Current Trends in Biodegradable PHAs

Journal of Bioscience and BioengineeringVOL. 110 No. 6, 621–632, 2010

www.elsevier.com/locate/jbiosc

REVIEW

Current trends in biodegradable polyhydroxyalkanoates

Suchada Chanprateep⁎

⁎ Tel.: +66 2E-mail add

1389-1723/$doi:10.1016/j

Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road, Patumwan, Bangkok 10330, Thailand

Received 5 April 2010; accepted 26 July 2010Available online 17 August 2010

The microbial polyesters known as polyhydroxyalkanoates (PHAs) positively impact global climate change scenarios byreducing the amount of non-degradable plastic used. A wide variety of different monomer compositions of PHAs has beendescribed, as well as their future prospects for applications where high biodegradability or biocompatibility is required. PHAscan be produced from renewable rawmaterials and are degraded naturally by microorganisms that enable carbon dioxide andorganic compound recycling in the ecosystem, providing a buffer to climate change. This review summarizes recent researchon 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 SUSTAINABLESTRATEGY FOR PROTECTING AGAINST CLIMATE CHANGE

Materials made from synthetic polymers are not biodegradableand are often improperly discarded. These materials are typicallyderived from petroleum-based plastics. Rapid progress in materialsscience technology has created new plastic products with favorablemechanical integrity and excellent durability. Nevertheless, plasticproducts usually have single-use applications, especially in foodpackaging and medical materials. Because these plastic products arenot biodegradable, they are extremely persistent and accumulate inthe ecosystem, resulting in a significant burden on solid wastemanagement. The total global capacity of commodity plastic produc-tion dramatically increased from 1.5 million tons in 1950 to245 million tons in 2008, an annual growth rate of 9%. The biggestpotential growth area is in the rapidly developing Asian countries(excluding Japan), where current plastics consumption per capita isonly around 20 kg (The compelling facts about plastics, TheAssociation of Plastic Europe, 2009). It is very difficult to reduce theconsumption of plastic products due to their versatile properties, butit is possible to replace petroleum-based plastics with alternativematerials that have polymer-like properties and that degrade afterbeing discarded.

Among the various types of biodegradable plastics, polyhydroxyalk-anoates (PHAs) are among the most well known, being recognized ascompletely biosynthetic and biodegradable with zero toxic waste, andcompletely recyclable into organic waste. They are microbial polyestersproduced by a wide range of microorganisms, mostly as intracellularstorage compounds for energy and carbon (1,2). PHAs are among themost fascinating and largest groups of biopolyesters, with over 150

218 5070; fax: +66 2 252 7576.ress: [email protected].

- see front matter © 2010, The Society for Biotechnology, Japan. All.jbiosc.2010.07.014

different types of monomer composition that provide differentproperties and functionalities (3–6). Their properties span a widerange, including materials that imitate thermoplastic properties andothers that possess electrometric properties. PHAs are efficientlydegraded in the environment because many microorganisms in soilsare able to secretepolyhydroxybutyrate (PHB)depolymerases, enzymesthat hydrolyze the ester bonds of a polymer into water-solublemonomers and oligomers. Microorganisms then metabolize thesedegradation products into water and carbon dioxide (7–9). There areseveral intensive academic studies of PHA production and applications,mostly based on seeking inexpensive carbon sources to reduce the costof production (10–17) and applying genetic engineering to improveproductivity (18–26). This knowledge is now at a bottleneck, beinglargely unused to bring products to themarket. Only a fewPHAs,mainlypolyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyva-lerate) (PHBV), and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)(PHBH), haveproceeded to theproduction stage in large quantities (27).This review aims to summarize the recent trends in biodegradablepolymers, based on the current situation of PHA-based plastic productsavailable on the market. The review also addresses global policy thatmay be useful for turning academic research towards the currentdemand for biodegradable plastics.

GLOBAL MARKETS AND POLICIES

Environmentally degradable polymers (biopolymers) are one of thepossible solutions to replace some petroleum-based polymers. Based onthedefinitiongivenby theEuropeanBioplasticsAssociation,biopolymersare based on renewable resources complyingwithASTMD-6866 and canbe degraded to comply with international standards such as EN13432,ASTM D6400, and ISO17088. In Europe, the criteria for biodegradabilityare set out within the standard EN13432, 2000, which is binding whenapplied to compostable packingunder the EUdirective on Packaging and

rights reserved.

622 CHANPRATEEP J. BIOSCI. BIOENG.,

Packaging Waste (94/62/EC). In North America, similar criteria fordefining compostability have been established by the Institute forStandards Research under ASTM D6400-99 Specification for Compo-stable Plastics. Synthetic components, such as additives, colorants, orglues, can be added to aid performance. Biopolymers can be classifiedinto three groups based on their biodegradable characteristics. The firstgroup consists of biopolymers that are not bio-based plastics but havebiodegradable or compostable properties. This group includes syntheticbiodegradable aliphatic–aromatic copolyesters, such as polybutyleneterephthalate adipate (PBTA), polybutylene terephthalate succinate(PBTS), and polybutylene terephthalate glutarate (PBTG). The secondgroup includes bio-based polymers that are biodegradable or compo-stable, for example (i) polylactide (PLA) (Ingeo™ by NatureWorks LLC,USA), (ii) starch-basedmaterials (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, TheChemical Company, Germany), and (v) PHAs (Mirel™ by Telles, USA).The third group is composed of bio-based resources that are manufac-tured into non-biodegradable polymers. This group includes bio-based1,3 propanediol (PDO) from corn sugar (Bio-PDO™ by DuPont Tate &Lyle Bio Products, LLC, USA), and bioethanol-based linear low-densitypolyethylene (LLDPE) (Dowlex™byDowChemical Company, USA) (27).

Alternatively, biopolymers can also be classified into four groupsbased on their components. The first group is produced directly byliving organisms (e.g., cotton, silk, wool, other natural fibers, cellulose,starch, lignin, oil proteins, natural rubber, and PHAs). The secondgroup is produced by polymerization of monomers that either exist innature 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 renewableresources with petrochemical-derived monomers (e.g., isosorbate-containing polycarbonates and soy-based urethanes). The last groupincludes polymers produced from blends of renewable resources andpetroleum-based materials, such as blends of starch and polyvinylalcohol (Eco Flo® by Green Light Products Ltd., UK) (28). Table 1summarizes the commercialized PHAs with their trade names andmanufacturing companies.

Members of the European Union have already established policiesagainst petroleum-based consumer packaging. For example, Belgiumhas established an eco-tax of €3 kg−1 on packaging such as shoppingbags,whereas compostable shoppingbags that conform to theEuropeanStandard (EN) 13432 for compostable packaging material will beexempted. The Netherlands has established a carbon-based packagingtax based on CO2 emissions from the production of packaging materialand the embedded carbon content of the packaging. France, Italy, andSpain are considering similar legislation. So far, two European countrieshave established legislative measures for bioplastics and their deriva-tives. Since June 2005, an interim regulation under the GermanPackagingDirective exempts compostablepackaging, enabling recycling

TABLE 1. The current and potential large volume m

Polymer Trade names M

PHB Biogreen® Mitsubishi Gas CPHB Mirel™PHB Biocycle® PHB IndusPHBV and PHB Biomer® BiomPHBV, PHBV+Ecoflex blend Enmat® Tianan BiPHBH Nodax™PHBH Nodax™ LianyPHBH Kaneka PHBH KanekaP(3HB-co-4HB) Green Bio Tianjin GrPolyhydroxyalkanoate from P&G Meredian M

a Bacteria to produce bioplastics, BIOPRO Baden-Württemberg GmbH, Septemberlang=en&artikelid=/artikel/04310/index.html, accessed on March 2010.

b Full-scale development of the world's first completely bio-based polymer with sohttp://www.kaneka.co.jp/kaneka-e/news/pdf/090310.pdf, accessed on March 2010. n/a

without specific supporting documentation or measures. Since 2006 inFrance, a regulation for the promotion of French agriculture requiresdisposable retail bags to be biodegradable by 2010 (Political strategiesand statutory regulation, The European Bioplastics Association, 2010).

In the U.S., the San Francisco Board of Supervisors approved thefirst-in-the nation legislation in 2007 that outlawed the use of non-biodegradable plastic bags in large supermarkets within 6 months andlarge chain pharmacies in about a year. Minnesota also bolstered itsban on the disposal of yard trimmings in landfills recently when thegovernor signed a bill forbidding the use of conventional plastic bagsfor collecting yard trimmings in the Twin Cities area. This bill alsorequires compostable bags to meet ASTM D6400 or be certified by anindependent organization. The law took effect on January 10, 2010(Plastic Bags Legislation, The Council of State Governments/EasternRegional Conference, 2010).

Among Asian countries, Japan is the biotechnology leader inbiodegradable plastic. After ratification of the Kyoto Protocol in 2002,the government announced two state measures, “BiotechnologyStrategic Scheme” and “Biomass Nippon Strategy,” to mitigate globalwarming by promoting the use of renewable resources and applyingrapid advances in biotechnology (29). The Japan Bioplastic Association(JBPA) classified bioplastics into two groups: (i) biodegradable plastics(GreePla®) with biodegradable characteristics and biodegradablesynthetic high-polymer materials with an average molecular weightof at least 1000 Da, including chemically modified starch- andpolyamino acid-based biodegradable high-polymer materials, and (ii)biomass-based plastics (BiomassPla®) producedwith either chemicallyor biologically synthesized materials containing renewable organicmaterials (excluding natural organic polymers that are not chemicallymodified). The JBPA has estimated that demand for biomass-basedplastics will reach 20% of total plastic consumption in 2020. Thus, theJBPA started the “Identification and Labeling System” in 2000 and hascertified about 900 biodegradable plastic products in Japan. The systemis based on a positive list system for all components, biodegradabilityspecifications based in Japanese Industrial Standards, safety certificationof all components, and proof of no hazardous effects to soil (http://www.jbpaweb.net/english/english.htm). Standards for the biodegrad-ability of plastics in the ISO have been proposed under TC61/SC5/WG22(30). Table 2 shows the global policies that have been enacted stating arequirement for biodegradable/compostable plastics.

CHALLENGES AND OPPORTUNITIES OFPOLYHYDROXYALKANOATES

Two of the most promising biopolymers are PLA and PHA. PLA hasreceived much attention as a potential alternative to existing materialswhile PHA is challenging and price-competitive with petroleum-basedpolymers. In the long term, bioplastics have significant potential for costreduction. The price of petroleum-based plastics could increase by 50–

anufacturers of polyhydroxyalkanoates (33).

anufacturers Capacity (tons) Price (kg−1)(in 2010)

hemical Company Inc. (Japan) 10,000 €2.5–3.0Telles (US) 50,000 €1.50a

trial Company (Brazil) 50 n/aer Inc. (Germany) 50 €3.0–5.0ologic, Ningbo (China) 10,000 €3.26P&G (US) 20,000–50,000 €2.50i Biotech (China) 2000 €3.70Corporation (Japan) 1000 n/ab

ee Bio-Science Co/DSM 10,000 n/aeredian (US) 272,000 (2013) n/a

24, 2009 available at http://www.bio-pro.de/magazin/thema/04308/index.html?

ft and heat resistant properties, Kaneka Corporation, March 10, 2009 available atmeans price is not able to be found.

TABLE 2. The global policies and measures.

Countries National policies and measures

Germanya German Packaging Directive has been in force (2005). The compostable packaging will be exempt from the requirements in § 6 of the DirectiveFrancea A law for the promotion of French agriculture has been in force (2006) stating a requirement for biodegradability of disposable retail carry bags by 2010Italyb,c Markets in Florence had been charging €0.10–0.20 per plastic bag (2009)Ireland, Scotland, Denmark

and SwedencThese countries have already imposed levies and taxes on non-degradable plastic bags

UKc,e In 2003, county Durham has been charging Ecotax per plastic bagUSd,e 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 yearCanadad Toronto City Council: retailers will be required to charge a minimum of 5 cents for each plastic retail shopping bag that customers take (2008)Japanf Law on Promoting Green Purchasing and Law on Recycling have been in force in 2001Indiad Plastic is officially banned in LadakhAustraliad Thin non-biodegradable plastic shopping bags have been prohibited in South Australia from 4 May 2009Bangladeshd From the beginning of January 2002, the Bangladesh government is banning the use of plastic bags in Dhaka

a The Association European Bioplastics, European Bioplastics e.V., Berlin, available at http://www.european-bioplastics.org/index.php?id=307, accessed on March 2010.b 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 inmotion, 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.

CURRENT TRENDS IN BIODEGRADABLE POLYHYDROXYALKANOA 623VOL. 110, 2010

80% in 2012, based on the increasing price of oil (The Plastics ExchangeLLC, 2010). Most manufacturers of biodegradable plastics are small-scale facilities (about 1000–20,000 tons per year of production), andthey lack the economy of scale of a single polyethylene unit with acapacity of 300,000 tons per year of production. The world's largestlactic acid manufacturing facility belongs to NatureWorks LLC in Blair,Nebraska, U.S.A. with a capacity of 140,000 tons. In 2003, NatureWorksLLC started to produce Ingeo™ PLA resin. Afterwards, PLA became thefirst bio-based plastic produced on a large scale. Among PHA-manufacturing companies, the main company with a large productionis the U.S. biotech company Metabolix, Inc. in Cambridge, Massachu-setts. In 2010, Telles, a joint venture company formed by the ArcherDaniels Midland Company (ADM) and Metabolix, Inc. opened the firstcommercial-scale plant to produce a corn syrup-based PHA resin,Mirel™, in Clinton, Iowa,U.S.A. It is expected tobegin shippingPHAresinin April of 2010 andwould 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. Graceand Company produced PHB in the U.S. for possible commercialapplications (Baptist, J.N., (Assignor to W.R. Grace & Co., New York)US Patent No. 3225766, 1965). However, the company shut down theprocess due to low production efficiency and a lack of suitablepurification methods. In 1970, PHBV was commercialized by ImperialChemical Industries Ltd. (ICI/Zeneca BioProducts, Bellingham, UK)under the trade name of Biopol™ (31). In 1996, the technology wassold to Monsanto and then to Metabolix, Inc. The U.S. Department ofCommerce Advanced Technology Program funded a project to re-engineer the central metabolism of Escherichia coli for more efficientconversion of renewable sugars into PHB. In 2008, Metabolix, Inc.announced the combined production of PHA Bio-based Polymers andBiomass Energy with a target to obtain PHA from switchgrass at a levelof 20% of dry-cell weight, 75% of which could be recovered. Thus, ifswitchgrass yields are 10 to15 tonsper acre, theneachacrewill yield1.5to 2.25 tons of PHA bio-based polymers or derived chemicals, and1 million acres will yield 3.3 to 5 billion pounds of PHAs (32).

Procter and Gamble, in partnership with Kaneka Corporation, TsingaUniversity in China, and the Riken Institute in Japan, has developed awide range of applications for PHB and PHBH (Nodax™) as fibers, non-woven materials, aqueous dispersions, and disposable products.However, Nodax technology was sold in 1993 (33). Recently, KanekaCorporation has announced its plan to launch the production of a plant-derived soft polymer called Kaneka PHBH in 2010, with a productioncapacity 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 PHBona commercial scalefor special applications. In 1993, Biomer acquired expertise andmicrobesfor PHB products from the Austrian company Petrochemia Danubia andregistered the trade name Biomer™ in 1995 (34). In Brazil, one of thelargest sugar-exporting countries, PHB Industrial S.A. (Serrana) usessugar cane tomanufacture PHB (Biocycle™) in a joint venture started in1992 between a sugar producer (Irmaoes Biagi) and an alcohol producer(the Balbo Group). The company has been running a pilot plant at50 tons per year and plans to increase production capacity to 3000 tonsper year (35). In Canada, Biomatera Inc. specializes in themanufacture ofPHA by fermentation of agricultural residues. The biopolymers are usedin the manufacture of creams and gels that are used as slow-releaseagents in drug manufacturing and as cosmetic agents and tissue matrixregeneration (36). In Japan, Mitsubishi Gas Chemical hasmade progresson the production of PHB from methanol fermentation (BioGreen™)(33). Table 3 presents physical, mechanical and thermal properties ofcommercialized PHAs, and Table 4 shows the potential of commercial-ized PHA to replace some petroleum-based plastics (37).

COST COMPETITIVENESS

In 2006, the cost of PHB was in the range of €10 per 12 kg−1. Thispricewasmuchhigher than that of starchpolymers and other bio-basedpolyesters due to high raw material costs, small production volumes,and high processing costs, particularly for purification (33). It wasconcluded that rawmaterial accounts for 30–40% of the total cost of PHB(38). The latest market price of Mirel™ is quoted at about €1.50 kg−1

(PlasticsNews, 10March2010). Thisproduct combinesbiodegradabilitywith premium pricing relative to most petroleum-based plastics. PHAswill soon enter the market and become potential candidates forcommodity materials. In 2010, the market potential of the totalbioplastics market in the EU will reach 200,000–500,000 tons. Themain 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 agriculturalsectors, includingmany durable applications (BioplasticsMarket TrendsandU.S.&E.U.Outlook, FUJ00024, p. 132, Fuji-Keizai,October 2007). Thesummary of market prices for PHAs in 2010 is given in Table 1.

SUSTAINABILITY AND ENVIRONMENTAL IMPACT OF PHAS BASEDON LIFE CYCLE ASSESSMENT

The sustainability of fossil resource depletion and its environmentalimpacts (such as greenhouse emissions) are important subjects of

TABLE 3. The physical, mechanical and thermal properties of some commercialized PHAs (37).

PHB PHB copolymers PHBV PHBHKanekaBiomer240 Biomer P226 MirelP1001 MirelP1002 Biocycle100 Biocycle24005

Application grade Injection mold Injection mold Extrusion and injection Foam mold

Physical propertiesMelt flow rate (g/10 min) 5–7 9–13 10–12 15–25 5–10Density (g/cm3) 1.17 1.25 1.39 1.30 1.22 1.20 1.2Crystallinity (%) 60–70 60–70 50–60

Mechanical propertiesTensile strength (MPa) 18–20 24–27 28 26 30–40 25–30 10–20Elongation (%) 10–17 6–9 6 13 2.5–6 20–30 10–100Flexural strength (MPa) 17 35 46 35Flexural modulus (GPa) 3.2 1.9 0.8–1.8

Thermal propertiesMelting temperature (°C) 170–175VICAT softening point (°C) 53 96 148 137 120–125

624 CHANPRATEEP J. BIOSCI. BIOENG.,

ongoing scientific and political argument. An attempt to developbiotechnological processes to produce chemical commodities fromrenewable agricultural resources is one advocated approach that hasrecently gained much attention. PHAs are currently included in theseconsiderations. Thus, it is necessary to ascertain whether the manufac-turing processes preserve non-renewable resources and benefit theenvironment as intended. Life Cycle Assessment (LCA) is a well-established approach for identifying best practices within the complex-ity of choices confronting society and industry. The LCA is an objectiveprocedure for evaluating the environmental impacts associated with aproduct 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 athome, and disposing of it or recycling it into a new product (39,40). TheLCA methodology has been standardized under the ISO-14040 series,and it distinguishes four phases: goal and scope definition, inventoryanalysis, impact assessment, and interpretation (41). LCA evaluationshave been investigated, for example, in a notorious argument betweenpaper and plastic (42,43). It was found that although LCA usually leadsto a definite conclusion, its results can easily be reversed under differentenvironmental impacts, different inventory parameters, and/or differ-ent boundaries of the study. For example, one may look at energy, solidwaste, or water. The number of published LCAs for PHAs is limited, andonly a few environmental analyses have been undertaken. Thus, thisarticle offers examples of LCAs that use different parameters to comparebetween PHAs and synthetic plastics (or other bioplastics) with respectto specific environmental impacts.

In earlier studies,most of thepublished LCAs of PHAs focused on theconsumption of fuel and non-renewable energy. In 1998, Heyde (44)reported that the energy requirements for PHB production can exceedthe energy requirements for conventional plastics of high densitypolyethylene (HDPE) and polystyrene (PS) (44). Likewise, Gerngross(45) conducted a cradle-to-grave analysis of theoretical large-scalePHA production from corn-based glucose versus a conventional PSprocess. It was concluded that the replacement of conventionalpolymers with fermentation-derived PHAs did not appear to be auseful approach if the sustainable production of polymers is the

TABLE 4. The potential of commercialized PHA to replace the petroleum-basedplastics (33).

LPDE PP HPDE PS HI-PS PVC PET PA PBT

Mirel™ ++ + ++ ± + ++ ± −Biomer® − ++ ++ + − − − − −Nodax™ + ++ ++ − − + + − −Biocycle® − ++ ++ + − − − − −

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

desired outcome. The biodegradability and biocompatibility benefits ofPHAs could justify the use of fossil resources; however, those benefitshave to be quantified and evaluated separately. A high level of energyconsumption from cradle-to-factory gate is an environmental burdenassociated with corn cultivation (45). This assessment has led toseveral LCA studies that adopted similar inventory analyses andenvironmental impact assessments. These studies noted the high levelof energy consumption and negative environmental impacts fromplant cultivation (46–48). Kim and Dale (49) suggested that someapproaches to reducing the environmental burdens of agriculturalprocesses are necessary to achieve better profiles for photochemicalsmog, acidification, and eutrophication associated with plant cultiva-tion (49). In 2008, Kim and Dale (50) conducted an LCA evaluationusing information from Telles on site-specific processes such as cornwetmilling, PHB fermentation, and recovery. In this specific case,mostof the energy used in cornwetmilling, PHB fermentation, and recoveryprocesses was generated in a cogeneration power plant in which cornstover is burned to generate electricity and steam. In this case, PHBderived from corn grain offers environmental advantages overpetroleum-derived polymers in terms of non-renewable energyconsumption and greenhouse gas emissions. Furthermore, PHBprovides greenhouse gas credits, and thus PHBuse reduces greenhousegas emissions compared to petroleum-derived polymers (50).

Pietrini et al. (51) pointed out a different challenge of cradle-to-grave environmental LCA comparisons of PHB-based composites withpetrochemical plastics. In this study, the end products were cathode raytubemonitor housings produced fromhigh-impact polystyrene and theinternal panels of an average car produced from glass-fiber-filledpolypropylene. The environmental impact was evaluated for non-renewable energy use and global warming potential over a 100-yeartime horizon. On a cradle-to-factory gate basis, all PHB compositesappeared to be environmentally superior to conventional polymers forboth chosen applications.When the analysiswas extended to cradle-to-grave (including the use phase, post-consumer waste incineration, andenergy recovery), PHB composites scored better only for the CRTmonitor housing. In the case of the automotive application, theweight ofthe functional unit becomes overriding, and no substantial savings areobserved regarding environmental impact (51).

Mixed bacterial culture PHA production has the potential to producelarge amounts of PHAs at low cost due to lower sterility, equipment andcontrol requirements, and the ability to utilize a wide range of cheapsubstrates (including industrial and agriculture wastes) (52–54).However, Rhu et al. (53) reported that the production cost of PHAfrom fermented foodwastewould be aboutUS $13per kg,meaning thatthe production costs would exceed the existing disposal costs, makingPHA production an unattractive option (53). Gurieff and Lant (55)

CURRENT TRENDS IN BIODEGRADABLE POLYHYDROXYALKANOA 625VOL. 110, 2010

conducted a comparative LCA and a financial analysis of mixed-culturePHA production. The analysis was performed for industrial wastewatertreatment technology, and the conditions used for mixed-culture PHAwere compared with biogas production that utilizes the same wasteresource. It was concluded that mixed-culture PHA production waspreferable to biogas production for treating the specified industrialeffluent, and it was financially attractive in comparison to pure-culturePHA production. In addition, the mixed-culture and pure-culture PHAproduction processes had similar environmental impacts that weresignificantly lower than HDPE production.

In 2009, Zhong et al. (56) investigated the environmental impacts ofthree manufacturing processes for PHA production. The processesstudied were microbial PHA manufacturing using (a) glucose as acarbon source, (b) cheese whey as a carbon source, and (c) PHAproduction using genetically engineered plants. These processes werecompared with respect to ten indicators under three categories (i)human health (carcinogenic effects, climate change, ozone layerdepletion, radiation, inorganic, and organic respiratory effects), (ii)ecosystem quality (acidification/nitrification and eco-toxicity), and (iii)sufficient supply of resources (fuels andminerals). Itwas concluded thatPHA manufacturing from transgenic plants was less beneficial over thefull cradle-to-gate life cycle than microbial PHA manufacturing withglucose andwhey. Themain cause of this high life cycle impactwas highsteam consumption. However, it was suggested that CO2 intake at thecorn forming stage canmore than compensate for its CO2 emissions, andits polymer extraction and compounding processes consumemuch lesspower than the fermentation and recovery processes in microbial PHAmanufacturing. Using a renewable energy resource would make PHAmanufacturing from transgenic plants significantly better. One recom-mended solution is using the residual corn stover, after extraction, forelectricity and steam (56). The available LCA results show that PHAscould have advantages over petrochemical polymers in severalenvironmental impact categories (cogeneration power plant, green-house gas credits etc.) but are less favorable in other categories(eutrophication, acidification, etc.). This is a common occurrence in LCAcomparisons of different raw materials and products. In addition, thedifferent environmental impact categories are usually not regarded asbeing of equal weight in terms of seriousness of the effect on theenvironment.Under the currentmanufacturing technology, a number ofLCA reports have indicated that petrochemical polymers can haveequivalent or better eco-profiles than PHAs. The main factors influenc-ing this assessmentwere energy for polymermanufacture, the effects ofthe number of recycling loops, and end-of-life disposal (especiallymethane generation in landfill). However, PHA manufacturing isgenerally small-scale at present. It is likely that as the scale increases,technical improvements and environmental economies of scale includ-ing recycling potential will accrue for PHAs. At this stage, PHAs couldcontribute to sustainability andhelp engage thepublic in environmentalawareness.

THE RESEARCH-TO-MARKET BOTTLENECK

PHA-producing microorganisms The microorganism of choicefor the industrial production of PHA varies depending on factors thatinclude the cell's ability to utilize an inexpensive carbon source (recentattention has been paid to agricultural wastes and industrial by-products), the cost of the medium, the growth rate, the polymersynthesis rate, the quality and quantity of PHAs, and the cost ofdownstream processes (57). Although more than 300 differentmicroorganisms synthesize PHAs (3–6), only a few, such as Cupriavi-dus necator (formerly known as Ralstonia eutropha or Alcaligeneseutrophus), Alcaligenes latus, Azotobacter vinelandii, Pseudomonas oleo-vorans, Paracoccus denitrificans, Protomonas extorquens, and recombi-nant E. coli, are able to produce sufficient PHA for large-scale production

(58). Some examples of patented PHA-producing bacterial strainscurrently used in industrial-scale production are reviewed below.

In 1970, ICI Ltd. initiated the production of PHB and PHBV on anindustrial 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 wascarried out in a two-step fed-batch culturewith glucose as the sole carbonsource, andphosphateas the limitingelement toenhancePHBproduction.The final biomass was 100 g/l with a productivity of 2.5 g/l h. The PHBVwas produced by providing amixed feed of glucose and propionic acid inthe 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 MonsantoCompany in the U.S., which stopped the production of PHB and PHBV atthe endof 1998 (62).Metabolix, Inc. developed recombinantE. coliK12 forthe overexpression of PHB, and this bacterial strain can produce 100 g/l ofPHB in 40 h (63).

Another industrial process for the production of PHB wasdeveloped at the Biotechnologische Forchungsgesellchaft Companyin 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 one-step fed-batch fermentation process using a mineral-salt mediumwith sucrose as the sole carbon source. Although a biomass density of60 g/l was achieved, the company stopped the production of PHB in1993 (65).

Several academic research groups have focused on producing PHAwith high productivity and high yield to reduce the overall costs. Fed-batch and continuous cultivation have been carried out to improveproductivity, either because of the difficulty of developing cultivationstrategies using unusual or expensive carbon substrates, or due to thelack of applications to justify the high production costs (66–71). Severalstrategies have been developed in academia for the efficient productionof PHA employing C. necator H16. By maintaining the glucoseconcentration at 10–20 g/l during a fed-batch culture, a total biomassof 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 beproduced by feeding a mixture of glucose and propionic acid (68).Several carbon sources other than glucose have also been used assubstrates for PHA production. Ethanol was tested for the production ofPHB by a mutant strain of C. necator, but the highest concentration ofPHB obtained was 47 g/l (72).

Several researchers (73–77) have tried to improve PHB productionby controlling the fermentation conditions. They developedmathemat-ical models to maximize PHB production and used suboptimalprocedures such as maintaining a constant nutrient concentration.The optimal feed profiles of glucose and ammonium hydroxide werecalculated using a model, and a final cell concentration of 141 g/l and aPHB concentration of 105 g/l in 40 h of fed-batch culture were achievedwith C. necator, for a productivity of 2.63 g/l h (74). To date, the highestPHB productivity (of 5.13 g/l h) was obtained in the short cultivationtime of 16 h using a fed-batch culture of A. latus DSM1123 undernitrogen limitation (78).

In addition to high productivity, one concern (particularly in PHBVproduction) is the rigorous control of HV content by the development ofan on-line feeding strategy for fed-batch cultivation. A series of studiesdeveloped a novel multivariable control strategy for alcohol (ethanoland n-pentanol) concentrations during the production of PHBV by P.denitrificans ATCC 1774. This development coincided with an improve-ment of thequality of PHAs produced. A simplemetabolic reaction (MR)model based on flux distribution analysis was constructed (75,76,79).Fig. 1 shows the metabolic map for PHBV biosynthesis in P. denitrificansATCC17741 based on the consumption rates of ethanol and n-pentanolas described previously (75). Next, a model predictive controller (MPC)was developed and used for the on-line estimation of the specific

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

626 CHANPRATEEP J. BIOSCI. BIOENG.,

consumption rate and control of the alcohol concentration in fed-batchcultures. TheMulti-Input andMulti-Output (MIMO) controller success-fully controlled alcohol concentration, and the 3HV mole fraction unitsreached a given set point in the range of 0–70 mol%. The MPCwas usedto optimize PHBV production with a target mole fraction of 3 HV units.The amount of PHBVwasmaximized with a givenmole fraction of 3 HVunits at the final cultivation step (76). Afterward, the random and blockpolymers of PHBV that produced in a range of 0–90 mol% using two

FIG. 2. Control results of ethanol and n-pentanol concentrations (a) and 3HV mole fractionmicrographs of the surface of film made of random PHBV consisting of 5 mol% 3HV units (c

feeding strategies were characterized using a non-isothermal kineticsstudy. The exponent function quantified by Osawa's equation in blockPHBV was similar to that for PHB because the majority of thecomponents in the block polymer were the 3HB unit. The rate of non-isothermal crystallization of block PHBVwas faster than that of randomPHBV but slower than that of PHB. Using the conventional method, thesimultaneous feeding of ethanol and n-pentanol and theMPC system tocontrol alcohol concentrations precisely, the availability of ethanol and

units (b) by Multi-Input and Multi-Output Controller under C/N of 50 (74). The SEM) and 3-D images obtained from AFM analysis (d) (81).

CURRENT TRENDS IN BIODEGRADABLE POLYHYDROXYALKANOA 627VOL. 110, 2010

n-pentanol can be maintained throughout the cultivation time. Therecent evidence from PHA samples produced by the optimizationmethod has tended to demonstrate the possibility of producing blockPHBV. The combined strategy may affect the polymerization step bynatural dynamic changes of preferential consumption between ethanoland n-pentanol due to the switching of the carbon to nitrogen (C/N)ratio and the feeding strategy (80,81). Finally, their biodegradation rateswere also compared in an in vitro study. Scanning electron microscopy(SEM) and trappingmodeatomic forcemicroscopy (AFM)wereused forhigh resolution imaging of the surface structure (Figs. 2 and 3). It wasobserved that the surfaces of block PHBV samples are remarkablydifferent from those of random PHBV samples. The highest degradationrate (of 2.6% per day) was observed for random PHBV produced by theconventional method consisting of 38 mol% of HV units. The degrada-tion rates of random PHBVs were about two times faster than those ofPHBVs produced by the optimized method (81). These observationsindicate that the development of a feeding strategy could affect thequality of PHA produced and in turn change the physical and biologicalproperties of the biopolymer.

Newbacterial strains identified in the laboratory have been reportedregularly, but research groups have struggled to optimize PHAproduction with inexpensive carbon sources. There have been a fewreports on the production of the terpolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-4-hydroxybutyrate), P(3HB-co-3HV-co-4HB).Chanprateep and Kulpreecha (82) were the first to report the pro-duction and characterization of terpolymer P(3HB-co-3HV-co-4HB) bythe newly isolated C. necator strain A-04. A transmission electronmicrograph of an ultra-thin section of C. necator strain A-04 containingPHB granules is shown in Fig. 4. The highest terpolymer content, 68%,was produced at 60 h. The terpolymer with the highest (93 mol%) 4HB

FIG. 3. Control results of ethanol and n-pentanol concentrations (a) and 3HV mole fractimicrographs of the surface of film made of block PHBV consisting of 7 mol% 3HV units (c) a

mole fraction units was produced when the cultivation time wasextended 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'smodulus of 127 MPa, similar to those of low-density polyethylene, asshown in Table 5. The terpolymer P(11%3HB-co-34%3HV-co-55%4HB)showed aYoung'sModulus of 618 MPa, similar to that of polypropylene,as shown in Table 6 (82). The kinetics of P(3HB-co-4HB) production byC. necator strain A-04 was also investigated in detail; the synthesis of4HB units was growth-associated under nitrogen-sufficientconditions. Fig. 5 shows the effect of the C/N ratio on the relationshipbetween specific growth rate and specific production rate of PHAwhenthe ratio of γ-hydroxybutyric acid in the feed was between 50 wt.% and95 wt.%. The synthesis of 3HB units was enhanced under nitrogen-deficient conditions. The mole fraction of 4HB units could be changedfrom 0 to 70 mol% by adjusting the ratio of substrate and the mole ratioof C/N. The total P(3HB-co-4HB) content was 71 wt.% with a 4HB unitmole fraction of 30 mol% in the copolymer (83).

SELECTION OF CARBON SOURCES

There are a number of literature reviews on the selection of suitablecarbon sources for efficient PHA production, for which the total cost ofbioprocessing must meet economic requirements (5,10,11,13). Themost frequently reported factor that influences the price of PHA is thecost of the carbon source. Fortunately, most microorganisms aresaprophytes that can metabolize a wide range of carbon sources.However, the selection of carbon sources should not focus only onmarket prices but also on availability and global price consistency. Inaddition, inexpensive carbon sources such as agricultural wastes andindustrial by-products may incur additional costs due to pre-treatment

on units (b) by a Model Predictive Controller under optimal strategy (74). The SEMnd 3-D images obtained from AFM analysis (d) (81).

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 μm. Bar, 1 μm. (b) C. necator strain A-04 containing PHB granules (white fractions). Bar, 200 nm (83).

TABLE 6. Mechanical properties of terpolymer produced by C. necator strain A-04comparing with P(3HB-co-3HV) and P(3HB-co-4HB) (82).

PHAs composition Toughness(MPa)

Young's modulus(MPa)

Tensile strength(MPa)

Elongation(%)

P(3HB) – 3500 40 0.4P(3HB-co-3HV)3% 3HV – 2900 38 –

9% 3HV – 1620 190 3714% 3HV – 1500 150 3520% 3HV – 1450 120 3225% 3HV – 1370 70 30

P(3HB-co-4HB)3% 4HB – – 28 4510% 4HB – – 24 24216% 4HB – – 26 44444% 4HB – – 10 51164% 4HB – 30 17 59190% 4HB – 100 65 1080

P(3HB-co-3HV-co-4HB)10% 3HB 40%3HV 50% 4HB

0.22 503 9 4

11% 3HB 34%3HV 55% 4HB

0.26 618 10 3

11% 3HB 23%3HV 66% 4HB

0.32 392 9 5

12% 3HB 12%3HV 76% 4HB

0.39 142 4 9

10% 3HB 6% 3HV84% 4HB

20 118 9 300

4% 3HB 3% 3HV93% 4HB

33 127 14 430

Petroleum based plasticPlactic bag (HDPE) 62 640 19 576Plactic bag (PP) 64 590 27 435Plactic bag (LDPE) 15 156 13 126UV degradable bag 60 674 24 384

628 CHANPRATEEP J. BIOSCI. BIOENG.,

steps, extended cultivation times, and purification. Simple carbonsources such as sugar and starch from crops seem to be superior tocomplex carbon sources, but they are also a primary source of humanfood and animal feed (84). Because the price of fossil fuels continues toincrease, using some of these carbohydrates for biofuel production isunavoidable. Given the limits on agricultural capacity and the increasingdemand for crops as bioenergy resources, researchers need to seekwaysof using non-food crops for biodegradable production (85). Below, Iprovide information on conventional raw materials and optional rawmaterials for PHA production. Table 7 shows the current global marketprices of substrates and their theoretical PHB yields.

Sugars Carbon sources are needed by all living organisms, andsugars 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%) Tg (°C) Tm (°C) MW MN PDI

PHB 10 177 – 7.82×105 1.8P(3HB-co-3HV)24% 3HV −6 138 – 1.38×105 1.945% 3HV −10 75 – 4.00×105 2.471% 3HV −13 87 – 2.54×105 2.0P(3HB-co-4HB)11% 4HB – 160 – 2.23×105 2.517% 4HB – 152 – 3.32×105 2.482% 4HB – 40 – 1.29×105 2.4P(3HB-co-3HV-co-4HB)10% 3HB 40% 3HV 50% 4HB −13.7 87.6 1.10×106 9.67×105 1.1311% 3HB 34% 3HV 55% 4HB −15.9 99.9 5.98×105 2.99×105 2.0011% 3HB 23% 3HV 66% 4HB −17.7 91.8 6.62×105 1.90×105 3.4612% 3HB 12% 3HV 76% 4HB −21.1 87.3 5.51×105 1.73×105 3.1810% 3HB 6% 3HV 84% 4HB −47.1 54.3 1.77×105 6.27×104 2.824% 3HB 3% 3HV 93% 4HB −51.6 54.8 4.15×105 1.33×105 3.11

carbon sources for large-scale PHB production, due to its availability inthe market. Metabolix, Inc. and ADM selected corn syrup to produceMirel™ resin from recombinant E. coli using a fully biological fermen-tation process. In academic research, the greatest success with PHAproduction from sugar has been reported by Kim et al. (86) They usedrecombinant 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 whilemaintaining the specific growth rate at 0.1 h−1. The kinetic information isfully available and ready for industrial-scale production (86).

Starches Starches are also an abundant carbon source. Manyresearch groups are interested in utilizing starch for PHA production.Starch has also been used as a bio-based polymer for various types ofbioplastics. However, microorganisms often display low productivitywhen grown directly on raw starch, and additional costs fromliquefaction and saccharification steps limit the potential use of starch.R. eutropha NCIMB 11599 was able to produce PHB from saccharifiedpotato starch waste with phosphate limitation. The biomass producedwas 179 g/l and 94 g/l of PHB for a productivity of 1.47 g/l h (87). Kimand 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, PHBcontent reached 44% with O2 limitation. In fed-batch cultures, 71 g/l ofbiomass with 20% PHB was obtained without O2 limitation, whereas54 g/l of biomass with 46% PHB was obtained with O2 limitation (38).Extruded rice branwas 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 arepeated fed-batch fermentation. In 2006, Huang et al. (88) reportedthat 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 producePHB. It was found that the isolated Bacillus cereus could secrete the

FIG. 5. The effect of C/N ratio on the relationship between μ and ρ when the ratio of γ-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 ofcopolymer of P(3HB-co-4HB) (83).

TABLE 7. The global market prices of substrates in 2010 and estimated substrate cost forproduction of PHA based on yield (59,98,101).

Carbon source Price per kg Yield of PHB (g g−1) Cost of C-source per kg of PHA

Sucrosea €0.35 0.40 €0.87Glucoseb €0.41 0.38 €1.07Ethanolc €0.31 0.50 €0.63Methanold €0.28 0.43 €0.58Cassava starche €0.19 0.20 €0.94Cane molassesb €0.10 0.42 €0.24Palm oil f €0.79 0.65 €1.22Soya oilg €0.92 0.70 €1.31

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

CURRENT TRENDS IN BIODEGRADABLE POLYHYDROXYALKANOA 629VOL. 110, 2010

enzyme amylase and simultaneously produce PHB at 0.48 g/l (89);however, industrial-scale production still awaits optimization of thesystem's productivity.

Alcohols Alcohols are sterile carbon substrates, and PHA fermen-tation processes using alcohols as substrates could possibly reduce thechance of contamination. There is considerable experience in methanolfermentation technology because methlylotrophic bacteria have beenconsidered for the large-scale industrial production of single-cellproteins. Some methalotrophic and methanotrophic bacteria arepromising for PHB production purposes. Pseudomonas sp. K wascultivated in a fed-batch process with nitrogen deficiency to a celldensity as high as 233 g/l dry-cell weight with a PHB content of 64%,resulting in a product yield of 0.2 g g−1 (48). P. extorquenswas grown tocell densities of 190 g/l dry-cell weight with a PHB content of 60% (67).The methlylotrophic bacteria P. denitrificans andM. extorquens are ableto synthesize copolyester PHBVwhenmethanol and n-amyl alcohol are

630 CHANPRATEEP J. BIOSCI. BIOENG.,

fed simultaneously in nitrogen-limited medium. The 3HV contentreached 91.5 mol% depending on the ratio of substrate used (76,81,90).P. denitrificansATCC 17741 can produce a homopolymer of PHV (24%w/w)whengrownonn-pentanol (91). Amicrocomputer-aided, automaticfed-batch culture system under potassium-limited conditions was setup for PHB production with M. organophilum using methanol as thesubstrate (92). However, alcohols have become very important asalternative energy sources to replace fossil oils.

Industrial by-products Molasses, an industrial by-product ofsugar production, is one potentially inexpensive carbon source for PHAproduction. However, molasses values over the past year have risen tounprecedented 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 explainthe surge in prices. At the same time, the expanding use of molasses infuel ethanol programs is boosting global molasses demand. Molassesproduction in Pakistan and Thailand for 2009–2010 is forecasted to riseonly slightly (Quarterly Market Outlook, International Sugar Organiza-tion, November 2009). Chen et al. (93) reported on the production ofPHB from beet molasses by an A. vinelandii UWD mutant in two-stagefed-batch cultures. The amount of PHB produced was 36 g/l withproductivity higher than 1 g/l h (93). Liu et al. (94) have successfullyreplaced glucose with beet molasses to produce PHB by a recombinantEscherichia coli strain (HMS174/pTZ18u-PHB). The final dry-cell weight,PHB content, and PHBproductivitywere 39.5 g/l, 80% (w/w), and1 g/l hafter 31.5 h of fed-batch fermentation with constant pH and dissolvedO2 content (94).

There are a number of reports on using cane molasses for PHAproduction. Gouda, et al. (95) reported that Bacillus megaterium wasable to produce PHB from cane molasses in shaken flask cultivation.Briefly, 46.2% PHBwas producedwhen 3% (w/v) of sugar canemolasseswas supplied (95). Kulpreecha et al. (96) reported an attempt toproduce PHB from sugar cane molasses by Bacillus sp. BA019. Theoptimal feeding medium in this system required a higher total sugarconcentration of 400 g/l and a C/N molar ratio of 10. Under theseconditions, thebiomasswas72.6 g/l andPHBproductivitywas1.27 g/l h(96). However, the cost of downstream decolorization processesbecomes the limiting factor for cost competitiveness. In addition, therecent trend toward utilizing molasses was based on projected profitsfrom bioethanol production.

Fatty acids and vegetable oils are also potential substrates for PHAproduction because it was reported that the theoretical yield of PHAproduction from fatty acids is 0.65 g g−1 (97), whereas the theoreticalyield of PHA production from glucose ranges between 0.30–0.40 g g−1

(98). It was reported that Rhodobacter sphaeroides (IFO 12203) couldutilize palm oil mill for PHA production, and a PHA yield of 0.50 g g−1 oforganic acid consumed was obtained, resulting in PHA content of 67%(99). Kahar et al. (100) reported the use of soybean oil for PHAproductionby C. necator H16 and its recombinant strain PHB-4 harboring the Aero-monas caviae PHA synthase gene. Theproductionof PHAbyC. necatorH16was 118–126 g/l, and the PHB content was 72–76%. The recombinantstrain could produce PHA from soybean oil with high cell density (128–138 g/l) and high PHA content (71–74%). The yield obtained from theseexperiments 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 itproduced 37.34% PHA that consisted of 3-hydroxyoctanoic, 3-hydro-xydecanonic, and 3-hydroxydodecanoic. Strain DR2 also could utilizevegetable oil as a carbon source for PHA production with a content of23.52% (16). Recently, waste glycerol, a by-product from the biodieselindustry, has received much interest from many researchers as aninexpensive organic source for PHAproduction. As reported byCavalheiroet al. (17),C. necatorwasused toproduce PHBusingwaste glycerol. A totalof 68.8 g/l of biomass was obtained with a PHB content of 38%. Finally, bydecreasing the biomass concentration, a PHB content of 50% with aproductivity of 1.1 g/l h was achieved (17). It is therefore reasonable to

develop bioprocesses based onwaste carbon resources. Finally, theworldmarket price of each substrate and the estimated substrate cost based onthe PHA production yield are listed in Table 7.

PHA is undoubtedly one of the potential candidates for replacingpetroleum-based plastics. However, only a few biodegradable plasticproducts made from PHAs are available on the market (Mirel™,Biocycle™, Biomer™, etc.). Although the current price of PHA is anongoing impediment to its widespread use, the additional cost provides acompletelybiodegradableproduct that leaves zerohazardouswaste in theenvironment. National polices and legal measures are also importantfactors that contribute to market initiatives for PHA. InternationalStandards (ISO) and Certified Labeling and Environmental ProductDeclarations (EPD) are required to communicate the environmentalcredentials of PHAs to consumers and businesses. Further research andtechnical development is needed to build awider range of applications, tocharacterize the greenhouse gas emissions fromPHAs, to obtain newdatafor LCA models of the environment impacts of PHAs as feedbackinformation for further research, and to develop new polymers that canbe recycled after a first use. Altogether, the prospects for PHAs look bright.The potential usefulness of PHAs should expand further as new marketniches open.

ACKNOWLEDGMENTS

The author wishes to express her appreciation to the Society forBiotechnology (Japan), which awarded the Young Asian Biotechnol-ogist Prize to her in 2009. The research done for this review wassupported in part by the “Joint Program in the Field of Biotechnology”under the National Research Council of Thailand, the National Scienceand Technology Development Agency of Thailand, and the JapanScience and Technology Agency. The author would like to extend herrespectful thanks to Professor Suteaki Shioya (Sojo University),Professor Hiroshi Shimizu (Osaka University), Professor Takuya Nihira(Osaka University), Professor Wattanalai Panbangred (MahidolUniversity), and Professor Yoshio Katakura (Kansai University) fortheir invaluable discussions and supports.

References

1. Steinbüchel, A.: PHB and other polyhydroxyalkanoic acids, in: H. J. Rehm, G.Reed (Eds.), Biotechnology, vol. 6, VCH Publishers, New York, pp. 405–464(1996).

2. James, B. W., Mauchline, W. S., Dennis, P. J., Keevil, C. W., and Wait, R.: Poly-3-hydroxybutyrate in Legionella pneumophila, an energy source for survival in low-nutrient environments, Appl. Environ. Microbiol., 65, 822–827 (1999).

3. Steinbüchel, A. and Valentin, H. E.: Diversity of bacterial polyhydroxyalkanoicacids, FEMS Microbiol. Lett., 128, 219–228 (1995).

4. Kim, Y. B. and Lenz, R. W.: Polyesters from microorganisms, in: T. Scheper (Ed.),Advances in Biochemical Engineering/Biotechnology, vol. 71, Springer-VerlagBerlin Heideberg, Germany, pp. 52–79 (2001).

5. Lenz, R. W. and Merchessault, R. H.: Bacterial polyesters: biosynthesis,biodegradable plastics and biotechnology, Biomacromolecules, 6, 1–8 (2005).

6. Hazer, B. and Steinbüchel, A.: Increased diversification of polyhydroxyalkano-ates by modification reactions for industrial and medical applications, Appl.Microbiol. Biotechnol., 74, 1–12 (2007).

7. Jendrossek, D. and Handrick, R.: Microbial degradation of polyhydroxyalk-anoates, Annu. Rev. Microbiol., 56, 403–432 (2002).

8. Lim, S. P., Gan, S. N., and Tan, I. K. P.: Degradation of medium-chain-lengthpolyhydroxyalkanaotes in tropical forest and mangrove soils, Appl. Biochem.Biotechnol., 126, 23–33 (2005).

9. Sridewi, J., Bhubalan, K., and Sudesh, K.: Degradation of commercially importantpolyhydroxyalkanoates in tropical mangrove ecosystem, Polym. Degrad. Stab., 91,2931–2940 (2006).

10. Lee, S. Y. and Chang, H. N.: Production of poly(hydroxyalkanoic acid), p. 27–58,in: A. Fiechter (Ed.), Advances in Biochemical Engineering/ Biotechnology, vol.71, Springer-Verlag Berlin Heideberg, Germany (1995).

11. Lee, S. Y., Choi, J., and Wong, H. H.: Recent advances in polyhydroxyalkanoateproduction by bacterial fermentation: mini review, Int. J. Biol. Macromol., 25,31–36 (1990).

CURRENT TRENDS IN BIODEGRADABLE POLYHYDROXYALKANOA 631VOL. 110, 2010

12. Wong, A. L., Chua, H., and Yu, P. H. F.: Microbial production of polyhydroxyalk-anoates by bacteria isolated from oil wastes, Appl. Biochem. Biotechnol., 84,843–857 (2000).

13. Tsuge, T.: Metabolic improvements and use of inexpensive carbon sources inmicrobial production of polyhydroxyalkanoates, J. Biosci. Bioeng., 94, 579–584(2002).

14. Dionisi, D., Carucci, G., Papini, M. P., Riccardi, C., Majone, M., and Carrasco, F.:Olive oil mill effluents as a feedstock for production of biodegradable polymer,Water Res., 39, 2076–2084 (2005).

15. Verlinden, R. A. J., Hill, D. J., Kenward, M. A., Williams, C. D., and Radecka, I.:Bacterial synthesis of biodegradable polyhydroxyalkanoates, J. Appl. Microbiol.,102, 1437–1449 (2007).

16. Song, J. H., Jeon, C. O., Choi, M. H., Yoon, S. C., and Park, W.: Polyhydroxyalk-anoate (PHA) production using waste vegetable oil by Pseudomonas sp. strainDR2, J. Microbiol. Biotech., 18, 1408–1415 (2008).

17. Cavalheiro, J. M. B. T., Almeida, M. C. M. D., Grandfils, C., and Fonseca, M. M. R.:Poly(3-hydroxybutyrate) production by Cupriavidus necator using waste glycerol,Process Biochem., 44, 509–515 (2009).

18. Madison, L. L. and Huisman, G. W.: Metabolic engineering of poly(3-hydroxyalk-anoates): from DNA to plastic, Microbiol. Mol. Rev., 63, 21–53 (1999).

19. Steinbüchel, A. and Hein, S.: Biochemical and molecular basis of microbialsynthesis of polyhydroxyalkanoates in microorganisms, in: T. Scheper (Ed.),Advances in Biochemical Engineering/Biotechnology, vol. 71, Springer-VerlagBerlin Heideberg, Germany, pp. 81–123 (2001).

20. Steinbüchel, A.: Perspectives for biotechnological production and utilization ofbiopolymers: metabolic engineering of polyhydroxyalkanoate biosynthesis path-ways as a successful example, Macromol. Biosci., 1, 1–24 (2001).

21. Sang, J. S., Snell, K. D., Kim, B. W., Rha, C. K., and Sinskey, A. J.: Increased poly-β-hydroxybutyrate (PHB) chain length by the modulation of PHA synthase activityin recombinant Escherichia coli, Biotechnol. Lett., 23, 2057–2061 (2001).

22. Aldor, I. S. and Keasling, J. D.: Process design for microbial plastic factories:metabolic engineering of polyhydroxyalkanoates, Curr. Opin. Biotechnol., 14,475–483 (2003).

23. Lee, S. Y.: Deciphering bioplastic production, Nat. Biotechnol., 10, 1227–1229(2006).

24. Pohlmann, A., Fricke, W. F., Reinecke, F., Kusian, B., Liesegang, H., Cramm, R.,Eitinger, T., Ewering, C., Pötter, M., and Schwartz, E., et al.: Genome sequence ofbioplastic-producing “Knallgas” bacterium Ralstonia eutropha H16, Nat. Biote-chol., 24, 1257–1262 (2006).

25. Kim, D. Y., Kim, H. W., Chung, M. G., and Rhee, Y. H.: Biosynthesis, modification,and biodegradation of bacterial medium-chain-length polyhydroxyalkanoates,J. Microbiol., 45, 87–97 (2007).

26. Parveez, G. K. A., Bohari, B., Ayub, N. H., Yunus, A.M.M., Rasid, O. A., Hashim, A. T.,Ishak, Z., Manaf, M. A. A., Din, A. K., York, G., Jo, Y. B., and Sinskey, A. J.:Transformation of PHB and PHBV genes driven bymaize ubiquitin promoter into oilpalm for the production of biodegradable plastics, J. Oil Palm Res., S2, 77–86 (2008).

27. Avella, M., Bonadies, E., and Martuscelli, E.: European current standardizationfor plastic packaging recoverable through composting and biodegradation, Polym.Test., 20, 517–521 (2001).

28. Averous, L.: Biodegradable multiphase systems based on plasticized starch,polymer reviews, J. Macromol. Sci. Polymer. Rev., C44, 231–274 (2004).

29. Sudesh, K. and Iwata, T.: Sustainability of biobased and biodegradable plastics,Clean-Soil Air Water, 36, 433–442 (2008).

30. Funabashi, M., Ninomiya, F., and Kunioka, M.: Biodegradability evaluation ofpolymer by ISO 14855-2, Int. J. Mol. Sci., 10, 3635–3654 (2009).

31. Holmes, P. A.: Applications of PHB — a microbially produced biodegradablethermoplastic, Phys. Tech., 16, 32–36 (1985).

32. Somleva, M. N., Snell, K. D., Beaulieu, J. J., Peoples, O. P., Bradley, R., Garrison,B. R., and Patterson, N. A.: Production of polyhydroxybutyrate in switchgrass, avalue-added co-product in an important lignocellulosic biomass crop, PlantBiotechnol., 6, 663–678 (2008).

33. Kosior, E., Braganca, R. M., and Fowler, P.: Lightweight compostable packaging:literature review, The Waste & Resource Action Program, INN003/26, pp. 1–48(2006).

34. Schmack, G., Jehnichen, D., Vogel, R., and Tandler, B.: Biodegradable fibers ofpoly(3-hydroxybutyrate) produced by high speed melt spinning and spindrawing, J. Polym. Sci. Part B, 38, 2841–2850 (2000).

35. Pessoa-Jr,A., Roberto, I. C.,Menossi,M., Santos,R.R., Filho,S.O., andPenna, T. C.V.:Perspectives on bioenergy and biotechnology in Brazil, Appl. Biochem. Biotechnol.,121, 59–70 (2005).

36. Archambault, É.: Canadian R&D Biostrategy, Towards a Canadian R&D Strategyfor Bioproducts and Bioprocesses, Prepared for the National Re-search Council ofCanada, Science-Metrix Canadian R & D Biostrategy, Canada (2004).

37. Shen, L., Haufe, J. and Patel, M.K.: Product overview and market projection ofemerging biobased plastics (PROBIP 2009). Commissioned by European Polysac-charide Network of Excellence (EPNOE) and European Bioplastics. Group Science,Technology and Society (STS), Copernicus Institute for Sustainable Developmentand Innovation, Utrecht University, Utrecht, The Netherlands, June 2009. ReportNo: NWS-E-2009-32 (2009).

38. Kim, B. S. and Chang, H. N.: Production of poly (3-hydroxybutyrate) from starchby Azotobacter chroococcum, Biotechnol. Lett., 29, 109–112 (1998).

39. Bishop, P. L.: Pollution prevention, Fundamentals and Practice, McGraw Hill,Boston, p. 717 (2000).

40. Giudice, F., Rosa, G. L., and Risitano, A.: Product Design for the Environment: LifeCycle Approach, CRC, Boca Raton, Taylor & Francis, FL (2006).

41. International Standard Organization (ISO): Environmental Management-LifeCycle Assessment-Principle and Framework, ISO14040, International StandardOrganization, Geneva (1997).

42. Franklin Associates Report: Resource and environmental profile analysis ofpolyethylene and unbleached paper grocery sacks, CSWS (800-243-5790)Prepared for Council for Solid Waste Solutions, Washington, DC (1990).

43. C. Chaffee, C.B. R. Yaros, B. R.: Life cycle Assessment for Three Types of GroceryBags — Recyclable Plastic; Compostable, Biodegradable Plastic; and Recycled,Recyclable Paper, Final Report Prepared for the Progressive Bag Alliance, BousteadConsulting & Associates Ltd. (2007).

44. M. Hyde, M.: Ecological considerations on the use and production of biosyntheticand synthetic biodegradable polymersPolym. Degrad. Stab., 59, 3–6 (1998).

45. Gerngross, T. U.: Can biotechnology move us toward a sustainable society? Nat.Biotechnol., 17, 541–544 (1999).

46. Grengross, T. U. and Slater, S. C.: How green are green plastics? Sci. Am., 283,36–41 (2000).

47. Akiyama, M., Tsuge, T., and Doi, Y.: Environmental life cycle comparison ofpolyhydroxyalkanoates produced from renewable carbon resources by bacterialfermentation, Polym. Degrad. Stab., 80, 183–194 (2003).

48. Harding, K. G., Dennis, J. S., von Blottnitz, H., and Harrison, S. T. L.:Environmental analysis of plastic production processes: comparing petroleum-based polypropylene and polyethylene with biologically-based poly-β-hydro-xybutyric acid using life cycle analysis, J. Biotechnol., 130, 57–66 (2007).

49. Kim, S. and Dale, B. E.: Lifecycle assessment study of biopolymer (polyhydroxyalk-anoates) derived from no-tilled corn, Int. J. Life Cycle Assess., 10, 200–210 (2005).

50. Kim, S. and Dale, B. E.: Energy and greenhouse gas profiles of polyhydrox-ybutyrates derived from corn grain: a life cycle perspective, Environ. Sci. Technol.,42, 7690–7695 (2008).

51. Pietrini,M., Roes, L., Martin, K., Patel,M. K., and Chiellini, E.: Comparative life cyclestudies on poly(3-hydroxybutyrate)-based composites as potential replacement forconventional petrochemical plastics, Biomacromolecules, 8, 2210–2218 (2007).

52. Reis, M. A. M., Serafim, L. S., Lemos, P. C., Ramos, A. M., Aguiar, F. R., and Vanloosdrecht, M. C. M.: Production of polyhydroxyalkanoates by mixed microbialcultures, Bioproc. Biosyst. Eng., 25, 377–385 (2003).

53. Rhu, D. H., Lee, W. H., Kim, J. Y., and Choi, E.: Polyhydroxyalkanoate (PHA)production from waste, Water Sci. Technol., 48, 221–228 (2003).

54. Khardenavis, A. A., Kumar, M. S., Mudliar, S. N., and Chakrabarti, T.:Biotechnological conversion of agro-industrial wastewaters into biodegradableplastic, poly β-hydroxybutyrate, Bioresour. Technol., 98, 3579–3584 (2007).

55. Gurieff, N. and Lant, P.: Comparative life cycle assessment and financial analysisof mixed culture polyhydroxyalkanoate production, Bioresour. Technol., 98,3393–3403 (2007).

56. Zhong, Z. W., Song, B., and Huang, C. X.: Environmental impacts of threepolyhydroxyalkanoate (PHA) manufacturing process, Mater. Manuf. Process., 24,519–523 (2009).

57. Lee, S. Y. and Choi, J.: Production of microbial polyester by fermentation ofrecombinant microorganisms, in: T. Scheper (Ed.), Advances in BiochemicalEngineering/Biotechnology, vol. 71, Springer-Verlag Berlin Heideberg, Germany,pp. 183–207 (2001).

58. S. Y. Lee, S. Y.: Plastic bacteria? Progress and prospects for polyhydroxyalkanoateproduction in bacteriaTrends Biotechnol., 14, 431–438 (1996).

59. Byrom, D.: Production of poly-β-hydroxybutyrate: poly-β-hydroxyvaleratecopolymers, FEMS Microbiol. Rev., 103, 207 (1992).

60. Schlegel, H. G. and Gottschalk, G.: Verwertung von Glucose durch eine Mutantevon Hydrogenomonas H16, Biochem. Z., 341, 249–259 (1965).

61. Byrom, D.: Industrial production of copolymer from Alcaligenes eutrophus, in: E. A.Dawes (Ed.), Novel Biodegradable Microbial Polymers, Kluwer, Dordrecht, p. 113(1990).

62. G. Q. Chen, G. Q.: Industrial production of PHAin: G. Q. Chen (Ed.), Plastics fromBacteria, Natural Functions and Applications, Microbiology Monographs, vol. 14,Springer-Verlag Berlin Heideberg, Germany, pp. 183–207 (2010).

63. S. Y. Lee, S. Y.K. M. Lee, K. M.H. N. Chan, H. N.A. Steinbüchel, A.: Comparison ofrecombinant Escherichia coli strains for synthesis and accumulation of poly-(3-hydroxybutyric acid) and morphological changesBiotechol. Bioeng., 44,1337–1347 (2004).

64. Hanggi, U. J.: Pilot scale production of poly(3HB) with Alcaligenes eutrophus, in:E. A. Dawes (Ed.), Novel Biodegradable Microbial Production, Kluwer,Dordercht, pp. 65–70 (1990).

65. O. Hrabak, O.: Industrial production of poly-β-hydroxybutyrateFEMS Microbiol.Rev., 103, 251–256 (1992).

66. Suzuki, T., Yamane, T., and Shimizu, S.: Mass production of poly-β-hydro-xybutyric acid by fully automatic fed-batch culture of methylotroph, Appl.Microbiol. Biotechnol., 23, 322–329 (1986).

632 CHANPRATEEP J. BIOSCI. BIOENG.,

67. Suzuki, T., Yamane, T., and Shimizu, S.: Mass production of poly-β-hydro-xybutyric acid by fed-batch culture with controlled carbon/nitrogen feeding,Appl. Microbiol. Biotechnol., 24, 370–374 (1986).

68. Kim,B. S., Lee, S. C., Lee, S. Y., Chang,H.N., Chang, Y.K., andWoo, S. I.:Productionofpoly(3-hydroxybutyric acid) by fed-batch cultures of Alcaligenes eutrophus withglucose concentration control, Biotechnol. Bioeng., 43, 892–898 (1994).

69. Kim, B. S., Lee, S. C., Lee, S. Y., Chang, H. N., Chang, Y. K., and Woo, S. I.:Production of poly(3-hydroxybutyric-co-3-hydroxyvaleric acid) by fed-batchculture of Alcaligenes eutrophus with substrate feeding using on-line glucoseanalyzer, Enzyme, Microbial. Technol., 16, 556–561 (1994).

70. Shimizu, H., Kozaki, Y., Kodama, H., and Shioya, S.: Maximum productionstrategy for biodegradable copolymer P(3HB-co-3HV) in fed-batch culture of Al-caligenes eutrophus, Biotechnol. Bioeng., 62, 518–525 (1999).

71. Choi, J. and Lee, S. Y.: Factors affecting the economics of poly hydroxyalkanoaesproduction by bacterial fermentation, Appl. Microbiol. Biotechnol., 51, 13–21 (1999).

72. Alderete, J. E., Karl, D. W., and Park, C. H.: Production of poly(hydroxybutryate)homopolymer and copolymer from ethanol and propanol in a fed-batch culture,Biotechnol. Prog., 9, 520–525 (1993).

73. Shimizu, H., Tamura, S., Shioya, S., and Suka, K.: Kinetic study of poly-D-(−)-3-hydroxybutyric acid (PHB)productionand itsmolecularweightdistributioncontrol ina fed-batch culture of Alcaligenes eutrophus, J. Ferment. Bioeng., 76, 465–469 (1993).

74. Jung, H. L., Juan, H., and Henry, C. L.: Experimental optimization of fed-batchculture for poly-β-hydroxybutyric acid production, Biotechnol. Bioeng., 56,699–705 (1997).

75. Chanprateep, S., Abe, N., Shimizu, H., and Shioya, S.: Multivariable control ofalcohol concentrations in the production of polyhydroxyalkanoates (PHAs) byParacoccus denitrificans, Biotechnol. Bioeng., 74, 116–124 (2001).

76. Chanprateep, S., Kikuya, K., Shimizu, H., and Shioya, S.: Model predictivecontrol for biodegradable polyhydroxyalkanoates production in fed-batchculture, J. Biotechnol., 95, 157–169 (2002).

77. Shimizu, H., Chanprateep, S., Kulpreecha, S., Boonruangthavorn, A., and Shioya, S.:Development of production processes of a biodegradable polymer, poly(3-hydro-xybutyrate-co-3-hydroxyvalerate): metabolic pathway analysis, quality control ofpolymer, and use of starch, in: T. Seki (Ed.), Biotechnology for Sustainable Utilizationof Biological Resources in the Tropics, 17, pp. 78–83 (2005).

78. F. Wang, F.S. Y. Lee, S. Y.: Poly(3-Hydroxybutyrate) production with highproductivity and high polymer content by a fed-batch culture of Alcaligenes latusunder nitrogen limitationAppl. Environ. Microbiol., 63, 3703–3706 (1997).

79. Shimizu, H.: Metabolic engineering — integrating methodologies of molecularbreeding and bioprocess systems engineering, J. Biosci. Bioeng., 94, 563–573 (2002).

80. Chanprateep, S., Kikuya, K., Seki, S., Takawa, S., Shimizu, H., and Shioya, S.: Non-isothermal crystallization kinetics of biodegradable random poly(3-hydroxybuty-rate-co-3-hydroxyvalerate) and block one, J. Chem. Eng. Jpn, 36, 639–646 (2003).

81. Chanprateep, S., Shimizu, H., and Shioya, S.: Characterization and enzymaticdegradation of microbial copolyester P(3HB-co-3HV)s produced by metabolicreaction model based system, Polym. Degrad. Stab., 91, 2941–2950 (2006).

82. Chanprateep, S. and Kulpreecha, S.: Production and characterization ofbiodegradable terpolymer poly(3-hydroxybutyrate-co-3-hydrobyvalerate-co-4-hydroxybutyrate) by Alcaligenes sp. A-04, J. Biosci. Bioeng., 101, 51–56 (2006).

83. Chanprateep, S., Katakura, Y., Shimizu, H., Visetkoop, S., Kulpreecha, S., andShioya, S.: Characterization of new isolated Ralstonia eutropha strain A-04 andkinetic study of biodegradable copolyester poly(3-hydroxybutyrate-co-4-hydro-xybutyrate) production, J. Ind. Microbiol. Biotechnol., 35, 1205–1215 (2008).

84. Langevelda, J. W. A., Dixonb, J., and Jaworskic, J. F.:Development perspectives ofthe biobased economy: a review, Crop Sci., 50, 142–151 (2010).

85. Mangan, C. L.: Non-food crops and non-food uses in EC research programs, FEMSMicrobiol. Rev., 16, 81–88 (2006).

86. Kim, B. S., Lee, S. C., Lee, S. Y., Chang, Y. K., and Chang, H. N.: High cell densityfed-batch cultivation of Escherichia coli using exponential feeding combined withpH-stat, Bioproc. Biosyst. Eng., 26, 147–150 (2004).

87. Haas, R., Jin, B., and Zepf, F. T.: Production of poly(3-hydroxybutyrate) fromwaste potato starch, Biosci. Biotechnol. Biochem., 72, 253–256 (2008).

88. Huang, T. Y., Duan, K. J., Huang, S. Y., and Chen, C. W.: Production ofpolyhydroxyalkanoates from inexpensive extruded rice bran and starch by Ha-loferax mediterranei, J. Ind. Microbiol. Biotechnol., 33, 701–706 (2006).

89. Halami, P. M.: Production of polyhydroxyalkanoate from starch by the nativeisolate Bacillus cereus CFR06, World J. Microbiol. Biotechnol., 24, 805–812(2008).

90. Ueda, S., Matsumoto, S., Takagi, A., and Yamane, T.: Synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) from methanol and n-amyl alcohol bythe methylotrophic bacteria Paracoccus denitrificans and Methylobacteriumextroquens, Appl. Environ. Microbiol., 58, 3574–3579 (1992).

91. Yamane, T., Chen, X. F., and Ueda, S.: Growth associated production of poly(3-hydroxyvalerate from n-pentanol by a methylotrophic bacterium Paracoccusdenitrificans, Appl. Environ. Microbiol., 62, 380–384 (1996).

92. Kim, S. W., Kim, P., Lee, H. S., and Kim, J. H.: High production of poly-β-hydroxybutyrate (PHB) from Methylobacterium organophilum under potassiumlimitation, Biotechnol. Lett., 18, 25–30 (1996).

93. Chen, G. Q. and Page, W. J.: Production of poly-β-hydroxybutyrate by Azotobactervinelandii in a two-stage fermentation process, Biotechnol. Techniques, 11,347–350 (1997).

94. Liu, F., Li, W., Ridgway, D., and Gu, T.: Production of poly-β-hydroxybutyrateon molasses by recombinant Escherichia coli, Biotechnol. Lett., 20, 345–348(1998).

95. Gouda, M. K., Swellam, A. E., and Omar, S. H.: Production of PHB by a Bacillusmegaterium strain using sugarcane molasses and corn steep liquor as sole carbonand nitrogen sources, Microbiol. Res., 156, 201–207 (2001).

96. Kulpreecha, S., Boonruangthavorn, A., Meksiriporn, B., and Thongchul, N.:Inexpensive fed-batch cultivation for high poly(3-hydroxybutyrate) produc-tion by a new isolate of Bacillus megaterium, J. Biosci. Bioeng., 107, 240–245(2009).

97. Yamane, T.: Yield of poly-D(−)-3-hydroxybutyrate from various carbon sources:a theoretical study, Biotechnol. Bioeng., 41, 165–170 (1993).

98. Babel, W., Ackermann, J. U., and Breuer, U.: Physiology, regulation, and limits ofthe synthesis of poly(3HB), in: T. Scheper (Ed.), Advances in BiochemicalEngineering/Biotechnology, vol. 71, Springer-Verlag Berlin Heideberg, Germany,pp. 125–157 (2001).

99. M. A. Hassan, M. A.Y. Shirai, Y.N. Kusubayashi, N.M. I. A. Karim, M. I. A.K.Nakanishi, K.K. Hashimoto, K.: Effect of organic acid profiles during anaerobictreatment of palm oil mill effluent on the production of polyhydroxyalkanoatesby Rhodobacter sphaeroidesJ. Ferment. Bioeng., 82, 151–156 (1996).

100. Kahar, P., Tsuge, T., Taguchi, K., and Doi, Y.: High yield production ofpolyhydroxyalkanoates from soybean oil by Ralstonia eutropha and its recombi-nant strain, Polym. Degrad. Stab., 83, 79–86 (2004).

101. Byrom, D.: Polymer synthesis by microorganisms: technology and economics,Tibtechnology, 5, 246–250 (1987).