**3.1 Structure and classification of PHA**

Recently, Zhang et al. [67] have reported about 150 different types of PHAs. They have various types and structures, this diversity is due to the number of carbon atoms, molecular structure, and chain lengths they have [68, 69]. However, when the carbon chain length is taken into account, three types of PHAs emerge. These are short-chain (scl-PHAs), medium-chain (mcl PHAs), and long-chain (lcl-PHAs) [18, 69, 70]. The most common and known member of the PHA family is poly-3-hydroxybutyrate (P3HB). It polymerizes to give a polymeric chain and consists of (R)-3HB repeating unit (monomer) [69–73]. Poly(3-hydroxybutyrate), poly(4-hydroxybutyrate), poly(3-hydroxyvalerate), and poly(3-hydroxybutyrateco-3-hydroxyvalerate) are known scl-PHAs, and they have three to four carbon atoms, and they usually can be used in food packaging and disposable products [69, 74–76].

In the preparation of these PHAs containing 3-hydroxyvalerate (3HV) or 4-hydroxybutyrate (4HB) monomers, copolymers containing a mixture of four carbon chain length subunits and bacteria synthesizing these polymers with valeric acid are used. The incorporation of HV into the PHB polymers results in a less hard and brittle poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [70, 77]. Medium-chain length PHAs with 6-14 carbon atoms are considered medium chain length. They consist of homopolymers such as poly(3-hydroxyhexanoate), poly(3 hydroxyoctanoate), or P(3HO) [69, 75]. They are synthesized by various bacteria with β-oxidation or novo biosynthesis pathway [78]. mcl-PHAs are flexible and elastic, having low crystallinity with low tensile strength and high elongation-to-break ratios [70]. On contrary to short-chain (scl-PHAs), they are rare, and less used in

process of bioplastics [69]. The difference between the two classes is mainly due to substrate specificity. While an eutrophus can polymerize 3HAs consisting of 3–5 carbon atoms, *Pseudomonas oleovorans* can only use 3HAs consisting of 6–14 carbon atoms in PHA synthesis [79, 80].

## **3.2 Properties of PHA**

Hydrophobicity, melting point, glass transition temperature, degree of crystallinity, and some mechanical properties differ depending on the composition of the monomer [80, 81]. Structural differences in the monomers that make up PHAs cause them to differ chemically as well. For example, poly3-hydroxybutyrate (PHB) has good moisture resistance compared to polypropylene, and barrier properties against gases. They also have a high degree of crystallinity, about 55–80%, and form fine crystals with melting points of about 175°C [82]. Considering its tensile and impact strength, UV resistance, and oxygen permeability, PHB is similar to isotactic polypropylene. This shows that it has the potential to be a packaging material [82]. Having good resistance to hydrolytic attack, PHAs are insoluble in water and resistant to UV [18]. In addition to these properties, they are biodegradable in nature [83]. The main reason why the degradation of PHAs depends on their species and composition is that they have chiral molecules [84]. The type of polymer, its composition, environmental conditions, and microorganism species are effective in the biodegradation of PHAs. It is known that microorganisms produce different PHA-depolymerase enzymes to decompose PHAs and thus they are effective on them [85].

Some thermal and mechanical properties, such as melting temperature, glass transition temperature, crystallinity, tensile strength, and percent elongation, determine the quality of PHA [69]. Some thermal properties of PHAs are crystallization and heat resistance, and they affect the polymer quality [86]. The glass transition temperature (Tg) for the amorphous phase and the melting temperature (Tm) for the crystalline phase are expressed [69]. Increasing the number of carbons in the side chain from one to seven causes Tg to decrease and Tm to increase. As a result, the melting temperature rises from 45 to 69°C [69, 74, 87]. Medium-chain length PHAs are crystalline, have more tensile strength, and have high elongation at break. Due to this feature, they show different mechanical properties as compared to short-chain length PHAs, which have a high crystallinity usually 60–80%. Short-chain length PHAs are more brittle and stiff compare to medium-chain length PHAs [69, 80]. The addition of different monomers or mixing with PHA are methods used to improve the fracture flexibility and elongation of the polymer [69, 88]. One of the purposes of these processes is to reduce the difficulties created by the lack of flexibility of PHAs in terms of food packaging uses [89]. Blending with other polymers can decrease brittleness but it is not enough to be competitive with fossil fuel-based polymers used for food packaging [89]. PHBs have similar water vapor permeability to thermoplastics, such as PVC or PET, and their properties are seen as potential for food packaging applications. The fact that they do not swell and have lower hydrophilicity is seen as an advantage compared to various biopolymers, such as starch and cellulose [89, 90]. In addition to these properties, PHAs also have good barrier properties to some organic solvents. They show relatively high permeability to the moderately polar solvents chloroform, acetone, and toluene, while they have lower permeability to methanol, n-hexane, and isopropyl ether [90, 91].

## **3.3 The microbial production of PHA**

The first discovered PHA, P3HB, is produced by *Bacillus magaterium*, and French researcher Maurice Lemoigne isolated it between 1923 and 1927 [69, 92, 93], also more than 300 species have been reported to produce these polymers. These species include various gram-positive and gram-negative bacteria, fungi, and microalgae [80, 94, 95]. Biodegradable PHAs can be produced by many bacterial species and their different strains [69, 96]. Although many bacterial species are capable of producing a variety of biopolymers, only a few have high productivity and high production rate [69, 97, 98]. Among these bacteria; *Wauteria eutropha*, *Azotobacter* spp. *Bacillus* sp., *Pseudomonas putida*, *Pseudomonas fuorescens*, *P. oleovorans*, *Ralstonia eutropha*, *Cupriavidus necator*, *Burkholderia* sp., *Halomonas* sp., *Haloferax* sp., *Aeromonas* sp., *Thermus thermophilus*, *Hydrogenophagobacter*, *Saxogradanobacteria* de *Saxogradia Erwinia* sp., and recombinant *E. coli* [80, 97, 98]. Buhwal et al. [99] isolated bacteria that accumulate polyhydroxyalkanoates (PHA) from pulp, paper, and wastewater. The isolates *Enterococcus* sp. NAP11 and *Brevundimonas* sp. NAC1 showed maximum PHA production between 79.27% and 77.63%, and they are considered good candidates for industrial production of PHB. Preusting et al. [100] investigated to high concentration of PHA and high productivity with *P. oleovorans* by fed-batch and continuous culture, and they were reached in a continuous mode, and the culture productivity was 11.6 g/l and 0.58 g/(l h), respectively.

Another study by Guo-Qiang et al. [101] found to *Pseudomonas stutzeri* 1317 synthesized a variety of PHAs when grown in glucose and/or fatty acids. The use of recombinant *E. coli* is common in PHA production, as in many areas. Recombinant *E. coli* has been used for PHA biosynthesis, to synthesize the biopolymer to extremely high intracellular levels, and to produce the P(3HB-co-3HV) copolymer. As PHB synthesis with recombinant *E. coli*, it depends on the amount of acetyl-CoA available. Some advantages of using recombinant E. coli for PHA production are rapid growth, high cell density, ability to use a few inexpensive carbon sources [79]. Masood et al. [102] investigated various parameters on the yield of PHAs produced by bacillus cereus. They determined that B. cereus was able to produce both PHAs copolymer and tercopolymer, and it is depending on the type of substrates. Some fungi, such as *Aspergillus fumigatus*, *Saccharomyces cerevisiae,* and *Yarrowia lipolytica,* can be producers [69]. Microalgae have the ability to produce pigments, carotenoids, proteins, enzymes, sugars, fatty acids, polysaccharides, and vitamins, as well as many bioactive compounds. They are ideal for PHA production, but the number of knowns is limited. Some of those are *Nostoc muscorum*, *Chlorella minutissima,* and *Botryococcus braunii* [103]. Cyanobacteria are known to produce PHA by oxygenic photosynthesis, also various studies show that some cyanobacteria have natural capabilities to store PHAs. Although species-specific, some cyanobacteria produce predominantly PHB [104, 105]. It is known to produce PHA in archaea, which needs salt to maintain its growth and can optimally tolerate 5% NaCl (*w*/*v*). First reported in 1970 from the Dead Sea, designated *Halobacterium marismortui* [105, 106] these halophilic archaea produce PHB under nutrient-abundant carbon sources [105, 107]. Different bacteria produce different types of PHAs [108], Fluorescent *Pseudomonas* species are preferred, because they have the ability of mcl-PHA synthases, and can synthesize of PHAs with 6–14 carbon atoms [109]. **Figure 2** shows PHA biosynthesis process scheme.

*Perspective Chapter: Development of Food Packaging Films from Microorganism-Generated… DOI: http://dx.doi.org/10.5772/intechopen.108802*

#### **Figure 2.** *PHA biosynthesis process scheme.*

### **3.4 Carbon sources for the production of PHAs**

The substrates used in the biosynthesis of PHAs, which are synthesized by bacteria through a metabolic process, are generally small molecules. This is due to the rigid cell walls of bacteria. Large molecules cannot be transported into the cell, they must undergo an extracellular transformation by the microorganism or by a chemical process in order to be used. The substrates that can be used are simple sugars (monosaccharides), triacylglycerol, and hydrocarbons. Most microorganisms use simple sugars. Triacylglycerol and hydrocarbon metabolism are less common. Different bacteria for the same substrate can produce PHAs with different compositions [110]. Monosaccharides and disaccharides do not need any hydrolysis to be used in the production of PHA, while polysaccharides cannot be fermented unless hydrolyzed first [110]. It has been reported by various researchers that *Azotobacter vinelandii*, *Alcaligenes latus,* and *Hydrogenophaga pseudoflava* can hydrolyze sucrose, consisting of a glucose unit linked to fructose, extracellularly to glucose and fructose, both of which can then be used for cell growth, and thus have the ability to produce PHA [110–114]; (Jiang et al., 2016). Lactose, a disaccharide composed of galactose and glucose, can be used by microorganisms. Whey is preferred as a source [110].

Whey, rich media that is suited for microbial growth, is obtained by precipitation and removal of milk casein during cheese-making processes [115]. Being a good PHA producer does not mean that the microorganism can produce PHA directly from whey. Vandamme and Coenye [116] stated that *C. necator*, *Wautersia eutropha,* or *Alcaligenes eutrophus* can accumulate up to 80% of its dry weight PHA. *Escherichia coli* cells, which can consume lactose as a solution for microorganisms that can produce high levels of PHA but cannot obtain it from whey, have been modified to express PHA biosynthesis genes from microorganisms that produce high PHA [115, 117]. Starch, the main component of maize, rice, and potatoes, is a polymer of D-glucose and must be enzymatically or acid hydrolyzed to fine maltose and then to glucose for the industrial production of PHAs [110]. Triacylglycerols are the main components of animal fats and plant oils [110], they were considered a cheap and viable source for the biosynthesis of PHAs [102]. In order for bacteria to use triacylglycerols in the production of PHA, they must be able to secrete lipase. The

use of triacylglycerol as a carbon source was determined by Shiotani and Kobayashi in 1993 with *Aeromonas caviae* [110, 118]. Several studies have reported that some gram-negative bacteria can produce PHA using waste glycerol [119], oleic acid [120], or palm kernel oil [121].
