polyhydroxyalkanoates

POLYHYDROXYALKANOATES 1. Introduction Most human day-to-day activities from domestic, agricultural, and industrial processes depend on the usage of petroleum-based plastics. The global petroleum-based plastic production in 2015 alone was about 300 million tons (1), with the expected increase to 810 million tons by the year 2050 (2). The reasons for the ever-growing demand for plastics are because commodity plastics are durable, cheap, and can be molded easily into any shape making them applicable in a wide range of applications. Plastic products are, however, recalcitrant to microbial degradation and their accumulation causes environmental pollution. Discarded plastic products become mosquito breeding space (3), they contaminate water bodies and soil through the leaching of chemical additives from the plastics and accumulate in the landfills all around the world. Incineration of the plastics causes emission of hazardous gases (4). Huge amounts of styrene and volatile hydrocarbons (benzene, ethylbenzene, xylene) are released into the atmosphere. On the other hand, plastic recycling is both time consuming and labor intensive. Recently, microplastics, such as microbeads and microfibers, released from plastics litters, clothes, and cosmetic products were found in water bodies and reported to endanger the marine biota (5). These problems among others have made synthetic plastics a growing concern, especially in recent years, in the wake of sustainable development goals. Therefore, there is a need for a more environment-friendly plastic that can at least replace some conventional plastics (Fig. 1). Figure 1 shows the drawbacks associated with the use of conventional plastics and the beneficial properties of bio-based polymers. The unique properties of biopolymers have attracted the attention of many researchers to embark on the production, characterization, and application of biopolymers for the replacement of some conventional plastics (6–9). The focus in this article is on one type of biobased and biodegradable polymer that is entirely synthesized by microorganisms. 1.1. Polyhydroxyalkanoates. PHAs are polyesters of hydroxyalkanoate units synthesized as an energy or carbon storage materials by bacterial cells such as Alcaligenes latus, Bacillus megaterium, Cupriavidus necator, as well as some cyanobacteria and archaea. PHAs are accumulated in the bacterial cell cytoplasm in the form of spherical granules of the diameter between 200 and 800 nm. There can be several PHA granules in the cells at any one time. The PHA granules inside the bacterial cells can be seen by using a microscope. Phase-contrast light microscope can be used to observe the intracellular light-refracting PHA granules without applying any dyes (Fig. 2). Special lipophilic dyes such as Nile red, Nile blue, and Sudan black B (10,11) can also be used to stain the granules for further confirmation because of the lipidlike properties of PHA. Transmission electron microscopy (TEM) can show finer details of the distinct PHA granules such as the presence of a membrane-like layer on the granule surface that forms a boundary between the hydrophilic cytoplasm and hydrophobic granule (7,12) as shown in Figure 3. PHA accumulation occurs under unfavorable growth conditions of excess carbon source but limited essential growth nutrient such as nitrogen, oxygen, phosphorous, and/or magnesium. However, there are some bacterial species that can accumulate PHA under normal growth conditions without any nutrient limitations. The accumulated PHA 1 Kirk-Othmer Encyclopedia of Chemical Technology. Copyright  2018 John Wiley & Sons, Inc. All rights reserved. DOI: 10.1002/0471238961.16151225200114.a01.pub3

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POLYHYDROXYALKANOATES

Nonrenewable Petroleumbased plastics

Synthetic

Mostly nonbiodegradable

Environmental problems

Mostly natural

Renewable Mostly biodegradable

Bio-based plastics

Environment-friendly

Fig. 1. The sustainability and disposal of petroleum-based plastics have become a serious problem that necessitate the development of renewable bio-based polymers.

granules are utilized by the microorganisms during starvation or limitation of carbon source supply. The higher the number of PHA granules within a bacterial cell, the longer is its survival. PHA accumulating C. necator H16 was reported to survive up to 600 days under low nutrient condition (13,14). The main functions of PHA are regulation of intracellular energy flow, protection against osmotic shock, ultraviolet irradiation, desiccation, oxidative stress, and channeling carbon compounds to metabolic pathways (15,16). PHA granules generally exist as an amorphous, water-insoluble material in the cell cytoplasm but tend to become

Fig. 2. Phase-contrast light microscope picture of recombinant Cupriavidus necator Re2058/pCB113 cultivated on palm olein in a fed-batch fermentation process showing the presence of light refracting granules in the cells.


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Fig. 3. TEM picture of Cupriavidus necator H16 cultivated on palm olein in a fed-batch fermentation process. The P(3HB) content in the cells is 64 wt% of the cell dry weight. The PHA granules appear white because they allow electron beam to pass through them.

crystalline when they are outside of the cell. The densities of the amorphous and crystalline P(3HB) are 1.18 and 1.26 g/cm3, respectively. The initial study proposed that PHA granule was covered by a 15–20 nm thick, stress delicate membrane composed of primarily proteins and phospholipid, which is useful for its formation, stabilization, and degradation. However, recent study by Bresan and co-workers proved the absence of phospholipid components in PHA granule (17). This means that the PHA granules are enveloped by a membrane-like layer that is composed of only proteins that have special functions in the metabolism of PHA. 1.2. Types of PHA. The classification of PHAs is based on the size of monomers: the short-chain-length (SCL) PHAs contain monomers of 3–5 carbon atoms like 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) whereas the medium-chain-length (MCL) PHAs are made up of monomers containing 6–14 carbon atoms. Examples of common MCL monomers are 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), and 3-hydroxydecanoate (3HD). P(3HB) was the first type of PHA isolated from B. megaterium and characterized in 1926 by Maurice Lemoigne (18) and remains as the most studied PHA to date. However, P(3HB) cannot be used for many applications due to its unfavorable properties like high brittleness and crystallinity (55–80%), low decomposition temperature (270°C), and high melting temperature (180°C). The properties of P (3HB) can be improved through copolymerization by incorporating or increasing the percentage of other MCL monomers. Polymers with improved properties, such as higher elasticity, flexibility, and with lower melting temperatures were obtained through this process and used for the production of surgical devices, matrices, and scaffolds for tissue engineering. The average molecular weight of P (3HB) is between 1 × 105 and 3 × 106 g/mol (18) and with a polydispersity index (PDI) of about 2 (3,19). However, several factors such as the type of microorganism and its growth conditions, the enzyme to substrate ratio, the intracellular concentration of the PHA synthase (PhaC), enzyme specificity, and the level of the PhaC expression determine the Mw of each synthesized PHA (19). 1.3. PHA Biosynthesis. Industrial PHA production requires large bioreactors for a high cell density fermentation. The cell cultivation time is between 24 and 96 h depending on the microorganism. The PHA production begins at the

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Fig. 4. Biosynthesis pathways of P(3HB)/MCL-PHA and intracellular mobilization of accumulated P(3HB). PhaA, β-ketothiolase; PhaB, NADPH-dependent acetoacetyl-CoA reductase; PhaC, PHA synthase; PhaZ, PHA depolymerase.

logarithmic phase of cell growth and ends at the late stationary phase (6). P(3HB) biosynthesis (Fig. 4) starts with the condensation of two molecules of acetyl-CoA to acetoacetyl-CoA, catalyzed by β-ketothiolase (PhaA). Acetoacetyl-CoA is then converted to (R)-3-hydroxybutyryl-CoA by an NADPH-dependent acetoacetylCoA dehydrogenase (PhaB). The (R)-3-hydroxybutyryl-CoA monomers are then polymerized by PhaC in the final reaction into P(3HB). The pathway occurs in over 250 different natural PHA-producing bacteria. PhaC (Fig. 4) is the most important among the three enzymes because it determines the type of PHA synthesized (3,20). More than 40 PHA synthase structural genes have been successfully cloned from different bacterial genera and classified into four classes (I, II, III, and IV) on the basis of substrate specificities, primary structures, and subunit compositions (21–23). All the PHA synthases have one conserved region (containing a cysteine residue), which is the active site necessary for polymerization reaction. Both Class I and Class II synthases have a single and common subunit (PhaC) of 60–70 kDa Mw. However, Class I synthases polymerize predominantly SCL monomers while Class II synthases are more specific toward MCL substrates such as CoA thioesters of various (R)-3-hydroxy fatty acids containing 6–14 carbons. The representative model Class I synthase is that of C. necator. Besides polymerizing 3HB and 3HV monomers, some Class I synthases can also polymerize 4-hydroxybutyrate (4HB) monomer units (24). Class II synthases are represented by that of Pseudomonas oleovorans that polymerizes MCL monomers efficiently. Class III synthases from Allochromatium vinosum are made up of two subunits, namely, the PhaC subunit of about 40 kDa Mw with amino acid sequence similarity of 21–28% to both classes I and II PHA synthases and the 40 kDa PhaE subunit (with no similarity to PHA synthases). Unlike Class II, PHA synthases in Class III prefer


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CoA thioesters of (R)-3-hydroxy fatty acids of 3–5 carbon atoms. Meanwhile, Class IV PHA synthases containing PhaC and PhaR subunits of 40 kDa Mw are found in B. megaterium (25,26). Sugars, plant oils, and fatty acids (27–29) are the most common carbon sources used for PHA production. However, plant oils produce higher cell biomass and PHA yield of about 0.6–0.8 g per gram of oil due to the presence of higher carbon content as compared to only 0.3–0.4 g of PHA/g from sugar substrates (20). P(3HB) production from fructose by C. necator in a 200,000 L bioreactor was first reported in the 1970s (7). Up to 80 wt% PHA content by C. necator was achieved under nitrogen limitation (3). Chee and co-workers (30) reported 70 wt% P(3HB) content in Burkholderia sp. USM (JCM15050) isolated from oil-polluted wastewater. Besides, Budde and co-workers (31) obtained 66 wt% and 71 wt% of P(3HBco-3HHx) in Re2058/pCB113 and Re2160/pCB113 recombinant strains of C. necator, respectively. Both strains were cultivated on palm oil as the carbon source. Another group of researchers have also successfully achieved high cell dry weight (118–126 g/L) and P(3HB) content (72–76 wt%) using wild-type and recombinant strains of C. necator harboring Aeromonas caviae PHA synthase gene phaCAc cultivated on soybean oil (32). High cell density cultures of recombinant C. necator strain grown on palm oil was achieved by Riedel and the team. As high as 139 g/L of CDW and 74 wt% PHA content containing 19 mol% 3HHx were obtained in that study (33). The type of PHA synthesized is generally determined by the bacterial strain and the type of carbon sources supplied (17). For example, a copolymer, poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)], containing 3HB and 4HB was synthesized by C. necator using precursors such as γ-butyrolactone, 4-hydroxybutyric acid, 1,6-hexanediol, and 4-chlorobutyric acid as carbon sources. On the other hand, P(3HB-co-3HV) was synthesized by the addition of either valeric acid or propionic acid to the PHA culture medium (34–36). However, Fukui and co-workers modified the metabolic pathway of C. necator genetically to synthesize P(3HB-co-3HP) from unrelated carbon source (37). Another study by Jeon and co-workers demonstrated that P(3HB-co-3HHx) was synthesized from butyrate as the sole carbon source (38). All the above examples show that various strategies can be used to synthesize a particular type of PHA. The final choice will depend on the availability of cheap carbon source and the regulatory requirements for the utilization of genetically modified organisms. 1.4. Degradation of PHA. The end products of PHA degradation are useful for several biological processes. For instance, PHA is completely biodegradable to water and carbon dioxide under aerobic conditions; as a result, the green plants could utilize the end products for regeneration of carbohydrates. Meanwhile, anaerobic degradation of PHA produces a useful biogas (methane). Degradation of PHA can be divided into intracellular and extracellular degradation. Degradation of the native PHA granules occurs intracellularly in the bacteria that produce the PHA. On the other hand, extracellular degradation of crystalline form of PHA is necessary to generate lower molecular weight water-soluble monomers that can be transported into the microbial cells. In this case, the extracellular PHA is hydrolyzed by depolymerase enzymes secreted by PHAdegrading microorganisms. The monomers are then taken up by the microorganisms as nutrients. Some lipases besides the extracellular depolymerase enzymes are also able to degrade crystalline P(3HB) (39). PHA degradation, enzyme

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activity, and the growth of PHA-degrading microorganisms are affected by environmental factors like pH, temperature, nutrient supply, and moisture level. The enzymes involved in PHA degradation show optimal activity at a pH of 7.5–9.8 (21). PHA depolymerases are specific to either SCL-PHA or MCL-PHA. However, some strains such as Comamonas sp. are capable of degrading both SCL-PHA and MCL-PHA. PhaZd1, PhaZd2, and PhaZa1–PhaZa5 are the known P(3HB) depolymerases in C. necator H16. Paucimonas lemoignei is a unique and extensively studied PHA-degrading bacterium because it produces up to seven extracellular PHA depolymerases (PhaZ1–PhaZ7) (40).

2. Industrial Production of PHA The research on PHA has been ongoing since the discovery of P(3HB). At present, the potential of PHA is well known and many industries are either interested or trying to develop cost-effective methods for the large-scale production of PHA. For instance, waste was successfully converted to PHA by a food and wastewater treatment plant. Up to 65 wt% PHA content was advantageously produced from volatile fatty acids by sludge fermentation (41). Furthermore, PHA composition could be altered to suit the desired application. Murugan and the team reported different thermal characteristics in copolymers containing varied 3HHx molar fractions (42). Nevertheless, there are still challenges that need to be overcome by the PHA industry in order to make PHA a sustainable and economically feasible material. 2.1. Advantages of PHA. PHAs are important and desirable sustainable thermoplastic materials having good attributes: 1. Nontoxicity and biocompatibility, hence suitable for food packaging and medical applications. 2. Water insoluble and anaerobically biodegradable beneath water. 3. Resistant to hydrolytic degradation. 4. High temperature stability of more than 100°C 5. Produced from renewable resources. 6. Enriches soil quality and microbial diversity because of its biodegradability (43). Despite all the advantages offered by PHA, there are numerous challenges faced by the PHA industry at the current state. 2.2. Challenges of Industrial PHA Production. Industrial production and commercialization of PHAs are still struggling to compete with conventional plastics in terms of price and to a certain extent properties. About 45% of production cost arise from the use of high purity substrates like sugars and large quantities of solvents for polymer extraction (44). These factors among others have prevented the successful operations of several biopolymer companies in the past. W. R. Grace and Company in the United States was the first to embark on commercial application of PHA in 1959. However, the firm was closed down due to lack of purification systems and low production efficiency. The


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commercialization of P(3HB-co-3HV) synthesized from C. necator by Imperial Chemical Industries Ltd. UK, also failed in 1970 and the technology was bought over by Monsanto in 1996 and then sold to Metabolix, Inc. The major setback in PHA commercialization is its high price compared to synthetic plastics. For instance, P(3HB) was sold at a high cost of €10/kg in 2006 and at €1.50/kg in 2010 by MirelTM Company. Companies like Kaneka Corporation in Japan, Biomer Inc. (Germany), Biomatera Inc. (Canada), Industrial S.A (Brazil), and Mitsubishi gas chemical in Japan have been producing PHA from vegetable oils and sugars at pilot or commercial scale and the PHA was priced between US$4.96 and 6.06/kg compared to US$1.32–1.92/kg for polypropylene and polyethylene (44,45). 2.3. Strategies to Improve the Production of PHA. Currently, there is an increasing demand for bio-based and biodegradable polymers in most developed countries. Some companies are also beginning to incorporate such “green” polymers into their products or investing in the research and development of ecofriendly materials. It is expected that the global PHA market will expand to US $93.5 million by 2021 (44). Hence, efforts are ongoing and various strategies were taken toward reducing the PHA production cost and enhancing its commercialization. The major approaches include the utilization of cheap carbon feedstocks like industrial and agricultural waste materials such as sugarcane bagasse and molasses, lignocellulosic, cheese whey, palm oil mill effluent, transgenic (45) plants, and waste animal fat (46). This approach has enabled the successful production of P(3HB) and P(3HB-co-3HHx) for applications in disposable products, aqueous dispersions, fibers, and nonwoven materials. High productivity of P(3HB), P(3HB-co-3HV), and P(3HHx-co-3HO) for medical and biomedical applications were also achieved in 2004. A breakthrough in P(3HB) production was achieved through the combination of bio-based polymers and biomass energy from switchgrass in 2008 (47). Second, genetically engineered bacterial strains were used to reduce the long generation time and low optimal growth temperature associated with natural PHA-producing bacteria for the production of PHAs with better properties. For example, P(3HB-co-3HHx) was produced from palm kernel oil using C. necator harboring the Aeromonas caviae PHA synthase gene (3). Other copolymers containing 3HB and 3HO monomers were produced by recombinant strains of Escherichia coli from soybean oil. In fact, up to 80–90 wt% PHA accumulation was achieved by recombinant E. coli harboring C. necator PHA synthase gene (48). The presence of compounds, such as glycogen, and proteins alongside PHA granules in bacterial cells may limit the PHA accumulation space. Hence, cellular modification by morphology engineering, such as gene deletion or expression, changing the cell size and morphology, is required to enhance PHA accumulation space, high cell density, and accelerate cell growth. Wang and the team have successfully achieved more than 100% P(3HB) accumulation by engineering a rod-shaped E. coli to filamentous shape (49). High production cost is associated with the current use of pure cultures in PHA production since it requires the use of energy for sterilization. Hence, utilization of mixed microbial culture could be another approach that would help to reduce the PHA production cost. Mixed microbial cultures are groups of unknown microbial populations that carry out specific cellular reactions and selected by the biological operational conditions. The system prevents the occurrence of some cellular primary metabolism in the selected cultures for high intracellular PHA storage capacity. Hence,

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the overall cost of PHA will be lowered here because volatile fatty acids and waste streams are used as carbon feedstocks in PHA production. This is a cost-saving approach since mixed microbial consortia would be grown in an open system without the need for sterilization. The method is carried out in two separate steps: (i) selection of high PHA storing microbial population in a sequential batch reactor and (ii) maximum PHA accumulation step (50). The concept of mixed microbial culture was adopted by Chua and the team. In their study, metropolitan wastewater in a sequential batch reactor was enriched with high PHA-accumulating microbes to achieve 30 wt% PHA content and 0.050 g PHA/g CDW/h (51). Other studies reported 56–65 wt% PHA content from mixed microbial cultures (52,53). Another important approach taken was the application of efficient fermentation strategies like fed-batch or continuous culture conditions for optimized PHA production. Fed-batch fermentation involves the cultivation of cells on sufficient nutrients for cellular multiplication, followed by the nutrient limitation stage for high cell density (33,54) and maximum PHA accumulation (50). In comparison to batch fermentation, fed-batch process gives higher PHA yield because it allows the control of the medium compositions. C. necator, Protomonas extorquens, and Protomonas oleovorans are the most suitable types of bacteria for fed-batch fermentation since they require excess carbon but limited amount of nutrient for growth and PHA accumulation. A mutant strain of Azotobacter vinelandii besides A. latus and recombinant E. coli could also provide higher PHA yield in continuous fermentation where both the growth and PHA accumulation occur simultaneously without the limitation of any nutrient (20).

2.4. Strategies to Improve the Purification and Recovery of PHA. The identification of suitable approaches for the extraction and purification of PHA granules from other cell components is an important aspect in PHA production. The proteins and nucleic acids associated with PHA granules are contaminants at this stage and must be removed. The impurities give PHA an unpleasant odor during the melting process. Hence, the need to disrupt the cells and get rid of the protein layer. As demonstrated in Figure 5, the major steps for PHA recovery are cell harvesting, pretreatment, and PHA extraction and purification. Cell

Harvested biomass

Pre-treatment

PHA extraction

Purification

Lyophilization

Solvent

Thermal drying

Supercritical fluid

Washing

Salt treatment

Chemical Enzymatic

Drying

Mechanical Physical

Precipitation

Polishing

Biological

Fig. 5. Downstream processes of PHA production.

Pure PHA


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harvesting is the retrieval of biomass from the cultures through centrifugation or filtration. The main objective of biomass pretreatment is to simplify PHA retrieval from cell biomass. The extraction step uses different techniques ranging from chemical, mechanical, physical to biological means to recover the PHA. The polymer is further purified by washing, grinding, and polishing at the last step. The solvent extraction method was first demonstrated by Lemoigne (1923–1951) and remained the commonly used method of PHA recovery in the laboratory. It involves the use of chloroform to break the cell membrane and subsequent dissolution of the PHA. Mixture of chloroform/methanol and dichloromethane/ethanol were used for dissolution and precipitation of PHAs and highly purified PHA was obtained with no reduction in molecular weight. Other effective solvents are simple halogenated and nonhalogenated solvents such as methylene chloride, chloropropane, and 1,2-dichloroethane. Although effective, these solvents are not suitable for industrial applications because the solvents are costly, hazardous, and required in large volumes (55). Sodium hypochlorite is an effective solvent to recover PHA with high purity, but it results in high PHA degradation and about 50% of molecular weight loss (56). Other methods are the enzymatic digestion (57) of the cell wall using common enzymes like protease and lysozyme for high PHA recovery with little or no Mw reduction. But this method is rather expensive for commercial scale production of PHA. Combination of mechanical cell disruption and biological methods of PHA extraction may result in a cost-effective and environment-friendly process. Biological method of PHA recovery was first reported by Kunasundari and coworkers (58). In their study, the freeze-dried cells of C. necator were fed to rats in the laboratory. The animal excreted white feces that was composed of 82–97 wt% P (3HB). Its molecular mass of 930 kg/mol is similar to the chloroform-extracted P (3HB) (950 kg/mol) (58). The method is easy and environment-friendly because laboratory production of PHA in kilograms was possible without the use of any solvent or expensive instruments. However, it is not practical to use rats in largescale production of PHA. Therefore, other more suitable animals were evaluated. The use of mealworm (Tenebrio molitor), the larvae of darkling beetles was found to be a potential biological system. Mealworms readily consumed the freeze-dried C. necator cells and the PHA granules were recovered from the feces. Close to 100% purity was obtained upon washing the PHA granules with water and sodium dodecyl sulfate with heat applied (59). Further scale-up studies are necessary to establish the economic feasibility of biological recovery of PHA.

3. Commercialization of PHA In general, the future of PHA can be very promising if the necessary policies and regulations are in place. PHA has been used to partly substitute some synthetic materials and this is currently being tested by various industries, especially in the pursuit of a greener environment. In order to make PHA more competitive economically, further efforts are necessary. 3.1. Economic Value of PHA. In this section, cost analysis studies are reviewed. One of the limitations of PHA is its high cost that needs to be reduced to make it more economically attractive. The major factors contributing to the

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production cost would be the PHA productivity of bacterial fermentation, PHA content in the cell, the cost for carbon substrate, and PHA yield and recovery method. As there is a “domino effect” in the chain, either one of the process will affect the cost of PHA in one way or another. The examples provided here are some of the initiatives taken by different groups of researchers in the attempt of analyzing the cost involved for the production of their desired PHA. As different groups produced different type of PHA, the cost analysis was merely based on the processes involved and thus no comparison of cost between PHAs were conducted. This section describes the overview of the cost analysis of the PHA produced previously. In 1997, the economic values of MCL-PHA from octane was determined. The study made a comparison on the MCL-PHA production systems from the aspect of volumetric productivity, cellular PHA content, and the polymer yield on carbon substrates. Based on the economic framework for the tabulation of PHA production cost, it showed that the cost for large-scale production of octane-based MCL-PHA (more than 1000 tonnes/year) could be below US$ 10/kg (60). In the same year, Choi and Lee reported the price of annual production of purified P(3HB), in which the P(3HB) was produced using C. necator and was further recovered through surfactant-hypochlorite digestion. For the production of 2850 tonnes of purified P (3HB)/year, the estimated price was US$5.58/kg, which is the lowest. In addition, the price of P(3HB) can be further reduced to US$4.75/kg when the production scale was increased to 1 million tonnes/year. It was suggested that the production cost could be lowered with the use of agricultural wastes such as whey and molasses (61). A proficient and cost-effective recovery of P(3HB) from E. coli using sodium hydroxide (NaOH) was then reported. The fermentation process yielded P (3HB) with the concentration of 157 g/L, 77 wt% of P(3HB) content, and productivity of 3.2 g/L/h, coupled with the recovery method of NaOH digestion gave rise to the production cost of P(3HB) to be as low as US$3.66/kg, which was 25% lesser than the cost of P(3HB) recovered from surfactant-hypochlorite digestion method (62). In 1998, the final production cost of P(3HB) was calculated according to the fermentation performance. It was proposed that direct fixed capital-dependent costs decreased with the increase of productivity. When the fermentation process gave rise to high P(3HB) concentration (98.7 g/L), with 88.3 wt% of P(3HB) content and favorable productivity (4.94 g P(3HB)/L/h), the final P(3HB) production cost was only US$2.6/kg P(3HB) (63). Apart from that, the overall cost of PHA production was affected significantly by the cost of carbon source. Given a scenario in which a cheaper carbon source such as hydrolyzed corn starch (US$0.22/kg) rather than glucose (US$0.5/kg) was used for the P(3HB) biosynthesis using E. coli with the same fermentation efficiency, the cost of production will reduce to US$3.72/kg as compared to US $4.91/kg when glucose was used (64). In 2003, Akiyama and colleagues estimated the production cost of large-scale fermentation of P(3HB-co-5 mol% 3HHx) copolymer using recombinant strain of C. necator, with soybean oil as the substrate (65). The cost of annual production of 5000 tonnes of P(3HB-co-5 mol% 3HHx) was forecasted to be as much as US$3.5–4.5/kg (according to the assumption of previous production performances). Meanwhile, the estimated cost for P(3HB) homopolymer production with the similar scale was predicted to be US$3.8–4.2/kg (65). Due to the growing biodiesel production and glycerol surplus, crude glycerol


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has been used as an alternative carbon source for PHA production. The raw material cost for crude and refined glycerol was US$0.118/kg and US$0.149/kg, respectively (66). By cultivating C. necator on crude glycerol and refined glycerol, the cost of PHA production was US$0.362/kg PHA and US$0.484/kg PHA, respectively. As a result, crude glycerol seems to be the most competitive substrate. Furthermore, both of these substrates gave a better yield. Apart from that, the use of waste cooking oil as the substrate was also economical in which the cost of PHA production was estimated to be US$1.18/kg PHA (67). In order to be competitive with the petroleum-based materials, the production of PHA needs to be optimized in various aspects. At the current stage, the cost of carbon source has contributed to greater than 50% of the raw material cost (68). Thus, the alternatives would be to use cheaper and renewable carbon sources. This will provide a cheaper technology for PHA production. In addition, the recovery and purification cost should also be taken into consideration. Ultimately, PHA can be sustainable and economically attractive if the cost of the feedstock and processes can be reduced. 3.2. Application of PHA. Even though PHA has faced numerous challenges, it has been produced to meet the industrial needs. PHA as described earlier, have shown great potential for various application due to its desirable properties. This has attracted the small and middle-sized companies to commercialize PHA. In the past, Biopol® (69), NodaxTM (70), and DegraPol® (71) managed to be commercialized in the market despite the challenges faced by the PHA industry. In addition, it was projected that the global market for PHA will increase from US$73.6 million in 2016 to US$93.5 million by 2021. Several companies are actively establishing PHA production such as Metabolix Inc. (U.S) (www .metabolix.com), Kaneka Corporation (Japan) (http://www.kaneka.co.jp), Bio-On Srl (Italy) (http://www.bio-on.it/), Shenzhen Ecomann Biotechnology Co., Ltd (China) (www.ecomann.com), and PolyFerm Canada, Inc. (Canada) (http://www .polyfermcanada.com) (72). At the current stage, PHA has been applied in high potential areas ranging from medical field to industrial application. These applications are further described in the following sections. Medical Application. In order to be used as a material for medical application, the PHA-based material has to be biocompatible and does not trigger any serious immune response when exposed to soft tissues or blood circulation of the host as well as during PHA degradation in the host’s body. In general, PHAs are degraded through the activity of natural nonspecific esterases and lipases (73). The degradation of PHA matrices in the tissues could also help in the release of bioactive compounds (antibiotic or antitumor drug) in the host over time. By altering the properties of the polymer through the variation of the type of PHA with different monomer side chains, it is possible to fine-tune the kinetics of dosing a compound from the PHA matrix (74). Some of the PHA monomers, such as 3HB, are a natural metabolite in human body associated with ketone body formation. Therefore, P(3HB) and other types of PHA are in general compatible and most likely will not result in an immune response in the host organism. One of the requirement of a biomaterial is the need to be sterilized. Sterilization of PHA-based materials has also shown that their molecular weight, tensile strength, and other properties were not affected (75). Such properties are essential especially if PHA is going to be used for surgical implants and

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sutures. In addition, PHA is also appropriate as scaffolding material in tissue engineering due to its biocompatibility. PHA has recorded some interesting success as a scaffolding material in tissue engineering. Bone defects were treated using the blends of P(3HB) and hydroxyapatite as scaffold. In that study, it was demonstrated that P(3HB)-based materials could generate a stable and promising bone tissue adaptation without any negative chronic inflammatory signs after the implantation. It was noteworthy that up to 80% of the implant surface was successfully formed near to the new bone (76). Besides, Kose and team did an in vitro investigation on bone formation by culturing the rat marrow stromal osteoblasts in the biodegradable, macroporous P(3HB-co-3HV) matrices for a duration of 60 days. It was discovered that the osteoblasts could propagate inside the matrices and resulted in mineralization subsequently. Thus, P(3HB-co-3HV) was shown to be a favorable polymeric matrix material for bone tissue engineering (77). Previous studies have shown that PHA films were favorable for propagation and connection of tissue cells; such as the adherence of NIH 3T3 fibroblast cells on PHA membranes (73), proliferation of mesenchymal stem cells on terpolymer poly (hydroxybutyrate-co-hydroxyvalerate-co-hydroxyhexanoate) [P(3HB-co-3HV-co3HHx)] (78), and attachment of mouse connective tissue fibroblasts on poly(3hydroxy-10-undecenoate) and poly(3-hydroxyoctanoate-co-3-hydroxy-10-undecenoate) (79). A copolymer of polyglycolic acid (PGA) and P(3HB) has also been used to generate pulmonary artery scaffolds and pulmonary valve leaflets in sheep (80). In another study, Novikov and colleagues showed that neuronal survival and regeneration in rats that have spinal cord injury could be supported via a graft matrix that was made up of the P(3HB) implants as a carrier scaffold coupled with fibronectin, alginate hydrogel, and Schwann cells (81). In 2003, Deng and colleagues successfully showed that P(3HB-co-3HHx) stimulate the building of extracellular matrix of rabbit articular cartilage chondrocytes in vitro. Their findings revealed a positive effect of P(3HB-co-3HHx) on extracellular matrix construction that was supported by the increased mRNA level of type II collagen of chondrocytes seeds on the P(3HB-co-3HHx) scaffolds (82). A similar study was also conducted by Wang and colleagues. The use of unblended P(3HB-co-3HHx) was effective in cartilage repair, whereby the P(3HBco-3HHx) scaffold with a pore size of 100 μm and a porosity of 90% was able to provide a favorable environment for the proliferation, migration, and differentiation state maintenance of the chondrocytes, both in vitro and in vivo. Full thickness cartilage repair in the rabbit articular cartilage defect model was successfully achieved over a period of 16 weeks, using the engineered cartilage constructs that were made of scaffolds seeded with the allogeneic chondrocytes (83). In another study to investigate the biocompatibility of PHA, the fibers made from P(3HB) or P(3HB-co-3HV) demonstrated a comparable tissue reaction to the implants made from silk or catgut, which are currently used in the surgical procedures. The functional characteristics of P(3HB)-based surgical threads were found to be as equally good as the conventional surgical materials (silk and catgut) (84). PHA is used in sutures due to its remarkable tensile strength. Surgical material made from poly(3-hydroxybutyrate-co-4-hydroxybutyrate) was shown to be stronger than the polypropylene sutures, 545 MPa and 410–460 MPa, respectively (85). Due to its excellent mechanical properties, Tepha Inc. (www.tepha.com)


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has manufactured several medical devices from PHA, including surgical meshes and films. One of the famous products is TephaFLEX® suture, which was fabricated from P(4HB) (86). It should be noted here that Tepha Inc. is the first company to be awarded FDA approval for its PHA-based sutures. Besides, P(3HB) and P(3HB-co3HV) sutures could also aid in the healing of muscle–fascial wounds (87). PHA can also be integrated in the drug delivery system driven by its biocompatibility and biodegradability in the host. Previous studies have shown that rifampicin and tetracycline were incorporated into P(3HB-co-3HV) and P (3HB), respectively, in an attempt to prepare a drug delivery device (88,89). However, the deliverance of antibiotics from P(3HB) microspheres was generally too rapid and hence, PHA with lower crystallinity was preferred for a timely release of drug into the surrounding tissue (90). In another study of antibiotic delivery, gentamycin was assimilated into the discs made of P(3HB-co-3HV) in which the deliverance of the drug was monitored for a period of time. The increment of the 3HV molar fraction from 8 to 12% showed that the release of the integrated antibiotic was elevated. In addition, there was no negative effect of the formulations on the blood cell integrity (91). Pouton and Akhtar suggested that the implementation of P(3HB) and P(3HB-co-3HV) as a drug release matrix was dependent on the design of the proper blends with other biocompatible polymers by taking into consideration the blending techniques that could control the porosity, erosion rate, and drug release kinetics (92). To this end, the application of PHA in the medical field seems to be the most promising in terms of the material cost. Agricultural Application. PHA materials have also been evaluated for their application in the area of agriculture in the form of mulching films, oneseason irrigation tubes, biodegradable flower pots, and biodegradable matrices for the controlled release of plant growth factors, including the nutrients and fertilizers as well as for pesticides and herbicides. Holmes has demonstrated that the controlled release of insecticides could be achieved by using P(3HB-co-3HV) (93). In this case, the insecticides were integrated into P(3HB-co-3HV) capsules and were seeded together with the crops. The release rate of the insecticides was dependent on the level of pest activity since the disintegration of the polymer by bacteria would be affected by the similar ecological environment as the soil pests (93). PHAs were also tested for enhancing the nitrogen fixation in plants in the form of bacterial inocula. The field experiment conducted in Mexico on wheat and maize has shown improved uniformity in growing the crop yield through the incorporation of peat inocula that were fixed with PHA-rich Azospirillum cells (94). Cirujeda and co-workers compared the use of black polyethylene film and biodegradable mulch for controlling the weed and yield of tomato in various parts of Spain (95). Their findings showed that biodegradable mulch resulted in good weed control and better harvest of tomato crop, indicating that biodegradable plastics are in principle a possible alternative to polyethylene although its cost remains as a challenge from the economic perspective. The formulations of nitrogen fertilizer (urea) loaded in a degradable matrix of P(3HB) in the form of films, pellets, and coated granules were investigated by Volova and co-workers (96). It was observed that the nitrogen release into the soil corresponded to the degradation of the polymer. The amount of nitrogen loaded into the carrier as well as the geometry of the carrier have a direct influence on the nitrogen release. The result showed that nitrogen release can last for 30 days or longer and that release

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rate could be controlled by varying the fabrication technique employed. P(3HB) with urea formulations have a positive effect on the soil microbial community. The use of embedded urea showed beneficial impact on the growth of creeping bentgrass (Agrostis stolonifera) and lettuce (Latuca sativa) (96). The same group of researchers has recently published their work on the slowrelease preparations of the herbicide metribuzin (MET) implanted in the polymer matrix of degradable P(3HB) in the form of films, microparticles, pellets, and microgranules. The MET release could be controlled by using different methods of making the formulations and varying the MET loading. The MET accumulation in the soil occurred progressively as the polymer started to degrade. The results of this study showed that all P(3HB)/MET formulations exhibited herbicidal activity. Therefore, P(3HB) can be viewed as a potential material for making slow-release formulations of the herbicide metribuzin for soil applications (97). All the above examples show that PHA has great potential as a suitable matrix for the development of slow or controlled release systems for various fertilizers and chemicals. This is not limited to soil applications only but can also be potentially used in water bodies since PHA degrades in almost any ecosystem that has microbial activities. Industrial Application. Due to the improved performance of PHA, especially its mechanical properties, including thermal stability and elasticity (depending on the molecular composition of the PHA), it has been demonstrated as a substitute for some petroleum-based commodity plastics. Prototypes such as single-use bottles for cosmetics and shampoo, cups, containers, and bags have been produced using PHA materials. Besides, PHA-coated materials such as paper cups and trays for holding foods and drinks were also developed. The most used PHA for packaging purposes are P(3HB) and P(3HB-co-3HV) (98). Besides, fibers and nonwoven fabrics, which are used for diapers and sanitary napkins, were also manufactured (98–100). The potential of P(3HB-co-3HV) to be used as hot melt adhesives for bookbinding, bag ending, as well as for case and carton sealing was also evaluated (101). The potential of using PHA in the cosmetics and skin care industry has also been studied. PHA cast films of P(3HB), P(3HB-co-3HV), and P(3HB-co-3HHx) were investigated as prospective facial oil blotting material (Fig. 6). In that study, all the PHA cast films were evaluated for its oil absorbability, retention, and oil-indication properties. Interestingly, all these PHA films showed similar

Fig. 6. The ability of PHA films to absorb oil can be used to develop eco-friendly cosmetic films for the removal of facial oil. (a) Before oil absorption. (b) After oil absorption.


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oil absorption characteristics and were able to absorb sebum on the skin even without the incorporation of lipophilic additives like zinc stearate and mineral oil, which are the additional components in commercial facial oil blotting films. In addition, the PHA films were able to absorb oil efficiently even after they were subjected to washing with detergent, and thus highlighting the reusability of these films (102). In the aspect of coatings and paints manufacturing industry, various applications are generally derived from resins that are made up of synthetic polymers such as polyacrylates, polyurethanes, and acid- or epoxy-functionalized polyesters. These applications include printing inks and primers for home decoration and automotive coatings. Unsaturated MCL-PHAs have been applied for manufacturing high solid alkyd-like paints. Application of MCL-PHA as the polymer binder in paints could help to decrease the viscosity of the currently applied synthetic alkyd resins and thus reduce the amount of organic solvent that is needed in these paints for optimal performance (103). In the past decade, PHA has also been evaluated as the solid substrate for the purpose of denitrification of water and wastewater. It was reported that PHA serves as solid matrices appropriate for the development of microbial film besides being the constant source of reducing power for denitrification (104). Due to its hydrophobic property, PHA has also been used for dye removal study. Decolorization of Batik dye wastewater with the incorporation of solar photocatalytic titanium dioxide (TiO2) nanoparticles immobilized on P(3HB) film was investigated. It was shown that the decolorization of the real industrial Batik dye wastewater was completed within 3 h by the photocatalytic films. In addition, the chemical oxygen demand (COD) of the wastewater was reduced by 80% (105). Besides, electrospun P(3HB) film was also tested for its role as a dye adsorbent agent. As much as 78% of dye was adsorbed from a 30 μM solution of malachite green dye, indicating that the electrospun P(3HB) has an excellent dye adsorption potential. Further improvement on the film was achieved by the incorporation of TiO2 photocatalyst to form a double action dye treatment system engaging both the adsorption and photocatalytic degradation simultaneously. Within 45 min under solar irradiation, the electrospun P(3HB)-50 wt% TiO2 was able to decolorize the malachite green solution completely, which corresponded to 58.7% COD removal (106). Apart from potential application in wastewater treatment, PHA has also been incorporated with other fibers such as kenaf to produce biocomposite (Fig. 7), which can be used in applications that require slow- to long-term biodegradation with good mechanical properties (107).

4. PHA from the Sustainability Point of View The concept of sustainability is acknowledged globally as an important issue because of the increasing awareness for the environment, to protect the planet and for the future of human civilization. The United Nations has listed 17 goals to transform our world. The development and use of biodegradable plastics fit nicely into some of these goals. To understand clearly the sustainability of PHA, it is necessary to look at it holistically by accessing the life cycle of PHA in a quantitative manner.

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Fig. 7. Example of biocomposite produced from mixture of kenaf and biologically recovered PHA. The sticks measure 120 mm in length, 12 mm in width and 3 mm in thickness.

4.1. Life Cycle Assessment (LCA) of PHA. There are two main challenges faced by the industries that want to produce PHA. First, it is the cost competitiveness of the bio-based polymers versus the petrochemical-based polymers (polystyrene, polypropylene, polyethylene). Second, it is about the environmental friendliness of the bio-based polymers. Is PHA really environment-friendly in view of fossil fuel consumption? This question was raised by Gerngross (108). He concluded that biopolymers will use more agricultural land, combust more fossil fuel in the production, cause more emission of greenhouse gasses, and more water consumption that eventually leads to more eutrophication of water as compared to the processes based on fossil raw materials. Furthermore, degradation of bioplastics leads to carbon dioxide and methane emission, which are greenhouse gasses. In this case, environment-friendly does not necessarily mean that the material is produced from natural or renewable resources or whether it can be degraded naturally. Such criteria might not be sufficient to be coined as environment-friendly material. In order to evaluate how “green” is PHA, it is vital to consider the life cycle of PHA. According to Rebitzer and colleagues (109), there is a need for the establishment of methods to evaluate the impact of human activities toward the environment while manufacturing the goods and products in order to ensure sustainable development. Acidification and eutrophication, exhaustion of resources, toxicological effects on human health and ecological unit, climate change, and stratospheric ozone depletion are some of the major impacts. As a result, LCA is recommended for assessing the prospective environmental impacts related to a product by examining its life cycle (109). The environmental impacts of every stage of a product’s life beginning from its raw nature till the end product is assessed. LCA can be divided into cradle-to-gate (assessment of a partial product life cycle from


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raw material to the factory gate) and cradle-to-grave (full life cycle assessment from raw material to use phase and disposal phase) (110). The purpose of LCA is to make a thorough assessment on the environmental impact of a product based on the cause and effect of the material flows to the environment (111). In the past years, LCA of PHA have been carried out by a few group of researchers. According to Yates and Barlow, the nonexistence of large-scale commercial facilities has resulted in LCA studies to be based on virtual reality and estimations from laboratory and small-scale facility (112). In 1998, Heyde reported that different fermentation efficiencies and substrate supply will give rise to different total energy efficiencies of the polymer production (113). It was shown that the P(3HB) system requires less energetically valuable resources than petroleum-based polymers under advantageous technological boundary conditions (the ecological considerations and technological opportunities during the use and production of a material). However, the energy demand of the P (3HB) reaches another level under the worst conditions. In terms of waste management, biodegradable polymers seem to contribute to global warming significantly than the petroleum-based polymers under the conventional waste management conditions (70% landfill, 30% incineration). On the other hand, the global warming potential could be lowered if the waste polymer is either incinerated or composted. In addition, there will be minor contribution to global warming if the polymer is produced from renewable substrate source (113). Meanwhile, there are a few studies that showed that P(3HB) can have lower global warming potential (GWP) and nonrenewable energy (NREU) than the petroleum-based plastics. The substrates for P(3HB) production ranges from vegetable oil, waste animal fats, and waste cooking oil to sugar and starch. Different starting material will yield different life cycle of the PHA. A study pertaining to the life cycle inventories (LCI) on the amount of energy used and the amount of carbon dioxide released for the large-scale fermentation of P(3HB-co-5 mol% 3HHx) from soybean oil were calculated and compared with P(3HB) that was produced from glucose. It was shown that the LCI figures for the amount of energy used and the amount of carbon dioxide released were lower for the soy-based PHA copolymer as compared to the glucose-based P(3HB) homopolymer. Furthermore, these values were significantly lower than the conventional petroleum-based polymers (65). Kim and Dale have attempted to estimate the environmental performance of corn-based PHA, starting from the agricultural production all the way through the PHA fermentation and recovery process with the concept of cradle-to-gate analysis (114). Their findings revealed that global warming associated with PHA produced from grain was 1.6–4.1 kg-CO2 eq./kg. On the other hand, the PHA produced from corn grain and corn stover showed –0.28 to –1.9 kg-CO2 eq./kg. The figure was dependent on the PHA fermentation technologies used, and the current result showed that PHA produced from corn grain seemed to have lesser environmental benefits than polystyrene. This could be due to the environmental burdens related with corn cultivation, in which it contributed to photochemical smog, acidification, and eutrophication. Nonetheless, if the PHA is produced in the integrated system (utilization of the corn stover as the starting material and incorporation of corn grain), it gave a favorable impact compared to polystyrene (114). In 2008, the authors expounded further on the impact of corn grain-based

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P(3HB) by evaluating its life cycle energy and greenhouse gas profiles. As a result, they found out that P(3HB) produced from corn grain was more advantageous than petroleum-based polymers. Sustainable practices in corn cultivation such as the use of no-till farming and winter cover crops were anticipated to lessen the environmental effects of P(3HB) as much as 72% (115). A similar study was conducted by Yu and Chen (116). They quantified the release of greenhouse gas and expenditure of fossil energy for the production of bioplastics based on the cellulosic biomass refinery using cradle-to-gate LCA. In their work, it was discovered that PHA could reduce the global warming potential up to 80% because as low as 0.49 kg CO2-eq. was being emitted when 1 kg of resin was produced in comparison to petroleum-based plastics that generated 2 3 kg CO2-eq. The fossil energy requirement per kg of bioplastics and petroleum-based plastics was 44 MJ and 78–88 MJ, respectively (116). Besides, a comparative LCA and financial analyses for PHA and biogas production from mixed culture were carried out based on the wastewater treatment industry. It was noteworthy that the mixed culture PHA was desirable in the biogas production for treating the industrial effluent. In addition, mixed culture PHA was economically more attractive than using pure culture to produce PHA. This approach showed lower environmental effects as compared to high density polyethylene (HDPE). It was concluded that mixed culture was more viable for PHA production and effective in treating industrial wastewater. Nonetheless, the financial and environmental costs can only be reduced through the optimization of the process (117). Most of the previous LCA of PHA was mainly focusing on the environmental impacts related to global warming and fossil fuel depletion. In one of the examples, a detailed comparison between polyethylene, polypropylene, and biologically based P(3HB) was carried out. In that study, the cradle-to-gate LCA of P(3HB) production was evaluated by assessing the emission of carbon dioxide and other effects to the environment. In terms of energy requirements, it was shown that P (3HB) was better than polypropylene and marginally lower than polyolefin. However, the impact of polyethylene on acidification and eutrophication was shown to be lower as compared to P(3HB) (118). Furthermore, polyethylene does not biodegrade and serve as a carbon source for microorganisms. In this case, it was obvious that each material has its own LCA and at certain step, one material could be better than the other or vice versa. In another case study, a bio-based bag that was produced from PHA in the United States was compared with a polyethylene plastic bag produced in Singapore in terms of their environmental impacts. Based on the cradle-to-gate LCA study, it was shown that when the production chain was supplied by naturally occurring hydrocarbon gas, the life cycle production impacts of polyethylene plastic bags are comparable to bio-based bags. On the other hand, if clean and renewable energy was used for the production, conventional plastic bags were shown to be 80% less environment-friendly than bio-based bags. As a result, the life cycle of biobased bags could only be deemed as “green” if renewable energy sources are supplied at every stage of the production process. Otherwise, conventional plastic bags remain more profitable (119). The term “green” here refers to less energy consumption that will indirectly contribute to the environmental impact. When less energy was used, it will also reduce the cost of electricity and eventually the


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product will become more profitable in terms of cost analysis and thus making it more sustainable and profitable. The above LCA studies give an overview of the environmental impact of PHA at the current stage. It is noteworthy that different biodegradable polymers will give different results and cannot be viewed as a global representative. Furthermore, in the LCA analysis, some components were either excluded or included, depending on the assessment method. Nevertheless, the fact that PHA is on par with conventional plastics proves that PHA with favorable outcome could be achieved with improved technology and economy. 4.2. PHA and the Concept of Industrial Symbiosis. In this section, the birth of industrial ecology and related terminology are introduced. There are six core modules for industrial ecology that are listed in Figure 8 (120). The industrial ecology concept is adopted from the biological ecology perspective, whereby the components in the biological ecosystem, such as sunlight, water, and minerals, were consumed by certain group of organisms as a form of energy and the waste and gasses produced were then converted into minerals and recycled back into the system. There is an intricate network involving all the players in the ecosystem and nothing is being wasted in this case. Likewise, in the industrial ecosystem, every process is interdependent and interrelated with one another. Industrial ecology is a new wave involving the product and process design as well as viable manufacturing application. It does not stand alone but involves the surrounding systems, in which it pursues the maximum potential of the total

Fig. 8. Core module of industrial ecology with six main components in which the concept of biological analogy brought forth the whole new system perspective, coupled with the advancement of technology and the involvement of companies with the vision of ecoefficiency and forward-looking research.

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materials cycle, beginning from the crude material to the final product and disposal (121). From the perspective of PHA industry, suitable bacterial strain and carbon sources derived from plants and biomass (biological component) are used in the fermentation system to produce PHAs that are beneficial and ecofriendly. The involvement of companies to improve the quality and production of PHA in a forward-looking research culture and practice are the driving force for a sustainable PHA industry in which different company plays different role in a continuous cycle (Fig. 8). All these components are essential in the industrial ecology and form a holistic system for an efficient and economical PHA production processes. Industrial symbiosis is a subgroup of industrial ecology that involves individual entity in a mutual system that benefits from the trading of materials, energy, water, and by-products. The favorable outcome of industrial symbiosis is dependent on the partnership and synergistic potentials offered within the geographical vicinity (122). The terminology of symbiosis was founded on the biological relationships in nature, whereby a mutual exchange of information, energy, and materials took place between two unrelated species (123). Similarly, in industrial symbiosis, there is a collective benefit achieved by different entities that will further advance the social relationships between the participants. Therefore, there are different tier of focus in industrial ecology that include the facility level, interfirm level, and at the district or worldwide level. One of the best example of industrial symbiosis was modeled by the ecoindustrial park at Kalundborg, Denmark. The water from various sources (ground, surface, and waste) as well as the steam and electricity were shared between the pharmaceutical plant, gypsum board facility, oil refinery, power station, and the city of Kalundborg. In addition, the residues produced were used as feedstocks for other processes. Such cooperation has proven that the existence of each partner increases the sustainability of others by considering the demands of the public for resource savings and environmental protection (124). The nature of the cooperation has created substantial benefits such as increased environmental and economic efficiency. Industrial symbiosis is definitely an ideal concept that will benefit more companies in the near future. As mentioned earlier, industry ecology focuses on three different levels. These levels include the following: (a) Facility or firm level, which is designed for the environment, focusing on pollution prevention and “green” accounting. (b) Interfirm level, which is the central for industrial symbiosis, involving the product life cycles and industrial sector initiatives. (c) Local and worldwide level, which emphasizes on the capitals and cycles, resources and energy flow studies that are known as industrial metabolism (122). All the above support the principles of sustainability. Besides sustainability, this concept also involves economical values. The schematic diagram showing the interrelation between the components are simplified in Figure 9. In this figure, it shows that once the natural resources are being processed and manufactured into


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Fig. 9. The networking in industrial symbiosis that revolves around the natural resources, production, and consumption chain.

goods, the products were then consumed by the consumers. At the same time, the by-products generated from the primary production were also used for other purposes. The used products were then recycled back into the chain for secondary production. At each step, there was minimal waste and fewer resources extracted. This whole idea made the industry more sustainable and profitable. The current workflow of producing PHA involves the fermentation process using robust bacterial strains and suitable carbon source in the bioreactor that has different volumetric capacity. Subsequent steps involving the recovery and purification process and the final end product will then be used for various applications. In order to make PHA a sustainable product, each processing step needs to be optimized. The ability to produce a product successfully and without waste, which involves the capability of a specific application or process to generate the specific outcome with a minimal amount of cost and energy has always been the ultimate goal for the industry. In order to achieve this goal, it is essential to adopt the concept of industrial symbiosis in the production line of PHA. Thus, a futuristic framework for PHA industry is envisioned and discussed further in the following section. 4.3. Framework for PHA Industry. As most of the items that we use daily originate from plastics, having a sustainable PHA industry would help to reduce the waste generated from plastics and eventually substituting these items with PHA-based materials. The application of industrial ecology model for the PHA industry would be beneficial to mankind and the environment in the future. A futuristic framework would be to setup the PHA production factory near to the

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source of raw materials for fermentation of PHA such as within the vicinity of plantation areas of, for example, oil palm, sugar cane, paddy, corn, and wheat. Furthermore, these crops could be processed immediately and the plant-based materials could be used directly as the substrate for fermentation. By using the renewable energy sources such as solar, wind, wave power, and geothermal heat, the whole process would be more economical since the energy is being replenished over time. Besides, waste cooking oil from the settlements as well as the plant biomass and fibers from the plantation and mills are potential substrates for PHA production, turning waste into value-added product. The cell biomass produced from the PHA fermentation can also serve as a feedstock for certain animals such as mealworms. Furthermore, mealworms are capable of selective digestion of cell biomass and function as the PHA partial purification machinery. Subsequently, the mealworms themselves become a feedstock for the fish and chicken farm. The effluent from the PHA industry could be treated at the wastewater treatment plant that in return provide the water supply for the industry and for the settlements. The partially purified PHA can be used as a fertilizer for the plantation while the purified PHA can be used for the industrial applications such as for making composites, biodegradable plastic bags, daily consumables, and medical applications. Since the product can degrade naturally, it also helps to reduce waste accumulation and pollution in the environment. A proposed framework for PHA industry is shown in Figure 10.

Fig. 10. The proposed concept of PHA industry in the future. This framework employs the concept of industry symbiosis where all the main components of PHA industry are interconnected, forming a sustainable networking.


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5. Summary PHA is definitely an interesting biopolymer with various benefits that can be developed to overcome plastic pollution as we move toward a greener and safer environment. Numerous studies have shown that PHA is on par with some petroleum-based plastics in terms of properties. Many commodity products that are currently made from petrochemical plastics can be made from PHA. The main factor that is hindering fully fledged commercialization is the cost of PHA that is still higher than that of commodity plastics. Therefore, new strategies must be developed in order to commercialize PHA successfully. These include the utilization of cheap industrial and agricultural by-products as feedstock, the development of superior bacterial strains, and simpler recovery methods. New government policies that are inline with the sustainable development goals set by United Nations are already providing an impetus to expedite the use of environmentally benign plastics in our daily life.

Acknowledgment The authors would like to acknowledge Long-Term Research Grant Scheme (LRGS) (203/PKT/6725001) for the financial support that has led to some of the findings used in this article. Idris Z-L. and S. Y. Ong would like to acknowledge Malaysian International Scholarship (MIS) and MyBrain15 scholarship from Ministry of Higher Education, respectively, for financial support. The authors would also like to thank Lee Joyyi for providing the biocomposite picture in Figure 7 for this work.

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ZAINAB-L IDRIS ONG SU YEAN SUDESH KUMAR Universiti Sains Malaysia, Penang, Malaysia