A. G., Salacinski H. J., Seifalian A. M.
2005. Interactions between endothelial cells and a poly(carbonate-silsesquioxane-bridge-
urea)urethane. Biomaterials 26: 6271-6279.
Qi, Q. S., Rehm B. H. A. 2001. Polyhydroxybutyrate biosynthesis in Caulobacter crescentus:
molecular characterization of the polyhydroxybutyrate synthase. Microbiol.-SGM 147: 3353-
3358.
Qu, X. H., Wu Q., Liang J., Qu X., Wang S. G., Chen G. Q. 2005. Enhanced vascular-
related cellular affinity on surface modified copolyesters of 3-hydroxybutyrate and 3-
hydroxyhexanoate (PHBHHx). Biomaterials 26: 6991-7001.
Raetz, C. R. H., Reynolds C. M., Trent M. S., Bisho p R. E. 2007. Lipid A modification
systems in Gram-negative bacteria. Annu. Rev. Biochem. 76: 295-329.
Ramsay, B. A. 1990. Recovery of poly-3-hydroxyalkanoic acid granules by a surfactant-
hypochlorite treatment. Biotechnol. Techn. 4: 221-226.
Ramsay, B. A., Ramsay J., Berger E., Chavarie C., B raunegg G.; Ecole Polytechnique
Montreal, 1992. Separation of poly-ß-hydroxyalkanoic acid from microbial biomass. US
patent 5110980.
Ramsay, J. A., Berger E., Voyer R., Chavarie C., Ra msay B. A. 1994. Extraction of poly-3-
hydroxybutyrate using chlorinated solvents. Biotechnol. Techn. 8: 589-594.
Randriamahefa, S., Renard E., Guérin P., Langlois V . 2003. Fourier transform infrared
spectroscopy for screening and quantifying production of PHAs by Pseudomonas grown on
sodium octanoate. Biomacromolecules 4: 1092-1097.
133 References
Rapthel, I., Lehmann O., Runkel D., Mayer T., Rauch stein K. D.; Buna A.G., Germany,
1993a. Manufacture of colorless polyhydroxyalkanoates by extraction of bacterial biomass
with acetic acid containing ß-butyrolactone. DE patent 4215860.
Rapthel, I., Lehmann O., Runkel D., Mayer T., Rauch stein K. D., Schaffer J.; Buna A.G.,
Germany, 1993b. Manufacture of colorless polyhydroxyalkanoates by extraction of bacterial
biomass with acetic acid containing acetic anhydride. DE patent 4215861.
Rehm, B. H. A. 2003. Polyester synthases: natural catalysts for plastics. Biochem. J. 376:
15-33.
Rehm, B. H. A. 2007. Biogenesis of microbial polyhydroxyalkanoate granules: a platform
technology for the production of tailor-made bioparticles. Curr. Issues Mol. Biol. 9: 41-62.
Ren, Q., Grubelnik A., Hoerler M., Ruth K., Hartman n R., Felber H., Zinn M. 2005.
Bacterial poly(hydroxyalkanoates) as a source of chiral hydroxyalkanoic acids.
Biomacromolecules 6: 2290-2298.
Renard, E., Poux A., Timbart L., Langlois V., Gueri n P. 2005. Preparation of a novel
artificial bacterial polyester modified with pendant hydroxyl groups. Biomacromolecules 6:
891-896.
Rietschel, E. T., Kirikae T., Schade F. U., Mamat U ., Schmidt G., Loppnow H., Ulmer A.
J., Zahringer U., Seydel U., Dipadova F., Schreier M., Brade H. 1994. Bacterial endotoxin
- molecular relationships of structure to activity and function. FASEB J. 8: 217-225.
Riis, V., Mai W. 1988. Gas chromatographic determination of poly-ß-hydroxybutyric acid in
microbial biomass after hydrochloric acid propanolysis. J. Chromatogr. 445: 285-289.
Rizk, S.; Tepha, Inc., 2005. Non-curling polyhydroxyalkanoate sutures. WO patent
2006015276.
Rochex, A., Lebeault J. M. 2007. Effects of nutrients on biofilm formation and detachment
of a Pseudomonas putida strain isolated from a paper machine. Water Res. 41: 2885-2892.
Rodrigues, M. F. D., Valentin H. E., Berger P. A., Tran M., Asrar J., Gruys K. J.,
Steinbüchel A. 2000. Polyhydroxyalkanoate accumulation in Burkholderia sp.: a molecular
approach to elucidate the genes involved in the formation of two homopolymers consisting of
short-chain-length 3-hydroxyalkanoic acids. Appl. Microbiol. Biot. 53: 453-460.
Steinbüchel A. 2000. Polyhydroxyalkanoate accumulation in Burkholderia sp.: a molecular
approach to elucidate the genes involved in the formation of two homopolymers consisting of
short-chain-length 3-hydroxyalkanoic acids. Appl. Microbiol. Biot. 53: 453-460.
References 134
Rotzsche, H. 1991. Gas chromatographic analysis of fatty acid salts. J. Chromatogr. 552:
281-288.
Rouxhet, L., Legras R., Schneider Y. J. 1998. Interactions between a biodegradable
polymer, poly(hydroxybutyrate-hydroxyvalerate), proteins and macrophages. Macromol.
Symp. 130: 347-366.
Runkel, D., Lehmann O., Mayer T., Rauchstein K. D., Schaffer J.; Buna A.G., Germany.
1993a. Manufacture of colorless polyhydroxyalkanoates by extraction of bacterial biomass
with acetic acid. DE patent 4215862.
Runkel, D., Lehmann O., Mayer T., Rauchstein K. D., Schaffer J.; Buna A.G., Germany,
1993b. Manufacture of colorless polyhydroxyalkanoates by extraction of bacterial biomass
with acetic anhydride. DE patent 4215864.
Ruth, K., Grubelnik A., Hartmann R., Egli T., Zinn M., Ren Q. 2007. Efficient production of
(R)-3-hydroxycarboxylic acids by biotechnological conversion of polyhydroxyalkanoates and
their purification. Biomacromolecules 8: 279-286.
Sabirova, J. S., Ferrer M., Lunsdorf H., Wray V., K alscheuer R., Steinbüchel A., Timmis
K. N., Golyshin P. N. 2006. Mutation in a "tesB-like" hydroxyacyl-coenzyme A-specific
thioesterase gene causes hyperproduction of extracellular polyhydroxyalkanoates by
Alcanivorax borkumensis SK2. J. Bacteriol. 188: 8452-8459.
Schlegel, H.G. and Gottschalk, G., 1962. Poly-beta-hydroxybuttersäure, ihre Verbreitung,
Funktion und Biosynthese. Angew. Chem. 74: 342-347.
Schmidt, J., Biedermann W., Schmiechen H.; Akademie der Wissenschaften der DDR,
Ger. Dem. Rep., 1985. Extraction of poly-ß-hydroxybutyric acid from bacterial biomass. DD
patent 229428.
Schmidt, J., Schmiechen H., Rehm H., Trennert M.; Akademie der Wissenschaften der
DDR, Ger. Dem. Rep., 1986. Verfahren zur Gewinnung von PHB aus getrockneten
Biomassen. DD patent 239609.
Schumann, D., Mueller R. A.; UFZ Umweltforschungszentrum Leipzig-Halle G.m.b.H.,
Germany, 2001. Method for obtaining polyhydroxyalkanoates (PHA) or the copolymers
thereof. WO patent 2001068892.
Schumann, D., Müller R. A.; UFZ Umweltforschungszentrum Leipzig-Halle G.m.b.H.,
Germany, 2003. Method for obtaining polyhydroxyalkanoates (PHA) and the copolymers
thereof. WO patent 0168892.
135 References
Seidel, H., Voigt B., Roethe K. P., Rosahl B., Moth es S.; Akademie der Wissenschaften,
Germany, 1991. Supercritical fluid extraction in polyhydroxybutyrate and ubiquinone
extraction for bacteria. DD patent 294280.
Sela, M., Metzner K.; Buna Sow Leuna Olefinverbund Gmbh, Germany, 1996. Use of ethyl
lactate as an extractant for poly(hydroxyalkanoic acids). DE patent 19533459.
Sendil, D., Gursel I., Wise D. L., Hasirci V. 1999. Antibiotic release from biodegradable
PHBV microparticles. J. Control. Rel. 59: 207-217.
Sevastianov, V. I., Perova N. V., Shishatskaya E. I ., Kalacheva G. S., Volova T. G. 2003.
Production of purified polyhydroxyalkanoates (PHAs) for applications in contact with blood. J.
Biomater. Sci. Polym. Ed. 14: 1029-1042.
Shaka, A. J., Barker P. B., Freeman R. 1985. Computer-optimized decoupling scheme for
wideband applications and low level operation. J. Magn. Reson. 64: 547-552.
Shangguan, Y. Y., Wang Y. W., Wu Q., Chen G. Q. 2006. The mechanical properties and in
vitro biodegradation and biocompatibility of UV-treated poly(3-hydroxybutyrate-co-3-
hydroxyhexanoate). Biomaterials 27: 2349-2357.
Shantha, N. C., Decker E. A., Hennig B. 1993. Comparison of methylation methods for the
quantitation of conjugated linoleic acid isomers. J. AOAC Int. 76: 644-649.
Shikanov, A., Kumar N., Domb A. J. 2005. Biodegradable polymers: an update. Israel J.
Chem. 45: 393-399.
Silva, J. A., Tobella L. M., Becerra J., Godoy F., Martínez M. A. 2007. Biosynthesis of
poly-β-hydroxyalkanoate by Brevundimonas vesicularis LMG P-23615 and Sphingopyxis
macrogoltabida LMG 17324 using acid-hydrolyzed sawdust as carbon source. J. Biosci.
Bioeng. 103: 542-546.
Silver, J. H., Lin J.-C., Lim F., Tegoulia V. A., C haudhury M. K., Cooper S. L. 1999.
Surface properties and hemocompatibility of alkyl-siloxane monolayers supported on silicone
rubber: effect of alkyl chain length and ionic functionality. Biomaterials 20: 1533-1543.
Slepecky, R. A., Law J. H. 1960. A rapid spectrophotometric assay of alpha, ß-unsaturated
acids and ß-hydroxy acids. Anal. Chem. 32: 1697-1699.
Sodian, R., Hoerstrup S. P., Sperling J. S., Daebri tz S., Martin D. P., Moran A. M., Kim
B. S., Schoen F. J., Vacanti J. P., Mayer J. E. 2000a. Early in vivo experience with tissue-
engineered trileaflet heart valves. Circulation 102: 22-29.
References 136
Sodian, R., Hoerstrup S. P., Sperling J. S., Martin D. P., Daebritz S., Mayer J. E.,
Vacanti J. P. 2000b. Evaluation of biodegradable, three-dimensional matrices for tissue
engineering of heart valves. Asaio J. 46: 107-110.
Sodian, R., Loebe M., Hein A., Martin D. P., Hoerst rup S. P., Potapov E. V., Hausmann
H. A., Lueth T., Hetzer R. 2002. Application of stereolithography for scaffold fabrication for
tissue engineered heart valves. Asaio J. 48: 12-16.
Sodian, R., Sperling J. S., Martin D. P., Egozy A., Stock U., Mayer J. E., Vacanti J. P.
2000c. Fabrication of a trileaflet heart valve scaffold from a polyhydroxyalkanoate
biopolyester for use in tissue engineering. Tissue Eng. 6: 183-188.
Stageman, J. F.; Imperial chemical industries, 1985. Extraction process. US patent
4562245.
Steeghs, L., de Cock H., Evers E., Zomer B., Tommas sen J., van der Ley P. 2001. Outer
membrane composition of a lipopolysaccharide-deficient Neisseria meningitidis mutant.
EMBO Journal 17: 6937–6945.
Steinbüchel, A., Valentin H. E. 1995. Diversity of bacterial polyhydroxyalkanoic acids.
FEMS Microbiol. Lett. 128: 219-228.
Stewart, J. C. M. 1980. Colorimetric determination of phospholipids with ammonium
ferrothiocyanate. Anal. Biochem. 104: 10-14.
Stock, U. A., Nagashima M., Khalil P. N., Nollert G . D., Herden T., Sperling J. S., Moran
A., Lien J., Martin D. P., Schoen F. J., Vacanti J. P., Mayer J. E. 2000a. Tissue-
engineered valved conduits in the pulmonary circulation. J. Thorac. Cardiovasc. Surg. 119:
732-740.
Stott, K., Keeler J., Van Q. N., Shaka A. J. 1997. One-dimensional NOE experiments using
pulsed field gradients. J. Magn. Reson. 125: 302-324.
Sun, Z. Y., Ramsay J. A., Guay M., Ramsay B. A. 2007. Fermentation process
development for the production of medium-chain-length poly-3-hyroxyalkanoates. Appl.
Microbiol. Biot. 75: 475-485.
Suriyamongkol, P., Weselake R., Narine S., Moloney M., Shah S. 2007. Biotechnological
approaches for the production of polyhydroxyalkanoates in microorganisms and plants - A
review. Biotechnol. Adv. 25: 148-175.
Sutcliffe, I. C., Shaw N. 1991. Atypical lipoteichoic acids of Gram-positive bacteria. J.
Bacteriol. 173: 7065-7069.
137 References
Svensson, M., Han L., Silfversparre G., Haggstrom L ., Enfors S. O. 2005. Control of
endotoxin release in Escherichia coli fed-batch cultures. Bioproc. Biosyst. Eng. 27: 91-97.
Szewczyk, E., Mikucki J. 1989. Poly-ß-hydroxybutyric acid in Staphylococci. FEMS
Microbiol. Lett. 61: 279-284.
Tajima, K., Igari T., Nishimura D., Nakamura M., Sa toh Y., Munekata M. 2003. Isolation
and characterization of Bacillus sp. INT005 accumulating polyhydroxyalkanoate (PHA) from
gas field soil. J. Biosci. Bioeng. 95: 77-81.
Tamer, I. M., Moo-Young M., Chisti Y. 1998a. Disruption of Alcaligenes latus for recovery of
poly(ß-hydroxybutyric acid): comparison of high-pressure homogenization, bead milling, and
chemically induced lysis. Ind. Eng. Chem. Res. 37: 1807-1814.
Tamer, I. M., Moo-Young M., Chisti Y. 1998b. Optimization of poly(ß-hydroxybutyric acid)
recovery from Alcaligenes latus: combined mechanical and chemical treatments. Bioprocess
Eng. 19: 459-468.
Taniguchi, I., Kagotani K., Kimura Y. 2003. Microbial production of poly(hydroxyalkanoate)
from waste edible oils. Green Chem. 5: 545-548.
Terada, M., Marchessault R. H. 1999. Determination of solubility parameters for poly(3-
hydroxyalkanoates). Int. J. Biol. Macromol. 25: 207-215.
Tesema, Y., Raghavan D., Stubbs J. 2004. Bone cell viability on collagen immobilized
poly(3-hydroxybutrate-co-3-hydroxyvalerate) membrane: Effect of surface chemistry. J. Appl.
Pol. Sci. 93: 2445-2453.
Tezcaner, A., Bugra K., Hasirci V. 2003. Retinal pigment epithelium cell culture on surface
modified poly(hydroxybutyrate-co-hydroxyvalerate) thin films. Biomaterials 24: 4573-4583.
Timm, A., Steinbüchel A. 1990. Formation of polyesters consisting of medium-chain-length
3-hydroxyalkanoic acids from gluconate by Pseudomonas aeruginosa and other fluorescent
Pseudomonads. Appl. Environ. Microbiol. 56: 3360-3367.
Trent, M. S., Stead C. M., Tran A. X., Hankins J. V . 2006. Diversity of endotoxin and its
impact on pathogenesis. J. Endotoxin Res. 12: 205-223.
Turakhia, M. H., Characklis W. G. 1989. Activity of Pseudomonas aeruginosa in biofilms -
effect of calcium. Biotechnol. Bioeng. 33: 406-414.
U.S. Department of health and human services, F. D. A. 1997a. Guidance for industry.
Rockville. 54 p.
U.S. Department of health and human services, F. D. A. 1997b. guideline on validation of
the Limulus amebocyte lysate test. Rockville. 54 p.
References 138
Ueda, H., Tabata Y. 2003. Polyhydroxyalkanoate derivatives in current clinical applications
and trials. Adv. Drug Deliv. Rev. 55: 501-518.
Valappil, S. P., Boccaccini A. R., Bucke C., Roy I. 2007a. Polyhydroxyalkanoates in Gram-
positive bacteria: insights from the genera Bacillus and Streptomyces. Anton. Leeuw. Int. J.
G. 91: 1-17.
Valappil, S. P., Peiris D., Langley G. J., Hemiman J. M., Boccaccini A. R., Bucke C., Roy
I. 2007b. Polyhydroxyalkanoate (PHA) biosynthesis from structurally unrelated carbon
sources by a newly characterized Bacillus spp. J. Biotechnol. 127: 475-487.
Valentin, H. E., Steinbüchel A. 1995. Accumulation of poly(3-hydroxybutyric acid co-3-
hydroxyvaleric acid co-4-hydroxyvaleric acid) by mutants and recombinant strains of
Alcaligenes eutrophus. J. Environ. Polym. Degrad. 3: 169-175.
van Hee, P., Elumbaring A., van der Lans R., van de r Wielen L. A. M. 2006. Selective
recovery of polyhydroxyalkanoate inclusion bodies from fermentation broth by dissolved-air
flotation. J. Colloid Interf. Sci. 297: 595-606.
Vanlautem, N., Gilain J.; Solvay & Cie., 1982. Process for separating poly-ß-
hydroxybutyrates from a biomass. US patent 4310684.
Vanlautem, N., Gilain J.; Solvay & Cie., 1987. Process for extracting poly-ß-
hydroxybutyrates by means of a solvent from an aqueous suspension of microorganisms. US
patent 4705604.
Viktor N. Vasilets, Artem V. Kuznetsov, Sevastianov V. I. 2004. Vacuum ultraviolet
treatment of polyethylene to change surface properties and characteristics of protein
adsorption. J. Biomed. Mater. Res. A 69A: 428-435.
Volova, T. G., Trusova M. Y., Kalacheva G. S., Kozh evnicov I. V. 2006. Physiological-
biochemical properties and the ability to synthesize polyhydroxyalkanoates of the glucose-
utilizing strain of the hydrogen bacterium Ralstonia eutropha B8562. Appl. Microbiol. Biot. 73:
429-433.
Wallen, L. L., Rohwedder W. K. 1974. Poly-ß-hydroxyalkanoate from activated sludge.
Environ. Science. Technol. 8: 576-579.
Walsem, H., Zhong L., Shih S.; Metabolix, 2004. Polymer extraction methods. US patent
2004013204.
Wang, F. L., Lee S. Y. 1997a. Poly(3-hydroxybutyrate) production with high productivity and
high polymer content by a fed-batch culture of Alcaligenes latus under nitrogen limitation.
Appl. Environ. Microb. 63: 3703-3706.
139 References
Wang, F. L., Lee S. Y. 1997b. Production of poly(3-hydroxybutyrate) by fed-batch culture of
filamentation-suppressed recombinant Escherichia coli. Appl. Environ. Microb. 63: 4765-
4769.
Wang, X. Y., Karbarz M. J., McGrath S. C., Cotter R . J., Raetz C. R. H. 2004a. MsbA
transporter-dependent lipid A 1-dephosphorylation on the periplasmic surface of the inner
membrane - Topography of Francisella novicida LpxE expressed in Escherichia coli. J. Biol.
Chem. 279: 49470-49478.
Wang, X. Y., McGrath S. C., Cotter R. J., Raetz C. R. H. 2006. Expression cloning and
periplasmic orientation of the Francisella novicida lipid A4 '-phosphatase LpxF. J. Biol. Chem.
281: 9321-9330.
Wang, Y. W., Wu Q. O., Chen G. Q. A. 2004b. Attachment, proliferation and differentiation
of osteoblasts on random biopolyester poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)
scaffolds. Biomaterials 25: 669-675.
Wang, Z. X., Itoh Y., Hosaka Y., Kobayashi I., Naka no Y., Maeda I., Umeda F.,
Yamakawa J., Kawase M., Yagi K. 2003. Novel transdermal drug delivery system with
polyhydroxyalkanoate and starburst polyamidoamine dendrimer. J. Biosci. Bioeng. 95: 541-
543.
Ward, A.C., Rowley, B.I. and Dawes, E.A. 1977. Effect of oxygen and nitrogen limitation on
poly-β-hydroxybutyrate biosynthesis in ammonium-grown Azotobacter beijerinckii. J. Gen.
Microbiol. 102: 61-68.
Williams, S. F., Martin D. P., Gerngross T., Horowi tz D. M.; Metabolix, Inc. Cambridge,
MA, 2001. Removing endotoxin with an oxidizing agent from polyhydroxyalkanoates
produced by fermentation. US patent 6245537.
Williams, S. F., Martin D. P., Horowitz D. M., Peop les O. P. 1999a. PHA applications:
Addressing the price performance issue I. Tissue engineering. Int. J. Biol. Macromol. 25:
111-121.
Witholt, B., Kessler B. 1999a. Perspectives of medium-chain-length
poly(hydroxyalkanoates), a versatile set of bacterial bioplastics. Curr. Opin. Biotechnol. 10:
279-285.
Yagmurlu, M. F., Korkusuz F., Gursel I., Korkusuz P ., Ors U., Hasirci V. 1999.
Sulbactam-cefoperazone polyhydroxybutyrate-co-hydroxyvalerate (PHBV) local antibiotic
delivery system: In vivo effectiveness and biocompatibility in the treatment of implant-related
experimental osteomyelitis. J. Biomed. Mater. Res. 46: 494-503.
References 140
Yamamoto, O., Myata Y., Yanagi S.; Denki Kagaku Kogyo Kk, Japan. 1995. Separation and
purification of poly(hydroxyalkanoates) from microorganisms using surfactants. JP patent
07079787.
Yan, Q., Du G. C., Chen J. 2003. Biosynthesis of polyhydroxyalkanoates (PHAs) with
continuous feeding of mixed organic acids as carbon sources by Ralstonia eutropha.
Process Biochem. 39: 387-391.
Yasotha, K., Aroua M. K., Ramachandran K. B., Tan I . K. P. 2006. Recovery of medium-
chain-length polyhydroxyalkanoates (PHAs) through enzymatic digestion treatments and
ultrafiltration. Biochem. Eng. J. 30: 260-268.
Yezza, A., Fournier D., Halasz A., Hawari J. 2006. Production of polyhydroxyalkanoates
from methanol by a new methylotrophic bacterium Methylobacterium sp. GW2. Appl.
Microbiol. Biot. 73: 211-218.
Yu, J., Chen L. X. L. 2006. Cost-effective recovery and purification of polyhydroxyalkanoates
by selective dissolution of cell mass. Biotechnol. Prog. 22: 547-553.
Yuasa, J. 2002. Pseudomonas aeruginosa under phosphorus-poor culture conditions.
Microb. Environ. 17: 75-81.
Zhang, J. P., Qun Wang T. R. S., William E. Hurst, and, Sulpizio T. 2005. Endotoxin
removal using a synthetic adsorbent of crystalline calcium silicate hydrate. Biotechnol. Progr.
21: 1220-1225.
Zinn, M., Hany R. 2005. Tailored material properties of polyhydroxyalkanoates through
biosynthesis and chemical modification. Adv. Eng. Mat. 7: 408-411.
Zinn, M., Weilenmann H. U., Hany R., Schmid M., Egl i T. 2003. Tailored synthesis of
poly([R]-3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/HV) in Ralstonia eutropha DSM 428.
Acta Biotechnol. 23: 309-316.
Zinn, M., Witholt B., Egli T. 2001. Occurrence, synthesis and medical application of
bacterial polyhydroxyalkanoate. Adv. Drug Del. Rev. 53: 5-21.
141
Curriculum Vitae
1976, 19. January Born in Bern, Switzerland
1991-1996 Grammar school, Bern
1996 Matura type C
1996-1999 Chemistry studies at ETH-Lausanne
1999-2002 Chemistry studies at university of Basel
2002 Diploma in chemistry, with physics as minor subject
2002-2003 Trainee in polymer analytics, Ciba specialty chemicals
Basel
2003-2007 PhD studies in bioprocess engineering at Empa St. Gallen
and ETH Zurich
2008 Project manager at Sika Technology
top related