Journal of Applied Microbiology ISSN 1364-5072

REVIEW ARTICLE

Bacterial synthesis of biodegradable

polyhydroxyalkanoates
R.A.J. Verlinden, D.J. Hill, M.A. Kenward, C.D. Williams and I. Radecka
School of Applied Sciences, University of Wolverhampton, Wolverhampton, UK

Keywords
bacterial synthesis, bioplastics, biopolymer,
PHAs, PHB, polyhydroxyalkanoates,
polyhydroxybutyrate.
Correspondence
I. Radecka, School of Applied Sciences,
University of Wolverhampton, Wulfruna
Street, WV1 1SB Wolverhampton, UK.
E-mail: i.radecka@wlv.ac.uk
2006 ⁄ 1623: received 21 November 2006,
revised 12 January 2007 and accepted 7 Feb-
ruary 2007
doi:10.1111/j.1365-2672.2007.03335.x
Summary
Various bacterial species accumulate intracellular polyhydroxyalkanoates
(PHAs) granules as energy and carbon reserves inside their cells. PHAs are bio-
degradable, environmentally friendly and biocompatible thermoplastics. Vary-
ing in toughness and flexibility, depending on their formulation, they can be
used in various ways similar to many nonbiodegradable petrochemical plastics
currently in use. They can be used either in pure form or as additives to oil-
derived plastics such as polyethylene. However, these bioplastics are currently
far more expensive than petrochemically based plastics and are therefore used
mostly in applications that conventional plastics cannot perform, such as med-
ical applications. PHAs are immunologically inert and are only slowly degraded
in human tissue, which means they can be used as devices inside the body.
Recent research has focused on the use of alternative substrates, novel extrac-
tion methods, genetically enhanced species and mixed cultures with a view to
make PHAs more commercially attractive.

gated the bacterial production of PHAs and a great effort is

Introduction
The accumulation of petrochemical plastic waste in the
environment is an increasing problem. In order to find
alternative materials, researchers have developed fully
biodegradable plastics, such as polyhydroxyalkanoates
(PHAs). PHAs extracted from bacterial cells show mater-
ial properties that are similar to polypropylene (Braunegg
et al. 1998). Many micro-organisms have the ability to
degrade these macromolecules enzymatically (Mergaert
et al. 1992). Other advantages of these materials over pet-
rochemical plastics are that they are natural, renewable
and biocompatible.
The occurrence of PHAs in bacteria has been known
since 1920s, when Lemoigne reported the formation of
poly(3-hydroxybutyrate) (PHB) inside bacteria (Lemoigne
1926).
However, the high cost of producing these bioplastics
and the availability of low-cost petrochemical-derived
plastics led to bioplastics being ignored for a long time.
Concern over petrochemical plastics in the environment
has created a renewed interest in biologically derived poly-
mers. During recent years, intensive research has investi-
underway to improve this procedure (Braunegg et al. 2004;
Khanna and Srivastava 2005b). Nonetheless, the PHA pro-
duction price is still far above the price of conventional
plastics (Salehizadeh and Van Loosdrecht 2004).
In order to make the process economically attractive,
many goals have to be addressed simultaneously. Recom-
binant microbial strains are being developed to achieve
both a high substrate conversion rate and close packing
of PHAs granules in the host cell (Taguchi et al. 2003;
Kahar et al. 2005; Agus et al. 2006b; Nikel et al. 2006;
Sujatha and Shenbagarathai, 2006). A more efficient fer-
mentation process (Grothe et al. 1999; Patwardhan and
Srivastava 2004), better recovery ⁄ purification (Jung et al.
2005) and the use of inexpensive substrates (Lemos et al.
2006) can also substantially reduce the production cost.
Additionally, further research is required to enhance the
physical properties of PHAs (Zinn and Hany 2005).
PHA synthesis in bacteria
PHAs are synthesized by many living organisms. The
main candidates for the large-scale production of PHAs

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Bacterial synthesis of PHAs R.A.J. Verlinden et al.

are plants and bacteria. Plant cells can only cope with low
yields [<10% (w ⁄ w) of dry weight] of PHA production.
High levels [10–40% (w ⁄ w) of dry weight] of polymer
inside the plant have a negative effect on the growth and
development of the plant. At present, this problem has
not been overcome (Bohmert et al. 2002). In contrast,
within bacteria, PHAs are accumulated to levels as high
as 90% (w ⁄ w) of the dry cell mass (Steinbüchel and
Lütke-Eversloh 2003).
Accumulating PHAs is a natural way for bacteria to
store carbon and energy, when nutrient supplies are
imbalanced. These polyesters are accumulated when bac-
terial growth is limited by depletion of nitrogen, phos-
phorous (Shang et al. 2003) or oxygen and an excess
amount of a carbon source is still present. While the most
common limitation is nitrogen, for some bacteria, such as
Azotobacter spp., the most effective limitation is oxygen
(Dawes 1990).
As PHAs are insoluble in water, the polymers are accu-
mulated in intracellular granules inside the cells. It is
advantageous for bacteria to store excess nutrients inside
their cells, especially as their general physiological fitness
is not affected. By polymerizing soluble intermediates into
insoluble molecules, the cell does not undergo alterations
of its osmotic state. Thus, leakage of these valuable com-
pounds out of the cell is prevented and the nutrient
stores will remain securely available at a low maintenance
cost (Peters and Rehm 2005).
The surface of a PHA granule is coated with a layer of
phospholipids and proteins. Phasins, a class of proteins,
are the predominant compounds in the interface of a
granule. The phasins influence the number and size of
PHA granules (Pötter et al. 2002; Pötter and Steinbüchel
2005). Expression of genes of phasins can be the 2 to clo-
sely packed granules in bacterial cells.
The first PHA to be discovered and therefore the most
studied is PHB. In their metabolism, bacteria produce
acetyl-coenzyme-A (acetyl-CoA), which is converted into
PHB by three biosynthetic enzymes (Fig. 1).
In the first step, 3-ketothiolase (PhaA) combines two
molecules of acetyl-CoA to form acetoacetyl-CoA. Acetoac-
ethyl-CoA reductase (PhaB) allows the reduction of aceto-
acetyl-CoA by NADH to 3-hydroxybutyryl-CoA. Finally,
PHB synthase (PhaC) polymerizes 3-hydroxybutyryl-CoA
to PHB, coenzyme-A being liberated. Only (R)-isomers are
accepted as substrates for the polymerizing enzyme (Tsuge
et al. 2005).
During normal bacterial growth, the 3-ketothiolase will
be inhibited by free coenzyme-A coming out of the Krebs
cycle. But when entry of acetyl-CoA into the Krebs cycle
is restricted (during noncarbon nutrient limitation), the
surplus acetyl-CoA is channelled into PHB biosynthesis
(Ratledge and Kristiansen 2001).
sugars
acetyl-CoA Krebs cycle
3-ketothiolase (PhaA)
acetoacetyl-CoA
acetoacetyl-CoA reductase (PhaB)
(R)-3-hydroxybutyryl-CoA
PHB synthase (PhaC)
PHB
Figure 1 Metabolic pathway to PHB.
Chemical structure of PHAs
Besides PHB, there are many other PHAs composed of 3-
hydroxy fatty acids. The pendant group (R in Fig. 2) var-
ies from methyl (C1) to tridecyl (C13). Fatty acids with
the hydroxy group at position 4, 5 or 6 and pendant
groups containing substituents or unsaturations are also
known. Within bacterial metabolism, carbon substrates
are converted into hydroxyacyl-CoA thioesters. As seen in
Fig. 2, the carboxyl group of one monomer forms an
ester bond with the hydroxyl group of the neighbouring
monomer. This polymerization reaction is catalysed by
the host’s PHA synthase.
In all PHAs that have been characterized so far, the
hydroxyl-substituted carbon atom is of the stereochemical
(R)-configuration. There is an enormous variation poss-
ible in the length and composition of the side chains.
This variation makes the PHA polymer family suitable for
an array of potential applications (Doi 1990).
The structure of PHAs composed of 3-hydroxy fatty
acids is shown in Fig. 3. The most common polymers,
with a structure given in Fig. 3, are shown in Table 1.
The value of n in Figs 3 and 4a,b depends on the pendant
group and the micro-organisms in which the polymer is
O
R H O PHA-synthase R H
n + n HSCoA
SCoA O
HO n
Figure 2 Synthesis of PHAs in bacteria using hydroxyacyl-CoA thio-
esters as precursor.

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R.A.J. Verlinden et al. Bacterial synthesis of PHAs

O Table 2 Properties of PHAs and polypropylene (PP). PHBV contains

R H
20% 3HV-monomers, PHB4B) contains 16% 4HB-monomers, PHBHx
O contains 10% 3HHx-monomers (Tsuge 2002)
n
Parameter PHB PHBV PHB4B PHBHx PP
Figure 3 Poly(3-hydroxyalkanoates).
Melting temperature (C) 177 145 150 127 176
Glass transition temperature (C) 2 )1 )7 )1 )10
Crystallinity (%) 60 56 45 34 50–70
Table 1 PHAs and corresponding R-groups
Tensile strength (MPa) 43 20 26 21 38
R-group Full name Short Extension to break (%) 5 50 444 400 400

CH3 Poly(3-hydroxybutyrate) PHB

CH2CH3 Poly(3-hydroxyvalerate)
CH2CH2CH3 Poly(3-hydroxyhexanoate)
produced. It is typically between 100 and 30 000 (Lee
1996).
PHB and PHV [poly(3-hydroxyvalerate)] form a class
of PHAs typically referred to as short-chain-length PHAs
(scl-PHAs). In contrast, medium-chain-length PHAs
(mcl-PHAs) are composed of C6 to C16 3-hydroxy fatty
acids (Bayari and Severcan 2005). It has been suggested
that PHB ‘homopolymer’, synthesized by bacteria, always
contains less than 1 mol% of 3-hydroxyvalerate mono-
mers (Sato et al. 2005b).
Copolymers of PHB are formed when mixed substrates
are used, such as a mix of glucose and valerate. The
micro-organisms convert the substrates into scl-PHAs like
poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) or
poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (PHB4B)
(Yan et al. 2005). In addition, PHBHx copolymers that
contain 3-hydroxyhexanoate units and other mcl-PHAs
are reported (Park et al. 2005a). When a mixture of sub-
strates is used, the resulting polymers are random copoly-
mers. However, when substrates are alternated overtime,
it is possible to obtain PHA block copolymers synthesized
by bacteria (Pederson et al. 2006). Figure 4a, b shows the
most common PHA copolymers. In this figure, x and y
are the number of respective monomeric units in the
copolymer.
PHV
PHHx Physical properties
Bacteria produce PHAs with average molecular mass
(Mn) of up to 4Æ0 · 106 Da with a polydispersity
(Mw ⁄ Mn) of around 2Æ0 (Agus et al. 2006a). The material
characteristics of these biopolymers are similar to conven-
tional plastics such as polypropylene (Marchessault and
Yu 2004; Sato et al. 2005a; Tsz-Chun et al. 2005).
The properties of PHB (homopolymer), PHBV, PHB4B
(scl-copolymers) and PHBHx (mcl-copolymer) are com-
pared with polypropylene (PP) in Table 2.
PHB homopolymer is a highly crystalline (Paderm-
shoke et al. 2005), stiff, but brittle material. When spun
into fibres it behaves as a hard-elastic material (Antipov
et al. 2006). Copolymers like PHBV or mcl-PHAs are less
stiff and brittle than PHB, while retaining most of the
other mechanical properties of PHB. Homopolymer PHB
has a helical crystalline structure, this structure seems to
be similar in various copolymers (Padermshoke et al.
2004).
Melting behaviour and crystallization of PHAs have
recently been studied by Gunaratne and Shanks (2005).
In this study, PHAs show multiple melting peak beha-
viour and melting–recrystallization–remelting.
When processing biopolymers, it is important to know
the point of thermal degradation. Carrasco et al. (2006)
recently determined that PHB (Biopol) decomposition
starts at 246Æ3C, while the value for PHBV (Biopol) is
260Æ4C. This indicates that the presence of valerate in
the chain increases the thermal stability of the polymer.

(a)
O R H
O Biodegradability
CH3 H
O Besides the typical polymeric properties described above,
O y
x an important characteristic of PHAs is their biodegrada-
bility. Micro-organisms in nature are able to degrade
(b) O PHAs by using PHA hydrolases and PHA depolymerases
CH3 H O (Jendrossek and Handrick 2002; Choi et al. 2004). The
O
O y
activities of these enzymes may vary and depend on the
x
composition of the polymer and the environmental con-
Figure 4 (a) Polyhydroxybutyrate copolymers. (b) Poly(3-hydroxybuty- ditions. The degradation rate of a piece of PHB is typic-
rate-co-4-hydroxybutyrate) (PHB4B). ally in the order of a few months (in anaerobic sewage)

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Bacterial synthesis of PHAs
Carbon sources
Energy
(sugars, lipids)
Oxygen
Water
Fermentation Plants
Extraction,
Purification
Moulding
PHAs Bioplastic products
(polyhydroxyalkanoates) (packaging, implants, etc.)
Recycling
to years (in seawater) (Madison and Huisman 1999). UV
light can accelerate the degradation of PHAs (Shangguan
et al. 2006).
PHAs have been proved biocompatible, which means
they have no toxic effects in living organisms (Volova
et al. 2003). Within mammals, the polymer is hydrolysed
only slowly. After a 6-month period of implantation in
mice, the mass loss was less than 1Æ6% (w ⁄ w) (Pouton
and Akhtar 1996).
Renewable nature and life cycle
Maybe even more important than biodegradability of
PHAs is the fact that their production is biological and
based on renewable resources (Braunegg et al. 2004). Fer-
mentative production of PHAs uses agricultural feeds
such as sugars and fatty acids as carbon and energy
sources (Kadouri et al. 2005). Consequently, the synthesis
and biodegradation of PHAs are totally compatible to the
carbon-cycle (as depicted in Fig. 5). Thus, while for some
applications the biodegradability is critical, PHAs receive
general attention because they are based on renewable
compounds instead of on fossil fuels (Gavrilescu and
Chisti 2005).
Studies into the life cycle of PHAs show concerns that
the production of these biopolymers may not be any bet-
ter for the environment than the production of conven-
tional polymers. According to those studies, more energy
would be needed during the life cycle of PHA, from crop
growing to moulding the final product, than in the life
cycle of conventional plastics. However, the fermentation
1440 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 1437–1449
R.A.J. Verlinden et al.
Sunlight
Carbon dioxide
Water
Composting
Figure 5 Life cycle of PHAs (Gross and Kalra
2002).
process to make PHAs is far from optimized, while the
production of petrochemical plastics is fully developed
(Gerngross 1999; Dove 2000; Stevens 2002; Kim and Dale
2005).
Material applications
The majority of expected applications of PHAs are as
replacements for petrochemical polymers. The plastics
currently used for packaging and coating applications can
be replaced partially or entirely by PHAs. The extensive
range of physical properties of the PHA family and the
extended performance obtainable by chemical modifica-
tion (Zinn and Hany 2005) or blending (Zhang et al.
1997; Avella et al. 2000; Lee and Park 2002; Wang et al.
2005; Gao et al. 2006; Kunze et al. 2006) provide a broad
range of potential end-use applications.
Applications focus in particular on packaging such as
containers and films (Bucci and Tavares 2005). In
addition, their use as biodegradable personal hygiene arti-
cles such as diapers and their packaging have already been
described (Noda 2001). PHAs have also been processed
into toners for printing applications and adhesives for
coating applications (Madison and Huisman 1999).
Composites of bioplastics are already used in electronic
products, like mobile phones (NEC Corporation and
UNITIKA Ltd. 2006). Potential agricultural applications
include encapsulation of seeds, encapsulation of fertilizers
for slow release, biodegradable plastic films for crop
protection and biodegradable containers for hothouse
facilities.
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R.A.J. Verlinden et al.
PHAs also have numerous medical applications. The
main advantage in the medical field is that a biodegrad-
able plastic can be inserted into the human body and
does not need to be removed again. PHA has an ideal
biocompatibility as it is a product of cell metabolism and
also 3-hydroxy butyric acid (the product of degradation)
is normally present in blood at concentrations between
0Æ3 and 1Æ3 mmol l)1 (Zinn et al. 2001). In pure form or
as composites with other materials, PHAs are used as
sutures, repair patches, orthopedic pins, adhesion barriers,
stents, nerve guides and bone marrow scaffolds. An inter-
esting aspect of PHA scaffolds is the fact that the tissue-
engineered cells can be implanted with the supporting
scaffolds. Research shows that PHA materials can be use-
ful in bone healing processes. PHA together with
hydroxyapatite (HA) can find an applications as a bioac-
tive and biodegradable composite for applications in hard
tissue replacement and regeneration (Chen and Wu
2005a). Polymer implants for targeted drug delivery, an
emerging medical application, can be made out of PHAs
(Chen and Wu 2005b; Park et al. 2005b). However,
because of the high level of specifications for plastics used
in the human body, not every PHA can be used in med-
ical applications (Vert 2005). PHA used in contact with
blood has to be free of bacterial endotoxins and conse-
quently there are high requirements for the extraction
and purification methods for medical PHAs (Sevastianov
et al. 2003).
Bacterial strains
PHAs are produced by many different bacterial cultures.
Cupriavidus necator (formerly known as Ralstonia eutro-
pha or Alcaligenes eutrophus) (Vandamme and Coenye
2004; Vaneechoutte et al. 2004) is the one that has been
most extensively studied. Imperial Chemical Industries
(ICI plc) were the first to use this bacterial strain for the
production of PHBV copolymer under the trade name
Biopol. Recently, Metabolix Inc. (USA) acquired the
Biopol patents. They currently produce 100 t per year
and plan to increase their capacity to 50 000 t per year in
2008 (presented on the Bioplastics Conference 2005,
Frankfurt am Main, Germany).
At present, bacterial fermentation of Cupriavidus neca-
tor seems to be the most cost-effective process and even if
production switches to other bacteria or agricultural
crops, these processes are likely to use Cupriavidus necator
genes. A few important other strains that were recently
studied include: Bacillus spp., Alcaligenes spp., Pseudo-
monas spp., Aeromonas hydrophila, Rhodopseudomonas
palustris, Escherichia coli, Burkholderia sacchari and Halo-
monas boliviensis. In Table 3 an overview is given of bac-
terial strains used to produce PHAs, including their
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Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 1437–1449
Bacterial synthesis of PHAs
corresponding initial carbon sources and produced (co)-
polymers.
From work of Łabu_zek and Radecka (2001) it is known
that spore-forming Bacillus strains are able to produce a
novel terpolymer. Because the environmental conditions,
which induce biopolymer production, are also favourable
for spore production, there is a conflict between the two
metabolic processes and biopolymer production may be
reduced. It is therefore promising to evaluate nonspore-
forming mutants of Bacillus for their potential to produce
PHAs.
Genetic engineering is a powerful tool in the optimiza-
tion of the microbial metabolism towards polymer pro-
duction. Escherichia coli strains (Park et al. 2005a) have
been genetically modified to produce PHB with an Mw
up to 107 Da from glucose. This so-called ultra high
molecular weight PHB (UHMW-PHB) can be processed
into very strong films (Kahar et al. 2005).
Fermentation process
The fermentative production of PHAs is normally oper-
ated as a two-stage fed-batch process (Doi 1990). An ini-
tial growth phase in nutritionally enriched medium yields
sufficient biomass, followed by a product formation phase
in nitrogen-depleted medium. Single fed-batch fermenta-
tions that are nitrogen limited lead to low amounts of
polymer, because there is not enough accumulation of
biomass (Katırcıoǧlu et al. 2003).
As Tanaka et al (1995) introduced the use of mixed
cultures for the production of PHAs, it has been assessed
that they can improve the efficiency of fermentation. The
use of open mixed cultures, such as activated sludge
(Satoh et al. 1999; Chua et al. 2003; Lemos et al. 2006),
can contribute to decrease the cost of PHAs and therefore
increase their market potential (Patnaik 2005).
While the production of PHAs in pure cultures is
limited by an external nutrient, production in mixed
cultures is induced by an intracellular limitation. When
cells are exposed to a medium with very little amounts
of nutrient for a long time, the bacteria are altered
physiologically (Daigger and Grady 1982). Sudden
increase of carbon substrate concentrations causes the
cell to change their physiology again. As PHA-synthesis
requires less adaptation than growth, the culture starts
producing polymer. This kind of fermentation is referred
to as ‘feast and famine’ (Dias et al. 2005; Lemos et al.
2006).
Modelling approaches
In the past 10 years, a number of mechanistic models for
the production of PHAs have been constructed. Models
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Bacterial synthesis of PHAs
Table 3 Overview of bacterial strains used to produce PHAs. The table includes initial carbon sources, produced polymers and reference. mcl-
PHAs: medium-chain-length polyhydroxyalkanoates, PHB: poly(3-hydroxybutyrate), PHBV: poly(3-hydroxybutyrate-co-valerate), UHMW: ultra high
molecular weight
Bacterial strain (s)
Aeromonas hydrophila
Alcaligenes latus
Bacillus cereus
Bacillus spp.
Burkholderia sacchari sp. nov.
Burkholderia cepacia
Caulobacter crescentus
Escherichia coli mutants
Halomonas boliviensis
Legionella pneumophila
Methylocystis sp.
Microlunatus phosphovorus
Pseudomonas aeruginosa
Pseudomonas oleovorans
Pseudomonas putida
Pseudomonas putida,
P. fluorescens, P. jessenii
Pseudomonas stutzeri
Rhizobium meliloti, R. viciae,
Bradyrhizobium japonicum
Rhodopseudomonas palustris
Spirulina platensis (cyanobacterium)
Staphylococcus epidermidis
Cupriavidus necator
Cupriavidus necator H16
for fermentation with only one type of culture (mostly
Cupriavidus necator) are frequently described (Patwardhan
and Srivastava 2004; Yan et al. 2005; Yu et al. 2005; Lee
1442
R.A.J. Verlinden et al.
Carbon source (s) Polymer (s) produced Reference
Lauric acid, oleic acid mcl-PHAs (Lee et al. 2000; Han et al. 2004)
Malt, soy waste, milk waste, PHB (Wong et al. 2004, 2005)
vinegar waste, sesame oil
Glucose, e-caprolactone, sugarbeet PHB, terpolymer (Labuzek and Radecka 2001; Yilmaz and
molasses Beyatli 2005; Valappil et al. 2007)
Nutrient broth, glucose, alkanoates, PHB, PHBV, (Katircioglu et al. 2003; Shamala et al.
e-caprolactone, soy molasses copolymers 2003; Tajima et al. 2003; Yilmaz et al.
2005; Full et al. 2006)
Adonitol, arabinose, arabitol, cellobiose, PHB, PHBV (Brämer et al. 2001)
fructose, fucose, lactose, maltose,
melibiose, raffinose, rhamnose,
sorbitol, sucrose, trehalose, xylitol
Palm olein, palm stearin, crude palm oil, PHB, PHBV (Keenan et al. 2004; Nakas et al. 2004;
palm kernel oil, oleic acid, xylose, Alias and Tan 2005; Çelik et al. 2005)
levulinic acid, sugarbeet molasses
Caulobacter medium, glucose PHB (Qi and Rehm 2001)
Glucose, glycerol, palm oil, ethanol, (UHMW)PHB (Mahishi et al. 2003; Kahar et al. 2005;
sucrose, molasses Park et al. 2005a; Nikel et al. 2006;
Sujatha and Shenbagarathai 2006)
Starch hydolysate, maltose, PHB (Quillaguaman et al. 2005, 2006)
maltotetraose and maltohexaose
Nutrient broth PHB (James et al. 1999)
Methane PHB (Wendlandt et al. 2005)
Glucose, acetate PHB (Akar et al. 2006)
Glucose, technical oleic acid, waste free mcl-PHAs (Hoffmann and Rehm 2004;
fatty acids, waste free frying oil Fernández et al. 2005)
Octanoic acid mcl-PHAs (Durner et al. 2000; Foster et al. 2005)
Glucose, octanoic acid, undecenoic acid mcl-PHAs (Tobin and O’Connor 2005;
Hartmann et al. 2006)
Glucose, aromatic monomers aromatic polymers (Tobin and O’Connor 2005; Ward and
O’Connor 2005; Ward et al. 2005)
Glucose, soybean oil, alcohols, mcl-PHAs (Xu et al. 2005)
alkanoates
Glucose, sucrose, galactose, mannitol, PHB (Mercan and Beyatli 2005)
trehalose, xylose, raffinose, maltose,
dextrose, lactose, pyruvate, sugar beet
molasses, whey
Acetate, malate, fumarate, succinate, PHB, PHBV (Mukhopadhyay et al. 2005)
propionate, malonate, gluconate,
butyrate, glycerol, citrate
Carbon dioxide PHB (Jau et al. 2005)
Malt, soy waste, milk waste, PHB (Wong et al. 2004, 2005)
vinegar waste, sesame oil
Glucose, sucrose, fructose, valerate, PHB, copolymers (Kim et al. 1995; Kichise et al. 1999;
octanoate, lactic acid, soybean oil Taguchi et al. 2003; Kahar et al. 2004;
Khanna and Srivastava 2005a;
Volova and Kalacheva 2005;
Volova et al. 2005)
Hydrogen, carbon dioxide PHB (Pohlmann et al. 2006)
and Gilmore 2006). However, Cupriavidus necator cannot
easily metabolize sugars, molasses, whey or starchy waste.
Consequently, mixed cultures of lactic acid producing
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R.A.J. Verlinden et al. Bacterial synthesis of PHAs

bacteria and Cupriavidus necator have been investigated.
The original substrates are converted into lactic acid first,
which is taken up by Cupriavidus necator to produce
PHAs (Patnaik 2005). Recently Dias et al (2005) con-
structed a mathematical model for a production process
of PHAs produced by mixed cultures.
Carbon substrates
In the bacterial cell, carbon substrates are metabolized by
many different pathways. The three most-studied meta-
bolic pathways are shown in Fig. 6. Sugars such as glu-
cose and fructose are mostly processed via pathway I,
yielding PHB homopolymer. If fatty acids or sugars are
metabolized by pathway II, III or other pathways, copoly-
mers are produced (Aldor and Keasling 2003; Steinbüchel
and Lütke-Eversloh 2003).
Currently efforts are being made to grow the bacteria
on different renewable vegetable oils and various waste
products (Koller et al. 2005; Lee and Gilmore 2006). The
use of these inexpensive carbon sources to produce PHAs
could lead to significant economical advantages (Quillag-
uaman et al. 2005).
Wong et al. (2004, 2005) studied the accumulation of
PHB by A. latus and Staphylococcus epidermidis grown on
several types of food waste. Dionisi et al. (2005) reported
PHAs from olive oil mill effluents. Quillaguaman et al.
(2005) were able to biosynthesize PHB with H. boliviensis
using starch hydrolysate as carbon source with a maxi-
mum yield of 56% (w ⁄ w). Fernández et al. (2005) pub-
lished their results on the ability of Pseudomonas
aeruginosa to feed on fatty acids and frying oil, with a
maximum production of 66% (w ⁄ w) PHA. Alias and Tan
(2005) were able to obtain PHAs [57Æ4% (w ⁄ w)] from
palm-oil-utilizing bacteria, while Mercan and Beyatli
(2005) were successful with bacteria feeding on sugar beet
molasses (56Æ31% (w ⁄ w) maximum yield).
Additionally, researchers discovered that not only
cyanobacteria can use gaseous carbon dioxide (Jau et al.
2005) to directly produce PHAs. Methylocystis sp. can
metabolize methane (Wendlandt et al. 2005) and Cupri-
avidus necator H16 can metabolize a mixture of hydrogen
and carbon dioxide (Pohlmann et al. 2006) to form
PHAs.
An overview of alternative carbon sources can be found
in Table 3.

Pathway I Pathway II
sugars fatty acids

Krebs cycle acetyl-CoA acyl-CoA

PhaA
fatty acid
3-ketoacyl-CoA enoyl-CoA
acetoacetyl-CoA β-oxidation
PhaB
(S)-3-hydroxyacyl-CoA
(R)-3-hydroxybutyryl-CoA FabG (?) PhaJ
PhaC

PHA (R)-3-hydroxyacyl-CoA
PhaC
PhaC PhaG
4-hydroxyacyl-CoA (R)-3-hydroxyacyl-ACP
FabG
fatty acid
carbon sources 3-ketoacyl-ACP enoyl-ACP
biosynthesis
Other pathways
acyl-ACP

malonyl-ACP

malonyl-CoA

acetyl-CoA

sugars
Figure 6 Metabolic pathways supplying
monomers for PHA synthesis (Tsuge 2002). Pathway III

ª 2007 The Authors

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 1437–1449 1443
Bacterial synthesis of PHAs
PHA recovery
After fermentation, bacterial cells containing PHAs are
separated from the medium by centrifugation. Most
methods to recover intracellular PHA involve the use of
organic solvents, such as acetone (Jiang et al. 2006), chlo-
roform, methylene chloride or dichloroethane. However,
the necessity of large quantities of solvent makes the pro-
cedure economically and environmentally unattractive
(Braunegg et al. 1998). For medical applications the sol-
vent extraction is a good method, because the resulting
PHAs have a high purity (Chen and Wu 2005a).
As an alternative to the unfavourable extraction with
organic solvents, aqueous enzymatic procedures (Holmes
and Lim 1984; Kapritchkoff et al. 2006; Lakshman and
Shamala 2006), treatments with ammonia (Page and
Cornish 1993) or digestion with sodium hypochlorite
and surfactants (Ramsay et al. 1990; Ryu et al. 2000) have
been proposed. Recently, supercritical fluid disruption
(Hejazi et al. 2003; Khosravi-Darani et al. 2004),
dissolved-air flotation (van Hee et al. 2006) and selective
dissolution of cell mass (Yu and Chen 2006) for the
recovery of PHAs were studied. A new cultivation
method allowes spontaneous release of up to 80% of the
intracellular PHB from E. coli (Jung et al. 2005). All of
these methods are promising alternatives to the solvent
extraction.
Outlook
Mineral oil prices will rise substantially in the next century,
forcing the world to consider alternatives for petrochem-
ical plastics. The renewable nature and biodegradability of
PHAs make them suitable materials to replace synthetic
plastics in many applications (Stevens 2002).
Currently their production is expensive, but these
plastics are only in their first stage of commercial deve-
lopment (Lee 1996). Further research on recombinant
microbial strains, mixed cultures, efficient fermentations,
recovery ⁄ purification and the use of inexpensive substrates
can substantially reduce the production cost.
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