Plastics from Bacteria and for Bacteria: Poly([I-Hydroxyalkanoates) as Natural, Biocompatible, and Biodegradable Polyesters Helmut Brandll*, Richard A. Gross 2 z~= Robert W. Lenzz, and R. Clinton Fuller 1 Department of Biochemistry, University of Massachusetts, Amherst, MA 01003, USA 2 Department of Polymer Science and Engineering, University of Massachusetts, Amherst, MA 01003, USA 1 Microbial Formation of Poly(13-Hydroxyalkanoates) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Structure of P H A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Occurrence of P H A in Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Environmental Conditions Affecting P H A Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.4 P H A as IntraceUular Inclusion Bodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 Characterization and Properties of P H A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1 Physical and Chemical Characterization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2 Analytical Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~. . . . 2.3 Functional P H A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 P H A as Biodegradable Plastics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 Industrial Production of P H A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Biodegradation of P H A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Biodegradable Plastics and Solid Waste Disposal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Ecological Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2 Disposal o f Plastics Waste 5 Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6 Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~. . . . . . .

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A wide variety of different types of microorganisms are known to produce intracellular energy and carbon storage products which have been generally described as being poly(~-hydroxybutyrate), PHB, but which are, more often than not, copolyrners containing different alkyl groups at the 13-position. Hence, PHB belongs to the family of poly(fl-hydroxyalkanoates), PHA, all of which are usually formed as intracellular inclusions under unbalanced growth conditions. Recently, it became o f industrial interest to evaluate P H A polyesters as natural, biodegradable, and biocompatible plastics for a wide range of possible applications such as surgical sutures or packaging containers. For industrial applications, the controlled incorporation of repeating units with different chain lengths into a series of copolymers is desirable in order to produce polyesters with a range of material properties because physical and chemical characteristics depend strongly on the polymer composition. Such "tailormade" copolymers can be produced under controlled growth conditions, in that if a defined mixture of substrates for a certain type o f microorganisms is supplied, a well defined and reproducible copolymer is formed.

* Present address: Institute of Plant Biology, ZollikerstraBe 107, 8008 Ziirich, Switzerland Present address: Department of Chemistry, University of Lowell, 1 University Ave., Lowell, M A 01854, USA

Advancesin BiochemicalEngineering/ Biotechnology,Vol.41 Managing Editor: A. Fiechter 9 Springer-VerlagBerlin Heidelberg1990

78

H. Brandl

1 Microbial Formation of Poly([~-Hydroxyalkanoates) 1.1 Structure of PHA A wide variety of different types of microorganisms are known to produce intracellular energy and carbon storage products which have been generally described as being poly(~3-hydroxybutyrate), PHB [1-4]. The formation of this type of polymers is limited to prokaryotic organisms, whereas eukaryotic cells are not known to produce PHB. This particular polymer belongs to the family of poly(J3-hydroxyalkanoates), (PHA), (1), which are usually formed as intracellular inclusions under stressed growth conditions; that is, in the presence of an excess of a carbon or energy source on the one hand and a limiting nutrient or growth factor on the other [1, 2, 5-8]. Because of these unbalanced growth conditions, reduction equivalents, which originate from metabolic oxidation processes, are stored in a water-insoluble, chemically and osmotically inert form of the following structure:

R

0

"JEO-CH-CH2-C-~

(t)

R = n-alkyl pendant group of variable chain length HB 13-hydroxybutyrate where R = methyl HV. [3-hydroxyvalerate where R = ethyl HC. [3-hydroxycaproate where R = n-propyl H H [3-hydroxyheptanoate where R = n-butyl HO 13-hydroxyoctanoate where R = n-pentyl HN ~-hydroxynonanoate where R = n-hexyl HD 13-hydroxydecanoate where R = n-heptyl HUD, [3-hydroxyundecanoate where R = n-octyl HDD, [3-hydroxydodecanoate where R = n-nonyl

1.2 Occurrence of PHA in Microorganisms The spectrum of PHA-producing microorganisms includes a variety of taxonomically different groups (Table 1). Most of the organisms are capable of accumulating PHA from 30 to 80 % of their cellular dry weight. However, under specific conditions, Alealigenes eutrophus N9A is known to contain 96 % PHA [9]. These storage polyesters are also found in cyanobacteria such as Aphanothece or MicroeoIeus [10], but usually their PHA content is relatively low. Interstingly, enterobacteria are among the organisms which do not form PHA. However, it has been demonstrated recently that the PHB biosynthetic pathway from A. eutrophus can be cloned and expressed in Eseherichia coli [11, 12]. Polymer contents of up 90 % of the cellular dry weight were obtained from E. coli. In addition, it has been shown that PHA is a structural component of cell membranes of E. eoli as well as Haemophilus influenzae, a strictly parasitic organism [13-16]. Besides some phototrophic microorganisms, Clostridium [17] and Syntrophomonas [18] are the only strictly anaerobic

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Plastics from Bacteria and for Bacteria

Table 1. The accumulation of poly(13-hydroxyalkanoates) in a variety of microorganisms known to form intracellular storage products Genus

Classification after Bergey's ManuaP

Maximum PHA content ( ~ dry wt)

PHA producing substrate

Reference

A cinetobacter Alcaligenes Aphanotheee Aquaspirillum Azospirillum Azotobacter Bacillus Beggiatoa Beijerinckia Caulobacter Chloroflexus Chlorogloea Chromatium Chromobacterium Clostridium Derxia Ectothiorhodospira Escherichiab Gamphosphaeria Haemophilusb Halobacterium Hyphomicrobium Lamprocystis Lampropedia Leptothrix Methylobacterium Methylocystis Methylosinus Micrococcus Microcoleus Microcystis Moraxella Mycoplana Nitrobacter Nitrococcus Nocardia Oceanospirillum Paracoccus Photobacterium Pseudomonas Rhizobium Rhodobacter Rhodospirillum Sphaerotilus Spirillum Spirulina Streptomyces Syntrophomonas Thiobacillus Thiocapsa

10 7 Cyanobactefia 6 6 7 15 2 7 4 1 Cyanobacteria t 8 15 7 1 8 Cyanobacteria 8 13 4 1 10 3 7 ND 7 14 Cyanobacteria Cyanobacteria 10 17 12 12 17 6 10 8 7 7 1 1 3 6 Cyanobacteria 17 9 12 1

Plastics from bacteria and for bacteria: poly(beta-hydroxyalkanoates) as natural, biocompatible, and biodegradable polyesters.

Hence, PHB belongs to the family of poly(beta-hydroxyalkanoates), PHA, all of which are usually formed as intracellular inclusions under unbalanced gr...
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