Summary
Various bacterial species accumulate intracellular polyhydroxyalkanoates (PHAs) granules as energy and carbon reserves inside their cells. PHAs are biodegradable, environmentally friendly and biocompatible thermoplastics. Varying 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 medical 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 extraction methods, genetically enhanced species and mixed cultures with a view to make PHAs more commercially attractive.
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 material 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 petrochemical 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 polymers. During recent years, intensive research has investigated 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 production 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. Recombinant 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 fermentation 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 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 bacterial growth is limited by depletion of nitrogen, phosphorous (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 accumulated 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 compounds 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 closely 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).
Metabolic pathway to PHB.
In the first step, 3‐ketothiolase (PhaA) combines two molecules of acetyl‐CoA to form acetoacetyl‐CoA. Acetoacethyl‐CoA reductase (PhaB) allows the reduction of acetoacetyl‐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) varies 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.
Synthesis of PHAs in bacteria using hydroxyacyl‐CoA thioesters as precursor.
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 possible 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 produced. It is typically between 100 and 30 000 (Lee 1996).
Poly(3‐hydroxyalkanoates).
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 |
| R‐group | Full name | Short |
|---|---|---|
| CH3 | Poly(3‐hydroxybutyrate) | PHB |
| CH2CH3 | Poly(3‐hydroxyvalerate) | PHV |
| CH2CH2CH3 | Poly(3‐hydroxyhexanoate) | PHHx |
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 |
| R‐group | Full name | Short |
|---|---|---|
| CH3 | Poly(3‐hydroxybutyrate) | PHB |
| CH2CH3 | Poly(3‐hydroxyvalerate) | PHV |
| CH2CH2CH3 | Poly(3‐hydroxyhexanoate) | PHHx |
(a) Polyhydroxybutyrate copolymers. (b) Poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate) (PHB4B).
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 monomers (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 substrates is used, the resulting polymers are random copolymers. 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 4·0 × 106 Da with a polydispersity (Mw/Mn) of around 2·0 (Agus et al. 2006a). The material characteristics of these biopolymers are similar to conventional 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 compared with polypropylene (PP) in 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 |
| 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 |
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 |
| 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 |
PHB homopolymer is a highly crystalline (Padermshoke 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 behaviour 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 246·3°C, while the value for PHBV (Biopol) is 260·4°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 biodegradability. 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 conditions. The degradation rate of a piece of PHB is typically in the order of a few months (in anaerobic sewage) 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 1·6% (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). Fermentative 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 better for the environment than the production of conventional 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 modification (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 articles 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.
PHAs also have numerous medical applications. The main advantage in the medical field is that a biodegradable 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 0·3 and 1·3 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 interesting 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 useful in bone healing processes. PHA together with hydroxyapatite (HA) can find an applications as a bioactive 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 medical applications (Vert 2005). PHA used in contact with blood has to be free of bacterial endotoxins and consequently 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 eutropha 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 necator 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., Pseudomonas spp., Aeromonas hydrophila, Rhodopseudomonas palustris, Escherichia coli, Burkholderia sacchari and Halomonas boliviensis. In Table 3 an overview is given of bacterial strains used to produce PHAs, including their corresponding initial carbon sources and produced (co)polymers.
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, ɛ‐caprolactone, sugarbeet molasses | PHB, terpolymer | (Labuzek and Radecka 2001; Yilmaz and Beyatli 2005; Valappil et al. 2007) |
| Bacillus spp. | Nutrient broth, glucose, alkanoates, ɛ‐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 | (Brämer 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; Çelik 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; Fernández 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 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, ɛ‐caprolactone, sugarbeet molasses | PHB, terpolymer | (Labuzek and Radecka 2001; Yilmaz and Beyatli 2005; Valappil et al. 2007) |
| Bacillus spp. | Nutrient broth, glucose, alkanoates, ɛ‐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 | (Brämer 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; Çelik 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; Fernández 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) |
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, ɛ‐caprolactone, sugarbeet molasses | PHB, terpolymer | (Labuzek and Radecka 2001; Yilmaz and Beyatli 2005; Valappil et al. 2007) |
| Bacillus spp. | Nutrient broth, glucose, alkanoates, ɛ‐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 | (Brämer 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; Çelik 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; Fernández 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 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, ɛ‐caprolactone, sugarbeet molasses | PHB, terpolymer | (Labuzek and Radecka 2001; Yilmaz and Beyatli 2005; Valappil et al. 2007) |
| Bacillus spp. | Nutrient broth, glucose, alkanoates, ɛ‐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 | (Brämer 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; Çelik 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; Fernández 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) |
From work of Łabużek 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 optimization of the microbial metabolism towards polymer production. 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 operated as a two‐stage fed‐batch process (Doi 1990). An initial growth phase in nutritionally enriched medium yields sufficient biomass, followed by a product formation phase in nitrogen‐depleted medium. Single fed‐batch fermentations 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 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 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) constructed 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 metabolic pathways are shown in Fig. 6. Sugars such as glucose 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, copolymers 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 (Quillaguaman 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 maximum yield of 56% (w/w). Fernández et al. (2005) published 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 [57·4% (w/w)] from palm‐oil‐utilizing bacteria, while Mercan and Beyatli (2005) were successful with bacteria feeding on sugar beet molasses (56·31% (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 Cupriavidus 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.
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), chloroform, methylene chloride or dichloroethane. However, the necessity of large quantities of solvent makes the procedure economically and environmentally unattractive (Braunegg et al. 1998). For medical applications the solvent 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 petrochemical 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 development (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.
