REVIEW ARTICLE

Bacterial synthesis of biodegradablepolyhydroxyalkanoatesR.A.J. Verlinden, D.J. Hill, M.A. Kenward, C.D. Williams and I. Radecka

School of Applied Sciences, University of Wolverhampton, Wolverhampton, UK

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-

gated the bacterial production of PHAs and a great effort is

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

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.

Journal of Applied Microbiology ISSN 1364-5072

ª 2007 The Authors

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 1437–1449 1437

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 (Steinbuchel and

Lutke-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 (Potter et al. 2002; Potter and Steinbuchel

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).

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

sugars

acetoacetyl-CoA

acetoacetyl-CoA reductase (PhaB)

(R)-3-hydroxybutyryl-CoA

PHB synthase (PhaC)

PHB

acetyl-CoA Krebs cycle

3-ketothiolase (PhaA)

Figure 1 Metabolic pathway to PHB.

n

HO

O

SCoA

PHA-synthase

+ n HSCoAO

O

n

R HR H

Figure 2 Synthesis of PHAs in bacteria using hydroxyacyl-CoA thio-

esters as precursor.

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

1438 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 1437–1449

ª 2007 The Authors

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.

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 246Æ3�C, while the value for PHBV (Biopol) is

260Æ4�C. This indicates that the presence of valerate in

the chain increases the thermal stability of the polymer.

Biodegradability

Besides the typical polymeric properties described above,

an important characteristic of PHAs is their biodegrada-

bility. Micro-organisms in nature are able to degrade

PHAs by using PHA hydrolases and PHA depolymerases

(Jendrossek and Handrick 2002; Choi et al. 2004). The

activities of these enzymes may vary and depend on the

composition of the polymer and the environmental con-

ditions. The degradation rate of a piece of PHB is typic-

ally in the order of a few months (in anaerobic sewage)

O

R H O

n

Figure 3 Poly(3-hydroxyalkanoates).

Table 1 PHAs and corresponding R-groups

R-group Full name Short

CH3 Poly(3-hydroxybutyrate) PHB

CH2CH3 Poly(3-hydroxyvalerate) PHV

CH2CH2CH3 Poly(3-hydroxyhexanoate) PHHx

yO

R H O

O

H O

x

yO

CH3

CH3

(a)

(b)H O O

Ox

Figure 4 (a) Polyhydroxybutyrate copolymers. (b) Poly(3-hydroxybuty-

rate-co-4-hydroxybutyrate) (PHB4B).

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

20% 3HV-monomers, PHB4B) contains 16% 4HB-monomers, PHBHx

contains 10% 3HHx-monomers (Tsuge 2002)

Parameter PHB PHBV PHB4B PHBHx PP

Melting temperature (�C) 177 145 150 127 176

Glass transition temperature (�C) 2 )1 )7 )1 )10

Crystallinity (%) 60 56 45 34 50–70

Tensile strength (MPa) 43 20 26 21 38

Extension to break (%) 5 50 444 400 400

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

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Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 1437–1449 1439

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

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.

Composting

PHAs(polyhydroxyalkanoates)

Extraction,Purification

Fermentation Plants

Recycling

Moulding

Bioplastic products(packaging, implants, etc.)

Carbon dioxide

SunlightCarbon sources(sugars, lipids)

WaterWater

Oxygen

Energy

Figure 5 Life cycle of PHAs (Gross and Kalra

2002).

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

1440 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 1437–1449

ª 2007 The Authors

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

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 (Katırcıoglu 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

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

ª 2007 The Authors

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 1437–1449 1441

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

and Gilmore 2006). However, Cupriavidus necator cannot

easily metabolize sugars, molasses, whey or starchy waste.

Consequently, mixed cultures of lactic acid producing

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) Carbon source (s) Polymer (s) produced Reference

Aeromonas hydrophila Lauric acid, oleic acid mcl-PHAs (Lee et al. 2000; Han et al. 2004)

Alcaligenes latus Malt, soy waste, milk waste,

vinegar waste, sesame oil

PHB (Wong et al. 2004, 2005)

Bacillus cereus Glucose, e-caprolactone, sugarbeet

molasses

PHB, terpolymer (Labuzek and Radecka 2001; Yilmaz and

Beyatli 2005; Valappil et al. 2007)

Bacillus spp. Nutrient broth, glucose, alkanoates,

e-caprolactone, soy molasses

PHB, PHBV,

copolymers

(Katircioglu et al. 2003; Shamala et al.

2003; Tajima et al. 2003; Yilmaz et al.

2005; Full et al. 2006)

Burkholderia sacchari sp. nov. Adonitol, arabinose, arabitol, cellobiose,

fructose, fucose, lactose, maltose,

melibiose, raffinose, rhamnose,

sorbitol, sucrose, trehalose, xylitol

PHB, PHBV (Bramer et al. 2001)

Burkholderia cepacia Palm olein, palm stearin, crude palm oil,

palm kernel oil, oleic acid, xylose,

levulinic acid, sugarbeet molasses

PHB, PHBV (Keenan et al. 2004; Nakas et al. 2004;

Alias and Tan 2005; Celik et al. 2005)

Caulobacter crescentus Caulobacter medium, glucose PHB (Qi and Rehm 2001)

Escherichia coli mutants Glucose, glycerol, palm oil, ethanol,

sucrose, molasses

(UHMW)PHB (Mahishi et al. 2003; Kahar et al. 2005;

Park et al. 2005a; Nikel et al. 2006;

Sujatha and Shenbagarathai 2006)

Halomonas boliviensis Starch hydolysate, maltose,

maltotetraose and maltohexaose

PHB (Quillaguaman et al. 2005, 2006)

Legionella pneumophila Nutrient broth PHB (James et al. 1999)

Methylocystis sp. Methane PHB (Wendlandt et al. 2005)

Microlunatus phosphovorus Glucose, acetate PHB (Akar et al. 2006)

Pseudomonas aeruginosa Glucose, technical oleic acid, waste free

fatty acids, waste free frying oil

mcl-PHAs (Hoffmann and Rehm 2004;

Fernandez et al. 2005)

Pseudomonas oleovorans Octanoic acid mcl-PHAs (Durner et al. 2000; Foster et al. 2005)

Pseudomonas putida Glucose, octanoic acid, undecenoic acid mcl-PHAs (Tobin and O’Connor 2005;

Hartmann et al. 2006)

Pseudomonas putida,

P. fluorescens, P. jessenii

Glucose, aromatic monomers aromatic polymers (Tobin and O’Connor 2005; Ward and

O’Connor 2005; Ward et al. 2005)

Pseudomonas stutzeri Glucose, soybean oil, alcohols,

alkanoates

mcl-PHAs (Xu et al. 2005)

Rhizobium meliloti, R. viciae,

Bradyrhizobium japonicum

Glucose, sucrose, galactose, mannitol,

trehalose, xylose, raffinose, maltose,

dextrose, lactose, pyruvate, sugar beet

molasses, whey

PHB (Mercan and Beyatli 2005)

Rhodopseudomonas palustris Acetate, malate, fumarate, succinate,

propionate, malonate, gluconate,

butyrate, glycerol, citrate

PHB, PHBV (Mukhopadhyay et al. 2005)

Spirulina platensis (cyanobacterium) Carbon dioxide PHB (Jau et al. 2005)

Staphylococcus epidermidis Malt, soy waste, milk waste,

vinegar waste, sesame oil

PHB (Wong et al. 2004, 2005)

Cupriavidus necator Glucose, sucrose, fructose, valerate,

octanoate, lactic acid, soybean oil

PHB, copolymers (Kim et al. 1995; Kichise et al. 1999;

Taguchi et al. 2003; Kahar et al. 2004;

Khanna and Srivastava 2005a;

Volova and Kalacheva 2005;

Volova et al. 2005)

Cupriavidus necator H16 Hydrogen, carbon dioxide PHB (Pohlmann et al. 2006)

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

1442 Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 1437–1449

ª 2007 The Authors

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; Steinbuchel

and Lutke-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). Fernandez 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

Pathway III

acyl-CoA

acyl-ACP

sugars

acetyl-CoA

malonyl-CoA

malonyl-ACP

3-ketoacyl-ACPfatty acid

biosynthesisenoyl-ACP

3-ketoacyl-CoA enoyl-CoA

(S)-3-hydroxyacyl-CoA

(R)-3-hydroxyacyl-CoA

FabG (?)

FabG

PhaC

PhaJ

PhaG

(R)-3-hydroxyacyl-ACP

fatty acidβ-oxidation

fatty acids

PhaA

acetoacetyl-CoA

(R)-3-hydroxybutyryl-CoA

4-hydroxyacyl-CoA

carbon sources

Other pathways

PhaB

PhaC

PhaC

PHA

acetyl-CoAKrebs cycle

sugars

Figure 6 Metabolic pathways supplying

monomers for PHA synthesis (Tsuge 2002).

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

ª 2007 The Authors

Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 102 (2007) 1437–1449 1443

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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