Accepted Manuscript Title: Bacterial production of the biodegradable plastics polyhydroxyalkanoates Author: Viviana Urtuvia Pamela Villegas Myriam Gonz´alez Michael Seeger PII: DOI: Reference:
S0141-8130(14)00384-5 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.06.001 BIOMAC 4402
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
3-3-2014 13-5-2014 3-6-2014
Please cite this article as: V. Urtuvia, P. Villegas, M. Gonz´alez, M. Seeger, Bacterial production of the biodegradable plastics polyhydroxyalkanoates, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.06.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Bacterial production of the biodegradable plastics polyhydroxyalkanoates Viviana Urtuvia, Pamela Villegas, Myriam González & Michael Seeger*
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Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química & Center for Nanotechnology and Systems Biology & Centro de Biotecnología, Universidad Técnica Federico Santa María, Avenida España 1680, Valparaíso, Chile
*For correspondence. E-mail: [email protected]; Tel: (+56-32) 2654236; Fax: (+56-32) 2654782
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Abstract
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Petroleum-based plastics constitute a major environmental problem due to their low biodegradability and accumulation in various environments. Therefore, searching for novel biodegradable plastics is of increasing interest. Microbial polyesters known as polyhydroxyalkanoates (PHAs) are biodegradable plastics. Life Cycle Assessment indicates that PHB is more beneficial than petroleum-based plastics. In this report, bacterial production of PHAs and their industrial applications are reviewed and the synthesis of PHAs in Burkholderia xenovorans LB400 is described. PHAs are synthesized by a large number of microorganisms during unbalanced nutritional conditions. These polymers are accumulated as carbon and energy reserve in discrete granules in the bacterial cytoplasm. 3-hydroxybutyrate and 3-hydroxyvalerate are two main PHA units among 150 monomers that have been reported. B. xenovorans LB400 is a model bacterium for the degradation of polychlorobiphenyls and a wide range of aromatic compounds. A bioinformatic analysis of LB400 genome indicated the presence of pha genes encoding enzymes of pathways for PHA synthesis. This study showed that B. xenovorans LB400 synthesize PHAs under nutrient limitation. Staining with Sudan Black B indicated the production of PHAs by B. xenovorans LB400 colonies. The PHAs produced were characterized by GC-MS. Diverse substrates for the production of PHAs in strain LB400 were analyzed.
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Keywords: polyhydroxyalkanoate, biodegradable plastic, polyhydroxybutyrate, Burkholderia xenovorans, pha gene
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1. Introduction
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The highly versatile petroleum-based plastics have made them valuable materials for modern life with a wide range of applications. However, due to their low biodegradability and accumulation in various environmental compartments they are major environmental pollutants. Searching for novel biodegradable polymers that possess similar physicochemical properties as conventional plastics is of increasing interest. PHAs are biodegradable plastics synthesized by diverse bacteria from a range of substrates including sugars and fatty acids. These biopolymers are stored as intracellular carbon and energy reserve granules by Gram-negative and Gram-positive bacteria under nutrient limitation. 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) are two common and widely studied PHA monomers. However, 150 different PHA monomers have been reported. Depending on the microorganism, the carbon source and culture conditions different PHA homopolymers, copolymers and combination thereof are synthesized. A number of companies in America, Asia and Europe produce PHAs at industrial scale. In this report bacterial production of PHAs and their industrial applications are reviewed and the synthesis of PHAs in the model aromatic-degrading bacterium B. xenovorans LB400 is described.
2. Petroleum-derived plastics: applications and pollution
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The versatility and low production costs of plastics derived from petroleum have allowed them to substitute other materials such as paper, glass and wood. Plastics are important for various applications in different industrial sectors. During the year 2008, 245 million tons were consumed at global level and a 9% annual growth in the plastic consumption was forecasted. Fig. 1 illustrates that in Chile, plastic consumption is mainly concentrated in the areas of packaging, mining, construction and agroindustry [1]. The remaining 9% is distributed among household, agriculture and transport. 844,000 tons of plastic consumption in Chile for 2012 was expected, with 51 kg plastic consumption per capita, placing Chile as the second largest plastic-consuming country in Latin America [2]. The plastic consumption per capita in European countries exceeds 100 kg per year [3]. Chemical and physical properties of plastics provide them with high durability and versatility for a wide range of applications. However, their properties convert plastics in pollutants when they are disposed in the environment. The plastics present very low degradation rates in the environment, with a half-life up to >500 years. The low degradability causes, among other drawbacks, a high accumulation of plastics in sanitary landfills, decreasing the life thereof. The plastic wastes are about 8% in weight and 25% in volume of the total solid urban wastes. The low degradability combined with the inadequate waste disposal and low environmental education have caused that plastics are accumulated in different environmental compartments. An example is the presence of plastic fragments in almost all oceans, which causes plastic ingestion by marine organisms with largely unknown consequences [4]. The implementation of environmental management politics to reduce the plastic wastes is crucial for the conservation of natural resources and the reduction of environmental pollution. The research and development of new biodegradable materials is of increasing importance. In recent decades plastic manufacturing using renewable feedstock has been promoted and the productions of biologically based plastics known as biopolymers, which could be biodegradable, are of increasing interest [5].
3. Biopolymers and polyhydroxyalkanoates
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The term biopolymer includes polymers of natural origin, but also high molecular mass molecules that have been polymerized by chemical and biological methods. Not all biopolymers are biodegradable [5]. The biodegradable plastics primarily are produced from renewable raw materials by aerobic synthesis and from waste management by composting and anaerobic digestion [6]. The biopolymers are classified according to their biodegradability or composition. PHAs and polylactic acid (PLA) are well-studied biopolymers due to their biodegradability and physicochemical properties [5,7]. PHAs are thermoplastic polyesters of R-hydroxyalkanoic acids. PHAs are synthesized and stored as intracellular carbon and energy reserve in Gram-negative and Gram-positive bacteria under nutrient limitation of nitrogen, phosphorus, oxygen or after pH shifts [3,8,9]. PHAs accumulate in cytoplasmic granules that typically have a diameter from 0.2 to 0.5 µm. These granules can be visualized by phase contrast microscopy due to their high refractivity or using staining dyes such as Sudan Black B and Nile red. Once the limiting nutrient is provided to the cell, these energy storage compounds are degraded and used as carbon source for bacterial growth [10]. Polyhydroxybutyrate (PHB) was discovered in 1926 by Maurice Lemoige in the bacterium Bacillus megaterium, which showed intracellular granules [11]. PHB is the most common and the best studied member of PHAs [8]. The presence of 3HB as a PHA monomer was reported in activated sludge [12]. The presence of 3HV as a major constituent and 3-hydroxyhexanoate (3HHx) as a minor constituent were described [13]. The oil crisis of the early 1970s prompted the search for new materials to replace petroleum-based plastics [9]. Fifty years after its discovery, PHB production was carried out at commercial scale. PHAs could have different composition depending on the microorganism, the carbon source and culture conditions (Table 1). The most common polymers are the short-chain-length PHA (PHASCL), which contain from 3 to 5 carbons atoms. The structural formulas of PHASCL are depicted in Fig. 2. These biopolymers are stiff, brittle and possess a high degree of crystallinity. Middle-chain-length PHA (PHAMCL) contain from 6 to 14 carbons atoms and are flexible and possess low crystallinity, tensile strength and high melting point [9,10,14,15]. Currently 150 different monomer constituents of PHAs that could be homopolymers, copolymers and combination thereof have been reported [10,16,17]. PHB is produced by bacteria from a wide range of substrates (Fig. 3). In contrast, specific substrates such as propionic acid, valeric acid and other organic acids are required for the synthesis of the PHV monomer [17,24]. The metabolic pathways for the synthesis of both monomers have been reported in the model PHA-producing bacterium R. eutropha strain H16, which synthesizes PHASCL [17,27–29]. PHAs could reach up to 80% of the bacterial cell dry biomass. Life Cycle Assessment (LCA) indicates that the energy requirements for PHB production are lower than the energy requirements for conventional high density plastics polyethylene and polypropylene [30]. In addition, the production of PHB is more beneficial for ozone layer protection, and reduces toxicity levels, abiotic depletion and global warming. However, PHB showed higher impacts than polyethylene on acidification and eutrophication. On a cradle-tofactory gate LCA, PHB is more beneficial than conventional polymers such as polypropylene [30]. 4. Substrates for bacterial PHA production and PHA production cost PHAs are produced by bacteria from a wide range of substrates such as renewable sources (e.g., sucrose, starch, cellulose, triglycerides, hemicellulose, wheat), sub-products (e.g., molasses, whey, glycerol, corn steep liquor, rice bran), organic acids (e.g., propionic acid, 4-hydroxybutyric acid), fossil resources (e.g., methane, mineral oil, lignite, hard coal) and wastes (e.g., wastewater, palm oil mill and activated sludge effluents) [15,24,27,31–33]. Substrates such as glucose and sucrose have been used for industrial PHA production. The raw materials showed a high impact on the PHA production cost. Therefore, cheap raw materials are attractive for industrial PHA production [8]. Xylose could be an interesting alternative substrate for
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PHA production (Fig 3). Xylose is the main sugar component of hemicellulose in Gramineae plants and hardwood. Hemicellulose is the third most abundant polymer in nature that is converted into fermentable sugars by chemical or enzymatic hydrolysis [31]. Hemicellulose hydrolysis releases xylose, mannose, galactose, arabinose and glucose. PHB production from xylose by Pseudomonas pseudoflava ATCC 33668, Burkholderia cepacia ATCC 17759 and recombinant Escherichia coli strains TG1(pSYL105) and W3110(pSYL107) has been reported [34–37]. The forestry, which produces high amounts of hemicellulose-containing sub-products, is an important activity in diverse countries (e.g., in Canada, Chile, Brazil and USA). Therefore, PHA production from sub-products containing xylose may be an attractive alternative. One of the most significant barriers for an increased industrial PHAs production is its high cost. The product price depends up to 50% on the raw material cost, mainly the carbon source. The biopolymer recovery process also contributes to higher PHA production cost [10,15, 35]. PHAs are still produced on a small-scale production volume from 1000 to 20,000 tons per year, which also increases the cost. In contrast, polyethylene is produced in quantities exceeding 300,000 tons per year, with an important benefit in the final price due to the economy of scale [7,38]. PHA price varies from 1.5 to 5 € per kg, whereas polypropylene price varies from 0.2 to 0.4 € per kg [7,10]. However, the versatility and biocompatibility of PHAs have made them interesting candidates for low volume products of high value, especially for biomedical applications [39]. For example, PHA nanoparticles for drug delivery and biocompatible porous implants made from poly-4hydroxybutyrate has been reported [8,39]. Various small and middle-sized enterprises around the world produce PHAs at small-scale. Forexample, P&G (USA), Biomer Inc. (Germany), Tianan Biologic (China) and PHB Industrial (Brazil) [7]. Bags, pencils, plates, glasses and various plastic parts are manufactured from these polymers. PHA resins are used for the manufacture of sustained release drugs, bone implants and sutures [39]. In addition, PHAs can be used as latex (for example, for paper-coating applications) or in foods [40]. However, to enhance the PHA production, the production cost should be decreased. Therefore, novel approaches for PHA production have to be established [5].
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5. PHA synthesis by Burkholderia xenovorans strain LB400
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The genus Burkholderia possesses high metabolic versatility and is adapted to different ecological niches including soil and aqueous environments. Burkholderia xenovorans LB400 is a model aerobic bacterium for the degradation of polychlorobiphenyls (PCBs) and a wide range of aromatic compounds. PCBs have been used as complex mixtures for diverse industrial applications. These compounds are toxic and tend to accumulate as persistent organic pollutants in soil and water sediment [41,42]. The large genome from B. xenovorans strain LB400 (9.73 Mbp) has been sequenced and characterized [43]. Strain LB400 possesses genes encoding enzymes of eleven central and more than twenty peripheral pathways for the degradation of aromatic compounds. For example, the hmgABC gene cluster encoding the homogentisate central pathway and the hpaG1G2EDFHI gene cluster encoding the homoprotocatechuate central pathway are located at chromosome 1 (C1) and chromosome 2 (C2), respectively. Additional hmg gene copies were identified within the LB400 genome [43,44]. The functionality of diverse catabolic pathways such as biphenyl, homogentisate, homoprotocatechuate, gentisate, protocatechuate and 2-aminophenol pathways has been reported [42,44–46]. B. xenovorans LB400 is able to grow on 3-hydroxyphenylacetate (3-HPA) and 4hydroxyphenylacetate (4-HPA) isomers as sole carbon and energy sources, indicating active peripheral and central catabolic pathways. The homogentisate and homoprotocatechuate central pathways are involved in 3-HPA and 4-HPA degradation in strain LB400. Both ring-cleavage pathways are used in aromatic amino acid metabolism in Bacteria and Eucarya. A number of bioinformatic tools are useful to reconstruct metabolic pathways in bacteria. The genes of the PHA anabolic pathway from the model PHA-producing bacterium Ralstonia eutropha
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strain H16 have been characterized. The genes for the synthesis and degradation of PHA were found in B. xenovorans strain LB400. Strain LB400 has two PHA anabolic pathways: i) the pathway that uses sugars as carbon sources and, ii) the pathway associated to fatty acids catabolic pathway (β-oxidation). In B. xenovorans strain LB400, the pha genes for the synthesis of PHAs are arranged in a gene cluster at the major chromosome [47]. The pha gene clusters from R. eutropha H16 and B. xenovorans LB400 have a similar organization, which are shown in Fig. 4. B. xenovorans strain LB400 has three copies of the phaC gene distributed in the major chromosome, minor chromosome and the megaplasmid. The PHA polymerase PhaC from strain H16 possesses fifteen functionally key amino acids [48,49].These amino acids are conserved in PhaC polymerase from strain LB400 [47]. Different compounds including sugars and alkanoates are substrates for the bacterial production of PHAs. B. xenovorans LB400 is able to grow on glucose, mannitol and xylose as sole carbon source. In contrast, strain LB400 was not able to grow on lactose and sucrose. To study the PHA synthesis by strain LB400 in a first approach staining of bacterial colonies with Sudan Black B dye was analysed [10,50]. LB400 colonies grown on mannitol (10 g L-1) were stained with Sudan Black B, indicating the intracellular accumulation of PHAs (Fig. 5). The synthesis of PHAs by strain LB400 was studied during growth on various sugars and fatty acids. PHAs were identified by gas chromatography and mass spectrometry. During growth on glucose the synthesis of the homopolymer PHB was observed. Searching for low cost renewable materials will be useful to increase the production of these biopolymers.
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6. Conclusions and future prospects
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The global trends of plastic consumption promote the production of novel biodegradable materials, replacing traditional plastics and increasing environmental protection and sustainable development. PHAs are attractive biopolymers due to their biodegradability and physicochemical properties. PHAs are synthesized and stored as intracellular carbon and energy reserve by bacteria under nutrient limitation. Diverse bacteria are able to synthesize PHAs from a wide range of substrates. The carbon sources have a high impact on the PHA production cost. Cheap raw materials are attractive to increase industrial PHAs production, which are currently produced by small and middle-sized companies. LCA indicates that PHB is environmentally more beneficial than conventional plastics such as polypropylene. Bioinformatics tools have been used for the reconstruction of metabolic pathways from bacteria. Our genome analyses have shown that B. xenovorans strain LB400 possesses the genes for the synthesis and degradation of polyhydroxyalkanoates. The pha gene cluster encoding enzymes for PHA synthesis in strain LB400 has the same gene organization as the pha genes in R. eutropha strain H16. PhaC polymerase from strain LB400 is closely related with class I type PhaC polymerase from other bacteria. B. xenovorans LB400 cultivated on sugars such as glucose and mannitol synthesizes PHAs. Xylose is a sugar released from hemicellulose, which is available in high amounts as by-products of forestry. This might enable in the future the use of xylose as a low cost substrate for large-scale production of polyhydroxyalkanoates.
Acknowledgments The authors gratefully acknowledge Gregorio Gomez and Luiziana Ferreira da Silva for their generous support. The authors acknowledge financial support from Ph.D CONICYT, Mecesup CD FSM1204 and Cyted PRIBOP fellowships (VU) and FONDECYT 1110992 (MS), USM 131109 & 131342 (MS, MG), Pie>A (PV,VU), CN&SB (MS) and Cyted PRIBOP (MS) grants. The funders had no role in study design, data collection and analyses, decision to publish, or preparation of the manuscript.
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Fig. 1. Plastic consumption in Chile in different areas. Adapted from reference 1.
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Fig. 2. Structural formulas of PHAs. General PHA structural formula (a). R derived from radical represents different substituents. PHB structural formula (b). Structural formulas of short-chainlength PHA monomers (c).
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Fig. 3. Biosynthesis of 3-hydroxybutyrate and 3-hydroxyvalerate from different substrates. 3HB is synthesized from sugars via the Entner-Doudoroff pathway and from few fatty acids. 3HV is synthesized from diverse fatty acids. Abbreviations: 3-hydroxyvalerate (3HV), 3-hydroxybutyrate (3HB), phosphate (P); Coenzyme A (CoA) and 2-keto-3-deoxy-6-phosphogluconate (KDPG).
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Fig. 4. The pha gene cluster organization in B. xenovorans strain LB400 and R. eutropha strain H16. The arrows indicate the PHA metabolic pathway genes wherein the black arrows indicate the PHA polymerase genes.
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Fig. 5. Synthesis of polyhdroxyalkanoates by B. xenovorans strain LB400. LB400 colonies grown on M9 medium with mannitol (10 g L -1) as sole carbon source and 10% N (NH4Cl 0.1 g L-1) were stained with Sudan Black B dye (a). Magnification of the image (b).
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Table 1. Production of polyhydroxyalkanoates by diverse bacteria. Carbon source
PHA
PHA content (%)
Reference
Ralstonia eutropha H16
Glucosa, fructose, acetic acid, valeric acid
PHB PHV
80 90
[14,18]
Burkholderia sp. DSMZ 9243
Sucrose or gluconate
PHB P(3HPEa)
Burkholderia cepacia ATCC 17759
Xylose:levulinic acidb
P(3HB-co3HV)
Burkholderia sacchari IPT189
Sucrose:propionic acidc
Burkholderia xenovorans LB400
Glucose
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[19,20]
49
[21,22]
P(3HB-co3HV)
30
[23,24]
PHB
ND
[25,26]
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Bacterial strain
3-hydroxy-4-pentanoic acid, bRatio (w/v in %) 2.2:(0.07 – 0.52), cRatio (w/w) 10:1, 19:1, 30:1, 61.5:1. ND: not determined.
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S0141-8130(14)00384-5 http://dx.doi.org/doi:10.1016/j.ijbiomac.2014.06.001 BIOMAC 4402
To appear in:
International Journal of Biological Macromolecules
Received date: Revised date: Accepted date:
3-3-2014 13-5-2014 3-6-2014
Please cite this article as: V. Urtuvia, P. Villegas, M. Gonz´alez, M. Seeger, Bacterial production of the biodegradable plastics polyhydroxyalkanoates, International Journal of Biological Macromolecules (2014), http://dx.doi.org/10.1016/j.ijbiomac.2014.06.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Bacterial production of the biodegradable plastics polyhydroxyalkanoates Viviana Urtuvia, Pamela Villegas, Myriam González & Michael Seeger*
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Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Departamento de Química & Center for Nanotechnology and Systems Biology & Centro de Biotecnología, Universidad Técnica Federico Santa María, Avenida España 1680, Valparaíso, Chile
*For correspondence. E-mail: [email protected]; Tel: (+56-32) 2654236; Fax: (+56-32) 2654782
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Abstract
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Petroleum-based plastics constitute a major environmental problem due to their low biodegradability and accumulation in various environments. Therefore, searching for novel biodegradable plastics is of increasing interest. Microbial polyesters known as polyhydroxyalkanoates (PHAs) are biodegradable plastics. Life Cycle Assessment indicates that PHB is more beneficial than petroleum-based plastics. In this report, bacterial production of PHAs and their industrial applications are reviewed and the synthesis of PHAs in Burkholderia xenovorans LB400 is described. PHAs are synthesized by a large number of microorganisms during unbalanced nutritional conditions. These polymers are accumulated as carbon and energy reserve in discrete granules in the bacterial cytoplasm. 3-hydroxybutyrate and 3-hydroxyvalerate are two main PHA units among 150 monomers that have been reported. B. xenovorans LB400 is a model bacterium for the degradation of polychlorobiphenyls and a wide range of aromatic compounds. A bioinformatic analysis of LB400 genome indicated the presence of pha genes encoding enzymes of pathways for PHA synthesis. This study showed that B. xenovorans LB400 synthesize PHAs under nutrient limitation. Staining with Sudan Black B indicated the production of PHAs by B. xenovorans LB400 colonies. The PHAs produced were characterized by GC-MS. Diverse substrates for the production of PHAs in strain LB400 were analyzed.
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Keywords: polyhydroxyalkanoate, biodegradable plastic, polyhydroxybutyrate, Burkholderia xenovorans, pha gene
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1. Introduction
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The highly versatile petroleum-based plastics have made them valuable materials for modern life with a wide range of applications. However, due to their low biodegradability and accumulation in various environmental compartments they are major environmental pollutants. Searching for novel biodegradable polymers that possess similar physicochemical properties as conventional plastics is of increasing interest. PHAs are biodegradable plastics synthesized by diverse bacteria from a range of substrates including sugars and fatty acids. These biopolymers are stored as intracellular carbon and energy reserve granules by Gram-negative and Gram-positive bacteria under nutrient limitation. 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) are two common and widely studied PHA monomers. However, 150 different PHA monomers have been reported. Depending on the microorganism, the carbon source and culture conditions different PHA homopolymers, copolymers and combination thereof are synthesized. A number of companies in America, Asia and Europe produce PHAs at industrial scale. In this report bacterial production of PHAs and their industrial applications are reviewed and the synthesis of PHAs in the model aromatic-degrading bacterium B. xenovorans LB400 is described.
2. Petroleum-derived plastics: applications and pollution
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The versatility and low production costs of plastics derived from petroleum have allowed them to substitute other materials such as paper, glass and wood. Plastics are important for various applications in different industrial sectors. During the year 2008, 245 million tons were consumed at global level and a 9% annual growth in the plastic consumption was forecasted. Fig. 1 illustrates that in Chile, plastic consumption is mainly concentrated in the areas of packaging, mining, construction and agroindustry [1]. The remaining 9% is distributed among household, agriculture and transport. 844,000 tons of plastic consumption in Chile for 2012 was expected, with 51 kg plastic consumption per capita, placing Chile as the second largest plastic-consuming country in Latin America [2]. The plastic consumption per capita in European countries exceeds 100 kg per year [3]. Chemical and physical properties of plastics provide them with high durability and versatility for a wide range of applications. However, their properties convert plastics in pollutants when they are disposed in the environment. The plastics present very low degradation rates in the environment, with a half-life up to >500 years. The low degradability causes, among other drawbacks, a high accumulation of plastics in sanitary landfills, decreasing the life thereof. The plastic wastes are about 8% in weight and 25% in volume of the total solid urban wastes. The low degradability combined with the inadequate waste disposal and low environmental education have caused that plastics are accumulated in different environmental compartments. An example is the presence of plastic fragments in almost all oceans, which causes plastic ingestion by marine organisms with largely unknown consequences [4]. The implementation of environmental management politics to reduce the plastic wastes is crucial for the conservation of natural resources and the reduction of environmental pollution. The research and development of new biodegradable materials is of increasing importance. In recent decades plastic manufacturing using renewable feedstock has been promoted and the productions of biologically based plastics known as biopolymers, which could be biodegradable, are of increasing interest [5].
3. Biopolymers and polyhydroxyalkanoates
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The term biopolymer includes polymers of natural origin, but also high molecular mass molecules that have been polymerized by chemical and biological methods. Not all biopolymers are biodegradable [5]. The biodegradable plastics primarily are produced from renewable raw materials by aerobic synthesis and from waste management by composting and anaerobic digestion [6]. The biopolymers are classified according to their biodegradability or composition. PHAs and polylactic acid (PLA) are well-studied biopolymers due to their biodegradability and physicochemical properties [5,7]. PHAs are thermoplastic polyesters of R-hydroxyalkanoic acids. PHAs are synthesized and stored as intracellular carbon and energy reserve in Gram-negative and Gram-positive bacteria under nutrient limitation of nitrogen, phosphorus, oxygen or after pH shifts [3,8,9]. PHAs accumulate in cytoplasmic granules that typically have a diameter from 0.2 to 0.5 µm. These granules can be visualized by phase contrast microscopy due to their high refractivity or using staining dyes such as Sudan Black B and Nile red. Once the limiting nutrient is provided to the cell, these energy storage compounds are degraded and used as carbon source for bacterial growth [10]. Polyhydroxybutyrate (PHB) was discovered in 1926 by Maurice Lemoige in the bacterium Bacillus megaterium, which showed intracellular granules [11]. PHB is the most common and the best studied member of PHAs [8]. The presence of 3HB as a PHA monomer was reported in activated sludge [12]. The presence of 3HV as a major constituent and 3-hydroxyhexanoate (3HHx) as a minor constituent were described [13]. The oil crisis of the early 1970s prompted the search for new materials to replace petroleum-based plastics [9]. Fifty years after its discovery, PHB production was carried out at commercial scale. PHAs could have different composition depending on the microorganism, the carbon source and culture conditions (Table 1). The most common polymers are the short-chain-length PHA (PHASCL), which contain from 3 to 5 carbons atoms. The structural formulas of PHASCL are depicted in Fig. 2. These biopolymers are stiff, brittle and possess a high degree of crystallinity. Middle-chain-length PHA (PHAMCL) contain from 6 to 14 carbons atoms and are flexible and possess low crystallinity, tensile strength and high melting point [9,10,14,15]. Currently 150 different monomer constituents of PHAs that could be homopolymers, copolymers and combination thereof have been reported [10,16,17]. PHB is produced by bacteria from a wide range of substrates (Fig. 3). In contrast, specific substrates such as propionic acid, valeric acid and other organic acids are required for the synthesis of the PHV monomer [17,24]. The metabolic pathways for the synthesis of both monomers have been reported in the model PHA-producing bacterium R. eutropha strain H16, which synthesizes PHASCL [17,27–29]. PHAs could reach up to 80% of the bacterial cell dry biomass. Life Cycle Assessment (LCA) indicates that the energy requirements for PHB production are lower than the energy requirements for conventional high density plastics polyethylene and polypropylene [30]. In addition, the production of PHB is more beneficial for ozone layer protection, and reduces toxicity levels, abiotic depletion and global warming. However, PHB showed higher impacts than polyethylene on acidification and eutrophication. On a cradle-tofactory gate LCA, PHB is more beneficial than conventional polymers such as polypropylene [30]. 4. Substrates for bacterial PHA production and PHA production cost PHAs are produced by bacteria from a wide range of substrates such as renewable sources (e.g., sucrose, starch, cellulose, triglycerides, hemicellulose, wheat), sub-products (e.g., molasses, whey, glycerol, corn steep liquor, rice bran), organic acids (e.g., propionic acid, 4-hydroxybutyric acid), fossil resources (e.g., methane, mineral oil, lignite, hard coal) and wastes (e.g., wastewater, palm oil mill and activated sludge effluents) [15,24,27,31–33]. Substrates such as glucose and sucrose have been used for industrial PHA production. The raw materials showed a high impact on the PHA production cost. Therefore, cheap raw materials are attractive for industrial PHA production [8]. Xylose could be an interesting alternative substrate for
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PHA production (Fig 3). Xylose is the main sugar component of hemicellulose in Gramineae plants and hardwood. Hemicellulose is the third most abundant polymer in nature that is converted into fermentable sugars by chemical or enzymatic hydrolysis [31]. Hemicellulose hydrolysis releases xylose, mannose, galactose, arabinose and glucose. PHB production from xylose by Pseudomonas pseudoflava ATCC 33668, Burkholderia cepacia ATCC 17759 and recombinant Escherichia coli strains TG1(pSYL105) and W3110(pSYL107) has been reported [34–37]. The forestry, which produces high amounts of hemicellulose-containing sub-products, is an important activity in diverse countries (e.g., in Canada, Chile, Brazil and USA). Therefore, PHA production from sub-products containing xylose may be an attractive alternative. One of the most significant barriers for an increased industrial PHAs production is its high cost. The product price depends up to 50% on the raw material cost, mainly the carbon source. The biopolymer recovery process also contributes to higher PHA production cost [10,15, 35]. PHAs are still produced on a small-scale production volume from 1000 to 20,000 tons per year, which also increases the cost. In contrast, polyethylene is produced in quantities exceeding 300,000 tons per year, with an important benefit in the final price due to the economy of scale [7,38]. PHA price varies from 1.5 to 5 € per kg, whereas polypropylene price varies from 0.2 to 0.4 € per kg [7,10]. However, the versatility and biocompatibility of PHAs have made them interesting candidates for low volume products of high value, especially for biomedical applications [39]. For example, PHA nanoparticles for drug delivery and biocompatible porous implants made from poly-4hydroxybutyrate has been reported [8,39]. Various small and middle-sized enterprises around the world produce PHAs at small-scale. Forexample, P&G (USA), Biomer Inc. (Germany), Tianan Biologic (China) and PHB Industrial (Brazil) [7]. Bags, pencils, plates, glasses and various plastic parts are manufactured from these polymers. PHA resins are used for the manufacture of sustained release drugs, bone implants and sutures [39]. In addition, PHAs can be used as latex (for example, for paper-coating applications) or in foods [40]. However, to enhance the PHA production, the production cost should be decreased. Therefore, novel approaches for PHA production have to be established [5].
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5. PHA synthesis by Burkholderia xenovorans strain LB400
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The genus Burkholderia possesses high metabolic versatility and is adapted to different ecological niches including soil and aqueous environments. Burkholderia xenovorans LB400 is a model aerobic bacterium for the degradation of polychlorobiphenyls (PCBs) and a wide range of aromatic compounds. PCBs have been used as complex mixtures for diverse industrial applications. These compounds are toxic and tend to accumulate as persistent organic pollutants in soil and water sediment [41,42]. The large genome from B. xenovorans strain LB400 (9.73 Mbp) has been sequenced and characterized [43]. Strain LB400 possesses genes encoding enzymes of eleven central and more than twenty peripheral pathways for the degradation of aromatic compounds. For example, the hmgABC gene cluster encoding the homogentisate central pathway and the hpaG1G2EDFHI gene cluster encoding the homoprotocatechuate central pathway are located at chromosome 1 (C1) and chromosome 2 (C2), respectively. Additional hmg gene copies were identified within the LB400 genome [43,44]. The functionality of diverse catabolic pathways such as biphenyl, homogentisate, homoprotocatechuate, gentisate, protocatechuate and 2-aminophenol pathways has been reported [42,44–46]. B. xenovorans LB400 is able to grow on 3-hydroxyphenylacetate (3-HPA) and 4hydroxyphenylacetate (4-HPA) isomers as sole carbon and energy sources, indicating active peripheral and central catabolic pathways. The homogentisate and homoprotocatechuate central pathways are involved in 3-HPA and 4-HPA degradation in strain LB400. Both ring-cleavage pathways are used in aromatic amino acid metabolism in Bacteria and Eucarya. A number of bioinformatic tools are useful to reconstruct metabolic pathways in bacteria. The genes of the PHA anabolic pathway from the model PHA-producing bacterium Ralstonia eutropha
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strain H16 have been characterized. The genes for the synthesis and degradation of PHA were found in B. xenovorans strain LB400. Strain LB400 has two PHA anabolic pathways: i) the pathway that uses sugars as carbon sources and, ii) the pathway associated to fatty acids catabolic pathway (β-oxidation). In B. xenovorans strain LB400, the pha genes for the synthesis of PHAs are arranged in a gene cluster at the major chromosome [47]. The pha gene clusters from R. eutropha H16 and B. xenovorans LB400 have a similar organization, which are shown in Fig. 4. B. xenovorans strain LB400 has three copies of the phaC gene distributed in the major chromosome, minor chromosome and the megaplasmid. The PHA polymerase PhaC from strain H16 possesses fifteen functionally key amino acids [48,49].These amino acids are conserved in PhaC polymerase from strain LB400 [47]. Different compounds including sugars and alkanoates are substrates for the bacterial production of PHAs. B. xenovorans LB400 is able to grow on glucose, mannitol and xylose as sole carbon source. In contrast, strain LB400 was not able to grow on lactose and sucrose. To study the PHA synthesis by strain LB400 in a first approach staining of bacterial colonies with Sudan Black B dye was analysed [10,50]. LB400 colonies grown on mannitol (10 g L-1) were stained with Sudan Black B, indicating the intracellular accumulation of PHAs (Fig. 5). The synthesis of PHAs by strain LB400 was studied during growth on various sugars and fatty acids. PHAs were identified by gas chromatography and mass spectrometry. During growth on glucose the synthesis of the homopolymer PHB was observed. Searching for low cost renewable materials will be useful to increase the production of these biopolymers.
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6. Conclusions and future prospects
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The global trends of plastic consumption promote the production of novel biodegradable materials, replacing traditional plastics and increasing environmental protection and sustainable development. PHAs are attractive biopolymers due to their biodegradability and physicochemical properties. PHAs are synthesized and stored as intracellular carbon and energy reserve by bacteria under nutrient limitation. Diverse bacteria are able to synthesize PHAs from a wide range of substrates. The carbon sources have a high impact on the PHA production cost. Cheap raw materials are attractive to increase industrial PHAs production, which are currently produced by small and middle-sized companies. LCA indicates that PHB is environmentally more beneficial than conventional plastics such as polypropylene. Bioinformatics tools have been used for the reconstruction of metabolic pathways from bacteria. Our genome analyses have shown that B. xenovorans strain LB400 possesses the genes for the synthesis and degradation of polyhydroxyalkanoates. The pha gene cluster encoding enzymes for PHA synthesis in strain LB400 has the same gene organization as the pha genes in R. eutropha strain H16. PhaC polymerase from strain LB400 is closely related with class I type PhaC polymerase from other bacteria. B. xenovorans LB400 cultivated on sugars such as glucose and mannitol synthesizes PHAs. Xylose is a sugar released from hemicellulose, which is available in high amounts as by-products of forestry. This might enable in the future the use of xylose as a low cost substrate for large-scale production of polyhydroxyalkanoates.
Acknowledgments The authors gratefully acknowledge Gregorio Gomez and Luiziana Ferreira da Silva for their generous support. The authors acknowledge financial support from Ph.D CONICYT, Mecesup CD FSM1204 and Cyted PRIBOP fellowships (VU) and FONDECYT 1110992 (MS), USM 131109 & 131342 (MS, MG), Pie>A (PV,VU), CN&SB (MS) and Cyted PRIBOP (MS) grants. The funders had no role in study design, data collection and analyses, decision to publish, or preparation of the manuscript.
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Fig. 1. Plastic consumption in Chile in different areas. Adapted from reference 1.
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Fig. 2. Structural formulas of PHAs. General PHA structural formula (a). R derived from radical represents different substituents. PHB structural formula (b). Structural formulas of short-chainlength PHA monomers (c).
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Fig. 3. Biosynthesis of 3-hydroxybutyrate and 3-hydroxyvalerate from different substrates. 3HB is synthesized from sugars via the Entner-Doudoroff pathway and from few fatty acids. 3HV is synthesized from diverse fatty acids. Abbreviations: 3-hydroxyvalerate (3HV), 3-hydroxybutyrate (3HB), phosphate (P); Coenzyme A (CoA) and 2-keto-3-deoxy-6-phosphogluconate (KDPG).
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Fig. 4. The pha gene cluster organization in B. xenovorans strain LB400 and R. eutropha strain H16. The arrows indicate the PHA metabolic pathway genes wherein the black arrows indicate the PHA polymerase genes.
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Fig. 5. Synthesis of polyhdroxyalkanoates by B. xenovorans strain LB400. LB400 colonies grown on M9 medium with mannitol (10 g L -1) as sole carbon source and 10% N (NH4Cl 0.1 g L-1) were stained with Sudan Black B dye (a). Magnification of the image (b).
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Table 1. Production of polyhydroxyalkanoates by diverse bacteria. Carbon source
PHA
PHA content (%)
Reference
Ralstonia eutropha H16
Glucosa, fructose, acetic acid, valeric acid
PHB PHV
80 90
[14,18]
Burkholderia sp. DSMZ 9243
Sucrose or gluconate
PHB P(3HPEa)
Burkholderia cepacia ATCC 17759
Xylose:levulinic acidb
P(3HB-co3HV)
Burkholderia sacchari IPT189
Sucrose:propionic acidc
Burkholderia xenovorans LB400
Glucose
us
an
45
[19,20]
49
[21,22]
P(3HB-co3HV)
30
[23,24]
PHB
ND
[25,26]
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Bacterial strain
3-hydroxy-4-pentanoic acid, bRatio (w/v in %) 2.2:(0.07 – 0.52), cRatio (w/w) 10:1, 19:1, 30:1, 61.5:1. ND: not determined.
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a
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