Journal of Bioscience and Bioengineering VOL. 110 No. 6, 621 632, 2010 www.elsevier.

com/locate/jbiosc

REVIEW

Current trends in biodegradable polyhydroxyalkanoates

Suchada Chanprateep
Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand Received 5 April 2010; accepted 26 July 2010 Available online 17 August 2010

The microbial polyesters known as polyhydroxyalkanoates (PHAs) positively impact global climate change scenarios by reducing the amount of non-degradable plastic used. A wide variety of different monomer compositions of PHAs has been described, as well as their future prospects for applications where high biodegradability or biocompatibility is required. PHAs can be produced from renewable raw materials and are degraded naturally by microorganisms that enable carbon dioxide and organic compound recycling in the ecosystem, providing a buffer to climate change. This review summarizes recent research on PHAs and addresses the opportunities as well as challenges for their place in the global market. 2010, The Society for Biotechnology, Japan. All rights reserved.
[Key words: Polyhydroxyalkanoates (PHAs); Microbial polyester; Biodegradable polymer]

THE NECESSITY OF BIODEGRADABLE POLYMER AS A SUSTAINABLE STRATEGY FOR PROTECTING AGAINST CLIMATE CHANGE Materials made from synthetic polymers are not biodegradable and are often improperly discarded. These materials are typically derived from petroleum-based plastics. Rapid progress in materials science technology has created new plastic products with favorable mechanical integrity and excellent durability. Nevertheless, plastic products usually have single-use applications, especially in food packaging and medical materials. Because these plastic products are not biodegradable, they are extremely persistent and accumulate in the ecosystem, resulting in a significant burden on solid waste management. The total global capacity of commodity plastic production dramatically increased from 1.5 million tons in 1950 to 245 million tons in 2008, an annual growth rate of 9%. The biggest potential growth area is in the rapidly developing Asian countries (excluding Japan), where current plastics consumption per capita is only around 20 kg (The compelling facts about plastics, The Association of Plastic Europe, 2009). It is very difficult to reduce the consumption of plastic products due to their versatile properties, but it is possible to replace petroleum-based plastics with alternative materials that have polymer-like properties and that degrade after being discarded. Among the various types of biodegradable plastics, polyhydroxyalkanoates (PHAs) are among the most well known, being recognized as completely biosynthetic and biodegradable with zero toxic waste, and completely recyclable into organic waste. They are microbial polyesters produced by a wide range of microorganisms, mostly as intracellular storage compounds for energy and carbon (1,2). PHAs are among the most fascinating and largest groups of biopolyesters, with over 150
Tel.: + 66 2 218 5070; fax: + 66 2 252 7576. E-mail address: suchada.cha@chula.ac.th.

different types of monomer composition that provide different properties and functionalities (36). Their properties span a wide range, including materials that imitate thermoplastic properties and others that possess electrometric properties. PHAs are efficiently degraded in the environment because many microorganisms in soils are able to secrete polyhydroxybutyrate (PHB) depolymerases, enzymes that hydrolyze the ester bonds of a polymer into water-soluble monomers and oligomers. Microorganisms then metabolize these degradation products into water and carbon dioxide (79). There are several intensive academic studies of PHA production and applications, mostly based on seeking inexpensive carbon sources to reduce the cost of production (1017) and applying genetic engineering to improve productivity (1826). This knowledge is now at a bottleneck, being largely unused to bring products to the market. Only a few PHAs, mainly polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH), have proceeded to the production stage in large quantities (27). This review aims to summarize the recent trends in biodegradable polymers, based on the current situation of PHA-based plastic products available on the market. The review also addresses global policy that may be useful for turning academic research towards the current demand for biodegradable plastics. GLOBAL MARKETS AND POLICIES Environmentally degradable polymers (biopolymers) are one of the possible solutions to replace some petroleum-based polymers. Based on the definition given by the European Bioplastics Association, biopolymers are based on renewable resources complying with ASTM D-6866 and can be degraded to comply with international standards such as EN13432, ASTM D6400, and ISO17088. In Europe, the criteria for biodegradability are set out within the standard EN13432, 2000, which is binding when applied to compostable packing under the EU directive on Packaging and

1389-1723/$ - see front matter 2010, The Society for Biotechnology, Japan. All rights reserved. doi:10.1016/j.jbiosc.2010.07.014

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J. BIOSCI. BIOENG., without specific supporting documentation or measures. Since 2006 in France, a regulation for the promotion of French agriculture requires disposable retail bags to be biodegradable by 2010 (Political strategies and statutory regulation, The European Bioplastics Association, 2010). In the U.S., the San Francisco Board of Supervisors approved the first-in-the nation legislation in 2007 that outlawed the use of nonbiodegradable plastic bags in large supermarkets within 6 months and large chain pharmacies in about a year. Minnesota also bolstered its ban on the disposal of yard trimmings in landfills recently when the governor signed a bill forbidding the use of conventional plastic bags for collecting yard trimmings in the Twin Cities area. This bill also requires compostable bags to meet ASTM D6400 or be certified by an independent organization. The law took effect on January 10, 2010 (Plastic Bags Legislation, The Council of State Governments/Eastern Regional Conference, 2010). Among Asian countries, Japan is the biotechnology leader in biodegradable plastic. After ratification of the Kyoto Protocol in 2002, the government announced two state measures, Biotechnology Strategic Scheme and Biomass Nippon Strategy, to mitigate global warming by promoting the use of renewable resources and applying rapid advances in biotechnology (29). The Japan Bioplastic Association (JBPA) classified bioplastics into two groups: (i) biodegradable plastics (GreePla) with biodegradable characteristics and biodegradable synthetic high-polymer materials with an average molecular weight of at least 1000 Da, including chemically modified starch- and polyamino acid-based biodegradable high-polymer materials, and (ii) biomass-based plastics (BiomassPla) produced with either chemically or biologically synthesized materials containing renewable organic materials (excluding natural organic polymers that are not chemically modified). The JBPA has estimated that demand for biomass-based plastics will reach 20% of total plastic consumption in 2020. Thus, the JBPA started the Identification and Labeling System in 2000 and has certified about 900 biodegradable plastic products in Japan. The system is based on a positive list system for all components, biodegradability specifications based in Japanese Industrial Standards, safety certification of all components, and proof of no hazardous effects to soil (http:// www.jbpaweb.net/english/english.htm). Standards for the biodegradability of plastics in the ISO have been proposed under TC61/SC5/WG22 (30). Table 2 shows the global policies that have been enacted stating a requirement for biodegradable/compostable plastics. CHALLENGES AND OPPORTUNITIES OF POLYHYDROXYALKANOATES Two of the most promising biopolymers are PLA and PHA. PLA has received much attention as a potential alternative to existing materials while PHA is challenging and price-competitive with petroleum-based polymers. In the long term, bioplastics have significant potential for cost reduction. The price of petroleum-based plastics could increase by 50

Packaging Waste (94/62/EC). In North America, similar criteria for defining compostability have been established by the Institute for Standards Research under ASTM D6400-99 Specification for Compostable Plastics. Synthetic components, such as additives, colorants, or glues, can be added to aid performance. Biopolymers can be classified into three groups based on their biodegradable characteristics. The first group consists of biopolymers that are not bio-based plastics but have biodegradable or compostable properties. This group includes synthetic biodegradable aliphaticaromatic copolyesters, such as polybutylene terephthalate adipate (PBTA), polybutylene terephthalate succinate (PBTS), and polybutylene terephthalate glutarate (PBTG). The second group includes bio-based polymers that are biodegradable or compostable, for example (i) polylactide (PLA) (Ingeo by NatureWorks LLC, USA), (ii) starch-based materials (Mater-Bi by Novamont S.p.A., Italy), (iii) cellulose-based materials (NatureFlex by Innovia Films Ltd., UK), (iv) PLA compounds and blends (Ecoflex and Ecovio by BASF, The Chemical Company, Germany), and (v) PHAs (Mirel by Telles, USA). The third group is composed of bio-based resources that are manufactured into non-biodegradable polymers. This group includes bio-based 1,3 propanediol (PDO) from corn sugar (Bio-PDO by DuPont Tate & Lyle Bio Products, LLC, USA), and bioethanol-based linear low-density polyethylene (LLDPE) (Dowlex by Dow Chemical Company, USA) (27). Alternatively, biopolymers can also be classified into four groups based on their components. The first group is produced directly by living organisms (e.g., cotton, silk, wool, other natural fibers, cellulose, starch, lignin, oil proteins, natural rubber, and PHAs). The second group is produced by polymerization of monomers that either exist in nature or are derived from materials that exist in nature (e.g., PLA, polytrimethylene glycol, soy-based polyols, and their derivatives). The third group contains combinations of monomers from renewable resources with petrochemical-derived monomers (e.g., isosorbatecontaining polycarbonates and soy-based urethanes). The last group includes polymers produced from blends of renewable resources and petroleum-based materials, such as blends of starch and polyvinyl alcohol (Eco Flo by Green Light Products Ltd., UK) (28). Table 1 summarizes the commercialized PHAs with their trade names and manufacturing companies. Members of the European Union have already established policies against petroleum-based consumer packaging. For example, Belgium has established an eco-tax of 3 kg1 on packaging such as shopping bags, whereas compostable shopping bags that conform to the European Standard (EN) 13432 for compostable packaging material will be exempted. The Netherlands has established a carbon-based packaging tax based on CO2 emissions from the production of packaging material and the embedded carbon content of the packaging. France, Italy, and Spain are considering similar legislation. So far, two European countries have established legislative measures for bioplastics and their derivatives. Since June 2005, an interim regulation under the German Packaging Directive exempts compostable packaging, enabling recycling

TABLE 1. The current and potential large volume manufacturers of polyhydroxyalkanoates (33). Polymer PHB PHB PHB PHBV and PHB PHBV, PHBV + Ecoflex blend PHBH PHBH PHBH P(3HB-co-4HB) Polyhydroxyalkanoate from P&G Trade names Biogreen Mirel Biocycle Biomer Enmat Nodax Nodax Kaneka PHBH Green Bio Meredian Manufacturers Mitsubishi Gas Chemical Company Inc. (Japan) Telles (US) PHB Industrial Company (Brazil) Biomer Inc. (Germany) Tianan Biologic, Ningbo (China) P&G (US) Lianyi Biotech (China) Kaneka Corporation (Japan) Tianjin Gree Bio-Science Co/DSM Meredian (US) Capacity (tons) 10,000 50,000 50 50 10,000 20,00050,000 2000 1000 10,000 272,000 (2013) Price (kg 1) (in 2010) 2.53.0 1.50 a n/a 3.05.0 3.26 2.50 3.70 n/a b n/a n/a

a Bacteria to produce bioplastics, BIOPRO Baden-Wrttemberg GmbH, September 24, 2009 available at http://www.bio-pro.de/magazin/thema/04308/index.html? lang=en&artikelid=/artikel/04310/index.html, accessed on March 2010. b Full-scale development of the world's first completely bio-based polymer with soft and heat resistant properties, Kaneka Corporation, March 10, 2009 available at http://www.kaneka.co.jp/kaneka-e/news/pdf/090310.pdf, accessed on March 2010. n/a means price is not able to be found.

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TABLE 2. The global policies and measures.

623

Countries

National policies and measures

German Packaging Directive has been in force (2005). The compostable packaging will be exempt from the requirements in 6 of the Directive Germany a France a A law for the promotion of French agriculture has been in force (2006) stating a requirement for biodegradability of disposable retail carry bags by 2010 Markets in Florence had been charging 0.100.20 per plastic bag (2009) Italyb,c Ireland, Scotland, Denmark These countries have already imposed levies and taxes on non-degradable plastic bags c and Sweden In 2003, county Durham has been charging Ecotax per plastic bag UKc,e d,e US San Francisco: in March 2007, the San Francisco Board of Supervisors approved first-in-the nation legislation that outlaws the use of non-biodegradable plastic bags in large supermarkets within 6 months and large chain pharmacies in about a year Canada d Toronto City Council: retailers will be required to charge a minimum of 5 cents for each plastic retail shopping bag that customers take (2008) Japan f Law on Promoting Green Purchasing and Law on Recycling have been in force in 2001 Plastic is officially banned in Ladakh India d d Australia Thin non-biodegradable plastic shopping bags have been prohibited in South Australia from 4 May 2009 d Bangladesh From the beginning of January 2002, the Bangladesh government is banning the use of plastic bags in Dhaka The Association European Bioplastics, European Bioplastics e.V., Berlin, available at http://www.european-bioplastics.org/index.php?id = 307, accessed on March 2010. Banning plastic bags, available at http://www.treehugger.com/files/2009/02/5-great-green-ways-us-cities-leading-by-example.php, accessed on March 2010. c Plastic carrier bag taxes in Europe, 13.05.04.09, Mepex Consult AS, Norway, available at The California Product Stewardship Council (http://www.calpsc.org/). d Seattle bag tax and Styrofoam ban, The Northwest economic policy seminar, available at http://www.seattlebagtax.org/california.html, accessed on March 2010. e Sand Francisco First city to ban plastic shopping bags, San Francisco Chronicle, Hearst newspapers, Hearst Communications Inc., available at http://www.sfgate.com/cgi-bin/ article.cgi?file=/c/a/2007/03/28/MNGDROT5QN1.DTL, accessed on March 2010. f Green purchasing and green procurement in motion, Japan for sustainability newsletter #004, December 2002, Japan for Sustainability (JFS), available at http://www.japanfs.org/ en_/newsletter/200212-2.html, accessed on March 2010.
b a

80% in 2012, based on the increasing price of oil (The Plastics Exchange LLC, 2010). Most manufacturers of biodegradable plastics are smallscale facilities (about 100020,000 tons per year of production), and they lack the economy of scale of a single polyethylene unit with a capacity of 300,000 tons per year of production. The world's largest lactic acid manufacturing facility belongs to NatureWorks LLC in Blair, Nebraska, U.S.A. with a capacity of 140,000 tons. In 2003, NatureWorks LLC started to produce Ingeo PLA resin. Afterwards, PLA became the first bio-based plastic produced on a large scale. Among PHAmanufacturing companies, the main company with a large production is the U.S. biotech company Metabolix, Inc. in Cambridge, Massachusetts. In 2010, Telles, a joint venture company formed by the Archer Daniels Midland Company (ADM) and Metabolix, Inc. opened the first commercial-scale plant to produce a corn syrup-based PHA resin, Mirel, in Clinton, Iowa, U.S.A. It is expected to begin shipping PHA resin in April of 2010 and would produce 50,000 tons per year at full capacity (Plastics Q6 News, 10 March 2010). The history of commercialized PHAs goes back to 1959. W. R. Grace and Company produced PHB in the U.S. for possible commercial applications (Baptist, J.N., (Assignor to W.R. Grace & Co., New York) US Patent No. 3225766, 1965). However, the company shut down the process due to low production efficiency and a lack of suitable purification methods. In 1970, PHBV was commercialized by Imperial Chemical Industries Ltd. (ICI/Zeneca BioProducts, Bellingham, UK) under the trade name of Biopol (31). In 1996, the technology was sold to Monsanto and then to Metabolix, Inc. The U.S. Department of Commerce Advanced Technology Program funded a project to reengineer the central metabolism of Escherichia coli for more efficient conversion of renewable sugars into PHB. In 2008, Metabolix, Inc. announced the combined production of PHA Bio-based Polymers and Biomass Energy with a target to obtain PHA from switchgrass at a level of 20% of dry-cell weight, 75% of which could be recovered. Thus, if switchgrass yields are 10 to 15 tons per acre, then each acre will yield 1.5 to 2.25 tons of PHA bio-based polymers or derived chemicals, and 1 million acres will yield 3.3 to 5 billion pounds of PHAs (32). Procter and Gamble, in partnership with Kaneka Corporation, Tsinga University in China, and the Riken Institute in Japan, has developed a wide range of applications for PHB and PHBH (Nodax) as fibers, nonwoven materials, aqueous dispersions, and disposable products. However, Nodax technology was sold in 1993 (33). Recently, Kaneka Corporation has announced its plan to launch the production of a plantderived soft polymer called Kaneka PHBH in 2010, with a production capacity of 1000 tons per year at Takasago City, Hyogo, Japan (http:// www.kaneka.co.jp/kaneka-e/news/pdf/090310.pdf). A German compa-

ny, Biomer Inc. (Kraaling, Germany) produces PHB on a commercial scale for special applications. In 1993, Biomer acquired expertise and microbes for PHB products from the Austrian company Petrochemia Danubia and registered the trade name Biomer in 1995 (34). In Brazil, one of the largest sugar-exporting countries, PHB Industrial S.A. (Serrana) uses sugar cane to manufacture PHB (Biocycle) in a joint venture started in 1992 between a sugar producer (Irmaoes Biagi) and an alcohol producer (the Balbo Group). The company has been running a pilot plant at 50 tons per year and plans to increase production capacity to 3000 tons per year (35). In Canada, Biomatera Inc. specializes in the manufacture of PHA by fermentation of agricultural residues. The biopolymers are used in the manufacture of creams and gels that are used as slow-release agents in drug manufacturing and as cosmetic agents and tissue matrix regeneration (36). In Japan, Mitsubishi Gas Chemical has made progress on the production of PHB from methanol fermentation (BioGreen) (33). Table 3 presents physical, mechanical and thermal properties of commercialized PHAs, and Table 4 shows the potential of commercialized PHA to replace some petroleum-based plastics (37). COST COMPETITIVENESS In 2006, the cost of PHB was in the range of 10 per 12 kg1. This price was much higher than that of starch polymers and other bio-based polyesters due to high raw material costs, small production volumes, and high processing costs, particularly for purification (33). It was concluded that raw material accounts for 3040% of the total cost of PHB (38). The latest market price of Mirel is quoted at about 1.50 kg1 (Plastics News, 10 March 2010). This product combines biodegradability with premium pricing relative to most petroleum-based plastics. PHAs will soon enter the market and become potential candidates for commodity materials. In 2010, the market potential of the total bioplastics market in the EU will reach 200,000500,000 tons. The main markets are short term application for packaging and agriculture. By 2020, the bioplastics market in the EU is forecast to increase to 2 5 million tons and to expand to the textile, automotive, and agricultural sectors, including many durable applications (BioplasticsMarket Trends andU.S.&E.U.Outlook, FUJ00024, p. 132, Fuji-Keizai,October 2007). The summary of market prices for PHAs in 2010 is given in Table 1. SUSTAINABILITY AND ENVIRONMENTAL IMPACT OF PHAS BASED ON LIFE CYCLE ASSESSMENT The sustainability of fossil resource depletion and its environmental impacts (such as greenhouse emissions) are important subjects of

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TABLE 3. The physical, mechanical and thermal properties of some commercialized PHAs (37). PHB Biomer240 Biomer P226 PHB copolymers MirelP1001 MirelP1002 Injection mold PHBV Biocycle100 Biocycle24005 Extrusion and injection 1012 1.22 5060 1525 1.20

J. BIOSCI. BIOENG.,

PHBH Kaneka Foam mold 510 1.2

Application grade Physical properties Melt flow rate (g/10 min) Density (g/cm3) Crystallinity (%) Mechanical properties Tensile strength (MPa) Elongation (%) Flexural strength (MPa) Flexural modulus (GPa) Thermal properties Melting temperature (C) VICAT softening point (C) 57 1.17 6070

Injection mold 913 1.25 6070

1.39

1.30

1820 1017 17

2427 69 35

28 6 46 3.2

26 13 35 1.9

3040 2.56

2530 2030

1020 10100 0.81.8

170175 53 96 148 137 120125

ongoing scientific and political argument. An attempt to develop biotechnological processes to produce chemical commodities from renewable agricultural resources is one advocated approach that has recently gained much attention. PHAs are currently included in these considerations. Thus, it is necessary to ascertain whether the manufacturing processes preserve non-renewable resources and benefit the environment as intended. Life Cycle Assessment (LCA) is a wellestablished approach for identifying best practices within the complexity of choices confronting society and industry. The LCA is an objective procedure for evaluating the environmental impacts associated with a product through every step of its life cycle starting from raw materials, making it in a factory, selling it in a store, using it in the workplace or at home, and disposing of it or recycling it into a new product (39,40). The LCA methodology has been standardized under the ISO-14040 series, and it distinguishes four phases: goal and scope definition, inventory analysis, impact assessment, and interpretation (41). LCA evaluations have been investigated, for example, in a notorious argument between paper and plastic (42,43). It was found that although LCA usually leads to a definite conclusion, its results can easily be reversed under different environmental impacts, different inventory parameters, and/or different boundaries of the study. For example, one may look at energy, solid waste, or water. The number of published LCAs for PHAs is limited, and only a few environmental analyses have been undertaken. Thus, this article offers examples of LCAs that use different parameters to compare between PHAs and synthetic plastics (or other bioplastics) with respect to specific environmental impacts. In earlier studies, most of the published LCAs of PHAs focused on the consumption of fuel and non-renewable energy. In 1998, Heyde (44) reported that the energy requirements for PHB production can exceed the energy requirements for conventional plastics of high density polyethylene (HDPE) and polystyrene (PS) (44). Likewise, Gerngross (45) conducted a cradle-to-grave analysis of theoretical large-scale PHA production from corn-based glucose versus a conventional PS process. It was concluded that the replacement of conventional polymers with fermentation-derived PHAs did not appear to be a useful approach if the sustainable production of polymers is the

TABLE 4. The potential of commercialized PHA to replace the petroleum-based plastics (33). LPDE Mirel Biomer Nodax Biocycle ++ + PP + ++ ++ ++ HPDE ++ ++ ++ ++ PS + + HI-PS PVC + + PET ++ + PA PBT

(++) Means probable; (+) means possible; () means doubtful; () means unlikely.

desired outcome. The biodegradability and biocompatibility benefits of PHAs could justify the use of fossil resources; however, those benefits have to be quantified and evaluated separately. A high level of energy consumption from cradle-to-factory gate is an environmental burden associated with corn cultivation (45). This assessment has led to several LCA studies that adopted similar inventory analyses and environmental impact assessments. These studies noted the high level of energy consumption and negative environmental impacts from plant cultivation (4648). Kim and Dale (49) suggested that some approaches to reducing the environmental burdens of agricultural processes are necessary to achieve better profiles for photochemical smog, acidification, and eutrophication associated with plant cultivation (49). In 2008, Kim and Dale (50) conducted an LCA evaluation using information from Telles on site-specific processes such as corn wet milling, PHB fermentation, and recovery. In this specific case, most of the energy used in corn wet milling, PHB fermentation, and recovery processes was generated in a cogeneration power plant in which corn stover is burned to generate electricity and steam. In this case, PHB derived from corn grain offers environmental advantages over petroleum-derived polymers in terms of non-renewable energy consumption and greenhouse gas emissions. Furthermore, PHB provides greenhouse gas credits, and thus PHB use reduces greenhouse gas emissions compared to petroleum-derived polymers (50). Pietrini et al. (51) pointed out a different challenge of cradle-tograve environmental LCA comparisons of PHB-based composites with petrochemical plastics. In this study, the end products were cathode ray tube monitor housings produced from high-impact polystyrene and the internal panels of an average car produced from glass-fiber-filled polypropylene. The environmental impact was evaluated for nonrenewable energy use and global warming potential over a 100-year time horizon. On a cradle-to-factory gate basis, all PHB composites appeared to be environmentally superior to conventional polymers for both chosen applications. When the analysis was extended to cradle-tograve (including the use phase, post-consumer waste incineration, and energy recovery), PHB composites scored better only for the CRT monitor housing. In the case of the automotive application, the weight of the functional unit becomes overriding, and no substantial savings are observed regarding environmental impact (51). Mixed bacterial culture PHA production has the potential to produce large amounts of PHAs at low cost due to lower sterility, equipment and control requirements, and the ability to utilize a wide range of cheap substrates (including industrial and agriculture wastes) (5254). However, Rhu et al. (53) reported that the production cost of PHA from fermented food waste would be about US $13 per kg, meaning that the production costs would exceed the existing disposal costs, making PHA production an unattractive option (53). Gurieff and Lant (55)

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conducted a comparative LCA and a financial analysis of mixed-culture PHA production. The analysis was performed for industrial wastewater treatment technology, and the conditions used for mixed-culture PHA were compared with biogas production that utilizes the same waste resource. It was concluded that mixed-culture PHA production was preferable to biogas production for treating the specified industrial effluent, and it was financially attractive in comparison to pure-culture PHA production. In addition, the mixed-culture and pure-culture PHA production processes had similar environmental impacts that were significantly lower than HDPE production. In 2009, Zhong et al. (56) investigated the environmental impacts of three manufacturing processes for PHA production. The processes studied were microbial PHA manufacturing using (a) glucose as a carbon source, (b) cheese whey as a carbon source, and (c) PHA production using genetically engineered plants. These processes were compared with respect to ten indicators under three categories (i) human health (carcinogenic effects, climate change, ozone layer depletion, radiation, inorganic, and organic respiratory effects), (ii) ecosystem quality (acidification/nitrification and eco-toxicity), and (iii) sufficient supply of resources (fuels and minerals). It was concluded that PHA manufacturing from transgenic plants was less beneficial over the full cradle-to-gate life cycle than microbial PHA manufacturing with glucose and whey. The main cause of this high life cycle impact was high steam consumption. However, it was suggested that CO2 intake at the corn forming stage can more than compensate for its CO2 emissions, and its polymer extraction and compounding processes consume much less power than the fermentation and recovery processes in microbial PHA manufacturing. Using a renewable energy resource would make PHA manufacturing from transgenic plants significantly better. One recommended solution is using the residual corn stover, after extraction, for electricity and steam (56). The available LCA results show that PHAs could have advantages over petrochemical polymers in several environmental impact categories (cogeneration power plant, greenhouse gas credits etc.) but are less favorable in other categories (eutrophication, acidification, etc.). This is a common occurrence in LCA comparisons of different raw materials and products. In addition, the different environmental impact categories are usually not regarded as being of equal weight in terms of seriousness of the effect on the environment. Under the current manufacturing technology, a number of LCA reports have indicated that petrochemical polymers can have equivalent or better eco-profiles than PHAs. The main factors influencing this assessment were energy for polymer manufacture, the effects of the number of recycling loops, and end-of-life disposal (especially methane generation in landfill). However, PHA manufacturing is generally small-scale at present. It is likely that as the scale increases, technical improvements and environmental economies of scale including recycling potential will accrue for PHAs. At this stage, PHAs could contribute to sustainability and help engage the public in environmental awareness.

THE RESEARCH-TO-MARKET BOTTLENECK PHA-producing microorganisms The microorganism of choice for the industrial production of PHA varies depending on factors that include the cell's ability to utilize an inexpensive carbon source (recent attention has been paid to agricultural wastes and industrial byproducts), the cost of the medium, the growth rate, the polymer synthesis rate, the quality and quantity of PHAs, and the cost of downstream processes (57). Although more than 300 different microorganisms synthesize PHAs (36), only a few, such as Cupriavidus necator (formerly known as Ralstonia eutropha or Alcaligenes eutrophus), Alcaligenes latus, Azotobacter vinelandii, Pseudomonas oleovorans, Paracoccus denitrificans, Protomonas extorquens, and recombinant E. coli, are able to produce sufficient PHA for large-scale production

(58). Some examples of patented PHA-producing bacterial strains currently used in industrial-scale production are reviewed below. In 1970, ICI Ltd. initiated the production of PHB and PHBV on an industrial scale (200,000 l) (59). The process utilized a mutant of C. necator, NCIB 11599 (60, Henman, T. J. and Holmes, P. A., ICI PLC (GB), European Patent Application EP0052946, 1982). The fermentation was carried out in a two-step fed-batch culture with glucose as the sole carbon source, and phosphate as the limiting element to enhance PHB production. The final biomass was 100 g/l with a productivity of 2.5 g/l h. The PHBV was produced by providing a mixed feed of glucose and propionic acid in the polymer accumulation phase. The content of 3-hydroxyvalerate (HV) was regulated by adjusting the ratio of the two substrates in the feed (61). In 1996, the PHA production process was acquired by The Monsanto Company in the U.S., which stopped the production of PHB and PHBV at the end of 1998 (62). Metabolix, Inc. developed recombinant E. coli K12 for the overexpression of PHB, and this bacterial strain can produce 100 g/l of PHB in 40 h (63). Another industrial process for the production of PHB was developed at the Biotechnologische Forchungsgesellchaft Company in Linz, Austria (64). The process was based on A. latus DSM1124, which can accumulate PHB at up to 80% of dry-cell weight in a onestep fed-batch fermentation process using a mineral-salt medium with sucrose as the sole carbon source. Although a biomass density of 60 g/l was achieved, the company stopped the production of PHB in 1993 (65). Several academic research groups have focused on producing PHA with high productivity and high yield to reduce the overall costs. Fedbatch and continuous cultivation have been carried out to improve productivity, either because of the difficulty of developing cultivation strategies using unusual or expensive carbon substrates, or due to the lack of applications to justify the high production costs (6671). Several strategies have been developed in academia for the efficient production of PHA employing C. necator H16. By maintaining the glucose concentration at 1020 g/l during a fed-batch culture, a total biomass of 164 g/l, a PHB concentration of 121 g/l, and a PHB content of 76% could be obtained in 50 h, resulting in a peak productivity of 2.42 g/l h. Using the same cultivation strategy, more than 110 g/l of PHBV could be produced by feeding a mixture of glucose and propionic acid (68). Several carbon sources other than glucose have also been used as substrates for PHA production. Ethanol was tested for the production of PHB by a mutant strain of C. necator, but the highest concentration of PHB obtained was 47 g/l (72). Several researchers (7377) have tried to improve PHB production by controlling the fermentation conditions. They developed mathematical models to maximize PHB production and used suboptimal procedures such as maintaining a constant nutrient concentration. The optimal feed profiles of glucose and ammonium hydroxide were calculated using a model, and a final cell concentration of 141 g/l and a PHB concentration of 105 g/l in 40 h of fed-batch culture were achieved with C. necator, for a productivity of 2.63 g/l h (74). To date, the highest PHB productivity (of 5.13 g/l h) was obtained in the short cultivation time of 16 h using a fed-batch culture of A. latus DSM1123 under nitrogen limitation (78). In addition to high productivity, one concern (particularly in PHBV production) is the rigorous control of HV content by the development of an on-line feeding strategy for fed-batch cultivation. A series of studies developed a novel multivariable control strategy for alcohol (ethanol and n-pentanol) concentrations during the production of PHBV by P. denitrificans ATCC 1774. This development coincided with an improvement of the quality of PHAs produced. A simple metabolic reaction (MR) model based on flux distribution analysis was constructed (75,76,79). Fig. 1 shows the metabolic map for PHBV biosynthesis in P. denitrificans ATCC17741 based on the consumption rates of ethanol and n-pentanol as described previously (75). Next, a model predictive controller (MPC) was developed and used for the on-line estimation of the specific

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J. BIOSCI. BIOENG.,

FIG. 1. Metabolic map for PHBV biosynthesis in Paracoccus denitrificans ATCC17741 based on consumption rates of ethanol and n-pentanol (75).

consumption rate and control of the alcohol concentration in fed-batch cultures. The Multi-Input and Multi-Output (MIMO) controller successfully controlled alcohol concentration, and the 3HV mole fraction units reached a given set point in the range of 070 mol%. The MPC was used to optimize PHBV production with a target mole fraction of 3 HV units. The amount of PHBV was maximized with a given mole fraction of 3 HV units at the final cultivation step (76). Afterward, the random and block polymers of PHBV that produced in a range of 090 mol% using two

feeding strategies were characterized using a non-isothermal kinetics study. The exponent function quantified by Osawa's equation in block PHBV was similar to that for PHB because the majority of the components in the block polymer were the 3HB unit. The rate of nonisothermal crystallization of block PHBV was faster than that of random PHBV but slower than that of PHB. Using the conventional method, the simultaneous feeding of ethanol and n-pentanol and the MPC system to control alcohol concentrations precisely, the availability of ethanol and

FIG. 2. Control results of ethanol and n-pentanol concentrations (a) and 3HV mole fraction units (b) by Multi-Input and Multi-Output Controller under C/N of 50 (74). The SEM micrographs of the surface of film made of random PHBV consisting of 5 mol% 3HV units (c) and 3-D images obtained from AFM analysis (d) (81).

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n-pentanol can be maintained throughout the cultivation time. The recent evidence from PHA samples produced by the optimization method has tended to demonstrate the possibility of producing block PHBV. The combined strategy may affect the polymerization step by natural dynamic changes of preferential consumption between ethanol and n-pentanol due to the switching of the carbon to nitrogen (C/N) ratio and the feeding strategy (80,81). Finally, their biodegradation rates were also compared in an in vitro study. Scanning electron microscopy (SEM) and trapping mode atomic force microscopy (AFM) were used for high resolution imaging of the surface structure (Figs. 2 and 3). It was observed that the surfaces of block PHBV samples are remarkably different from those of random PHBV samples. The highest degradation rate (of 2.6% per day) was observed for random PHBV produced by the conventional method consisting of 38 mol% of HV units. The degradation rates of random PHBVs were about two times faster than those of PHBVs produced by the optimized method (81). These observations indicate that the development of a feeding strategy could affect the quality of PHA produced and in turn change the physical and biological properties of the biopolymer. New bacterial strains identified in the laboratory have been reported regularly, but research groups have struggled to optimize PHA production with inexpensive carbon sources. There have been a few reports on the production of the terpolymer poly(3-hydroxybutyrateco-3-hydroxyvalerate-co-4-hydroxybutyrate), P(3HB-co-3HV-co-4HB). Chanprateep and Kulpreecha (82) were the first to report the production and characterization of terpolymer P(3HB-co-3HV-co-4HB) by the newly isolated C. necator strain A-04. A transmission electron micrograph of an ultra-thin section of C. necator strain A-04 containing PHB granules is shown in Fig. 4. The highest terpolymer content, 68%, was produced at 60 h. The terpolymer with the highest (93 mol%) 4HB

mole fraction units was produced when the cultivation time was extended to 96 h. The terpolymer P(4%3HB-co-3%3HV-co-93%4HB) showed an elongation of 430%, a toughness of 33 MPa, and a Young's modulus of 127 MPa, similar to those of low-density polyethylene, as shown in Table 5. The terpolymer P(11%3HB-co-34%3HV-co-55%4HB) showed a Young's Modulus of 618 MPa, similar to that of polypropylene, as shown in Table 6 (82). The kinetics of P(3HB-co-4HB) production by C. necator strain A-04 was also investigated in detail; the synthesis of 4HB units was growth-associated under nitrogen-sufficient conditions. Fig. 5 shows the effect of the C/N ratio on the relationship between specific growth rate and specific production rate of PHA when the ratio of -hydroxybutyric acid in the feed was between 50 wt.% and 95 wt.%. The synthesis of 3HB units was enhanced under nitrogendeficient conditions. The mole fraction of 4HB units could be changed from 0 to 70 mol% by adjusting the ratio of substrate and the mole ratio of C/N. The total P(3HB-co-4HB) content was 71 wt.% with a 4HB unit mole fraction of 30 mol% in the copolymer (83). SELECTION OF CARBON SOURCES There are a number of literature reviews on the selection of suitable carbon sources for efficient PHA production, for which the total cost of bioprocessing must meet economic requirements (5,10,11,13). The most frequently reported factor that influences the price of PHA is the cost of the carbon source. Fortunately, most microorganisms are saprophytes that can metabolize a wide range of carbon sources. However, the selection of carbon sources should not focus only on market prices but also on availability and global price consistency. In addition, inexpensive carbon sources such as agricultural wastes and industrial by-products may incur additional costs due to pre-treatment

FIG. 3. Control results of ethanol and n-pentanol concentrations (a) and 3HV mole fraction units (b) by a Model Predictive Controller under optimal strategy (74). The SEM micrographs of the surface of film made of block PHBV consisting of 7 mol% 3HV units (c) and 3-D images obtained from AFM analysis (d) (81).

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TABLE 6. Mechanical properties of terpolymer produced by C. necator strain A-04 comparing with P(3HB-co-3HV) and P(3HB-co-4HB) (82). PHAs composition P(3HB) P(3HB-co-3HV) 3% 3HV 9% 3HV 14% 3HV 20% 3HV 25% 3HV P(3HB-co-4HB) 3% 4HB 10% 4HB 16% 4HB 44% 4HB 64% 4HB 90% 4HB P(3HB-co-3HV-co-4HB) 10% 3HB 40% 3HV 50% 4HB 11% 3HB 34% 3HV 55% 4HB 11% 3HB 23% 3HV 66% 4HB 12% 3HB 12% 3HV 76% 4HB 10% 3HB 6% 3HV 84% 4HB 4% 3HB 3% 3HV 93% 4HB Petroleum based plastic Plactic bag (HDPE) Plactic bag (PP) Plactic bag (LDPE) UV degradable bag Toughness Young's modulus (MPa) (MPa) 0.22 0.26 0.32 0.39 20 33 3500 2900 1620 1500 1450 1370 30 100 503 618 392 142 118 127 Tensile strength (MPa) 40 38 190 150 120 70 28 24 26 10 17 65 9 10 9 4 9 14 Elongation (%) 0.4 37 35 32 30 45 242 444 511 591 1080 4 3 5 9 300 430

FIG. 4. Transmission electron micrograph of ultra-thin section of C. necator strain A-04 (a) typical short rod cells ranging from 0.6 to 1.0 m. Bar, 1 m. (b) C. necator strain A04 containing PHB granules (white fractions). Bar, 200 nm (83).

62 64 15 60

640 590 156 674

19 27 13 24

576 435 126 384

steps, extended cultivation times, and purification. Simple carbon sources such as sugar and starch from crops seem to be superior to complex carbon sources, but they are also a primary source of human food and animal feed (84). Because the price of fossil fuels continues to increase, using some of these carbohydrates for biofuel production is unavoidable. Given the limits on agricultural capacity and the increasing demand for crops as bioenergy resources, researchers need to seek ways of using non-food crops for biodegradable production (85). Below, I provide information on conventional raw materials and optional raw materials for PHA production. Table 7 shows the current global market prices of substrates and their theoretical PHB yields. Sugars Carbon sources are needed by all living organisms, and sugars are among the simplest. Pure sucrose is one of the most suitable

TABLE 5. Thermal properties of terpolymer comparing with copolymers produced by C. necator strain A-04 (82). PHAs composition (mol%) PHB P(3HB-co-3HV) 24% 3HV 45% 3HV 71% 3HV P(3HB-co-4HB) 11% 4HB 17% 4HB 82% 4HB P(3HB-co-3HV-co-4HB) 10% 3HB 40% 3HV 50% 4HB 11% 3HB 34% 3HV 55% 4HB 11% 3HB 23% 3HV 66% 4HB 12% 3HB 12% 3HV 76% 4HB 10% 3HB 6% 3HV 84% 4HB 4% 3HB 3% 3HV 93% 4HB Tg (C) 10 6 10 13 13.7 15.9 17.7 21.1 47.1 51.6 Tm (C) 177 138 75 87 160 152 40 87.6 99.9 91.8 87.3 54.3 54.8 MW 1.10 106 5.98 105 6.62 105 5.51 105 1.77 105 4.15 105 MN 7.82 105 1.38 105 4.00 105 2.54 105 2.23 105 3.32 105 1.29 105 9.67 105 2.99 105 1.90 105 1.73 105 6.27 104 1.33 105 PDI 1.8 1.9 2.4 2.0 2.5 2.4 2.4 1.13 2.00 3.46 3.18 2.82 3.11

carbon sources for large-scale PHB production, due to its availability in the market. Metabolix, Inc. and ADM selected corn syrup to produce Mirel resin from recombinant E. coli using a fully biological fermentation process. In academic research, the greatest success with PHA production from sugar has been reported by Kim et al. (86) They used recombinant E. coli K12 (ATCC 23716) harboring the plasmid pSYL104, which contains 8.8 kb of a PHB operon and an ampicillin-resistant gene, and grew the bacterial cultures to 101 g/l of biomass on glucose while maintaining the specific growth rate at 0.1 h1. The kinetic information is fully available and ready for industrial-scale production (86). Starches Starches are also an abundant carbon source. Many research groups are interested in utilizing starch for PHA production. Starch has also been used as a bio-based polymer for various types of bioplastics. However, microorganisms often display low productivity when grown directly on raw starch, and additional costs from liquefaction and saccharification steps limit the potential use of starch. R. eutropha NCIMB 11599 was able to produce PHB from saccharified potato starch waste with phosphate limitation. The biomass produced was 179 g/l and 94 g/l of PHB for a productivity of 1.47 g/l h (87). Kim and Chang (38) investigated PHB production from starch in flask, batch, and fed-batch cultures of Azotobacter chroococcum. In flask cultures, PHB content reached 74% of dry-cell weight. In batch cultures, PHB content reached 44% with O2 limitation. In fed-batch cultures, 71 g/l of biomass with 20% PHB was obtained without O2 limitation, whereas 54 g/l of biomass with 46% PHB was obtained with O2 limitation (38). Extruded rice bran was utilized to obtain a cell concentration of 140 g/l, a PHA concentration of 77.8 g/l, and a PHA content of 55.6 wt.% in a repeated fed-batch fermentation. In 2006, Huang et al. (88) reported that when extruded corn starch was used as the major carbon source, 62.6 g/l of biomass was obtained with 24.2 g/l of PHA (38.7 wt.%) (88). Halami screened for bacterial strains that could utilize starch to produce PHB. It was found that the isolated Bacillus cereus could secrete the

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FIG. 5. The effect of C/N ratio on the relationship between and when the ratio of -hydroxybutyric acid in the feed was 50 wt.% (a), 75 wt.% (b), and 95 wt.% (c), respectively. Symbols: closed square, specific production rate of 4HB monomer unit; open triangle, specific production rate of 3HB monomer unit; closed circle, specific production rate of copolymer of P(3HB-co-4HB) (83).

enzyme amylase and simultaneously produce PHB at 0.48 g/l (89); however, industrial-scale production still awaits optimization of the system's productivity. Alcohols Alcohols are sterile carbon substrates, and PHA fermentation processes using alcohols as substrates could possibly reduce the chance of contamination. There is considerable experience in methanol fermentation technology because methlylotrophic bacteria have been considered for the large-scale industrial production of single-cell proteins. Some methalotrophic and methanotrophic bacteria are promising for PHB production purposes. Pseudomonas sp. K was cultivated in a fed-batch process with nitrogen deficiency to a cell density as high as 233 g/l dry-cell weight with a PHB content of 64%, resulting in a product yield of 0.2 g g1 (48). P. extorquens was grown to cell densities of 190 g/l dry-cell weight with a PHB content of 60% (67). The methlylotrophic bacteria P. denitrificans and M. extorquens are able to synthesize copolyester PHBV when methanol and n-amyl alcohol are

TABLE 7. The global market prices of substrates in 2010 and estimated substrate cost for production of PHA based on yield (59,98,101). Carbon source Sucrose a Glucose b Ethanol c Methanol d Cassava starch e Cane molasses b Palm oil f Soya oil g
a b c d e f g

Price per kg Yield of PHB (g g 1) Cost of C-source per kg of PHA 0.35 0.41 0.31 0.28 0.19 0.10 0.79 0.92 0.40 0.38 0.50 0.43 0.20 0.42 0.65 0.70 0.87 1.07 0.63 0.58 0.94 0.24 1.22 1.31

London Daily Sugar Price. USDA Economic Research Service. CME Fuel Ethanol Futures. Methanex methanol price sheet. The Tapioca Trade Association (TTTA). Crude, cif North West Europe. Dutch, fob ex-mill.

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J. BIOSCI. BIOENG., develop bioprocesses based on waste carbon resources. Finally, the world market price of each substrate and the estimated substrate cost based on the PHA production yield are listed in Table 7. PHA is undoubtedly one of the potential candidates for replacing petroleum-based plastics. However, only a few biodegradable plastic products made from PHAs are available on the market (Mirel, Biocycle, Biomer, etc.). Although the current price of PHA is an ongoing impediment to its widespread use, the additional cost provides a completely biodegradable product that leaves zero hazardous waste in the environment. National polices and legal measures are also important factors that contribute to market initiatives for PHA. International Standards (ISO) and Certified Labeling and Environmental Product Declarations (EPD) are required to communicate the environmental credentials of PHAs to consumers and businesses. Further research and technical development is needed to build a wider range of applications, to characterize the greenhouse gas emissions from PHAs, to obtain new data for LCA models of the environment impacts of PHAs as feedback information for further research, and to develop new polymers that can be recycled after a first use. Altogether, the prospects for PHAs look bright. The potential usefulness of PHAs should expand further as new market niches open. ACKNOWLEDGMENTS The author wishes to express her appreciation to the Society for Biotechnology (Japan), which awarded the Young Asian Biotechnologist Prize to her in 2009. The research done for this review was supported in part by the Joint Program in the Field of Biotechnology under the National Research Council of Thailand, the National Science and Technology Development Agency of Thailand, and the Japan Science and Technology Agency. The author would like to extend her respectful thanks to Professor Suteaki Shioya (Sojo University), Professor Hiroshi Shimizu (Osaka University), Professor Takuya Nihira (Osaka University), Professor Wattanalai Panbangred (Mahidol University), and Professor Yoshio Katakura (Kansai University) for their invaluable discussions and supports.

fed simultaneously in nitrogen-limited medium. The 3HV content reached 91.5 mol% depending on the ratio of substrate used (76,81,90). P. denitrificans ATCC 17741 can produce a homopolymer of PHV (24% w/ w) when grown on n-pentanol (91). A microcomputer-aided, automatic fed-batch culture system under potassium-limited conditions was set up for PHB production with M. organophilum using methanol as the substrate (92). However, alcohols have become very important as alternative energy sources to replace fossil oils. Industrial by-products Molasses, an industrial by-product of sugar production, is one potentially inexpensive carbon source for PHA production. However, molasses values over the past year have risen to unprecedented levels at both origin and destination. The price of U.S. blackstrap cane molasses is approximately double its historical level. Sharply reduced production of molasses in India and Pakistan explain the surge in prices. At the same time, the expanding use of molasses in fuel ethanol programs is boosting global molasses demand. Molasses production in Pakistan and Thailand for 20092010 is forecasted to rise only slightly (Quarterly Market Outlook, International Sugar Organization, November 2009). Chen et al. (93) reported on the production of PHB from beet molasses by an A. vinelandii UWD mutant in two-stage fed-batch cultures. The amount of PHB produced was 36 g/l with productivity higher than 1 g/l h (93). Liu et al. (94) have successfully replaced glucose with beet molasses to produce PHB by a recombinant Escherichia coli strain (HMS174/pTZ18u-PHB). The final dry-cell weight, PHB content, and PHB productivity were 39.5 g/l, 80% (w/w), and 1 g/l h after 31.5 h of fed-batch fermentation with constant pH and dissolved O2 content (94). There are a number of reports on using cane molasses for PHA production. Gouda, et al. (95) reported that Bacillus megaterium was able to produce PHB from cane molasses in shaken flask cultivation. Briefly, 46.2% PHB was produced when 3% (w/v) of sugar cane molasses was supplied (95). Kulpreecha et al. (96) reported an attempt to produce PHB from sugar cane molasses by Bacillus sp. BA019. The optimal feeding medium in this system required a higher total sugar concentration of 400 g/l and a C/N molar ratio of 10. Under these conditions, the biomass was 72.6 g/l and PHB productivity was 1.27 g/l h (96). However, the cost of downstream decolorization processes becomes the limiting factor for cost competitiveness. In addition, the recent trend toward utilizing molasses was based on projected profits from bioethanol production. Fatty acids and vegetable oils are also potential substrates for PHA production because it was reported that the theoretical yield of PHA production from fatty acids is 0.65 g g 1 (97), whereas the theoretical yield of PHA production from glucose ranges between 0.300.40 g g 1 (98). It was reported that Rhodobacter sphaeroides (IFO 12203) could utilize palm oil mill for PHA production, and a PHA yield of 0.50 g g 1 of organic acid consumed was obtained, resulting in PHA content of 67% (99). Kahar et al. (100) reported the use of soybean oil for PHA production by C. necator H16 and its recombinant strain PHB-4 harboring the Aeromonas caviae PHA synthase gene. The production of PHA by C. necator H16 was 118126 g/l, and the PHB content was 7276%. The recombinant strain could produce PHA from soybean oil with high cell density (128 138 g/l) and high PHA content (7174%). The yield obtained from these experiments was as high as 0.72 to 0.76 g g 1 (100). The Pseudomas sp. strain DR2 isolated by Song et al. (16) was able to utilize corn oil, and it produced 37.34% PHA that consisted of 3-hydroxyoctanoic, 3-hydroxydecanonic, and 3-hydroxydodecanoic. Strain DR2 also could utilize vegetable oil as a carbon source for PHA production with a content of 23.52% (16). Recently, waste glycerol, a by-product from the biodiesel industry, has received much interest from many researchers as an inexpensive organic source for PHA production. As reported by Cavalheiro et al. (17), C. necator was used to produce PHB using waste glycerol. A total of 68.8 g/l of biomass was obtained with a PHB content of 38%. Finally, by decreasing the biomass concentration, a PHB content of 50% with a productivity of 1.1 g/l h was achieved (17). It is therefore reasonable to

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