LITERATURE REVIEW : Polymers
Literature on the use of Bacillus for the production of biodegradable polymers is scant. Further research on the use of the bacteria Bacillus as a producer of large quantities of the polymer should be conducted. It is only recently that this bacterium is found to be producing biodegradable polymers.
Biodegradable polymers are divided into two classifications: natural and synthetic. Natural polymers are isolated pure polymers with no physical or chemical modifications. The most widespread natural polymers are the polysaccharides, proteins and polyesters such as polyhydroxyalkanoates (2000).
Synthetic polyesters made from aliphatic diols and aliphatic acids are also biodegradable. Such polymers include polyglycolic acid, polylactic acid and polycaprolactone. Other biodegradable synthetic polymers include polyamides, polyanhydrides, polyamide-enamines, polyurethanes, polyethers, polyacetals, polyphosphazones and other condensation polymers (2000).
Polyhydroxyalkanoates (PHA) are polyester compounds which are produced by a wide range of microorganisms, such as bacteria and algae. However, their inherent biodegradability has been hampered by their thermal instability. Poly-3-hydroxybutyrate (PHB) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) have been studied, but PHB degrades at elevated temperatures and both polymers are very difficult to process into films suitable for backsheet applications due to their very slow crystallization rate (1997).
The observation of (1983) that a strain of Bacillus megaterium accumulates a polymer called poly-beta-hydroxybutyrate (PHB) under nutrient stress led to investigations of the role of these polymers in the physiology of bacteria. The polymer was thought to be a simple polyester of beta-hydroxybutyrate monomers. A wide variety of prokaryotic organisms have been shown to accumulate this polymer, including numerous heterotrophic and autotrophic aerobic bacteria, photosynthetic anaerobic bacteria, gliding bacteria, Actinomycetes spp., cyanobacteria and recently, an anaerobic, fatty acid-oxidizing, gram-negative bacterium (2003).
The notion of using bacteria, which exist in such abundance and diversity, to make materials may be an idea whose time has come. The field of biomimetics -- in which scientists emulate nature's own processes for fashioning high-quality compounds -- is emerging with force. Increasingly, scientists are turning to biology for innovative ideas about how to make natural materials. From the nails on our fingers to the bones in our hands to the seashells on a beach, nature has proved to be an extraordinary material manufacturer. Slowly but surely, though, scientists are learning to imitate nature's tricks (1994).
Biodegradable polymers are mostly studied in the medical field. But the medical field is not the only area where interest in biodegradable polymers is growing. Ecological issues are increasingly attracting interest, including substantial commercial interest because clinical and regulatory questions are not so significant in this area ( 1996).
Polyhydroxyalkanoates (PHAs) are thermoplastic polyesters naturally produced by various microorganism (especially bacteria) and are considered leading candidates for the 'clean plastic' material eagerly sought by many environmentalists. These polyester substances are part of the natural biosynthesis/biodegradation cycle, hence they respond to present environmental requirements in various countries for biodegradable materials. The first of these polyesters was discovered by Lemoigne of the Pasteur Institute in 1925. The biopolyesters are presently produced on a relatively small scale by fermentation technology. The most available commercial version is a copolymer, produced by the bacterium Alcaligenes eutrophus fed with glucose and proprionic acid (1995).
PHAs are 'a broad and versatile family of polymers that range in properties from rigid to elastic, and can be converted into moulded and thermoformed goods, extruded coatings and film, blown film, fibers, adhesives and many other products'. They have excellent shelf life and resistance even to hot liquids, greases and oils, yet they biodegrade in aquatic, marine and soil environments and under anaerobic conditions, such as found in septic systems and municipal waste treatment plants. They can be both hot and cold composted ( 2006).
Unlike polymers comprising 2-hydroxyacids such as polyglycolic acid and polylactic acid, polyhydroxyalkanoates normally are comprised of 3-hydroxyacids and, in certain cases, 4-, 5- and 6-hydroxyacids. Ester linkages derived from these hydroxyacids are generally less susceptible to hydrolysis than ester linkages derived from 2-hydroxyacids ( 2001).
Poly-3hydroxybutyric acid (PHB) is one of the most well studied polyhydroxyalkanoates (PHA), which are polyesters of various 3-, 4- and 5-hydroxyalkanoic acid found to accumulate in various prokaryotes and eukaryotes. They exhibit thermoplastic properties, are biodegradable and can be produced from renewable carbon sources. Hence, there has been considerable interest in the commercial exploitation of these biodegradable polyesters. The search for truly biodegradable plastics has led scientists into the arms of bacteria and plants, as they make headway to 'natural polymer factories' (1997).
Many species of bacteria accumulate polyhydroxyalkanoates (PHA) as energy storage compounds in response to nutritional stress. Some PHA polymers are commercially valuable as biodegradable plastics. Polyhydroxybutyrate is a PHA produced in R. eutropha via a three-enzyme biosynthetic pathway consisting of [beta]-ketothiolase, acetoacetyl-CoA reductase, and PHB synthase. These enzymes are encoded by phbA, phbB, and phbC, respectively ( 2002).
The monomeric composition of PHA polymers is determined by the substrate specificities of [beta]-ketothiolase and PHB synthase and by the availability of acetyl-CoA and propionyl-CoA. In the case of 4- and 5-carbon PHA precursors, when [beta]-ketothiolase is encoded by phbA, a homopolymer of PHB is produced. When 13-ketothiolase is encoded by bktB, a copolymer of PHB/V is produced. Because the PHB/V copolymer has better physical properties than PHB, it is preferred for most commercial applications. Presently, the bacterially produced polymers are not cost-competitive with nonbiodegradable plastic polymers derived from petroleum. By genetically engineering crop plants to produce PHA, a less expensive source of these polymers could become available (2002).
Desirable properties like durability and resistance to degradation have made plastic materials an integral part of contemporary life. The standard plastic formulations used today include polyolefins, polyesters and polyurethanes, all of which are petroleum based and non-degradable. In addition, hazardous chemicals are needed for their production as well as their disposal (1997).
The burden of accumulating plastic waste has stimulated research and development of degradable polymers (2000). More than 20 years there were some experiments with a new so-called biodegradable polymer. There was a great deal of commercial interest in this polymer because it was developed at the time of one of the first global oil crises and had considerable potential. Apart from its assumed biodegradability, it was derived from nonpetrochemical sources, being the product of bacterial metabolism. The polymer was polyhydroxybutyrate (PHB) ( 2005). Polyhydroxybutyrates are considered the most useful of all the microbially derived biodegradable plastics ( 1997).
PHB is a biodegradable thermoplastic which can be extracted from a wide range of bacteria. PHB is an important molecule on cytoplasm and cell walls. Industrial interest in PHB has flagged because of two major drawbacks: poor melt stability and extreme brittleness.
PHBs are aliphatic, polyester homopolymers which are stored as an energy reserve in the cells of many bacteria including Alcaligenes eutropus. When deprived of nitrogen, phosphate, magnesium or sulphate, A. eutropus produces poly(3-hydroxybutyrate) at up to 90% of its dry weight. As a polymer, PHB compares well with polypropylene in terms of molecular mass, melting point, crystallinity and tensile strength. However, its major drawback is its low impact strength. This can be overcome by inducing the bacteria to produce a copolymer (PHB-V) of PHB and polyhydroxy valerate (PHV) (1997).
The most important features of PHB include its ability to resist 120°C high temperature, high molecular weight, crystalline, brittle and not soluble in water. Important properties in this regard include porosity for cell in growth, pores are very important for the cells to enter and grow as well as capillary section of tissue fluid and blood. They also provide very large surface area for the invading cells or for interaction with cell (2003).
PHB stays flexible from sub-zero temperatures up to 130°C, and completely breaks down into water and carbon dioxide in a few months. Results on biodegradation rates of PHB and PHB-V showed that the copolymer degraded faster than the homopolymer. Both are degraded by a wide variety of microorganisms flourishing in the soil including Gram-negative bacteria, Gram-positive Bacilli and Streptomyces, as well as moulds ( 1997).
A group reported that changing the lamellar morphology of PHB, by using a simple annealing treatment (heat treatment), leads to a toughening of PHB. The morphological change increases the relaxation properties of the amorphous regions in PHB, thus improving the fracture behavior. This discovery may significantly widen the scope of possible industrial applications of PHB. PHB was said to age because of secondary crystallization ( 1995). The polymer which provides a reserve of carbon and energy, accumulates as intracellular granules.
Bacillus species have been shown to accumulate PHB during the sporulation of bacterial growth. The PHB production capacities of bacteria have been investigated for possible application in industry. During the 1970’s, in the aftermath of the first oil crisis, that the British chemical giant, Imperial Chemical Industries (ICI) began investigating the polymer-forming properties of bacteria. Alcaligenes lotus and A. eutrophus are presently utilized by ICI to produce a PHB-PHV copolymer under the trade name “Biopol” ( 2003).
Petroleum derived plastics are widely used in our daily lives, but they cause environmental pollution because they are persistent for hundred of years. Because of this, biodegradable polymer production (microbial thermoplastics) has gained importance. Furthermore the continuous depletion of petroleum sources has placed more emphasis on the need for biodegradable microbial plastics. PHB is an important raw material for microbial plastics. Today, most research efforts in this field concentrate on the isolation of PHB producing microorganisms from different sources and improvement of PHB production abilities of microorganisms (2003).
Today's plastics are tailored with little consideration for disposability or the impact on resources used to make them. Synthetic polymers were originally developed for their durability and resistance to all forms of degradation, including biodegradation. They were also chosen for performance characteristics achieved through molecular weight control, functionality and morphology (2000).
In cases where, as in agricultural films, biodegradability is important, it is difficult to use traditional plastics derived from petroleum, although plastics such as polycaprolactone are biodegradable. It is often misconstrued, however, that plastics from renewable sources are always biodegradable. This is certainly not the case, and in many instances, the end use demands that the plastic is not naturally biodegradable. Automotive paneling or construction materials are end uses which require the materials not to naturally biodegrade. In fact, the biodegradable plastics market is relatively small, and many of the opportunities for plastics from renewable sources are in the non-biodegradable plastics sector (2004).
Biodegradable plastics offer an alternative to traditional non biodegradable petroleum-based polymers. The principal driving force behind the technology is the solid waste problem with regard to the decreasing availability of landfills. In the last decade there has been renewed interest in developing materials that mimic plastics but have a significant component from agricultural commodities (2000).
The argument was, therefore, that if bacteria could be made to synthesize and store PHB under large-scale culture conditions, and if the PHB could then be extracted from these bacteria, a high molecular weight polymer could be produced that would be biodegradable under conditions when the intracellular enzymatic processes were replicated outside of these cells (2005).
This became an even more attractive proposition when it was realized that this polymer was capable of being processed into various forms, including fibers, powders and films, and that it was possible to create blends and copolymers with other organic materials to modify properties.
It is still unclear which of the variations on PHB-based materials will make the most impact in tissue engineering, but there is much evidence that they could become the preferred biodegradable scaffold materials, in preference to the more classical medical device based polyesters such as polylactic and polyglycolic acid based materials (2005).
Many bacteria synthesize and store natural, biodegradable polyesters, such as poly-3- hydroxybutyrate (PHB). However, large-scale bacterial production of this class of natural polyesters seemed impractical and costly, in part because fermentation equipment the size of several breweries would be needed to produce commercially significant amounts. But only three genes are required to make PHB ( 1998).
The high cost of PHA, so far, has cooled the interest of corporations which market personal products where biodegradability is desirable and must be relatively affordable. The production of PHB in plants is an attractive alternative to bacterial fermentation (1995).
The presence of PHB has also been used in bacterial taxonomy for classification and identification. SDS-Page, based on total protein profiles, is used in bacterial taxonomy to the levels of the species and. Therefore, it can used to distinguish between mutants ( 2003).
Fascinated by the physical properties of these bacteria -- their hearty cell walls made mostly from the two polymers peptidoglycan and teichoic acid. Bacteria make their cell walls from a curious material. Electrically active and physically resilient, the spongy, porous gel can shrink or swell, bend, twist, stretch, or shear, then resume its normal shape (1994).
An affinity for charged particles, enables the walls to retain mineral deposits and form crystalline structures. Macrofibers and threads arise when normal bacterial cell growth hits a glitch. Cell walls fail to cleave normally after cell division, and the bacteria link to form chains. As cells keep dividing, their cylindrical bodies warp, winding into helical coils --"like a twisted telephone cord” (1994).
Each macrofiber goes through a primitive life cycle. A single cell spawns a chain, which winds into a double stranded helix. That coil bends and twists into a four-stranded, then eight stranded, then 16-stranded fiber, coalescing into tightly bundled, rope-like strands. A closed loop completes each end. Through experimental manipulation, different strains of the same bacterial species have grown, controlling the degree of twist and whether the helix turns to the left or right ( 1994).
That nature repeatedly creates materials that twist and supercoil -- from tiny proteins and DNA to the yarns, ropes, and cables spun from natural filaments -- suggests that some fundamental process may affect how these diverse structures form. Such questions have brought mathematicians together with physical chemists to look at the "knot topology" of these biological shapes. If common physical processes do engender such related forms, then researchers as varied as cellular biologists and materials scientists may together find such knowledge valuable and illuminating (1994).
Durability and resistance to degradation are desirable properties when the plastics are in use, but they pose problems for disposal when out of use. A steady increase in plastics production and their persistence in the environment long after their intended use pose serious problems to the environment (1997).
The environmental problems mean that there is an increased demand for biodegradable polymers - materials which can be degraded by enzymes. In essence, to be biodegradable a plastic needs to have chemical groups at the ends of its polymer chains that are susceptible to oxidation by microbial enzymes.
Initial efforts to develop biodegradable plastics were focused on incorporating new materials into conventional plastics to make the final product degradable by generating these oxidizable chain ends. Based on this principle photodegradable polymers (which degrade when exposed to the sun's UV light) and copolymers of a plastic with cellulose or starch were developed (1997).
Poly-(R)-3-hydroxybutyrate has been attracting interest as a biodegradable thermoplastic. It is usually generated in fermenters using cultures of bacteria such as Bacillus megaterium. A new and simple synthetic route has now been proposed. The regioselective anionic polymerisation of (S)-[Beta]-butyrolactone can be initiated by the sodium salt of (R)-3-hydroxybutyric acid in the presence of acrown ether.
A living polymerisation, either in bulk or in solution, then proceeds by regioselective attack of the carboxylate and occurs with inversion of configuration. The resulting polymer is highly isotactic and crystalline. Its molar mass is easily scaled up to 20,000 g/mol, as determined by the monomer-to-initiator ratio ( 1998).
PHB production in plants was first demonstrated in transgenic Arabidopsis thaliana (L.) Heynh.. Poly-[beta]-hydroxybutyrate is tolerated by plants if it is synthesized in chloroplasts and PHB levels ranging between 0.1 and 14% DW have been reported with minimal effects on plant growth. Although various approaches for producing PHB in plants have been reported, most of these utilize Arabidopsis. Recently, production of PHB/V was achieved in the seed of oilseed rape (Brassica napus L.), demonstrating that it is possible to produce PHA in crop plants ( 2002).
In recent years, alginate and chitosan have found widespread use as novel biomaterials. As natural polymers, they are biocompatible, biodegradable, nontoxic and in abundant supply Alginate exists widely in brown seaweeds and chitosan can be extracted from many species of fungi and the cuticular or exoskeletons of crustaceans and insects (2004).
Alginate fibers have been successfully used as a moist-healing material Wound dressings made of alginate fibers have occupied a significant position in the high-tech wound dressing market in Western Europe and North America. Similarly, chitosan fibers have novel wound-healing properties and in recent years many efforts have been made to use chitosan as a tissue-engineering material, utilizing its excellent biocompatibility and biodegradability ( 2004).
One of the main applications of alginate and chitosan fibers is in wound-dressing materials. Because they are hydrophilic polymers, alginate and chitosan fibers are known to have high absorption capacities and are able to retain moisture. Alginate fibers and dressings (the carboxylic acid in alginate exists normally as a calcium salt) have ion exchange properties whereby calcium ions in the fibers are replaced by the sodium ions in the body fluid when the dressing is in contact with the wound exudates. As a result of this ion-exchange process, some of the calcium ions in the fibers are replaced by the sodium ions. Because sodium alginate is water soluble, water is drawn into the fiber and a fibrous gel is formed as a result (2004).
As a polymeric acid, alginate can form salt with metal ions. Most divalent metal ions can form water insoluble salt with alginate, hence alginate fibers can be made by extruding water-soluble sodium alginate solution into aqueous solutions containing divalent metal ions. Many metal ions are useful for wound healing; for example, zinc is useful for zinc-deficient patients, and silver has antimicrobial properties. Sodium alginate can be extruded into an aqueous zinc chloride solution to form zinc alginate fibers. Alternatively, a calcium alginate fiber can be treated with solutions containing zinc or silver. On ion exchange, the zinc and silver in the solution replace the calcium ions on the fiber to result in alginate fibers containing zinc and silver ions. Chitosan fibers can bind metal ions via chelation with the amine groups. It has been shown that chitosan fibers can chelate up to 6.2% of zinc ions. The degree of acetylation has a significant effect on the chelating abilities of the chitosan fibers. In a study with copper ions, it has been shown that when fully acetylated, the chitosan fibers lose their ability to chelate (2004).
Both alginate and chitosan fibers are known to have antimicrobial properties. When fluid is absorbed into an alginate wound dressing, the fibers swell after the ion-exchange process. As the fibers swell, the spaces between them dose and any bacteria that are carried in the wound exudates are trapped in the wound dressing. This can help reduce the spread of bacteria. The antimicrobial mechanism for chitosan is different. As a polymeric amine, chitosan is positively charged when wet, and because the cell walls of the bacteria are negatively charged, bacteria adhere to the chitosan fibers. As a result of the different electric charges, the bacteria cell walls can burst, resulting in the containment of the bacteria (2004).
Alginate and chitosan have many novel properties, making them useful biomaterials. When made into fibers, alginate and chitosan can be further processed into nonwoven, woven, knitted and other two--or three-dimensional structures. Alginate and chitosan have an acid and an amine group on their molecules respectively, making it easy for them to form novel materials with metal ions or through chemical modifications. Alginate fibers have already been successfully used as a raw material for making advanced wound dressings. Silver containing alginate fibers have been made into wound dressings that have good antimicrobial properties (2004).
At present, the synthesis of PHB as a single end-product in crops is economically challenging. Poly-[beta]-hydroxybutyrate levels of 15 % DW are needed for practical large-scale commercial production. Recently, Arabidopsis plants with PHB levels of 40% DW were obtained, but the plants were stunted and infertile. However, plants producing PHA at levels compatible with growth could be used for multiple purposes to increase overall crop value ( 2002).
Production of bioplastics in alfalfa leaves could be combined with use of alfalfa for feed and energy. In such a system, alfalfa leaves could be harvested to produce PHB, leaf by-product could be processed into a feed, and stems could be used for producing either electricity by gasification or ethanol by fermentation. As a start towards this goal, the objectives of this study were to genetically modify alfalfa to produce PHB and to initiate transfer of this trait into germplasm developed for high biomass production ( 2002).
The scientists say a controlled degradation rate of less than one year under physiological conditions is preferable. The degradation rates of the polymers can be manipulated through the addition of various components, as well as selection of the chemical composition, molecular weight, processing conditions and form of the final polymeric product. The chemical composition can be altered through selection of monomers which are incorporated into the polymer, by alteration of the linkages, chemical backbone or pendant groups and/or by manipulation of the molecular weight ( 2001).
Biodegradable polyhydroxyalkanoates can be synthesized by synthetic chemically or biologically based methods. A chemical approach involves the ring-opening polymerization of -lactone monomers. The catalysts or initiators used can be a variety of materials such as aluminoxanes, distannoxanes or alkoxy-zinc and alkoxy-aluminium compounds. Such PHAs can be processed into a wide range of plastic articles, including films, sheets, fibers, foams, moulded articles, nonwoven fabrics, elastomers and adhesives. The PHAs can be melt processed into films using either cast or blown film extrusion methods (1997).
Naturally occurring polymers such as polysaccharides, proteins and cellulose are easily biodegraded because many micro-organisms that produce the enzymes required to metabolize these compounds are readily available in nature. For the biodegradation process to function there is a need for a well-tuned environment where the desired micro-organisms can thrive. Some of these factors include appropriate temperature range, moisture level, salt, oxygen, trace metals, pH, redox potential, environmental stability or flux and pressure (2000).
Over the last few years several standard test methods have been developed to assess biodegradability. They include enzyme assays, plate tests, biological oxygery, simulated laboratory scale accelerated systems and exposure to natural environments (2000).
While natural polymers are generally inexpensive, they are difficult to process into useful end products. For example, products made from starch or proteins are brittle, inflexible and moisture sensitive. Because synthetic polyesters are eight to 10 times as expensive as their natural counterparts, blending the two materials could reduce costs while improving functionality. However, starch and plastics do not mix easily so combining them results in products with reduced physical properties (2000).
PHA and PHB are also widely used in the development of medical applications. The logic in the search for medical applications for this material and its derivatives was straightforward. It is the polymer of 3-hydroxybutyric acid, otherwise known as 3-hydroxybutanoic acid. It is produced within certain types of microorganisms at the time of excess nutrients and is utilized as an energy store because it can be broken down during periods of relative nutrient deficiency to release this energy.
The synthesis of the polymer is enzyme driven and, depending on the conditions, will have a molecular weight of between 60 000 and 250 000. When exogenous energy sources are unavailable and the environmental conditions favorable, PHB is degraded via a series of enzyme-mediated reactions to acetoacetyl-CoA, which is used by the cell as a source of energy (2005).
The main efforts with the PHBHHx-based materials have been in the area of musculoskeletal tissue engineering. The signs look good. Undoubtedly, an acceptable level of mechanical properties can be obtained. The relatively poor ductility and elasticity of the P(3HB) have been replaced with values of over 300% elongation at break for P(4HB) and some of the PHBHHx-based materials, and tensile moduli reduced to acceptable levels below 1 GPa for soft-tissue applications. Tensile strengths can be in excess of 100 MPa. There are still relatively little substantiated data on the in vivo degradation and resorption rates for these materials and the biocompatibility remains unclear in some situations, although no significant difficulties have been observed. It also has to be said that some resurgence of interest in the use of P(3HB), for example, in nerve conduits, may be suggesting that the use of these later varieties may not be necessary in all of these applications (2005).
At the moment, while speciality packaging uses up the much greater volume of biodegradable polymers, medical applications account for the higher value, lower volume end of the market. These applications include: surgical fixation, such as sutures, clips, bone pins and bone plates; wound healing, such as temporary skin substitutes and ulcerated wound scaffolding; and controlled drug delivery ( 1996).
Apart from adsorbable surgical sutures, controlled release applications are the most important, and potentially the most versatile, for these materials. The conventional approach to drug release is to incorporate the drug into a polymer matrix and allow diffusional processes to maintain release. However, this simple mechanism is too slow for large molecules, so enhanced release relies on the development of erosional release mechanisms. This problem presents a particular challenge ( 1996).
Implantable devices are regarded as very useful for the delivery of new peptides and proteins. However, although much attention has been paid to the ideal of site-specific drug delivery, there has not been much progress. A more fundamental approach "which addresses adsorption, adhesion and other interfacial processes in biological environments' is required, and particularly the ultimate fate of carrier molecules" ( 1996).
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