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Biochem. J. (2009)418, 219232 (Printed in Great Britain) doi:10.1042/BJ20081769 219

REVIEW ARTICLE

The second green revolution? Production of plant-based biodegradableplastics

Brian P. MOONEY1

University of Missouri, Interdisciplinary Plant Group, Division of Biochemistry, and Charles W. Gehrke Proteomics Center, 214 Christopher S. Bond Life Sciences Center,1201 Rollins St, Columbia, MO 65211, U.S.A.

Biodegradable plastics are those that can be completely degradedin landfills, composters or sewage treatment plants by the actionof naturally occurring micro-organisms. Truly biodegradableplastics leave no toxic, visible or distinguishable residuesfollowing degradation. Their biodegradability contrasts sharplywith most petroleum-based plastics, which are essentially indes-tructible in a biological context. Because of the ubiquitous use ofpetroleum-based plastics, their persistence in the environment and

their fossil-fuel derivation, alternatives to these traditional plasticsare being explored. Issues surrounding waste management oftraditional and biodegradable polymers are discussed in the con-text of reducing environmental pressures and carbon footprints.The main thrust of the present review addresses the developmentof plant-based biodegradable polymers. Plants naturally producenumerous polymers, including rubber, starch, cellulose and stor-age proteins, all of which have been exploited for biodegradable

plastic production. Bacterial bioreactors fed with renewable re-sources from plants so-called white biotechnology have alsobeen successful in producing biodegradable polymers. In additionto these methods of exploiting plant materials for biodegradablepolymer production, the present review also addresses theadvances in synthesizing novel polymers within transgenic plants,especially those in the polyhydroxyalkanoate class. Althoughthere is a stigma associated with transgenic plants, especially

food crops, plant-based biodegradable polymers, produced asvalue-added co-products, or,from marginal land (non-food), cropssuch as switchgrass (Panicum virgatumL.), have the potential tobecome viable alternatives to petroleum-based plastics and anenvironmentally benign and carbon-neutral source of polymers.

Key words: biodegradable plastic, natural polymer, poly-hydroxyalkanoate, transgenic plant.

INTRODUCTION

The term plastic is defined as any of numerous organic syntheticor processed materials that are mostly thermoplastic or thermoset-

ting polymers of high molecular mass and that can be made intoobjects, films or filaments (MerriamWebster Dictionary defini-tion). The majority of plastics are synthetic, using petroleum bothas feedstock and as energy during manufacture. With the priceof oil rising and easily accessible reserves dwindling, alternativesources of fuel and of oil-based commodities such as plastics arebeing explored across the world. The environmental concerns ofthe oil-based economy are also being widely voiced as companiesand individuals attempt to reduce their carbon footprints.

Plastics are truly a huge industry. Data for first-quarter2008 U.S. production show that total output of all petroleum-based plastics exceeded 11.34 billion kg (25 billion lb; AmericanChemistry Council press release April 2008). With this volume ofproduction, the economies of scale become evident [for example,

polypropylene or polyethylene cost approx. 40 U.S. cents/lb or $1(or approx. 1.5/1.3)/kg]. With a market value of $100 billionper year, packaging accounts for almost one-third of plastic usein the United States, followed by construction and consumerproducts [1]. The simple paper or plastic decision one makes inthe supermarket eventually has consequencesfor the environment.These consequences have been recognized in the form of bans onplastic bags in many of theEU (EuropeanUnion) countries, China,

Australia, the City of San Francisco and an attempted ban in theState of California.

The search for alternatives to traditional petroleum-basedplastics is progressing to the point that not just the source,

but also the downstream consequences, are being addressed inthe form of biodegradable plastics. Although photodegradableplastics continue to be explored [2], these alternatives must beconstantly exposed to sunlight and so are not suitable for landfilldisposal. Biodegradability in composters or municipal landfillsis the goal. Even large chemical companies, like BASF with itsEcoflex product, are touting their biodegradable-plastics effortsto address the downstream consequences on the environment ofconventional plastics.

In this context, production of biodegradable plastics in plantsis an enviable goal. Plants naturally produce many polymers,such as starch or cellulose, and these have been exploitedfor plastics production. Additionally, novel plastics, like thePHAs (polyhydroxyalkanoates), are also being synthesized in

plants. Plants can be considered as solar-driven biofactories withthe potential for being renewable, sustainable, scaleable andrelatively environmentally benign sources of edible vaccines orplantibodies [3,4], novel oils and fatty acids (reviewed in [5]and [6]) and biodegradable plastic. The present review will detailthe efforts to produce biodegradable plastics in plants and will beextended to include the broader topics of biodegradable polymersderived from plant materials, including starch and cellulose.

Abbreviations used: ADM, Archer Daniels Midland; ASTM, American Society for Testing and Materials; CaMV, cauliflower mosaic virus; CAP,

cellulose acetate phthalate; CEN, European Committee for Standardization; ER, endoplasmic reticulum; EU, European Union; GMO, genetically

modified organism; HB, hydroxybutyrate; HV, hydroxyvalerate; IPP, isopentenyl diphosphate; PA, protective antigen; PCLH, polycaprolactone

(polyhexanolactone)/hexamethylene di-isocyanate; PDC, pyruvate dehydrogenase complex; PLA, poly(lactic acid); PHA, polyhydroxyalkanoate; PHB,

polyhydroxybutyrate; PHBV, PHB-co-polyhydroxyvalerate; SPI, soy protein isolate; UTR, untranslated region.1

email [email protected]

c The Authors Journal compilation c 2009 Biochemical Society

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220 B. P. Mooney

WASTE MANAGEMENT

Studies on the ecological footprint of humanity have suggestedthat sustainability of human pressures on the worlds ecologicalresources shifted from 70% of global regenerative capacity in1961 to 120% in 1999 [7]. This translates to a clear and simplefact: that we consume more than can be regenerated by thebiosphere. A clear example is crude oil, which obviouslycannot beregenerated on a human timescale. Petroleum-based plastics addto this ecological imbalance, both from a source point of view andfrom a waste management standpoint. Traditional plastics havebeen engineered to be stable in many types of environments andto persist for many years. This central purpose of plastics andtheir cost-effective production (i.e. that they are cheap) has led totheir ubiquitous use and is also the main reason that their disposalpresents such problems. The ubiquitous use of plastics has ledto their comprising about 12% of the 227 tonnes/metric tons(250 million short tons) of municipal waste produced annuallyin the United States. Recycling recovered about 30% of thisplastic from the wastestream, but that still allows plastics toaccumulate in the environment at a rate in excess of 18.2 metrictons (20 million short tons) per year (source: http://www.epa.gov/

epaoswer/non-hw/muncpl/msw99.htm). Similar values forplastic waste apply in the EU, where the typical generation ofsolid waste in 2001 was 520 kg/year per person, of which approx.1015% was plastics. Most of the EUs municipal waste is sentto landfills (45%), but almost 40% is recycled or composted,and a further 18% is used for energy production by incin-eration (source: http://www.eea.europa.eu/themes/waste/about-waste-and-material-resources). Most of the common types ofpetroleum-based plastic (e.g. polyethylene, PVC, polypropyleneand polystyrene) are non-degradable, although incorporatingstarch into polyethylene allows breakdown of the polymerstate (more correctly called disintegration rather than truebiodegradation) in composting environments [8]. Additionally,municipalsolidwaste(including plastics)is being testedfor power

generation due to a relatively new EU landfills directive [9]. InAsia, plastic waste is also being explored as a source of fuels forpower generation; for example, in Korea, polyethylene has beentested as an additive to coal-burning blast furnaces [10].

Although efforts have been explored in source reduction (i.e.replacing plastics at the manufacturing/packaging stage), in mostcases recycling and disposal are the main ways in which plasticsare dealt with post-consumer. A clear example of one of the morerecent increases in use of plastics is bottled-water sales. AnnualU.S. sales of bottled water increased almost threefold between1997 and 2006, growing from a $4 billion to a $10.8 billionindustry. In the space of 20 years (19872006), U.S. per capitaconsumption of bottled water increased from 21.6 to 104.5 litres(5.7 to 27.6 U.S. gallons) [11]. Unfortunately, many water and

soda bottles are used on the go and so are more likely toend up in ordinary rubbish collections rather than separated forrecycling. Although there have been some ingenious strategiesfor separation of different types of plastics using electrostatics[12] and hydraulics [13], consumer separation of classes ofplastics and recycling them in bulk is typically the best courseof action. However, even with recycling, traditional plastics havea finite re-usability and eventually are only suitable for bulkapplications such as engineered lumber, WPC (wood plasticcomposite) [14] or additives to concrete [15].

Clearly a source of biodegradable plastics is an obviousalternative. A number of countries have implemented certificationstandards for biodegradability, with concomitant labelling basedon their degradability and with an emphasis on composting

environments (Figure 1). North America, Europe, and some

Figure 1 Labelling standards for biodegradable plastics

The United States, the European Union and countries in Asia have specific requirements forbiodegradable polymer testing and certification. Companies producing biodegradable polymerscan submit the products for independent testing and certification through the US CompostingCouncil or the Biodegradable Products Institute in New York, Vincotte in Brussels, DIN(Deutsches Institut fur Normung) Certco in Berlin, the European Bioplastics Association andthe Biodegradable Plastic Society in Japan. Their labels are based on the ASTM 6400-99and EN 13432 standards for biodegradable and compostable plastics. As examples, the Figureshows logos issued by Vincotte in Brussels, the European Bioplastics Association and theUS Composting Council/the Biodegradable Products Institute, and are reproduced with theirpermission.

countries in Asia have enforced standard product testing to achievecertification for labelling based on the ASTM/CEN (AmericanSociety for Testing and Materials/European Committee forStandardization) system of classification. The ASTM 6400-99 designation covers standard specification for compostableplastics and EN 13432 covers proof of compostability of plasticproducts. Two pertinent definitions from the ASTM standard areas follows:

biodegradable plastic: a degradable plastic in which thedegradation results from the action of naturally occurringmicro-organisms such as bacteria, fungi and algae

compostable plastic: a plastic that undergoes degradationby biological processes during composting to yield CO2,water, inorganic compounds, and biomass at a rate consistentwith other compostable materials and leaves no visible,distinguishable, or toxic residue [16]

The residue clause in the second definition is an importantdistinction that favours truly biodegradable plastics rather thanblended materials that disintegrate or partially biodegrade.

PLANT-BASED BIODEGRADABLE PLASTICS

Although plant-based biodegradable plastics are not new, thecurrent interest in green technologies has lead to a renewed

interest in using plantsfor a number of applications. Many of these


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Plant-based biodegradable plastics 221

Figure 2 Natural polymers: rubber and proteins

(A) Natural rubber is a polymer of isoprene units arranged incis-1,4 linkages. Natural rubber(unlike synthetic) also has minor protein, lipid, carbohydrate and mineral components thatare responsible for its properties of resistance to abrasion and impact, elasticity, efficient heatdispersal, and resilience and malleability at cold temperatures. (B) Gluten is a polymer ofglutenin that has inter- and intra-disulfide bonds that promote elasticity of the gluten polymer.The gliadin proteins are thought to intercalate within the glutenin matrix and are held in placeby hydrophobic interactions and hydrogen bonding.

applicationsare traditionally based on crop plants, be it for ethanolproduction, biomass production for power generation and sourcesof novel compounds such as pharmaceuticals. Additionally, withpressures on land use for farming, the concomitant use of ferti-lizers and pesticides, and various (perceived?) threats of GMOs

(genetically modified organisms) to the human food chain, non-food crops are also being explored as biofactories.

For plastics production, plant materials can be harvested andused directly (natural rubber is a clear example), or plant polymerscan be derivatized to produce plastics. Plants can be usedindirectly as a nutrient source in bioreactors for biodegradableplastic production. Plants can also be genetically modified todirectly synthesize novel polymers.

NATURAL PLANT POLYMERS

Plants naturally produce a number of structural and carbon-reserve polymers. Polysaccharides are estimated to make upapprox. 70% of all organic matter. Cellulose accounts for about40% of total organic matter and is the most abundant macromo-lecule on earth. Lignin comprises 1525% of a typical woodyplant. Starch is also a major component of global biomass, andthe ability to digest starchy foods is thought to have played a rolein human evolution and its success over other species [17]. Anumber of natural polymers have been exploited for commoditymanufacturing, and most of these products retain the inherentbiodegradability of their carbohydrate building blocks.

Rubber

Rubber, a polymer of isoprene (cis-1,4-polyisoprene; Figure 2A),is the most widely used natural plant polymer. All commercialproduction of rubber comes from the Brazilian rubber tree

(Hevea brasiliensis). Despite the plants common name, more

than 90% of theworlds natural rubbersupply now actually comesfrom South-East Asia. Although natural rubber production onlyaccounts for 40% of demand (the remainder is synthetic), naturalrubber is superior to synthetic, owing to its molecular structureand high-molecular mass (>1 MDa), which confers resistanceto abrasion and impact, elasticity, efficient heat dispersal andresilience and malleability at low temperatures. These propertieshave not been duplicated in synthetic rubber because of theunique, somewhat undefined, secondary compounds (proteins,lipids, carbohydrates and minerals) in natural rubber.

So although synthetic-rubber production has reached a plateau,natural-rubber production continues to increase, particularly inChina and Vietnam [18]. The main reason production hasswitched to Asia from South America is the South Americanleaf blight fungal infection caused by Microcyclus ulei, whichoriginates in the Amazon region. All attempts at commercial-scale rubber cultivation in South and Central America have beenthwarted by this, as yet, uncontrolled fungus. More recently,molecular-breeding approaches have been explored to attempt toconfer resistance traits, and, with strict quarantine, plantations inAsia have been unaffected by the blight. Rubber plantations arecomposed of clonal trees; in fact, the commercial rubber tree

is one of the most genetically restricted crops grown, making theHevea rubber tree particularly susceptible to pathogen attack [19].

Natural rubbers isoprene monomers are derived almostexclusively from IPP (isopentenyl diphosphate). IPP is derivedfrom cytosolic acetyl-CoA through the mevalonate pathway. Poly-merization of theisoprenyl units is catalysedby rubber elongationfactor [20] or particle-bound rubber transferase. This enzymeis a cis-prenyltransferase, which adds isoprenyl units from IPPto form the polymer. Two transferase cDNAs were cloned from

H. brasiliensis, then expressed in, and purified from,Escherichiacoli. Although low-molecular-mass rubber (

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for its Yulex Natural Rubber product based on guayule latex andthey plan to couple latex production with cellulosic biomass forbiofuels and power generation.

Russian dandelion produces a higher-molecular-mass polymerthan eitherHeveaor guayule (about 2 MDa), which accumulatesin lactifers in the roots. It was discovered in Kazakhstan in the1930s and was used as a source of motor-tyre rubber during WorldWar II by a number of countries, including the United States andthe U.K. Although production yields are poor and large-scalecultivation is hampered by cross-breeding with weedy speciesand labour-intensive crop maintenance, it can be useful as a modelspecies for rubber biosynthesis because, in part, of its relativelyshort life cycle (altered rubber phenotypes can be screened within6 months) compared with guayule andHevea. A further advantageis the double-cropping potential of Russian dandelion, both asa source of rubber and its fructose-based storage sugar, inulin,which accumulates at 2540% of root dry weight and could beused in bioethanol production [26].

Proteins

Proteins can be considered polymers of amino acids that arecombined in various combinations that confer function on thebasis of their side-chain structure and on the arrangementof the amino acid monomers within a protein for tertiary/quaternary structure. Although plants are being used to synthesizenovel proteins (see the subsequent section), a number of naturallyoccurring proteins have been exploited as plastics. For example,proteins from wheat (Triticum aestivum), maize (Zea mays) andsoybean (Glycine max) , particularly zeins and glutens, have beenused as the basis for biodegradable polymers.

Gluten is a composite of the proteins glutenin and gliadin (withother globulin and albumin components). Two-dimensional gel-electrophoretic analysis shows multiple spots corresponding tomultiple isoforms of glutenin [28]. The gluten fraction comprises

about 80% (w/w) of the wheat seed protein and about 815%of seed dry weight. Gluten is easily harvested from seed bywashing away soluble components (mainly starch) to producean essentially pure protein isolate [28]. Gluten is used as a proteinsource in a number of food products, for example in preparingfibrous meat analogues composed of gluten, soy protein andstarch [29]. Gluten confers elastic properties on dough, owingto the presence of disulfide-linked glutenin chains [28], gliadinintercalating with the glutenin chains (Figure 2B). This propertyhas led to research in using gluten and zein/gluten compositesas plastics. Gluten coated with zein has been used to produce aplastic that is biodegradable and yet has a compressive strengthsimilar to that of polypropylene. Production of this polymer isvery simple; gluten and zein are mixed in ethanol and then formed

in moulds. Essentially the zein forms a glue, which binds thematrix (gluten) and causes aggregation; pressure moulding thenremoves the ethanol and allows the polymer to form [30]. Maizegluten has been used in the manufacture of wood composites. Inthis process, wood fibres are mixed with maize gluten, plasticizedusing glycerol, water and ethanol, and then extrusion-mouldedinto pots that are reasonably water-resistant and biodegradable[31]. Gluten-based polymers are very efficiently degraded insoil and in liquid environments, being degraded completely after50 days in soil and about ten times more quickly in liquid [32].Medical uses for gluten-based polymers also exist. When gliadinis purified from wheat gluten using ethanol extraction, it canbe spun into fibres that allow adhesion and growth of musclecells. Using plant proteins for this purpose (rather than collagen)

is postulated to be superior, owing to the mechanical properties

of the gliadin fibre and because it obviates the risk of diseasetransmission [33].

Zein is an alcohol-soluble protein extracted from maize glutenmeal. Zein accounts for approx. 65% of the protein content ofthe meal. Zein is a member of the prolamin family, which areproteins characterized by a high percentage of hydrophobic aminoacid residues, including proline [34]. Its purification schemeand its use as a plastic resulted in the award of a number ofpatents in the late 19th and early 20th Centuries. Commercialproduction of zein started in the 1930s and was used in themanufacture of buttons, fibres, adhesives etc., with productionpeaking in the 1950s at around 6.8 million kg (15 million lb)/year(reviewed in [35]). More recently, production has fallen to about0.45 million kg (1 million lb)/year, and it is produced by twocompanies, one in the United States (Freeman Industries) anda second in Japan (Showa Sangyo Corp.). Its current priceranges from $10 to $40/kg, depending on the purity and so isconsidered a value-added by-product of maize [36]. Zein fibreswere used for clothing and furniture stuffing, and were marketedunder the brand name VicaraTM in the 1950s (reviewed in [36]).Zein has also been used for film production, for example foruse as a grease-resistant coating for paper that is resistant tomicrobial attack [37,38]. Addition of food-grade antimicrobialagents, such as lysozyme and the peptide nisin, enhanced theapplication for food-packaging films [39]. Pure zein polymerstend to be brittle and hygroscopic [40], but the addition ofvarious plasticizers has allowed production of useful plastics.PCLH [polycaprolactone (polyhexanolactone)/hexamethylene di-isocyanate] was used to coat a zein matrix by the additionof 10% zein to about 50% PCLH. Through interactionswith glutamate, glutamine, tyrosine and histidine side chains,a polymer with superior mechanical properties (specificallyflexibility) was produced [41]. Porous zein three-dimensionalscaffolds with the potential for use in bone-tissue engineeringhave been produced by mixing zein with NaCl crystals. Oncethis polymer is pressure-moulded, the salt is removed in hot

water. Following freeze-drying, the zein forms a porous polymerwith interconnecting pores; this interconnection allows blood-vessel proliferation during bone growth, but the mechanicalproperties (i.e. brittleness) of the polymer would limit it tonon-weight-bearing applications [42]. An improved polymer wassubsequently achieved by coating the zein with hydroxyapatite[43]. Multiple uses of zein microspheres for sustained or targeteddrug delivery have also been reported [4446]. These reports statethat simply mixing the drug and purified zein in aqueous ethanolsolutions results in the production of microspheres that have anaverage diameter of about 1 m and allow gradual release of thedrug.

An early use of plasticized soy protein was by the Ford MotorCompany. In 1940, Henry Ford applied for patent protection on

his invention for automobile body construction, which stated thatthe object of my invention is to provide a body construction inwhich plastic panels are employed, not only for the doors and sidepanels, but also for the roof, hood and all other exposed panelson the body. The panels were made from soy meal (about 50%protein) that was cross-linked with formaldehyde with the additionof phenol or urea to increase strength and resistance to moisture.The panels were layered over a unique (at the time) tubularsteel cage that provided the structural rigidity for the car, whichwas actually the focus of the patent application. The patent wasissued on 13 January 1942 (Automobile body construction, U.S.Patent 2269451). Henry Ford was photographed demonstratingthe strength of the panels by swinging an axe at the car(although the photograph fails to show the impact or any damage

associated with it). The prototype was never put into production,


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Figure 3 Natural polymers: cellulose and starch

Cellulose and starch can be modified to produce biodegradable polymers with various R groups in place of hydrogen. Nitrocellulose is cast as films/paper, and cellulose acetate and CAP havebeen used for both films and nanospheres. Derivatization is achieved by chemical means from the isolated plant material. Starch has been used directly for polymer production and also throughderivatization with caprolactone, cellulose and various vinyl alcohols. The degradation properties of starch polymers are dependent on the relative proportion of pure starch to secondary compounds.The degradation of the Mater-BiTM starch polymers is shown in the bottom left-hand panel.

partlybecause the plastic was susceptible to microbial degradation(it was biodegradable!) and was not adequately moisture resistant,and apparently the car smelled strongly of formaldehyde [47].

SPI (soy protein isolate) is a minimum of 90% protein (by

dry weight) and is purified from defatted soy flour. The glycininand conglycinin seed storage proteins are the bulk of the proteincontent. Although SPIhas been widelyused as a food ingredient, italso has been used as a basis for plastic production. Heat-inducedcross-linking of the proteins creates a thermoplastic polymerbased on three-dimensional networks of disulfide bonds, as wellas hydrophobic interactions and hydrogen bonding. Addition ofplasticizers, such as glycerol, improves the structural propertiesof the film. Combining 5% (w/v) SPI and 3% (w/v) glycerolin water and then adjusting the pH outside the pI of the majorstorage proteins to prevent aggregation/coagulation (pH 6), followed by heating to 80 C, allowed casting of filmson a levelling table. Alkaline-cast films are more flexible thanacid-cast ones. Although these films are poor moisture barriers,

they are good oxygen barriers and so can be used as a layer ina multilayer sheet to prevent oxidation of packaged food [48].Using -irradiation for protein cross-linking in film productionhas the added benefit of being a common sterilization technique.-Irradiation causes the production of free radicals in the proteinsolution, and cross-linking through biphenolic compounds isachieved. These films have superior puncture strength and de-formation properties than have the thermoplastic SPI films, andthis could be further improved by the inclusion of carboxymethyl-cellulose or poly(vinyl alcohol) [49]. More recently, compositesof SPI and chitin or cellulose microfibres (whiskers) havebeen produced that have superior properties compared withpure SPI-based plastics [50,51]. Cellulose whiskers with averagedimensions of 1.2m long 90 nm diameter were prepared from

cotton by hydrolysis in sulfuric acid. Mixing SPI, whiskers and

glycerol in water was followed by heating and pressure mouldingto produce the polymer. The SPIcellulose composites have asuperior moisture resistance, tensile strength, thermal stabilityand flexibility to SPI-based polymers, but retain biodegradability

[51]. Similar improvements in the quality of SPI-based plasticshave been achieved by using polylactide in the composite [52].

Cellulose

Although much research using starch and cellulose reserves isbased on their use for biofuels (reviewed in [53]), they have alsobeen used for plastic production through derivatization.

Cellulose is a linear polymer of -1,4-linked D-glucose(Figure 3). Although cellulose cannot be thermally processedinto plastics, owing to decomposition of its hydrogen-bondedstructure, derivatized cellulose has been employed for plasticsproduction. ParkesineTM (named after its inventor, AlexanderParkes) was an early pressure-mouldable form of nitrocellulose

(Figure 3) used as a replacement for ivory in the late 19th Century.The cellulose was derived from cotton fibres solubilized withnitric acid and ethanol. This solution, called collodion, couldbe cast in sheets or pressure-moulded. Parkesine was eventuallyupdated and replaced by celluloid, which used camphor, a toughgummy volatile aromatic crystalline compound (C10H16O), asa plasticizer. The addition of the camphor plasticizer madecelluloid more flexible and mouldable and less likely to fracturecompared with Parkesine. Celluloid was also used as a substitutefor ivory, specifically for billiard balls and, towards the endof the 19th Century, was used as a photographic film for stillphotographs and movie films and even as windshields [47].Cellulose acetate (Figure 3), another derivative of cellulose,is prepared by dissolving cellulose in acetic acid and acetic

anhydride in the presence of sulfuric acid. Sheets are then cast, or


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fibres spun, typically from an acetone solution. Cellulose acetatewas widely used as film stock in the early 20th Century.

There are a number of medical uses reported for a furtherderivative of cellulose acetate, namely CAP (cellulose acetatephthalate) (Figure 3). CAP is widely used as a coating for pills, butmore specialized uses also exist. For example, CAP microsphereshave been used to improve the retention of anti-diabetic drugsin the gut. The microspheres float in the stomach and so arenot evacuated as quickly and therefore aid drug release [54].Controlled release of drugs at sites of bone regeneration is also anapplication of CAP [55]. Additionally, there are reports of the useof CAP as an anti-HIV1 agent [56,57]. Cellophane is restructuredcellulose prepared from wood pulp using NaOH to break down thecrystalline structure, followed by gelling with acid [47]. Despitebeing mostly replaced by synthetics, it is still used in some food-packaging applications, where, for example, the cellophane isimpregnated with an antibacterial peptide, nisin, and used to wrapfresh meat [58], and some specialized medical uses, for exampleby preventive adhesion of intestine and abdominal wall tissues toallow proper healing [59]. Additionally, cellophane is still widelyused for packaging.

Starch

Starch is one of the cheapest and most abundant agriculturalproducts and is completely degradable in a number of environ-ments [60]. These properties have lead to exploring starch as apolymer for various applications. Thermoplastic starch is preparedby temperature and pressure extrusion and/or moulding. Thispure-starch polymer degrades very quickly in a compostingenvironment(the process lasting about a month), but it tends to agepoorly and is not moisture-resistant [61]. Production of variousthermoplastic starch composites is based on mixing starch withvinyl alcohols, and these types of polymers tend to be more stable.However, the biodegradability of these composites is inversely

proportional to their starch content [62]. An Italian company,Novamont, has commercialized four starch-based biodegradableplastic composites that vary in their additives and are trademarkedMater-BiTM (Figure 3). Their output is about 36 300 metric tons(40000 short tons) of biodegradable plastic pellets/year, whichare supplied to a number of packaging companies. Their V-class polymer is about 85% thermoplastic starch, degrades veryquickly in compost and is used as a polystyrene replacement.Their Z-class polymer is composed of starchand polycaprolactone(a synthetic polyester), is biodegradable in compostingenvironments (2045 days) and is mainly used for bags and films.The Y-class polymer can be injection-moulded, is composedof starch and modified cellulose and lasts about 4 months incomposting environments. Finally the A-class polymer, which is

a mixture of starch and ethylene vinyl alcohol, is their most stableform of polymer, which is not compostable, but does biodegrade(in about 2 years) in a simulated sewage- sludge treatmentplant. The A-class polymer can be used for rigid and expandeditems [63]. The Mater-BiTM-based products include garden-waste(leaves,twigsetc.)and rubbish bags, nappies (diapers)and organicfood packaging used by the U.K. supermarket chain Sainsburys.

Medical uses forstarch-based polymers arealso being explored,for example as scaffolds for bone-tissue engineering because oftheir biocompatibility, biodegradability and porous nature, whichallows blood-vessel proliferation during bone growth. The SEVA-C type polymer used is a 1:1 (w/w) blend of cornstarch andethyl vinyl alcohol, which is mouldable, non-toxic and doesnot inhibit cell growth [64]. Additionally, implantation of the

SEVA-C polymer in rats did not produce a severe inflammatory

Figure 4 Polymers produced by bacteria using plant materials

Using plant carbon reserves for industrial biotechnology feedstocks typically sugar, starch,or oils various polymers can be produced by bacterial fermentation. (A) PLA is producedby bacteria of the genus Lactobacillus, and often D -glucose (maize dextrose) is the carbonsource. It is a high-molecular-mass polymer (>1 MDa) that can be used for films and moulded.(B) PHB and (C) PHBV are in the class of PHAs that naturally occur in many bacteria as carbonreserves. Typically, highly refined sources of glucose derived from plants are used in batch-fedbacterial cultures; however, use of industrial wastewater and plant oils have also shown promisefor PHA production through fermentation.

response, demonstrating it as a good candidate for implants forbone regeneration [65].

BIOCONVERSION OF PLANT POLYMERS: FERMENTATION

Currently, bioethanol production is a major focus for the useof plant starch and sugar reserves. Attempts at increasing theamounts of reserves accumulated have been addressed throughaltering the activity of the ADP-glucose pyrophosphorylaseenzyme, altering source-sink allocation of carbon or alteringcarbon partitioning within storage organs in favour of starchsynthesis. These and other approaches were reviewed recentlyby Smith [66]. Use of plant carbon reserves for plastics hasalso been addressed through fermentation using various strainsof bacteria for different types of polymer production, such asPLA [poly(lactic acid)] and polyhydroxyalkanoates.

PLA [Poly(lactic acid)]

PLA (also known as polylactide) is a polymer of lactic acid(2-hydroxypropionic acid) that was first developed by DowChemicals in the 1950s. However, because of the high cost ofproduction, its use was limited to specialized medical devicessuch as sutures, soft-tissue implants etc. Figure 4(A) showsthe structure of the PLA polymer. More recent advances infermentation have resulted in a dramatic cost reduction tothe point that PLA is widely used in packaging. PLA isproduced by naturally occurring lactic acid bacteria of thegenus Lactobacillus, which ferment hexose sugars. Lactatedehydrogenase converts pyruvate into lactate, with concomitantoxidation of NADH (reviewed in [67]). Production of PLA is

classed as heterofermentative or homofermentative. The former


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method produces less than 1.8 mol of lactic acid/mol of hexose,along with a number of other metabolites, such as acetic acid,ethanol, glycerol, mannitol and CO2. The latter homofermentativemethod produces an average of 1.8 mol of lactic acid/mol ofglucose and only minor amounts of other metabolites, whichallows about a 90% conversion rate of glucose into lactic acid(reviewed in [68]). Polymerization of lactic acid to PLA bychemical synthesis produces a large amount of racemic D,L-lacticacid, but is very expensive. The method employed by DowCargill to produce their NatureWorks PLA product involvescondensation of lactic acid to lactide (circularized lactic acid; twomonomers both D-lactic acid, both L-lactic acid or a mixture of thetwo enantiomers), followed by ring-opening polymerization [69]to produce high-molecular-mass (>100 kDa) PLA. NatureWorksLLC uses dextrose maize sugar (D-glucose), from Cargill, as acarbon source for fermentation. They produce over 136 million kg(300 million lb)/year in their plant in Blair, Nebraska, U.S.A. Over70 companies are using their PLA for various film and mouldedapplications, because of its high gloss, high transparency andphysical properties similar to thoseof poly(ethylene terephthalate)(source: http://www.natureworksllc.com). PLA is compostableand degrades in a two-stage process that involves reduction in mo-lecular mass by hydrolysis, followed by biodegradation by micro-organisms [68]. For example, a number of filamentous funginaturally present in soil can biodegrade hydrolysed PLA [70].In the United States, NatureWorks LLC also runs a buy-backprogram for PLA-based products as part of their commitment toproducing an environmentally friendly plastic. A recent advancein PLA production has been made by utilizing a microbialfactory instead of ring-opening polymerization for generationof high-molecular-mass PLA. A Japanese group (includingresearchers for the Toyota Motor Corporation) has modified apolyhydroxyalkanoate-synthesizingenzyme to facilitatepolymer-ization of lactic acid, thereby allowing polymer synthesis withinthe microbe without the need for chemical polymerization [71].

PHAs

PHAs are hydroxyalkanoate polyesters of various chainlengths. PHAs are carbon and energy reserves stored as insolubleinclusion bodies in most bacteria under nutrient-limiting andcarbon-excess conditions [72]. The inclusion bodies were firstobserved in the late 19th Century, and the composition ofPHA was first examined in the 1920s by Maurice Lemoigneat the Pasteur Institute in Paris (reviewed in [73]). PHB(polyhydroxybutyrate; Figure 4B) has properties that resemblethose of polypropylene and is amenable to injection moulding,extrusion blow moulding and fibre spray-gun moulding. p(3HB-co-3HV) or simplyPHBV, the co-polymer of hydroxybutyrate andhydroxyvalerate (Figure 4C), is similar to the PHB homopolymer,

although more flexible [72]. PHAs have been used for themanufacture of films, coated paper and board, compost bags anddisposable flatware and can also be moulded for the productionof bottles and razors and are completely biodegradable (to CO2and water). These properties make PHAs an attractive source ofnon-polluting renewable plastics and elastics [74]. A considerablebody of research has gone into production of various PHAs intransgenic plants (see the subsequent section), but the use of plantproducts as carbon sources for bacterial bioreactors has also beenexplored.

Fermentative production of PHAs using the bacteria Ralstoniaeutropha and Alcaligenes latus has been established for almost40 years. The R. eutropha PHA synthase operon encodes athiolase (phaA),a reductase (phaB) anda synthase (orpolymerase,

phaC) [75]. Although wild-typeR. eutropha can only use fructose

as a carbon source, a glucose-utilizing mutant ofR. eutrophawasengineered and used in the production of Biopol. Biopol

was originally developed by ICI (Imperial Chemical Industries),which began worldwide commercialization of a family ofPHBV polymers in the 1980s. In 1990, the agricultural andpharmaceutical divisions of ICI were spun off as Zeneca Ltd,and then Biopol was bought by Monsanto in 1996. However,

soon thereafter, Monsanto closed its bioplastics division asit refocused on agricultural applications of biotechnology andmoved away from industrial applications. The patent rights werethen bought by a Cambridge, (MA, U.S.A.) company calledMetabolix (source: press release 16 May 2001, Metabolix.com).

In addition to the numerous naturally occurring PHA-producingprokaryotes, other bacteria that do not normally produce PHAscan be modified to do so. For example, cloning the PHAoperon into E. coli has also allowed various types of PHAs tobe produced. Specifically, the co-polymer PHBV is producedby adding glucose and propionic acid to the culture medium(reviewed in [76]). Figure 4(C) shows the structure of the co-polymer. Growing modifiedE. colistrains under specific growthconditions can be used to alter the structure of the co-polymer,specifically the relative abundances of the HB (hydroxybutyrate)

and HV (hydroxyvalerate) monomers. Adding valine to theglucose-based culture medium results in 2% HV co-polymer,and including threonine along with valine results in 4% HV[77]. Typically PHAs are produced using pure cultures of definedbacterial isolates and supplying highly purified carbon sourceslike glucose. This type of production yields an average cost of$14/kg (9/kg; 7.9), considerably more than petroleum-basedplastics (e.g. polyethylene). However, production of PHA frommixed cultures using various wastestreams has been proposedas an effective way to reduce these costs [78]. Yu et al. [79]described using industrial food wastewater as a carbon sourcefor production of PHA by A. latus. They investigated the use ofsoy wastes from a milk dairy and malt wastes from a breweryas carbon sources. The malt-waste-fed bacteria produced 70%

polymer (on a cell weight basis), whereas the soy-waste-fedand sucrose-fed cultures produced a little over 30% polymer.A recombinant strain ofE. coli was used to produce PHB withwhey and maize steep liquor as the main carbon and nitrogensources respectively. The whey liquor is a by-product of thecheese industry and maize steep liquor is a by-product of wet-milled maize.E. coliwas transformed with a plasmid containingthe phaABCgenes expressed from the phage T5 promoter andtwo lac operator sequences. PHB polymer accumulated to over70% of the cell dry weight, although this amount tended to de-crease as the cultures aged, a phenomenon attributable to plasmidloss during growth. The PHB obtained was very similar tothe naturally produced PHB, with a high molecular mass anda similar crystalline structure [80]. Interestingly, Metabolix

and ADM (Archer Daniels Midland) have co-operated to producea bacterially produced PHA with the trade name MirelTM. Theyare in the process of constructing a commercial-scale productionplant adjacent to ADMs wet maize mill in Clinton, Iowa,and plan to provide polymer in the second quarter of 2009 with aproduction capacity of 49.8 million kg (110 million pounds)/year(source: http://www.mirelplastics.com/).

Plant oils have also been successfully used to produce thetypical thermoplastic PHAs. PHB and the PHBV co-polymercan be produced by wild-type A. eutrophus and a modifiedstrain containing the synthase gene from Aeromonas caviaerespectively. Wild-typeA. eutrophusaccumulates 80% of its cellweight as PHB when fed olive, maize or palm oils, and similaramounts of PHBV are produced by the recombinant strain using

the same plant-oil carbon sources. Using these cheap oils as a


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226 B. P. Mooney

renewable carbon source could increase the economic viability offermentative PHA production [81].

Despite these advances in using relatively cheap plant oilsand carbohydrates or industrial effluents as carbon sources,the limitations of bioreactor production still exist. PHA orPLA production in bioreactors requires careful monitoring ofthe conditions within the bioreactor, including the cell density,nutrient reserves, accumulation of waste products etc. Bioreactorsalso have certain size and production capacity limits [82].Additionally, pure culture production of PHA is thought toconsume more fossil-fuel resources than petroleum-based plastics[83,84]. Although mixed-culture PHA fermentative productionis proposed to have significantly less environmental impactthan traditional petroleum-based plastics such as high-densitypolyethylene [85], using plants forde novosynthesis of polymerscould theoretically prove an economically viable option, either asa value-added co-product of food crop plants or perhaps as a noveluse for some marginal land species such as switchgrass (PanicumvirgatumL.).

DE NOVOSYNTHESIS OF POLYMERS WITHIN PLANTS

Traditional plant breeding to produce valuable traits has beenemployed since prehistory, when selection of visual phenotypesled to the domestication of the first crop plants. Wheat, forexample, is thought to have been domesticated about 10 000 yearsago in the Near East [86]. However, it was not until the late20th Century that molecular manipulation to produce the firsttransgenic plants was conducted. Altering plant metabolism toproduce value-added products by transgenic methods has slowlybegun to yield some promising results for plastics production.

Proteins

A number of hurdles need to be tackled before commercial-scale production of value-added heterologous proteins (and other

polymers) can be achieved in plants. The DNA encoding theforeign protein must be stable across a number of generations,and synthesis of the protein from mRNA, through processing,translation and, addition of post-translation modifications, mustbe considered. Additionally, the type of crop used and the site ofaccumulation must be carefully considered to allow high levelsof expression and ease of purification (reviewed in [87]).

Among the number of novel proteins being expressed intransgenic plants, many of these confertraitson the plant itself, forexample theBacillus thuringiensiscry toxin for insect resistance(see [88]). However, in the context of polymer production,proteins with an inherent commercial value following purificationfrom the plant are also being produced. Typically, these are high-value proteins with specialized uses, for example as vaccines.

Production of heterologous proteins within plants has numerousadvantages over production in animal or bacterial systems. Plant-based therapeutic proteins, for example, are theoretically saferfrom a disease point of view than those purified from animalsources (whether native or recombinant). Also, incorporationof post-translational modifications such as glycosylation canbe achieved by in planta production, in contrast with bacterialsystems. Although eukaryotic culture systems such as insect andmammalian cell cultures can be used in many cases to produceviable proteins, an additional advantage of plants is that they arerenewable and scaleable and require less energy and nutrient inputthan bioreactors, which should make plants cost-effective for bulkapplications (reviewed in [89]).

Heterologous protein accumulation within plants can be

achieved through standard transgenic approaches (i.e. nuclear

transformation), through plastid transformation and also throughviral vectors. The latter approach typically leads to transientexpression most commonly targeted to the leaves [90,91] and,thus, cannot be considered a sustainable approach. Nucleartransformation, typically mediated by Agrobacterium infection,allows incorporation of the heterologous genes into the nucleargenome, (often) followed by stable expression and heritability ofthe trait. A number of crop plants, including maize, soybean,rice and cabbage (Brassica) species, have been successfullytransformed using this technique. In terms of heterologousprotein expression for purification purposes, the goal, obviously,is to achieve high levels of expression. Avidin was producedin transgenic maize using a constitutive ubiquitin promoter tolevels exceeding 2% of aqueous-soluble protein from seed,where more than half of the protein accumulated within theembryo. The extracted avidin could be highly purified usingaffinity chromatography and was identical with the native egg-white protein. The study reported that 150300 mg of purifiedavidin could be extracted/kg of seed, and one high-expressing linecontained 20 g of avidin in 100 kg of maize seed, which was theequivalent of 900 kg of eggs. The authors postulated that withthelevels of accumulation, thestabilityand viabilityof theprotein,and the heritability of the trait, that commercial-scale productionwas quite feasible [92]. Nandi et al. [93] reported production inrice grain of human lactoferrin, a protein which is involvedin iron absorption and has properties ranging from antimicrobialto immune-system modulation and promotion of cell growth. Itwas demonstrated that stable expression over nine generationscould be achieved with production amounts ranging from 4.5 to5.5 g/kg (about 5% of the rice flour dry weight). The recombinantprotein was very similar to the native protein from human milk,although the glycosylation patterns were not identical. Processsimulations suggestedthat 1 g of lactoferrin would cost $6 (4.64,4.06) to produce, assuming a 5% (dry weight) level of expressionand using a single cation-exchange purification step [93]. Thereare potential problems with nuclear transformation, however; for

example, in the case of avidin production in maize, the plantsexhibited male sterility [92]. Additionally, silencing of transgenesis also a frequent problem in plants. Gene silencingcan occur fromposition effects the influence of the local environment on thechromosome around the site of transgene insertion, which canaffect, for example, the rates of transcription. Additionally, whenthe transgene and native genes are similar in sequence, homology-dependent gene silencing of the transgene and/or co-suppressionof transgene and native gene can occur. Achieving highlevels of heterologous protein expression that is stable over anumber of generations can be severely hampered by these events,although there are strategies to mitigate silencing of nuclear-transformed plants (reviewed in [94]).

Production of spider silk proteins in plants genetically modified

by Agrobacterium-mediated nuclear transformation has beenexplored. Spider silk proteins used in web construction tend tobe elastic Lycra-like fibres, whereas the dragline (lifeline) silksare Kevlar-like and are stronger than steel on a per-weight basis.Silk proteins are composed of GPGXX and GGX (one-letteramino acid code) motifs, along with [GA]n repeat sequences thatconfer elasticity and high tensile strength respectively. Transgenictobacco (Nicotiana tabacum), potato (Solanum tuberosum) andthale cress (Arabidopsis thaliana) have been used for silkproduction (reviewed in [95]). Yang et al. [96] reported productionof DP1B, a synthetic analogue of spider dragline silk protein, in

A. thaliana. Expression was driven from the CaMV (cauliflowermosaic virus) 35 S promoter introduced by the standard floral-dip method for nuclear transformation by Agrobacterium. When

protein accumulation was targeted to the ER (endoplasmic


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reticulum) lumen of leaf tissue, up to 6.7% total soluble proteinwas produced. This level could be increased to 8.5% in theleaf apoplast and as high as 18% in seed-targeted expression.In tobacco and potato, the CaMV 35 S promoter was used todrive expressionin nuclear-transformed plantsand expressionwastargeted to the ER using the KDEL (one-letter amino acid code)ER retention signal [97]. These workers concentrated on a silkelastin fusion protein and achieved laboratory-scale productionof 80 mg of pure protein/kg of tobacco leaves using a simplebuffer-extraction procedure followed by heating to 95 C for 1 htocause aggregation of most proteins, leaving the silk protein in thesupernatant. Purification of the protein, however, does not conferthe structural properties of the silks, as this is accomplished by thecorrect assembly of the proteins during the process of spinning inspecialized organs in the spider (i.e. spinnerets), and this naturalprocess has not been replicated for the recombinant proteins [98].

Generating transgenic plants through plastid genome(plastome) transformation has a number of advantages. Highlevels of transgene expression are typically achieved due to theplastome copy number and the presence of multiple chloroplastsper cell [99]. For example, cholera toxin B was expressed at verylow levels by nuclear transformation. However, 4003000-fold

increases were observed when chloroplasts were used as the siteof transformation/expression [100]. Another advantage of thetransplastomic approach is that multiple genes can be inserted ina single event [101] without problems of gene silencing, sincegenes are targeted to a specific locus on the plastid genomeby homologous recombination. Plastome transformation has theadded advantage of maternal inheritance, so that transmission oftransgenes through pollen is eliminated, resulting in improvedcontainment of the transgene in the environment [102]. Tobaccohas been widely used for expression of proteins from the plastomeas have soybean, cotton (Gossypium hirsutum) and carrot(Daucuscarota) [103]. Lutz et al. [104] produced a guide to aid selectionof vectors for plastid transformation that include marker genes toallow selection of transformants and also to follow insertion and

expression of the transgene of interest.Human growth hormone, or somatotropin, is one of the many

proteins produced in tobacco through plastid transformation.Chloroplasts were shown to accumulate soluble active anddisulfide-bonded somatotropin. Additionally, use of a fusion-protein approach (N-terminal ubiquitin) facilitated production ofa functional protein lacking the N-terminal methionine residue;the native protein starts with a phenylalanine residue followingsignal sequence removal in the pituitary gland. High levels ofsomatotropin protein accumulation within tobacco chloroplastswas achieved by driving expression of the gene from the plastidpsbA promoter and other domains in the UTRs (untranslatedregions) [105]. The psbA gene encodes the D1protein, part of thereaction centre of Photosystem II. Using the psbA promoter and

other sequences in the genes UTRs was previously demonstratedto confer high expression of foreign genes in light-exposed leaves[106]. The amount of somatotropin produced was >7% oftotal soluble protein, which corresponded to a 300-fold increaserelative to levels achieved by nuclear transformation [105].

Attempts at producing an anthrax vaccine have also beenmade by plastid transformation of tobacco leaves. The BacillusanthracisPA (protective antigen) was expressed in chloroplasts,also under the control of the psbA promoter. The widely usedanthrax vaccine derived from B. anthracis itself has other minorcomponents in addition to PA that cause side effects whenadministered. Using tobacco leaves as a source for a recombinantPA should obviate these reactions. During normal day/night lightregimes PA wasproducedat between 1.7and 2.7% of total soluble

protein from mature leaves. However, using a continuous light

regime up to a ten-fold increase in expression wasobserved. About170 mg was harvested from a single plant, equivalent to 2.5 mg/gfresh weight. The authors extrapolated this expression level to asupply of about 990 million doses of vaccine/hectare (400 milliondoses/acre) using their experimental cultivar and possibly morethan 18-fold higher using a commercial cultivar, which would bethe equivalent of a world supply of vaccine from just a few acresof transgenic tobacco [107].

Although extensive research has gone into using plants forheterologous-protein expression, apart from those that impart aspecific valuable trait on the crop plant itself, few have made theleap to commercial/agricultural-scale production. In fact, this canbe said of transgenic plants as a whole [108].

PHAs

The chemical properties of various types of PHAs impartmultiple potential applications. Additionally, these polymers arecompletely biodegradable. As a result, PHAs have been proposedas alternatives to conventional petroleum-based plastics. Thebacterial genes responsible for polymer synthesis have beenstudied in great detail, andnumerousrecombinant bacterialstrainshave been generated to facilitate PHA production. However,production of PHAs in transgenic plants has been proposed as aneconomically viable alternative to bioreactor-based or chemicalsynthesis.

Production of the PHB homopolymer in transgenic plants wasfirst achieved by Chris Somervilles group (then at Michigan StateUniversity in East Lansing, U.S.A.). These workers generated

A. thaliana plants that were transformed with the R. eutrophagenes encoding the acetoacetyl-CoA reductase and PHB synthase.Polymer synthesis was initiated by a cytosolic -oxothiolasenaturally present inA. thaliana along with the introduced bacterialenzymes (under the control of the CaMV 35 S promoter) thatfacilitated production of high-molecular-mass PHB polymer

in the cytosol, nucleus or vacuoles. However, the amount ofPHB produced was quite low (about 0.1% dry weight) [109],and this level of accumulation was not improved by cytosolicproduction in other transgenic plants such as tobacco [110].Additionally, the transgenic plants showed serious side effectsof PHB accumulation, namely a notable decrease in growth andseed production. The precursor for PHB production is acetyl-CoA(Figure 5), and diversion of much of this toward PHB productionand away from natural pathways, such as those for flavanoid andphytosterol production and fatty acid elongation, has beenpostulated as the likely cause of the deleterious side effects [95].

Plastids were considered as a good site for polymer productionfor a number of reasons. Plastids contain large quantities ofacetyl-CoA destined for fatty acid and amino acid synthesis,

so the metabolic flux should be sufficient for PHA production.Plastids also naturally accumulate bulky starch grains; thereforethey should tolerate accumulation of PHAs with less deleteriouseffects. However, plastids do not contain the -oxothiolase, sothis must be included in the generation of transgenic plants. Asdiscussed above, nuclear-transformation-mediated heterologous-protein production has been demonstrated to be far inferior toplastome transformation in terms of accumulation levels. Losslet al. [111] introduced the R. eutropha PHA-synthesis operoninto the plastome under the control of the psbA promoter bybiolistic (gene gun) transformation. The average amount ofPHB synthesized by their transplastomic tobacco lines was715 p.p.m. on a dry-weight basis (

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228 B. P. Mooney

Figure 5 Scheme for production of PHBV in plants

Accumulation of PHBV within plastids uses the endogenous PDC for generation of the twoprecursors of the polymer acetyl-CoA and propionyl-CoA. Introduction of the bacterial PHAsynthase operon comprising three enzymes (blue italics) allows accumulation of the polymerwithin the plastid. The plastidial -oxobutyrate (-ketobutyrate) is produced from threonine byan introduced threonine deaminase (not shown).

growth. Additionally, the transformed plants were male-sterile,and maintenance of the transgenes could only be achievedthroughfertilization with wild-type pollen [111]. Unlike the numeroussuccess stories for protein production by transplastomic plants,

in this case PHA production was poor when driven from theplastome.

An alternative to plastome transformation is to target theenzymes to the plastids post-translationally by incorporating N-terminal plastid-transit peptides. Nawrath et al. [112] inserteda plastid-transit peptide sequence derived from the pea (Pisumsativum) small subunit of RuBisCO (ribulose 1,5-bisphosphatecarboxylase/oxygenase) at the N-terminus of the three bacterialgenes necessary for PHA synthesis. The three chimaericenzymes were introduced into A. thaliana plants separately andthen a triple-hybrid plant generated by cross-pollination. PHBproduction in the hybrids of 14% (by dry weight; 10 mg/gby fresh weight) was achieved without affecting growth orseed set. Additionally, it appeared that the amount of PHB

produced was limited by the (bacterial) enzyme activities ratherthan limiting acetyl-CoA [112]. In addition to leaf chloroplasts,seed leucoplasts in rapeseed (canola; Brassica napus) wereused as the site of PHB accumulation by introduction of allthree bacterial genes on a single multigene vector. The authorspostulated that, becauseB. napusproduces large amounts of oil,the flux of acetyl-CoA should be more than sufficient to supportPHB accumulation and perhaps prove superior to the previous

A. thaliana experiments. Although their highest producing lineaccumulated 7.7% (by fresh weight), over 90% of their linesproduced< 3%. They did confirm, by electron microscopy, thatthe ultrastructure of the plastids changed in response to PHBaccumulation (they expanded), proving that plastids are suitableas a site of accumulation of PHAs and suggesting that expansion

of plastids is a direct result of accumulation of polymer and

not a specific response to starch-grain growth [113]. The highestaccumulation of PHB reported to date was achieved in transgenic

A. thaliana shoots, in which 4% (by fresh weight; approx. 40%by dry weight) was PHB polymer [114]. Numerous other plants(e.g. cotton, potato, maize and tobacco) have been examinedfor PHB production, both cytoplasmic and plastidic, but nonehas resulted in improving the levels of accumulation seen in

A. thaliana leaf chloroplasts [115119].Most recently, switchgrass has been employed as a source

of the PHB polymer. Switchgrass is a warm-season grass andone of the dominant species of the central North Americantall-grass prairie. Switchgrass is seen as a valuable biomasscrop with the potential for use in bioethanol production withconcomitant reduction in greenhouse-gas emissions comparedwith gasoline/petrol [120]; it was recently targeted for fundingas one of the useful bioenergy crops by the U.S. Department ofEnergy [121]. In addition to their bacterially produced polymer,researchers at Metabolix are also exploring plant-based PHAs.They used both constitutive and light-inducible promoters to driveexpression of PHB synthesis genes appended with N-terminalchloroplast transit peptides. Greenhouse-grown switchgrass wasfound to accumulate almost4% (by dry weight) PHB in the leavesand 1.2% (by dry weight) in whole tillers. Even their highestproducing lines [3.72% (bydry weight) PHBin leaves] developednormally and accumulated biomass at levels similar to those of thewild-type. Additionally, stable high-level PHB accumulation wasmaintained in the subsequent generation. Although the amountsof PHB produced were below commercially viable levels, this wasthe first successful expression of a functional multigene pathwayin switchgrass and bodes well for future engineering of the plantsfor value-added-product formation [122]. The hope eventuallywould be to produce a double-crop for both PHAs and cellulosicethanol production.

Polymers consisting of PHB tend to be crystalline and brittle,whereas PHBV, the co-polymer of HB and HV, is considerablymore flexible. PHBV precursors are acetyl-CoA and propionyl-

CoA, and, although acetyl-CoA is present in the cytosol andplastids, propionyl-CoA is restricted to the mitochondria. Analternative route for production of propionyl-CoA is introductionof a threonine deaminase, which converts threonine into -oxo-butyrate. The -oxobutyrate can then be utilized by theendogenous plastid PDC (pyruvate dehydrogenase complex) toproduce propionyl-CoA (Figure 5). Slater et al. [123] employedthis route to propionyl-CoA synthesis in plastids of B. napusandA. thaliana. This required introduction of the three bacterialPHA synthesis genes, along with the threonine deaminase theilvA gene from E. coli. Although the presence of the threoninedeaminase did raise the levels of propionyl-CoA relative to plantswithout it, the amounts were still 46-fold less than acetyl-CoA.Additionally, the poor efficiency of the plastid PDCfor conversion

of-oxobutyrate into propionyl-CoA resulted in accumulation of

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Figure 6 Using plants for production of biodegradable polymers

The present review discusses three main themes for synthesis of biodegradable polymers. There are a number of naturally occurring plant polymers that have been exploited by direct harvestingfor biodegradable plastics rubber, starch, proteins and cellulose sometimes followed by derivatization. Industrial or white biotechnology using plant extracts as carbon sources for bioreactorfermentative production of PLA and PHA polymers is an alternative. Through engineering of bacteria, a number of valuable polymers can be produced with various monomers based on the nutrientinput. Finally, using transgenic technologies, plants can be engineered to produce novel polymers, including value-added proteins and PHAs, and therefore function as solar-driven biofactories. Inall cases, the polymers produced are biodegradable, so the carbon cycle is maintained the transgenic route to plastics is considered the closest to a carbon-neutral method for polymer production.

polymer has been set by Metabolix as a commercially viablelevel of accumulation [122], and some of the research into the

slightly less desirable PHB polymer have clearly shown accu-mulation in excess of this (e.g. 40%; [114]). Unfortunatelyaccumulation is only part of the process; extraction of the polymeris probably an even greater component for commercialization ina cost-effective and environmentally friendly manner to allowplant-based biodegradable polymers to replace petroleum-basedplastics. Bacterial culture systems for PHA can accumulate 5085% (by dry weight) polymer, with 80 g/litre and 2 g/h perlitre capacities relatively easily met in fed-batch cultures [125].Laboratory-scale purification of PHAs from bacterial cells can beachieved by simple mechanical (20 h constant stirring) organic-solvent extraction using chloroform and methanol, which exploitsthe solubility of PHA in chloroform and insolubility in methanol,allowing fractionation of the PHA [126]. An alternative approach

is to use a cocktail of enzymes and detergents to removeproteins, nucleic acids and cell walls, leaving the PHA intact[127]. However, a low-cost, highly efficient and environmentallyfriendly commercial-scale PHA extraction process has yet tobe implemented. Two cost-effective and more environmentallyfriendly approaches have been developed recently, namelysupercritical CO2extraction and cell-mass dissolution by protonsin aqueous solution (sulfuric acid extraction at high temperature).Recovery efficiencies of 9095% with very high purity wereachieved with these methods respectively (reviewed in [82]).Whether these methods can be used for PHA extraction fromplants has not been tested. There are a number of patent filingsfor PHA extraction from plants based on non-halogenated solventextraction (U.S. Patent 6043063) at high-temperature (U.S. Patent

6087471) and those that allow simultaneous extraction of PHAs

and oils from oilseed crops using differential solvent extraction(U.S. Patent 6709848). None of these methods seem particularly

environmentally friendly, and none have been published to datefor commercial-scale extraction from plants.

CONCLUDING REMARKS

As discussed in the present review, the production of plant-basedpolymers can be approached in a number of ways (Figure 6).Some progress has been made in commercialization of plant-based materials, for example, the starch-based Mater-BiTM rangefrom the Italian company Novamont and U.S. NatureWorksLLC PLA polymer. However, commercialization of transgenicplants specifically designed to synthesize polymers has not beenachieved. Parameters affecting commercialization of transgenic

plants for bioplastics include the time and costs associated withtheir development (often 1012 years and tens of millions of U.S.dollars in total) [128], the typically poor accumulation levels ofnovel polymers (compared with bacterial systems), the lackof proven processing and extraction methods, the price perunit of polymer from plants, andthe stigmaassociated with GMOsin many parts of the world.

Many of these problems can be addressed by more research.Building on current seed stocks of plants that have alreadybeen developed for PHB production, such as those fromMetabolix, for example, through cross-pollination or furthergenetic manipulation, could conceivability reduce the timelinefor development and might, in part, reduce the costs associatedwith regulatory testing if the original plants already have a GRAS

(generally recognized as safe) designation. As more knowledge


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230 B. P. Mooney

is gained on carbon flux in plants, perhaps, through emergingmetabolomics methods for example, this will give us a betterunderstanding of how to increase polymer accumulation. Judgingfrom the numerous patent awards, processing technologies arebeing developed for transgenic plant polymers. In terms ofprice per unit polymer, fermentative PHA production couldproduce $2 (1.55, 1.35)/kg prices, which is still about twicethe price of polyethylene [129], but using crop plants in adouble-cropping value-added manner could allow exploitationof agronomic plants for polymer production. Additionally thescalability and renewable nature of plants should address priceissues. Finally the acceptance of GMOs is still a point ofcontention. The StarLink maize incident in 2000 gained nationaland worldwide attention for the problems of segregation of foodcrops [130]. Utilizing non-food crops, like switchgrass, mightameliorate the stigma associated with transgenic crops, althoughfood-chain contamination is only one of the issues that are usedto argue against GMOs. Having said all this, the pressures onfossil fuels, seen in rising oil prices and environmental concerns,could soon precipitate a revolution against traditional plasticsin favour of more environmentally conscious and, theoreticallycarbon-neutral, plastics such as those derived from plants.

ACKNOWLEDGMENTS

We thank Melody Kroll (Interdisciplinary Plant Group, University of Missouri, Columbia,MO, U.S.A.), Emeritus Professor Douglas D. Randall (Department of Biochemistry,University of Missouri, Columbia, MO, U.S.A.) and Dr Jan Miernyk (Plant GeneticsUnit, USDA/ARS, Columbia, MO, U.S.A., and Department of Biochemistry, Universityof Missouri, Columbia, MO, U.S.A.) for critical reading of the manuscript before itssubmission.

FUNDING

Thisresearch receivedno specificgrant fromany funding agency in thepublic,commercialor not-for-profit sectors.

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