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CHAPTER ONE
INTRODUCTION
1.1 Introduction:
The number of operational landfills in the United States has been steadily decreasing, while the amount of landfill material being deposited each year continues to increase. In 1990 alone, 84% of the 270 million tons of municipal solid waste generated in the United States was buried in landfills[1]. Discarded plastics contribute roughly 10% of the solid waste disposal material[2] in landfills, and contribute significantly to roadside litter[3]. In the next 5 to 10 years, the available landfill space in more than half the United States will be gone. As this occurs, more and more space must be made available for solid waste disposal, yet few new sites are currently being approved[2]. This lack of landfill space creates a waste disposal problem costing about $15 billion per year. Alternative approaches to ease the growing waste disposal problem include recycling and reusing nonbiodegradable synthetic polymers, incineration, and the chemical destruction of plastics[4]. Biodegradable plastics is another alternative to ease the growing waste disposal problem.
The use of biodegradable plastics must be studied and evaluated as a viable alternative to the petroleum-based plastics currently in use. By definition, biodegradable plastics must be susceptible to attack by microorganisms native to landfills, composts, and other environments in which refuse collects.
1.2 Degradation of plastics:
The introduction of photodegradable and biodegradable materials into traditionally nondegradable plastics are examples of the many attempts being made to produce degradable plastics. Photodegradation works by the introduction of photolabile chemical groups (e.g., carbonyl moieties, benzophenone) into polymers to accelerate their ultraviolet light-catalyzed depolymerization in the environment via a free radical process[4]. Though photodegradable plastics offer an attractive alternative to more traditional materials, degradation does not occur in polymers buried in soil, or coated with a light obstructing material[4] such as paint or lettering. The introduction of biodegradable materials, such as a starch filler, into nondegradable materials (e.g., low density polyethylene) is another example of the efforts being made to produce biodegradable plastics[3]. However (P. Barak) observed that only the starch degraded, leaving undegraded pieces of polyethylene behind.
Poly(3-hydroxybutyrate) (P3HB) and polyhydroxybutyrate(co-hydroxyvalerate) (PHBV) are two widely studied biodegradable polymers under consideration as alternative plastics.
1.3 Poly(3-hydroxybutyrate):
Poly(3-hyroxybutyric acid), or P3HB (Fig. 1.1 A), was detected as a constituent of bacterial cells 70 years ago[11]. For almost 60 years it was the only known member of the polyhydroxyalkanoic acids (PHA) polyester family[5]. P3HB is a well-known, naturally occurring intercellular polymer, which accumulates as a storage reserve for carbon and energy[6-8], and is synthesized by a wide variety of bacteria (Gram-negative and Gram-positive) and some types of fungi[5,6,9]. Natural P3HB (nat-P3HB) is a biodegradable material that can be completely broken down (mineralized) to naturally occurring and environmentally benign molecules[10]. The first step of biodegradation is depolymerization, or chain cleavage. In the first step, the polymer is cleaved into sufficiently small size oligomeric fragments that can be transported into the cell where they are broken down and assimilated[4]. Mineralization, a process from which cells derive metabolic energy (ATP) while converting the substrate into biogases (e.g., CO2, CH4, N2), water, salts, and minerals, is the second stage of biodegradation[4]. The by-products of mineralization, especially the biogases CO2 and CH4, can be measured to quantify the amount of degradation that has taken place.
P3HB and PHBV are synthesized by bacteria and fungi alike; for example, Alcaligenes eutrophus[11], Bacillus megaterium[12], Penicillium cyclopium[13], and Physarum polycephalum[14]. These organisms produce and accumulate P3HB (in the cells) in a three-step process when carbon is provided in excess, but cell proliferation is impaired owing to the lack of one or more essential nutrients such as nitrogen, sulfate, phosphorous, iron, magnesium or potassium[5]. In the three-step P3HB-biosynthetic pathway, acetyl-CoA is converted to P3HB (Fig. 1.2). A biosynthetic -ketothiolase catalyses the formation of a carbon-carbon bond by a biological Claisen condensation of two acetyl-Co-A moieties[12]. A NADPH-dependent acetoacetyl-CoA reductase catalyses the stereoselectivity reduction of acetoacetyl-CoA formed in the first reaction to D(-)-3-hydroxybutyryl-moiety to an existing polyester molecule by an ester bond[5]. This pathway is one of several that have been described for the biosynthesis of P3HB, and is referred to as the Alcaligenes eutrophus PHA-biosynthetic pathway[5]. The type of P3HA produced varies as a function of carbon source. PHBV (Fig. 1.1 B) is produced if the organism is cultured on propionic acid as the sole carbon source, or in combination with other carbon sources[5]. PHBV is made at an industrial scale and is distributed under the trade name BIOPOL and is used in the manufacturing of biodegradable films and bottles[15-17], e.g., shampoo bottles in Germany[18]. P3HB (or other PHA) is produced when n-alkanoic acids are used as the carbon source[19]. PHA’s currently are being produced by ICI Ltd. for the purpose of evaluating their manufacturing and marketing potential[19].
Nat-P3HB is a thermoplastic polyester with a melting point (Tm) of about 180C and a glass transition temperature (Tg) of about 5C[20,21]. The thermoplastic properties of P3HB and PHBV were recognized in the early 1960s and formed the basis for patents assigned to W.R. Grace & Co.[22,23]. The chain molecules of nat-P3HB are completely linear and the chiral centers possess only the [R]-steriochemical configuration, which implies that the natural polymer is completely isotactic and capable of crystallization[20]. The excessive brittleness of melt crystallized and solution cast nat-P3HB films, which increases upon aging at room temperature, have retarded the development of nat-P3HB products for commercial applications. It is of industrial interest to evaluate these polyesters as biodegradable thermoplastics with a wide range of possible applications, including surgical sutures, drug carriers, molded plastics, and films[24-26]. Methods of improving the physical properties of natural-origin P3HB include (i) the microbial synthesis of co-polymers, such as random co-polyesters of [R]-3HB with [R]-3-hydroxy valerate (3HV) or 4-hydroxybutyrate[27], (ii) and the blending of P3HB with other biodegradable polymers[28,29], and (iii) plasticization[30]. Blending synthetic P3HB with iso-P3HB is another method under consideration for improving the commercial value of P3HB[31].
Synthetic P3HB has been produced in the laboratory from racemic -butyrolactone using dibutyl tin dimethoxide as a catalyst (Fig. 1.1 C)[32]. Syn-P3HB possesses commercially favorable attributes, such as a lack of brittleness after solution casting of films, qualities that are intrinsic to the non-biodegrading petroleum based plastics in wide use and acceptance today[33]. Syn-P3HB has a different stereochemistry then that of nat-P3HB, therefore it can not be assumed that it is biodegradable in whole or part[31]. To date, few studies have been conducted into the biodegradability of syn-P3HB and blended nat-/syn-P3HB polymers[34-39].
1.4 Penicillium funiculosum:
Penicillium funiculosum (ATCC 9644, reclassified as Penicillium pinophilum) is one of an enormous genus of fungi[40,41]. Penicillium funiculosum strain ATCC 9644 was isolated in Australia by K.L. Jensen (classified SN 41), and sent to ATCC by W.H. Weston[40]. Fungi are eucaryotic microorganisms possessing a cell wall, liquid-filled intracellular vacuoles, microscopically visible streaming cytoplasm and (almost universal) lack of motility. They do not contain photosynthetic pigments and are chemoorganotrophic heterotrophs - obtaining their energy from the oxidation of organic substances[42]. Fungi are characterized as growing best in dark, moist habitats such as soil, where most are native, and may constitute from 1% to 20% of the culturable population in a soil environment[42]. Penicillium funiculosum colony morphology, when grown on a solid substrate (agar), has a characteristic white surface at first, then becoming very powdery, bluish green, with a white border[43]. At a microscopic level, septum divided hyphae with branched or unbranched conidiophores that have secondary branches known as metulae can be seen[43]. On the metulae, arranged in spirals, are flask-shaped sterigmata that bear unbranched chains of round conidia[43]. The entire structure forms the characteristic “penicillus” or “bush” appearance[43].
Like many types of bacteria, fungi can secrete hydrolytic enzymes that digest external substrates and can then absorb the soluble products, which in turn are used as a source of carbon, electrons, and energy[42]. Bacteria such as Alcaligenes faecalis[33] and Pseudomonas lemoignei[44], and fungi such as Aspergillus niger[45] and P. funiculosum[46] are recognized as microorganisms that produce depolymerases which allow complex carbon sources to be broken down into usable substrates needed to meet the organism’s resource requirements (depolymerization and mineralization).
Fungi play a major role in the depolymerization and mineralization of biodegradable materials. They are very flexible nutritionally and can aerobically degrade resistant substrates such as pectin, lignin, chitin, keratin, latex, and aromatic compounds[42]. P. funiculosum is of particular interest because of its ability to degrade, and use as its sole carbon source, different types of natural poly(3-hydroxybutyrate)[47,48].
1.5 Goals and Objectives:
There have been few studies into P. funiculosum’s ability to degrade and use as its sole carbon source P3HB’s of differing molecular weight and tacticity. This study investigated the ability of P. funiculosum to degrade and consume natural and synthetic P3HB polymers. Morphological characteristics of P. funiculosum cultured on the different polymers, as well as depolymerase production and activity, cellular growth and total protein production were studied. Degradation of solvent-cast P3HB films was examined in growing cultures of P. funiculosum in a basal salt medium solution[49], on agar plates[33] and in a soil environment[*].
* ASTM Methods UML-7645. 1994.
In addition, the depolymerases responsible for degradation were isolated, characterized[47,48], and used for degradation studies of the solvent cast films prepared from iso- and syn-P3HB blends[50]. This study demonstrated how the organism behaves differently when cultured on nat-P3HB, syn-P3HB, and PHBV sources, and how the depolymerases responsible for degradation work at different rates in the biodegradation of solvent-cast nat- and syn-P3HB films.