Polyhydroxyalkanoates, the bioplastics of microbial origin: Properties, biochemical synthesis, and their applications

Chemosphere 294 (2022) 133723
Contents lists available at ScienceDirect
Chemosphere
journal homepage: www.elsevier.com/locate/chemosphere
Polyhydroxyalkanoates, the bioplastics of microbial origin: Properties,

biochemical synthesis, and their applications
Shivananda Behera, Monika Priyadarshanee, Vandana, Surajit Das *
Laboratory of Environmental Microbiology and Ecology (LEnME), Department of Life Science, National Institute of Technology, Rourkela, 769 008, Odisha, India
H I G H L I G H T S G R A P H I C A L A B S T R A C T
• PHA is intracellularly accumulated

molecules in microbial cells.
• Chemo-mechanical properties of
biocompatible PHA resemble synthetic
plastics.
• phaCAB operon is responsible for the
synthesis of PHA.
• PHAs are widely used in the industrial,
environmental and biomedical sectors.
A R T I C L E I N F O A B S T R A C T
Handling Editor: Mathimani Thangavel The rising plastic pollution deteriorates the environment significantly as these petroleum-based plastics are not
biodegradable, and their production requires natural fuels (energy source) and other resources. Poly
Keywords: hydroxyalkanoates (PHAs) are bioplastic and a sustainable and eco-friendly alternative to synthetic plastics.
Polyhydroxyalkanoate PHAs can be entirely synthesized using various microorganisms such as bacteria, algae, and fungi. These value-
Bioplastic
added biopolymers show promising properties such as enhanced biodegradability, biocompatibility, and other
Biopolymer
chemo-mechanical properties. Further, it has been established that the properties of PHA polymers depend on the
Metabolites
Genetic regulations substrates and chemical composition (monomer unit) of these polymers. PHAs hold great potential as an alter
native to petroleum-based polymers, and further research for economic production and utilization of these
biopolymers is required. The review describes the synthesis mechanism and different properties of microbially
synthesized PHAs for various applications. The classification of PHAs and the multiple techniques necessary for
their detection and evaluation have been discussed. In addition, the synthesis mechanism involving the genetic
regulation of these biopolymers in various microbial groups has been described. This review provides infor
mation on various commercially available PHAs and their application in multiple sectors. The industrial pro
duction of these microbially synthesized polymers and the different extraction methods have been reviewed in
detail. Furthermore, the review provides an insight into the potential applications of this biopolymer in envi
ronmental, industrial, and biomedical applications.
* Corresponding author.
E-mail addresses: [email protected], [email protected] (S. Das).
https://doi.org/10.1016/j.chemosphere.2022.133723
Received 24 September 2021; Received in revised form 13 January 2022; Accepted 20 January 2022
Available online 24 January 2022
0045-6535/© 2022 Elsevier Ltd. All rights reserved.
S. Behera et al. Chemosphere 294 (2022) 133723
1. Introduction microbial sources (Kavitha et al., 2018; Grigore et al., 2019). However,
the current review comprehensively summarizes the biosynthesis of
In recent years, the increasing awareness towards environmental PHA from microbial sources, properties of these biopolymers, com
sustainability and green chemistry has played a significant role in mercial production, different extraction procedures, and application of
developing the next generation of materials, products, and technologies. these value-added products. The comprehension of extraction proced
The long-term persistence of plastics in the environment has drawn the ures and screening of efficient PHA producing microbes will be further
attention of researchers towards the development of sustainable poly helpful for synthesis of these eco-friendly biopolymers.
mers from renewable resources. Petroleum-based plastics are considered
a serious environmental threat, and the global production of these 2. Classification of polyhydroxyalkanoates (PHA)
plastics have reached 359 million metric tons in 2018 (Chia et al., 2020).
The global market of PHA was evaluated as US$73.6 million and is 2.1. General structure and composition
projected to reach US$93.5 million by 2021 (Khatami et al., 2021).
These plastics have been used for various purposes in day-to-day life. PHAs are polyesters of hydroxyalkanoate (HA), synthesized by
However, the major drawback of these polymers is their various microorganisms, habituating different ecological niches. These
non-biodegradable nature (Shen et al., 2020). Thus, to tackle the current biogenic polyester compounds typically range from 0.2 to 0.5 μm in
scenario, several studies aimed at producing environmentally friendly diameter and are stored as insoluble inclusion bodies in the cytoplasm
alternatives to petrochemical plastics. The requirement for such an (Obruca et al., 2020). PHAs are synthesized in the cell in adverse con
alternative led to the emergence of various value-added biopolymers. ditions, such as in scarcity of oxygen and essential nutrients like phos
These biopolymers include polyhydroxyalkanoates (PHA), polylactic phorous or nitrogen. However, the presence of a carbon source is a
acid (PLA), polycaprolactone (PCL), polyethylene, and polyesteramide, prerequisite for the biosynthesis of PHAs (Sagong et al., 2018). The
etc. (Kabir et al., 2020). PHAs are also known as bioplastic, and unlike microbial cells producing PHAs are osmotolerant towards these poly
petroleum-derived plastics, they are derived from renewable resources meric materials and thus store them at high concentrations (Anderson
such as vegetable oil, starch, and proteins. The applications of these and Dawe s, 1990). Unlike other biopolymers such as polylactic acid
biopolymers over petroleum-based plastics can be advantageous for (PLA) or polyethylene terephthalates (PET), PHAs are biodegradable
conserving fossil resources and reducing CO2 emissions to the environ and highly resemble synthetic plastics in terms of their materialistic
ment, thus making them an essential innovation of sustainable devel properties. PHAs have been extensively studied because their properties
opment (Bugnicourt et al., 2014). make them the most potential biopolymers (Riaz et al., 2021).
The synthesis and accumulation of PHAs occur intracellularly by a
wide range of microorganisms in high concentrations as reserve mate 2.2. Diversity of PHA
rials for carbon and energy under nutrient-deficient conditions (Abd
El-malek et al., 2020). In some cases, the spontaneous production of A wide range of microorganisms produces PHAs. However, their
these biopolymers has been observed in a few microorganisms. These molecular weight and composition vary greatly depending on the
biopolymers can be synthesized by Gram-positive and Gram-negative growth condition and the source organism (Elmowafy et al., 2019).
bacteria, fungi, yeast, and algae (Anitha and Srivastava, 2021). In Depending upon the number of carbon atoms constituting the mono
addition, the use of seaweed and plants for the production of PHA has meric units, PHAs are categorized into two major groups, namely, (i)
been explored. The use of red seaweed (Kappaphycus alvarezii and Geli Short-chain length (SCL) PHAs, and (ii) Medium-chain length (MCL)
dium amansii), and green macroalgae like Ulva for PHA production has PHAs (Raza et al., 2018). PHAs with a monomeric unit having up to 5
been established (Ghosh et al., 2019; Sudhakar et al., 2020). Apart from carbon atoms are classified as short-chain length (SCL) PHAs such as P
this, the commercial production of PHA has been carried out using oil (3HB)/PHB (Wang et al., 2016). P (3HB)/PHB is a homopolymer char
from the Camelina seed (Bustamante et al., 2019). However, there are acterized by a monomeric unit (3-Hydroxybutyrate) of 4 carbon atoms.
various challenges associated with the large-scale microbial production The other well-known biopolymers of this group are poly (4-Hydrox
of these biopolymers. These challenges include the cost of the carbon ybutyrate) and poly (3-Hydroxyvalerate). Alternatively, the PHAs hav
sources metabolized by the microbes for PHA production and the higher ing monomer units containing 6 to 14 carbon atoms are known as
energy requirement of the process (Fradinho et al., 2013). Various Medium-chain-length (MCL) PHAs. These include the poly (3-hydrox
renewable resources such as organic wastes, agro wastes, wastewater, yhexanoate), P (3HHx), poly (3-hydroxyoctanoate), P (3HO), and het
plant oil, animal fats, waste cooking oil, etc., have been used (Sathya eropolymers such as poly (3-hydroxybutyrate-co-3-hydroxyvalerate),
et al., 2018). Moreover, various advanced energy-efficient and (PHBV), and poly (3-hydroxyhexanoate-co-3-hydroxyoctanoate), P
eco-friendly technologies for the extraction and purification of PHA (3HHx-co-3HO) (Keshavarz and Roy, 2010). Apart from these two
have been employed (Kourmentza et al., 2017). groups, PHAs having monomer units containing more than 14 carbon
Among the various PHAs, poly (3-hydroxybutyrate)/poly atoms are known as long-chain-length PHAs.
hydroxybutyrate known as P3HB/PHB was first reported in different The highly diverse “R” group in PHAs is the prime reason for their
microbial strains, and subsequently, other monomer constituents were structural diversity. This “R” group can either be an aromatic or an alkyl
discovered (Albuquerque and Malafaia, 2018). About 150 different group (Grigore et al., 2019). Further, the saturation pattern in these
monomers constitute various PHAs (Sharma et al., 2021). The polymeric groups is not constant as some are saturated while others are highly
PHAs share similar properties with petroleum-based polymers, such as unsaturated (Luef et al., 2015). Apart from the structural diversity of
high melting temperature and high tensile strength. The other properties PHAs, SCL and MCL-PHAs have remarkable differences based on the
like biodegradability, biocompatibility, and piezoelectricity make the purity and recovery rate during the isolation process. For example,
PHA an attractive biopolymer worldwide for their use in biomedical solvent extraction of PHAs with acetone results in MCL PHAs (80–90%
applications. This review article aims at understanding the diversity and pure) with a recovery in between 60 and 80%, whereas the same process
properties of these microbially synthesized PHAs. Besides, the biosyn yields SCL-PHAs with much higher purity (98.4%) and recovery (96.8%)
thesis and genetic regulation of these value-added biopolymers have (Tan et al., 2014). In the natural environment, the microbial community
been elaborated. Various environmental, industrial, and biomedical can produce different types PHAs with structural diversity. These
applications of PHAs are also described in detail. Microbial PHAs are a polymers are intracellularly accumulated in the form of insoluble
family of biodegradable and biocompatible polyesters that are sustain granules inside the cytoplasm. An organized layer of proteins is found
able and eco-friendly alternatives to petroleum-based plastics. There are surrounding these granules, known as granule-associated proteins
various reports on the biosynthesis and production of PHAs from (Fig. 1). The vast diversities of PHAs are due to the monomeric units,
2
Fig. 1. PHA granules synthesized inside the prokaryotic cell. In a microbial community, the synthesis of PHAs is dependent upon the microorganism involved in its
synthesis. In the intracellular PHA granules, the granule-associated protein Phasin is the most abundant as structural proteins. The other proteins include PHA
synthase, PHA depolymerase, and regulator proteins associated with the PHA granules. Microbially synthesized PHAs with different chemical structures are (A)
Polyhydroxybutyrate (P3HB), (B) Polyhydroxyvalerate (P3HV), (C) Polyhydroxyhexanoate (P3HHx), (D) Poly (3-hydroxyoctanoate) P (3HO), (E) Poly (3-hydrox
ybutyrate-co-3-hydroxyvalerate) (PHBV) (F) Poly (3-hydroxyhexanoate-co-3-hydroxyoctanoate) P (3HHx-co-3HO).
functional groups, chain length, and molecular weight. The diversified employed for structural and conformational characterization of PHAs
structure and composition offer these polymers a wide range of physical (Table 1). These techniques are faster and more reliable than conven
properties which can be exploited for various applications. tional ones (Koller and Rodríguez-Contreras, 2015). The identification
and precise quantification are necessary for PHA purification so that the
purified polymer can be utilized directly or linked to another monomeric
2.3. Techniques involved in the characterization of PHA unit to form a copolymer.
Although various methods have been used to extract and estimate 3. Biosynthesis of PHA
PHA content in microorganisms, many of them are challenging, tedious,
and expensive, requiring multiple purification steps with organic sol Although both chemical and biological approaches can be applied for
vents. These methods decrease the efficacy of the downstream pro PHA synthesis, PHAs with higher molecular weight can be easily pro
cessing, and also they are not eco-friendly. Strazzullo et al. (2008) duced by biological means compared to chemical approaches (Chen,
proposed a simplified and highly efficient method for PHA extraction. 2010). The metabolic pathway for PHA production varies significantly
This method included the ultrasound shaking of cell biomass dissolved among different microbial groups. These biopolymers are synthesized in
in distilled water for their complete dispersion. Further, the dispersed both stationary and exponential phases of microbial growth. In the
biomass is digested using Sodium Dodecyl Sulphate (SDS) and thermal exponential phase, PHAs are produced under favorable balanced growth
treatment. Various advanced techniques including, Fourier Transform conditions. In the stationary phase, the limitation of nutrients like ni
Infrared (FTIR), Nuclear Magnetic Resonance (NMR), X-ray Diffraction trogen, phosphorous, oxygen, and excess carbon sources leads to the
analysis (XRD), Differential Scanning Calorimetry (DSC), have been
Table 1
Techniques and principles for detection and determination of PHAs.
Techniques Principles Qualitative or Required Distinguish References
Quantitative duration between PHAs
Optical microscopy Microscopic observation of cells on a galss slide after staining Qualitative Very less No Morgan-Sagastume
with Sudan Black and counter stain with safranin (2016)
Fluorescence Incubation of microbial colonies on agar medium containing Qualitative Less No Akinmulewo and Nwinyi
microscopy Nile blue stain (2019)
Solvent extraction Lysis of cell using sodium hypochlorite and extraction of PHA Quantitative High No Chavan et al. (2021)
method using chloroform
UV spectroscopy Conversion of PHA into crotonic acid by addition of hot Quantitative Less No Chavan et al. (2021)
H2SO4, Determination of OD at 235 nm
FTIR Study the IR-spectrum of PHA in a spectral range Qualitative and Semi Less No Tufail et al. (2017)
of 400–4000 cm− 1 quantitative
NMR 1H:for SCL-PHA Qualitative and High Yes Tripathi et al. (2013)
13C:for MCL-PHA Quantitative
GCMS Detection of PHA by MS Quantitative High Yes Samrot et al. (2021)
HPLC Separation of PHAs by reverse phase column using MS Quantitative High Yes Tripathi et al. (2013)
XRD Report crystal and mechanical properties of PHA Quantitative High Yes Cha et al. (2016)
DSC Determination of Thermal stability of PHA Quantitative High Yes Stanley et al. (2020)
3
synthesis and accumulation of PHAs. Excess nutrients are stored by the Pathway II of PHA synthesis is concerned with the utilization of fatty
microbial cells in the form of PHAs, and they are mobilized at the advent acids by microorganisms. After the β-oxidation of fatty acid, the pro
of favorable growth conditions. Moreover, the presence of a carbon duced acyl-CoA synthesizes PHA monomers. In this pathway, enzymes
source is a prerequisite for microbial PHA synthesis. including 3-ketoacyl-CoA reductase, epimerase, (R)-enoyl-CoA
hydratase/enoyl-CoA hydratase I, acyl-CoA oxidase (putative) are
involved. The 3-hydroxyacyl-CoA molecule functions as a precursor
3.1. Common pathways for biosynthesis of PHA
molecule for the synthesis of PHA. Using this pathway, various micro
organisms, including Pseudomonas putida, P.aeruginosa, and Aeromonas
PHB, the most common homopolymer among PHAs, has been stud
hydrophila synthesize MCL-PHAs (Manoli et al., 2020). Pathway III for
ied extensively in various microorganisms. The utilization of glucose for
PHA synthesis requires two key enzymes, 3-hydroxyacyl-ACP-CoA
PHA biosynthesis is the most common pathway in microorganisms. This
transferase (PhaG) and malonyl-CoA-ACP transacylase (FabD). These
pathway is followed by the generation of acetyl-CoA and NADPH via
enzymes supply the precursor (3-hydroxyacyl-ACP), which is further
glycolysis and pentose phosphate pathways. Further, the acetyl-CoA is
converted into 3-hydroxyacyl-CoA. The next step is catalyzed by PHA
converted into acetoacetyl-CoA utilizing the enzyme β-ketothiolase
synthase to synthesize PHA (Zhang et al., 2020). Apart from this, some
(PhaA). The NADPH plays a critical role in the next step, as it is used as a
other pathways involving a wide range of proteins are also found in
cofactor to reduce acetoacetyl-CoA to 3-hydroxybutyryl-CoA by
various microbes (Table 2).
acetoacetyl-CoA dehydrogenase enzyme (PhaB). The last step of the PHB
synthesis involves the polymerization of 3-hydroxybutyryl-CoA into
PHB, and it is catalyzed by P (3HB) polymerase (PhaC) (Mohapatra 3.2. Interaction of associated proteins with PHA
et al., 2017). In addition, the PHA synthesis increases with an increase in
the ratio of NADPH to NADP+ (Alsiyabi et al., 2021). Studies involving The structural composition of PHA revealed that the isolated PHA
the PHA biosynthesis revealed mainly three different pathways for mi granules are composed of 97.5% PHA and a small concentration of
crobial synthesis of PHA (Fig. 2). proteins and phospholipid (Jendrossek and Pfeiffer, 2014). The most
Pathway I of PHA biosynthesis is mainly governed by three enzymes common granule-associated proteins are phasins (a group of low mo
such as β-ketothiolase, NADPH dependent acetoacetyl-CoA reductase, lecular weight proteins). These metabolically active regulatory proteins
and PHA synthase. These enzymes are encoded by phaA, phaB, and phaC, associated with PHA granules organize the biopolymer into complex
respectively (Philip et al., 2007). Ralstonia eutropha was reported to subcellular entities designated as carbonosomes (Jendrossek, 2009).
synthesize PHA following this pathway (Raberg et al., 2018). The The most important enzyme involved in PHA synthesis is PHA
Fig. 2. Major pathways for biosynthesis of PHA in microorganisms. Pathway I converts sugar molecules into PHA by utilizing PhaA, PhaB, and PhaC enzymes.
Pathway II involves FabD, FabG, PhaG, and PhaC enzymes to convert sugar into PHA. Pathway III utilizes the fatty acid molecules and FabG, PhaJ, and PhaC to
produce PHA. PhaC is the crucial enzyme required in all three pathways and catalyzes the formation of PHA.
4
Table 2 conditions. Depolymerases responsible for PHB degradation is among

Pathways and the main enzymes involved in PHA biosynthesis. the most extensively studied enzymes. Depolymerases activity has been
Pathways Enzymes Organisms References assayed in various laboratories using PHB granules artificially coated
with detergent (aPHB) (Jendrossek, 2007). However, aPHB is not a
Pathway I β-Ketothiolase (PhaA), Ralstonia eutropha Philip et al.
NADPH dependent (2007); Chen natural substrate for depolymerase enzyme, and assay involving this
acetoacetyl-CoA (2010) may not be a replica of the in vivo condition. Hence the physiological
reductase (PhaB), function of this enzyme is not yet completely understood and demands
PHA synthase (PhaC) further studies.
Pathway 3-Ketoacyl-CoA Pseudomonas putida Chen (2010);
II reductase, KT2442, Manoli et al.
Epimerase (FabG), Aeromonas hydrophila (2020)
(R)-Enoyl-CoA hydrataseI 4AK4, 3.3. Role of phasins in PHA metabolism
(Pha J) Pseudomonas
PHA synthase (PhaC) aeruginosa The most abundant granule-associated proteins found on the PHA
Pathway Malonyl-CoA-ACP Pseudomonas Zhang et al.
granules are phasins (Mezzina and Pettinari, 2016). Pieper-Fürst et al.
III transacylase (FabD), mendocina (2020); Chen
3-hydroxyacyl-ACP-CoA (2010) (1994) identified these phasins in the PHA granules of Rhodococcus
transferase (PhaG), ruber. They found a predominant protein (GA14) with low molecular
PHA synthase (PhaC) weight, forming a layer at the surface of the PHA granules. The protein
Pathway NADH-dependent Rhizobium (Cicer) sp. Tan et al. was named phasin (PhaP), which was analogous to another protein
IV acetoacetyl-CoA CC 1192 (2014)
reductase (PhaB)
known as oleosins, located on the surface of triacylglycerol in plants.
Succinic semialdehyde Phasin has been found in association with all the naturally synthesized
dehydrogenase (SucD) PHA in microorganisms. Several phasins were found in various bacterial
Pathway Succinic semialdehyde Clostridium kluyveri Cal et al. strains, such as R. eutropha (Pötter and Steinbüchel, 2005), Bacillus
V dehydrogenase (SucD) (2021)
megaterium (Kihara et al., 2017), R. ruber (Maestro and Sanz, 2017), etc.,
4-Hydroxybutyrate
dehydrogenase (4hbD) as well as in the archaea (Haloferax mediterranei) (Cai et al., 2012). The
4-Hydroxybutyrate-CoA: phasins from H. mediterranei showed similarity with other recognized
CoA transferase (OrfZ) archaeal phasins, but no resemblance was found with the bacterial
Pathway Lactonase, putative Mutants and Sathya et al. phasins.
VI Hydroxyacyl-CoA recombinant of (2018)
synthase, putative Alcaligenes eutrophus
Early investigation on phasins suggested that these proteins are not
Pathway Alcohol dehydrogenase, Aeromonas hydrophila Xie and Chen part of a highly conserved family. The number of phasin reported earlier
VII putative 4AK4 (2008) is used to define various protein motifs. Four phasin-related families are
Pathway Cyclohexanol Acinetobacter sp. Yan et al. found in pfam database, and these protein families are differentiated
VIII dehydrogenase (ChnA) SE19, (2017)
from each other based on their characteristic domain and sequence
Cyclohexanone Brevibacterium
monooxygenases (ChnB) epidermidis HCU similarity (Table 3). Although most of the reported phasins have been
Caprolactone hydrolase obtained from Proteobacteria (PF09650), the classification of phasins
(ChnC) provides information about the phylogenetic origin of these proteins and
6-Hydroxyhexanoate the type of associated PHA (Maestro and Sanz, 2017). In Pseudomonas,
dehydrogenase (ChnD)
two families such as PF09650 and PF09361 contain phasins that bind to
6-Oxohexanoate
dehydrogenase (ChnE) SCL-PHA, whereas those associated with MCL-PHA are categorized as
PF05597 family (Mezzina and Pettinari, 2016).
PHA granules consist of hydrophobic polyester core surrounded by
synthase which catalyzes the polymerization of hydroxyacyl-coenzyme an amphiphilic layer (Obruca et al., 2020). The purification and char
A (CoA) to PHA and free CoA (Mohapatra et al., 2017). Several PHA acterization of phasin (GA14) isolated from R. ruber suggested that the
synthases from various sources have been identified and investigated two hydrophobic domains present in the C-terminus of the protein
biochemically. It has been observed that the PHA synthases are not interact with the PHA (Pieper-Fürst et al., 1994). Later, it was found that
homologous to any other proteins available in the data banks, but they many phasins do not possess any hydrophobic domain, and the amphi
are homologous to each other. Thus, based on the primary structures and philic helices present in these proteins help in interaction with the PHA
substrate specificity, they have been categorized into a separate class of granules (Mezzina et al., 2015). Hence, the amphipathic nature of these
homologous proteins (Sudesh et al., 2000). A conserved cysteine residue proteins is essential not only for their interaction with PHA but also for
acting as the active site of the enzyme is shared among all the PHA interaction with other granule-associated proteins.
synthases (Singh et al., 2018). Among other conserved residues, histi Phasins have been found to play a critical role in the metabolism
dine and aspartate are recognized as the most important, and with the (biosynthesis of PHA, its accumulation, and utilization) of PHA granules
conserved cysteine, these form the catalytic triad of the enzyme. This (Pötter and Steinbüchel, 2005). The genetic analysis of phasin was
catalytic triad is similar to the esterases, suggesting an α/β hydrolase
fold in the PHA synthases (Jendrossek, 2009).
Table 3
The prime objective of accumulating PHAs in the microbial cells is to Classification of Phasins based on the Pfam database.
provide them with nutrients (carbon) and energy required for various
Family name Type of protein Organisms Dominant Phasins
physiological activities during the nutrient-deficient condition (Koller (Protein)
et al., 2011). The utilization of PHAs by microbes is governed by
Phasin_2 Phasin protein Ralstonia PhaP from
depolymerization of PHA, which is regulated by an enzyme is known as
(PF09361) Ralstonia eutropha
intracellular PHA (iPHA) depolymerase (PhaZs) (Anis et al., 2017). This PhaP_Bmeg Polyhydroxyalkanoic Bacillus PhaP from Bacillus
iPHA depolymerase was first reported in P. oleovorans, and the micro (PF09602) acid inclusion protein megaterium
organisms used it for the depolymerization of MCL-PHAs (Jendrossek, PHA_gran_rgn Putative Proteobacteria Uncharacterized
2009). (PF09650) polyhydroxyakanoic acid group of phasins
system protein
Although in vivo study of PHA depolymerase is difficult due to their Phasin Poly (hydroxyalkanoate) Pseudomonas PhaF from
amorphous nature and outer protein layer, recent advanced studies re (PF05597) granule associated Pseudomonas
ported the activity of iPHA depolymerase under defined in vitro protein putida
5
performed for the first time in R. eutropha to explore their role in PHA control the release of drugs. The use of these polymers as a packaging
metabolism (Wieczorek et al., 1995). It has been observed that the and coating material has also been reported (Fabra et al., 2016).
amount of PhaPRe in microbial cells is highly dependent upon the con
centration of PHA granules present in the cells. If the PHA synthesis is 4.1. Thermal properties
hindered because of some unfavorable condition or mutation in the PHA
synthesizing gene, the concentration of phasins also reduces. Hence, it The biopolymeric PHAs exhibit various properties like high tensile
can be inferred that the synthesis of phasins is positively correlated with strength (30–35 MPa) and high melting point (175 ◦ C), which are similar
PHA synthesis. PhaPRe can also influence class II PHA synthase activity to that of the polymers composed of petroleum by-products like poly
by regulating phaC1 and phaC2 genes (Qi et al., 2000). Mutation in phaP propylene (El-Hadi et al., 2002). As PHAs are partially crystalline, the
gene reduced the number and size of the PHA granules and significantly thermal properties of the amorphous phase of these polymers are studied
modified the morphology of the granules. In phaP mutant, a single PHA in terms of glass transition temperature (Tg). However, the thermal
granule was formed, which was more significant in size than the small properties of the crystalline phase are described in terms of melting
and numerous granules synthesized in overexpressed phaP. Another temperature (Tm) (Grigore et al., 2019). The melting temperature of
study involving a mutant Synechocystis sp (strain PCC 6803) showed that PHA polymers is directly proportional to the number of carbon atoms in
the absence of phasin (PhaPSp) reduces the activity of PHB synthase the side chain of the polymer. In contrast, Tg is inversely proportional to
(Hauf et al., 2015). This analysis further revealed that phasins could the length of the side chain. Further, it was reported that an increase in
influence the size (surface-to-volume ratio) and number of PHA in the number of carbons from C4 to C7 in the side-chain (3HA) led to a
R. eutropha. 24 ◦ C increase in its Tm (45 ◦ C–69 ◦ C). However, unlike Tm, a decrease
Genetic regulation study on PhaPRe reported that phaP is regulated in Tg was observed with increased carbon atoms in the side chain (from
by another protein (PhaR) associated with PHA granules which bind to 1 to 7) (Gopi et al., 2018).
the upstream of phaP. It was proposed that during the biosynthesis of The SCL-PHAs have a lower Tg value than the MCL-PHAs, which
PHA, the PhaR protein remains bound to the granules and allows its further contributes to the increased crystallinity and brittleness of SCL-
synthesis. In contrast, when there is a high accumulation of PHA, the PHAs. For instance, the SCL-PHA, such as P (3HB), has a glass transition
PhaR becomes separated from the PHA and binds upstream of phaP, temperature of 4 ◦ C and a melting temperature of 180 ◦ C and is highly
inhibiting its transcription (Pötter et al., 2002). In several other bacteria, crystalline, stiff, and brittle (Martin and Williams, 2003). The SCL-PHAs
there are various proteins analogous to the PhaR, for example, PhaQ in have spherulite domains which are the prime reason for their brittleness.
B. megaterium (Biedendieck et al., 2021) and PhaFPp in Pseudomonas On the other hand, the MCL-PHAs have a Tg value between − 25 ◦ C and
(Tarazona et al., 2020). The archaeon, H. mediterranei, also possesses the − 65 ◦ C and Tm value ranging from 42 ◦ C to 75 ◦ C (Larrañaga and Liz
PhaR protein, but the pattern of regulation of phaP by PhaR is different undia, 2019). These PHAs are less crystalline than the SCL-PHAs due to
from that of the bacteria. PhaR protein regulates the transcription of irregular side groups in MCL-PHAs that hinder the tight packing in the
phaR and phaP genes (Cai et al., 2015). Further, the in vitro studies polymers (Sánchez et al., 2003). The Tg and the crystalline structure are
involving R. eutropha revealed that phasins could form both responsible for the elastomeric property of these polymers. Among all
homo-oligomers and hetero-oligomers. It was reported that phasins the microbially synthesized biopolymers, only MCL-PHAs have proper
interact with various other proteins, for instance, PhaR and PhaZ1 (PHB ties similar to thermoplastic elastomers (Grigore et al., 2019). However,
depolymerase) (Jendrossek and Pfeiffer, 2014). Another phasin (PhaM), these polymers exhibit elastomeric behavior within a narrow tempera
isolated from R. eutropha, was reported to be the physiological activator ture range due to their low Tm. These thermoplastic polymers become
of the PHB synthase (Sharma et al., 2008). fluid and easy to mold at a temperature above their Tm, and become
Besides the structural role, various phasins are also involved in completely amorphous (Wecker et al., 2015). Moreover, a decrease in
accumulating and degrading these granules. For example, the PhaP the Tg of MCL-PHAs was observed with the increase in the average side
phasin in R. eutropha present on the surface of the PHA granule plays a chain length (Rai et al., 2011). This observed characteristic was due to
crucial role in the degradation of these biopolymers. Further analysis of the increase in mobility of polymer chains. The thermal properties of the
this phasin suggested that PhaP may help in the degradation of PHA PHAs are also dependent on the substrate used for their synthesis (Lorini
directly (by interacting with PHB depolymerases) or indirectly (allowing et al., 2021). For instance, the MCL-PHAs produced by P. putida using
access of the PHB depolymerase to the granular surface of the PHB) coconut oil as substrate demonstrated a Tg of − 43.7 ◦ C. In contrast,
(Kuchta et al., 2007). when the same strain was used to produce PHAs using linseed oil, a
decrease of 18 ◦ C was observed, resulting in Tg of − 61.7 ◦ C (Rai et al.,
4. Properties of PHA 2011).
PHAs are thermoplastic polymers synthesized naturally by microbes, 4.2. Mechanical properties
and their properties differ depending on the chain length and chemical
composition (homopolyesters or copolyesters) (Muhammadi et al., The mechanical properties of the PHAs vary according to the poly
2015). Naturally, the degradation of these polymers resulted in carbon mer chain length. Properties like elongation at break and tensile strength
dioxide and water by various microbes under aerobic conditions. In vary significantly among the MCL-PHAs and the SCL-PHAs (Gopi et al.,
contrast, the anaerobic degradation resulted in the release of carbon 2018). MCL-PHAs demonstrate higher flexibility and elasticity than
dioxide and methane (Rudnik, 2012). The general characteristics of PHA SCL-PHAs that are brittle and highly crystalline (Anjum et al., 2016).
include biocompatibility, complete biodegradability, high processabil However, these properties are not universal, and certain exceptions are
ity, non-toxicity, and structural diversity (Mokhtarzadeh et al., 2016a, prevalent. For example, P (3HB) has Young’s modulus value of 3.5 GPa
b). Besides, the artificial modification of PHB by adding other monomers and tensile strength of 40 MPa, while the corresponding value for P
like polypropylene (PP) can be helpful to retain some materialistic (4HB) is 0.15 GPa and 104 MPa. Similarly, the elongation at break of P
properties such as moisture resistance and aroma barrier properties (3HB) and P (4HB) was observed as 3% and 1000%, respectively (Meng
(Bugnicourt et al., 2014). Based on these properties, the Food and Drug and Chen, 2017). The significant difference between these two polymers
Administration (FDA) approved the usage of these polyesters as bio is the position of the R-group in their side chains which alters their 3D
materials for resorbable sutures, scaffolds for tissue engineering, sub polymeric structure and mechanical properties (Zhang et al., 2018).
stitutes for bone implants, and cardiovascular devices In addition, the flexibility of the PHA can be improved by blending
(Tanadchangsaeng, 2014; Kalia et al., 2019). In addition, PHAs are also other co-monomers and polymers in different ratios with these bio
used for dressings and drug carrier devices as nano- and micro-spheres to polymers for various biomedical and industrial applications (Raza et al.,
6
2018). For instance, the mechanical properties of P (3HB) such as copolymer Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). The
hardness, rigidity, and flexibility differ significantly from that of the P PHBV/OVS composites showed increased crystallization temperature
(3HB-co-3HHx) copolymer (Sudesh, 2013). These properties depend and a decreased half crystallization time (Xiang et al., 2018). The
considerably on the 3HHx units. The copolymer containing a 5.9% mol crystallinity of PHA polymers can also be enhanced by various inorganic
fraction of 3HHx was reported to have the highest value for flexibility materials, including carbon nanotubes, ceramics, hydroxyapatite, or
(elongation at break 163%). Similarly, when the mol fraction of 3HHx bioactive glass (Volova et al., 2017). The cross-linking of these inorganic
units was 2.5% in the copolymer, the highest value for both tensile particles results in significant nucleation at the cross-linking point, thus,
strength (25.7 MPa) and Young’s modulus (631.3 MPa) was obtained reducing the crystallite size (Chen et al., 2017). The crystallinity of the
(Liao, 2010). Another study was performed using mixtures of P (3HB) polymer is mainly dependent on the functional group and chain length
and P (3HB-co-3HHx) to produce scaffolds and biopolymeric films to of the monomeric unit. The monomeric unit with longer side chains has
analyze their mechanical properties (Lim et al., 2017). The flexibility of lesser crystallinity. Hence, the flexibility of the polymer can be increased
the blend remarkably increased (from 15% to 106%), and the tensile by reducing the crystallinity and increasing the chain length.
strength was slightly decreased with an increase in the ratio of P
(3HB-co-3HHx) in the blend from 40 to 60%. In addition, it was 4.4. Biocompatibility
observed that the scaffolds containing 60% of P (3HB-co-3HHx) support
the escalated growth and rapid proliferation of chondrocytes on its The polymeric biomaterials used in the clinical field are generally
surface. Thus, it can be inferred that the suitable blending of PHAs can placed inside the tissues of any organism. These biomaterials do not
significantly enhance the effect of PHA biopolymer in biomedical and induce any negative response from the organism, differentiating them
industrial applications. from other polymers (Williams, 2008). Currently, biomaterials are pri
marily popular for their use in a vast array of medical and biological
4.3. Crystallinity fields like tissue engineering, drug delivery, nanotechnology, biotech
nology, etc. (Kovalcik et al., 2019). Biopolymers like PHAs have become
The type and size of the side chain in PHAs govern the crystallinity of the focus of research because of their biocompatibility and biodegrad
biopolymers. An increase in the length of the side chains (MCL) can ability. FDA approved one of the members of PHAs, i.e., P (4HB), for
inhibit the crystallization property. Thus, the degree of crystallinity of their use in resorbable sutures (Akaraonye et al., 2010). Since then, a
SCL and MCL-PHAs differs significantly. The crystalline property of MCL significant increase in the use of PHAs has been observed in clinical
has been reported to be lower than that of the SCL. SCL PHA has a science.
crystallinity ranging from 60 to 80% (Sanhueza et al., 2019). However, The biocompatibility of PHAs originates from their monomeric units,
the crystallization rate in these polymers is relatively low compared to incorporated in the polymers, which also exist naturally in the human
synthetic polymers (Tanadchangsaeng, 2014). body. For instance, in the polymer P (3HB), the monomeric unit is 3-Hy
The crystallization of PHAs is also influenced by the functional group droxybutyric acid (3HB) which is a normal metabolite found in human
present in the polymers. The functional group of some copolymers of blood (Mierziak et al., 2021). These biomaterials do not induce any
MCL-PHAs cannot crystallize due to the irregularity caused by these antigenic response or thrombosis even after their long-term use in
groups in the polymeric structure (Visakh, 2014). A study revealed that contact with blood in the human body. The compatibility of both P
the MCL-PHAs exhibit a crystallinity of only 24% (Rai et al., 2011). In (3HB) and P (3HB-co-3HHx) for the human body was assayed, and it was
addition, the crystallinity of the copolymer P (3HB-co-3HHx) was re found that these biopolymers reduce the adhesion of blood platelet and
ported to be dependent on the amount of 3HB present in the polymer. In thrombogenicity when in contact with blood (Grigore et al., 2019).
contrast, the 3HHx units were not involved in the crystallization Further, utilizing the articular cartilage from the rabbit, it was found
(Tanadchangsaeng, 2014). A model was proposed to predict the crys that the scaffolds produced by the mixture of P (3HB) and P
talline structure that assumes the co-crystallization of 3HB units and (3HB-co-3HHx) support the 3D growth of the chondrocytes (Koller et al.,
3HV units simultaneously in any crystalline network. Hence, the 2007). The biocompatibility property of PHAs has also been experi
isomorphism observed in P (3HB-co-3HV) polymers was confirmed mentally proven by using melt-spinning of mixtures of poly (3-hydrox
because of the geometrically similar structure of the two subunits, 3HB ybutyrate-co-3-hydroxyvalerate) (PHBHV) and poly (lactic acid) (PLA)
and 3HV (Grigore et al., 2019). (Hufenus et al., 2012). The biomaterials (fibers) obtained from this
Similarly, another study focussing on the degree of crystallization of study revealed an increased tensile strength. In addition, studies
P (3HB-co-3HHx) showed a significant reduction in crystallization by employing human fibroblast cells showed enhanced biocompatibility
the 3HHx units. This confirms that the random position of 3HHx units (well-proliferated cells) and biodegradability (progressing degradation)
minimizes the crystallization rate of polymer (Tanadchangsaeng, 2014). of these biopolymers (Hufenus et al., 2012). Conclusively, the biocom
Similarly, a study demonstrated that the crystallization rate of P (3HB) is patibility of PHA helps in the rapid growth and proliferation of tissues on
much higher than the copolyester P (3HB-co-3HHx). This suggested that its surface. Thus, PHA used for biomedical applications should be highly
incorporating 3HHx units in P (3HB-co-3HHx) significantly decreases purified and devoid of harmful substances.
the degree and rate of crystallization of the polymer (Volova et al.,
2021). Besides, the fragility and rigidity of the P (3HB) also increase due 4.5. Biodegradability
to the high crystallization rate. To reduce the fragility in P (3HB), 3HV
was incorporated (Leong et al., 2014). Similarly, other monomers can The biodegradability of PHAs in different environmental conditions
also be added to reduce the crystallinity of the polymer. such as soil, seawater, and lake water makes them unique from other
The copolymers with lower crystallinity can also be produced by polymers. The biodegradation is dependent on various factors like the
utilizing less crystalline and more degradable polymers. The films of the population of microorganisms, pH, temperature, humidity, oxygen and
copolymer, P (3HB-co-3HHx), showed an increase in degradability when nutrient availability in the environment, and the various properties of
blended with gelatin in different ratios (Rai et al., 2011). In addition, the the biopolymer (Fernandes et al., 2020). In any natural environment, the
rise in gelatin content has also decreased the polymer crystallinity. A microbial population significantly influences the biodegradation of the
study involving the mouse osteoblast cells revealed better cellular PHA. The microorganisms synthesize extracellular enzymes like PHA
viability on copolymeric blends than pure copolymers P (3HB-co-3HHx) depolymerases or PHA hydrolases which specifically act on the PHA and
(Koller, 2018). Similarly, the crystallinity of PHA polymers can be degrade the biopolymer (Zaheer and Kuddus, 2018). In nature, micro
increased by the blending of a suitable agent. In a study, Octaviny bial families including Pseudonocardiaceae, Streptosporangiaceae, Micro
loctasilasesquioxane (OVS) was used as a cross-linking agent for the monosporaceae, Streptomycetaceae, and Thermomonosporaceae degrade P
7
(3HB) predominantly (Verlinden et al., 2007). Generally, these enzymes thermoplastic has been thoroughly studied and investigated for its
are secreted extracellularly to solubilize the biopolymer. However, some various applications. Bacterial species, including R. eutropha, Azoto
of the bacteria can also degrade the PHA intracellularly. Hence, the bacter sp., Bacillus sp., Pseudomonas sp., Burkholderia sp., Halomonas sp.,
degradation of PHAs will be faster in regions where a large population of and Haloferax sp., has been widely used for their higher accumulation of
PHA degrading microbes is present. In addition to the microbial popu PHAs (Kumar et al., 2020). Apart from this, both wild and recombinant
lation, environmental conditions like pH, temperature, and hydration strains of these bacteria have also been employed for PHA production.
level significantly impact the degradation of PHAs. The biodegradation The genetic engineering in these bacteria results in PHA production from
increases considerably with an increase in temperature from 15 ◦ C to cheap and renewable resources, such as molasses, waste lipids, agri
30 ◦ C and reaches the maximum at around 60 ◦ C with 55% humidity cultural wastes, etc. (Favaro et al., 2019).
(Rudnik, 2012). Further, halophilic bacteria like H. mediterranei, Vibrio sp., V. harveyi,
Apart from the environmental factors, properties of the biopolymer V. natriegens, and V. nereis were reported to have PHA accumulated in
like composition, molecular weight, crystallinity, additives, lamellar their cells (Wei et al., 2011). The genes regulating the biosynthesis of
thickness, and surface area also affect the biodegradation process (Philip PHA, polyhydroxyalkanoic acid synthase, were identified in two halo
et al., 2007). The monomeric composition of the PHA affects their philic bacteria, such as V. cholerae and V. parahemolyticus (Kavitha et al.,
biodegradation. The crystallinity and Tm of the biopolymer vary 2018). The isolation of PHAs from these bacteria provides an advantage
inversely with the chain length. Thus, with the increase in the chain of getting MCL-PHAs with better biodegradability and biocompatibility
length, the crystallinity, and Tm of the biopolymer decrease, increasing than SCL-PHAs produced by other bacteria. Hence, the halophilic PHAs
their biodegradability. The biodegradability of copolymers with 4HB can be a potential candidate for various industrial applications. Among
monomeric unit [P (3HB-co-4HB)] is higher than the copolymers with different bacteria from the Halobacteriaceae family, H. mediterranei, is
3HB monomeric unit [P (3HB-co-3HV)] (Brigham and Sinskey, 2012). the best-studied organism that can produce PHAs (Quillaguamán et al.,
Similarly, the biodegradation rate of PHA copolymer containing 4HB 2010).
was shown to be much faster than the P (3HB) or copolymer containing Actinomycetes are characterized as Gram-positive bacteria having
3HB (Akaraonye et al., 2010). high GC content in their genetic material. The PHA synthesizing
Biodegradation is the essential property required for the application mechanism in actinomycetes is poorly understood, and only a few pre
of PHA in tissue engineering. The rate of degradation of scaffold used in liminary studies suggested the synthesis of PHB in Streptomyces
tissue engineering should be equal to the tissue regeneration rate that (Krishnan et al., 2017). Streptomyces are members of actinomycetes, and
has been studied by various researchers in both in vitro and in vivo their ability to synthesize both primary and secondary metabolites
conditions. The degradation study for P (3HB-co-3HV) fibers was con makes these bacteria a potential candidate for PHB production. The
ducted under buffer incubation at pH 7.4 and 37 ◦ C (Grigore et al., production of PHB in Streptomyces was investigated by taking twelve
2019). It was found that the molar mass of the fibers started decreasing different strains. Estimation using gas chromatograph suggested that
after 80 days, and after six months, it reached 64% of the initial mass. PHB accumulation in all the strains ranged from 1.5% to 11.8% of the
Another critical aspect is to determine an appropriate sterilization dry cell weight (Verma et al., 2002). In another study, Streptomyces
method for the scaffolds. Sterilization of these polymers using steam and coelicolor A3 (2) M145 was examined for its ability to synthesize PHB
gamma radiation was found to have various adverse effects like reducing (Valappil et al., 2004). A probable correlation between accumulation of
their molecular weight and altering the mechanical property (Freier, PHB and production of antibiotics was proposed where PHBs act as a
2006). This problem can be solved by using ethylene oxide or gaseous carbon source for the production of these metabolites. Thus, bacteria can
formaldehyde to sterilize these biopolymers (Kiyotake et al., 2019). In synthesize a wide variety of antibiotics; however, they can also be
addition, the degradation of poly (3-hydroxyoctanoate-co-3-hydrox exploited as a great source of PHAs (Matias et al., 2009).
yhexanoate) [P (3HO-co-3HHx)] was enhanced by gelatin addition,
which makes the surface more porous and exposed to attack by the 5.2. Genetic basis of PHA formation in bacteria
hydrolytic enzymes (Rai et al., 2011). The biocompatibility and biode
gradability properties of these biopolymers depend on their monomeric The regulation of biosynthesis of PHA differs from one microor
composition. However, the carbon source used by the microbes has no ganism to another. In bacteria, the distinct features of the genes and
role in it. Thus, the desired property can be incorporated into the bio enzymes involved in the PHA formation are strain-dependent (Mar
polymers by modifying the monomeric composition. cos-García et al., 2017). The specific PHA secretion depends upon the
substrate specificity of the enzyme. Cloning of PHA synthase genes and
5. Production and genetic regulation of PHA in various their determination by genome sequencing provides valuable informa
microorganisms tion like subunit composition and substrate specificity of the enzyme
(Zou et al., 2017). In addition, based on the substrate specificity, the
The increasing cost of petroleum-based plastics and their adverse PHA synthases have been classified into four classes (I, II, III, and IV)
effect on the environment demands an inexpensive and eco-friendly (Grage et al., 2009).
substitute for these synthetic polymers. The production of PHAs from The PHA synthases of class I are composed of a single type of subunit,
various microorganisms is widespread nowadays, owing to its biode i.e., (PhaC). PHA synthases of R. eutropha are representative of this class.
gradability and cost-effective manufacturing process. These bioplastics The molecular weight of these enzymes ranges from 60 to 73 kDa, and
can be easily produced by inducing multiple enzymatic reactions in these enzymes catalyze the polymerization of SCL monomers (Tsuge,
microbes using different feedstocks (Pakalapati et al., 2018). Since they 2016). The phaCAB operon in this class, constituted by phaA (β-keto
exhibit similar properties as that of petroleum-based plastics thus, thiolase), phaB (NADPH dependent acetoacetyl-CoA reductase), and
increased efforts are now being made to produce different biopolymers phaC (PHA synthase) gene, are responsible for the synthesis of PHA
commercially by employing the recombinant strains. Among various biopolymer (Li et al., 2017).
microbes, actinomycetes, algae, and marine bacteria are the prime focus The Class II PHA synthases are composed of a single subunit and are
of research for PHA production (Singh Saharan et al., 2014). regulated by two genes, phaC1 and phaC2 (Zou et al., 2017). The
representative species for this class of PHA synthase is P. aeruginosa. The
5.1. Production of PHA in eubacteria enzyme ranges from 60 to 65 kDa, and the two genes, phaC1, and phaC2
regulate the PHA synthesis by polymerizing the CoA thioesters of MCL
Bacteria are the largest group of microorganisms that exhibit the monomers (6–14 carbon atoms). The genetic regulation for PHA meta
ability to produce PHA. After its discovery, this biodegradable bolism in this class is regulated by various genes including phaZ, phaD,
8
phaI, and phaF. phaZ gene is present between the phaC1 and phaC2 and which is regulated by a common genetic regulation consisting of three
encodes the PHA depolymerase enzyme (Tan et al., 2020). In contrast, genes β-ketothiolase (phaA), acetoacetyl-CoA reductase (phaB), and PHA
the phaD gene in the synthase operon is located downstream of these synthase (phaC). These three genes constitute the phaCAB operon
three genes and regulates the number and size of PHA granules produced (Fig. 3). Various studies have suggested that the structural organization
inside the cell (Tarazona et al., 2020). The other two genes, phaI, and of the operon varies among the microbial species (Muneer et al., 2020).
phaF are situated collinearly with phaD and are associated with the In bacterial species including Acinetobacter sp., Agenes latus,
regulation of phasins (Ren et al., 2009). Another function of phaF gene is P. acidophila, and R. eutropha, the phaCAB genes are in tandem but not
to regulate the phaC1 gene expression, thus regulating the PHA syn necessarily in the same order on the chromosome. However, in Para
thesis. In unfavorable environmental conditions, lack of substrate leads coccus denitrificans, Rhizobium meliloti, and Zoogloea ramigera, the genes
to interruption of PHA synthesis, but the synthesis of granule-associated phaAB and phaC are separated and not linked to each other (Umeda
protein (PhaF) remains unaffected. This results in the binding of freely et al., 1998).
moving PhaF protein to the promoter region of the operon, thereby The PHA synthesizing bacteria have a longer generation time and
suppressing the expression of the phaC1 gene. However, in the advent of require a relatively lower temperature for optimal growth. Apart from
favorable conditions, the PHA synthesis resumes, and the PhaF protein this, the cell lysis in these bacteria is very difficult, increasing the cost of
binds to the PHA granule, and phaC1 gets expressed (Mitra et al., 2021). PHA purification. The recombinant bacteria having lower generation
The Class III PHA synthase contains PhaC and PhaE, which are het time and higher optimal growth temperature have been developed to
erodimers without any homology between the two protein subunits, overcome this situation. These bacteria are easy to lyse, and their short
each having a size of 40 kDa (Tsuge, 2016). The representative species of generation time helps them accumulate PHA in large quantities, making
this class are Allochromatium vinosum and Thiocapsa pfenningii. The two them highly efficient and economically feasible (Bhattacharyya et al.,
subunits form a PhaEC complex in which PhaC is the catalytic subunit, 2019).
and PhaE is essential for polymerization (Mezzolla et al., 2018). This The metabolic engineering technology has been extensively used to
class of PHA synthase usually polymerizes short chain length CoA commercially enhance PHA production and produce various novel
thioesters, except in T. pfenningii, which polymerizes MCL-PHAs (Grage PHAs. Recombinant strains of Escherichia coli (K24K), bearing PHA
et al., 2009). The synthase operon in this class also includes phaP, phaA, biosynthetic genes of Azotobacter sp. strain FA8, have been engineered
and phaB, apart from the PHA synthase gene. phaP encodes the struc for high productivity (Nikel et al., 2006). E. coli can metabolize a large
tural protein associated with the PHA granule called phasins. It has been number of carbon sources, such as glucose, sucrose, lactose, and xylose.
reported that these proteins regulate the number and size of the PHA In addition, by providing these microbes with cheap and renewable
(Mitra et al., 2021). phaA and phaB genes have a similar function as the feedstocks like molasses, whey, and hemicellulose hydro-lysate, the
class I PHA synthase. production cost of PHA can be minimized to a large extent. This pro
The class IV PHA synthases include heterodimers like the classIII, but cedure can also be followed using other bacteria to increase the overall
the two protein subunits differ in size (40 kDa and 20 kDa). The two feasibility of large-scale PHA production. Besides, the PHA with the
subunits, PhaC and PhaR, are regulated by the genes phaC and phaR, desired composition can also be produced via the metabolic engineering
respectively. B. megaterium is the representative species of this class, of microbes.
where the enzyme catalyzes the polymerization of SCL-HACoAs (Zou
et al., 2017). The phaRBC operon of class IV PHA synthases regulates the
PHA synthesis. The gene phaB is located between the phaC and phaR 5.3. Production and genetic regulation of PHA in microalgae
genes. The phaP and phaQ genes upstream of the operon regulate the size
and number of the biopolymer produced inside the cell. phaP gene is Biopolymeric PHAs produced from microalgae have been the focus
responsible for synthesizing phasins protein (Mitra et al., 2021). In of research for a long time. Microalgae rapidly use carbon dioxide
contrast, the gene phaQ negatively controls and regulates the expression (greenhouse gas) and energy from sunlight and convert these into eco-
of both phaQ and phaP (Biedendieck et al., 2021). friendly bioplastics (Costa et al., 2019). Cyanobacteria can intracellu
Earlier investigations on 45 different bacterial species revealed that larly produce and accumulate the PHB granules. PHB in cyanobacteria
the number of genes associated with PHA synthesis could be greater than was reported for the first time in Chlorogloea fritschii (Carr, 1966). Since
59 (Mohapatra et al., 2020). The number of PHA synthase genes varies then, many cyanobacterial species, including Spirulina sp., Gloeothece
among the bacteria, but usually, each bacterium contains at least two sp., Aphanothece sp., and Synechococcus sp, have been reported to possess
copies of the gene. PHB has been studied extensively in most bacteria, the PHB granules in their cytoplasm (Carpine et al., 2020). In addition,
PHB isolated from Spirulina LEB-18 was added to nanofibres to improve
Fig. 3. Genetic regulation of phaCAB operon. phaA synthesizes β-ketothiolase enzyme, which converts Acetyl-CoA to Acetoacetyl-CoA. Further, Acetoacetyl-CoA is
converted to Hydroxyacyl-CoA by phaB encoded Acetoacetyl-CoA reductase. phaC synthesizes the PHA polymerase enzyme, which catalyzes the conversion of
Hydroxyacyl-CoA to PHA.
9
the mechanical properties (elasticity, tensile strength, conductivity) of by competing pathways, or a combination of these factors.
these fibers, making it better than the commercial PHB (Morais et al.,
2015). Alternatively, in the diatom, Phaeodactylum tricornutum, bacterial 5.3.2. Extraction methods of PHA from microalgae
(R. eutropha U16) biosynthesis pathways for PHB were implemented, PHA extraction from microalgae can be described as a process of
which produced PHB up to 10.6% of its dry weight biomass (Hempel separating these biopolymers from biomass after their cultivation.
et al., 2011). The microalgae also secrete many polysaccharides used as Microalgae cultivation plays a vital role in determining the amount of
a blending material for PHB. Blending algal fibers with biopolymers like biosynthesized biopolymers. The microalgae can mainly be cultivated
PHB and PCL significantly increase their biocompatibility. by two systems such as the open pond cultivation system and the closed
The issues associated with the commercial production of PHA by photobioreactors (PBR) system (Troschl et al., 2017). However, the
bacteria are the higher cost of the carbon sources and the energy closed PBR system has various advantages over the open pond system as
required for the fermentation processes. In contrast, microalgae are it provides a controlled environment for the optimum growth of these
mostly autotrophic and require inorganic nutrients like CO2, nitrogen, microorganisms (Onen Cinar et al., 2020). Apart from cultivation, the
phosphorus, etc., for growth and metabolism (Markou and Nerantzis, harvesting and downstream processing of microalgal cultures is crucial
2013). The types of polymeric and copolymeric PHA synthesized by as these microorganisms have much lower cell densities than the bac
these microorganisms are highly dependent on the availability of nu teria. The harvesting of biomass is mainly performed by sedimentation,
trients like nitrogen and phosphorous (Chu et al., 2013). Another study filtration, or centrifugation (Milledge and Heaven, 2013). In addition,
found that the free coenzyme A released by the Krebs cycle inhibits the mixed microbial cultures (microalgae and bacteria) have also been uti
β-ketothiolase during normal microbial growth. However, the limitation lized for PHA production. In a study, a mixed culture of bacteria and
of nutrients increases the acetyl-CoA concentration which is used for the algae was cultivated in a feast and famine (FF) regime (Fradinho et al.,
PHA synthesis (Costa et al., 2019). A study observed that the limitation 2013). It was found that the bacteria consumed the carbon source in the
of nitrogen (30.7%) results in the accumulation of a higher percentage of feast phase and produced PHA. In the famine phase, the electron
PHA compared to the limitation of phosphorous (14.1%). In addition, released by algal photosynthesis is utilized to oxidize these PHA
the nitrogen limitation can increase the carbon reserve (PHA, lipid) molecules.
inside the cell via the degradation of various macromolecules (Coelho PHA extraction procedure from microalgae is crucial for the large-
et al., 2015). In contrast, the limitation of phosphorus directly influences scale production of these biopolymers. Various methods, such as
the synthesis of nucleotide, ATP, and phospholipid molecules (Saman chemical methods, non-PHA cell mass (NPCM) removal-based methods,
taray and Mallick, 2015). Thus, nitrogen limitation is crucial for PHA and mechanical methods, have been utilized to extract PHAs (Kurian
accumulation in microalgal cells. and Das, 2021). However, the selection of these methods depends on the
cellular properties of the microalgae, such as cell size and cell wall
5.3.1. Genetic regulation of PHA in microalgae structure (S. S. Costa et al., 2018). The chemical method involves using
The genetic regulation for PHA biosynthesis in microalgae is similar various solvents for the extraction of PHAs, known as the solvent
to that of bacteria. Various studies suggested that the limitation of ni extraction method. These solvents are mostly halogenated solvents such
trogen plays a crucial role in the biosynthesis of PHA in these micro as chloroform and sodium hypochlorite (Roja et al., 2019). The purity
algae. The most common form of PHA accumulated by the microalgae is and recovery rate of extracted PHAs by chemical methods are very high.
PHB, and the microalga, Synechocystis PCC6803, has been widely stud However, this approach should be limited to the lab scale as it is not
ied for PHB production (Tanweer and Panda, 2020). In this microalga, economical. In addition, the solvents like chloroform and sodium hy
the biosynthesis of PHB is mainly governed by four genes such as phaA, pochlorite required in this process can cause environmental problems.
phaB, phaC, and phaE. These genes are present as a two-component PHA Besides, other environmental friendly solvents such as non-halogenated
synthase. The phaA and phaB genes are organized in one operon solvents (cyclohexanone and γ-butyrolactone) (Jiang et al., 2018), green
responsible for the synthesis of β-ketothiolase and acetoacetyl-CoA solvents (Dimethyl carbonate) (Koller, 2020), and alkali like NaOH and
reductase. The phaE and phaC genes are organized in another operon KOH (Umesh and Mamatha, 2018) have also been utilized.
which synthesizes the two subunits of PHA synthase (Mitra et al., 2020). Apart from the chemical method, the NPCM removal and mechanical
The phaC, phaA, and phaB are the three genes crucial for producing PHAs methods mainly focus on the digestion of the cell biomass. The NPCM
in microalgae. The cotransformation of phbB and phbC confirmed this in can easily be removed by chemical, enzymatic or biological treatment
the green alga Chlamydomonas reinhardtii (Mal et al., 2021). phaA is methods (Kourmentza et al., 2017). The chemical treatment involves the
intrinsic to the C. reinhardtii genome. The cotransformation of phaB and use of chemicals like sodium hypochlorite, sodium hydroxide, and so
phaC genes leads to the production of PHB by the algae. However, the dium dodecyl sulphate (Jiang et al., 2015). The chemicals used in this
cotransformation of either of the genes individually inhibits PHB method may generate halogenated end products, which can cause severe
production. problems in the environment (Kurian and Das, 2021). In the enzymatic
The metabolism of PHA in microalgae is mainly governed by four treatment, purified proteolytic enzymes and cell wall degrading en
pathways. There are three main enzymes involved in Pathway I, such as zymes are used to disrupt cell material. This method requires a lower
β-ketothiolase and NADPH- dependent acetoacetyl-CoA reductase and energy requirement; however, the production cost of purified enzymes is
PHA synthase (Philip et al., 2007). Pathway II involves fatty acid too high to make this process feasible (Kachrimanidou et al., 2016). The
oxidation, and it is the most common pathway found in microalgae. biological method involves using biological agents such as viruses and
Moreover, the biosynthesis of PHA in microalgae is predominantly bacteria for the extraction of PHA by the lysis of cell biomass. In addi
mediated by the fatty acid production pathway (J. A. V. Costa et al., tion, another cost-effective approach was developed by Yu and Chen
2018). The acyl CoA molecules produced by the β-oxidation of fatty acid (2006), where sulphuric acid was used as a source of proton for the
are converted to 3-hydroxyacyl-CoA. The PHA synthase enzyme then digestion of cellular protein. The mechanical method involves disrup
catalyzes the binding of this 3-hydroxyacyl-CoA molecule to an existing tion by bead mill, High-pressure homogenization (HPH), disruption of
polymer via an ester linkage. In Pathway III, the substrates are converted cells by ultrasonic sonicator, and use of air classifiers (Madkour et al.,
to 3-hydroxy-acyl-CoA by the two enzymes 3-hydroxyacyl-ACP-CoA 2013).
transferase and malonyl-CoA-ACP tranacylase (Zhang et al., 2020). Industrial production of PHA mainly utilizes the sugar molecules
Pathway IV involves the utilization of NADPH-dependent acetoace (sucrose, beet sugar, cane sugar, lactose, starch, lignocellulose, etc.),
tyl-CoA reductase to oxidize (S)-(+)-3-hydroxybutyryl-CoA. These lipids (animal fats and plant oils), and fatty acids as carbon sources for
pathways are mainly regulated by specific environmental conditions, the production of PHA. In a study, it has been observed that the yield of
activation of enzymes by metabolic intermediates, inhibition of enzymes PHA using lipids (0.6–0.8 g/g of plant oil) as a carbon source is higher
10
than the carbohydrates (0.3–0.5 g/g of glucose). However, the carbo production of these polymers. Many studies conducted on various fungal
hydrates showed higher productivity (2.42 gL− 1h− 1) as compared to the species such as Rhodotorula minuta, Yarrowia lipolytica, and Wick
lipids (0.96 gL− 1h− 1) (Jiang et al., 2016). At present, glucose and erhamomyces anomalus reported the synthesis of PHAs (Desuoky et al.,
vegetable oil are mainly being used for the commercial production of 2007; Haddouche et al., 2011; Ojha and Das, 2018).
PHA. In addition, nowadays the biorefinery concepts are being incor The production of PHA from fungi has not yet been studied in detail,
porated for the production of PHA (Das et al., 2018). Although the in and only a few studies are available to date. However, some studies
dustrial use of microalgae for PHA production has not been economical suggested that PHA production can be enhanced by utilizing genetically
to date, various efforts are being made to reduce the cost of the process. modified yeast species. Another study involving the transgenic yeast,
The development of advanced production systems with lower energy S. cerevisiae, revealed the peroxisomal PHA synthesis in these organisms
requirements and selection of high PHA accumulating strains can help in via fatty acid metabolism (Zhang et al., 2006). Similar results were
the sustainable production of these value-added polymers. obtained when the PHA synthase from P. aeruginosa was transformed in
Pichia pastoris. In transformed P. pastoris, the production of MCL-PHA
5.4. Production and genetic regulation of PHA in fungi (1% per gram of dry weight) was induced via peroxisomal metabolism
(Poirier et al., 2002). Moreover, the production of PHAs utilizing
The biosynthesis of PHA has also been reported in various species of genetically modified yeasts can be an efficient way for the large-scale
fungi. Most studies involving PHA synthesis in fungi have been carried production of these polymers. The high surface area to volume ratio of
out on yeasts. Saccharomyces cerevisiae was reported first to accumulate the fungal filament may also increase PHA production.
the hydroxybutyrate in the form of PHB (Nuti and Lepidi, 1974). The
ability of fungi to produce PHAs by utilizing cheap organic wastes like
molasses make these organisms a potential candidate for the economical
Fig. 4. Application of microbially synthesized PHAs produced from renewable sources such as organic wastes. The industrial applications include the use of PHAs as
packaging and coating materials and for animal foods. The biocompatibility nature of these polymers is beneficial for their use in biomedical sectors, including bone
grafting, tissue engineering, and drug-delivery systems. The biofouling in hulls can also be prevented by PHA coating. Microbially synthesized value-added products
from organic wastes help in the recycling of these wastes.
11
6. Applications of PHA 3-hydroxyhexanoate, 3-hydroxyoctanoate, and 3-hydroxydecanoate.

The patent for Nodax™ was sold to Danimer scientific. This polymer
The characteristic features such as biodegradability, biocompati can be used to manufacture medical-surgical garments, upholstery,
bility, non-toxicity, and other mechanical properties make the PHAs carpet, packaging, compostable bags, and lids or tubs for thermoformed
suitable for a wide range of applications in various sectors. The indus articles (Anjum et al., 2016). PHA-based latex can also make
trial application of these value-added biopolymers includes their use as water-resistant surfaces to cover paper and cardboards (Vijayendra and
packaging and coating material and animal foods. Biomedical applica Shamala, 2014). The pioneers in the commercial production of PHA
tions include PHA for implants, drug carriers, tissue engineering, etc. were a Biogreen® (Mitsubishi Gas Chemical Company Inc, Japan),
(Masood et al., 2015). The continuous use of synthetic plastics has been Mirel™ (Telles (ADM/Metabolix), USA), Biomer® (Biomer Inc, Denver),
one of the main reasons for environmental pollution. Thus, PHAs can be GreenBio® (Tianjin Green Bio-Science Co., China/DSM, Netherlands),
an excellent substitute for these plastics and can be used in packaging or BIOPOL® (Metabolix Inc., USA now Yield10 bioscience), TephaFLEX®
coating of materials, polymeric films (Akaraonye et al., 2010). These (Tepha Inc., USA), VersaMer PHA (PolyFerm Canada, Canada), and
polymers are also used as antifouling and antimicrobial agents (Kavitha Procter & Gamble (USA) (Gurunathan et al., 2015). Currently, various
et al., 2018). Fig. 4 describes various applications of PHA polymers. other industries are also commercially producing PHA with a wide range
of applications (Table 4).
6.1. Industrial applications

6.2. Medical applications
PHAs have been used widely in the industrial sectors, and to meet the
requirements, these polymers are industrially produced with different 6.2.1. Biomedical implants
monomer compositions. For instance, Metabolix, a USA-based company PHAs have been used widely as implant materials such as sutures,
manufactures a polymer by blending P (3HB) and P (3HO), which the screws, staples, bone plates, stents, adhesion barriers, articular cartilage
FDA has already approved as a food additive (Muneer et al., 2020). repair devices, nerve guides, etc., in the human body (Chen, 2010). The
Another polymer marketed as BIOPOL®, a copolymer of P properties like biodegradability, biocompatibility, tendency to stimulate
(3HBco-3HV), is also produced by Metabolix. It has a wide range of bone growth, and wound healing make the PHA a potential candidate
applications, including packaging, shampoo bottles, disposable razors, for application in various biomedical fields (Ray and Kalia, 2017). Be
disposable cups, etc. (Anjum et al., 2016). The industrial-scale produc sides, the mechanical properties (Elasticity modulus and tensile
tion of P (3HB-3HHx) has been accomplished by Tsinghua University strength) of the PHAs are pretty similar to that of the bone (Koller,
(China) in collaboration with Guangdong Jiangmen Center for Biotech 2018). At present, only a few PHAs are adequately produced to be used
Development (China), KAIST (Korea), and Procter & Gamble (USA) as implant materials. The mechanical properties and degradability,
using A. hydrophila (Możejko-Ciesielska et al., 2019). The polymer including the degradation rates of these implant materials, can be
produced is used to manufacture various products, including flexible altered by varying their composition (Sharma et al., 2021).
packaging, synthetic paper, and medical devices. Another instance of It has been reported that the members of both SCL and MCL-PHAs
industrial production of these polymers is the production of P (3HB) (PHB, PHBV, P3HB4HB, PHBHHx, and PHBVHHx) have shown great
polymer by a German company, Biomer, utilizing A. latus. Various potential as biomedical implants (Peng et al., 2011). These biopolymers
day-to-day products, including combs and pens, have been manufac have been known to enhance cell proliferation and tissue regeneration.
tured using this polymer (Philip et al., 2007). A PHA, developed by The additional benefit of using PHA-based implants is that they do not
Procter and Gamble marketed as, Nodax™, is industrially produced by change the pH where they are implanted even during their degradation.
combining 3HB with a small quantity of MCL units such as Thus, they are tolerated by the host immune system (Koller, 2018). In
Table 4
Commercially available PHAs, their thermal and mechanical properties and applications.
Company Product Density Vicat Tm [◦ C] Tg Xcr ϵ σ Sources Applications References
[g/cm3] softening [◦ C] [%] [%] [MPa]
Temp [◦ C]
Ecomann EM50000 1.22 75 110–130 – – 800 9 Microbial Biodegradable Cataldi et al.

Biotechnology fermentation of plastic, Injection (2020)
Co. Ltd agricultural molding, Food
waste contact
Biomer Biomer P209 1.20 134 >195 – 60–70 8–15 15–20 Utilization of Biodegradable Follain et al.
Biopolyesters glucose and waterproof (2014)
propionate by materials, injection
bacteria molding
thermoplastic
Ercros SA Ercros® 1.20 62 124 1 – 21 – Renewable Packaging material, Ivorra-Martinez
PH110 biobased raw cosmetics, injection et al. (2020)
materials such as molding
the sugar cane
TianAn Biologic ENMAT™ 1.25 166 175 1.1 88.5 2 39 Fermentation of Injection molding, Raho et al.
Materials Co., Y1000P D- Glucose and thermoforming, (2020)
Ltd. propionic acid by blown films,
C. necator extrusions
Kaneka PHBHX151A 1.19 – 126 0 – 320 26 Plant oil Cutlery, Shopping Miyahara et al.
Corporation utilization by bags, Food (2021)
bacteria packaging
Metabolix, Inc. Mirel P1003 1.40 123.9 160–165 – – 4 25 Fermentation of Injection molding, Sarasini (2017)
renewable packaging material,
carbon source by disposable items
microbes
Tm = melting temperature, Tg = glass transition temperature, Xcr = crystallinity, ϵ = elongation at break, and σ = tensile strength.
12
vivo studies of the films and scaffolds of maleated PHBHHx for in vivo and in vitro applications (Kavitha et al., 2018). Nowadays,
(Ma-PHBHHx) showed enhanced growth and proliferation of L929 PHAs for tissue engineering have become a common practice. The
mouse fibroblasts and human microvascular endothelial cells (HMEC). development of novel polymeric and copolymeric materials will
Apart from this, the in vivo study of these biopolymeric implants using enhance the efficiency of these products.
bone marrow cells, human mesenchymal stromal cells (hMSCs), human
bone marrow mesenchymal stem cell (hBMSC), and NG108-15 neuronal 6.3. Aquaculture
cells showed higher biocompatibility as compared to other polymers
(Chen and Zhang, 2018). The application of PHA in aquaculture for controlling the outbreak of
the disease is highly promising. An early investigation suggested that
6.2.2. Therapeutic carrier short-chain fatty acids like PHB inhibit the pathogenic V. campbellii
The first pharmaceutical application of PHA was the use of these (Defoirdt et al., 2007). The study involving Artemia franciscana (model
polymers as drug carriers. They conducted various biocompatibility organism for aquaculture) revealed the anti-adhesive properties of PHB
experiments using PHA extracted from A. eutrophus H16 (Koller, 2018). (Sapkota et al., 2008). Later, it was found that the PHB had less cell
The drug-delivery systems, composed of a polymeric matrix, are used to adhesion due to the surface accumulation of methyl groups (Lee et al.,
deliver therapeutics (antibiotics, immunogens, contraceptives, hor 2013). Another study was conducted to control the Vibrio infection in
mones, and other active substances) in a controlled manner to the target shrimp aquaculture using PHB polymer produced from a marine bac
site. They also increase the bioavailability of the drugs and escape the terium, Brevibacterium casei MSI04 (Kiran et al., 2014). It was found that
immune system easily without causing any immune response or thera the polymer disrupts biofilm development and diminishes biomass for
peutic toxicity (Masood et al., 2015). mation instead of inhibiting growth. Thus, it could help to control the
The commercially available and most commonly used drug delivery growth of pathogens. Various other studies reported the antiadhesive
systems are composed of homopolymers and copolymers of lactate and property of these biopolymers against shrimp pathogens such as
glycolate (Chen, 2010). However, the release of drugs using these V. alginolyticus, V. harveyi, V. fischeri, V. vulnificus and
polymers is not fully controlled due to the bulk hydrolysis of glycolate V. parahaemolyticus. In the case of Nile tilapia larvae, PHB increases the
and lactate copolymers (Jem and Tan, 2020). Initially, SCL-PHAs were survival of the larvae infected with the pathogen Edwardsiella ictaluri.
used for drug delivery. However, later it was found that the MCL-PHAs (Kavitha et al., 2018). Besides, PHB can be used as an anti-infective
deliver the drugs more efficiently because of their lower crystallinity strategy in aquaculture. PHB can also be used as feed in fish culture as
(Grigore et al., 2019). In another study, two copolymers, P it is an excellent source of energy and reduces the process cost (Asiri and
(3HB-co-3HV) and P (3HB-co-4HB), were compared, and it was observed Chu, 2020).
that P (3HB-co-4HB) is more efficient as a drug carrier due to its lower
rigidity (Türesin et al., 2001). 6.4. Antifouling agent
The biocompatibility, non-toxicity, and biodegradability nature of
PHA-based drug delivery systems make these most efficient for drug Biofouling is the colonization and accumulation of microbes (algae,
release (Mendes et al., 2012). These drug delivery systems include mi bacteria, fungi) and other plants and animals on various underwater
crospheres, microparticles, and nanoparticles (Francis et al., 2011). surfaces. This undesired growth of organisms can cause severe compli
PHA-based nanoparticles increase the bioavailability of hydrophobic cations in operation and negatively impact the economy of many in
therapeutics and also help in controlled drug release (Lu et al., 2011). dustries. The most affected sectors by biofouling include shipping
Further, the use of micro and nanoparticles for drug delivery can industries, desalination plants, and underwater pipelines (Inbakandan
enhance tissue selectivity because of the selective uptake of nano et al., 2013). Traditionally, various toxicants, such as copper and trib
particles in target tissues (Rezaie Shirmard et al., 2017). Among various utyltin, were incorporated into a paint matrix that leaches from the
PHAs, only PHB and PHBV have been explored for controlled drug biocide to prevent the settlement (Kyei et al., 2020). Incorporating
release. However, various other PHAs can also be applied to release biogenic antifouling compounds isolated from actinomycetes, bacteria,
drugs in a controlled manner. algae, and sponges in a coating material can be a non-toxic strategy to
deal with this problem. However, these biogenic compounds have not
6.2.3. Tissue engineering yet been industrially produced. The microbial synthesis of PHB nano
The primary purpose of tissue engineering is to restore the damaged composite can be an effective way to delineate the effects of biofouling.
tissues by promoting the growth of new tissue using cells, various PHB nanocomposites can be used on vessel hulls to prevent the settle
signaling molecules, and biomaterials. Tissue engineering includes ment of various microbes on the surface of hulls (Sirohi et al., 2020). As
restoration, maintenance, and improvement of body tissues such as these polymers are biodegradable and prevent biofouling, they can be
vascular tissues, heart valves tissue, skin tissue, nerve tissue, bone and effectively used as an environmental-friendly antifouling agent.
cartilage tissue, and periodontal tissue engineering (Masood et al.,
2015). 7. Conclusion and future perspectives
PHA composites have become a valuable asset for their application in
cardiovascular patches, stents, pericardial patches, atrial septal defect PHAs have emerged as a potential substitute for synthetic plastics in
repair devices, and vein valves (Dai et al., 2009). Similarly, SCL and the last few years. These biopolymers have gained broad interest in
MCL-PHAs are involved in scaffold formation used in various tissue scientific research and commercial uses. The commercial production of
engineering processes (Dwivedi et al., 2020). A composite scaffold was these value-added biopolymers has been achieved by utilizing micro
fabricated using polyglycolic acid (PGA) and poly-4-hydroxybutyrate organisms such as bacteria, microalgae, fungi. The production and
(P4HB), which supports the enhanced growth and proliferation of extraction of microbially produced PHAs are dependent on the substrate
human pediatric aortic cells (Mokhtarzadeh et al., 2016a). Another and types of PHAs, respectively. There are various methods for the
interesting copolymer was developed by Adamus et al. (2012) to coat extraction and purification of these biopolymers, and the purity of the
the vascular prosthesis. It was a di-block copolymer, and when applied PHA depends on the method followed in their production. The chemo-
to the outer surface of the prosthesis, it made the surface impermeable. mechanical properties of these biopolymers are very similar to that of
Thus, this copolymer was found suitable for their use in cardiovascular synthetic plastics, and these properties can be altered by copolymeri
tissue engineering. PHAs have also been used to regenerate skin, muscle, zation. Hence, various PHAs exhibiting modified physical properties can
and blood vessels and repair cartilages and nerves (Masood et al., 2015). be synthesized by varying their chemical composition. In addition,
DegraPol, a block-copolyesterurethane, showed good biocompatibility compared to petroleum-based plastics, PHAs serve as an eco-friendly
13
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Lee, C.C., 2021. Production of polyhydroxyalkanoate copolymers containing 4-
The authors declare that they have no known competing financial hydroxybutyrate in engineered Bacillus megaterium. Int. J. Biol. Macromol. 168,
86–92. https://doi.org/10.1016/j.ijbiomac.2020.12.015.
interests or personal relationships that could have appeared to influence
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The authors acknowledge the National Institute of Technology Cataldi, P., Steiner, P., Raine, T., Lin, K., Kocabas, C., Young, R.J., Bissett, M., Kinloch, I.
A., Papageorgiou, D.G., 2020. Multifunctional biocomposites based on
Rourkela, Odisha, India, for providing laboratory and other facilities to polyhydroxyalkanoate and graphene/carbon nanofiber hybrids for electrical and
carry out the research. S.B. acknowledges the Ministry of Education thermal applications. ACS Appl. Polymer MAt. 2 (8), 3525–3534. https://doi.org/
(MoE), Govt. of India, for providing financial support. M.P. received 10.1021/acsapm.0c00539.
Cha, S.H., Son, J.H., Jamal, Y., Zafar, M., Park, H.S., 2016. Characterization of
INSPIRE Award (No. DST/INSPIRE Fellowship/2017/IF170195) for the
polyhydroxyalkanoates extracted from wastewater sludge under different
doctoral study from the Department of Science and Technology, Govt. of environmental conditions. Biochem. Eng. J. 112, 1–12. https://doi.org/10.1016/j.
India. Vandana receives fellowship from the Department of Science and bej.2015.12.021.
Technology, Govt. of Odisha (No. 1203/ST- (Bio)- 02/2017). Chavan, S., Yadav, B., Tyagi, R.D., Drogui, P., 2021. A review on production of
polyhydroxyalkanoate (PHA) biopolyesters by thermophilic microbes using waste
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Chemosphere 294 (2022) 133723

Contents lists available at ScienceDirect

Polyhydroxyalkanoates, the bioplastics of microbial origin: Properties,

• PHA is intracellularly accumulated

Table 2 conditions. Depolymerases responsible for PHB degradation is among

6. Applications of PHA 3-hydroxyhexanoate, 3-hydroxyoctanoate, and 3-hydroxydecanoate.

6.1. Industrial applications

Ecomann EM50000 1.22 75 110–130 – – 800 9 Microbial Biodegradable Cataldi et al.

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