Technical Overview Biopolymer Field B

Bblocks.

TechnicalOverview ofBiopolymer

Field 2copolymers can be produced through a variety ofmechanisms. They can be derived from microbialsystems, extracted from higher organisms such as plants,or synthesized chemically from basic biological buildingA wide range of emerging applications rely on all three

of these production techniques. Biopolymers are being devel-oped for use as medical materials, packaging, cosmetics, foodadditives, clothing fabrics, water treatment chemicals, industrialplastics, absorbents, biosensors, and even data storage elements.This chapter identifies the possible commercial applications anddescribes the various methods of production of some of the morepromising materials. Table 2-1 provides a partial list of thebiopolymers now in use. ’

SELECTED POLYMERSMICROBIAL SYSTEMS

PRODUCED BY

In recent years, considerable attention has been given tobiopolymers produced by microbes. It is on the microbial levelwhere the tools of genetic engineering can be most readilyapplied. A number of novel materials are now being developedor introduced into the market. In the following sections, three

I A comprehensive treatment of biopolymer compounds is beyond the scope of thisBackground Paper. The categories of biopolymers described here are designed toillustrate the diverse physical characteristics and the broad application range of thesematerials. For a more detailed discussion of biopolymers, see David Byrom (cd.),Biomaterials: Novel Materia1sj70m Biological Sources (New York NY: Stockton Press,199 1); Roger Rowell, Tor Schultz, and Ramani Narayan (eds.), Emerging Technologies

for Materials and Chemicals from Biomass (Washington, DC: American ChemicalSociety, 1992); David Kaplan et al., ‘‘Nahmally Occurring Biodegradable Polymers,’ G.Swift and R. Narayan (eds.), Polymer Systemdynthesis and Utility (New York+ NY:Hanser Publishing, forthcoming 1994).

19

20 I Biopolymers: Making Materials Nature’s Way

Table 2-l—A Snapshot of the Biopolymer Family

Polyesters Polysaccharides (plant/algal)Polyhydroxyalka noates Starch (amylose/amylopectin)Polylactic acid Cellulose

Proteins AgarSilks AlginateCollagen/gelatin CarrageenanElastin PectinResilin KonjacAdhesives Various gums (e.g., guar)Polyamino acids Polysaccharides (animal)Soy, zein, wheat gluten, casein, Chitin/chitosanSerum albumin Hyaluronic acid

Polysaccharides (bacterial) Lipids/s urfactantsXanthan Acetoglycerides, waxes, surfactantsDextran EmulsanGelIan PolyphenolsLevan LigninCurd Ian TanninPolygalactosamine Humic acidCellulose (bacterial) Specialty polymers

Polysaccharides (fungal) ShellacPullulan Poly-gamma-glutamic acidElsinan Natural rubberYeast glucans Synthetic polymers from natural fats and oils (e.g.,

nylon from castor oil)

SOURCE David L Kaplan et al , “Naturally Occurring Biodegradable Polymers,” G Swift and R Narayan (eds ), Po/ymer.Systems-Synthesis and Utility (New York, NY Hanser Publishing, forthcoming 1994)

different classes of microbially derived bio-polymers are profiled: polyesters, proteins, andpolysaccharides.

Microbial Polyesters:Polyhydroxyalka noates

Much of the current interest in biopolymersstems from the growing concern about the envi-ronmental impacts of synthetically producedmaterials. In particular, the highly publicizeddisposal problem of traditional oil-based thermo-plastics has promoted the search for biodegrada-ble alternatives (about 17 billion pounds ofthermoplastic packaging material was producedin the United States in 1991). Apart from theagriculturally derived biopolymers (e.g., starch)being investigated for their biodegradable proper-ties, there is a class of natural thermoplasticmaterials that is drawing much attention. Polyhy -

droxyalkanoates (PHAs) area family of microbialenergy reserve materials that accumulate asgranules within the cytoplasm of cells. They aregenuine polyester thermoplastics with propertiessimilar to oil-derived polymers (melting tempera-tures between 50 to 180”C). Their mechanicalcharacteristics can be tailored to resemble elasticrubber or hard crystalline plastic.3 The prototypeof this family, polyhydroxybutyrate (PHB), wasfrost discovered in 1927 at the Pasteur Institute inParis. Commercialization of PHB was first at-tempted by W.R. Grace Co. in the 1950s. Morerecently, a British company, Zeneca Bio Products(formerly ICI Bio Products), initiated commercialproduction of a series of PHA copolymers underthe trade name BIOPOLTM. Several companiesand government research organizations, particu-larly in Europe and Japan, have active researchand development (R&D) programs focusing onthese materials.

2 ~emopbtics we ~IPm tit ww ~eatedly soften when heated and Mden whm ~1~.3 y. hi, ‘‘~crobiat synthesis and Properties of Polyhydroxyalkanoates, Materials Research Bulletin, vol. 17, No. 11, November 1992,

pp. 39-42.

Production of PHAs is carried out throughfermentation. The general process is illustrated infigure 2-1. In their final stages of preparation,they can be processed by standard extrusion andmolding techniques. Careful control of the carbonsources (starting materials) and the choice ofproduction organism enables the production of anentire family of PHA copolymers with differentproperties. The PHB homopolymer is producedby a variety of bacteria that use it as a source ofcarbon and energy. The homopolymer is a brittlematerial that is difficult to use and is thermallyunstable. However, by combining polyhydroxyval-erate (PHV) with PHB, a nonbrittle copolymer—polyhydroxybutyrate-polyhydroxyvalerate (PHBV)--can be created.4 Other PHA copolymers have alsobeen produced.5 Currently, Zeneca’s productionof PHBV (BIOPOL) uses the bacterium Alcali-genes eutrophus, which occurs widely in soil andwater. PHBV is formed when the bacterium is feda precise combination of glucose and propionicacid. 6 It has properties similar to polypropyleneand polyethylene, including excellent flexibilityand toughness. The discovery and developmentof PHBV and other PHA copolymers have provento be a major step forward in expanding thepotential industrial utility of the PHAs.

PHAs biodegrade in rnicrobially active envi-ronments. Since PHAs function as an intracellularenergy and carbon source, bacteria can degradePHAs and use them as reserve materials. Microor-ganisms attack PHBV by secreting enzymes(depolymerases) that break down the polymer

Chapter 2–Technical Overview of

Figure 2-I—PHA Polymer

Raw materialsv

Media preparation

vFermentation

vCell disruption

vWashing

vCentrifugation

vDrying

P H A P r o d u c t

Biopolymer Field 21

Production Process

1. Carbon source

2. Growth of bacteriaand accumulationof polymer

3. Purification of polymer

SOURCE: Biolnformation Associates, Boston, MA.

into its basic hydroxybutyrate (HB) and hy -droxyvalerate (HV) constituents. The HB and HVfragments are then consumed by the cells tosustain growth. Under aerobic conditions, thefinal biodegradation products are water andcarbon dioxide; under anaerobic conditions, meth-ane is produced as well. The degradation ofPHBV can be quite rapid in biologically activesystems (see figure 2-2).7 A range of soil microor-

4 William D. Luzier, ‘Materials Derived from Biomass/Biodegradable Materials,’ National Academy of Sciences, vol. 89, Februmy 1992,pp. 839-842.

5 For example, researchers at the Tokyo Institute of Technology have produced a versatile PHA copolymer of 3-hydroxybutyrate and4-hydroxybutyrate. This material has been extracted and fermented from the bacterium AfcaJigenes eutrophu under starvation conditions. Byaltering the ratio of the copolymers, the elasticity and strength of the material can be varied (Doi, op. cit., footnote 3).

6 Glucose is derived from agricultural feedstocks such as sugar beets and cereal crops, whereas propionic acid can be produced fi-ompetroleum derivatives or by fermentation of wood pulp waste.

7 When 50 bottles made of PHBV material were inserted into a compost heap (6 cubic meters of organic waste) for a period of 15 weeks,at temperatures between 60 and 70”C, only about 20 percent (by weight) of the PHBV material remained . It is important to note, however, thatPHBV and other biopolymers will not degrade in sanitary Iandfiis, because they are essentially biologically inactive systems. See Petra Piichnerand Wolf-Rudiger Miiller, “Aspects on Biodegradation of PHA,” H.G. Schlegel and A. Steinbiichl (eds.), Proceedings InternationalSymposium on Bacterial Polyhydroxyalbnoates 1992 (GOttingen: GoI-Druc~ 1993).

22 Biopolymers: Making Materials Nature’s Way

Figure 2-2—Biodegradation of PHBV Polymers

100 —ru< 4 0

/’

L ‘-- -i‘== i20 i /L------- ~—-— ~ –-–--— ------ ,I o~o 10 20 30 40 50 0 5 10 15 20 25 30 35

Time (weeks) Time (days)

Degradation of PHBV landfill simulation Anaerobic degradation of PHBV 27% copolymer

PHBV can degrade relatively quickly in biologically active systems. in a simulated landfill environment with an elevated moisturecontent, PHBV showed a 40-percent weight loss In 40 weeks (left). Under anaerobic sewage conditions, where biodegradabilityis measured by gas production, the PHBV polymer decomposed nearly 80 percent in 30 days (right). Other data indicates thatPHBV readily degrades under aerobic conditions.

SOURCES: Petra Pochner and Wolf-Rudiger Muller, “Aspects on Biodegradation of PHA,” H.G. Schlegel and A. Steinb(khl (eds.), Proceedingshterrtafional Symposium on Bacterial Po/yhydroxyakarmates 7992 (G6ttingen, Goltze-Druck: 1993); William D. Luzier, “Materials Derived fromBiomass/Biodegradable Materials,” Proceedings iVationa/kademy of Sciences, vol. 89, February 1992, pp. 839-842.

ganisms, both bacterial and fungal, can utilizePHAs as a source of carbon and energy.

CURRENT AND POTENTIAL APPLICATIONSPHAs have many possible uses. The inherent

biocompatibil i ty 8 of these bacterial materialssuggests several medical applications: controlleddrug release, surgical sutures, bone plates, andwound care. PHAs could also be used as structuralmaterials in personal hygiene products and pack-aging applications. At present, higher-volumecommodity plastic applications are limited be-cause of economic constraints. PHBV, for exam-ple, costs $8 to 9 per pound. However, costs havefallen from about $800 per pound in 1980, and itis believed that the price of PHBV can be broughtto around $4 per pound by 1995. (Petroleum-based polyethylene and polypropylene polymerscost about 50 cents per pound.) PHBV is nowbeing used in a variety of molding applications in

the personal care sector (e.g., biodegradablecosmetic containers-a market where the cost ofthe container is almost negligible in relation to thecost of the contents). In addition, Zeneca is in theprocess of commercializing films and papercoatings from BIOPOL resin. Mitsubishi Kasei(Japan) is actively involved in the development ofPHAs for use as a biodegradable replacement ofmonofilament fishing nets. Because of the desira-ble environmental characteristics of PHAs, thenumber of such niche markets is likely tomultiply.

Over the past 5 years, there has been asubstantial increase in the number of publicationsdealing with the biosynthesis, fermentation, andcharacterization of the PHA family of biopoly -mers. It soon may be possible for these polymersto compete as specialty plastic products. Theability to genetically engineer the different spe-

a The final degradation product of one type of PHB is a normal constituent of human blood.

cies of bacteria used to produce PHAs (e.g., bymodifying the enzymes inside the bacteria) couldresult in the creation of highly customized poly-mers. Researchers have made significant progressin unraveling the biosynthetic pathways involvedin the production of PHAs. The genes encodingthe enzymes involved in PHA production havebeen isolated and cloned, and thus scientists cannow tailor the biosynthesis process to producepolymers with different properties. Over the longterm, there is the possibility that these materialscan be made economically in plant species.9

Recently, PHB was successfully synthesizedby using a genetically engineered experimentalplant (Arabidopsis thaliana).10 Researchers arenow exploring how to produce PHAs by modify-ing the enzyme machinery of corn or potatoes.11

Although significant technical challenges remain,the PHA family could potentially become a majoragricultural commodity, either as a fermentationproduct using raw materials (e.g., glucose) fromthe starch industry or, in the longer term, as a newcrop.

Recombinant Protein PolymersProteins are polymers composed of amino

acids. The specific amino acids used and thesequence of amino acids in a protein polymerchain are determined by the corresponding DNAtemplate. Many proteins are of commercial inter-est because of their catalytic (enzymatic) orpharmaceutical properties. However, nature has

Chapter 2–Technical Overview of Biopolymer Field 23

Coatings based on corn protein (zein) have goodmoisture and grease barrier properties and are beingused to replace polyethylene and wax-coated paperand paperboard.

also provided a vast array of proteins whoseprincipal function is to form structural materialsin living organisms. Some of the more familiarprotein materials include wool, leather, silk, andgelatin (jello is a simple, modified form of theprotein collagen12). Although many of thesestructural proteins have been used throughouthistory, the advances in recombinant DNA tech-nology have presented new approaches and op-portunities for the design and synthesis of proteinmaterials. In addition, many traditional proteinsprepared by the extraction of animal (e.g., colla-gen) or plant (e.g., soy or zein from corn) tissueare being chemically or physically modified for

g The genetic manipulation of bacteria could also alter the economics of PHA production. However, the manufacturing costs for PHAs aredetermined primarily by the purification steps rather than the bacterial production steps (see figure 2-l).

10 ~thou@ ~mly work ~ ~s mm is encowa@g, achievfig contro~~ gene expression of H iII plants is a formidable ~d~g.

Inserting the genes that encode the PHB-producing enzymes is relatively straightforward. However, regulating the PHB enzymes and theexisting plant enzymes is a more difficult challenge. See Yves Poirier, Douglas Dennis, Karen Klomparens, and Chris SomemiUe,‘ ‘Polyhydroxybutyrate, a Biodegradable Thermoplastic, Produced in Transgenic Plants,” Science, vol. 256, Apr. 24, 1992, pp. 520-523.

I I Robert Pwj, c{rn Search of the pktic Potato, ” Science, vol. 245, Sept. 15, 1989, pp. 1187-1189.

12 Collagen is a fi~ous prote~ that is the principal comwnent of animal connective tissue. It is the most abundant of all proteti fo~d ~mammals, typically accounting for more than 30 percent of body protein. The arrangement of collagen fibers depends on the nature of the tissue.For example, in tendons, fibers are arranged parallel to one another to give a structure with the tensile strength of a light steel wire. In sk@where strength and flexibility are required, collagen fibers are randomly oriented and woven together like felt.

2 4 Biopolymers: Making Materials Nature’s Way

Table 2-2—Repeat Units Found in Protein Materials

Protein Source Amino acid repeat unita

Silk Silk worm GAGAGS

Collagen Mammals GPPb

Adhesin Mussel AKPSYPPTYK

Elastin Pig VPGVG

Synthetic Chemically synthesized genes VariousaThere are 20 different amino acids, designated here in short form: A = alanine; G = 91Ycine; K = Iysine; p =

proline; S = Serine; T = threonine; V = valine; and Y = tyrosine. For a full list of amino acid symbols, see AlbertLehninger, Biochemistry (New York, NY: Worth Publishers, 1975), p. 72.baen one of the Prolines in collagen is hydrOWfJrOline.

SOURCES: Biolnformation Associates, Boston, MA; Office of Technology Assessment.

new applications in the biotechnology and foodindustries. 13 The discussion here focuses onprotein polymers that are being developed byusing the methods of recombinant biotechnology.

PRODUCTION OF RECOMBINANTPROTEIN POLYMERS

The extraordinary functional diversity of natu-ral proteins underscores the potential advantagesassociated with harnessing the genetic code. Intheory, proteins can be designed to have virtuallyany structure, and thus specific physical andchemical properties. The fact that one-dimensional genetic sequences can specify pro-teins having complex three-dimensional struc-tures over distances of hundreds of nanometersreveals the power of nature’s material synthesisprocesses. Current chemical synthesis techniquesare essentially limited to creating polymers in onedimension only,14 with lengths of less than 10nanometers .15

A number of proteins that form importantstructural materials in various organisms havebeen studied extensively. The fibrous proteinssuch as collagen and silk have been the subject ofconsiderable attention. More specialized proteins,such as the adhesive material that bonds the seamussel to the ocean bed and proteins that contrib-ute to the formation of ‘ceramic-like’ materials(e.g., oyster shells and teeth), are also beingactively studied. A common feature of many ofthese proteins is the presence of repeated aminoacid sequences in the polymer product (see table2-2). These proteins, or specific regions of theseproteins, have structural features similar to blockcopolymers (see figure l-l).

Some of the structural proteins have beenchemically synthesized. In this approach, specificpeptides (sequences of amino acids) are createdand then linked together to form a polypeptide(the protein polymer). However, chemical synthe-sis of proteins can be quite expensively and

rimarily as coatings for paper and paperboard. Soy paper coatings impart a smoother surface and improve13 SOY and corn proteins ~ ~~ psurface appearance. Zein has excellent grease and moisture barrier properties. Soy proteins are also being used as structuring agents inwater-based inks, bemuse they are regarded as environmentally preferable to solvent-based inks. See Thomas L. Krinski, “Emerging PolymericMaterials Based on Soy Prote@” Rowell et al. (eds.), op. cit., footnote 1.

14 men ~Ve ~n some tiv~ces recently in the synthesis of two-dimensional synthetic polymers. However, this work k still smewhtprelimhmry. See Scientific American, “Flat Chemistry,” April 1993, p, 26.

15 See Joseph @@lo, ‘‘Genetic Production of Synthetic Protein Polymers,” Materials Research Bulletin, October 1992, pp. 4S-53.IS ~ a few CmS, thou~ some fm have been able to develop relatively inexpensive approaches for synthesizing complex polypeptides.

For example, Monsanto developed comme~ial chemical methods for making sornatotrop~ a complicated polypeptide, for about $15 perpound.


Figure 2-3—Production of Recombinant Protein Polymers

-—-.— ..—————/ - L – . - . - - . - - i . – – ‘— — - - - J - . , Create a synthetic gene encoding the protein

I Genetic contruct ( D N A ) I\ –..A

polymer of Interest (regions coding for individualrepeat units are indicated by the hatched boxes).

I Introduce the genetic material Into a suitableproduction organism.

v—.

/-” A//’ 1 >r~y”,) Grow large volumes of the recombinant

\ (DNA ‘ ,, CIIIZ Protein polymerorganism and switch on production ofthe protein.

U--&-J--u

‘ \..-. -..-...-F?/\

Purify the recombinant

“4 [~,{ I \

El -L.-n ~

protein.

Protein polymer product


produced or investigated by using recombinanttypically does not yield products with sufficientphysical and chemical uniformity.

In contrast, the recombinant DNA approachallows for the production of protein polymers thathave high purity, specific molecular weights, andexcellent lot-to-lot uniformity. This is possiblebecause the exact sequence of amino acids in thepolymer and the desired molecular weight arespecified by the DNA sequence of the gene. In thelong term, recombinant DNA technology couldbecome an important strategy for producinghigh-value, ‘‘knowledge-intensive’ materials thatare custom-designed for specific applications.The steps involved in producing recombinantproteins are illustrated in figure 2-3, Currently, allof the proteins shown in table 2-2 are being

DNA techniques. The fact that it is now possibleto chemically synthesize genes encoding anyrepeat unit suggests almost limitless possibilitiesfor creating novel protein polymers with uniquephysical and functional properties. Althoughresearchers are addressing major technical prob-lems such as genetic instability, toxicity inforeign hosts, and metabolic incompatibility,considerable progress in genetically engineeredprotein synthesis has occurred in recent years.17

Initial work in the synthesis of artificial genes hasled to the creation of proteins that could be usedas coatings and adhesives, membrane separators,and medical and optical materials.18

17 Baause foreign DNA im~ses a metabolic burden on host cells, in some cases these DNA segments cm k destroyed. h ptic~m, hehighly repetitive gene sequences that are necessary to create artificial protein polymers are frequently unstable in microorganisms. In additio~novel proteins that are encoded by .Synthetic genes maybe toxic to the host cells, thus causing cell death before the polymer an be accumulatedin useful quantities. See David A. Tirrell et al., ‘ ‘Genetic Engineering of Polymeric Materials, ’ Materials Research Bulletin, July 1991, pp.23-28; see also Joseph Cappello, op. cit., footnote 15.

10 See R&d) Magazine, “Bioderived Materials, ” June 1990, pp. 58-64.

330-076 0 - 93 - 3 : QL 3


The marine mussel, Mytilus edulis, is shown suspendedin water by threads attached with adhesive to a glassplate. Biotechnology research on adhesives producedby rnarine organisms is leading to new adhesives thatcan be used for many applications such as surgery andundersea structures.

CURRENT AND POTENTIAL APPLICATIONSMore than 25 genetically tailored proteins have

been synthesized in microorganisms. Many ofthese materials are being transformed into films,gels, and fibers. One of the first genetically

engineered protein polymers to be introducedcommercially, ProNectin FTM,19 was designed toserve as an adhesive coating in cell culturevessels. The polymer was customized to have twodistinct peptide blocks: one block possesses thestrong structural attributes of silk; the other hasthe cell-binding properties of the human proteinfibronectin. 20 The peptide blocks were chosenafter analyzing which particular structures couldprovide the desired physical, chemical, and bio-logical properties. ProNectin F has demonstratedexcellent adhesion to plastic surfaces such aspolystyrene and thus can be used to attachmammalian cells to synthetic substrates.21

A similar application of recombinant DNAtechnology has led to the development of abioadhesive based on a protein from the sea

22 Researchers have genet-mussel Mytilus edulus.ically modified yeast cells to produce the basicmussel protein. An enzyme-catalyzed process(the enzyme-a tyrosinase-modifies the tyro-sine amino acids in the protein) was also devel-oped to convert this recombinant protein into atrue adhesive.23 This polymer could be used as amarine coating, as a wetting agent for fibers incomposite materials, or as a dental or surgicaladhesive.24 For example, it might be employed asa sealant during eye surgery .25

A number of other protein polymers are beinginvestigated by using biotechnology methods.One intriguing new material is a polypeptidebased on the natural protein elastin found in cows

19 me prote~ 15 produced by protein Polymer Technologies, bC., San Diego, CA.

20 Cappello, op. cit., footnote 15.

21 wi~ ce~ &P5 of ce~s, ProNcxfi F displayed adhesive characteristics superior to that of XMhmd athchment proteins inchK@fibronectin. Ibid.

22 Enzon Corp. (formerly Genex Corp.) developed the genetically designed adhesive undez the name AdheraCell. See Chen”cal andEngineering News, “Biotechnology Providing Springboard to New Functional Materials,” July 16, 1990, pp. 26-32.

23 ~5 enzyme.~~yz~ pr~~me is bOw w‘ ‘post-h~htio~’ modi.fkation. The sea mussel performs this rntikation titerpmtein

biosynthesis.

~ Chem’ca[ ad Engineering News, op. Cit., fOOb20te 22.

25 R~ Magazine, op. cit., footnote 18.

and pigs.26 This rubberlike material responds to

changes in temperature and is able to convertchemical energy into mechanical energy .27 As aconsequence, the material could be used as areplacement for ligament tissue, blood vessels, orany other tissues requiring the contractile proper-ties of elastin.28 Because the peptide is similar incomposition to natural elastin, the polymer is notexpected to elicit an allergic reaction or unfavora-ble immune system response.

Another interesting material is the light-sensitive protein bacteriorhodopsin (BR). BR is aretinal protein consisting of 248 amino acids.When subjected to photons (light) of varyingenergy intensity, BR undergoes reversible colorchanges. Through substitution of specific aminoacids by genetic engineering, the photochemicalproperties of BR can be precisely modified, whichmeans color changes can be carefully controlled.The unique properties of BR could lead toapplications in optical data storage, image proc-essing, light switching (computational process-ing), and holography. Some initial work indicatesthat the optical performance of BR is comparableto conventional materials such as liquid crys-tals.29


Recombinant biotechnology is also being usedto modify materials that have been utilized forthousands of years. Silk has always been amaterial of great fascination. Spiders can processsilk protein into a material that has a tensilestrength 16 times greater than that of nylon30 anda very high degree of elasticity .31 Researchershave manipulated the genetic code to createsilklike materials with a variety of elasticities.32

Because of their exceptional tensile strength andelastic properties, these polymers could be usedas fibers for reinforced plastics and other ad-vanced composite materials. In the medical area,the polymers could potentially be used as wounddressings, artificial ligaments, and skin or as abiocompatible coating for prosthetic devices.However, the yields of genetically modified silkpolymers from microorganisms have thus farbeen fairly 10W.33

Most protein polymer research is focused onhigh-technology applications, such as elastomers,adhesives, bioceramics, and electro-optical ma-terials. To date, commercial applications havebeen limited to the use of genetically engineeredadhesives for fixing mammalian cells to culturevessels. Because of extremely high productioncosts, these products will probably have limited

24 Elastin fibers are elastic, load-bearing protein polymers found in connective tissue such as ligaments. Another protein similar to elastinis resilim a rubberlike polymer found in insects.

‘7 See Science, “Heeding the Catl of the Wild, ” vol. 253, Aug. 30, 1991, pp. %5-%8.ZB R~ Magazine, op. cit., fOO?210te 18.

29 See N. tipp, C. Brauchle, and D. Oesterhe16 “Mutated Bactenorhodopsins: Competitive Materials for Optical InformationProcessing?” Materials Research Bulletin, vol. 17, No. 11, November 1992, pp. 56-60.

so silkworm silk is about two to three timeS stronger than nylon.

31 Spider dragline silk not only ~s=sses great streng~ but alSO has the ability to “sup~con~act.’ Silk fibm wifl con~act to less @ @

percent of its original length when wet, This results in a nearly thousandfold decrease in the elastic modulus and an enhanced ability to extendwhen necessary. This property allows the spider web to tighten each day when wetted with dew, while still maintaining its shape and tension.Although some human-made materials can sup-contract in organic solvents, no synthetic materials can supercontract-as spider silk does-inwater alone. The web is constructed of several different silks, each of which is produced in a different spider gland. Some of the silk fiberscontain a number of water-soluble compounds that keep the fibecs wetted, allowing them to stretch and entangle prey that hit the web. SeeRandolf V. Lewis, “Spider Silk: The Unraveling of a Mystery, “ Accounts of Chem”cal Research, vol. 25, No. 9, 1992, pp. 392-398.

32 ~otein po]ymer Technologies, Inc. has produced recombinant polymers based on silkworm silk. The company k developed m ~~lci~silk gene that appears to be stable in host Escherichia coli cells. However, the stability of cloned silk genes in host systems still remains asignificant problem (R&D Magazine, op. cit., footnote 18; Chemical and Engineering News, op. cit., footnote 22. Also see David L. Kaplanet al., “Biosynthesis and Processing of Silk Proteins, ’ Materials Research Bulletin, October 1992, pp. 4147.

J~ Lewis, op. cit., footnote 31.

. . . . .


Figure 2-&The Structure of Cellulose

CH20H

o o

H oHOH H

HO HH OH

L

YFoH ‘\!

O+OH H/”

H OH

H C H20 H

D

oH

o OH H

H OHn

H,OH

The cellulose molecule is composed of glucose units connected by B(1-4) bonds (see Figure 1 -3). Starchhas an identical chemical composition to cellulose except for its connecting bonds-a(l -4). The differentlinkages in starch molecules endow them with a greater water volubility than cellulose. In humans, starchcan be digested while cellulose cannot be digested.SOURCE: Office of Technology Assessment, 1993.

success (e.g., experimental quantities of thegenetically derived sea mussel adhesive were atone time selling for about $45 per milligram, or$20 million per pound). However, over the longterm, genetic techniques may allow production tobe scaled up significantly at reasonable cost.Once programmed with the proper genetic in-structions, bacterial cells can work in parallel bythe billions to produce polymer materials.34

Although some biotechnology companies havebeen involved in protein polymer research for 10years, most recombinant protein materials are stillin early stages of development.

Nevertheless, this area of research is one of themost active and better funded in the biopolymerfield. In addition to providing new materials,genetic engineering is now enabling scientists tostudy how biological systems transform proteinsinto final products. It is remarkable that livingorganisms are able to produce sophisticatedmaterials under mild processing conditions (i.e.,

low temperature and pressure in water-basedenvironments), without creating toxic bypro-ducts. This is certainly not the case for a varietyof human-made materials.35 Spiders and silk-worms, for example, can transform water-solubleprotein droplets into globally aligned insolublefibers. 36 The fibers are spun with very littleenergy consumption. Thus, protein polymer re-search could also lead to the development ofradically new industrial processing methods thatpose little threat to the environment.

The Microbial PolysaccharidesBACTERIAL CELLULOSE

Cellulose is the most abundant component ofbiomass and the basic feedstock of the paper andpulp industries. Traditionally extracted from planttissue (trees, cotton, etc.), cellulose can also beproduced by certain bacterial species by ferment-ation, yielding a very pure cellulose product withunique properties.

34 s~i~~ce, op. cit., f00b30te 27.

3S my ~vmced mat~~s ~ sw~es~~ at exhemely high temperature and pressure, and require toxic substi~s at various s@es of

processing. See U.S. Congress, Office of Technology Assessmen~ Advanced Materials by Design, OTA-E-351 (Washington, DC: U.S.Government Printing Office, June 1988).

36 Sik fibers ~ pu~ed from spiders, not forced out by pressure. The fibers are formed as they travel down a tubular duct kid@ from tiegland to the exit valve. The key chemical and physical events that change the soluble proteins into solid fibers occur during this journey. Asthe protein molecules travel down the duct, they align themselves into regular arrays. It appears that the mechanical and frictional forces at workin the duct facilitate the transformation of the soluble protein droplets into solid fibers. See Lewis, op. cit., footnote31; Kaplau op. cit., footnote32.


Cellulose is a polysaccharide consisting oflinear glucose chains (see figure 2-4). Bacterialcellulose is synthesized in a process whereby thepolymer material is extruded from the bacterialcells. Most cellulose-producing bacteria (e.g.,Acefobacter) extrude cellulose as a ribbonlikeproduct from a single fixed site on the cell surface.This results in the formation of a network ofinterlocking fibers.

Bacterial cellulose is produced under condi-tions of agitated fermentation. High polymerproduction rates occur when the growth mediumcontains glucose, salts, corn steep liquor, ironchelators, and various productivity enhancers.Current yields are more than 0.2 gram of celluloseper gram of glucose, and production has beendemonstrated in commercial 50,000-gallon fer-menters. After fermentation, the bacterial cellsare destroyed during a hot caustic treatment.Bacterial cellulose is a water-insoluble materialthat has a very large surface area because of itslarge network of fibers, Bacterial fibers haveroughly 200 times the surface area of fibers fromwood pulp.37 This, coupled with their ability toform hydrogen bonds, accounts for their uniqueinteractions with water. Bacterially derived cellu-lose materials can absorb up to six times theirweight of water, and when used as suspensions,they have pseudoplastic thickening properties.Sheets prepared from bacterial cellulose haveexcellent mechanical properties.

CURRENT AND POTENTIAL APPLICATIONSConsiderable progress has been made in the

field of bacterial cellulose synthesis in the pastfew years. Bacterial cellulose is now available inlimited quantities from Weyerhaeuser in theUnited States and Ajinomoto in Japan. The mostprevalent applications of bacterial cellulose ex-

Cellulon@ engineered bacterial cellulose fiber is aproduct under development at WeyerhaeuserCompany. The unique reticulated network of finefibers gives the biopolymer its thickening, binding,and coating properties. The fibers have a typicaldiameter of 0.1 microns (0.1 millionth of a meter),while some wood pulp fibers have a diameter of25 to 35 microns.

ploit its very large surface area and its ability toabsorb liquids. Consequently, very low concen-trations of bacterial cellulose can be used to createexcellent binding, thickening, and coating agents.Because of its thickening properties, many appli-cations in the food industry are possible. Paperthat is coated with bacterial cellulose is extremelysmooth and protects the underlying fibers frommoisture. End uses in oil and gas recovery,mining, paints, adhesives, and cosmetics are also

envisioned. This material is currently used bySony Corporation in the production of high-endaudio speaker systems because of its excellentacoustic properties.38 In the last 10 years, at least

37 Weyerbeuser’s bactefi~ ~ll~ose fibers (the product is called Cellulon) have a typical diameter of 0.1 ~crons ~ oPPosed to some wood

pulp fibers that are 25 to 35 microns across. The small fiber diameter results in an extraordinarily high surface area and is responsible for muchof the fiber’s functionality as a thickener and binder. A. Robert Winslow, Cellulon Fiber Business Marketing Manager, WeyerhaeuserCompany, personal communication, Aug. 31, 1993.

38 me ~gh.fideliq headphones employ bacterial cellulose as diaphragms. The headphones retail fOr about $$,000 a wt.


Figure 2-&The Structure of Xanthan Gum

AcOCH2 Iw—o

I OH O

OH

co*I) ‘---- o ,u,/‘ OH

c o * , OCHL‘\\\ 8’ ,/- --00

c ’ \ ~ OH

n

--@

4Ac M

n

CHEMICAL STRUCTURE BLOCK STRUCTURAL REPRESENTATION

The Xanthan gum repeat unit is made of 5 sugar groups: two glucose (G) groups, two mannose (M) groups, and one gluouronicacid (GA) unit. Pyruvate (Pyr) and acetyl (Ac) units are also present in the mannose structures. The degree of pyruvate and acetatesubstitution varies with the specific fermentation conditions. The charged pyruvate molecule alters xanthan’s electrical properties,while the acetate serves to stabilize the conformation or spatial arrangement of xanthan.SOURCE: Biolnformation Associates, Inc.., Boston, MA.

50 patents on the production and applications ofbacterial cellulose have been filed. Currently,bacterial cellulose sells from $35 to $50 perpound. If the material is to be used in commodityas opposed to niche applications, production costswill have to drop. A Japanese consortium wasrecently formed to reduce the manufacturingcosts of bacterial cellulose.39

XANTHANXanthan gum, a complex copolymer produced

by a bacterium, was one of the first commerciallysuccessful bacterial polysaccharides to be pro-duced by fermentation. The xanthan polymerbuilding blocks or “repeat units” contain fivedifferent sugar groups (see figure 2-5). Thexanthan-producing bacterium, Xanthomonas cam-pestris, is one of the frost bacterial polysaccharide

w The comemi~ venture in bacterial cellulose technology is supported fmrmcially by the Japan Key TectioIogY Center, a jointorganization under the Ministry of International Trade and Industry and the Ministry of Post and Telecommunications, along with six privatesector companies: Ajinomoto, Shimizu Construction N- Mitsubishi Paper, Niklciso, and Nakarnori Vinegar. The venture is calledBiopolymer Research Co., Ltd, and its principal focus will be the development of mass production techniques for cellulose that is made byfermentation (Japan Chemical Daily, Apr. 13, 1992).

.-


production systems targeted for genetic engineer-ing. Under certain conditions, genetic modifica-tion of Xanthomonas by using recombinant DNAtechnology has increased the rate of xanthanproduction by more than 50 percent.40 In thefuture, recombinant DNA technology may enableentirely new xanthan biosynthetic pathways to becreated in host organisms.

Xanthan gum is produced by large-scale fer-mentation of X. campestris using a number ofdifferent feedstocks including molasses and cornsyrup. The gum is extruded from the bacteriaduring the polymerization process and can berecovered by alcohol precipitation followingremoval of the bacterial cells. For some applica-tions such as enhanced oil recovery, the crudeculture broth can be used directly followingsterilization. Probably the most significant tech-nical problem in the production of xanthan is thefact that as the polymer is produced, the fermenta-tion medium becomes increasingly viscous. Thisincreases the energy required for the mixingprocess that feeds oxygen to the bacterial cells.

CURRENT AND POTENTIAL APPLICATIONSThe unusual physical and mechanical proper-

ties of xanthan gum make it an attractive polymerfor industrial and biological use. It is usedextensively in both the food and the nonfoodindustries. Examples of industrial applicationsinclude oil recovery (provides viscosity control indrilling mud fluids), mineral ore processing (usedas a biocide), paper manufacturing (used as amodifier), agriculture (acts as plant growth stimu-lator), pharmaceuticals (being evaluated for sus-

tained drug release), and cosmetics (controls dustrelease). Food applications include gelling agentsfor cheese spreads, ice creams, puddings, andother deserts. More recently, xanthan has beenused in the new clear-gel toothpastes. Reading thelabels on many of the processed foods in thesupermarket should give one a clear picture of thewide use of this material. Good examples arepacket soups and many of the fat-free foods thathave recently become available.

In terms of production volume, xanthan gum isthe most widely used microbial polysaccharide.Worldwide production is currently in the range of10,000 to 20,000 tons. Companies such as ADMand Merck have recently announced the expan-sion of their xanthan production facilities. About60 percent of the xanthan produced is used infoods, with the remaining 40 percent used inindustrial applications. Food-grade xanthan costsabout $8 to $10 per pound, while non-food gradessell for about $5 per pound. Thus far, onlyexperimental samples of genetically modifiedxanthan have been produced.41

DEXTRANSDextran is the generic name of a large family of

microbial polysaccharides that are assembled orpolymerized outside the cell by enzymes calleddextran sucrases. This class of polysaccharides iscomposed of building blocks (monomers) of thesimple sugar glucose and is stored as fuel inyeasts and bacteria. Dextrans are produced byfermentation or enzymatic conversion of thefeedstock sucrose, a product of the sugar beet andsugarcane industries. Most commercial dextran

@ See T.J. Pollack and J. Thorne, SphW COW., ‘‘Enhanced Manufacture of Xant.han Gum with Genetically Modif”ed Xandwnonas,”European Patent Application EP 287,367. CA: 9358z; and M. Yalpani, Polysaccharide Synthesis, Modification, and StructurelPropertyRelations, Studies in Organic Chemistry 36 (Amsterdam: Elsevier, 1988).

q I TWO SIIMI1 biotecholog companies, Syntro (San Diego, CA) and Synergen (Boulder, CO), worked on genetically ememed x~tigums in the 1980s. Neither fii was successful in marketing these xanthan products because their manufacturing costs were much higher thanconventional xanthan production methods. The relatively high production costs of genetically moditled polymers is a major problem that couldseriously constrain biopolymer commercialization efforts. ‘‘Conventioml wisdom’ holds that biopolymer production costs must fafl below$20 per pound for most niche market applications, and below $5 per pound for more general applications. Because of these manufacturinghurdles, some producers of genetically derived biopolymer compounds are focusing on low-volume, high value-added applications such asmedical materials or specialized industrial adhesives. David Many~ President, Adheron Corporation personal communication Aug. 24, 1993.


production uses the microorganism Leuconstocmesenteroides. Dextran can be synthesized byusing either large-scale industrial fermentors orenzymatic filtration methods. The latter approachis generally favored since it results in an enhanceddextran yield and a uniform product quality,which allows the product to be readily purified.Both of these production methods permit systemconditions to be adjusted so as to control themolecular weight range of the products. Thisfeature is an integral requirement for polysaccha-ride biosynthesis.

CURRENT AND POTENTIAL APPLICATIONSDextran polymers have a number of medical

applications. Dextrans have been used for woundcoverings, in surgical sutures, as blood volumeexpanders, to improve blood flow in capillaries inthe treatment of vascular occlusion, and in thetreatment of iron deficiency anemia in bothhumans and animals. Dextran-hemoglobin com-pounds may be used as blood substitutes that haveoxygen delivery potential and can also function asplasma expanders. Chemically modified dextranssuch as dextran sulfate have both antiulcer andanticoagulant properties. Other modified dex-trans such as Sephadex@ are used extensively inthe separation of biological compounds.

In the industrial area, dextrans are beingincorporated into x-ray and other photographicemulsions. This results in the more economicalusage of silver compounds and at the same timereduces surface gloss on photographic positives.Dextrans are used extensively in oil drilling mudsto improve the ease and efficiency of oil recovery.They also have potential use in agriculture as seeddressings and soil conditioners. The protectivepolysaccharide coatings are found to improvegermination efficiencies under suboptimal condi-tions.

Although many applications have been pro-posed for dextrans, only a small number of thesehave been realized and developed on a large scale.There is considerable potential for using low-molecular-weight dextrans in the biomedical industry in

surgery and drug delivery systems. However,low-molecular-weight dextrans sell for about $80per pound. As new and higher-volume applica-tions for these materials are developed, large-scale production of dextrans may represent amajor new market for the sugarcane and sugarbeet industries.

PULLULANPullulan is a water-soluble polysaccharide

produced outside the cell by several species ofyeast, most notably Aureobasidium pullulans.Pullulan is a linear polymer made up of mono-mers that contain three glucose sugars linkedtogether (see figure 2-6).

For more than a decade, a Japanese firm,Hayashibara Biochemical Laboratories, has useda simple fermentation process to produce pullu-lan. A number of feedstocks are used for thisprocess, including waste streams containing sim-ple sugars. Pullulan can be chemically modifiedto produce a polymer that is either less soluble orcompletely insoluble in water. The thermal andionic (electrical) properties of pullulans can alsobe altered.

CURRENT AND POTENTIAL APPLICATIONSPullulan and its derivatives have a number of

useful properties and consequently have manypossible applications. Hayashibara BiochemicalLaboratories currently sells three different pullu-lan grades: industrial grade ($6.50 per pound),food grade ($11 per pound), and medical grade($15 per pound). Current efforts to increase theyields of pullulan from the fermentation ofvarious strains of A. pullulans suggest lowerproduction costs are likely in the future. Pullulancompounds are biodegradable in biologicallyactive environments, have high heat resistance,and display a wide range of elasticities andsolubilities. This versatility allows them to beutilized in many different ways.

Pullulan has many uses as an industrial plastic.It can be formed into compression moldings thatresemble polystyrene or polyvinyl chloride in


Figure 2-6-The Structure of Pullulan

CH2 CH20H CH2OH

}--- ------o }-- - -0 &——- o

K&LWo’tioOH OH OH

CH2 CH20H CH20H

~o ,~() L. ._()

Pullulan is made up of glucose sugars linked together in groups of three. The three member repeat units areconnected together in a branched fashion.

SOURCE: Office of Technology Assessment, 1993.

transparency, gloss, hardness, strength, and tough-ness, but is far more elastic. It decomposes above200’C, apparently without the formation of toxicgases. 42

A completely different application of pullulancan be found in the food industry. It can be usedas a food additive, providing bulk and texture. Itis tasteless, odorless, and nontoxic. It does notbreak down in the presence of naturally occurringdigestive enzymes and therefore has no caloriccontent. Consequently, it can be used as a foodadditive in low-calorie foods and drinks, in placeof starch or other fillers. In addition, pullulaninhibits fungal growth and has good moistureretention, and thus can be used as a preservative.

Pullulan can also be used as a water-soluble,edible film for the packaging of food products. Itis transparent, impermeable to oxygen, and oil-and grease-resistant. Foods can be either im-mersed in a solution of pullulan or coated by apolymer spray. After the pullulan coating is dried,an airtight membrane is formed. The membranecan be used in the packaging of drugs andsupplements, as well as oil-rich food products that

are vulnerable to oxidation, such as nuts and friedfoods. It is not necessary to remove the pullulancoating before eating or cooking. In the packagingof tobacco, pullulan enhances product longevityand retention of aroma. It also protects againstoxidative degradation as well as attack by mold.Water insoluble coatings may be made by usingthe esterified or etherified forms of pullulan.

Ester and ether derivatives of pullulan haveadhesive qualities similar to those of gum arabic.Viscosity and adhesive properties depend on thedegree of polymerization. Modified pullulan canbe used as a stationary paste that gelatinizes uponmoistening. Fibers can be made from concen-trated solutions of pullulan having high viscosity.The fibers have a gloss resembling rayon and atensile strength similar to that of nylon fibers.Pullulan and its ester or ether derivatives can alsobe used as binders, in conjunction with othermaterials, for the production of nonwoven fab-rics. With or without the addition of othervegetable pulps, it can be used to make paper thatis suitable for printing and writing. Because it is

42 However, it should be not~ that partial combustion of carbohydrates can produce cMbOn monofide.


an antioxidant, pullulan can substitute for gumarabic in lithographic printing.

There are a plethora of other applications.Pullulan can be used as a binding agent for solidfertilizers, allowing time-released fertilizationand thereby avoiding the burning of crops bycontrolling the release of nitrogen in the fertilizer.As a binding agent in sand molds used for metalcasting, pullulan prevents the generation of dustor toxic fumes. The biopolymer can be used as aflocculating or aggregating agent for the precipi-tation of potash clays, uranium clays, and ferrichydroxide from slurries used in the beneficiationof mineral ores. (Currently, synthetic chemicalsare primarily employed in mineral processing.)Pullulan can be used as an additive to resins andpaints, where its preservative and antioxidationproperties help retain color and gloss. Also,pictures or illustrations printed on pullulan filmwith edible ink can be transferred onto foodproducts. In the medical area, pullulan acts as aplasma extender without undesired side effects.After metabolic turnover, it is completely ex-creted. Pullulan compounds can also serve asdrug carriers, and can be used as medical adhe-sives.

Although markets for many of the applicationslisted here are still relatively small, with someapplications only in the exploratory stage, pullu-lan appears to have long-term commercial poten-tial. In sum, pullulan’s many disparate uses mayentitle it to become known as a biopolymer‘‘wonder material.

GLUCANSGlucans are, by definition, any homopolymer

of the simple sugar glucose. This large group

includes cellulose,However, the term

pullulan, and yeast glucan.‘‘glucan’ is commonly used

to describe the glucan component of the yeast cellwall. A common source for this glucan is baker’syeast, Saccharomyces cerevisiae, although it isalso found in a number of other sources (bacteria,fungi, lichen, and higher plants, e.g., barley).

Large supplies of inexpensive yeast are availa-ble from both the baking and the brewing(brewer’s yeast) industries. Glucans are the mostabundant polymers in yeast, making up approxi-mately 12 to 14 percent of the total dry cellweight. Glucan is readily purified from yeast cellsby using hot alkali treatment to remove all othercellular materials, thereby allowing recovery ofthe insoluble glucan material. Yeast glucan parti-cles purified by this method contain both highmolecular weight and lower molecular weightpolymers.

CURRENT AND POTENTIAL APPLICATIONSGlucans have a number of potential medical

uses. 43 Glucans that are extracted from yeast cell

walls are found to markedly increase the ability ofsome organisms (e.g., mice) to resist invadingforeign bodies. Because of its action as animmunomodulator, or enhancer of the immunesystem, a number of studies have been performedexploring the use of glucan as an anti-infectiousagent. Glucan is also effective as an antiviralagent in plants. For example, one form of glucanis very effective in protecting many species oftobacco plant against invasion by the tobaccomosaic virus and tomato black ring virus. Plantscan be either injected or sprayed with the glucanpolymer.

43 s~ce polysacc~des play an important role in cell ad.lmsionand asmolecdar reCO@tiOn el~ents (JJlyCOprOtefi), tieypotm~y ~vea number of medical applications. Recent developments in the cloning and expression of enzymes known as oligosaccharyl transferases couldfacilitate the creation of new carbohydrate-based pharmaceuticals. See ChemicaZ andEngineering News, “Race Is on ‘h Develop Sugar-BasedAnti-inft ammatory, Antitumor Drugs, ” Dec. 7, 1992, pp. 25-28. Apart from genetic engineering methods, some of these enzymes can also bechemically isolated. A new enzyme isolation method is being used to simplify the synthesis of novel polysaccharide CQmpounds. Some of thenew compounds have been found to prevent bacterial pneumonia in animals and are also being evaluated as anti-infective drugs. Ihe enzymescould also be used to create polysaccharides that act as industrial coatings (e.g., as coatings on ship hulls to improve fuel efllciency and preventmarine corrosion). Stephen RotlL Neose Pharmaceuticals, personal communication, Sept. 3, 1993. Also see Chemical and Engineering News,“Patent Grants Broad Protection to Enzymatic Carbohydrate Synthesis,” Mar. 29, 1993, pp. 24-27.

Chapter 2–Technical Overview of Biopolymer Field I 35

Several studies using different tumor models inmice and rats have revealed that glucans caninhibit tumor growth. The less toxic, soluble formof glucan is effective as an antitumor agent,although it is slightly less effective than theparticulate form. Many antitumor glucans arecurrently being used in Japan on human subjects.In the United States, one company (Alpha BetaTechnology of Worcester, Massachusetts) hasyeast glucans that are undergoing clinical trials asimmune-stimulating agents. Another interestingproperty of glucans is that they are radio-protective (i.e., they appear to enhance survivalby preventing death due to postirradiation infec-tion). This enhances the survival of test animalsafter otherwise lethal doses of radiation.

Although glucans are being exploited princi-pally for their antitumor, anti-infectious, andradioprotective properties, they also have non-medical applications. Glucans resist breakdownwhen attacked by digestive enzymes, and thus canbe used as noncaloric food thickeners. Otherpossible applications include use in sustained-release tablets, encapsulation of oxygen for masstransfer in fermentation reactions, and as a solidsupport material for chromatographic separa-tions.

GELLANIn addition to xanthan, pullulan, and glucan, a

number of other microbial polysaccharides arebeing investigated for applications as thickeningagents. These include succinoglycan, sclero-glucan, and the three structurally related poly-mers rharnsan, welan, and gellan. Among these,gellan gum is the most recent microbial polysac-charide to be given Food and Drug Administra-tion (FDA) approval for applications in foodproducts (September 1990). Gellan is a complexpolysaccharide having a four sugar repeat unit

(glucose-glucuronic acid-glucose-rhamnose). Itis produced by the bacterium Pseudomonaselodea, which is derived from plant tissue.

The production of gellan follows essentiallythe same fermentation process described forxanthan. The properties of gellan can be easilymodified. A hot caustic treatment of gellan yieldsa polymer that has the desirable characteristic oflow viscosity at high temperature. Cooling gellanin the presence of various cations (e.g., calcium)results in the formation of strong gels.

CURRENT AND POTENTIAL APPLICATIONSGellan gum represents the newest member of

the microbial polysaccharides to be developedcommercially. Developed and produced by theKelco Division of Merck under the trade namesKelcogel and Gelrite, this polymer has applica-tions in the food industry as a gelling agent infrostings, glazes, icings, jams, and jellies. GelIancurrently sells for about $5 per pound.

SELECTED POLYMERS OF PLANTS ANDHIGHER ORGANISMS”

StarchStarch is the principal carbohydrate storage

product of higher plants. The term starch actuallyrefers to a class of materials with a wide range ofstructures and properties. Starch polymers can beextracted from corn, potatoes, rice, barley, sor-ghum, and wheat. The principal source of starchfor industrial and food purposes is corn. In theUnited States, about 4.5 billion pounds of corn-starch is used annually for industrial applica-tions.45 Starches are mixtures of two glucanpolymers, amylose and amylopectin.46 Thesepolymers are accumulated in plants as insolubleenergy storage granules, with each granule con-taining a mixture of the two polymers. Plant

~ Higher ~rga~sm (l.e,, eqotes) refer to all organisms except viruses, bacteri& and bhle-gr~ ~gae.

45 see us Congtis, Ofilce of Te~-moloW Assessmen6 Agn’culmral Cowdities as Indusvial Raw Materials, OTA-F-476 ~aS~tOrl+

DC: U.S. Government Printing Office, May 1991).46 @ylose is a /inear ~lmer of glucose tits, w~e amylop~~ is a branched polymer of glucose W& (~ figure 1-3).


breeding techniques have been used to producenew strains with altered ratios of amylose toamylopectin (e.g., waxy corn contains only 0.8percent amylose compared with natural corn,which contains 28 percent amylose, and amylo-maize can contain up to 80 percent amylose). Theability to manipulate the ratio of amylose toamylopectin by strain development has drasti-cally reduced the economic costs associated withphysical separation of the two polymers. This isimportant because amylose and amylopectin havedifferent properties and applications.

CURRENT AND POTENTIAL APPLICATIONSBecause of its low cost and widespread availa-

bility, starch has been incorporated into a varietyof products. Chemical modification of starchpolymers can lead to a number of useful deriva-tives. Current U.S. production of ethanol requiresabout 400 million bushels of corn. An additionalseveral billion pounds of cornstarch is used fornonfuel purposes. Approximately 75 percent ofthe industrial cornstarch produced is transformedinto adhesives for use in the paper, paperboard,and related industries. Because cornstarch canabsorb up to 1,000 times its weight in moisture, itis used in disposable diapers (about 200 millionpounds annually), as a treatment for burns, and infuel filters to remove water. Cornstarch polymersare also used as thickeners, stabilizers, soilconditioners, and even road deicers.47

In recent years, starch has attracted consider-able attention as a biodegradable additive orreplacement material in traditional oil-based com-modity plastics. Although a number of starch-plastic material blends have been used in differentproducts, particularly packaging and garbagebags, there has been considerable controversy as

to whether these starch-polymer composites canbiodegrade.48 Starch itself degrades readily, andis in fact one of main components of thebiological food chain. When added to petroleum-derived polymers such as polyethylene, starchcan in theory accelerate the disintegration orfragmentation of the synthetic polymer chains.Microbial action consumes the starch, therebycreating pores in the material, which weakens itand enables it to break apart. Many have incor-rectly characterized this process as a form ofbiodegradation. Independent tests of polyethylene-starch blends show that starch may biodegrade,but that the overall polymer formulation will notbiodegrade at any significant rate. Distintegrationof polyethylene-starch blends is not the same asbiodegradation. Moreover, studies indicate thatdegradation rates vary considerably under differ-ent temperature, oxygen, and moisture condi-tions. In landfills, for example, degradation rates,even for readily degradable materials, are ex-tremely s10W.49 Under optimal conditions, break-down of starch-plastic blends that contain lessthan 30 percent starch is quite slow. Someresearch indicates that the starch compositionneeds to exceed about 60 percent before signifi-cant material breakdown occurs.50

These problems have led to the development ofa new generation of biodegradable materials thatcontain very high percentages of starch. Undercertain conditions, starch can be combined withwater and other compounds to create a resin thatis somewhat similar to crystalline polystyrene.Warner-Lambert has recently introduced theNOVON@ family of polymers that contain from40 to 98 percent starch and readily dissolve inwater. The NOVON resins combine starch withother biodegradable materials, and when dis-

47 U.S. Congress, op. cit., fmtnote 45, pp. ~W.

48 See “Dega&ble Plastics Generate Controversy h Solid WaStC Issues, ’ Chemical & Engineering News, June 25, 1990, pp. 7-14; and“Degradable Plastics,” R&D Magazine, March 1990, pp. 51-56.

49 Safiw landfills me essentiwy biologically inactive environments. See the testimony of Joan Ham on “Degradable Plastics andMunicipal Solid Waste Management” before the Senate Committee on Governmental Affairs, July 18, 1989.

so us. Congess, Offlm of TedmoIogy Assessment op. cit., f~~ote 45) P. 96.


posed in biologically active environments such ascompost facilities and wastewater treatment sys-tems, display degradation characteristics similarto lignocellulosic materials (e.g., leaves, wood-chips, and paper) .51

Properties of these new materials can be variedas the composition of starch and other materialcomponents change. They can replace traditionalplastics used in food service, food packaging,personal health care, agricultural, and outdoormarkets. Early applications of NOVON polymersinclude compost bags, degradable golf tees,loose-fill packaging, cutlery, pharmaceutical cap-sules, and agricultural mulch films. The companyopened a 100-million-pound NOVON manufac-turing facility in 1992. The materials are beingtargeted for markets where the benefits of theirbiodegradability can be clearly demonstrated. Tofully take advantage of their environmental char-acteristics, however, a coordinated compost infra-structure will have to be established.52 Althoughthese starch-based resins cost two to four timesmore than commodity resins ($1.50 to $3 perpound), their novel properties might lead to thecreation of new specialty markets.

Plant CelluloseAs mentioned previously, cellulose is one of

the most abundant constituents of biologicalmatter. 53 It is the principal component of plantcell walls. Among the plant cellulose, cottonfiber is the most pure, containing around 90percent cellulose. Wood, on the other hand,consists of about 50 percent cellulose. Celluloseserves as an important material feedstock formany industries. U.S. production of cellulosefibers amounted to 485 million pounds in 1991.By adding various functional groups to the basic

4New starch-based thermoplastic biopolymers such asthe AMYPOLTM family of resins are water resistantand can be extruded, pelletized, and injection-moldedinto industrial and consumer products and films.

glucose building blocks of cellulose (see figure2-4), a range of useful derivatives (cellulosics)can be created.

CURRENT AND POTENTIAL APPLICATIONSChemically modified plant cellulose are used

in a remarkably diverse set of applications.Cellulose derivatives are used to forma variety offibers, thickening solutions, and gels. For exam-ple, carboxymethylcellulose (CMC) is used as athickener, binder, stabilizer, suspending agent, orflow control agent. The major markets for CMCare detergents, food, toothpaste, shampoo, skinlotions, textiles, paper, adhesives, ceramics, andlatex paints. In the biotechnology area, CMC gelsare used for separating molecules. Hydroxyeth-ylcellulose (HEC) is a water-soluble compoundthat has major applications in the oil industry.HEC is used as a thickener in drilling fluids andas a fluid-loss agent in cementing. Hydroxypro-pylcellulose (HPC) has excellent surface proper-

51 me ~ompmy rePf15 tit we end pr~ucts of these new materials are carbon dioxide, water, and bio~s, ~~ no Persistent syn~eticresidues. See the testimony of Ken Tracy, Vice-President of Environmental Technology, Warner Lambefl Company, before t.be SenateCommittee on Environment and Public Works, June 5, 1991.

52 For more on ~5 ~bj~t, SW us. congres5, off~~~ of T~~olo~ Assessment Facing Ameficu’~ Trash: Wh@N~t~or MUnicipa/ SO~id

Waste? OTA-O-424 (Washington, DC: U.S. Government Printing Oflice, October 1989).

53 B~ause of cell~ose’s abundance it sells for about 30 to W Cents per pound.

330-076 0 - 93 -4 : QL 3


A photomicrograph shows modified ethyl cellulosefilms magnified by a factor of 2,000. The materials arebeing evaluated as carriers for the controlled-releaseof nitrogen fertilizers.

ties and forms highly flexible films. It is used incoating pharmaceutical tablets, in molding opera-tions, in paper coatings, and as a suspending agentin inks, cleaners, and polishes. In the medicalarea, hydroxypropylmethylcellulose (HPMC) hasshown considerable promise as an agent forlowering blood cholesterol levels.54

There are many other useful derivatives. Cellu-lose acetate is a plastic-grade material that iswidely used in packaging, particularly for blis-ters, skins, transparent rigid containers, andwindows in folding or setup boxes.55 In addition,cellulose acetate is used in some fabrics and as awrite-on pressure-sensitive tape (e.g., for credit

card receipts). Methylcellulose, created by treat-ing cellulose fibers with methyl chloride, hasexcellent absorption properties and is a goodthickener. It has been used in a variety of foodproducts, including salad dressings, pie fillings,and baked goods. Nonfood applications includeadhesives, agricultural chemicals, tile cements,plywood glues, printing inks, and cosmetics.

Cellulose is also receiving considerable atten-tion as a potential feedstock for liquid fuels,particularly ethanol. By either acid or enzymatictreatment (biological enzymes break down thecellulose into its basic sugars), cellulose can beconverted to fermentable glucose and then dis-tilled to remove ethanol. Although not currentlycompetitive with ethanol derived from corn orsugarcane, the economic attractiveness of cellulose-derived fuel could very well change with ad-vances in biotechnology .56

Cellulose will no doubt continue to be a majormaterial feedstock for a wide spectrum of indus-tries. Future research is likely to focus on thedevelopment of new chemical derivatives and thecreation of composites that combine cellulosewith other biodegradable materials.

LigninLignin is a polymer found in woody and

herbaceous plants. Its principal function is toprovide structural support in plant cell walls.Lignin consists of phenylpropane building blocksand belongs to the polyphenol family of poly-mers. Along with cellulose and hemicellulose,lignin is one of the three chemically distinctcomponents occurring in plant tissue. Typically,woody and herbaceous biomass consists of 50percent cellulose, 25 percent hemicellulose, and

% see Genetic Engineering New$* “High Molecular Weight Cellulose Derivative Shown to Lower CholestemL” July 1993, p. 26.

55 However, cellulose esters such as cellulose acetate are not degmdable.

56 us. conue~s, office of Te~hnology Assmsm~~ Fueling DeVelOp~flt: Energy TeC/WWIOgieS for Developing c0U?lm”e5, OTA-E516

(Washington DC: U.S. Government Printing Office, April 1992), pp. 226-227.57 ~addition t. ~ese we ~ficip~ bio~s compo~nts, - ~ounts of o~er compounds CmbC prescntdependingon tkphlt Sp~itX.

Common examples include fatty acids, waxes, tannins, and more specialized compounds such as terpene (used as a substitute forchlorofluorocarbons in electronics manufacturing) and taxol (a compound being explored as an anticancer dreg).


25 percent lignin.57 Wood is a complex lingocel-luolosic composite,58 Lignin polymers are highlyamorphous, three-dimensional structures that areassociated with hemicellulose and play a key rolein preventing decay of the lignocellulosic ma-terial. Lignin is generated in great quantities as abyproduct of wood pulping processes and conse-quently is relatively inexpensive. The most com-mon commercial form of lignin is lignosulfonate,a compound derived from sulfite pulping. Higher-purity lignin can be obtained from “kraft”pulping, but this process is more costly .59

CURRENT AND POTENTIAL APPLICATIONSAt present, most of the lignin that is isolated

from pulping processes is burned as an on-sitefuel source. However, the material is increasinglybeing used in nonenergy applications.60 Becauselignin acts as a natural adhesive holding cellulosefibers together in plant cell walls, many of itscommercial applications take advantage of thisproperty. Millions of pounds of lignosulfonatesare used annually for road dust control, Lignosul-fonates are also employed as binding agents inmolding applications and in animal feed. Ligninderivatives are beginning to be used as phenolicadhesives that can replace formaldehyde-basedcompounds in applications such as industrialpackaging and tape.

The ionic properties of lignosulfonates andkraft lignins allow them to act as dispersants.They are being used to prevent mineral buildup inboilers and cooling towers, as thinning agents inoil drilling muds and concrete admixtures, and as

dispersing agents in pesticide powders. Somemajor chemicals are also produced from ligninprecursors. For example, vanillin, the principalingredient in artificial vanilla, is derived from thearomatic components of lignin. In addition, chem-ically modified lignins are being explored forpossible pharmaceutical applications. The devel-opment of specialized lignin compounds, such aselectrically conducting polymers and engineeringplastics, is an area of considerable research.

ChitinChitin, a polysaccharide, is one of the most

ubiquitous polymers found in nature. It is almostas common as cellulose, and possesses many ofthe structural and chemical characteristics ofcellulose (see figure 2-7). Chitin is an importantstructural component of the exoskeleton of a greatnumber of organisms such as insects and shell-fish. It also serves as a cell wall component offungi and of numerous plankton and other smallorganisms in the ocean (see box 2-A for examplesof other marine polysaccharides). Because of thedifferent biological requirements of these variousspecies, chitin is an extremely versatile naturalpolymer. Chitin and its most important deriva-tive, chitosan, have a number of useful physicaland chemical properties, including high strength,biodegradability, and nontoxicity. 61 Currently,the principal source of chitin is shellfish waste,but given the seasonal fluctuation of shellfishharvests, genetically engineered microbial sys-

58 Apm from the~ &aditio@ use m wood products, Lignocellulosic fibers are being used tO CNate h@h-PrfOITIMInCe s~c~~ ~t~~s

(see Roger Rowell, “Opportunities for Lignocelhdosic Materials and Composites,” Rowell, et al. (eds.), op. cit., footnote 1).59 ~ tie ~~t p~p~g prWe\s, fi~ is isolated from the rest of the woody tissue by s~~ hy~xide treatment. Lignin can alSO be broken

down by enzymatic means-a process that is cleaner than kraft or sulfite pulping, but considerably more expensive.

@ See Robert Northey, “Low-Cost Uses of Lignin+” R. Rowel~ T. Schultq and R. Narayan (eda.), Emerging Techmdogiesfor Materialsand Chemicalsfiom Bionmss (Washington DC: American ChemicaJ Society, 1992).

61 whm c~~ a~tyl SOUPS (CH3CO) are replaced by hydrogen to form amino groups (NHJ, chitosan iscreat~ (fiWe 2-7). When exposedto acids, these% groups attract hydrogen ions, forming (NHgH, impardng a net positive charge to the chitosanpolymer. This enables chitosmto remove negatively charged compounds and contaminants from wastewater. The positively charged chitosan forms solid precipitates withthese negatively charged compounds. Chitosan is one of few natural polysaccharides that has this “ionic” property (i.e., a positive charge).


Figure 2-7-Chitin and Its Polysaccharide Relatives

CH20H CH2OH CH2OH CH20Hk—o ~o ~o +0

NHCOCH3

CH20HLo

B ti”NH2

CH20Hk–—o

c JQ”OH

NH COCH3 NHCOCH3 NHCOCH3 ~

CH20H CH20H CH20H

Q’”o a’”o 0°NH2 NH2 NH2

CH20H CH20H CH20HA—o ~o ~o

U“°Kd 0 w’ 0

OH OH OH

Although polysaccharides are made up of simple sugars, slight medications can lead to dramatically differentchemical and physical properties. Shown here are the structures of: A) chitin, B) chitosan, and C) cellulose. Theonly difference between cellulose and chitosan is that cellulose has hydroxide (OH) groups instead of amino(NH,) groups. Chitin is identical in composition except for the acetylated amine groups (NHCH,OH).

SOURCE: Office of Technology Assessment, 1993.

terns might be used to provide a stable supply ofhigh-grade chitin compounds.62

CURRENT AND POTENTIAL APPLICATIONSThe chitin family of polymers is being widely

used in medicine, manufacturing, agriculture, andwaste treatment. In the biomedical area, chitosanis incorporated into bandages and sutures inwound-healing treatment, because it forms atough, water-absorbent, oxygen permeable, bio-compatible film. It can be used to accelerate tissuerepair and can be applied directly as an aqueoussolution to treat burns. Because of its high oxygen

permeability, chitosan is used as a material forcontact and intraocular lenses. Chitosan has alsobeen found to expedite blood clotting. The factthat chitin compounds are biodegradable (thehuman body breaks chitin down into simplecarbohydrates, carbon dioxide, and water) makesthem particularly appropriate for use in drugdelivery systems. Chitosan carriers can releasedrugs slowly. This property is extremely valuablein cancer chemotherapy since the agents are oftenhighly toxic and require long periods of time foradministration. A chitosan compound is also

62 Ceti ~gae, for ex~ple, produce a relatively pure form of chitin fiber that can be readily extracted and processed. However, these algaegrow quite slowly. The application of biotechnology may lead to the development of fast-growing strains of algae that produce large quantitiesof chitin. In additiow some fimgi produce chitosan directly. By modifying these stmins, greater amounts of chitosan could be produced,eliminating the need for chemical treatment of chitin. See New Scientisf, ‘‘Life after Death for E~ty Shells, ’ Feb. 9, 1991, pp. 464.S.


Box 2-A–Polymers oft he Sea

As major repositories of the earth’s genetic diversity, the oceans are a rich source of proteins,polysaccharides, and other polymeric compounds. Because marine organisms live in a variety of

different environments-some of them extremely harsh-they have developed polymers with a wide

range of properties. For example, the hard calcium carbonate shell of the abalone is held together by

a glue composed of proteins and sugars, and ocean species in polar climates are able to surviveextremely cold temperatures by producing antifreeze proteins. Other proteins regulate the mineraliza-tion processes involved in the creation of shells and crystals (e.g., the calcite crystals of sea urchinspines). Polysaccharides serve as structural components in crustaceans (e.g., chitin), and in a numberof algal species such as kelp.

Chitin and some algal polysaccharides such as agar, alginate, and carrageenan are widely usedin industry and medicine. The market for these marine polymers is several hundred million dollarsannually. Agar, a major component of the cell walls of certain red algae, is used as a photographicemulsifier; a gel for cosmetics, toothpaste, and medical ointments; an inert drug carrier, a corrosioninhibitor, an adhesive, and a thickening agent in confectioneries and dairy products. Alginate is aprincipal structural constituent of brown algae (rockweeds and kelps), and carrageenan is extractedfrom red seaweed. Like agar, these two natural sugars have excellent gelling and colloidal (suspending)properties. They are used extensively in the production of ice cream and other dairy items, as well asin the textile, paper, printing, and biomedical (e.g., wound dressing and dental impression) industries.

Other marine-derived compounds are being used to formulate new drug agents, such as theanti-inflammatory compound Fucoside B. Marine organisms provide a vast range of biologicalprocesses and substances that could be genetically modified for novel medical and manufacturingpurposes. The Federal investment in marine biotechnology research was about $44 m ill ion in f iscal year1992.

SOURCES: Federal Coordinating Council for Science, Engineering, and Technology, Committee on Life Sciencesand Health, Biotechnology for the 27st Century (Washington, DC: U.S. Government Printing Office, February 1992);Office of Technology Assessment, 1993.

being investigated as an inhibitor of the AIDS strength of paper fibers, particularly when wet.v i r u s .6 3

The high moisture retention and film-formingcharacteristics of chitosan have resulted in anumber of applications in the cosmetics andpersonal care areas. Chitosan is being utilized inhair spray, skin cream, shampoo, soap, nailpolish, toothpaste, and personal hygiene prod-ucts.64 In paper manufacturing, the addition Of 1

percent chitin by weight greatly increases the

Thus, chitin has been incorporated into diapers,shopping bags, and paper towels.65 In addition topaper-chitin composites, some researchers havedeveloped complexes of chitin and cellulose thathave excellent water-resistant properties.66 Thematerial can be molded or made into biodegrada-ble plastic films. Eventually, high-strength chitinpolymers could be used in food packaging. Onecompany, Technics of Japan, in trying to replicate

63 mis research is being led by Ruth Ruprecht at Harvard University, See “Chitin Craze,” Science News, VO1. 144, Jdy 31, 1993, pp. 72-74.

~ New Scientist, op. cit., footnote 62.

65 Ibid.

~~ J, Hosokawa et al., ‘ ‘Biodegradable Film Derived from Chitosan and Homogenized Cellulose,’ Indusm”al Engineering ChemicalRese~rch, vol. 29, 1990, pp. 800-805.


the acoustic properties of crickets’ wings, haseven constructed audio speaker vibrators fromchitosan materials.67

In terms of actual sales, agricultural end usesconstitute the largest and most successful marketfor chitin and chitosan polymers. The fungi-resistant properties of chitosan have resulted in itsapplication as a fertilizer, soil stabilizer, and seedprotector. It is a yield-enhancing agent for wheat,barley, oats, peas, beans, and soybeans.68 Thus,chitosan is used both as a seed coating and as aplant growth regulator. Chitosan is also used torecover protein wastes, particularly dairy prod-ucts such as cheese whey, that are subsequentlyadded to animal feed.

Because of its binding and ionic properties(i.e., in solution, the chitosan polymer carries apositive charge), chitosan can be used as aflocculating agent to remove heavy metals andother contaminants from wastewater.69 Currentapplications in this area include treatment ofsewage effluents, paper mill wastes, metal finish-ing residues, and radioactive wastes. In severalcountries, particularly Japan, chitosan is used topurify drinking water. Chitosan is also beingevaluated for use in the bioremediation of toxicphenolic compounds.70 This could greatly im-prove the efficiency of pharmaceutical and plasticmanufacturing, by eliminating phenolic contami-nates.

Chitin and its various derivatives have becomeimportant constituents in a number of diverseproducts and industrial processes. Pharmaceutical-grade chitin sells for about $32 per pound, whileindustrial grade chitin/chitosan compounds rangefrom about $7 to $27 per pound (specific costdepends on the application). The unique chemical

properties and biodegradability of this family ofbiopolymers presage an even wider range ofapplications in the future.

Hyaluronic AcidHyaluronic acid (HA) is a natural product that

is found throughout vertebrate tissue. It alsooccurs as an extracellular polysaccharide in avariety of bacteria. HA plays an important physio-logical role in many organisms. Research indi-cates that HA aids tissue formation and repair,provides a protective matrix for reproductivecells, serves as a regulator in the lymphaticsystem, and acts as a lubricating fluid in joints.Currently, most of the HA used for research andcommercial purposes is extracted from roostercombs. In the future, it is likely that this bio-polymer will be produced from fermentationbroths of Streptococcus and other bacteria.

Hyduronic acid, discovered in 1934, is a long,unbranched polysaccharide chain, composed ofrepeating twin sugar units. Because of the highdensity of negative charges along the polymerchain, HA is very hydrophilic (has a strongaffinity for water) and adopts highly extended,random-coil conformations. This structure occu-pies a large volume relative to its mass and formsgels even at very low concentrations. It isextremely flexible and has a high viscosity.

CURRENT AND POTENTIAL APPLICATIONSSince hyaluronic acid plays an important role

in many developmental and regulatory processesof the body, it has been used principally inbiomedical applications. It is an extremely attrac-tive polymer material because it is a natural

67 New scientist, op. cit., footnote 62.68 ~~ p~c~ c~tow product is ~keted under the tradename YEA by Bentech bbomtorics. An es-te of b Potenw ~ket for

YEA is about $160 million amually (BioInformation Associates, Inc., “Technology and Commercial Opportunities in BiodegradablePolymers, ” Boston, MA).

@ See Chemical and Engineen”ng News, “Chitin Removes Textile Dyes from Wastewater, ” July 12, 1993.TO ~s application is being developed by Mfizyme Corp. (Hanover, MD) in collaboration with researchers at the University Of -tid.

Science News, op. cit., footnote 63.


Table 2-3—Biomedical Uses of Hyaluronic Acid

Area Application

Disease indicator Identifies presence of liver cirrhosis, arthritis, scleroderma (tissue disease), andtumors.

Ear surgery Scaffold material for ear surgery, healing of tympanic membrane perforations

Eye surgery Protects corneal tissue; used In retinal reattachment, and in glaucoma surgery

Wound healing Stimulates tissue repair.

Tendon surgery Repair of flexor tendon lacerations, degenerative joint disease in animals

Antiadhesion General surgery.

Scar control General surgery

SOURCE Blolnformatlon Associates, Boston, MA

product that degrades into simple sugars. Pres-ently, its major uses are in eye surgery, treatmentof arthritis, and wound-healing preparations; it isalso being used in some cosmetic products.Various biomedical applications for HA are listedin table 2-3.

The unique physiochemical and structuralcharacteristics of hyaluronic acid make it anexcellent candidate for applications that requirebiocompatibility. However, the prices of HA areextremely high, more than $100,000 per kilo-gram. A recent survey estimates a total market forHA of $425 million by 1996.71 The largestsegment of this market is for surgery. Thesmallest segment is for cosmetics. Because of itsutility in surgical treatment, the demand forhyaluronic acid is likely to expand. Some firmsare now investigating genetically engineered HAproduced by microbes. While the application ofgenetic techniques may produce HA that hasgreater polymer uniformity, switching to bacte-rial production (from rooster comb extraction)probably will not lower total production costsbecause of the need for expensive purificationsteps .72

POLYMERS PRODUCED BY CHEMICALPOLYMERIZATION OF BIOLOGICALSTARTING MATERIALS

Polymers that are created by the chemicalpolymerization of naturally occurring monomersare attracting considerable commercial interest.Although these polymers are not produced bybiological systems, the fact that they are derivedfrom basic biological building blocks confers onthem many of the same properties as microbiallyor plant-derived biopolymers. These may includenontoxicity, biodegradability, and biocompatibil-ity. In addition, these polymers are by definitiondrawing on feedstocks that are renewable. Whilethere are several different classes of chemicallysynthesized biopolymers, two particular groupsstand out. One is the family of polymers producedfrom lactic acid, a molecule used extensively inthe food industry. The other is the growingensemble of polyamino acid polymers.

Polymers Synthesized from Lactic AcidLactic acid (lactate) is a natural molecule that

is widely employed in foods as a preservative anda flavoring agent.73 It is also used in biomedical

71 See Genefic Engineering News, ‘‘Complex Carbohydrate Therapeutic Products: ‘Ibmorrow’s Billion $ Market, ” January 1993, pp. 6-7.

72 James Bro~, Division D&ctor, MOIWUIU and Cellular Biosciences, National Science Foundation perSOfKil COfnmUniCatiOn, JUIY 28,1993.

73 hctic acid ca exist in NVO different forms: l-lactic acid and d-lactic acid. These two compounds are chemically identical except that theyare mirror images of each other. Such mirror image structures are called stereoisomers. Because the stereoisomers have different spatialconfigurations, they have different reactive properties. Only the l-lactide is found in animals, whereas both the d and 1 forms of lactic acid arefound in lower organisms, such as the Luctobucilli, a class of bacteria used for centuries in the dairy industry.


Figure 2-8-Commodity-Scale Production ofPolylactide

Raw material .-

“ - - 1

MonomerAgricultural wasteWhey (dairy Industry)Starch (potato processing)

‘ermentat’On>E

Chemicalcondensation ~

r—‘- “–—------- --w-.–,

A, Low molecular weight/’ PLA

Thermal /’ — ---–——/treatment,,

//

,/’Cyclic dimer-L’

Lactide 1o\ o Product

‘ c “ “CH 3J-

Rlng opening –

High molecular weight

HC A polymerization 11--- - ‘ L A - - - - -‘ o ” ‘ o

CH 3(catalyzed)


applications in intravenous and dialysis solutions.It is the main building block in the chemicalsynthesis of the polylactide family of polymers.This family includes polylactide homopolymers(PLA) and copolymers with glycolic acid (PLA-PGA). (Glycolic acid occurs naturally in sugar-cane syrup and in the leaves of certain plants, butis generally synthesized chemically).

Lactic acid is found in blood and muscle tissue,where it is a product of the metabolic processingof glucose. Although it can be synthesizedchemically, lactic acid is produced principally bythe microbial fermentation of sugars such asglucose or hexose. These sugar feedstocks aredrawn from agricultural (potato skins and corn)and dairy wastes. The lactic acid monomersproduced by fermentation can be used to create

either low or high molecular weight polylactidepolymers (figure 2-8). Variation of the reactionconditions, such as temperature and choice ofcatalyst, provides control over the molecularweight of the polymer. In this way, the physicaland chemical properties of polylactide can beadapted for different applications.

CURRENT AND POTENTIAL APPLICATIONSPolylactide polymers are the most widely used

biodegradable polyester materials. Although theirprincipal area of application has been in the healthcare field, agricultural and manufacturing useshave been found as well. Polylactides are fre-quently used in combination with polyglycolicacid. PLA-PGA copolymers are employed asmedical materials and as platforms for the sus-tained release of agricultural chemicals. In theindustrial sector, PLA commodity polymers arebeing developed for use as pulping additives inpaper manufacturing and as degradable packag-ing materials.74 Commodity-grade polylactidesells for about $5 per pound, but manufacturersexpect to bring the price down to the $2 to $3range. Medical grade polylactide prices rangefrom $100 to $1,000 per pound. The properties ofthese materials are being tailored to meet a varietyof different needs.

Most applications of PLA-PGA materials havebeen for therapeutic purposes. Devices made ofPLA-PGA copolymers have been used for thecontrolled release of antibiotics, anticancer andantimalarial agents, contraceptives, hormones,insulin, narcotic antagonists, and proteins.75 Thecopolymers have been molded into microsphereor microcapsules, pellets, implants, and hollowfibers.

There are several other important medicalapplications. Polylactide sutures are widely used

w c~~l, ~c. ~ b~d~ aw~ac- facility that will be capable of producing IOtion Pourlds of Polykctide ~~Y. me ~t~*

will be used as biodegradable plastics. See Wall Street Journal, “Concern WU Expand Move into Biodegradable Plastic,” May 20, 1993, p.A7. Also, Dupont Chemical and ConAgra have formed a joint venture, like that is developing polylactide polymers.

75 PLA.PGA copolymms UC @ as drug delivery vehicles. Since protein-based drugs are quickly degraded in tie body, tirn~-rel-delivery by PLA-PGA devices can decrease the rate of drug degradation.


in surgery because they degrade within the bodyafter the incision has healed. Commercial absorb-able sutures such as Vicryl@ are made of copoly-mers containing 90 percent PLA and 10 percentPGA. This ratio creates fibers that have excellentdurability, absorbency, and tensile strength. Su-tures made from PLA-PGA copolymers are strongerand absorb faster than sutures, such as Dexon@,made only of PGA. A considerable amount ofongoing research is focused on the application ofPLA-PGA copolymers as sutures, clips, staples,and reinforcement materials.

PLA-PGA copolymers have also been success-fully applied in experimental orthopedic surgery.Compression-molded copolymers have been usedas plates or screws for the treatment of fracturesand to fill in bone defects. The materials have alsobeen used as scaffolding to facilitate the forma-tion of new cartilage material in the body.76

Copolymers of PLA and PGA are more usefulthan homopolymers of PLA and PGA becausetheir rate of degradation can be adjusted. Anadvantage of using prostheses made of PLA-PGAcopolymers is their biocompatibility and nontox-icity. The breadth of current research effortssuggests that the range of biomedical applicationsfor PLA-PGA materials will expand considerablyin coming years.

Polyamino AcidsPolyamino acids are an important class of

synthetic polymers produced by chemical polym-erization of the same amino-acid building blocksfound in naturally occurring proteins.77 Polyam-

ino acid chains are sometimes referred to aspolypeptides. Approximately 20 amino acids canbe found in proteins, and from these basicbuilding blocks a variety of homopolymers andcomplex copolymers have been synthesized. Be-cause of the great chemical diversity of amino-acid monomers—anionic, cationic, hydrophobic,polar, nonpolar, thermally stable-polyaminoacids can be envisioned for virtually all types ofpolymer applications.78

Currently, two principal chemical proceduresare used to produce polyamino acids. One ap-proach specifically links amino-acid monomerunits together by amide bonds. These are the sametype of bonds that exist in natural proteins. Thisparticular type of chemical synthesis is a fairlycomplex process and is used primarily to createthe high-purity materials needed for biomedicalapplications. In the future, this method of synthe-sis may be supplanted by bacterial fermentation79

or recombinant DNA techniques. One type ofbacterial species, for example, can produce poly -glutamic acid, a polypeptide that has man y

therapeutic uses. As highlighted earlier, recombi-nant DNA technology is being used to develop anew class of polymers based on spider silk (silkis a natural polyamino acid).

Polymer chains consisting of glutamic acid,aspartic acid (a component of NutrasweetT M) ,leucine, and valine are the polypeptides mostfrequently used for biomedical purposes. Lysineand methionine are also important amino-acidbuilding blocks in polypeptide polymers.80 Glu-tamic acid is the sodium salt form of monosodium

76 See Robefl ~nger and Joseph Vacanti, ‘‘Tissue Engineer ing,” Science, vol. 260, May 14, 1993, pp. 920-925.

77 As ~1~ lactlc acid (foo~ote 73), ~. acids ~so ~c~ as stereoisomers; tit is, ~ righ~ndti (d) or lefthanded (1) mirror image

structures. One of the distinguishing features of proteins is that they are constructed only from left-handed amino acids. The synthetic polyaminoacids discussed here are for the most part derived ffom l-amino acids. Polymers that contain only l-isomers or only d-isomers are known ashomochiral structures. Like the phenomenon of DNA self-replication homochirality is a unique property of living systems. See V. Avetisovet al., “Handedness, Origin of Life, and Evolutioq” Physics To&ry, July 1991, p. 33.

78 cc~o~c’ monomers are negatively Charged; “cationic” monomers are positively charged.

w New bacteri~ fmen~tion Prwesses tit tivolve immobilized enzymatic reactions could POtentirdly lower the cost of PolY*o acid

production.80 Lyslne ~dme~onine me widely us~ m food and animal feed additives. Methionine k prOdUCed chemically, wh~~s IYs~e is ~ct~a~Y

produced.


Polyamino acid microsphere can be used toencapsulate bugs and agricultural chemicals,thereby ensuring that such active agents can bereleased in a controlled fashion. The microsphereshown here have a diameter of about 1.5 microns(1.5 millionth of a meter). Microsphere occurnaturally and figure prominently in a theory ofthe origin of living cells over 3 billion years ago.

glutamate (MSG) and is a major product of thefermentation industry, with an annual productionlevel of around 600 million pounds. A majoradvantage of using glutamic acid is its low costand relative abundance. Glutarnic acid and aspar-tic acid are hydrophilic (they have high wateraffinity), whereas leucine and valine are hydro-phobic (low water affinity). When these hydro-philic and hydrophobic building blocks are com-bined, copolymers with vastly different rates of

biodegradation can be created. This allows thecopolymers to be used as delivery systems for avariety of different drugs. The fact that ho-mopolymers and copolymers of these simpleamino acids are nonimmunogenic (i.e., they donot produce an immune response when injectedinto animals) makes them particularly attractivefor medical applications. Polyamino acid micro-spheres—spheres ranging in size from 50 nanom-eters (billionth of a meter) to 20 microns (mil-lionth of a meter)--are currently being developedfor oral drug administration.81 (Polyamin o acidmicrosphere can also be used for the controlledrelease of fertilizers.) In addition to drug deliverysystems, polyamino acids are being investigatedfor use as biodegradable sutures, artificial skin,and orthopedic support materials.

A less complex chemical procedure is used toproduce polyamino acids for commodity-scaleindustrial applications. Polypeptides are made bythermal polymerization of amino-acid buildingblocks at moderately high temperature (160 to240oC), followed by a mild alkaline treatment at60 to 80°C to open ring structures that may form.This process is not very specific and yields apolymer product in which the monomers arelinked by two different types of bonds. However,the advantage of this method is that large quanti-ties of polypeptides can be synthesized at lowcost.

This approach has been used to create polyas-partate polymers from aspartic acid.82 The poly-aspartate polymers are analogues of naturalproteins, particularly the aspartate-rich proteinsfrom oyster shells that play a key role in

81 fiotein-based drugs we exkmely diflicult to insert into the human body. Because proteins are easily tiokm down by digestive wzymesand cannot readily pass through the skiq they are currently administered by injection. By eneasing protein drugs in polyarnino acid materials,researchers are trying to protect the proteins long enough so that they can slip past digestive enzymes in the stomach and be absorbed into thebloodstream. This would allow oral administration of genetically administered proteins. However, in animal studies ecmducted thus far, onlyabout IOpercent of an orally administered drug dose makes it to the bloodstream. One company involved in this area is Ehnisphere Tedmologiesof Hawthorne, NY. See “Stand and Deliver: Getting Peptide Drugs into the Body,” Science, vol. 260, May 14, 1993, pp. 912-913.

82 Asp~c acid is fairly inexpensive as it em be produced from -ofi and mdeic anhy~de.

...— ———


Table 2-4-Possible Applications of Industrial Polyaspartates

Water treatment Antiscaling, anticorrosion, flocculation. Cooling towers, evaporators (forexample in pulp processing), desalinators, boilers

Dlspersants Detergents, paint pigments, drilling mud, portland cement, etc

Air pollution control Remove sulfur dioxide by preventing deposition of Insoluble sulfates

Ceramics Promotion of crystallization of specific minerals (e g insulators)

Oilfield applications Prevent mlneralization and corrosion in well holes

Fertilizer preparation Prevent calcification of phosphate slurrles

Mineral pocesslng Antiscalants used to keep ores at an optimum size after grlnding.

Textile Industry Addition of crystallization regulators results in better fibers

Antifouling Prevent growth of calcified organisms on marine/freshwater surfaces

Superabsorbents Diapers

Bioplastics At high molecular weights, polyaspartates become solid materials that mayhave a number of uses,

Dental treatment Tartar control agents (toothpaste)

Biomedical devices Prosthetic devices (heart valves), prevention of pathological calcification,microencapsulation for drug delivery, surface coatings for Implants topromote crystallization

SOURCE Steven Sikes, Department of Biological Sciences, University of South Alabama, personal communication, Aug. 11, 1993

regulating mineralization,83 Consequently, these

polymers have intrinsic antiscalant and anticorro-sive properties that can prevent the buildup ofmineral deposits on the surfaces of ships, coolingtowers, heat exchangers, and other industrialequipment. Since the polyaspartate compoundsare derived from natural precursors, they arebiodegradable 84 and can be used to replacepetroleum-derived polymers such as polyacrylateand polyacrylamide.85 Because of their uniquemineralization and ionic properties, there existsan enormous range of possible applications forthe polyaspartate materials (see table 2-4).

At present, markets for these synthetic bio-polymers are only now being identified. Never-theless, there exist opportunities to use polyaspar-tates for a variety of specialized biomedicalpurposes, as well as a number of high-volumeapplications such as flocculants, dispersants, andsuperabsorbents. Industrial polypeptides, particu-larly water treatment chemicals, are likely tobecome commercially available in the near fu-ture.86 It is expected that these compounds willcost from one-third to twice the price of existingsynthetic chemicals such as the polyacrylamides(about $1.30 to $2 per pound).

83 M(nx proteins from oysta shells me powerful regulators of mineral formation. These polypeptides CWI inhibit mineral deposition whereit is not wanted and, under certain conditions, can promote crystallization where it is needed. The formation of minerals in solution (e.g., thecalclum carbonate structure of oyster shells) is a fundamental process of living and nonliving systems, It is apparent that nature has producedsome extraordinary polymers to regulate the mineralization process. By investigating the properties of these natural polymers, researchers aretrying to develop new biopolymers that can be tailored for a variety of industrial applications. See C. S, Sikes and A. P. Wheeler, ‘ ‘Regulatorsof Biomineraliz.ation,” Cherntech, October 1988, pp. 620-626.

84 ~eliminm tests of polya.spartate degradability are pmmishg. See A.P. Wh=lm and L.p. Kos~, “Large Scale Thermally SynthesizedPolympartate as a Substitute in Polymer Applications, ” Materials Research Society Symposium Proceedings, vol. 292, pp. 277-283.

85 Swtietic Polymas ~ving mu][iple positive or negative charges, such m Polyacrylrite ~d @YaWlti@ ~ ~dely us~ m wat~treatment additives, components in paper manufacturing, active ingredients in detergents, and tartar control agents in toothpaste, Theirworldwide use amounts to billion of pounds annually, They are excellent materials in terms of their chemical activity and cost. However, theyare not biodegradable.

86 scver~ chemical and bio[echno]om Conlpafies ~ve ongoing R&D prog~ involving poly~~o acids. Steve Sikes, Department Of

Biological Sciences, University of South Alabama, personal communicatio~ July 9, 1993,


SUGGESTED FURTHER READING Microbial Polyesters:Polyhydroxyalkanoates

Sumner A. Barenberg, John L. Brash, RamaniNarayan, and Anthony E. Radpath, DegradableMaterials: Perspectives, Issues and Opportuni-ties (Boca Raton, LA: CRC Press, 1990).

James H. Brown, ‘‘Research Opportunities forBiologists in Bimolecular Materials,” NationalScience Foundation, Division Director, Molecu-lar and Cellular Biosciences, internal report, June1992.

Yoshiharu Doi, Microbial Polyesters (NewYork, NY: VCH Publishers, 1990).

Oliver P. Peoples and Anthony J. Sinskey,“Polyhydroxybutyrate (PHB): A Model Systemfor Biopolymer Engineering II,” E.A. Dawes(cd.), Novel Biodegradable Microbial Polymers,NATO Advanced Science Institute Series (Hol-land: Kluwer Academic Publishers, 1990), vol.186, pp. 191-202.

I Recombinant Protein Polymers

James H. Brown, ‘‘Research Opportunities forBiologists in Bimolecular Materials,” NationalScience Foundation, Division Director, Molecu-lar and Cellular Biosciences, internal report, June1992.

D.R. Filpula, S.-M. Lee, R.L. Link, S.L.Strausberg, and R.L. Strausberg, “Structure andFunctional Repetition in a Marine Mussel Adhe-sive Protein, ” Biotechnology Progress, vol. 6,1990, pp. 171-177.

S.D. Gorham, “Collagen,” D. Byrom (cd.),Biomaterials: Novel Materials from BiologicalSources (New York, NY: Stockton Press, 1991),pp. 55-122.

D.L. Kaplan, S.J. Lombardi,, W.S. Muller, andS.A. Fossey, ‘Silks, ’ D. Byrom (cd.), Biomateri-als: Novel Materials from Biological Sources(New York, NY: Stockton Press, 1991), pp. 1-53.

D. McPherson, C. Morrow, D.S. Minehan, J.Wu, E. Hunter, and D.W, Urry, “Production andPurification of a Recombinant Elastomeric Pol-

ypeptide, G-(VPGVG)19-VPGV from E.. coli,”Biotechnology Progress, vol. 8, 1992, pp. 347-352.

Bacterial CelluloseP. Ross, R. Mayer, and M. Benziman, “Cellu-

lose Biosynthesis and Function in Bacteria,”Microbiological Revues, vol. 55,1991, pp. 35-58.

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XanthanR.A. Hassler and D.H. Doherty, “Genetic

Engineering of Polysaccharide Structure: Produc-tion of Variants of Xanthan Gum, Xanthomonascampestris, Biotechnology Progress, vol. 6, 1990,pp. 182-187.

J.F. Kennedy and I.J. Bradshaw, “Productionand Applications of Xanthan, ” M.E. Bushell(cd.), Progress in Indutrial Microbiology, vol.19, 1984, pp. 319-371

DextransR.M. Alsop, “Industrial Production of Dex-

trans,’ M.E. Bushell (cd.), Progress in IndustrialMicrobiology (Amsterdam: Elsevier, 1983), pp.1-44.

C. Larsen, “Dextran Prodrugs-Structure andStability in Relation to Therapeutic Activity,”Adv.Drug Delivery Review, vol. 3, No. 1, pp.103-154.

PullulanChemical and Engineering News, “Japanese

Develop Starch-Derived Plastic, ” vol. 24, De-cember 1973, p. 40.


A. Jeanes, “Dextrans and Pullulans: Industri-ally Significant Alpha-D-Glucans,” P.A. Sand-ford and A. Laskin (eds.), Extracellular Micro-bial Polysaccharides, ACS Symposium Series,vol. 45, 1977, pp. 284-298.

Y.C. Shin, Y.H. Kim, H.S. Lee, S.J. Cho, andS.M. Byun, “Production of ExopolysaccharidePullulan from Inulin by a Mixed Culture ofAureobasidium Pullulans and KluyveromycesFragilis,’ Biotechnology & Bioengineering, vol.33, 1989, pp. 129-133.

GlucansE. Cabib, R. Roberts, and B. Bowers, ‘Synthe-

sis of the Yeast Cell Wall and Its Regulation, ’Annual Review of Biochemistry, vol. 5, No. 17,1982, pp. 63-793.

N.R. Di Luzio, D.L. Williams, R.B. McNamee,B.F. Ewards, and A. Kitahama, “ComparativeTumor-Inhibitory and Anti-Bacterial Activity ofSoluble and Particulate Glucan,” InternationalJournal of Cancer, vol. 24, 1978, pp. 773-779.

P.W.A. Mansell, G. Rowden, and C. Hammer,“Clinical Experiences with the Use of Glucan,”M.A. Chirigos (cd.), Immune Modulation andControl of Neoplasia by Adjuvant Therapy (NewYork, NY: Raven Press, 1978), pp. 255-280.

D.L. Williams, E,R. Sherwood, LW. Browder,R.B. McNamee, E.L. Jones, and N.R. Di Luzio,“The Role of Complement in Glucan-InducedProtection Against Septic Shock,” CirculatoryShock, vol. 25, 1988, pp. 53-60.

Ge l lanV.J. Morris, ‘ ‘Science, Structure and Applica-

tions of Microbial Polysaccharides, ” G.O. Phil-lips, D.J. Wedlock, and P.A. Williams (eds.),Gums and Stabilizers for the Food Industry, vol.5, 1990, pp. 315-328.

StarchR.L. Whist ler , J .N. BeMiller , and E.F.

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R.P. Wool and S.M. Goheen, ‘‘DegradationMechanisms in Polymer-Starch Composites,”International Symposium on Biodegradable Po[y-mers, Biodegradable Plastic Society, October1990, pp. 137-143.

LigninW. Glasser and S. Sarkanen (eds.), Lignin:

Properties and Materials (Washington, DC: Amer-ican Chemical Society, 1989).

Fred Boye, Utilization of Lignins and LigninDerivatives (Appleton, WI: Institute of PaperChemistry, 1985).

Robert Northey, “Low-Cost Uses of Lignin,”R. Rowell, T. Schultz, and R. Narayan (eds.),Emerging Technologies for Materials and Chem-icals from Biomass (Washington, DC: AmericanChemical Society, 1992).

ChitinG.G. Allan, L.C. Alturan, R.E. Bensinger, D.K.

Ghost, Y. Hirabayashi, A.N. Neogi, and S. Neogi,“Biomedical Applications of Chitin and Chito-sari, ’ J.P. Zikakis (cd.), Chitin, Chitosan andRelated Enzymes (New York, NY: AcademicPress, 1984), pp. 119-135.

A. Naude, “Drug and Cosmetic Uses Targetedfor Chitin Products,’ Chemical Marketing Re-porter, Dec. 11, 1989, pp. 24-25.

E.R. Pariser and D.P. Lombardi, Chitin Source-book: A Guide to the Chitin Research Literature(New York, NY: John Wiley & Sons, 1989).

Hyaluronic AcidA. Scheidegger, “Biocosmetics in Japan: New

Wrinkles Beneath the Make-Up, ” Trends inBiotechnology, vol. 7, 1989, pp. 138-144.

J. van Brunt, “More to Hyaluronic Acid thanMeets the Eye, ’ Biotechnology, vol. 4, 1986, pp.780-782.

Polymers Synthesized from Lactic AcidS.J. Holland, B.J. Tighe, and P.L. Gould,

‘‘Polymers for Biodegradable Medical Devices,


the Potential of Polyesters as Controlled Macro-molecular Release Systems, ’ Journal of Con-trolled Release, vol. 4, 1986, pp. 155-180.

M. Vert, P. Christel, M. Audion, M. Chanavaz,and F. Chabot, “Totally Bioresorbable Compos-ites Systems for Internal Fixation of Bone Frac-tures, ’ E. Chiellini et al. (eds.), Polymers inMedicine II (New York, NY: Plenum Press, 1985,pp. 263-275.

R.H. Wehrenberg, “Lactic Acid Polymers:Strong, Degradable Thermoplastics,” Mechani-cal Engineering , September 1981, pp. 63-66.

Polyamino AcidsJ.M. Anderson, K.L. Spilizewski, A. and

Hiltner, “Poly Alpha-Amino Acids as Biomedi-cal Polymers, ’ D. F. Williams (cd.), Biompatibil-ity of Tissue Analogs, vol. 1 (Boca Raton, FL:CRC Press, 1985), pp. 67-88.

C.S. Sikes and A.P. Wheeler, “Regulators ofBiomineralization,’ Chemtech, October 1988,pp. 620-626.

Technical Overview Biopolymer Field B

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