January/February 2005

Published by the American Chemical Society

Volume 6, Number 1

Copyright 2005 by the American Chemical Society

Reviews
Bacterial Polyesters: Biosynthesis, Biodegradable Plastics and Biotechnology
Robert W. Lenz*, and Robert H. Marchessault
Polymer Science & Engineering Department, University of Massachusetts, Amherst, Massachusetts 01003-4530, and Department of Chemistry, McGill University, 3420 University St., Montreal, QC, H3A 2A7 Canada Received May 21, 2004; Revised Manuscript Received September 23, 2004

The discovery and chemical identification, in the 1920s, of the aliphatic polyester: poly(3-hydroxybutyrate), PHB, as a granular component in bacterial cells proceeded without any of the controversies which marked the recognition of macromolecules by Staudinger. Some thirty years after its discovery, PHB was recognized as the prototypical biodegradable thermoplastic to solve the waste disposal challenge. The development effort led by Imperial Chemical Industries Ltd., encouraged interdisciplinary research from genetic engineering and biotechnology to the study of enzymes involved in biosynthesis and biodegradation. From the simple PHB homopolyester discovered by Maurice Lemoigne in the mid-twenties, a family of over 100 different aliphatic polyesters of the same general structure has been discovered. Depending on bacterial species and substrates, these high molecular weight stereoregular polyesters have emerged as a new family of natural polymers ranking with nucleic acids, polyamides, polyisoprenoids, polyphenols, polyphosphates, and polysaccharides. In this historical review, the chemical, biochemical and microbial highlights are linked to personalities and locations involved with the events covering a discovery timespan of 75 years. In 1982, Imperial Chemical Industries Ltd. (ICI) in England announced a product development program on a new type of thermoplastic polyester which was totally biodegradable and could be melt processed into a wide variety of consumer products including plastics, films, and fibers.1 The polymer was to be manufactured by a largescale fermentation process not unlike the brewing of beer but which, in this case, involved the production of the polymer inside the cells of bacteria grown in high densities and containing as much as 90% of their dry weight as polymer. The bacterium capable of performing this feat was Alcaligenes eutrophus, since renamed Ralstonia eutropha (more recently changed again to Wautersia eutropha) and
* To whom correspondence should be addressed. Tel.: 1-413-545-3060. Fax: 1-413-545-0082. E-mail: rwlenz@polysci.umass.edu. University of Massachusetts. McGill University.

the commercial polyester product, tradenamed Biopol, was a copolyester containing randomly arranged units of [R]-3-hydroxybutyrate, HB, and [R]-3-hydroxyvalerate, HV:2

Discovery of Bacterial Polyesters That bacteria could produce polyesters was unknown to polymer chemists before 1960 and even to most biochemists and microbiologists before 1958, although their presence in bacterial cells in isolable amounts, their chemical composition, and even the fact that they were polymers, were reported

10.1021/bm049700c CCC: $30.25 2005 American Chemical Society Published on Web 11/23/2004

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Figure 1. Photograph of Maurice Lemoigne, Head of Services de Fermentation at Institut Pasteur, Paris 1949. Courtesy of Institut Pasteur Archives, with permission.

in the literature as early as 1926. These natural polyesters remained unknown to a wider scientific community for so long because their discoverer, Maurice Lemoigne, published his results in little-read French journals at a time when microbiologists had no interest in lipids, as they were referred to by Lemoigne, that were not ether soluble, and many organic chemists refused to believe that there were such things as polymers. Lemoigne (Figure 1) was a bacteriologist with training in analytical chemistry, and his series of papers published over the five-year period from 1923 to 1927 are remarkable for their breadth of research and prescience.3-8 The polyester that Lemoigne isolated and characterized was poly-3-hydroxybutyrate, PHB, shown below. PHB is the reserve polymer found in many types of bacteria, which can grow in a wide variety of natural environments and who have

the ability to produce and polymerize the monomer, [R]-3hydroxybutyric acid:

As indicated in this structure, the repeating unit of PHB has a chiral center, and Lemoigne reported that the polymer is optically active.4 In fact, PHB is only the parent member of a family of natural polyesters having the same three-carbon backbone structure but differing in the type of alkyl group at the or 3 position. These polymers are referred to in general as polyhydroxyalkanoates, PHAs, and all such natural polyesters have the same configuration for the chiral center

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at the 3 position, which is very important both for their physical properties and for the activities of the enzymes involved in their biosynthesis and biodegradation.1,9 At the time of his discovery of PHB in bacteria, Lemoigne was the Director of the Fermentation Laboratory of the Pasteur Institute in Lille, France. He became involved with PHB in an attempt to determine the cause of the acidification of aqueous suspensions of the bacterium Bacillus megaterium when it was kept under an oxygen-free atmosphere. In 1923, Lemoigne reported that the acid produced by the bacteria was 3-hydroxybutyric acid,3 and in 1927, he described the isolation of a solid material obtained from the cell which he characterized as a polymer of 3-hydroxybutyric acid.8 He came to that conclusion by carefully hydrolyzing the solid into a series of water-soluble oligomers of 3-hydroxybutyric acid, which he characterized for molecular weight and melting point. He named the source of the acid lipide-hydroxybutyrique, and, remarkably for his time, he even suggested that the polymer was produced within the cell by a dehydration polymerization.7 Lemoigne published these observations and interpretations at the time when Herman Staudinger at the University of Freiburg, Germany was being ridiculed by his colleagues in organic chemistry in Europe for proposing the existence of high molecular weight molecules or polymers, which he termed macromolecules. Fortunately, Lemoigne was free of such prejudices, and he was probably familiar with the work of Emil Fischer, who demonstrated as early as 1906 that proteins are large molecules of enchained amino acid units or polypeptides, a term he originated.10 Eventually Staudingers concepts about synthetic polymers won out, but not until the 1930s and the publication of the definitive research of Wallace Carothers at duPont Experimental Station, Wilmington, Delaware, on the synthesis and characterization of aliphatic polyesters and polyamides. In 1953, Staudinger was awarded the Nobel Prize in Chemistry for his work on polymer synthesis and for his staunch defense of the concept of macromolecules.11 There is no indication that either Staudinger or Carothers were ever aware of Lemoignes discovery of natures polyesters, which remained hidden from organic and polymer chemists for over 30 years even though PHB was described in biochemistry textbooks, where, however, it was referred to as a lipid not a polyester. Lemoigne and co-workers reported on their PHB studies in 27 publications from 1923 until 1951, and in their later work they found that the cells of B. megaterium could contain as much as 44% of their dry weight of PHB depending on growth conditions.12,63 Lemoigne was the first to describe an analytical method for quantifying PHB, and he showed that PHB could be cast into a transparent film like the then well-known cellulose nitrate material, collodion.7 In followup studies, he and co-workers also reported that a variety of bacteria could produce PHB, but apparently he never became involved in determining the function of such polyesters in cell metabolism even though he labeled it as a reserve material. It was not until microbial physiologists recognized, in the late 1950s, the important role that PHB played in the overall metabolism of bacterial cells that the significance of Lemoignes earlier discoveries was realized.

Rediscovery of PHB The rediscovery of PHB occurred simultaneously and was published independently in 1957 and 1958 by microbiologists in Great Britain and the United States. At the University of Edinburgh in Scotland, Wilkinson and co-workers became interested in the relationship between the presence of the intracellular lipid granules in bacteria, which had been known since 1901, and the large amounts of PHB found in some species as reported by Lemoigne and co-workers, and the function of PHB in the cells.13 During the same time period at the University of California in Berkeley, Stanier, Doudoroff and co-workers found that PHB was the primary product of the oxidative and photosynthetic assimilation of organic compounds by phototropic bacteria, and they attempted to detail the biosynthesis and breakdown mechanism of PHB in the cells.14 Well before these studies, Weibull in 1953 had isolated the granules of B. megaterium by dissolution of the cell wall with a lysozyme, and he confirmed the claim made by Lemoigne in 1944 that PHB was the major constituent of the granules.15 A typical example of such granules inside a cell is shown in Figure 2 for the bacterium A. Chrococcum. In 1958, Wilkinson and co-workers obtained morphologically intact granules from Bacillus cereus by disrupting the cells with alkaline hypochlorite solution and determined the amount of PHB in the granules, but this reagent, the wellknown eau de javelle laundry bleach, was later shown by Lundgren and co-workers at Syracuse University to degrade these polyesters and yield, only low molecular weight polymers.16,17 Only cells that were chloroform extracted yielded high molecular weight PHB when reliable methods were used later, starting in 1965.17a In 1961, Doudoroff and Merrick isolated what they described as native PHB granules of two chemoheterotropic bacteria, Rhodospirillum rubrum and B. megaterium. Native granules are intact granules which are carefully isolated from the cell and purified to retain the active synthase.18 R. rubrum native granules also retain the depolymerase enzyme that can degrade PHB to the monomer, but the B. megaterium native granules have only the associated synthase. Doudoroff and co-workers also studied the enzyme-catalyzed hydrolysis of PHB extracted from the cell and free of all proteins by the extracellular depolymerases which are excreted by a variety of bacteria that can use the polymer as a carbon source as discussed below.19 Biosynthesis of PHB Stanier and Wilkinson and their co-workers determined that the PHB granules in bacteria serve as an intracellular food and energy reserve and that the polymer is produced by the cell in response to a nutrient limitation in the environment in order to prevent starvation if an essential element becomes unavailable. The nutrient limitation activates a metabolic pathway, which shunts acetyl units from the tricarboxylic cycle into the production of PHB. The latter is ideal as a carbon-storage polymer because it is water insoluble, chemically and osmotically inert, and can be readily reconverted to acetic acid by a series of enzymatic reactions inside the cell.20

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The reactions involved in the metabolic pathway responsible for the biosynthesis of PHB from acetic acid were first identified by Stanier and co-workers in 1959 in their studies on PHB formation in R. rubrum. However, the specific enzymes which catalyzed the reactions for the synthesis of 3-hydroxybutyric acid, the monomer for PHB, were not identified until 1973, when Schlegel at the University of Gottingen, Germany and Dawes at the University of Hull, England, working independently, were able to isolate and characterize those enzymes.21,22 Schlegel and co-workers carried out their investigations on the metabolic cycle for PHB production in Alcaligenes eutrophus while Dawes and co-workers studied the cycle for PHB production in Azotobacter beijerinckii. Schlegel first began his research on A. eutrophus in the late 1950s as part of a study of the oxidation of molecular hydrogen by Hydrogenomonas bacteria. In 1961, they observed that A. eutrophus, a member of this group, could accumulate very large amounts of PHB during growth in nitrogen-limited media.23 Coincidentally, Dawes became interested in PHB while preparing a review on microbial metabolism in 1962 before joining the University of Hull. He began his research program there with a study of the accumulation of PHB in A. beijerinckii, which he found capable of accumulating as much as 70% of its dry weight of polymer.24 In the same 1973 issue of Biochemical Journal, Schlegel and Dawes published simultaneously their discoveries on the identification of the two enzymes involved in the reactions for converting acetic acid to 3-hydroxybutyric acid in the two different bacteria.21,22 For both bacteria, the enzymes were a ketothiolase (1), which catalyzes the dimerization of the Coenzyme A derivative of acetic acid, acetyl-CoA, to acetoacetyl-CoA, and a reductase (2), which catalyzes the hydrogenation of the latter to [R]-3-hydroxybutyryl-CoA, the monomer that is polymerized to PHB by a synthase (3), as shown in the following reaction scheme:

existence and role of the synthase in their work on the isolation and characterization of the active native PHB granules from these bacteria. These granules could be used in an aqueous suspension for the in vitro polymerization of [R]-3-hydroxybutyryl-CoA to PHB. With their native PHB granules, Merrick and co-workers were also able to carry out kinetic studies on the polymerization reaction to determine the Michaelis-Menten constants for the reaction. They even proposed in their 1968 studies that the active site of the synthase contains a cysteine unit which provides a thiol group that covalently bonds to the growing polymer chain as a thioester.26 A detailed mechanism for the polymerization reaction, which was based on Merricks suggestion, was proposed by Ballard and co-workers at ICI in 1987 and further elaborated by Doi and co-workers at the RIKEN Institute in Japan in 1992.27,28 They proposed a mechanism in which two thiol groups are involved in the active site for both the initiation and propagation reactions of the polymerization. For initiation, the two thiol groups form thioesters with two molecules of monomer, which then undergo a thioester-oxyester interchange reaction at the active site to form a dimer and release one of the thiol groups for the propagation reaction, as follows:

As discussed above, this cycle becomes activated when acetyl-CoA is restricted from entering the tricarboxylic acid cycle because of a deficiency in nutrients (generally either phosphorus, nitrogen or oxygen) needed by the cell to further metabolize acetyl-CoA for cell growth. Although the first two enzymes, the ketothiolase (1) and the reductase (2) which are responsible for monomer synthesis, were not fully identified and characterized until the investigations of Schlegel and Dawes in 1973, the enzyme responsible for the polymerization process, the synthase or polymerase (3), was initially recognized by Doudoroff, Merrick and co-workers as early as 1964, and it was characterized by Merrick and co-workers in 1968 in their studies on the production of PHB in both R. rubrum and B. megaterium.25,26 They made their initial recognition of the

Propagation ensues by bonding another monomer to the free thiol group of the active site followed by another thioester-oxyester exchange reaction to form the trimer, and so on. These reactions are thermodynamically favorable because of the higher bond strength of the oxyester compared to the thioester. The synthase, therefore, both initiates and catalyzes the polymerization process, which proceeds by a continuous series of insertion reactions in much the same manner as in the stereoregular polymerization of olefins by Ziegler-Natta catalysts. In this case, the enzyme is specific for monomers with the [R] configuration and will not polymerize identical compounds having the [S] configuration as initially reported by Dawes and co-workers in 1989,29 so as a result, all natural PHAs are completely isotactic. Biotechnology As mentioned at the start of this review, bacterial polyesters became an article of commerce when ICI began their production of Biopol in 1982, but Biopol was not PHB. PHB has a high melting point (180 C) and forms highly crystalline solids which crystallize slowly and form large spherulitic structures that impart poor mechanical properties in molded plastics and films, although, addition of nucleating agents and suitable posttreatment after extrusion

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or casting can lead to much improved properties.30 Because of its high melting point, PHB is also susceptible to thermal degradation during melt processing by ester pyrolysis of the aliphatic secondary esters of the repeating units. These deficiencies were partly eliminated when it was found that, when A. eutrophus is grown on a mixture of glucose and propionic acid, the storage polyester formed is a random copolyester of HB and HV units which has a lower melting point.30 As a result, the copolymers have better processing characteristics and considerably improved mechanical properties for use as plastics as shown in Figure 3. Nevertheless, like PHB, the copolymer is fully biodegradable in a wide variety of natural environments as well as in waste disposal facilities, especially in municipal compost sites. The ability of bacteria to produce storage polyesters with compositions other than PHB was not realized until 1974 when Wallen and Rohwedder at the USDA Northern Regional Research Laboratory reported that a polyester isolated from activated sludge contained both HB and HV units, but they were not able to identify the microbial species in sludge which produced the polyester.31 In 1983, White and co-workers at Florida State University demonstrated that the hydroxyalkanoic acid units present in polyesters extracted from bacteria in marine sediments included even more than HB and HV units.32 They analyzed the ethyl esters of the units, which were obtained by ethanolysis of the polyester, by gas chromatography and showed the presence of at least 11 types of repeating units, including both linear and branched 3-hydroxyalkanoic acids with compositions varying from four to eight carbon atoms. White and co-workers also showed that Lemoignes original bacterium B. megaterium could produce polyesters containing at least six different types of units, although HB units still comprised approximately 95% of the contents. In 1983, Witholt and coworkers at the University of Groningen, The Netherlands, found that Pseudomonas oleoVorans grown on alkanes produced a large number of granules containing polyesters with units of 6-10 carbon atoms.33 These bacterial polyesters have low glass transition temperatures and much lower crystallinities than PHB, and as a result, they display elastomeric properties. In 1988, Doi and co-workers obtained PHAs with 4-hydroxybutyric acid repeat units from bacteria grown on carbon substrates having these structures.34 ICI became involved in the commercial development of bacterial polyesters after terminating a program on the largescale production of single cell proteins, SCP, by bacteria for use as fodder.35 After evaluating a variety of methods for that purpose, they had concentrated on the use of methylotropic bacteria to produce SCPs from methanol, but the project was terminated in 1976 because of consumer resistance. The company then turned instead to the possible large-scale production of PHB by the same bacteria for their entree into industrial biotechnology and bioprocessing.36 In this case, they were partially motivated by the major petroleum crisis of the 1970s, which made the production of plastics from renewable resources economically attractive.35 During the 1950s, Schlegel had also studied the production of SCPs by bacteria, eventually selecting the Hydrogenomas

bacterium A. eutrophus for that purpose. In those investigations, he and co-workers observed that this bacterium was capable of producing very large amounts of PHB under selected growth conditions, and in 1979, Schlegel provided samples of several strains of A. eutrophus to ICI for their PHB process development program.23 ICI selected one of these strains for intensive study after Holmes and co-workers in their research laboratory found that the bacterium could produce up to 80% of its dry weight of the HB/HV copolymer when grown on a mixture of glucose and propionic acid as mentioned above.35 Furthermore, the composition of the copolymer could be varied over a wide range by varying the composition of the feed mixture. ICI was not the first company to consider the commercial development of bacterial polyesters for consumer products. Their efforts were preceded by a much earlier attempt at the W. R. Grace Company in Maryland, where Baptist and Werber initiated a similar program in 1960.37 Baptist had joined the Research Division of Grace in 1959 after a postdoctoral in biochemistry at the University of Michigan, where he had learned about bacterial production of PHB. Baptist and Werber recognized that PHB was a stereoregular polymer with a melting point close to that of polypropylene, which suggested to them that PHB might be able to compete with polyolefins as a thermoplastic but with the added advantage of being biodegradable. Baptist used a sample of Rhizobium obtained from Hayward and co-workers at the Colonial Microbiological Research Institute in Trinidad, who reported in 1958 that their strain of that bacterium could produce PHB to 58% of its dry cell weight.38 With this bacterium Baptist was able to produce large quantities of PHB for evaluation, initially for molded plastics and later for absorbable sutures. The latter subject was of interest because PHB, as a natural polyester, was assumed to be a biocompatible polymer in humans, which it has been found to be in more recent studies, but it is very slowly resorbable. They improved the mechanical properties of PHB plastics when they found that PHB was compatible with a variety of plasticizers, which greatly improved its processing and solid-state properties. Nevertheless, the project was terminated in 1962 because of the poor thermal stability of PHB, and their work on the synthesis and properties of PHB was reported in 1964 in the Transactions of the Society of Plastics Engineers.37 However, Chemistry and Engineering News in the March 18, 1963, issue published an extensive research report titled: Bacteria Produce Polyester Thermoplastic, which was apparently the first time that most polymer chemists became aware of this thermoplastic biopolyester, which occurred as inclusions in bacterial cells (Figure 2). Coincidentally, in 1962, Marchessault and co-workers began a program at the State University of New York in Syracuse on characterization of the structure and properties of PHB both in the solid state and in solution.12,39 Samples of the polymer were provided to them by Lundgren, who had been studying the presence of PHB in bacteria for several years in the Microbiology Department of Syracuse University. Issues such as obtaining high molecular weight polymers, optical rotation, and X-ray crystal structure were settled

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Figure 2. Transmission electron micrograph of ultrathin section of Azotobacter chroococcum cell treated with phenylacetic acid. From Nuti et al., ref 17a, herein, with permission.

in definitive experiments. Also, at about the same time, Merrick joined Lundgrens Department and continued his studies on the composition and activity of the native PHB granules of B. megaterium.26,40 They observed that the carefully purified, or native, granules (cf. Figure 2) were surrounded by a protein membrane. They also continued and expanded the earlier studies of Merrick and Doudoroff on the depolymerization of PHB by soluble depolymerases, which they obtained from a variety of bacteria that produce and release these enzymes and are capable of utilizing PHB as a sole carbon source.19,41,42 The protein membranes covering the PHA granules of P. oleVorans were studied in detail by Fuller and co-workers at the University of Massachusetts, Amherst. In 1995, they reported that the granules are enclosed in two separate protein membranes, and the synthase is associated with the inner membrane.42 Biodegradable Polymers Because PHB is stored by bacteria for eventual breakdown and utilization as a carbon source when extracellular carbon is no longer available, there must be an effective and rapid mechanism within the cell for the biodegradation of this high molecular weight polyester into simple organic compounds. As discussed above, Lemoigne had been led into his study of PHB by finding that [R]-3-hydroxybutyric acid is released by B. megaterium in an aqueous environment. In 1958, Wilkinson and co-workers also observed the release of both acetoacetic acid and acetic acid during the utilization of PHB reserves by that bacterium.43 Subsequently, in 1962, Merrick and co-workers demonstrated that native granules from B. rubrum were self-hydrolyzing, and they isolated the enzyme responsible for this reaction which they referred to as a depolymerase or hydrolase (1).44 In 1967, Williamson and co-workers identified a specific dehydrogenase (2) that converted [R]-3-hydroxybutyric acid to acetoacetic acid,45 and in 1973, Dawes and co-workers identified an enzyme for the conversion of acetoacetic acid to acetic acid (3),22 so the entire intracellular pathway for the reconversion of PHB to acetic acid was established to include the following steps:

PHB can also be rapidly hydrolyzed to the monomer by extracellular depolymerase enzymes secreted by a wide variety of bacteria and fungi that can utilize this compound after it is liberated by the death and lyses of bacteria in which it is stored. Initial observations made by Schlegel and Chowdhury in 1963 with strains of Pseudomonas obtained from soil and compost samples established this concept,46 and in 1965 Delafield, Doudoroff and co-workers isolated and characterized a number of pseudomonads capable of utilizing extracellular PHB as their sole source of carbon and energy.47 It is now known that microorganisms exist in all natural environments that are capable of degrading PHB and metabolizing [R]-3-hydroxybutyric acid by enzyme-catalyzed reactions, so by definition, PHB is a biodegradable polymer. In more recent studies, depolymerases have also been found for the PHAs with long alkyl chains.48 As mentioned above, these polyesters have much different physical and mechanical properties, and they can also be utilized as biodegradable polymers in applications such as elastomers and adhesives. Many different types of intracellular and extracellular polyester depolymerases have now been isolated and characterized. As reported by Doi and co-workers, all of these enzymes consist of a single polypeptide chain in the molecular weight range of approximately 40 000-60 000.49 The structural genes of a large number of extracellular depolymerases of different microorganisms have been isolated and analyzed, and they appear to have three characteristics in common along the polypeptide chain, including (1) a catalytic domain (termed a lipid box), (2) a substratebinding domain, and (3) a linking region connecting these two domains. In that manner, they have the same features as the depolymerizing enzymes for insoluble polysaccharides such as cellulose and chitin. Genetic Engineering In 1988, Dennis and co-workers at James Madison University cloned the entire set of genes in R. eutropha for the three enzymes involved in the synthesis of PHB from acetyl CoA as described above.50 The three genes are clustered in one operon, and Dennis and co-workers were able to introduce this operon into E. coli. The genetically engineered E. coli containing the operon can express all three enzymes and can synthesize PHB in large quantities from a wide range of organic compounds. Some recombinant strains of E. coli can also produce the HB/HV copolymer,51 or alternatively as reported by Sinskey and co-workers at the Massachusetts Institute of Technology in 1994, strains containing only the synthase gene can express this protein in sufficiently large quantities for isolation and purification.52

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utilized by plants as a food and energy reserve, one has to be surprised that the view of hundreds or thousands of glucose molecules joined together by glucosidic bonds into long chains could have remained unchallenged because it is improbable that a plant in converting sugar to a reserve substance from which it might soon have to be recovered would perform such complex work as would be required in the build-up of a polyglucoside. 56 Had he known about PHB, Karrer would undoubtedly have made the same argument against Lemoignes contention of the existence of high molecular weight reserve polyesters in bacteria. So much for conventional wisdom. Outlook
Figure 3. Moulded PHB objects for various applications. In soil burial or composting experiments, such objects biodegrade in about three months.

Figure 4. PHB granules in the choloroplast of Arabidopsis thaliana. With permission from Yves Poirier.

The purified enzyme is stable in aqueous solution and has been used for in vitro polymerization reactions of a wide variety of 3- and 4-hydroxyalkanoate-CoA monomers.53 Lenz and co-workers at the University of Massachusetts reported in 2000 that these in vitro polymerization reactions can form living polymers, which means that the polymerization process has no polymer chain termination reaction, so the propagating end group remains active indefinitely and very high molecular weight polymers can be prepared in vitro.54 In another application of genetic engineering for bacterial polyester synthesis, Somerville and co-workers at Michigan State University reported in 1992 that the reductase and synthase genes of A. eutrophus can be inserted into a plant, Arabidopsis thaliana, which can also produce acetoacetylCoA, and the transgenic plant can then accumulate PHB granules, Figure 4, to approximately 14% of its dry weight.55 PHB and the Macromolecule Controversy Reserve polymers also played a role in the controversy between Staudinger and his colleagues in organic chemistry in Germany during the 1920s over the very existence of macromolecules. In his book on the history of polymer science, Morawetz discusses how organic chemists at that time considered starch, which is a reserve polymer for plants, and cellulose to be colloidal aggregates of glucose molecules rather than long chain polymers.56 A leading German organic chemist at that time, Karrer, reasoned that, because starch is

Despite the 75 years, on and off, of research on PHAs and 20 years of intense industrial interest, PHAs still appear to be far removed from large scale production. At this writing, two development programs on these biopolymers are receiving attention, namely (1) a joint program by the Proctor & Gamble Co. and Kaneka Corp. on a family of short and medium chain copolymers, especially on poly(3hydroxybutyrate-co-3-hydroxyhexanoate), and (2) a program at Metabolix Inc. on PHAs for medical applications. The lack of commercialization of the initially promising bacterial poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymers has been generally attributed to the high investment for the fermentation and product recovery processes on a large scale and to the cost of the substrates. To reduce the latter limitation, alternative substrates are receiving much attention, including starch and vegetable oils, but no major breakthroughs in this area have been announced. Nevertheless, in the long run, it is possible that advances in our understanding and control of the genetic pathways involved in the biosynthesis of PHAs in microorganisms and plants could make the industrial scale production of these biopolymers competitive with oil-based synthetic polymers. As for the agricultural production of PHAs, the feasibility of this route has been demonstrated in small plants such as Arabidopsis thaliana,57 but the transfer of this technology into crops such as canola with acceptable production levels is still in the research stage. On the other hand, the chemical modification of medium chain PHAs produced by bacteria is a promising approach to the commercialization of highvalue polymers for specialty applications.58-60 Indeed, by either direct bacterial synthesis or by the chemical modification of bacterially produced PHAs, polyesters with more than one hundred different types of repeating units have been identified and characterized.61 Very recently, it was even found possible to produce a thioester analogue of the PHAs with bacteria,62 so it is apparent that there is still much more to be discovered about the synthesis of bacterial polyesters. Acknowledgment. We wish to recognize the important contributions by Professors Schlegel, Dawes, Merrick, and Fuller for the factual details herein. In addition, Dr. Bernard Hautecoeur and Archives of the Institut Pasteur provided historical background concerning Professor Maurice Lemoigne as surveyed by Rene Dujarric de la Riviere.63 Dr. ` Francis Werber kindly informed us on the development of

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PHB at W.R. Grace Company, where he was V.P. research. Finally, we are grateful to Professor Alexander Steinbuchel who provided a critical review of our manuscript. References and Notes
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