Polyhydroxyalkanoates (PHAs) synthesis and degradation by microbes and applications towards a circular economy

Overusing non-degradable plastics causes a series of environmental issues, inferring a switch to biodegradable plastics. Polyhydroxyalkanoates (PHAs) are promising biodegradable plastics that can be produced by many microbes using various substrates from waste feedstock. However, the cost of PHAs production is higher compared to fossil-based plastics, impeding further industrial production and applications. To provide a guideline for reducing costs, the potential cheap waste feedstock for PHAs production have been summarized in this work. Besides, to increase the competitiveness of PHAs in the mainstream plastics economy, the influencing parameters of PHAs production have been discussed. The PHAs degradation has been reviewed related to the type of bacteria, their metabolic pathways/enzymes, and environmental conditions. Finally, the applications of PHAs in different fields have been presented and discussed to induce comprehension on the practical potentials of PHAs.


Introduction
To meet the trend of sustainable development and economic competitiveness, environmentally friendly technologies have been considered in extensive research (Jourdin and Burdyny, 2021).Microorganisms as cell factories have unprecedented potential in settling environmental issues and industrial applications (Khatami et al., 2021).Besides, microbial products like biopolymers have many excellent properties compared with polymers produced from chemicals and fossil fuels, such as biocompatibility, biodegradability, and structural diversity.Hence, polyhydroxyalkanoates (PHAs) as biodegradable microbial polyesters have been regarded as potential candidates to replace traditional petrochemical-derived plastics in a circular economy (Moradali and Rehm, 2020).
Currently, research on PHAs attracts more attention due to potentially wide applications based on the numerous properties of different PHAs types (Nanda et al., 2022).The upwards trend of PHAs research can be seen in Web of Science (Fig. S1).The type of variable PHAs is mainly based on the category of carbon sources (Kourmentza and Kornaros, 2016), and the mixed microbial community (MMC) involved in the utilization of carbon sources (Albuquerque et al., 2013).Low-cost renewable feedstock and waste streams are prioritized to lessen the cost of PHAs production (Sabapathy et al., 2020).Numerous value-added products can be converted from wood, grass, and agricultural residues via multiple conversion processes (Dietrich et al., 2019).Along these lines, most trash has the potential to be turned into treasure (Gross, 2012).Hence, the proposed renewable feedstock and the pre-treatment approaches open doors to ensure a new supply of sustainable carbon sources and create a more circular economy (Bellini et al., 2022).
PHAs granules can serve as energy and storage sources in microbes, and microbes play a leading role in PHAs production and degradation (Park et al., 2012).To improve the efficiency of PHAs production, different feeding regimes have been employed to enrich the high productivity of PHAs-producing cultures (Colpa et al., 2020).However, there are still some bottlenecks for PHAs production.Single strains require highly sterilized conditions and complicated operational processes (Koller and Mukherjee, 2022).PHAs-producing MMCs can overcome the limited number of carbon sources and complex operational processes, but it is challenging to ensure a stable situation of PHAs production with an MMC (Zhou et al., 2022).Besides, the process conditions significantly influence PHAs production (Johnson et al., 2010).Hence, the optimal conditions are essential to facilitate PHAs productivity.PHAs degradation has been investigated on lab scale and in natural environments ( Emadian et al., 2017;Shah et al., 2008).The degradation in production environments has yet to be revealed, to better understand the balance between PHAs production and degradation.
Generally, a majority of microorganisms have the ability of PHAs storage and PHAs degradation owing to the presence of PHAs synthase (PhaC) and PHAs depolymerase (PhaZ) (Chen et al., 2020).Many studies have been performed with mesophilic PHAs-degrading bacteria, while only a few studies on thermophilic bacteria have been published (Takeda et al., 1998).Most research focused on PHAs production and how to avoid internal degradation (Ren et al., 2010).Preventing enzymatic degradation could provide a new approach to overcome the issue of PHAs degradation in the production process (Hou et al., 2021).Meanwhile, the possibility of extracellular PHAs degradation in nature will benefit the development of a circular economy ("Science-Based Solutions to Plastic Pollution," 2020).Exploring the related enzymes and potential pathways is essential to understand the PHAs production and degradation.
This work intends to systematically outline the latest developments in PHAs synthesis, degradation, and potential applications for providing a guideline to eco-circular production.Possible renewable feedstock, the PHAs producers and degraders, the influencing parameters of production and degradation, and the perspective applications of PHAs will be discussed.

The production of PHAs
To ensure that PHAs have more competitiveness in the plastic mainstream market, many efforts have been undertaken in synthesizing a wide variety of PHAs (Ganesh Saratale et al., 2021).Many laboratories have studied PHAs production using different carbon sources showing that the type of PHA is mainly dependent on the provided carbon sources (Możejko-Ciesielska and Kiewisz, 2016).The commonly used carbon sources are grouped into five categories: gases (including methane and carbon dioxide), n-alcohols (methanol, ethanol, glycerol, and octanol), carbohydrates (glucose, fructose, sucrose, maltose, lactose, xylose, starch, and cellulose), n-alkanoic acids (including acetic acid, propionic acid, butyric acid, valeric acid, hexanoic acid, lauric acid, and oleic acid), and n-alkanes (hexane, octane, and dodecane).Considering the broad range of carbon sources, PHAs are becoming one of the most popular bio-plastics for waste management, packaging, and agriculture applications (Chen et al., 2020).
Up to now, over 150 PHAs have been identified in terms of their unique monomer units.More compositions and physicochemical properties are continuously being discovered (Choi et al., 2020).Generally, PHAs are divided into short-chain length (SCL, C3-C5) and medium-chain-length (MCL, >C5) based on the number of carbon atoms (Zheng et al., 2020).PHAs can be classified as homopolymer and copolymer according to their blends.The general structure can be seen in Fig. 1.PHAs are accumulated intracellular as a reserve energy source in the cytoplasm of the PHAs-producing microbes under unbalanced growth conditions or synthesized to resist environmental stress (e.g., high temperature, osmotic shock, and UV radiation) (Obruca et al., 2018).Poly (3-hydroxybutyrate) (PHB) is the most common and the first industrially produced homopolymeric PHA.Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHX) and poly (-3-hydroxybutyrate-co-4-hydroxybutyrate) (P3HB4HB) are the mainly produced PHAs copolymers (Chen and Wu, 2005).Different PHAs have their own unique properties and applications that attract more attention for production on industrial scale.However, the costs of substrates in PHAs production is one of the major reasons why PHAs cannot win the competition in the oil-based plastic market.To reduce the costs of carbon sources, a series of pre-treatments (e.g., hydrolysis, enzymolysis, pyrolysis, and fermentation) have been performed to transfer cheap, raw feedstock into the "food" for PHA-producing microbes (Nielsen et al., 2017).

The potential feedstocks of PHAs synthesis
The concept of "transfer waste into treasure" is prevalent, which results in agricultural residues and wastes are considered into being reused to generate valuable products (Harrison et al., 2023).Their applicability lessens the burden on waste management and make a valuable resource in a closed-loop flow (De Donno Novelli et al., 2021).PHAs production from waste streams reduces the need for virgin materials and minimized waste, which enables a closed-loop system in a circular economy.The general closed-loop of PHAs production from waste feedstock is shown in Fig. 2. Generally, the complex organic sources from waste streams can be directly converted into sugars and subsequently into volatile fatty acids (VFAs) through fermentation processes, e.g. in an anaerobic environment (Lu et al., 2009).However, pretreatment technologies increase the costs and may generate some toxic substances (e.g., furfural), which have a negative effect on PHAs synthesis (Berson et al., 2005).In order to obtain a sustainable supply of sugars and VFAs production from waste streams, the process operating parameters must be optimized (Jankowska et al., 2018).Besides, acidogenic inhibition should be avoided or minimized in fermentation (Zhou et al., 2018).Hence, a green, sustainable, and cost-effective pretreatment is required in a large-scale conversion process.W. Zhou et al.To protect the ecological environment, using waste feedstock to produce PHA provides a renewable and sustainable approach.In Table 1 a summary has been provided to better understand the potential waste feedstock for PHA production, the carbon sources derived from the waste stream, the respective PHA-producing microbes, the type of produced PHA, and the PHA concentration.Many studies have been done to use different waste streams from industry, agriculture, and our daily life for PHA production, such as sugar cane molasses, pulp and paper mill, and multiple types of wastewater.Carbon sources are obtained from waste streams, and then they are subsequently metabolized by enriched cultures or mixed microbial cultures (MMCs) to produce different PHAs (Kumar et al., 2018).Bacterial mono-cultures have been employed in industrial production with PHAs yielding up to 90 wt% of dry cell weight, while they used pure substrates resulting in high production costs (Koller, 2018).Many renewable feedstocks are complex, containing a range of substances that single strains of bacteria can not completely use as carbon source (Steinbüchel and Füchtenbusch, 1998).MMCs contain a variety of microorganisms that can make better use of these complex substrates.As a result, the usage of MMCs would reduce complicated operations in the process of PHAs production (Montiel-Jarillo et al., 2017).A techno-economic analysis also indicated that MMCs had a promising prospect in large-scale production (Wang et al., 2022).MMCs play a vital role in PHAs production, especially in using waste feedstock (Morgan-Sagastume, 2016).PHB and PHBV are the main types of produced PHAs from waste streams.Therefore, effectively using waste streams will be crucial in reducing the costs of PHA production.

The producers of PHAs
To date, more than 300 microbial species, including bacteria and Archaea, have demonstrated the capability of PHAs production (Yadav et al., 2021).Many strains have been reported for their high efficiency (more than 80 wt% of the dry cell weight) of PHAs production on a lab scale (Kourilova et al., 2020).Most of them can produce different types of PHAs based on the utilization of carbon sources (Bhatia et al., 2021).Herein, Table 2 summarizes the utilization of carbon sources from renewable feedstock by common producers of PHAs production and their feedstock.Methanotrophs, highly specialized microbes, have been identified to produce PHA when methane is the sole carbon source (Koller, 2020).Cupriavidus necator (former name: Ralstonia eutropha and Alcaligenes eutrophus) and Paracoccus denitrificans belong to hydrogen oxidizing bacteria having the ability of PHAs production using CO 2 as the sole carbon source (Volova et al., 2013), which can contribute to climate change mitigation in a circular economy (Lee et al., 2021).In addition, C. necator is a well-known strain in PHAs production that can utilize various common carbon sources, including acetate, butyrate, propionate, glucose, and fructose (Tan et al., 2014).P. denitrificans is frequently used in wastewater treatment, especially in nitrate removal (Zhang et al., 2020).High cell density cultivations of Methylobacterium extorquens using methanol as carbon source yields 70 g/L dry cell weight containing 35 wt% PHB (Mokhtari-Hosseini et al., 2009).Increasing cell biomass also provides a promising method to achieve a higher yields of PHA.Crude glycerol is a common by-product from the biodiesel industry, which has frequently been tested for PHAs production (Porras et al., 2017).When hexanoate was the sole carbon source, PHBVHHX, a kind of PHAs with superior properties was produced (Haywoodt et al., 1991), which offers more possibilities in PHA applications.
Sugars are the main carbon sources for PHAs production, and most microbes can use them as substrates to produce PHAs (Jiang et al., 2016).The prices of sugars like glucose, sucrose, and xylose are low, therefore many companies use sugars as the main substrate for PHAs production (Chanprateep, 2010).Meanwhile, most sugars can be obtained from agricultural residues and natural wastes through bio-refineries (Liu et al., 2019).Haloferax mediterranei can use glucose as the sole carbon source and yeast extract as the nitrogen source to produce 48.6 wt% PHBV on 85.8 g/L dry cell weight under fed-batch fermentation onditions (Don et al., 2006).Another advantage is that H. mediterranei belongs to the haloarchaea group (Archaea domain), surviving under extreme saline conditions (Simó-Cabrera et al., 2021), which reduces the costs for required sterile operations.Thermophilic microbes can also be used in the fermentative production of PHA, avoiding contamination by mesophilic microflora due to high-temperature conditions (Chavan et al., 2021).Schlegelella thermodepolymerans has been investigated for its capability of high PHA production at 55 • C (Kourilova et al., 2020), and its capability in degrading poly (3-hydroxybutyrate-co-3-mercaptopropionate) (Elbanna et al., 2003).Sucrose has been employed for PHA production using Alcaligenes latus on an industrial scale, with more than 10,000 tons of production capacity per year (Jiang et al., 2016).Polymeric carbohydrates such as starch and cellulose can also be directly used as a carbon source for PHB production, although the productivity is lower than on other simple sugars (Halami, 2008) (Sawant et al., 2017).Although Escherichia coli is  Note: MMC means mixed microbial culture, VSS presents Volatile suspended solids."-" stands for no information.
W. Zhou et al. not able to produce PHA, recombinant E. coli shows its aptitude for economical and efficient PHA production (Li et al., 2007).Salamanca-Cardona et al. ( 2014) showed that recombinant E. coli can produce PHAs from beechwood xylan.

The enzymes involved in PHAs biosynthesis
Many enzymes are related to PHAs synthesis, as shown in Fig. 3 (data based on the model strain: Ralstonia eutropha H16) and their names and functions are listed in Table S1.Up to now, more and more enzymes have been identified with a role in PHA synthesis, degradation, and regulation (Mitra et al., 2022), as listed in Table S2.Acetyl-CoA c-acetyltransferase (encoded by phaA), acetoacetyl-CoA reductase (encoded by phaB), and PHAs synthase subunit (encoded by phaC) are the main enzymes in the PHAs biosynthesis process (Stubbe and Tian, 2003).The three enzymes are encoded by three genes organized in an operon.The operon phaCAB is a class I biosynthetic pathway of PHAs, which can increase PHAs production through overexpressing (Jiang and Chen, 2016).Meanwhile, the transcriptional regulator of phasin expression (phaR) is a regulatory protein involved in the elongation process of PHAs (Choi et al., 2020).PHAs depolymerase encoded by phaZ can degrade PHAs, which results in a decrease of PHAs production (Papaneophytou et al., 2009).PhaZ is concomitantly expressed together with phaC as reported by Ren et al. (2010).The knockout of phaZ has shown its positive influence on PHAs accumulation in Pseudomonas putida KT2442 (Cai et al., 2009).Overexpressing phaC or inhibiting the enzyme PhaZ activity is an approach to enhance PHAs production in monocultures.
Except for specific enzymes in PHAs biosynthesis, many intermediates of other metabolic pathways are also involved in PHAs accumulation (Choi et al., 2020).Pyruvate dehydrogenase complex (pdh) and acetyl-CoA carboxylase (acc) are the two most prominent enzymes in the metabolic flux of PHAs biosynthesis, especially acc (Liu et al., 2018).Both of them are linked with the initial enzyme of PHAs biosynthesis (PhaA).Carbon sources are converted to pyruvate and acetyl-CoA, and then to PHA mainly depending on nutrient conditions (García et al., 2021).PhaA can be inhibited due to high coenzyme A activity under nutrient conditions (Tan et al., 2014).The rate of PHA accumulation is primarily based on the formation of acetyl-CoA (Filipe et al., 2001).The green color balls in Fig. 3, show that other enzymes including 3-oxoacyl-[acyl-carrier-protein] synthase (FabB), 3-oxoacyl-[acyl-carrier protein] reductase (FabG), and [acyl-carrier-protein] s-malonyltransferase (FabD) have been reported with roles in cell growth and PHA production.They are primary enzymes in the fatty acid synthesis and β-oxidation associated with PhaB.Reducing β-oxidation activity significantly influenced the structure of produced PHAs (Liu et al., 2011).Protein-protein interaction (PPI) networks are complicated in PHA biosynthesis.How to accurately control the type of PHA and efficiently enhance PHA production through the regulation of gene expression and enzyme activity will be interesting and challenging.Besides, unraveling new PHAs biosynthetic pathways also will be crucial for the generation of novel PHAs.

Parameters influencing PHAs production
In order to perform sustainable PHAs production, an MMC or single strain with the capability to accumulate a high concentration of PHAs is indispensable (Johnson et al., 2009).However, no industrial PHAs production process uses MMCs due to the instability of the community in time, which is crucial for downstream processes.Many environmental parameters also influence the productivity of PHAs, which can bring a series of challenges in further scale-up of the production process (Sabapathy et al., 2020).The biggest challenge is to ensure the stability of the microbial consortia in the PHA production process using MMCs.The quality of the produced PHAs by MMCs is also an important property in plastic applications, particularly the produced PHAs from waste streams.Meanwhile, a green and efficient extraction method is a bottleneck in obtaining a high purity and quality of PHAs from bacterial cells.Hence, a suitable culturing strategy, optimal growth conditions, and efficient extraction methods are essential for improving PHAs yield in the case of MMCs.There are three main stages to obtain PHAs from complex substrates, including culture selection, PHAs production, and PHAs extraction.The general process is presented in Fig. 4. Besides, the influencing parameters for PHAs production using MMC has been presented in Table S3.

The enrichment of PHAs-producing MMCs
In recent years, open mixed cultures have been studied to characterize sustainable PHA production (Paul et al., 2021).Employing Note: "-" means that there was no information.
W. Zhou et al. enriched MMCs with wastewater streams as substrates showed an improved PHAs accumulation compared with PHA accumulation without an enrichment step (Estévez-Alonso et al., 2021).To maximize PHAs production using MMCs, the selection of PHAs-producing MMC/bacteria is important (Corsino et al., 2022).Applying a feast-famine regime is a well-known method that has been widely utilized to enrich a PHAs-producing culture in a sequencing batch reactor (SBR).The selection method is composed of a short period of carbon source sufficiency and a long period of carbon deficiency (Huang et al., 2017).Microbes that have the capability of PHAs accumulation can survive, while strains that do not have PHAs stored will be eliminated under the long famine phase (Corsino et al., 2022).As reported by Johnson et al. (2009), up to 89 wt% PHA of dry cell weight can be obtained using MMCs after culture enrichment through the feast-famine regime.The ratio of feast to famine plays an important role in obtaining a stable selection of PHA-producing MMCs (Cruz et al., 2022).
Other selective pressures also affect the performance of the enriched MMCs.A constant solid retention time (SRT) can be a selection factor to remove non-PHA-producing microorganisms and can drive changes in microbial community performance (Moretto et al., 2020).Meanwhile, to enrich a high PHA-producing MMC, the number of cycles and cycle length per dilution needs to be minimized (Jiang et al., 2011b).The development of mixed culture PHAs production is composed of optimizing the enrichment of PHAs-producing cultures, harvesting of the biomass, and extraction of PHAs (Janarthanan et al., 2016).Certainly, the growth conditions and fermentation processes need to be taken into consideration to improve the yield of PHA production.

The growth conditions
The total yield on PHAs using MMCs depends on the PHAs content in the cells and the biomass accumulation of MMCs (Huang et al., 2017).Hence, the improvement of biomass accumulation should be considered in increasing the productivity of PHAs using MMCs.Generally, the control of carbon and nitrogen sources is key in PHAs production (Patnaik, 2005).The strategy of nitrogen limitation has been employed extensively in obtaining high PHAs production (Guerra-Blanco et al., 2018).Phosphorus limitation also has shown its effect on the content and types of PHAs production (Wen et al., 2010) (Jiang et al., 2011b).Many studies indicated that alkaline conditions are better than acidic and neutral conditions for PHAs production by MMCs.Extensive efforts have been performed to optimize the growth conditions and the composition of microbial community.

Fermentation conditions for optimal PHAs production
An advantage of using MMCs is that the complex substrates from the waste streams can be directly fermented into mixtures of multiple organic acids, especially VFAs (Wang, 2022).Nutrient feeding strategy and fermentation parameters can affect PHAs production levels in bacteria (Mokhtari-Hosseini et al., 2009).The type of carbon source and concentration influence PHAs production and composition (Shen et al., 2022).Butyrate has been identified as the preferred substrate for PHB production when an enrichment culture with Plasticicumulans acidivorans as the dominant species was employed (Marang et al., 2013).However, a too-high substrate concentration has proven to be detrimental to cell growth (De Donno Novelli et al., 2021).To reduce the extra costs and improve the content of PHAs production, the optimal carbon source concentration should be utilized.
In order to reduce the inhibition of high substrate concentrations, different feeding regimes have been studied (Rajesh Banu et al., 2021).A fed-batch reactor has been widely used to harvest PHAs from PHAs-producing cultures in an SBR.Serafim et al. ( 2004) compared continuously fed and pulse-wise feeding modes, and showed that a higher PHAs content could be obtained with three pulses of substrate supply to prevent inhibition by a high substrate concentration.Chen et al. (2015) presented that a continuous feeding mode at low substrate loading rates was better than the pulse-wise feeding mode in PHAs production.The reason was that PHAs were consumed at the end of each substrate pulse.Hence, it is necessary to determine the rate of substrate consumption and the maximum endurance of substrate to ensure the optimal feeding strategy before scaling up production.On the other hand, maintaining the dominant PHAs-producing strains within the MMCs in the PHAs production process is also essential.The continuous culture mode shows its superiority in ensuring the stability of PHAs productivity and reducing the sensitivity of MMCs to the inhibitors (Paul et al., 2021).A crucial requirement for a fed-batch feeding strategy is online monitoring of the substrate concentration and automatically supplying substrate before it is depleted (Chen et al., 2013).
Dissolved oxygen (DO) is linked to substrate and oxygen consumption, and the influence of DO on PHAs synthesis also has been investigated in many microorganisms (Bhatia et al., 2021).When the concentration of substrate and oxygen consumption decreases, DO concentration increases (Colpa et al., 2020).Therefore, DO can be used as an indicator in the PHAs production process when a bioreactor is employed (Morgan-Sagastume et al., 2020).A low DO concentration inhibits PHA production due to the reduction of oxygen-requiring enzymes, such as propionyl-coenzyme A (Lefebvre et al., 1997).In contrast, Ling et al. ( 2018) reported that the ratio of NADH/NAD + increased for promoting PHA accumulation under oxygen-limitation conditions.Anaerobic-aerobic feeding regime (short-time anaerobic duration and aerobic operation cycle) enhanced PHAs production from rubber wood waste (Li et al., 2022).Modeling the fermentation process is also an alternative approach for the optimization of mixed substrate consumption and PHAs production by MMCs (Jiang et al., 2011a).

Degradation of PHAs
The superiority of PHAs compared to other bio-plastics is their complete biodegradability, especially in marine environments (Lee, 1996).As Wang et al. reported (2021), the biotic degradation of PHAs is estimated to be 8-20 times faster than abiotic degradation.Up to now, numerous bacteria and fungi have proven their capability of PHAs degradation (Kim and Rhee, 2003).The isolated PHAs degraders have been found in different environments, including soil, compost, and marine (Alshehrei, 2017).It can be degraded into carbon dioxide and water under aerobic conditions (e.g., soil and marine), and degraded into carbon dioxide and methane under anaerobic conditions (e.g., sediments and landfills).The predicted pathway of PHAs degradation is presented in Fig. 5. Microbial exo-enzymes are essential in breaking down the completely non-soluble PHAs into smaller water-soluble molecules (like oligomers) in the initial degradation process.The oligo and monomers are carbon and energy sources for further complete degradation by the same or other microorganisms (Meereboer et al., 2020).There is a carbon cycle for PHAs production and degradation to solve the issues of plastic pollution, which provides a foundation in a circular economy.

PHAs-degrading microbes
Based on the location of PHA, there are two different biophysical PHA degradation pathways: intracellular PHA degradation and extracellular degradation (Mas-Castellà et al., 1994).Intracellular PHA degradation by PHA depolymerase is necessary to release the stored carbon in PHA-accumulating microorganisms.Extracellular PHA degradation can be performed by PHA-producing and non-PHA-producing organisms such as fungi and yeast (Kim and Rhee, 2003).A list of PHAs-degrading microorganisms in diverse environments is summarized in Table 3.Both anaerobic and aerobic bacteria can degrade PHAs, including microorganisms present in activated sludge, soil, seawater, anaerobic sludge, lake water, and marine.Alcaligenes faecalis is the first discovered PHAs-degrading bacterium in activated sludge (Tanio et al., 1982).Ilyobacter delafieldii is the first isolated anaerobic PHAs degrading strain (Mas-Castellà et al., 1995).The PHAs producing and degrading Schlegelella thermodepolymerans is a thermophilic strain that can survive at a maximal 60 • C (Elbanna et al., 2003).Fungi play an important role in decomposing organic matter in the soil ecosystem, which has been comprehensively reviewed by Kim and Rhee (2003) and Ekanayaka et al. (2022).Aspergillus fumigatus is a thermophilic fungus isolated from soil that can degrade PHB and PHBV at higher temperatures (40 • C) (Iyer et al., 2000).The yeast Candida guilliermondii, Debaryomyces hansenii, Polyporus circinatus, and Rhodosporidium sphaerocarpum were isolated from deep sea samples that could degrade PHB from atmospheric pressure up to 20 MPa but were able to degrade PHB above 30 MPa (Gonda et al., 2000).Generally, the majority of fungi are mesophilic microbes that degrade PHAs in soil, like Fusarium oxysporium, Paecilomyces funiculosum, and Verticillium leptobactrum.

Enzymes
PHAs can be degraded by various enzymes, depending on the specific polymer structure and the microorganisms involved in the degradation process.The comparison of intracellular and extracellular enzymes involved in PHAs degradation is shown in Table 4. Generally, PHAs depolymerases are classified into intracellular and extracellular based on their mode of reaction towards the substrates (Chen and Jiang, 2018).Besides, PHAs depolymerases can be divided into SCL-PHAs depolymerase and MCL-PHA depolymerase depending on the substrate specificity (SCL-PHAs or MCL-PHAs) (Mas-Castellà et al., 1994).Extracellular PHA depolymerase is the key enzyme for the PHA degradation in nature, and many of them have been isolated from various microbes, including thermophilic strains, Archaea, and fungi (Knoll et al., 2009).Contrary to extracellular depolymerases, intracellular PHAs depolymerases only are produced by PHA-producing microorganisms.Extracellular PHA depolymerases can hydrolyze a wide range of PHA polymers, while the intracellular depolymerases are specific to the type of the stored PHAs inside the cells.PHAs serve as energy and carbon sources for microorganisms, their monomers can then be assimilated in the cells by a series of enzymes (Vigneswari et al., 2015).Intracellular PHA degradation is to release its monomers by PHA depolymerase and oligomer hydrolase.The monomer is further metabolized by  CoA-transferase and the β-oxidation pathway (Kawaguchi and Doi, 1992;Tajima et al., 2016;TANAKA et al., 1981).Extracellular PHA degradation is mainly based on the extracellular PHA depolymerase that attach to water-insoluble PHA and hydrolyze the polymer into water-soluble oligomers and monomers with a random-scission.Meanwhile, lipases help to cleave the ester bond of PHA polymer (Jendrossek and Handrick, 2002;Tarazona et al., 2020;Sharma et al., 2019).
The amino acid sequence of PHAs depolymerase from different microorganisms is aligned using MEGA6, and the results are presented in Fig. S2.Many depolymerases are endo-type hydrolases that have a hydrophobic site that binds to the polymer substrate, like the PHB depolymerase from A. faecalis (Tanio et al., 1982).As Kim and Rhee (2003) stated, fungal PHB depolymerases have less specific hydrolase activity compared to bacterial PHB depolymerases, which can increase the chain scission.The PHAs depolymerase of anaerobic bacteria is different from others.

Factors that influence PHA degradation
The rate of PHAs degradation is determined by several factors, including environmental conditions like temperature, oxygen, and salinity, and PHAs properties, such as monomeric composition and crystallinity (Sridewi et al., 2006;Numata et al., 2009).Mergaert et al. (1994) compared the PHAs degradation of homopolymers and copolymers in soil and showed that copolymer degradation rate was faster than homopolymers degradation.Due to their high crystallinity, homopolymers are more difficult to be degraded by microbes.In contrast, copolymers have low crystallinity and a porous surface, which facilitate the adsorption of microorganisms to the surface (Dartiailh et al., 2021).Volova et al. (2017) investigated the degradation rate based on the different chemical positions of the hydroxyl group in PHAs and found that the rate of degradation is as follows: P (3HB/4HB) > P (3HB/3HHX) > P (3HB/3HV) > P (3HB).Wang et al. (2004) concluded that an increase of 3HHX monomers in PHAs copolymers is favorable for the degradation rate.(2008) showed that PHAs degraded faster in aerobic conditions.A better understanding of the influencing factors in PHAs degradation will contribute to controlling the rate of PHAs degradation and provides more opportunities to offer suitable PHAs for specific applications.

Applications of PHAs
The broader use of bio-plastics in daily life will solve the increasing plastic waste problem.The use of biodegradable plastics will also decrease the dependency on fossil fuels and support sustainable resource use in a circular economy.One of the main drivers for the increase in PHA research is biodegradability and its composition versatility (Lee, 1996).The number of different PHA-polymers with different compositions, each with its properties and usage, is considerable.Generally, SCL-PHAs, like PHB, have poor tensile strength and high crystallinity properties and can be used in disposable utensils and straws.While MCL-PHAs, like PHBV and PHBHHX, have low crystallinity and low melting points and have a soft and elastic appearance.The material properties allow PHA to be used in various industries, the most notable being agriculture and medicine.The PHAs-based composites also provide alternative opportunities in industrial applications and versatility in a circular economy (Eesaee et al., 2022;Park et al., 2012).Fig. 6 presents some applications of PHAs in multiple fields that have been previously mentioned.The increasing demand for biodegradable plastic worldwide means a wide range of markets for PHA exists.Governmental regulations aimed at phasing out non-biodegradable plastics make PHA even more invaluable in the future.

Medical applications
Many studies have been performed to investigate PHAs applications in drug delivery, tissue engineering, and medical devices over the past years (Zhang et al., 2022).As Chen and Wu (2005) reported, the usage of PHAs has no toxic influence on the human body.Due to their superiority in biocompatibility and biodegradability, PHAs, particularly MCL-PHAs, occupy the majority of PHAs in medical applications (Reddy et al., 2022).Besides, PHAs have a high flexibility in structure based on the abundance of monomers.Soft MCL-PHAs have been used as a scaffold for skin regeneration (Guo et al., 2022), like PHBV.PHBHHX is more flexible than PHBV and has been employed in bone tissue engineering (Rekhi et al., 2022).Moreover, PHBHHx showed its potential to accelerate chondrogenic differentiation when bound to granule-binding protein (PhaP) in cartilage tissue engineering (Guo et al., 2022).P3HB4HB, an amorphous and elastomeric material, can be applied in heart valves and blood vessels (Rai et al., 2011).Owing to the hydrophobicity of PHAs, they can be applied in drug delivery (Zhang et al., 2022).One of the most attention-drawing applications of PHA is its ability to detect cancer cells (O'Connor et al., 2013).Sabarinathan et al. (2018) mentioned that breast cancer cells adhere to PHB with the help of specific target proteins, while normal cells lack this ability.Hence, PHA can be employed as a novel cancer detection tool.Besides, PHAs can be implanted in the body without causing inflammations showing their biocompatibility.Some possible bioplastics applications include biodegradable carriers that demonstrate the ability to deliver drugs for a given time within the human body, surgical needles, suture materials, bone tissue replacement, etc. PHAs and the composites of PHAs can have an important future role in medical applications due to their biodegradability and non-toxicity.These unique properties of PHA can be a driving force for developing the future PHA economy.

Disposable daily applications
The number of environmental issues caused by single-use plastics is increasing, driving the government and researchers to reconsider the importance of biodegradable plastics.PHAs, as an alternative plastic to petroleum-based plastics, demonstrate comparably good performances against oxygen and water and complete degradability (Nanda et al., 2022).With the plastic ban implementation, there is a great opportunity for PHAs.To date, many reports have mentioned PHAs applications in Fig. 6.PHAs applications in various fields (Wang and Chen, 2017;Zhang et al., 2022;Olayiwola Sirajudeen et al., 2021).
W. Zhou et al. various disposable items, including plastic bags, food packages, cosmetic containers, drinking straws, cups, lids, forks, utensils, etc (Gupta et al., 2022;Ullah et al., 2022;Bátori et al., 2018).The convenient disposal of PHAs after usage also attracts attention due to easier management of the PHAs end of life.Regardless of the price limitation, PHAs will have a bigger market leading to a sustainable and circular economy.

Agricultural applications
PHA also can find a high degree of usefulness as mulch in agriculture.PHAs-based mulching is good for soil structure, which helps in retaining water, preventing contamination, and thus benefits the crop yield (Sintim et al., 2021).Generally, high-density polyethylene (HDPE) and low-density polyethylene (LDPE) are the most conventional plastics used for mulch, which cause soil pollution due to their end up in landfills (Tocchetto, 2001).Increasingly stringent regulations amplify the search for alternatives.Compared to conventional plastics, biodegradable PHAs show their advantages in protecting soil (Kasirajan and Ngouajio, 2012).In addition, the low oxygen permeability of PHAs allows further applications for forming films and coatings.Another exciting application in agriculture is growth bags (Rekhi et al., 2022).PHAs-based growth bags have significant advantages over traditional growth bags, including lower toxicity for the environment, lower change of root deformity, and allowing plants to grow faster and develop better immunity against pathogens (El-malek et al., 2020).To conclude, the main properties of PHA that make it suitable for agriculture are its biodegradability and its friendliness to the environment.

Future perspective
In the introduction an ongoing increase in research efforts on PHA are laid bare.Scientists are more and more aware of the environmental problems that are caused by traditional plastics.Bioplastics like PHA are increasingly being recognized as a solution to these problems.The main issue associated with the emergence of PHA in the global plastic market is high production costs.This obstacle could be bypassed by using waste streams as feedstock.Additionally, the production process should be optimized to increase yields and decrease costs.MMC can be taken as a prime example of optimization of the production process.Innovations like these should be cherished, as they provide the gateway to an economically viable production process.To conclude, a more fundamental understanding of the mechanisms in PHAs production and degradation will allow the design of suitable plastics for multifarious industrial needs.The wide array of PHAs with varying properties gives rise to an unparalleled flexibility granting PHAs access to various niche markets.The possibility of large-scale extraction in industrial applications should be the main focus in future research.The use of PHAs will play an important role for reducing plastic waste, supporting sustainable resources, providing versatility, mitigating climate change, and forming a closed-loop system in a circular economy.Addressing the limitations of PHAs produced from waste streams will require a multifaceted approach.More research on extraction methods would allow for a cleaner production process to improve the purity and quality of PHAs.

Conclusion
This review involved the production and degradation of PHAs.The required carbon sources can be obtained from various waste streams to produce low-cost PHAs.The key producers and degraders present in different environments have the ability to produce PHAs synthase and extracellular depolymerase, respectively.Many enzymes and proteins in a strain interact for PHAs production and degradation.It is evident that many factors affect PHAs production and degradation.Understanding the mechanism of PHAs production and degradation will benefit further research to design/synthesize a suitable plastic for industrial needs and circular economy.Opportunities and challenges exist at the same time in the development of PHAs applications.

Fig. 1 .
Fig. 1.General chemical structures of polyhydroxyalkanoates (PHAs), including different categories: based on carbon atoms within the monomeric unit and the overall PHA structure, respectively.The picture of the PHAs granules inside the microbe cell is our unpublished research work.

Fig. 3 .Fig. 4 .
Fig. 3.The Protein-Protein Interaction (PPI) networks of PHA production based on the proteins of Ralstonia eutropha H16.Network nodes present proteins.Different colors balls present distinct clusters based on kmeans clustering in the String website (http s://string-db.org/cgi/network).The blue cluster shows the PHA biosynthetic pathway; the green cluster stands for fatty acid β-oxidation; the red cluster shows pyruvate complex activity.(For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) . As Xu et al. (2019) reported, sufficient nitrogen benefited the accumulation of cells but restricted PHA production.Meanwhile, as Wen et al. (2010) and Zhou et al. (2022) previously studied, excess nitrogen limitation negatively influences PHA production and cell growth.How to control the limited nutrient conditions for balancing cell growth and PHAs accumulation is still a challenge.The pH and temperature also have their effect on PHAs production, especially considering the range of different PHAs accumulating microorganisms (Stouten et al., 2019).Short-term temperature change influences the dominant PHAs-producing species within MMCs due to the dominant species was sensitive to temperature

Fig. 5 .
Fig. 5.A simplified representation of extracellular PHA degradation under anaerobic and aerobic conditions.
Deroiné et al. (2014) compared the PHBV degradation rate using natural seawater at different temperatures (4 • C, 25 • C, and 40 • C), and demonstrated that a high temperature (40 • C) increased degradation efficiency.PHAs can be degraded under anaerobic and aerobic conditions (Çetin, 2009).Siracusa et al. (2008) mentioned that PHAs degraded faster in anaerobic conditions, but Voinova et al.

Table 2
PHA producers and their used carbon sources.

Table 3
PHAs biodegradation by microbes under different conditions.

Table 4
The list of the related enzymes involved in intracellular and extracellular PHAs degradation.