Chapter 2

Polyhydroxyalkanoates: The Application of Eco-Friendly Materials

G.V.N. Rathna*, Bhagyashri S. Thorat Gadgil, and Naresh Killi

Polymer Science and Engineering, CSIR- National Chemical Laboratory, Pune, India

*Corresponding author: rv.gundloori@ncl.res.in

Abstract

Polyhydroxyalkanoates (PHAs) are gaining a lot of attention in several areas of research. Biocompatibility, biodegradability and mechanical strength are the major properties of PHAs that are disclosing previously overlooked potential for changing how polymers may be used across a wide range of applications. In the biomedical arena, drug delivery systems, implants, tissue engineering, development of scaffolds, packing material have adapted uses of this major polymer. Increasing numbers of publications and patents in this area prove its potentiality. This chapter reviews this wide-ranging application of of PHAs with the aim of further illuminating their versatility and future capabilities.

Keywords: Polyhydroxyalkanoate, biomedical applications, agriculture applications, drug delivery, implants and scaffolds

2.1 Introduction

Polyhydroxyalkanoates (PHAs) are a group of polymers obtained from bacteria. Some of the fungi that can count for as much as 80% of the dry weight of the cell [1]. The very first step in developing study of PHAs may have been the observation by Beijerinck in 1888 of PHA as granules inside bacteria. Chemically speaking: these are the polyesters comprising -3,-4,-5 hydroxycarboxylic acids as a basic units. PHAs of bacterial origin are currently in the research limelight due to the excellence of PHAs' excellent biocompatibility, biodegradability, mechanical strength and other fundamental properties. Discovered by Lemoigne in 1926, one of the PHAs, namely PHB (poly (3-hydroxybutyrate)), although less flexible, revealed other characteristics similar to polyethylene and polypropylene as well as properties similar to conventional thermoplastics such as polypropylene [2-3]. Due to their non-elasticity and high production cost, these polymers are used less for certain applications. Nevertheless, PHAs - whose environmental impact is far more benign than petroleum-based alternatives - are being investigated extensively in a bid to displace the petroleum-based alternatives. Therefore, research regarding PHAs mainly focuses on some major frontiers like improvement of the quality of different PHAs by modifying its functional groups or surface properties, copolymerizing, grafting it with different polymers to achieve desired properties, blending or making composites, adding suitable plasticizer or filler so as to make it more useable. In our earlier chapter 11, of book entitled, "Biopolymers: Biomedical and Environmental Applications Vol. I, we have described about the various polymers obtained from microbes. In one of the section 2.1 we have mentioned their origin and how the different PHAs are being synthesized by various microbes [4-5]. Hence, in this chapter we briefly account their occurrence and applications of different PHAs in various areas.

2.2 Natural Occurrence

PHAs are the natural energy and carbon reserves for the organisms facing starvation condition, particularly when availability of other elements like nitrogen, phosphorus or oxygen sources is in short supply [6]. Depending upon the type of organism and growth conditions, the molecular weight of the polymer chains produced by the micro-organisms range from 2× 105 to 3×106Da,. The diameter of the PHAs granules ranges from 0.2-0.5 µm are localized in the cell cytoplasm. They are more refractive and hence clearly seen by simple staining by sudan black under light microscope and as well under fluorescent microscope or phase contrast microscope (Figure 2.1-a, b, c). Different PHAs are being synthesized by various microbes. The mode of synthesis could be natural or biosynthetic. Among various PHAs, the production of PHB is high in cost, inspite, it is extensively studied and well characterized due to its better properties such as, it is easily mold able, spun into fibers, cast into films and is usable just like the conventional plastics. These properties of PHB attracted the attention of researchers so as to discover about 150 different PHAs till date [7-8]. These are the storage material of different bacteria and are produced under different growth conditions. Few of the important PHAs are enlisted in Table 2.1.

Figure 2.1 PHA granules as inclusion bodies in bacterial cells under different microscopes (a) light microscope (b) florescent microscope (c) phase contrast microscope (d) schematic representation of structure of granule in the bacterial cell, PHA core covered with lipid layer and associated proteins.

(courtesy- Kabilan et al. [11] and Masood et al. [12].)

Table 2.1 List of PHAs which are used commonly.

PHA granules are usually found associated with some proteins known as granule associated proteins (GAPs), which surround the main polyester core and consist of enzymes like PHA polymerase, PHA depolymerase, phasins and some regulatory proteins (Figure 2.1d) [9]. These enzymes are supposed to be the controlling factor for formation or utilization of the PHA according to external environmental conditions. Moreover they help the PHA to maintain amorphous nature in the cytoplasm. It is shown that though the nature of isolated PHA is crystalline; in cytoplasm it is present in mobile amorphous phase. Even a mild treatment like centrifugation can remove this coating leading to loss of degradability by depolymerase enzyme and initiating crystallization process. So, this is one of the external factors for initiating crystallinity of PHAs [10].

The mechanical properties of PHB depend on the degree of crystallization, therefore with increase in crystallization the mechanical property increases. It was observed that, the crystallinity of PHB increase on storing at room temperature. Efforts are being made to reduce the crystallinity of PHB, so as to decrease brittleness. Major properties of the PHB can be enlisted as follows:

• Water insoluble and less susceptible to hydrolysis
• Sinks in water and more prone to anaerobic degradation in the sediments
• Resistant to UV but not to acids and alkalis
• Nontoxic and biocompatible so suitable for biomedical applications
• Soluble in chloroform and other chlorinated solvents
• On melting less sticky than other conventional polymers

The different PHAs consisting of various lengths of hydroxy alkanoate as a basic unit are: scl (short chain length) (eg.- PHB, PHV, PHBV), mcl (middle chain length C6-C14 (eg.- PHO, PHN, PHHx, PHHp), and lcl (long chain length) PHAs [13]. Properties of, and behavioral response to, biological surroundings of PHAs depend on the chain length. Accordingly they have been designated as scl, mcl, and lcl. Most scl- PHAs show high crystallinity, but poor mechanical properties, rigidness and brittleness. This makes them difficult to use in biomedical or packaging applications. The exception is scl of P(4HB), which is strong, pliable thermoplastic produced by biosynthetic route. The mcl-PHAs are produced by variety of Gram Negative bacteria and disclose useful mechanical and biosuitable properties. mcl-PHAs show low crystallinity, Tg and Tm. Their tensile strength is low but elongation at break is high [13]. They show very narrow temperature range of elasticity below Tg, above which they loss their flexibility and crystallinity. To attain the required properties, copolymers of the mcl and scl PHAs were tailor made by engineered organisms [14].

2.3 Bio-Synthetic/Semi-Synthetic Approach

The major constraint to use the PHAs on large scale is its high production coast by natural way of microbial metabolism. To reduce the cost researchers are finding alternative methods right from genetic modifications of PHA producing organisms to fermentation techniques using different kind of parameters to get high yield with desired polymer chain length and properties. Accordingly, PHA producing micro- organisms are modified to produce the polymer of interest, for example; polymer type, polymer chain length, hybrid polymers, protein embedded polymers, etc. Along with the genetic modification various fermentation techniques are developed for growing and down streaming the desired product with more superior and accurate techniques of characterization. For example, harvesting time is crucial for any fermentation based industry. Detection of optimum production of PHA in living cell could be done during the fermentation process by a very new direct approach of real time NMR (Nuclear Magnetic Resonance) technique, which is more sensitive than any microscopic or optical detection method. Biopol is one of the commercial biopolymer of PHAs produced on industrial scale by Monsento using fermentation method [15].

Table 2.2 Modified PHAs and their significance.

There are several PHA-producing sources that can be used for industrial-scale mass production of PHAs. PHAs produced by algae have advantages over those produced by bacteria, including: high product yield, flexible response to a wide range of environmental conditions and the potential use of by-products of the process as bio-fuel. The production of PHAs by microorganisms depends on the basic units incorporated in the polymer of raw material [16]. Different plant oils and animal fats are efficiently utilized by the bacteria and are readily translated into unsaturated fatty acids. For example, inexpensive castor oil is...