Introduction

Plastic is a flexible product and has the ability to adapt to different functional activities easily. It is of low cost and easy to produce in large numbers. It has numerous applications in various industries such as food, agricultural, and pharmaceutical¹. However, the usage has resulted in wide problems over the last decade; incineration and fabrication of plastics have made air, water, and soil pollution worse. Waste management and treatment of plastics have become a major problem. A study states that per year 31.9 million tons of plastics had been dumped in the environment². Also, a survey explains that over 60 million metric tons of plastics were produced in 1980, followed by 187 million metric tons in 2000, in 2010 was 265 million, and in 2017, 348 million were produced³. These issues have led scientists and researchers to find an immediate alternative to synthetic plastics, which have less or no effect on the environment.

Polymer materials have dominated global industries for the past 50 years due to their adaptability, durability, and low cost, so that we cannot imagine a product without them. In contrast, many synthetic polymers are made from petroleum and coal as raw materials, making them incompatible with the environment since they cannot be recycled naturally. Since synthetic polymers have a negative impact on the environment, a solution could include combining different types and sources of biological materials called biopolymers, including starch, cellulose, chitin, chitosan, zein, and gelatin, which could be gradually replaced by synthetic polymers for these purposes. Biopolymers, including those obtained naturally and those synthesized, are much more eco-friendly, user-friendly, and cost-effective than petroleum-based polymers. Biopolymers have certain advantages over petroleum-based polymers based on life cycle assessments. Additionally, petroleum-based plastics are more environmentally damaging than biopolymers. It has been demonstrated that biopolymers have advantages over synthetic polymers in medical, tissue engineering, military, and environmental applications⁴.

Biopolymers are composites of monomers that are derived from living things. Due to its distinct characteristics, it has drawn the attention of researchers. They are structures that resemble chains and might deteriorate in the environment, also, their significance is derived from how they interact with other polymers. They make excellent gas and vapour sensors since they are widely available and biocompatible. Biopolymers are divided into three categories: natural, synthetic, and microbial biopolymers, depending on where they are produced. Biopolymers are molecules made up of carbohydrates, amino acids, and hydroxy fatty acids, which are linked together by enzymes to produce high molecular weight molecules. Biopolymers such as polysaccharides, polyesters, polyamides, and polyphosphates can be synthesized by bacteria. Polysaccharides such as cellulose and starch are also biopolymers produced by microorganisms incuding bacteria and fungi. Usually, polyhydroxybutyrates (PHB) are produced by microorganisms under unbalanced or nutrient-limited conditions. Other than acting as storage molecules, PHB also has other functions such as increasing the bacteria’s resistance against the abiotic stresses and also serves as a protection against hydroxyl-radicals⁵. Despite these many advantages, there are some drawbacks in large scale production and usage of biopolymers in industries. It is because of the high cost of production that is considered as a major drawback for the production of biopolymers in large scale. Another drawback is that PHB alone cannot be used to produce a product. It must be blended with other ecofriendly materials to make the product more stable and to extend its lifetime. This is why the cost of production of the biopolymer is high. The researchers are very keen to overcome this drawback and replace all polypropylene or petroleum-based plastics with bioplastics or biopolymers. To retain the available resources and to avoid further damage to the environment, these alternatives must be developed as soon as possible.

Polyhydroxyalkanoates

PHAs, a type of polyester that is commonly used as a compound for the production of biopolymers, are well known as PHBs. PHBs, Polyhydroxyvalerates (PHVs), poly-4-hydroxybutyrates (P4HBs), Polyhydroxyhexanoates (PHHs), and polyhydroxyoctanoates (PHOs) are a few examples of polyhydroxyalkanoates which are biodegradable and environmentally non-toxic. It has a variety of qualities that can be altered according to the requirement.

PHAs produced by bacteria are classified based on their structural characteristics into three groups: short chain length (scl) PHAs (3-5 carbon atoms), medium chain length (mcl) PHAs (6-14 carbon atoms), and long chain length (lcl) PHAs⁶. The quantity of production is higher in scl-PHAs than mcl-PHAs⁷. The cost of PHA in the market is currently €5 per kilogram. It is 6 times more expensive than normal plastics which are already in use⁸. The carbon sources such as olive oil, fermented molasses, date syrup, pomace, and effluents from paper mills and palm oil mills have accumulated biodegradable PHAs of around 40 and 70%⁹.

Polyhydroxybutyrates

There have been various microorganisms that have been found to produce PHBs, a copolymer of poly (3- hydroxybutyrate-co-hydroxy-valerate) utilizing biomasses consisting of lignocellulosic molecules¹⁰. PHBs produce granules that are not soluble in water and act as a storage unit of energy under stress conditions. Both Gram-positive and Gram-negative bacteria synthesize these PHBs intracellularly only when there is an excess of carbon and a lesser amount of other nutrients¹¹.

In 1925, the first PHB (polyester poly-3-hydroxybutyrate) was discovered by Lemoigne. It has a linear chain structure in both crystalline and amorphous phases, with high crystallinity and availability in both pure polymer and also as copolymers. It is mainly produced as a carbon storage unit under stress in most bacterial strains¹². A sufficient amount of carbon supply (simple sugars such as glucose, fructose, mannose, galactose, sucrose, and xylose or polysaccharides such as starch), lipids (oleates and glycerides), and nitrogen is the key component for the growth of microorganisms that produce PHBs. Nitrogen provides proteins, nucleic acid, and co-enzymes like vitamins for the growth of these microorganisms¹³. In 1983, researchers found that Pseudomonas oleovorans produce poly-beta-hydroxyoctanoate granules when grown on octane. That was the first time a PHA other than PHBs was identified¹⁴.

PHB is a member of the PHA family, which is distinguished by an ester linkage group (-COOR) and methyl functional group (-CH₃). Functional groups present in the PHBs are the main reason for the material’s hydrophobic nature, high crystallinity, thermoplastic, and brittle properties. Melting temperature (T_m) and glass transition temperature (T_g) are two temperatures that explain the thermal properties of the material¹⁵.

Many bacterial species that produce PHBs have been identified and used. Bacillus cereus¹⁶, Cupriavidus necator^17, Pseudomonas aeruginosa¹⁸, Azospirillum ruburum¹⁹, Brevundimonas spp., and Enterococcus spp are few bacterial species isolated from the wastes of the cardboard industry with the ability to produce PHBs²⁰. Few other species, such as Burkholderia cepacian²¹ and Pseudomonas putida²² isolated from the biodiesel-glycerol and vegetable oil wastes, respectively, also have the ability to produce PHBs. Recent reports also explain that Bacillus megaterium, Bacillus siamensis, Bacillus subtilis, Staphylococcus aureus²³, Paraburkholderia spp.²⁴, Methulocystis spp., Rhizobium spp.^25;26, Aeromonas hydrophia, Burkholderia sacchari, Acinetobacter spp., Halomonas boliviensis, Sphingobacterium spp., Caulobacter spp., Brochothrix spp., Ralstonia spp., and Yokenella spp.²⁷ also have the potential to produce PHB. Recently, it was reported that eukaryotic algae such as Chlorella²⁸ and Botryococcus²⁹ have the ability to produce PHB naturally.

Mechanism of Phb Production and Regulation

Microorganisms produce PHBs through various mechanisms. The production mechanism mainly depends on whether the chief source is structurally similar or not similar to the PHB structure. For example, hydroxyalkanoic acids are structurally related, while glucose is structurally unrelated to the PHB structure.

The most common pathway observed in most microorganisms is the formation of PHB from two acetyl-CoA molecules. There are three main phases involved in this biochemical mechanism through which PHBs are produced. The first phase involves the formation of acetoacetyl-coenzyme A. Utilizing 3-ketothiolase (PhaA), two molecules of acetyl-CoA are condensed to form acetoacetyl-coenzyme A (CoA). PhaA enzyme is an acetyl-CoA acetyltransferase, also known as acetoacetyl-CoA thiolase, which assists in the condensation of two units of acetyl-CoA to acetoacetyl-CoA. The second phase involves the formation of 3-hydroxybutyryl-CoA. In this step, acetoacetyl-CoA is reduced by nicotinamide adenine dinucleotide (PhaB) to generate 3-hydroxybutyryl-CoA. Here, one molecule of NADPH is oxidized to NADP⁺. The third phase involves the formation of PHB by utilizing the PHB synthase (PhaC). 3-hydroxybutyryl-CoA formed in the previous step is polymerized, releasing CoA to form PHB³⁰. Microorganisms such as Aeromonas hydrophila produce PHB using both acetyl-CoA and beta-oxidation pathways simultaneously³¹.

PhaR/PhaP gene is the main regulatory gene involved in the regulation of PHB production. To observe the regulatory conditions, the phaR gene was mutated and compared to the PhaP1 and PhaP4 double mutated genes. The results showed that under impaired conditions, PhaR gene showed less PHB production when compared to PhaP1 and PhaP4 double mutated genes. Other than that, PhaR negatively regulated the PhaP1, PhaP2, and other genes such as PhaA1, PhaA2, PhaC1, and PhaC2 genes involved in the PHB production. By these, the researchers understood that PhaR gene, by controlling the expression of phasins and biosynthetic enzymes, regulates the PHB granule formation³².

Proteins Associated with PHBs

PHBs have spherically shaped, highly organized structures known as PHB granules or carbonosomes. The proteins related to PHB granules are named granule-associated proteins (GAP), located on their surface. It has many roles, such as structural, biosynthetic, catabolic, and regulatory functions. Other than GAP, low-molecular weight proteins named phasins are also attached to the surface of PHB granules³³. Unlike GAP, phasins are rare proteins that avoid interaction with other proteins and shield the hydrophobic PHB surface from hydrophilic cytoplasm. An example of phasin is PhaP1, which is present in Cupriavidus necator³⁴.

GAP also has other functions than covering the granule surface. PhaM, a granule associated protein present in C. necator, binds to PhaC chromosomal DNA and plays a role in the equal distribution between the daughter cells. PhaM influences the PHB production by activating the PHB synthase (PhaC1)³⁵.

Application of PHB In Various Industries

PHB is the most widely and popularly used alternative for non-biodegradable plastics such as polypropylene. PHB has properties such as tensile strength, tensile modulus, and melting temperature, similar to polypropylene. However, PHBs are biodegradable, while polypropylene doesn’t have that property. Unlike polypropylene, they are biocompatible and doesn’t release any toxic substances³⁶. They are more suitable to replace polypropylene in many industries, but a few limitations make them difficult to use. The limitations include high-cost production, low thermal stability, high degree of crystallinity, brittleness, and hydrophobicity^37;38.

The PHB can be biodegraded in soil, water, and both aerobic and anaerobic environments. It can be degraded by the microorganisms containing extracellular depolymerases. The aerobic degradation of PHB will lead to the release of CO₂ and H₂O. In anaerobic degradation, in addition to CO₂ and H₂O, methane is also released into the environment. The degradation activity depends on different parameters such as pH, temperature, moisture, microbial activity, and PHB molecular weight³⁹.

PHB are combined with other materials to overcome their limitations. Some examples include hyaluronic acid (HA), polycaprolactone (PCL), polylactic acid (PLA), polyethylene glycol (PEG), chitosan, and other material³⁸. PHB finds its application in various biomedical, pharmacology, packaging, and agricultural industries.

PHB in Medicine and Drug Delivery

The inflammation caused by macrophages exposes PHB to extracellular liquids and cells, resulting in the degradation of the polymer into monomers and oligomers of 3-hydroxybutyrate⁴⁰. This degradation property has made PHB a good candidate for the delivery of drugs⁴¹. The PHB was incorporated with an inhibiting agent called ursolic acid against tumor proliferation, producing antitumor PHB nanoparticles. This is done to increase the activity, availability, and delivery of the ursolic acid in PHB nanoparticles against HeLa cells. This study also reveals that the ursolic acid release is more efficient at 96hr, and the number of dead cells was high at that time⁴².

Parsian⁴³ had designed PHB coated magnetic nanoparticles, which are loaded with gemcitabine (GEM-PHB-MNPs) to treat breast cancer. Also observed that the gemcitabine is released from the PHB coated nanoparticles only during an acidic environment i.e., during the presence of tumor cells, the nanoparticles are not cytotoxic to normal cells.

Extended-spectrum antibiotics loaded in PHB microspheres and PHB nanospheres are used to prevent infections caused by surgeries. Antibiotics such as sulbactam ampicillin or cefoperazone and gentamicin are loaded in the PHB for drug delivery⁴⁴.

Natural and synthetic polymers such as PHB are investigated by researchers for the production of fibrous materials⁴⁵. The researcher successfully blended collagen with PHB to form fibrous scaffolds using TFA co-solvent, which can be used in cartilage engineering. They also found that the material produced had high hydrophilicity and a high weight loss rate with suitable mechanical properties⁴⁶.

Zhou⁴⁷, by electrospinning technique, constructed a biocomposite using chitosan and PHB. Through this research, it was found that with different percentages of PHB and chitosan, medical devices can be constructed with controlled degradation rate.

The use of biopolymers can also lead to promising medical applications such as implant materials which are immune to human immune responses. ⁴⁸. It had become an ideal material for the delivery of antimicrobial compounds to a target site and nano-entrapment⁴⁹. PHB also provides an antimicrobial effect to titanium loaded with antibiotic implants, which is used to prevent infections caused by synthetic implants⁵⁰.

As PHBs are well tolerated by the immune system, they can be widely used to create surgical mesh, medical devices, orthopedic pins, surgical sutures, stents, repair patches, heart valves, staples, and screws⁵¹. Microcapsules made of PHB can be utilized to enclose Langerhans cells to restore insulin production and release; as a result, it can also be used as packing material for tablets^52;53.

PHB in Tissue Engineering:

An extracellular matrix of rabbit chondrocytes was grown on polyhydroxybutyrate-co-hydroxyhexanoate (PHB-co-PHH) scaffolds⁵⁴. Researchers also observed an increased production of collagen in PHB-co-PHH than in normal PHB. For wound dressing and ocular implants, PHB based composites were used, as well as scaffolds for bone implants⁵⁵. Other than these, artificial tissues of retinal, tendon, bone, cartilage, and muscle have been developed using PHB-based composites^56;57.

PHB and Piezoelectric Materials in Bone Tissue Regeneration:

The use of polyhydroxybutyrate-co-valerate (PHBV) based piezoelectric material has shown a great result in bone tissue engineering. It has amazing biocompatibility and has the ability to make the material attractive in some tissue engineering related applications, such as the construction of functional scaffolds⁵⁸.

The researchers had developed PHBV or chitosan nanocomposite scaffolds embraced with Nano-Hydroxyapatite (nHA), which had been proven to be an alternative for bone tissue engineering due to its osteoconductivity and biocompatibility⁵⁹. These scaffolds are further fabricated using 3D printing technology, which has been used to improve osteogenic differentiation and cell proliferation for bone tissue regeneration⁶⁰. To improve bone tissue regeneration and osteoblast proliferation, bioactive glass nanoparticles embraced with PHBV scaffolds have been used⁶¹.

PHB in Food Packaging

PHB acts as a barrier against water vapour, oxygen, and carbon dioxide and is stable, flexible, and highly resistant, which makes them suitable for use in food packaging⁶². They are also biodegradable and non-toxic to the environment, unlike polypropylene, widely used in bottles and jars manufacturing⁶³. Coconut Fibers blended PHB composites have exhibited better thermal stability and good tensile properties, which has made them better for use as a plastic bag that can be recovered as seeds and planted⁶⁴.

PHB in Other Industries

Biomaterials can be produced by fabricating biopolymers, which can be used for many new industrial based application purposes⁶⁵. As environmental pollution has become an increasingly serious issue in today’s world, biopolymers have largely been used to tackle various challenges that have yet to be resolved⁶⁶. To improve the functional and physical properties of biopolymers, materials such as essential oils, nanomaterials, bioactive components, and nanomaterials are used⁶⁷. Few biopolymer-based biomaterials are microfibers and nanofibers, which are used in textile industries⁶⁸.

Due to its biodegradable properties, PHB can be used for foils and films production. It has become a flawless material for packing materials such as diapers, sanitary towels, shampoo bottles, disposable hygiene products, milk cartons, and razors due to its water-resistant property⁶⁹.

In recent years, PHBs have replaced commercial plastics to produce eco-friendly grow bags, protection nets, and compostable greenhouse films⁷⁰. Based on the PHA copolymer of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), biodegradable mulch has been produced and patented by Danimer Scientific^71;72.

To improve crop growth and for the protection of crops from insects, birds, and natural climate fluctuations, biodegradable PHBs based agriculture nets are produced and used. It also helps the crop from overheating. Unlike normal plastics, these are biodegradable plastics that can be directly disposed of in the soil⁷³. It also works as a microbial growth matrix that is friendly to roots and helps in water denitrification⁷⁴.

PHB In Synthetic Biology

PHB production in the microorganisms can be enhanced by using some of the synthetic biology and genome editing approaches. Ribosome-binding (RBS) optimization, cell morphology engineering, promoter engineering, chromosomal integration, downstream processing, and cell growth behaviour reprogramming are some synthetic biology approaches used to increase PHB production. The most recent approach used to increase the PHB synthetic pathways is CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) or CRISPR-associated protein 9 (Cas9)⁷⁵.

RBS and promotor engineering of genes in phbCAB operon, which is responsible for the PHB accumulation, has been proven to improve the transcription levels that directly influence the PHB production in the microorganisms⁷⁶. RBS is of high accuracy and can be used to control gene expression using the calculator designed specifically for this method⁷⁷. Using this method, a clone of phbCAB operon has been produced from Cupriavidus necator, which was used to optimize PHB production pathway in Escherichia coli. The cell dry weight has been in the range of 0% to 92% in the E. coli, which was genetically engineered by the RBS method, indicating the efficiency of this method⁷⁸.

Other than the RBS method, the promoter engineering method can also be used to regulate gene expression. It is one of the most common and powerful tools used to promote gene expression in the microorganisms next to the RBS method. Inducible hybrid promoters such as P_lacl and P_trp were developed using this method⁷⁹. These inducible promoters were successful in promoting gene expression in E. coli; however, they were not able to promote gene expression in some organisms, such as Halomonas bluephagenesis⁸⁰. By this method, the researchers were able to synthesize P3HB4HB, which has resulted in the >100 g/l cell weight with the productivity of 1.59 g/(l^-h) that contains 80% poly(3-hydroxybutyrate-co-4-hydroxybutyrate)⁸¹.

CRISPR/Cas9 Method For PHB Synthesis

CRISPR/Cas9 is the most widely used technique for genomic editing in recent times. It is not only used for genomic editing but also for gene deletion, insertion, and also for the replacement of genes in some eukaryotic and bacterial cells⁸⁰. It has been extensively used in both model and non-model organisms such as E. Coli, Bacilli subtilis, Clostridium beijerinckii, Corynebacterium glutamicum, Lactocoocus lactis, Streptomyces spp., Cupriavidus necator, Klebsiella pnemoniae, and Halomonas bluephagenesis^{82;83;84;85;86;87}. A model organism E. Coli has been modified using CRISPR/Cas9. The genes such as pflb, IdhaA, adhE, and fnr, which are related to the by-product formation, have been deleted. With the pntAB overexpression, which catalyzes the conversion of NADH and NADPH increased, the PHB production resulted in cell growth⁸⁸. Cupriavidus necator and H. bluephagenesis, which are non-model organisms, were used by the researchers in Cupriavidus necator. They were able to edit five genes with an efficiency range of 78% to 100% using inducible pBAD promotor⁸⁹. In H. bluephagenesis, they were able to reach an efficiency of 100% using CRISPR/Cas9. Deleting the prpC gene from H. bluephagenesis using CRISPR/Cas9 resulted in the production of PHB in H. bluephagenesis⁹⁰. Recently, it has created more attention and also led to the development of inclusion bodies. Researchers had changed the cell morphology from rod to sphere shaped by eliminating mreB, an actin-like protein gene⁹¹. The other proteins that are involved in the cell division, such as FtsZ, SulA, and Ftsz inhibitor MinCD, have also been engineered to manipulate and control PHB production⁹².

Genome editing techniques such as RBS, promotor engineering method, and CRISPR/Cas9 play a vital role in the synthetic PHB production pathways and their regulation. These techniques help microbes to produce PHB quickly and also increase production when compared to normal microbes. Other than these techniques, more methods can be developed which will be useful for the PHB production industries⁸⁹.

A major drawback of the use of PHB in developing a product is its cost. The cost of microbial PHB production depends on both upstream and downstream processes. To reduce the cost of upstream processes, many techniques, such as the usage of low-cost carbon sources, economically synthetic pathway development, and many ingenious techniques for concentration, purification, and formulation of the products, were developed⁹³. For downstream processing, some changes in the behaviour of cell growth and shapes have been developed using synthetic and genome editing techniques.

Increased PHB production and easy downstream recovery of product through sedimentation or filtration can be done when the FtsZ gene is inhibited. The inhibition of this gene leads to abnormal cell growth and filamentous cell formation, which will directly lead to more intracellular space for product accumulation⁹⁴. Another example is the change in cell division patterns in E. coli caused by disrupting the MinC and MinD cell regulators. By disrupting the cell regulators, the binary fission is changed into multiple fission, which leads to a higher accumulation of PHB⁹⁵.

Advantages and Drawbacks of Biopolymers

Biopolymers are widely used instead of synthetic plastics due to its eco-friendly and easily degradable nature. It has become an alternative to petroleum-based plastics in a short period of time. The researchers have also identified that these biopolymers have the potential to reduce global warming. The rate of recycling of biopolymers is high, and it releases fewer toxins into the environment when compared to synthetic plastics. Due to its biocompatible and biodegradable nature, it is widely used in many industries, such as food and agriculture. These biopolymer-based products are widely used in the medical field as implants, material for drug delivery, scaffolds, and dressings. The greater advantage of these microbial biopolymers is that it can be easily manipulated according to the needs of the industries.

In industrial scale, commercial PHA production requires expensive raw materials and chemicals as sources of organic matter. The costs associated with fossil-fuel plastic production must be offset in order for this technology to be economically viable⁹⁶. At lab-scale, many operating alternatives have been proposed to increase profitability and make the system easier to implement in the plastics market. Using industrial by-products and waste streams to create PHA has the advantage of being a more environmentally friendly method. Examples include agriculture feedstock, waste plant oils, and wastewater^97,98.

However, there are a few drawbacks to completely replacing synthetic plastics with biopolymers. Due to the biopolymers limited mechanical properties, high-cost production, and low processing capabilities, they are not widely used in industries as an alternative to synthetic plastics. The PHBs must be blended with other materials to make it flexible to use, which increases their cost of production. Due to high production, many industries are unable to completely replace synthetic plastics with biopolymers and researchers are working and trying to figure out methods to reduce productivity costs.

Conclusion

Synthetic plastics were most widely used and disposed of in the environment without proper treatment. These disposed wastes remain in the environment for longer periods of time, and accumulate in the soil for a prolonged time. This accumulation has led to many environmental concerns, causing harm to human life. The only way is either to identify how to degrade these synthetic plastics or to identify an alternative for the synthetic plastics. Researchers are also keen to identify an alternative. As a result, biopolymers were identified, which can be produced from different biological sources. The production of polyhydroxyalkonates (PHAs), such as polyhydroxybutyrates (PHBs) from microbes, represents a promising and sustainable avenue in this field. This has several advantages, which include environmental friendliness and renewable resource utilization, and it also aids in reducing dependence on fossil fuels.

As a biodegradable alternative to traditional petroleum-based plastics, PHB holds immense potential to address the escalating environmental concerns associated with plastic waste. Industries and consumers increasingly prioritize sustainable practices, and the production of biopolymers from microbes offers a viable solution to reduce the ecological footprint of plastic materials. Collaborative efforts between academia, industry, and policymakers will play a pivotal role in accelerating the adoption of microbial-based biopolymer production, fostering a more sustainable and circular economy. In the quest for eco-friendly alternatives, paving the way for a greener and more sustainable future.

Acknowledgement

The authors thank the Management and the Principal, Dwaraka Doss Goverdhan Doss Vaishnav College, Arumbakkam, Chennai for providing the necessary support to carry out this work.

Conflict of Interest

The authors declare no conflict of interests.

Funding Sources

The authors did not receive any financial support.

Data Availability Statement

All data and materials are available with corresponding authors.

Ethics Statement

This research did not involve human participants, animal subjects, or any material that requires ethical approval.

Authors’ Contribution

All authors contributed to the conception and design of this study. Material preparation, data collection and analysis were performed by Balakumaran MD and Swetha J. Manuscript was proof read and approved by Balakumaran MD, Uma A, Ananth C, Nithya K, Swetha J. 

References

1. Frias JP, Nash R. Microplastics: Finding a consensus on the definition. Pollut. Bull. 2019; 138:145-7.
   CrossRef
2. Bucci K, Tulio M, Rochman CM. What is known and unknown about the effects of plastic pollution: A meta-analysis and systematic review. Appl. 2020; 30(2):e02044.
   CrossRef
3. Chalmin P. The history of plastics: from the Capitol to the Tarpeian Rock. Field actions science reports. Field Actions Sci. Rep. 2019; 1(Special Issue 19):6-11.
4. Gowthaman NSK, Lim HN, Sreeraj TR, Amalraj A, Gopi S. 2021. Advantages of biopolymers over synthetic polymers: social, economic, and environmental aspects. In Biopolymers and their industrial applications (pp. 351-372). Elsevier.
   CrossRef
5. Koskimäki JJ, Kajula M, Hokkanen J, Ihantola EL, Kim JH, Hautajärvi H, Hankala E, Suokas M, Pohjanen J, Podolich O, Kozyrovska N. Methyl-esterified 3-hydroxybutyrate oligomers protect bacteria from hydroxyl radicals. Chem. Biol. 2016; 12(5):332-8.
   CrossRef
6. Kim BS. Production of medium chain length polyhydroxyalkanoates by fed-batch culture of Pseudomonas oleovorans. Lett. 2002; 24:125-30.
7. Sun Z, Ramsay JA, Guay M, Ramsay BA. Fermentation process development for the production of medium-chain-length poly-3-hyroxyalkanoates. Microbiol. Biotechnol. 2007; 75:475-85.
   CrossRef
8. Khatami K, Perez-Zabaleta M, Owusu-Agyeman I, Cetecioglu Z. Waste to bioplastics: How close are we to sustainable polyhydroxyalkanoates production? Waste Manag. 2021; 1; 119:374-88.
   CrossRef
9. Abd El-malek F, Khairy H, Farag A, Omar S. The sustainability of microbial bioplastics, production and applications. J. Biol. Macromol. 2020; 15; 157:319-28.
   CrossRef
10. Muneer F, Rasul I, Azeem F, Siddique MH, Zubair M, Nadeem H. Microbial polyhydroxyalkanoates (PHAs): efficient replacement of synthetic polymers. Polym. Environ. 2020; 28:2301-23.
    CrossRef
11. Możejko-Ciesielska J, Kiewisz R. Bacterial polyhydroxyalkanoates: Still fabulous? Res. 2016; 192:271-82.
    CrossRef
12. McAdam B, Brennan Fournet M, McDonald P, Mojicevic M. Production of polyhydroxybutyrate (PHB) and factors impacting its chemical and mechanical characteristics. Polym. 2020; 4;12(12):2908.
    CrossRef
13. Allikian K, Edgar R, Syed R, Zhang S. Fundamentals of fermentation media. Ferment Technol. 2019:41-84.
    CrossRef
14. de Smet MJ, Eggink G, Witholt B, Kingma J, Wynberg H. Characterization of intracellular inclusions formed by Pseudomonas oleovorans during growth on octane. Bacteriol. 1983;154(2):870-8.
    CrossRef
15. Grigore ME, Grigorescu RM, Iancu L, Ion RM, Zaharia C, Andrei ER. Methods of synthesis, properties and biomedical applications of polyhydroxyalkanoates: a review. Biomater. Sci. Polym. Ed. 2019 13;30(9):695-712.
    CrossRef
16. Narayanan M, Kandasamy S, Kumarasamy S, Gnanavel K, Ranganathan M, Kandasamy G. Screening of polyhydroxybutyrate producing indigenous bacteria from polluted lake soil. 2020; 1;6(10).
    CrossRef
17. Narayanan M, Kumarasamy S, Ranganathan M, Kandasamy S, Kandasamy G, Gnanavel K, Mamtha K. Production and characterization of polyhydroxyalkanoates synthesized by E. coli Isolated from sludge soil. Mater. Today Proc. 2020; 1; 33:3646-53.
    CrossRef
18. Aramvash A, Akbari Shahabi Z, Dashti Aghjeh S, Ghafari MD. Statistical physical and nutrient optimization of bioplastic polyhydroxybutyrate production by Cupriavidus necator. IJEST. 2015; 12:2307-16.
    CrossRef
19. Javed H, Jamil N. Utilization of mustard oil for the production of Polyhydroxyalkanoates by Pseudomonas aeruginosa. microbio., biotechnol. food sci. 2015; 1; 2021:412-4.
    CrossRef
20. Jin H, Nikolau BJ. Evaluating PHA productivity of bioengineered Rhodosprillum rubrum. PloS one. 2014;19;9(5): e96621.
    CrossRef
21. Bhuwal AK, Singh G, Aggarwal NK, Goyal V, Yadav A. Isolation and screening of polyhydroxyalkanoates producing bacteria from pulp, paper, and cardboard industry wastes. J. Biomater. 2013.
    CrossRef
22. Zhu C, Nomura CT, Perrotta JA, Stipanovic AJ, Nakas JP. Production and characterization of poly-3-hydroxybutyrate from biodiesel-glycerol by Burkholderia cepacia ATCC 17759. Prog. 2010; 26(2):424-30.
    CrossRef
23. Gatea IH, Abbas AS, Abid AG, Halob AA, Maied SK, Abidali AS. Isolation and characterization of Pseudomonas putida producing bioplastic (Polyhydroxyalkanoate) from vegetable oil waste. Pak J Biotechnol. 2018; 15:469-73.
24. Vijay R, Tarika K. Microbial production of polyhydroxyalkanoates (PHAs) using kitchen waste as an inexpensive carbon source., Biotech. Res. Asia. 2019; 30;16(1):155-66.
    CrossRef
25. Sriyapai T, Chuarung T, Kimbara K, Samosorn S, & Sriyapai P. Production and optimization of polyhydroxyalkanoates (PHAs) from Paraburkholderia PFN 29 under submerged fermentation. Electron. J. Biotechnol. 2022; 56, 1–11. https://doi.org/10. 1016/j.ejbt.2021.12.003
    CrossRef
26. Abd El-malek F, Khairy H, Farag A, Omar S. The sustainability of microbial bioplastics, production and applications. J. Biol. Macromol. 2020; 157:319-28.
    CrossRef
27. Snell K D, & Peoples O P. PHA bioplastic: A value added coproduct for biomass biorefineries. 2009. 3(4), 456–467. https://doi.org/10.1002/bbb. 161
    CrossRef
28. Mascarenhas J, Aruna K. Screening of polyhydroxyalkonates (PHA) accumulating bacteria from diverse habitats. global. biosci. 2017;6(3):4835-48.
29. Cassuriaga AP, Freitas BC, Morais MG, Costa JA. Innovative polyhydroxybutyrate production by Chlorella fusca grown with pentoses. Technol. 2018; 265:456-63.
    CrossRef
30. Kavitha G, Kurinjimalar C, Sivakumar K, Palani P, Rengasamy R. Biosynthesis, purification and characterization of polyhydroxybutyrate from Botryococcus braunii kütz. J. Biol. Macromol. 2016; 89:700-6.
    CrossRef
31. Ross G, Ross S, Tighe BJ. Bioplastics: new routes, new products. Brydson’s Plastics Materials. 2017:631-52.
    CrossRef
32. Lee SY, Wong HH, Choi JI, Lee SH, Lee SC, Han CS. Production of medium-chain-length polyhydroxyalkanoates by high-cell-density cultivation of Pseudomonas putida under phosphorus limitation. Bioeng. 2000; 68(4):466-70.
    CrossRef
33. Quelas JI, Mesa S, Mongiardini EJ, Jendrossek D, Lodeiro AR. Regulation of polyhydroxybutyrate synthesis in the soil bacterium Bradyrhizobium diazoefficiens. 2016; 82(14):4299-308.
    CrossRef
34. Mezzina MP, Pettinari MJ. Phasins, multifaceted polyhydroxyalkanoate granule-associated proteins. AEM. 2016; 82(17):5060-7.
    CrossRef
35. Pfeiffer D, Jendrossek D. Localization of poly (3-hydroxybutyrate)(PHB) granule-associated proteins during PHB granule formation and identification of two new phasins, PhaP6 and PhaP7, in Ralstonia eutropha J. Bacteriol. 2012; 194(21):5909-21.
    CrossRef
36. Pfeiffer D, Jendrossek D. PhaM is the physiological activator of poly (3-hydroxybutyrate) (PHB) synthase (PhaC1) in Ralstonia eutropha. 2014; 80(2):555-63.
    CrossRef
37. Chai JM, Amelia TS, Mouriya GK, Bhubalan K, Amirul AA, Vigneswari S, Ramakrishna S. Surface-modified highly biocompatible bacterial-poly (3-hydroxybutyrate-co-4-hydroxybutyrate): a review on the promising next-generation biomaterial. Polym. 2020; 13(1):51.
    CrossRef
38. Majerczak K, Wadkin Snaith D, Magueijo V, Mulheran P, Liggat J, Johnston K. Polyhydroxybutyrate: A review of experimental and simulation studies of the effect of fillers on crystallinity and mechanical properties. Int. 2022; 71(12):1398-408.
    CrossRef
39. Raza ZA, Khalil S, Abid S. Recent progress in development and chemical modification of poly (hydroxybutyrate)-based blends for potential medical applications. J. Biol. Macromol. 2020; 160:77-100.
    CrossRef
40. Rajan KP, Thomas SP, Gopanna A, Chavali M. Polyhydroxybutyrate (PHB): a standout biopolymer for environmental sustainability. of ecomater. 2019;4.
    CrossRef
41. Bonartsev AP, Bonartseva GA, Reshetov IV, Kirpichnikov MP, Shaitan KV. Application of polyhydroxyalkanoates in medicine and the biological activity of natural poly (3-hydroxybutyrate). Acta Naturae. 2019;11(2 (41)):4-16.
    CrossRef
42. Shishatskaya E I, Voinova O N, Goreva A V, Mogilnaya O A, Volova T G. Biocompatibility of polyhydroxybutyrate microspheres: In vitro and in vivo evaluation. Mat. Sci. 2008, 19, 2493–2502.
    CrossRef
43. Pandian SR, Kunjiappan S, Pavadai P, Sundarapandian V, Chandramohan V, Sundar K. Delivery of ursolic acid by polyhydroxybutyrate nanoparticles for cancer therapy: in silico and in vitro studies. Drug Res. 2022; 72(02):72-81.
    CrossRef
44. Parsian M, Mutlu P, Yalcin S, Gunduz U. Characterization of gemcitabine loaded polyhydroxybutyrate coated magnetic nanoparticles for targeted drug deliver. Anti-cancer Agents Med. Chem. (Formerly Current Medicinal Chemistry-Anti-Cancer Agents). 2020; 20(10):1233-40.
    CrossRef
45. Martínez MD, Urzúa LS, Carrillo YA, Ramírez MB, Morales LJ. Polyhydroxybutyrate Metabolism in Azospirillum brasilense and Its Applications, a Review. Polym. 2023; 15(14):3027.
    CrossRef
46. Sadeghi D, Karbasi S, Razavi S, Mohammadi S, Shokrgozar MA, Bonakdar S. Electrospun poly (hydroxybutyrate)/chitosan blend fibrous scaffolds for cartilage tissue engineering. Appl. Polym. Sci. 2016; 133(47).
    CrossRef
47. Khoroushi M, Foroughi MR, Karbasi S, Hashemibeni B, Khademi AA. Effect of Polyhydroxybutyrate/ Chitosan/Bioglass nanofiber scaffold on proliferation and differentiation of stem cells from human exfoliated deciduous teeth into odontoblast-like cells. Mater. Sci. Eng. C. 2018; 89:128-39.
    CrossRef
48. Zhou Y, Li Y, Li D, Yin Y, Zhou F. Electrospun PHB/chitosan composite fibrous membrane and its degradation behaviours in different pH conditions. Funct. Biomater. 2022; 13(2):58.
    CrossRef
49. Shrivastav A, Kim HY, Kim YR. Advances in the applications of polyhydroxyalkanoate nanoparticles for novel drug delivery system. Res. Int. 2013.
    CrossRef
50. Basnett P, Ching KY, Stolz M, Knowles JC, Boccaccini AR, Smith C, Locke IC, Roy I. Aspirin-loaded P (3HO)/P (3HB) blend films: Potential materials for biodegradable drug-eluting stents. Bioinspired Biomim. Nanobiomaterials. 2013; 2(3):141-53.
    CrossRef
51. Rodriguez-Contreras A. Recent advances in the use of polyhydroyalkanoates in biomedicine. 2019; 6(3):82.
    CrossRef
52. Oryan A, Alidadi S, Moshiri A, Maffulli N. Bone regenerative medicine: classic options, novel strategies, and future directions. Orthop. Surg. Res. 2014; 9:1-27.
    CrossRef
53. Peng Q, Sun X, Gong T, Wu CY, Zhang T, Tan J, Zhang ZR. Injectable and biodegradable thermosensitive hydrogels loaded with PHBHHx nanoparticles for the sustained and controlled release of insulin. Acta Biomater. 2013; 9(2):5063-9.
    CrossRef
54. Poltronieri P, Kumar P. Polyhydroxyalkanoates (PHAs) in industrial applications. of ecomater. 2017; 4:2843-72.
    CrossRef
55. Deng Y, Lin XS, Zheng Z, Deng JG, Chen JC, Ma H, Chen GQ. Poly (hydroxybutyrate-co-hydroxyhexanoate) promoted production of extracellular matrix of articular cartilage chondrocytes in vitro. Biomater. 2003 Oct 1;24(23):4273-81.
    CrossRef
56. Sharma N. Polyhydroxybutyrate (PHB) production by bacteria and its application as biodegradable plastic in various industries. J. Polym. Sci. 2019, 2, 001–003.
    CrossRef
57. Lee CW, Horiike M, Masutani K, Kimura Y. Characteristic cell adhesion behaviors on various derivatives of poly (3-hydroxybutyrate) (PHB) and a block copolymer of poly (3-[RS]-hydroxybutyrate) and poly (oxyethylene). Degrad. Stab. 2015; 111:194-202.
    CrossRef
58. Garcia-Garcia D, Ferri JM, Boronat T, López-Martínez J, Balart R. Processing and characterization of binary poly (hydroxybutyrate)(PHB) and poly (caprolactone)(PCL) blends with improved impact properties. Bull. 2016; 73:3333-50.
    CrossRef
59. Li Y, Liao C, Tjong SC. Electrospun polyvinylidene fluoride-based fibrous scaffolds with piezoelectric characteristics for bone and neural tissue engineering. 2019; 9(7):952.
    CrossRef
60. Amaro L, Correia DM, Martins PM, Botelho G, Carabineiro SA, Ribeiro C, Lanceros-Mendez S. Morphology dependence degradation of electro-and magnetoactive poly (3-hydroxybutyrate-co-hydroxyvalerate) for tissue engineering applications. Polym. 2020; 12(4):953.
    CrossRef
61. Gorodzha SN, Muslimov AR, Syromotina DS, Timin AS, Tcvetkov NY, Lepik KV, Petrova AV, Surmeneva MA, Gorin DA, Sukhorukov GB, Surmenev RA. A comparison study between electrospun polycaprolactone and piezoelectric poly (3-hydroxybutyrate-co-3-hydroxyvalerate) scaffolds for bone tissue engineering. Colloids and Surf B: Biointerfaces. 2017; 160:48-59.
    CrossRef
62. Jiao H, Song S, Zhao K, Zhang X, Tang Y. Synthesis and properties of porous piezoelectric BT/PHBV composite scaffold. Biomater. Sci. Polym. Ed. 2020; 31(12):1552-65.
    CrossRef
63. Rhim JW, Park HM, Ha CS. Bio-nanocomposites for food packaging applications. Polym. Sci. 2013; 38(10-11):1629-52.
    CrossRef
64. Popa MS, Frone AN, Panaitescu DM. Polyhydroxybutyrate blends: A solution for biodegradable packaging? J. Biol. Macromol. 2022; 207:263-77.
    CrossRef
65. Hosokawa MN, Darros AB, Moris VA, Paiva JM. Polyhydroxybutyrate composites with random mats of sisal and coconut fibers. Res. 2016; 20:279-90.
    CrossRef
66. Roy S, Siracusa V. Multifunctional Application of Biopolymers and Biomaterials. J. Mol. Sci. 2023; 24(12):10372.
    CrossRef
67. Baranwal J, Barse B, Fais A, Delogu GL, Kumar A. Biopolymer: A sustainable material for food and medical applications. 2022;14(5):983.
    CrossRef
68. Ebrahimzadeh S, Biswas D, Roy S, McClements DJ. Incorporation of essential oils in edible seaweed-based films: A comprehensive review. Trends Food Sci. Technol. 2023
    CrossRef
69. Deng Z, Wang T, Chen X, Liu Y. Applications of chitosan-based biomaterials: a focus on dependent antimicrobial properties. 2020; 2(4):398-413.
    CrossRef
70. Singh G, Kumari A, Mittal A, Yadav A, Aggarwal NK. Poly β-hydroxybutyrate production by Bacillus subtilis NG220 using sugar industry waste water. Res. Int. 2013.
    CrossRef
71. Shamsuddin IM, Jafar JA, Shawai AS, Yusuf S, Lateefah M, Aminu I. Bioplastics as better alternative to petroplastics and their role in national sustainability: a review. Biosci. Bioeng. 2017; 5(4):63.
    CrossRef
72. Chen GQ. Plastics completely synthesized by bacteria: polyhydroxyalkanoates. Plastics from bacteria: natural functions and applications. 2010:17-37.
    CrossRef
73. Scientific D. Danimer scientific film resins. Compostable Options for a Wide Range of Products. 2018.
74. Guerrini S, Borreani G, Voojis H. Biodegradable materials in agriculture: case histories and perspectives. Soil degradable bioplastics for a sustainable modern agriculture. 2017:35-65.
    CrossRef
75. Amelia TS, Govindasamy S, Tamothran AM, Vigneswari S, Bhubalan K. Applications of PHA in agriculture. Biotechnological applications of polyhydroxyalkanoates. 2019:347-61.
    CrossRef
76. Zhang X, Lin Y, Wu Q, Wang Y, Chen GQ. Synthetic biology and genome-editing tools for improving PHA metabolic engineering. Trends in biotechnol. 2020; 38(7):689-700.
    CrossRef
77. Shen R, Yin J, Ye JW, Xiang RJ, Ning ZY, Huang WZ, Chen GQ. Promoter engineering for enhanced P (3HB-co-4HB) production by Halomonas bluephagenesis. ACS Synth. Biol. 2018; 7(8):1897-906.
    CrossRef
78. Zhang S, Zhao X, Tao Y, Lou C. A novel approach for metabolic pathway optimization: Oligo-linker mediated assembly (OLMA) method. Biol. Eng. 2015; 9:1-0.
    CrossRef
79. Li T, Ye J, Shen R, Zong Y, Zhao X, Lou C, Chen GQ. Semirational approach for ultrahigh poly (3-hydroxybutyrate) accumulation in Escherichia coli by combining one-step library construction and high-throughput screening. ACS Synth. Biol. 2016; 5(11):1308-17.
    CrossRef
80. Tan D, Wu Q, Chen JC, Chen GQ. Engineering Halomonas TD01 for the low-cost production of polyhydroxyalkanoates. Eng. 2014; 26:34-47.
    CrossRef
81. Chen X, Yu L, Qiao G, Chen GQ. Reprogramming Halomonas for industrial production of chemicals. JIMB. 2018; 45(7):545-54.
    CrossRef
82. Beckers V. Screening of endogenous strong promoters for enhanced production of medium-chain-length polyhydroxyalkanoates in Pseudomonas mendocina, 2019. NK-01. Sci. Rep. 9, 1798
    CrossRef
83. Qin Q, Ling C, Zhao Y, Yang T, Yin J, Guo Y, Chen GQ. CRISPR/Cas9 editing genome of extremophile Halomonas spp. Metab. Eng. 2018; 47:219-29.
    CrossRef
84. Cho S, Choe D, Lee E, Kim SC, Palsson B, Cho BK. High-level dCas9 expression induces abnormal cell morphology in Escherichia coli. ACS Synth. Biol. 2018; 7(4):1085-94.
    CrossRef
85. Tao W, Lv L, Chen GQ. Engineering Halomonas species TD01 for enhanced polyhydroxyalkanoates synthesis via CRISPRi. Cell fact. 2017; 16:1-0.
    CrossRef
86. Woolston BM, Emerson DF, Currie DH, Stephanopoulos G. Rediverting carbon flux in Clostridium ljungdahlii using CRISPR interference (CRISPRi). Eng. 2018; 48:243-53.
    CrossRef
87. Xiong B, Li Z, Liu L, Zhao D, Zhang X, Bi C. Genome editing of Ralstonia eutropha using an electroporation-based CRISPR-Cas9 technique. Biofuels bioprod. 2018; 11:1-9.
    CrossRef
88. Wang J, Zhao P, Li Y, Xu L, Tian P. Engineering CRISPR interference system in Klebsiella pneumoniae for attenuating lactic acid synthesis. Microb. Cell fact. 2018; 17:1-2.
    CrossRef
89. Jung HR, Yang SY, Moon YM, Choi TR, Song HS, Bhatia SK, Gurav R, Kim EJ, Kim BG, Yang YH. Construction of efficient platform Escherichia coli strains for polyhydroxyalkanoate production by engineering branched pathway. 2019; 11(3):509.
    CrossRef
90. Zhang X, Lin Y, Wu Q, Wang Y, Chen GQ. Synthetic biology and genome-editing tools for improving PHA metabolic engineering. Trends Biotechnol. 2020; 38(7):689-700.
    CrossRef
91. Qin Q, Ling C, Zhao Y, Yang T, Yin J, Guo Y, Chen GQ. CRISPR/Cas9 editing genome of extremophile Halomonas spp. Eng. 2018; 47:219-29.
    CrossRef
92. Elhadi D, Lv L, Jiang XR, Wu H, Chen GQ. CRISPRi engineering coli for morphology diversification. Metab. Eng. 2016; 38:358-69.
    CrossRef
93. Wu H, Chen J, Chen GQ. Engineering the growth pattern and cell morphology for enhanced PHB production by Escherichia coli. Appl. Microbiol. Biotechnol. 2016; 100:9907-16.
    CrossRef
94. Wang Y, Ling C, Chen Y, Jiang X, Chen GQ. Microbial engineering for easy downstream processing. Adv. 2019; 37(6):107365.
    CrossRef
95. Wang Y, Wu H, Jiang X, Chen GQ. Engineering Escherichia coli for enhanced production of poly (3-hydroxybutyrate-co-4-hydroxybutyrate) in larger cellular space. Eng. 2014; 25:183-93.
    CrossRef
96. Wu H, Chen J, Chen GQ. Engineering the growth pattern and cell morphology for enhanced PHB production by Escherichia coli. Microbiol. Biotechnol. 2016; 100:9907-16.
    CrossRef
97. Rehm, Bernd HA. “Bacterial polymers: biosynthesis, modifications and applications.” Nature Reviews Microbiology 8, no. 8 (2010): 578-592.
    CrossRef
98. Madkour MH, Heinrich D, Alghamdi MA, Shabbaj II, Steinbüchel A. PHA recovery from biomass. Biomacromolecules. 2013 Sep 9;14(9):2963-72.
    CrossRef
99. Narodoslawsky M, Shazad K, Kollmann R, Schnitzer H. LCA of PHA production–Identifying the ecological potential of bio-plastic. Chemical and biochemical engineering quarterly. 2015 Jul 18;29(2):299-305.
    CrossRef