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Polyhydroxyalkanoate (PHA): Properties and Modifications
Journal Pre-proof Polyhydroxyalkanoate (PHA): Properties and their Modifications Vibhuti Sharma, Rutika Sehgal, Reena Gupta PII:
S0032-3861(20)30986-1
DOI:
https://doi.org/10.1016/j.polymer.2020.123161
Reference:
JPOL 123161
To appear in:
Polymer
Received Date: 16 September 2020 Revised Date:
13 October 2020
Accepted Date: 18 October 2020
Please cite this article as: Sharma V, Sehgal R, Gupta R, Polyhydroxyalkanoate (PHA): Properties and their Modifications, Polymer, https://doi.org/10.1016/j.polymer.2020.123161. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Ltd. All rights reserved.
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Graphical abstract
Polyhydroxyalkanoate (PHA): Properties and their Modifications Vibhuti Sharma, Rutika Sehgal and Reena Gupta* Department of Biotechnology, Himachal Pradesh University, Summerhill, Shimla-171005, INDIA *Corresponding Author Dr. Reena Gupta, Professor Department of Biotechnology, Himachal Pradesh University, Summerhill, Shimla-171005, INDIA
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Phone: 91-177-2831948
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Fax: 91-177-2831948 E-mail: [email protected]
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First Author
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Vibhuti Sharma
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Department of Biotechnology, Himachal Pradesh University, Summerhill, Shimla-171005, INDIA
Fax: 91-177-2831948
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Phone: 7876713762
Second Author
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Rutika Sehgal
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E-mail: [email protected]
Department of Biotechnology, Himachal Pradesh University, Summerhill, Shimla-171005, INDIA Phone: 8146062390 Fax: 91-177-2831948 E-mail: [email protected]
Author contributions Rutika Sehgal: Conceptualization, Validation, Visualization, Investigation Vibhuti Sharma: Writing-Original draft preparation, Writing-Review and Editing, Data curation Dr. Reena Gupta: Supervision, Validation
Abstract There are a wide range of biopolymers produced by a number of different microorganisms. Polyhydroxyalkanoates are also a diverse group among these biopolymers. In recent times, PHA is gaining a lot of attraction due to its properties like biodegradability, biocompatibility, hydrophobicity, etc. but on the other hand, these biopolymers also possess some disadvantages which limit their competition with synthetic polymers. Therefore, to overcome these limitations, PHAs are being modified to improve their properties for their potential applications in different fields. This review comprises different PHA modification methods which include physical
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bending using natural and synthetic polymers, different approaches of chemical modification and
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some biological modification methods. The aim of this review is to represent new approaches and ideas to improve the degradable properties of PHAs and to present them as one of the
fields. Polyhydroxyalkanoates,
Biocompatibility,
Biodegradibility,
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Hydrophobicity, Modifications
Biopolymers,
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Keywords:
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valuable and eco-friendly biopolymer, so that, they can contribute their potentials in various
1. Introduction
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Plastics have become a very important part of human life due to their useful properties
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like strength, light-weight, flexibility, etc. but the non-degradable and non-disposable properties of plastic have led to a fall in its reputation from last many years. Plastic lasts forever in the environment, therefore its accumulation in the environment is becoming the most visible form of environmental pollution which is causing a lot of problems related to human health and other living organisms [1]. The estimated rate of plastic accumulation in environment and expected rate of accumulation in 2050 is shown in Figure 1 [2]. To overcome this problem, the main focus of researchers is to find out some safer alternatives to replace synthetic plastic. Polyhydroxyalkanoates are such sustainable biopolymers which can be used as a good alternate to synthetic plastics [3].
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Figure 1 - Expected increase in rate of plastic accumulation
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Polyhydroxyalkanoates (PHA) are naturally occurring biopolymers, accumulated by different
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microorganisms in the form of granules as shown in Figure 2 [4], in presence of excess of carbon and other nutrients deficient conditions [5]. Archaea and various bacterial strains like Gram
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positive bacteria, Gram negative bacteria, photosynthetic bacteria and mixture of different
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microorganisms have been identified to accumulate PHA both aerobically and anaerobically.
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More than 150 different types PHA have been identified till now [6].
Figure 2 - Microscopic image of PHA granules PHA has favourable properties such as small pore size with high propensity to get recycled, high volume to surface ratio, biodegradability and biocompatibility. PHA has recently been the point of attention due to their various advantageous properties like easy processing, good resistance to UV rays, insolubility in water, etc. [7]. These advantageous properties make them worth to substitute materials like poly (lactic acid), polyethylene terephthalate, biopolyamide and other non-biodegradable bio-based materials that are used in different areas like
agriculture, food, medical, etc. Despite their high potential for commercial applications, most of the PHA based biomaterials face many research gaps, especially crystalline short chain length PHA with higher monomeric compositions of 3-hydroxybutyric acid like poly(3hydroxybutyrate) (PHB) are highly hydrophobic, exhibit a low heat distortion temperature and poor gas barrier properties. These disadvantages result in their slow degradability [8]. The cost of PHA production is much high as compared to other bio-based plastics. Thus, these kinds of biopolymers fail to meet the industrial demands. Therefore, various approaches are devised to enhance the physical and chemical properties of the PHA in order to overcome these
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shortcomings by modifying the biopolymers. In order to extend PHA applications in various
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fields, they are modified either physically, chemically or biologically [9]. For example: chemically modified PHA with folic acid could efficiently serve as a cancer drug carrier. The
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PHA’s modifications can also enhance the rate of degradation of these polymers.
2.1 Basic structure of PHA
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2. Basic Structure and Properties of PHA
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A PHA molecule consists of the monomer units of (R)-hydroxy fatty acid. The basic
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structure of PHA is shown in Figure 3 [10]. The monomeric units are connected to each other by ester bond [11]. Each monomeric unit has a side chain R group i.e. saturated alkyl group or
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unsaturated alkyl groups, substituted alkyl groups and branched alkyl groups.
Figure 3 – The basic structure of PHA: R- acyl group that can contain 1-13 carbons; m-1, 2 or 3; n-100 to many thousands
PHA are categorized into different types on the basis of their structural chain length : 2.1.1 Short chain length PHA (scl-PHA) They consist of monomeric building blocks of 3-5 carbons. For example: poly (3hydroxyvalerate) (PHV); poly (3-hydroxybutyrate) (PHB) are produced by Cupriavidus necator. Short chain length PHA are too rigid and brittle and lack the superior mechanical properties required for biomedical and packaging film applications [12]. 2.1.2 Medium chain length PHA (mcl-PHA)
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They consist of monomeric units of 6-14 carbons. For example: poly (3-
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hydroxyoctanoate) (PHO) is produced by Pseudomonas mendocina. Medium chain length PHA
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are elastomeric but have very low mechanical strength which limits the application of these PHA
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[13] 2.1.3 Long chain length PHA
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They contain monomer building blocks of 15 carbons or more than 15 carbons. For
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2.2 Properties of PHA
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example: poly (3-hydroxypentadecanoate) is produced by Pseudomonas aeruginosa.
PHA generally exhibits similar properties to petroleum-based polymers [14]. Some
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physical properties of PHA include their insolubility in water, good resistance to UV rays, stiffness, high degree of polymerization, biodegradability and thermoplasticity, etc. 3. Biosynthesis of PHA The catalytic mechanism for production of PHA by microorganisms involves different pathways which are linked to the various metabolic pathways of microorganisms such as glycolysis, β-oxidation, etc. Generally, in bacteria, there are three naturally occurring PHA biosynthetic pathways. 3.1 Pathway 1 In this pathway, two molecules of acetyl-CoA (from Tricarboxylic Acid Cycle) are condensed into a molecule of acetoacetyl-CoA with the help of β-ketothiolase enzyme.
Acetoacetyl-Co-A is then converted into 3-hydroxybutyryl-CoA by enzyme NADPHacetoacetyl-CoA reductase; followed by the action of PHA synthase which finally catalyzes the ester bond formation in 3-hydroxybutyryl-CoA to generate poly (3HB) [15]. 3.2 Pathway 2 In this pathway, the substrates originate from the β-oxidation pathway of fatty acids because fatty acids are suitable carbon source for PHA production [16]. The fatty acid metabolism generates different hydroxyalkanoate monomers by the action of (R)-enoyl-CoA
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hydratase, acyl-CoA oxidase and 3-ketoacyl-CoA reductase. Then enzyme, PHA synthase
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catalyzes the polymerization of hydroxyalkanoate monomers [15].
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3.3 Pathway 3
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Simple inexpensive carbon sources like glucose,
sucrose, lactose etc. that
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microorganisms obtain from the environmental sources like wastewater, activated sludge, animal fats, hydrocarbons etc. are used to convert (R)-hydroxy acyl intermediates from their acyl carrier by the action of acyl-ACP-CoA transacetylase and then
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protein form to Co-A form
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hydroxyalkanoate monomers are eventually polymerized by PHA polymerase [17].
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4. Modifications of PHA
The structure of a PHA can be altered physically, chemically or biologically to produce a new altered polymer with foreseeable variations in their functions and molecular weight. These kinds of modifications may alter their mechanical properties, amphiphilic character, surface structure and rate of degradation to accomplish the requirements for their specific applications in different fields. For example: The rate of hydrolysis of PHA can be modified to become relatively faster by incorporating more hydrolysis-prone chemical groups into the polymeric backbone thereby modulating the reactivity of the ester linkage [18]. Different methods of modifications of PHA are shown in Figure 4 [12].
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4.1.1 Blending
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4.1 Physical modifications
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Figure 4 – Different methods of modifications of PHA
Blending is very effective and simple approach for producing new polymeric materials
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with improved properties. The major advantage of this process is that it can suppress the drawbacks of the parent component. The properties of polymers can be improved by the selection of appropriate starting materials, by changing the conditions required for blending and varying the composition of the blends. PHA can be blended with synthetic biodegradable polymers as well as with natural raw materials. Molecular structures of some biodegradable polymers used in PHA blends are shown in Figure 5 [12]. The blending adds some interesting properties to PHA. These blends have attracted great attention because they employ conventional technology at low cost and have high potential applications in various fields.
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Figure 5 - Molecular structures of some biodegradable polymers used in PHA blends 4.1.1.1 Blending of PHA with natural raw materials
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There are many low-cost natural raw materials such as cellulose, starch, lignin, etc. that can be used in the blending of PHA.
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PHA blending with cellulose derivatives
Cellulose derivatives have gained great interest as blending components with PHA
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because of their compatibility with PHA and their ability to enhance the rate of PHA degradation. PHA blended with cellulose has many applications in different fields. Cellulose derivatives such as cellulose acetate butyrate (CAB), ethyl cellulose, cellulose propionate are major biomaterials that are used as carriers for poorly soluble drugs, act as blood coagulant, used in pharmaceutical tablet coating [19]. Many scientists investigated the properties like crystallization, miscibility, melting behavior and phase morphology of blends of ethyl cellulose and PHB. An increase in glass transition temperature was observed with a decrease in the PHB concentration in the blends in a study by Zhang et al. [20]. Ei-Shafee et al. [21] investigated the properties of a blend of PHB and cellulose acetate butyrate (CAB). The dispersion of CAB in the PHB-crystallized phase induces the improved mechanical properties with an elongation at break that increased from 2.2 to 7.3%. PHA blending with starch
Starch is one of the most promising natural polymers due to its inherent biodegradability and abundance. The blending of PHA with starch or starch derivatives (starch acetate) showed some ability in reducing the production cost of PHA with improved mechanical properties.These blends do not form intact films and are brittle in nature due to the incompatibility between the PHA matrix and starch. Lai et al. [22] investigated the properties of modified corn-starch and PHB blend. After blending PHB with the corn starch, only a single glass transition temperature was detected for all PHB and corn starch blends, which increased from 2 to 37°C with an increase in the concentration of corn starch, which gives the indication about the compatibility of
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PHB with corn-starch.
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PHA blending with lignin
Lignin is a macromolecule that is composed of repeated phenyl propane units and
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possesses aliphatic and aromatic hydroxyl groups, as well as carboxylic acid groups. The
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presence of these functional groups makes lignin a valuable material for blending purpose.
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Mousavioun et al. [23] investigated the rheology and thermo-physical properties of blends of PHB and lignin. Lignin was employed as a blending component to improve the properties of
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PHB. The results indicated that lignin can improve the total thermal stability of PHB. 4.1.1.2 Blending of PHA with synthetic biodegradable polymers
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PHA blending with Poly (lactic acid)
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Poly (lactic acid) (PLA) is a chemically synthesized biodegradable polymer from lactic acid. Poly (lactic acid) is environment friendly, linear aliphatic polyester. Several studies have been done that focused on the study of blending of PHA and PLA. Takagi et al. [24] blended a series of epoxy-group-modified PHA (e-PHA) and mcl-PHA with PLA. Furukawa et al. [25] compared the properties of PHB and PLA blends. They observed that PHB and PLA blends were immiscible and highly compatible. PHA blending with Poly (ε-caprolactone) Poly (ε-caprolactone) (PCL) is semicrystalline aliphatic polyester which is biodegradable in nature. PCL possesses high mechanical strength and ductility. Lim et al. [26] blended PCL with poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). The blending of polymer with PCL may cause PHBHHx to exhibit enhanced toughness with substantial elasticity. Chiono et al. [27] developed a blend of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and
PCL in chloroform. The fibers resulted from these blends exhibited a low degree of bulk and surface porosity. 4.2 Chemical modifications Polyhydroxyalkanoates can be modified by adding a chemical group in the structure of PHA. These chemically modified PHA can be utilized as multifunctional materials. For example, the hydrolytic rate of PHA can be modified by incorporating hydrolysis-prone chemical groups. There are number of methods in which chemical modifications of the PHA can be done, these
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methods are shown in Figure 6 [28] and are briefly described as follows:
Figure 6 - Chemical modifications of PHA 4.2.1 Carboxylation PHA modification by carboxylation is the addition of carboxylic (-COOH) functional group to the substrate. The carboxylation of PHA enhances the hydrophilicity of the polymer through better water penetration. The incorporated carboxylic group acts as active binding site for biologically active moieties like hydrophilic components [29]. Kurth et al. [30] carboxylated
the double bonds of poly (3-hydroxyoctanoate-co-3-hydroxyundecenoate) (PHOU) in the presence of NaHCO3 using KMnO4 as an oxidation agent. Crown ether was used as the phase transfer and dissociating agent for the KMnO4 to carboxylate the unsaturated PHOU. The oxidative carboxylation of unsaturated PHA using KMnO4 as an oxidation agent is shown in
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Figure 7 [28]. Around 70 PHAs had been identified that have such -COOH groups [31].
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4.2.2 Halogenation
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Figure 7 - Oxidative carboxylation of unsaturated PHA using KMnO4 as an oxidation
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Halogenation of PHA is considered as one of the excellent methods in improving the properties, functions and applications of polymers. Halogen atoms (chlorine, bromine and fluorine) are added to unsaturated PHA and saturated PHA through an addition and substitution reaction respectively. Chlorination is one kind of halogenation. The process of chlorination involves the introduction of chlorine to a substrate. Chlorination makes the soft and sticky mclPHA; crystalline, hard and brittle. Arkin and Hazer [32] demonstrated chlorination of PHA which was stiff and crystalline polymer at approximately 54 wt% chlorination. They performed an experiment by adding excess HCl to KMnO4 in a drop wise fashion to generate chlorine gas. Reaction set-up for the chemical modification of PHA via chlorination is shown in Figure 8 [28]. Then they subsequently passed the gas into a solution of sticky unsaturated PHA obtained from Pseudomonas putida. Depending on the amount of chlorine passed, the resulting PHA exhibited higher melting and glass transition temperatures.
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Figure 8 - Reaction set-up for the chemical modification of PHA via chlorination
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4.2.3 Hydroxylation
The properties of PHA can be altered by hydroxylation method [33, 34]. Acid or base
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catalyzed reactions are used in the PHA modification in the presence of low molecular weight mono or diol compounds by the process of hydroxylation. Different approaches to PHA
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modifications by hydroxylation are shown in Figure 9 [28]. Timbart et al. [35] reported the
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production of monohydroxylated oligomers of poly (3-hydroxyoctanoate) (PHO) and poly (3hydroxyoctanoate-co-3-hydroxyundecenoate (PHOU) using both base and acid catalyzed hydrolyses. In base catalysed hydrolysis, the alcoholic NaOH was used to catalyze the hydrolysis at pH 10–14 and the reaction was stopped by adding concentrated aqueous hydrochloric acid. On the other hand, the acid hydrolysis was performed using two different approaches: (i) a reaction catalyzed by para-toluenesulfonic acid monohydrate (PTSA) at 120ᵒC, which was stopped by cooling the mixture in an ice bath; (ii) monohydroxylation by acidic methanolysis at 100ᵒC to yield the respective 3-hydroxymethyl esters bearing a methyl protected carboxylic acid group. It was found that the modified PHO showed lower glass transition and melting temperature than original PHO.
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Figure 9 - Different approaches to PHA modification by hydroxylation
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4.2.4 Epoxidation
The epoxidation of PHA can modify its properties. The high reactivity and easy
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conversion of epoxy groups to anionic and polar groups, even in mild environments, makes
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epoxidation an important strategy for mcl-PHA [28]. Epoxidation of mcl-PHA with mchloroperoxybenzoic acid (m-CPBA) is shown in Figure 10 [28]. Bear et al. [36] performed the
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epoxidation of poly (hydroxyoctanoate-co-10-undecanoates) (PHOUs) in the presence of vinyl group which easily undergoes epoxidation reaction and copolymers were obtained with the help
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of m-CPBA. With increase in epoxy groups, an increase in the thermal stability was also
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observed in the final modified PHA.
Figure 10 - Epoxidation of mcl-PHA using m-CPBA There have been several reports on the epoxidation process. Park et al. [37] epoxidized PHOU with a limited amount of olefinic bonds using m-CPBA. With increasing conversion of
olefinic bonds to epoxy bonds, a decrease in the melting temperature and melting enthalpy was observed. 4.2.5 Grafting Grafting is a PHA modification method in which there exists a covalent bond between polymer chain and other monomers to obtain desired valuable properties. Two different polymer units make up a copolymer.One or more side chains are attached to the main chains in case of grafting. Grafting is an excellent way to synthesize a copolymer with minimum loss of original properties and with addition of some new properties of the polymer [38]. Different types of
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grafting methods are as follows:
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4.2.5.1 Radiations based grafting
Radiations based grafting follows the irradiation of a biopolymer through high energy source or
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by ionizing radiations. PHB is chemically inactive polyester, therefore, chitosan could be grafted
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onto PHB using gamma irradiation along with some different solvents (acetone, ethyl acetate and
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acetic acid) [39]. Gamma irradiations along with different solvents were also used to graft poly (vinyl alcohol) (PVA) onto PHB. The grafted PHB showed alterations in different properties
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such as surface roughness formation of cavities and formation of waves. 4.2.5.2 Free radical based grafting
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A growing chain of polymer has an unpaired electron which acts as site of reaction (a radical site) to react with the unsaturated part of new coming monomer. In this way the unpaired
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electron of growing polymer chain gets transferred to the coming monomer. Free radicals are generated and are transferred to the substrate to react with the monomer which results in a grafted copolymer. Nguyen and Marchessault [40] successfully synthesized a graft copolymer of poly (methyl methacrylate) (PMMA) and PHB using free radical based grafting mechanism. This copolymer had a shape of a comb and exhibited huge alterations in its glass transition temperature (Tg) from 100°C to 3°C. 4.2.5.3 Ionic graft polymerization Ionic grafting is a specific ion based grafting which includes cationic and anionic polymerization reactions. In anionic polymerization, the growing chain of polymer possesses either a negative or a positive charge. Anionic grafting
Anionic graft polymerizations are the grafting methods in which the growing polymer chain contains a negative charge. Anionic catalyst or polymerization initiators are required to start anionic graft polymerization reactions.The most commonly used initiators include Grignard reagents, alkali metal suspensions, Ziegler-Natta catalyst, organic radical anions and metalocene. Anionic grafting offers some narrow molecular weight distribution, block copolymers and stereo control grafted copolymers. The major disadvantage of anionic grafting includes requirements of low temperature and this method is only feasible for limited number of polymers. Cationic grafting
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Cation graft polymerization occurs in growing chain of polymer which contains a positive
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charge. Cationic initiators or cationic catalyst plays very important role in these polymerization reactions. Strong protonic acids, Lewis acids and their complexes are mostly used as initiators.
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The main advantage of cationic grafting is controlling the molecular weight of a grafted
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copolymer. But the main disadvantage of grafting process is that it is moisture sensitive, requires
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low temperature to start the reaction and is limited only to the olefins [38]. 4.2.5.4 Enzyme grafting
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Enzymatic grafting method is more promising due to its less hazardous nature as compared to the risky chemical approaches. One example of enzymatic grafting is laccase based grafting of ethyl
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cellulose onto the surface of PHB. It was demonstrated that enzymatic grafting is an energy saving and environment friendly process for grafting and it provides a suitable environment for
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polymer grafting which results in products with multiple functions [41]. 4.3 Biological modifications of PHA PHA can be modified biologically in following ways: 4.3.1
Co-adding two substrates in culture medium
Various PHA can be modified by co-feeding substrates in the culture medium of bacteria. For example, when cells of Halomonas bluephagenesis are supplied with glucose and propionic acid or other propionogenic carbon sources under nitrogen limited conditions, it produces PHBV copolymer depending upon their concentrations [6]. 4.3.2 Feeding the culture with functional group containing substrates PHA can be modified biologically by feeding the bacterial culture medium with different substrates containing functional groups. For example, Pseudomonas putida when cultivated in
the presence of ω-phenoxyalkanoates produces PHA with phenoxy groups in the side chains [42]. 5. Applications of PHA Biodegradable nature and other properties of polyhydroxyalkanoates such as high temperature stability, low degree of surface porosity, enhanced toughness, elasticity, etc. results in their applications in various fields like medical, agriculture, etc. Some applications of PHA are
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as follow 5.1 Medical Applications
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PHA has properties quite similar to those of synthetic plastics. The unique properties like
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biodegradability, biocompatibility and non-toxicity make them desirable materials for biomedical applications. PHA has been found suitable for many applications in medical field.
Drug delivery
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Some medical applications of PHA are as follows
Technique of drug delivery is emerging as an interdisciplinary science aimed at improving the
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health of human. The major goal in the drug delivery system is that the drugs should be delivered in a controlled and targeted manner in order to improve their efficacy. Hence, PHA, due to their
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origin from natural sources, is used as raw material for producing tablets, nano-particles, etc. [43,
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44]. For example: PHB, PHVB and their copolymers are used for delivery of antibiotics. Tissue engineering
PHA with little modifications contributes very much in the field of tissue engineering. PHA can be used to produce scaffolds which promote cell growth by supplying nutrition, have higher mechanical strength. These scaffolds are available in the form of pins, films, sutures, etc. For example: 3D scaffolds developed by using PHA nanofibers are used for cartilage repair [45]. 5.1.3
Dressings
Wound dressing is carried out with the help of volatile solutions of PHA [46]. These materials form a thin film over the wound to prevent contamination. Dressing based on non-woven fibrous material of P (3HB) can be used as swabs, fleece, lint, etc. There are many other medical applications of these polymers which include soft tissue regenerations, nerve repair, cardiovascular treatments, etc. [45].
5.2 Agricultural applications PHA has many applications in the field of agriculture. PHA improves the nutritional properties and shelf life of the plants under stressful environments. One of the specialized applications of P (3HB) in agriculture is the controlled release of pesticides and insecticides. Potential applications of PHAin agriculture include: biodegradable plastic films for crop protection, seeds encapsulation, encapsulation of fertilizers, etc. [47].
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5.3 Market applications
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PHA bioplastics possess properties making them suitable replacements for many of the applications currently served by petroleum‐based plastics, thus providing tremendous market
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potential. The versatility of PHA results in their wide applications in market. The main uses of
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PHA are seen in packaging and food services due to their degradable nature, non solubility, non permeable nature and flexibility [48, 49]. PHA are developed as packaging films mainly for use
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as shopping bags, paper coatings, cups, diapers, carpets, etc. Many products are now available in
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6. Drawbacks of PHA
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the form of PHA coated boxes, paper, sheets, boards, etc. [50].
These days, PHA are gaining lot of attention due to their biodegradable nature but their
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high cost and requirement of complex substrates for their production leads to a decline in their demands in different fields. These are about 15 times more expensive than the petroleum derived polymers [51]. One of the major limitations of the industrial production of PHA is to maintain the optimal bacterial growth conditions. Most fermentation processes do not allow maximum synthesis of PHA granules at the end of the cultivation. 7. Strategies for reduction on PHA production cost 7.1 Inexpensive substrate High cost of PHA is related to the substrates that account for 30-40% of the total production costs. PHA can be produced from cheap substrates such as sucrose, glucose, lignin, waste material, etc. New pathways to utilize cheap substrates can be established for PHA
production [52]. For example, by integrating genes encoding aqua glyceroporin and glycerol kinase from E.coli into chromosome of Cupriavidus necator H16, the resulting recombinant could produce large amount of PHB from glycerol [53]. Many other cases have been reported, such as co-productions of PHA with amino acids, proteins, alcohols, biosurfactants and other chemicals, etc. 7.2 Developing better bacterial strains Using transgenically modified bacterial strains is another strategy to decline the price of
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PHA production and to increase the yield of PHA. For example: Metabolically engineered
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mutant strain, C. necator expresses genes that encodes L-arabinose catabolic enzymes, and
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therefore can use L-arabinose as a carbon substrate to accumulate high level of PHB [54].
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7.3 Reduction on cost of separation and purification
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The downstream processing of PHA mainly includes cell and broth separation, product purification. The separation requires expensive centrifugation, micro-filtration and other time-
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consuming processes [55]. Therefore, it is necessary to develop methods that permit the recovery of PHA by a simple, efficient and less polluting process. Some of the processes are: Self-flocculation
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7.3.1
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Self-flocculation refers to the ability of some microorganisms to display controlled aggregation, leading to the formation of extensive but highly compact cell flocs; this reduces the difficulty of recovering biomass from a culture [56]. Self-flocculation brings benefits of recycling the fermentation media. After the collection of precipitated cell mass, the supernatant can be again used for the growth of the net batch without sterilization or inoculation. 7.3.2
PHA purification by animals
An effective biological recovery approach for PHA was developed using Tenebrio molitor, a mealworm, which was shown to readily consume the freeze-dried C. necator cells and excrete the PHA granules in the form of whitish feces. Further purification using water, detergent and heat resulted in almost 100% pure PHA granules [57, 58]. This significantly simplifies the purification process. 7.3.3 Balancing power reduction
The accumulation of PHA requires vast energy consumption. Continuous fermentation process is one way to reduce the power cost for PHA production. It also reduces the manpower, material resources consumption and can also enhance the production efficiency [59]. 8. Future perspective Polyhydroxyalkanoates are produced by different microorganisms under stress conditions. PHA are easily biodegradable and sustainable, thus, are considered as the plastics of future.These bioplastics have the potential to replace the synthetic plastics, which can reduce the chances of
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development of junk of synthetic plastics. Many PHA possess objectionable properties such as
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crystallinity, brittleness, etc. which can be reduced by different methods of modifications. As
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very low-cost substrates like starch, PLA, cellulose, etc. are used in the PHA production, these low-cost polymers also play role in modifications of PHA which can lead to the production of
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high yield of PHA at very reasonable prices. In the future, production of these high-valued
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polyhydroxyalkanoates and other metabolites from the microorganisms using domestic, industrial, agricultural wastes as substrates may help us to overcome the issues related to
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environmental pollution, its commercialization and high cost and could enhance its implementation at industrial scale. PHA will become more prominent in the coming years and
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9. Conclusion
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will increase the growth rate of various industries.
This review comprises of a brief outlook of poyhydroxyakanoates, their modifications and applications of PHA in various fields. PHA has drawn much attention due to their biological origin, biodegradable and biocompatible properties. These properties of biopolymers can be improved. There are many different modification approaches that have been employed to alter the polymer and its properties. This review summarizes the well-established modification approaches which include blending, chemical and biological modifications. These modifications can be further improved and new cheaper modification strategies can be adopted to minimize the poor properties of PHA. One approach for which is by replacing the expensive substrates with novel waste materials for modifications. The use of these modified biopolymers limit carbon dioxide emissions during creation and degrade to organic matter after disposal. Their degradation
products are harmless to the natural environment and to the in vivo systems. They offer great potential values in various fields like medical, agriculture, cosmetics, pharmacy, etc. Acknowledgements The fellowship granted to Ms. Rutika Sehgal in the form of SRF from Council of Scientific and Industrial Research (CSIR) is thankfully acknowledged. The financial support from Department of Biotechnology, Ministry of Science and Technology, Govt. of India, to Department of Biotechnology, Himachal Pradesh University, Shimla, India, is also thankfully
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acknowledged.
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Highlights •
Replacement of conventional synthetic plastics with PHA
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PHA possesses properties like biodegradability, renewability, biocompatibility, etc.
•
Modifications of PHA using physical, chemical and biological methods for their
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efficient production
Declaration of interests ☑ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:
S0032-3861(20)30986-1
DOI:
https://doi.org/10.1016/j.polymer.2020.123161
Reference:
JPOL 123161
To appear in:
Polymer
Received Date: 16 September 2020 Revised Date:
13 October 2020
Accepted Date: 18 October 2020
Please cite this article as: Sharma V, Sehgal R, Gupta R, Polyhydroxyalkanoate (PHA): Properties and their Modifications, Polymer, https://doi.org/10.1016/j.polymer.2020.123161. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Elsevier Ltd. All rights reserved.
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Graphical abstract
Polyhydroxyalkanoate (PHA): Properties and their Modifications Vibhuti Sharma, Rutika Sehgal and Reena Gupta* Department of Biotechnology, Himachal Pradesh University, Summerhill, Shimla-171005, INDIA *Corresponding Author Dr. Reena Gupta, Professor Department of Biotechnology, Himachal Pradesh University, Summerhill, Shimla-171005, INDIA
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Phone: 91-177-2831948
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Fax: 91-177-2831948 E-mail: [email protected]
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First Author
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Vibhuti Sharma
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Department of Biotechnology, Himachal Pradesh University, Summerhill, Shimla-171005, INDIA
Fax: 91-177-2831948
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Phone: 7876713762
Second Author
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Rutika Sehgal
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E-mail: [email protected]
Department of Biotechnology, Himachal Pradesh University, Summerhill, Shimla-171005, INDIA Phone: 8146062390 Fax: 91-177-2831948 E-mail: [email protected]
Author contributions Rutika Sehgal: Conceptualization, Validation, Visualization, Investigation Vibhuti Sharma: Writing-Original draft preparation, Writing-Review and Editing, Data curation Dr. Reena Gupta: Supervision, Validation
Abstract There are a wide range of biopolymers produced by a number of different microorganisms. Polyhydroxyalkanoates are also a diverse group among these biopolymers. In recent times, PHA is gaining a lot of attraction due to its properties like biodegradability, biocompatibility, hydrophobicity, etc. but on the other hand, these biopolymers also possess some disadvantages which limit their competition with synthetic polymers. Therefore, to overcome these limitations, PHAs are being modified to improve their properties for their potential applications in different fields. This review comprises different PHA modification methods which include physical
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bending using natural and synthetic polymers, different approaches of chemical modification and
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some biological modification methods. The aim of this review is to represent new approaches and ideas to improve the degradable properties of PHAs and to present them as one of the
fields. Polyhydroxyalkanoates,
Biocompatibility,
Biodegradibility,
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Hydrophobicity, Modifications
Biopolymers,
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Keywords:
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valuable and eco-friendly biopolymer, so that, they can contribute their potentials in various
1. Introduction
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Plastics have become a very important part of human life due to their useful properties
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like strength, light-weight, flexibility, etc. but the non-degradable and non-disposable properties of plastic have led to a fall in its reputation from last many years. Plastic lasts forever in the environment, therefore its accumulation in the environment is becoming the most visible form of environmental pollution which is causing a lot of problems related to human health and other living organisms [1]. The estimated rate of plastic accumulation in environment and expected rate of accumulation in 2050 is shown in Figure 1 [2]. To overcome this problem, the main focus of researchers is to find out some safer alternatives to replace synthetic plastic. Polyhydroxyalkanoates are such sustainable biopolymers which can be used as a good alternate to synthetic plastics [3].
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Figure 1 - Expected increase in rate of plastic accumulation
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Polyhydroxyalkanoates (PHA) are naturally occurring biopolymers, accumulated by different
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microorganisms in the form of granules as shown in Figure 2 [4], in presence of excess of carbon and other nutrients deficient conditions [5]. Archaea and various bacterial strains like Gram
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positive bacteria, Gram negative bacteria, photosynthetic bacteria and mixture of different
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microorganisms have been identified to accumulate PHA both aerobically and anaerobically.
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More than 150 different types PHA have been identified till now [6].
Figure 2 - Microscopic image of PHA granules PHA has favourable properties such as small pore size with high propensity to get recycled, high volume to surface ratio, biodegradability and biocompatibility. PHA has recently been the point of attention due to their various advantageous properties like easy processing, good resistance to UV rays, insolubility in water, etc. [7]. These advantageous properties make them worth to substitute materials like poly (lactic acid), polyethylene terephthalate, biopolyamide and other non-biodegradable bio-based materials that are used in different areas like
agriculture, food, medical, etc. Despite their high potential for commercial applications, most of the PHA based biomaterials face many research gaps, especially crystalline short chain length PHA with higher monomeric compositions of 3-hydroxybutyric acid like poly(3hydroxybutyrate) (PHB) are highly hydrophobic, exhibit a low heat distortion temperature and poor gas barrier properties. These disadvantages result in their slow degradability [8]. The cost of PHA production is much high as compared to other bio-based plastics. Thus, these kinds of biopolymers fail to meet the industrial demands. Therefore, various approaches are devised to enhance the physical and chemical properties of the PHA in order to overcome these
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shortcomings by modifying the biopolymers. In order to extend PHA applications in various
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fields, they are modified either physically, chemically or biologically [9]. For example: chemically modified PHA with folic acid could efficiently serve as a cancer drug carrier. The
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PHA’s modifications can also enhance the rate of degradation of these polymers.
2.1 Basic structure of PHA
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2. Basic Structure and Properties of PHA
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A PHA molecule consists of the monomer units of (R)-hydroxy fatty acid. The basic
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structure of PHA is shown in Figure 3 [10]. The monomeric units are connected to each other by ester bond [11]. Each monomeric unit has a side chain R group i.e. saturated alkyl group or
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unsaturated alkyl groups, substituted alkyl groups and branched alkyl groups.
Figure 3 – The basic structure of PHA: R- acyl group that can contain 1-13 carbons; m-1, 2 or 3; n-100 to many thousands
PHA are categorized into different types on the basis of their structural chain length : 2.1.1 Short chain length PHA (scl-PHA) They consist of monomeric building blocks of 3-5 carbons. For example: poly (3hydroxyvalerate) (PHV); poly (3-hydroxybutyrate) (PHB) are produced by Cupriavidus necator. Short chain length PHA are too rigid and brittle and lack the superior mechanical properties required for biomedical and packaging film applications [12]. 2.1.2 Medium chain length PHA (mcl-PHA)
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They consist of monomeric units of 6-14 carbons. For example: poly (3-
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hydroxyoctanoate) (PHO) is produced by Pseudomonas mendocina. Medium chain length PHA
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are elastomeric but have very low mechanical strength which limits the application of these PHA
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[13] 2.1.3 Long chain length PHA
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They contain monomer building blocks of 15 carbons or more than 15 carbons. For
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2.2 Properties of PHA
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example: poly (3-hydroxypentadecanoate) is produced by Pseudomonas aeruginosa.
PHA generally exhibits similar properties to petroleum-based polymers [14]. Some
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physical properties of PHA include their insolubility in water, good resistance to UV rays, stiffness, high degree of polymerization, biodegradability and thermoplasticity, etc. 3. Biosynthesis of PHA The catalytic mechanism for production of PHA by microorganisms involves different pathways which are linked to the various metabolic pathways of microorganisms such as glycolysis, β-oxidation, etc. Generally, in bacteria, there are three naturally occurring PHA biosynthetic pathways. 3.1 Pathway 1 In this pathway, two molecules of acetyl-CoA (from Tricarboxylic Acid Cycle) are condensed into a molecule of acetoacetyl-CoA with the help of β-ketothiolase enzyme.
Acetoacetyl-Co-A is then converted into 3-hydroxybutyryl-CoA by enzyme NADPHacetoacetyl-CoA reductase; followed by the action of PHA synthase which finally catalyzes the ester bond formation in 3-hydroxybutyryl-CoA to generate poly (3HB) [15]. 3.2 Pathway 2 In this pathway, the substrates originate from the β-oxidation pathway of fatty acids because fatty acids are suitable carbon source for PHA production [16]. The fatty acid metabolism generates different hydroxyalkanoate monomers by the action of (R)-enoyl-CoA
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hydratase, acyl-CoA oxidase and 3-ketoacyl-CoA reductase. Then enzyme, PHA synthase
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catalyzes the polymerization of hydroxyalkanoate monomers [15].
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3.3 Pathway 3
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Simple inexpensive carbon sources like glucose,
sucrose, lactose etc. that
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microorganisms obtain from the environmental sources like wastewater, activated sludge, animal fats, hydrocarbons etc. are used to convert (R)-hydroxy acyl intermediates from their acyl carrier by the action of acyl-ACP-CoA transacetylase and then
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protein form to Co-A form
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hydroxyalkanoate monomers are eventually polymerized by PHA polymerase [17].
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4. Modifications of PHA
The structure of a PHA can be altered physically, chemically or biologically to produce a new altered polymer with foreseeable variations in their functions and molecular weight. These kinds of modifications may alter their mechanical properties, amphiphilic character, surface structure and rate of degradation to accomplish the requirements for their specific applications in different fields. For example: The rate of hydrolysis of PHA can be modified to become relatively faster by incorporating more hydrolysis-prone chemical groups into the polymeric backbone thereby modulating the reactivity of the ester linkage [18]. Different methods of modifications of PHA are shown in Figure 4 [12].
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4.1.1 Blending
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4.1 Physical modifications
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Figure 4 – Different methods of modifications of PHA
Blending is very effective and simple approach for producing new polymeric materials
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with improved properties. The major advantage of this process is that it can suppress the drawbacks of the parent component. The properties of polymers can be improved by the selection of appropriate starting materials, by changing the conditions required for blending and varying the composition of the blends. PHA can be blended with synthetic biodegradable polymers as well as with natural raw materials. Molecular structures of some biodegradable polymers used in PHA blends are shown in Figure 5 [12]. The blending adds some interesting properties to PHA. These blends have attracted great attention because they employ conventional technology at low cost and have high potential applications in various fields.
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Figure 5 - Molecular structures of some biodegradable polymers used in PHA blends 4.1.1.1 Blending of PHA with natural raw materials
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There are many low-cost natural raw materials such as cellulose, starch, lignin, etc. that can be used in the blending of PHA.
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PHA blending with cellulose derivatives
Cellulose derivatives have gained great interest as blending components with PHA
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because of their compatibility with PHA and their ability to enhance the rate of PHA degradation. PHA blended with cellulose has many applications in different fields. Cellulose derivatives such as cellulose acetate butyrate (CAB), ethyl cellulose, cellulose propionate are major biomaterials that are used as carriers for poorly soluble drugs, act as blood coagulant, used in pharmaceutical tablet coating [19]. Many scientists investigated the properties like crystallization, miscibility, melting behavior and phase morphology of blends of ethyl cellulose and PHB. An increase in glass transition temperature was observed with a decrease in the PHB concentration in the blends in a study by Zhang et al. [20]. Ei-Shafee et al. [21] investigated the properties of a blend of PHB and cellulose acetate butyrate (CAB). The dispersion of CAB in the PHB-crystallized phase induces the improved mechanical properties with an elongation at break that increased from 2.2 to 7.3%. PHA blending with starch
Starch is one of the most promising natural polymers due to its inherent biodegradability and abundance. The blending of PHA with starch or starch derivatives (starch acetate) showed some ability in reducing the production cost of PHA with improved mechanical properties.These blends do not form intact films and are brittle in nature due to the incompatibility between the PHA matrix and starch. Lai et al. [22] investigated the properties of modified corn-starch and PHB blend. After blending PHB with the corn starch, only a single glass transition temperature was detected for all PHB and corn starch blends, which increased from 2 to 37°C with an increase in the concentration of corn starch, which gives the indication about the compatibility of
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PHB with corn-starch.
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PHA blending with lignin
Lignin is a macromolecule that is composed of repeated phenyl propane units and
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possesses aliphatic and aromatic hydroxyl groups, as well as carboxylic acid groups. The
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presence of these functional groups makes lignin a valuable material for blending purpose.
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Mousavioun et al. [23] investigated the rheology and thermo-physical properties of blends of PHB and lignin. Lignin was employed as a blending component to improve the properties of
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PHB. The results indicated that lignin can improve the total thermal stability of PHB. 4.1.1.2 Blending of PHA with synthetic biodegradable polymers
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PHA blending with Poly (lactic acid)
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Poly (lactic acid) (PLA) is a chemically synthesized biodegradable polymer from lactic acid. Poly (lactic acid) is environment friendly, linear aliphatic polyester. Several studies have been done that focused on the study of blending of PHA and PLA. Takagi et al. [24] blended a series of epoxy-group-modified PHA (e-PHA) and mcl-PHA with PLA. Furukawa et al. [25] compared the properties of PHB and PLA blends. They observed that PHB and PLA blends were immiscible and highly compatible. PHA blending with Poly (ε-caprolactone) Poly (ε-caprolactone) (PCL) is semicrystalline aliphatic polyester which is biodegradable in nature. PCL possesses high mechanical strength and ductility. Lim et al. [26] blended PCL with poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx). The blending of polymer with PCL may cause PHBHHx to exhibit enhanced toughness with substantial elasticity. Chiono et al. [27] developed a blend of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and
PCL in chloroform. The fibers resulted from these blends exhibited a low degree of bulk and surface porosity. 4.2 Chemical modifications Polyhydroxyalkanoates can be modified by adding a chemical group in the structure of PHA. These chemically modified PHA can be utilized as multifunctional materials. For example, the hydrolytic rate of PHA can be modified by incorporating hydrolysis-prone chemical groups. There are number of methods in which chemical modifications of the PHA can be done, these
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methods are shown in Figure 6 [28] and are briefly described as follows:
Figure 6 - Chemical modifications of PHA 4.2.1 Carboxylation PHA modification by carboxylation is the addition of carboxylic (-COOH) functional group to the substrate. The carboxylation of PHA enhances the hydrophilicity of the polymer through better water penetration. The incorporated carboxylic group acts as active binding site for biologically active moieties like hydrophilic components [29]. Kurth et al. [30] carboxylated
the double bonds of poly (3-hydroxyoctanoate-co-3-hydroxyundecenoate) (PHOU) in the presence of NaHCO3 using KMnO4 as an oxidation agent. Crown ether was used as the phase transfer and dissociating agent for the KMnO4 to carboxylate the unsaturated PHOU. The oxidative carboxylation of unsaturated PHA using KMnO4 as an oxidation agent is shown in
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Figure 7 [28]. Around 70 PHAs had been identified that have such -COOH groups [31].
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4.2.2 Halogenation
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Figure 7 - Oxidative carboxylation of unsaturated PHA using KMnO4 as an oxidation
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Halogenation of PHA is considered as one of the excellent methods in improving the properties, functions and applications of polymers. Halogen atoms (chlorine, bromine and fluorine) are added to unsaturated PHA and saturated PHA through an addition and substitution reaction respectively. Chlorination is one kind of halogenation. The process of chlorination involves the introduction of chlorine to a substrate. Chlorination makes the soft and sticky mclPHA; crystalline, hard and brittle. Arkin and Hazer [32] demonstrated chlorination of PHA which was stiff and crystalline polymer at approximately 54 wt% chlorination. They performed an experiment by adding excess HCl to KMnO4 in a drop wise fashion to generate chlorine gas. Reaction set-up for the chemical modification of PHA via chlorination is shown in Figure 8 [28]. Then they subsequently passed the gas into a solution of sticky unsaturated PHA obtained from Pseudomonas putida. Depending on the amount of chlorine passed, the resulting PHA exhibited higher melting and glass transition temperatures.
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Figure 8 - Reaction set-up for the chemical modification of PHA via chlorination
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4.2.3 Hydroxylation
The properties of PHA can be altered by hydroxylation method [33, 34]. Acid or base
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catalyzed reactions are used in the PHA modification in the presence of low molecular weight mono or diol compounds by the process of hydroxylation. Different approaches to PHA
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modifications by hydroxylation are shown in Figure 9 [28]. Timbart et al. [35] reported the
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production of monohydroxylated oligomers of poly (3-hydroxyoctanoate) (PHO) and poly (3hydroxyoctanoate-co-3-hydroxyundecenoate (PHOU) using both base and acid catalyzed hydrolyses. In base catalysed hydrolysis, the alcoholic NaOH was used to catalyze the hydrolysis at pH 10–14 and the reaction was stopped by adding concentrated aqueous hydrochloric acid. On the other hand, the acid hydrolysis was performed using two different approaches: (i) a reaction catalyzed by para-toluenesulfonic acid monohydrate (PTSA) at 120ᵒC, which was stopped by cooling the mixture in an ice bath; (ii) monohydroxylation by acidic methanolysis at 100ᵒC to yield the respective 3-hydroxymethyl esters bearing a methyl protected carboxylic acid group. It was found that the modified PHO showed lower glass transition and melting temperature than original PHO.
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Figure 9 - Different approaches to PHA modification by hydroxylation
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4.2.4 Epoxidation
The epoxidation of PHA can modify its properties. The high reactivity and easy
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conversion of epoxy groups to anionic and polar groups, even in mild environments, makes
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epoxidation an important strategy for mcl-PHA [28]. Epoxidation of mcl-PHA with mchloroperoxybenzoic acid (m-CPBA) is shown in Figure 10 [28]. Bear et al. [36] performed the
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epoxidation of poly (hydroxyoctanoate-co-10-undecanoates) (PHOUs) in the presence of vinyl group which easily undergoes epoxidation reaction and copolymers were obtained with the help
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of m-CPBA. With increase in epoxy groups, an increase in the thermal stability was also
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observed in the final modified PHA.
Figure 10 - Epoxidation of mcl-PHA using m-CPBA There have been several reports on the epoxidation process. Park et al. [37] epoxidized PHOU with a limited amount of olefinic bonds using m-CPBA. With increasing conversion of
olefinic bonds to epoxy bonds, a decrease in the melting temperature and melting enthalpy was observed. 4.2.5 Grafting Grafting is a PHA modification method in which there exists a covalent bond between polymer chain and other monomers to obtain desired valuable properties. Two different polymer units make up a copolymer.One or more side chains are attached to the main chains in case of grafting. Grafting is an excellent way to synthesize a copolymer with minimum loss of original properties and with addition of some new properties of the polymer [38]. Different types of
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grafting methods are as follows:
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4.2.5.1 Radiations based grafting
Radiations based grafting follows the irradiation of a biopolymer through high energy source or
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by ionizing radiations. PHB is chemically inactive polyester, therefore, chitosan could be grafted
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onto PHB using gamma irradiation along with some different solvents (acetone, ethyl acetate and
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acetic acid) [39]. Gamma irradiations along with different solvents were also used to graft poly (vinyl alcohol) (PVA) onto PHB. The grafted PHB showed alterations in different properties
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such as surface roughness formation of cavities and formation of waves. 4.2.5.2 Free radical based grafting
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A growing chain of polymer has an unpaired electron which acts as site of reaction (a radical site) to react with the unsaturated part of new coming monomer. In this way the unpaired
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electron of growing polymer chain gets transferred to the coming monomer. Free radicals are generated and are transferred to the substrate to react with the monomer which results in a grafted copolymer. Nguyen and Marchessault [40] successfully synthesized a graft copolymer of poly (methyl methacrylate) (PMMA) and PHB using free radical based grafting mechanism. This copolymer had a shape of a comb and exhibited huge alterations in its glass transition temperature (Tg) from 100°C to 3°C. 4.2.5.3 Ionic graft polymerization Ionic grafting is a specific ion based grafting which includes cationic and anionic polymerization reactions. In anionic polymerization, the growing chain of polymer possesses either a negative or a positive charge. Anionic grafting
Anionic graft polymerizations are the grafting methods in which the growing polymer chain contains a negative charge. Anionic catalyst or polymerization initiators are required to start anionic graft polymerization reactions.The most commonly used initiators include Grignard reagents, alkali metal suspensions, Ziegler-Natta catalyst, organic radical anions and metalocene. Anionic grafting offers some narrow molecular weight distribution, block copolymers and stereo control grafted copolymers. The major disadvantage of anionic grafting includes requirements of low temperature and this method is only feasible for limited number of polymers. Cationic grafting
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Cation graft polymerization occurs in growing chain of polymer which contains a positive
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charge. Cationic initiators or cationic catalyst plays very important role in these polymerization reactions. Strong protonic acids, Lewis acids and their complexes are mostly used as initiators.
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The main advantage of cationic grafting is controlling the molecular weight of a grafted
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copolymer. But the main disadvantage of grafting process is that it is moisture sensitive, requires
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low temperature to start the reaction and is limited only to the olefins [38]. 4.2.5.4 Enzyme grafting
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Enzymatic grafting method is more promising due to its less hazardous nature as compared to the risky chemical approaches. One example of enzymatic grafting is laccase based grafting of ethyl
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cellulose onto the surface of PHB. It was demonstrated that enzymatic grafting is an energy saving and environment friendly process for grafting and it provides a suitable environment for
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polymer grafting which results in products with multiple functions [41]. 4.3 Biological modifications of PHA PHA can be modified biologically in following ways: 4.3.1
Co-adding two substrates in culture medium
Various PHA can be modified by co-feeding substrates in the culture medium of bacteria. For example, when cells of Halomonas bluephagenesis are supplied with glucose and propionic acid or other propionogenic carbon sources under nitrogen limited conditions, it produces PHBV copolymer depending upon their concentrations [6]. 4.3.2 Feeding the culture with functional group containing substrates PHA can be modified biologically by feeding the bacterial culture medium with different substrates containing functional groups. For example, Pseudomonas putida when cultivated in
the presence of ω-phenoxyalkanoates produces PHA with phenoxy groups in the side chains [42]. 5. Applications of PHA Biodegradable nature and other properties of polyhydroxyalkanoates such as high temperature stability, low degree of surface porosity, enhanced toughness, elasticity, etc. results in their applications in various fields like medical, agriculture, etc. Some applications of PHA are
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as follow 5.1 Medical Applications
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PHA has properties quite similar to those of synthetic plastics. The unique properties like
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biodegradability, biocompatibility and non-toxicity make them desirable materials for biomedical applications. PHA has been found suitable for many applications in medical field.
Drug delivery
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5.1.1
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Some medical applications of PHA are as follows
Technique of drug delivery is emerging as an interdisciplinary science aimed at improving the
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health of human. The major goal in the drug delivery system is that the drugs should be delivered in a controlled and targeted manner in order to improve their efficacy. Hence, PHA, due to their
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origin from natural sources, is used as raw material for producing tablets, nano-particles, etc. [43,
5.1.2
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44]. For example: PHB, PHVB and their copolymers are used for delivery of antibiotics. Tissue engineering
PHA with little modifications contributes very much in the field of tissue engineering. PHA can be used to produce scaffolds which promote cell growth by supplying nutrition, have higher mechanical strength. These scaffolds are available in the form of pins, films, sutures, etc. For example: 3D scaffolds developed by using PHA nanofibers are used for cartilage repair [45]. 5.1.3
Dressings
Wound dressing is carried out with the help of volatile solutions of PHA [46]. These materials form a thin film over the wound to prevent contamination. Dressing based on non-woven fibrous material of P (3HB) can be used as swabs, fleece, lint, etc. There are many other medical applications of these polymers which include soft tissue regenerations, nerve repair, cardiovascular treatments, etc. [45].
5.2 Agricultural applications PHA has many applications in the field of agriculture. PHA improves the nutritional properties and shelf life of the plants under stressful environments. One of the specialized applications of P (3HB) in agriculture is the controlled release of pesticides and insecticides. Potential applications of PHAin agriculture include: biodegradable plastic films for crop protection, seeds encapsulation, encapsulation of fertilizers, etc. [47].
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5.3 Market applications
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PHA bioplastics possess properties making them suitable replacements for many of the applications currently served by petroleum‐based plastics, thus providing tremendous market
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potential. The versatility of PHA results in their wide applications in market. The main uses of
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PHA are seen in packaging and food services due to their degradable nature, non solubility, non permeable nature and flexibility [48, 49]. PHA are developed as packaging films mainly for use
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as shopping bags, paper coatings, cups, diapers, carpets, etc. Many products are now available in
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6. Drawbacks of PHA
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the form of PHA coated boxes, paper, sheets, boards, etc. [50].
These days, PHA are gaining lot of attention due to their biodegradable nature but their
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high cost and requirement of complex substrates for their production leads to a decline in their demands in different fields. These are about 15 times more expensive than the petroleum derived polymers [51]. One of the major limitations of the industrial production of PHA is to maintain the optimal bacterial growth conditions. Most fermentation processes do not allow maximum synthesis of PHA granules at the end of the cultivation. 7. Strategies for reduction on PHA production cost 7.1 Inexpensive substrate High cost of PHA is related to the substrates that account for 30-40% of the total production costs. PHA can be produced from cheap substrates such as sucrose, glucose, lignin, waste material, etc. New pathways to utilize cheap substrates can be established for PHA
production [52]. For example, by integrating genes encoding aqua glyceroporin and glycerol kinase from E.coli into chromosome of Cupriavidus necator H16, the resulting recombinant could produce large amount of PHB from glycerol [53]. Many other cases have been reported, such as co-productions of PHA with amino acids, proteins, alcohols, biosurfactants and other chemicals, etc. 7.2 Developing better bacterial strains Using transgenically modified bacterial strains is another strategy to decline the price of
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PHA production and to increase the yield of PHA. For example: Metabolically engineered
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mutant strain, C. necator expresses genes that encodes L-arabinose catabolic enzymes, and
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therefore can use L-arabinose as a carbon substrate to accumulate high level of PHB [54].
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7.3 Reduction on cost of separation and purification
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The downstream processing of PHA mainly includes cell and broth separation, product purification. The separation requires expensive centrifugation, micro-filtration and other time-
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consuming processes [55]. Therefore, it is necessary to develop methods that permit the recovery of PHA by a simple, efficient and less polluting process. Some of the processes are: Self-flocculation
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7.3.1
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Self-flocculation refers to the ability of some microorganisms to display controlled aggregation, leading to the formation of extensive but highly compact cell flocs; this reduces the difficulty of recovering biomass from a culture [56]. Self-flocculation brings benefits of recycling the fermentation media. After the collection of precipitated cell mass, the supernatant can be again used for the growth of the net batch without sterilization or inoculation. 7.3.2
PHA purification by animals
An effective biological recovery approach for PHA was developed using Tenebrio molitor, a mealworm, which was shown to readily consume the freeze-dried C. necator cells and excrete the PHA granules in the form of whitish feces. Further purification using water, detergent and heat resulted in almost 100% pure PHA granules [57, 58]. This significantly simplifies the purification process. 7.3.3 Balancing power reduction
The accumulation of PHA requires vast energy consumption. Continuous fermentation process is one way to reduce the power cost for PHA production. It also reduces the manpower, material resources consumption and can also enhance the production efficiency [59]. 8. Future perspective Polyhydroxyalkanoates are produced by different microorganisms under stress conditions. PHA are easily biodegradable and sustainable, thus, are considered as the plastics of future.These bioplastics have the potential to replace the synthetic plastics, which can reduce the chances of
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development of junk of synthetic plastics. Many PHA possess objectionable properties such as
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crystallinity, brittleness, etc. which can be reduced by different methods of modifications. As
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very low-cost substrates like starch, PLA, cellulose, etc. are used in the PHA production, these low-cost polymers also play role in modifications of PHA which can lead to the production of
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high yield of PHA at very reasonable prices. In the future, production of these high-valued
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polyhydroxyalkanoates and other metabolites from the microorganisms using domestic, industrial, agricultural wastes as substrates may help us to overcome the issues related to
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environmental pollution, its commercialization and high cost and could enhance its implementation at industrial scale. PHA will become more prominent in the coming years and
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9. Conclusion
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will increase the growth rate of various industries.
This review comprises of a brief outlook of poyhydroxyakanoates, their modifications and applications of PHA in various fields. PHA has drawn much attention due to their biological origin, biodegradable and biocompatible properties. These properties of biopolymers can be improved. There are many different modification approaches that have been employed to alter the polymer and its properties. This review summarizes the well-established modification approaches which include blending, chemical and biological modifications. These modifications can be further improved and new cheaper modification strategies can be adopted to minimize the poor properties of PHA. One approach for which is by replacing the expensive substrates with novel waste materials for modifications. The use of these modified biopolymers limit carbon dioxide emissions during creation and degrade to organic matter after disposal. Their degradation
products are harmless to the natural environment and to the in vivo systems. They offer great potential values in various fields like medical, agriculture, cosmetics, pharmacy, etc. Acknowledgements The fellowship granted to Ms. Rutika Sehgal in the form of SRF from Council of Scientific and Industrial Research (CSIR) is thankfully acknowledged. The financial support from Department of Biotechnology, Ministry of Science and Technology, Govt. of India, to Department of Biotechnology, Himachal Pradesh University, Shimla, India, is also thankfully
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acknowledged.
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Highlights •
Replacement of conventional synthetic plastics with PHA
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PHA possesses properties like biodegradability, renewability, biocompatibility, etc.
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Modifications of PHA using physical, chemical and biological methods for their
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efficient production
Declaration of interests ☑ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: