Industries and societies are facing problems with the sustainable production of value-added chemicals while seeking reductions in greenhouse gas emissions (Ai-Yaeeshi et al., 2020). Currently, fossil fuels fulfill the need for energy and chemicals and play a major role in the global economy (Abe et al., 2019). Such resources are present in limited quantities, and their excessive use lead to an environmental pollution (Ai-Yaeeshi et al., 2020). In addition to anthropogenic activities, industries are also emitting waste gases, including carbon dioxide (CO₂), methane (CH₄), and carbon monoxide (CO) into the atmosphere every year (Choi et al., 2020a). Several approaches, such as physical, chemical methods, are employed to reduce pollution however; physio-chemical methods require intensive energy. To overcome this problem, these waste gases (C1) can be valorized to produce value-added chemicals by biological approaches (Dürre and Eikmanns, 2015). Some microbes can grow on these C1 compounds, including methanol (CH₃OH). Therefore, they would help to mitigate waste gases from the environment while simultaneously exploiting them to produce value-added bioproducts such as polyhydroxyalkanoates (PHAs) a biodegradable biopolymer (Dürre and Eikmanns, 2015) (Figure 1). Once realized on a large scale, this could be a paradigm approach towards the valorization of waste gases to biopolymers. Such a waste biorefinery strategy offers a great potential towards a sustainable economy (Kumar et al., 2016). PHA production from C1 carbon sources have more advances such as i) direct bioconversion process ii) fermentation approach without sterilization.

Synthetic polymers are irreplaceable materials in our daily life and are commonly used in packaging, electronic devices, and the transportation of household materials. Globally, annual plastic production has reached 311 million tons and by 2050, it is anticipated to reach up to 500 million metric tons (Liu et al., 2020). The overuse of synthetic plastics leads to a prodigious amount of waste generated in the environment. The primary disadvantage is its non-biodegradable nature and accumulation in the environment in enormous quantities. When exposed to solar radiation, synthetic plastics generate greenhouse gases such as CH₄ and ethylene. This scenario has encouraged many researchers to search for alternative polymers. Green alternatives by replacing synthetic plastics with biodegradable polymers such as PHAs are considered sustainable approaches (Raza et al., 2018). Microorganisms accumulate PHAs as carbon reservoirs under nutrient limiting conditions (Ray and Kalia, 2017a,b; Kumar et al., 2015). They have attractive physio-chemical properties such as elastomeric, piezoelectric, biodegradability, non-toxicity, and biocompatibility (Steinbüchel and Lutke-Eversloh, 2003; Chen and Wu, 2005; Bugnicourt et al., 2014; Ray and Kalia, 2017c). The physical properties of PHA depend on its monomeric composition, which is classified according to the number of carbon chain lengths such as short-chain length (scl, C₂-C₅), medium-chain length (mcl, C₆-C₁₄), and long-chain length in the monomer (lcl, C₁₅-C₂₀) (Ray and Kalia, 2017c). To date, one hundred sixty monomers of PHAs have been identified. Depending on the monomeric composition, PHAs have various applications (Singh et al., 2015; Choi et al., 2020b). Several homopolymers and co-polymers of PHAs are used in industrial and biomedical applications such as packaging, food additives, films, fibers, drug delivery, medical implants, scaffold preparation, tissue engineering, memory enhancers, and biofuels (Ray et al., 2018). However, higher substrate cost is the limiting factor for large-scale production.

Biowastes have been a key player in the production of value added bio-products, but the availability, difficulty in pretreatment has become an issue (Kumar et al., 2013). On the other hand, anthropogenic activities including industries lead to the emission of waste gasses, which cause environmental pollution. To reduce pollution, some gas-fermenting microbes can grow on C₁ compounds to produce PHAs and, lead to the elimination of the consumption of biowastes (Dürre and Eikmanns, 2015). Several Gram-negative bacteria are capable of producing PHAs such as Azotobacter, Comamonas, Pseudomonas, Ralstonia, Cupriavidus, Haloferax, and Klebsiella, while some Gram-positive bacteria accumulate PHAs such as Streptomyces, Rhodococcus, Clostridium, Staphylococcus, Nocardia, Microlunatus, and Bacillus from diverse substrates (Kumar et al., 2016; Ray and Kalia, 2017a).

C1 gases play a major role in global warming. Commonly, C1 gases can be generated from household wastes, industrial wastes and agricultural wastes. Thus, they can be exploited by microbes for the production of valuable bio-products such as PHAs. This approach could help to alleviate the environmental crises of global warming and plastic waste. In this present review, recent advances in PHA production approaches from carbon dioxide (CO₂), methane (CH₄), methanol (CH₃OH), carbon monoxide (CO), and formate are discussed. Utilization of C1 gases by autotrophs, acetogens and methanotrophs are discussed briefly. Several approaches to enhance PHA production such as metabolic engineering, process engineering etc. are discussed. These approaches help to establish a complete closed-loop PHA production from C1 gases and its effective degradation. This review articles encompasses the utility of PHA produced by C1 sources in various biotechnological applications.

Since the industrial revolution, CO₂, a strong greenhouse gas emitter which has reached up to 43%, and by the year 2100, it is expected to increase by 60% (Kumar et al., 2016). The microbial approach towards CO₂ sequestration and fixation are sustainable strategies for CO₂ mitigation and are more energy-efficient methods as compared to catalytic conversion (Claassens, 2017). Ribulose-1,5-bisphosphate and carbonic anhydrase help microbes to capture and store CO₂ thereby reducing greenhouse gases in the atmosphere (Kumar et al., 2016). Hydrogen-oxidizing bacteria, cyanobacteria, and algae are capable of consuming CO₂ from industrial flue gas as the sole energy source or in the form of sodium bicarbonate (NaHCO₃) or sodium carbonate (NaCO₃). They produce several valuable bioproducts including PHA and also treat wastewater by removing organic matter, and heavy metals (Oswald et al., 1957; Rosgaard et al., 2012; Nozzi et al., 2013; Lee et al., 2015).

A technological approach based on the cyanobacterial treatment of wastewater was successfully employed for treating municipal and industrial wastewaters (Arias et al., 2020). Chlorella vulgaris, Chlorella pyrenoidosa, Rhodovulum viride, Scenedesmus obliquus, and Thermosynechococcus elongatus can tolerate high CO₂ concentrations. Cyanobacterial CO₂ fixation is mainly influenced by physical parameters, such as type of cultivation, type of bioreactors, pH, nutrients, temperature, and light (Salehizadeh et al., 2020). However, under nutrient-limiting conditions, cyanobacteria produce different types of storage compounds, such as glycogen, polyhydroxybutyrate (PHB) (carbon-rich), cyanophycin (nitrogen-rich), and polyphosphate (Price et al., 2020) (Figure 2 and Figure 3).

Through the oxidative pentose pathway, glycogen is synthesized in the presence of sunlight and CO₂ (Shinde et al., 2020). When compared to glycogen, PHAs could serve as long-term storage material within the cell. Chlorogloea fritschii was the first reported cyanobacteria to produce PHA by utilizing acetate in a photoautotrophic environment (Carr, 1966; Singh et al., 2017). Fatty acid biosynthesis and nitrogen assimilation are proposed biosynthetic pathways for the production of PHAs (Mozejko-Ciesielska et al., 2018; Price et al., 2020). Cyanobacteria are capable of performing oxygenic photosynthesis via Calvin-Benson-Bassham (CBB) cycle (Figure 2). They produce ATP and NADPH by capturing the thylakoid membrane from sunlight. These two intermediates are utilized for essential nutrient assimilation. The CBB pathway contains 3 phases: carboxylation, reduction, and regeneration. In carboxylation, 6 molecules of 3-phosphoglycerate (PGA) were produced by combining by 3 molecules of CO₂ and 6 molecules of ribulose 1, 4-bisphosphate (RuBP). Subsequently, the production of NADPH and ATP occurs to reduce PGA to triose phosphate and dihydroxyacetone phosphate (DHAP). 5 molecules of PGA are utilized to reproduce 3 molecules of RuBP. Cyanobacteria uptakes Ci, CO₂, and HCO₃ ⁻ inside the cell by CO₂ concentrating mechanism. There are five types of mechanisms; 3 for the uptake of HCO₃ ⁻ (BicA, SbtA, and BCT1) and 2 for the uptake of CO₂ (NDH-I₃ and NDH-I₄) in cyanobacteria. CO₂ transport occurs through Ci transporters at the plasma membrane. Bicarbonate transporters help to transport intracellular HCO₃ ⁻ ions across the plasma membrane, chloroplast, and periplasmic carbonic anhydrase convert HCO₃ ⁻ to CO₂ (Durall and Lindblad, 2015).

Furthermore, the remaining 3-PGA is utilized for the synthesis of cellular material depending upon nutrient availability. RuBisCO, phosphoribulokinase, and sedoheptulose bisphosphatase are the major enzymes of the Calvin-Benson-Bassham cycle (Kumar et al., 2018). Under nutrient-depleted conditions, 3-PGA diverts the pathway towards PHA biosynthesis (Figure 2). Several cyanobacterial species, such as Spirulina maxima, Aphanocapsa sp, Synechocystis sp. UNIWG, Synechocystis sp. PCC 6803, Nostoc muscorum, and S. platensis accumulate PHAs (Singh and Mallick, 2017).

The synthesis of PHA governed by the following enzymes:

a) ß-ketothiolase (encoded by phaA) catalyzes the conversion of acetyl CoA to acetoacetyl-CoA, b) acetoacetyl-CoA reductase (encoded by phaB) help to reduces acetoacetyl-CoA to 3-hydroxybutyryl-CoA, and c) polymerization of 3-hydroxybutyryl-CoA to PHB carried out by PHA synthase (encoded by phaEC) (Taroncher-Oldenburg et al., 2000; Carpine et al., 2017). PHA synthase can also incorporate other hydroxy acid monomers within the PHA. PHA synthase is categorized into four groups: 1) Class I PhaC subunit contains 60–73 kDa in Ralstonia eutropha, 2) Class II PhaC subunit contains 60–65 kDa in Pseudomonas oleovorans, 3) Class III PhaC and PhaE subunit contains 40 kDa each in Allochromaticum vinosum, and Thiocapsa pfennigii, and 4) Bacillus megaterium contains 40 kDa and 22 kDa of Class IV (PhaC and PhaR) (Ray and Kalia, 2017b). Only Class III PHA synthase has been observed in cyanobacterial species (Singh and Mallick, 2017). Cyanobacterial gene locations are slightly different from other bacteria. In bacteria, all four genes are present in one single operon, but in cyanobacteria, two separate operons are present. PhaA and PhaB are putatively co-expressed and present in the first loci while PhaEC, present in the second loci (Price et al., 2020).

PHB production from CO₂ is gaining interest because of its low cost of production (Carpine et al., 2017). Under nitrogen-limiting conditions, Synechocystis PCC6803 produced 4.1% PHB of the total cell dry weight (Wu et al., 2001). Synechococcus MA19, a thermophilic organism, was reported to produce 55% PHB (Nishioka et al., 2001). However, non-thermophilic cyanobacterial species produced PHB up to 20–25% (Troschl et al., 2017a; Troschl et al., 2017b) (Table 1). However, some bacterial species such as Cupriavidus eutrophus B-10646, Bacillus cereus SS105 produced 55–85% of PHA in a batch and continuous stirred tank reactor (Table 1). Supplementation of CO₂ to R. eutropha B5786 led to the production of 51% of PHA in a batch fermentation system (Table 1). Some cyanobacteria produce PHB through nitrogen fixation. Calothrix species produced PHB from 17 to 25% of total cell dry mass through nitrogen fixation (Price et al., 2020). Arthrospira subsalsa is a species of filamentous cyanobacteria that produces up to 14.7% PHB under increased salinity (Shrivastav et al., 2010). Acetate addition to the system led to the enhancement, in PHB content up to 40% by another group of cyanobacteria Nostoc. Further addition of propionate and valerate to the CO₂-containing medium resulted in an enhanced P (3HB-co-3HV) content (Bhati Malick, 2015). By contrast, under phosphorous limiting conditions, co-utilization of acetate and fructose enhanced the PHB production up to 38% (w/w) of cell dry mass of Synechocyctis PCC6803 (Panda and Malick, 2007). Other cyanobacteria, such as Calothrix scytonemicola TISTR 8095 produced PHB up to 356.6 mg/L in 44 days under nitrogen limitation (Kaewbai-ngam et al., 2016). Microbial mutualism have the potential role in the bioproduction of value-added byproducts. In one study, Azotobacter vinelandii, a diazotroph and S. elongates PCC7942 co-cultured for the production of PHB by fixing CO₂ from the atmosphere. Here, ¹³C bicarbonate was added to increase the cell growth and produced ¹³C labeled PHB. This study proved the model for cross feeding between two organisms (Smith Francis, 2016). Co-cultures containing S. elongatus cscB and P. putida cscAB utilized CO₂ for the production of PHA in two-phase systems. In the first phase, S. elongates cscB fixes and converts CO₂ to sucrose via the Calvin-Benson-Bassham cycle pathway. Sucrose will then release into the culture supernatant through heterologous sucrose permease cscB activity. P. putida cscAB used sucrose as a carbon source to produced PHA up to 23.8 mg/L/h with a yield of 156 mg/L after 16 days. Here, 3-hydroxydecanoic acid was found to be the major monomer (Löwe et al., 2017). The first report of mcl-PHA production was found in co-cultures containing S. elongates and P. putida. Here P. putida was utilized as chassis for the generation of PHA by operating metabolic pathways (Löwe et al., 2017). Some photosynthetic bacteria utilize CO₂ to produce PHA such as purple sulfur bacteria and purple non-sulfur bacteria. PHB homopolymer was found in purple sulfur bacteria while co-polymers such as 3HB and 3HV were found in purple non-sulfur bacteria (Higuchi-Takeuchi et al., 2016). However, from GPC analysis it was observed that photosynthetic purple bacteria produced PHAs with high molecular weight (3,000–994,000 g mol⁻¹) which will be favorable for industrial application. The polydispersity index range was found between 1.5 and 6.7 Mn (Higuchi-Takeuchi et al., 2016). A Horizontal tubular photobioreactor was installed in an Energie-Versorgung Niederosterreich AG power company present in Austria for PHB production. Here, one ton of CO₂ was used as feed for cyanobacteria and produced 115 kg PHB and biogas up to 320 m³ (Salehizadeh et al., 2020). Thus, this strategy will enhance CO₂ assimilation capacity and could be a promising solution towards large-scale PHB production. Cyanobacteria can efficiently adapt to any environmental condition, which suggests that this group of microorganisms does not require any energy-rich source for their growth, and therefore, they can be exploited for the generation of valuable byproducts such as PHAs and carbohydrates (Parmar et al., 2011; Arias et al., 2020).

Hydrogen-oxidizing bacteria are the defined group of bacteria that utilize H₂ and O₂ as an electron donor and acceptor with the help of ribulose biphosphate for fixing CO₂ within the bacterial cell (Salehizadeh et al., 2020). A. eutropha is capable of converting reduce form of CO₂ to PHA under different H₂/CO₂ stochiometric ratios. In addition, R. eutropha can produce PHB up to 75% (Khosravi-Darani et al., 2013) (Table 1). The autotrophic culture of C. necator was also checked for PHB production. The utilization of CO₂ led to the production of up to 72% PHB with 60 g/L of dry cell mass (Garcia-Gonzalez and De Wever, 2018). Fed-batch cultivation of the PHA copolymer resulted in 65 g/L cell dry weight and PHBV of up to 27 mol% (Garcia-Gonzalez and De Wever, 2018). The co-utilization of CO₂ and valeric acid produced 1.14 g/L of P(3HB-co-3HV) by C. necator DSM 545 under mixotrophic conditions. Here, CO₂ was continuously sparged to prevent the accumulation of substrate in the medium (Ghysels et al., 2018). Acetic acid can indeed act as an indirect reservoir of CO₂ for the production of both PHA homo and co-polymer. The consumption of acetic acid was found to be favorable for C. necator growth and PHA production.

Worldwide, CH₄ stands as the second most greenhouse gas agent which contributes 20% towards climate change and traps atmospheric heat about 25 times higher than CO₂ (Mounasamy et al., 2020). Sixty-three percent of CH₄ is generated from anthropogenic activities and the remainder from coal mining, land filling, anaerobic wastewater treatment (Strong et al., 2016). In natural gas, methane counts for 90%. Due to the current world wide demand it is estimated to increase up to 44.0% by 2040 (Liu et al., 2020). Currently, CH₄ accounts for 18% of the total greenhouse gas emissions (EU-28) (Cantera et al., 2018a). This situation raises concerns about global warming and has compelled researchers to look for novel approaches that can help to reduce greenhouse gases and environmental pollution. The production of PHB from CH₄ may act as a sequestration process, and the biotechnological approach could be the best solution for the abatement of methane because it is cost-effective and has a low impact on the environment. Such approaches are mainly based on the biocatalytic action of microorganisms known as methanotrophs that convert CH₄ into CO₂ and H₂O using O₂ as an electron acceptor (Cantera et al., 2018a; Cantera et al., 2018b). Methanotrophs can grow on C₁ carbon compounds, and some can utilize multicarbon sources as an energy source in specific environments (Semrau, 2011). The well-studied methanotrophs are aerobic methane-oxidizing bacteria that are categorized as γ-proteobacteria (Type I) and α-proteobacteria (Type II). For carbon assimilation, they follow the ribulose monophosphate pathway (RUMP) and the serine cycle (Pieja et al., 2017). The key enzyme for CH₄ oxidation is methane monooxygenase which can be found in soluble or particulate forms. Methanogenesis requires two reducing equivalents for the oxidation of CH₄ into CH₃OH, where anaerobic methanotrophic archaea and non-methanotrophic bacterial consortia conduct the anaerobic methane oxidation.

Methanol can be generated not only from industrial waste gas but also from biomass and CO₂ hydrogenation process (Borisut and Nuchitprasittichai, 2019). CH₃OH can be used as feedstock in both biological and chemical industries by methylotrophs for the sustainable production of value-added chemicals (Liao et al., 2016). Under environmental conditions, CH₃OH is present in liquid form, thus, easy to handle and bio convert to PHA production. The application of methylotrophic bacteria facilitates PHA production from CH₃OH.

Among others, Methylobacterium spp. is the most widely studied organism for PHB synthesis through the serine pathway. NH₄ ⁺, Mg²⁺, PO₄ ^3-, and K⁺ are the major deficiencies studied during the accumulation of PHB (Khosravi-Darani et al., 2013). Methylobacterium extorquens were studied for PHB and its co-polymer production from CH₃OH (Bourque et al., 1992). The optimization of fed-batch fermentation led to a cell dry weight up to 115 g/L and a polymer content of up to 46% (w/w) (Bourque et al., 1992) (Table 2). The maximum PHB production using CH₃OH as carbon feedstock was found to be 0.18 g/g. A recent study suggested that the addition of a co-substrate such as valeric acid, n-amyl, or propionic acid to CH₃OH could be helpful in the higher production of PHB and its co-polymer (Strong et al., 2016). Enhanced PHB production of up to 136 g/L was achieved by using computer-controlled fed-batch fermentation of CH₃OH with 66% cell dry weight using was obtained. It has been proposed that genetic engineering may enhance PHB productivity and yield. Methylobacterium extorquens DSMZ 1340 utilized methanol for the production of PHB. The deficiency of MgSO₄ and (NH₄)₂SO₄ in the medium stimulates PHB production, which increased up to 62.3% of total dry cell mass by Methylobacterium extorquens DSMZ 1340. After 35 h of fed-batch fermentation, the yield of PHB was 2.8 g/L/h and 0.98 g/L/h (Mokhtari-Hosseini et al., 2009a). In contrast, the growth of Methylobacterium trichosporium IMV3011 on CH₃OH was tested at a feeding rate of 0.1% five times during fermentation. Hence, the concentration of PHB increased to 47.6% with 2.91 g/L cell dry weight. The addition of malic acid at the rate of 0.2 g/L enhanced to 58.5% of PHB production, with 3.32 g/L dry cell mass. Various reports have suggested that PHB production can be enhanced after the addition of acetyl-CoA, malic acid, and citric acid (Xin et al., 2011; Khosravi-Darani et al., 2013). Similarly, the addition of 0.12 g/L of NH₄ ⁺ produced 7.04 g/L of PHB by Methyloligella halotolerans from methanol with a molecular weight of 8,000–10,000 kDa (Ezhov et al., 2017). Under the nutrient limitations of nitrogen and magnesium, Methylobacterium extroquens DSMZ 1340 produced 9.5 g/L of PHB with a cell dry weight of 65.3% and a yield of 0.16 g/g by utilizing CH₃OH (Mokhtari-Hosseini et al., 2009b) (Table 2).

Moreover, Methylobacterium extorquens DSMZ produced 44% (w/w) of PHB by fed-batch cultivation. Recombinant technology helped to produce PHA copolymers such as 3HB, 3HV, and 3HHx within the polymer in Methylobacterium extorquens AM1 (Orita et al., 2014) (Table 2). The present study reported that C_O ²⁺ concentration in the medium influences cell growth and PHA productivity. A low concentration of C_O ²⁺ helped to achieve 3HV content up to 6 mol%. Furthermore, the pccA gene deletion increased the 3HV fraction in terpolymers (Orita et al., 2014) (Table 2).

Methylobacterium extorquens AM1 utilized CH₃OH as a sole source of carbon and produced PHB up to 149 g/L (Suzuki et al., 1986). Methylobacterium extorquens ATCC55366 synthesized up to 46% (w/w) PHB by utilizing CH₃OH. Moreover, under oxygen-limiting conditions and addition of 0.01 g/L of CH₃OH led to the production of PHB with high molecular weight (900–1,800 kDa). The composition of media was found to be a significant approach for higher cell biomass and PHB production. The cheaper cost of methanol could, therefore, reduce the cost of PHA production when compared to pure sugar, but the yield is low for other substrates (Höfer et al., 2010) (Table 2). Moreover, the tolerance of organisms should be improved for industrial applications (Zhag et al., 2018).

Several thermal power plant and steel plant industries generate large quantities of carbon monoxide. For example, a South Korean steel company (POSCO) generates 60 million Nm³ of CO/day (Hwang et al., 2020). CO can be utilized to produce value added products. However, the processes have several limitations. Being an inert gas, the efficiency of this process is low, and the risk of the explosion has limited the use of CO (Choi et al., 2020a). It is toxic to several microorganisms but some microbes can utilize CO.

Acetobacterium woodii produced PHB using CO as a substrate in a two-phase whole-cell bio-catalytic operating system. A. woodii utilized CO and produced 53.1–60.7 mM of formate in the first step. In the second step, engineered Methylobacterium extorquens subsequently utilized formate, and the PHB content improved by up to 2.24%. The two-stage concept for the bioconversion of CO to PHB offers an effective solution for CO utilization (Hwang et al., 2020). Although, the interest in bioconversion of CO to PHA has increased recently, however, it is still in a nascent stage. Thus, much insight is required about the underlying mechanism. Once deciphered, it would be an asset for the biotechnological industries (Figure 3).

Bioconversion of C1 feed substrates to value-added compounds have attracted researcher owing to the environmental pollution and the shale gas revolution (Durre and Eikmanns, 2015; Humphreys and Minton, 2018; Sun et al., 2019). Electrochemical reduction of carbon dioxide or syngas hydration lead to the generation of formate (HCOO⁻) which is regarded as environmentally friendly and sustainable microbial feed substrate (Agarwal et al., 2011; Schuchmann and Muller, 2013; Hwang et al., 2015; Shinagawa et al., 2018; Hwang et al., 2020). CO₂ is reduced to formate, methane, carbon monoxide, and ethylene (Whipple et al., 2010). Formate can be used as fuel in fuel cells (Joo, 2008; Enthaler et al., 2010). CO₂ reduction by the electrochemical reaction can produce formic acid with high faradaic efficiency. Also, one oxygen-stable whole-cell biocatalyst is used to produce formate (Kortlever et al., 2015). It can act as a good electron mediator that has stability, non-toxicity, good permeability. Methylotrophs, non-pigmented Pseudomonas, facultative autotrophs, heterotrophs, and hypomicrobia are capable of growing on formate through serine pathway or ribulose monophosphate (RuMP) pathway. (Hwang et al., 2015) (Figure 3).

In bacteria, formate is oxidized to carbon dioxide by generating NADH (Berrios-Rivera et al., 2002; Yishai et al., 2016; Barin et al., 2018). Several reports are found on the utilization of formate by Escherichia coli using formate assimilating pathways. However, due to slow bacterial growth, the industrial application has been limited (Kim et al., 2019; Bang et al., 2020; Kim et al., 2020). On the other hand, Methylorubrum extorquens can able to utilize formate through the tetrahydrofolate pathway and serine cycle (Yishai et al., 2016; Hwang et al., 2020). Metabolic engineering of Methylorubrum extorquens has enhanced the product yield of various bioproducts from C1 feed such as PHAs, proteins, dicarboxylic acids (Bélanger et al., 2004; Choi et al., 2008; Hofer et al., 2010; Orita et al., 2014; Sonntag et al., 2014, 2015). One study reported that Methylobacterium extorquens utilized formate and produced P(3HB-co-3HV) with 8.9% HV content. Here, bktB, phaJ1, and phaC2 were heterologously expressed and effectively converted propionyl-CoA into 3-HVCoA (Yoon et al., 2021). Methylobacterium extorquens utilizes formate through the Foc A transporter (Lü et al., 2011; Hwang et al., 2020).

The effect of PHA-producing organisms on a commercial scale is indicated by the PHA yield, organism growth rate, and rate of PHB accumulation. The productivity of PHA is directly proportional to the price of the polymer. The conversion of CO₂ to PHA depends mainly on the bacterial cell growth rates and cell densities of the organisms. The unavailability of a mass cultivation system is hindering the growth of commercial production of PHA from CO_2. Cyanobacterial mediated PHA production is generally influenced by abiotic factors, and operational parameters (Singh et al., 2017; Singh et al., 2019). The cyanobacterial production of PHA varies significantly, and there are high chances of the simultaneous production of other value added byproducts for example, proteins and pigments. The process parameters should be constant to achieve a high PHA yield. The commercial cultivation of PHA can be achieved in two ways: 1) Photobioreactor (closed system) and 2) pond culture systems (open) (Koller and Marsalek, 2015). Some challenges remain, for example, 1) improved photoautotrophic PHA production, 2) enhanced bacterial cell biomass in the photobioreactor, 3) photobioreactors modification for scale-up purpose, 4) Optimized downstream processing, and 5) CO₂ uptake capacity (Drosg et al., 2015). An ideal photobioreactor system can maintain a phototrophic culture system for the production of valuable bio-products. An advanced photon system was operated for cultivation system with an average temperature range of 15–60°C (Singh et al., 2017; Singh and Mallick, 2017). Maintaining a monoculture and its productivity is the main problem of the system. Also, compared to open pond systems photobioreactors are more expensive. There are several uncertainties due to the underdevelopment of photobioreactors and open pond systems (Singh et al., 2017). The complexity found in the harvesting and processing of biomass limits the growth of PHA towards commercialization. Harvesting is an approach that creates a bottleneck for commercial scale. Different techniques have been employed for harvesting cyanobacterial biomass, for example, filtration, centrifugation, flocculation, and gravity settling. However, because of the small size, low concentration, and colloidal stability of cyanobacterial biomass, the harvesting process is difficult to recover (Xia et al., 2017). Furthermore, the harvesting cost is 20–30% of the entire process. After the harvesting process, it is necessary to eliminate water from the wet biomass, which helps to further store cyanobacterial feedstock, and therefore, intensive energy is required for the cyanobacteria drying process. A study has reported that drying alone costs 20% of the total production cost. Air-drying is also possible for cyanobacterial biomass production, but it requires a longer time and additional storage place. However, the air-drying process could be replaced by solar and wind energy (Parmar et al., 2011).

The biotechnological approach based on CH₄ bioconversion is promising and imminent, but there are some physical and biological limitations. Being a hydrophobic gas pollutant, CH₄ has poor solubility in water, which results in a low elimination rate and poor biomass concentration. Uptake of CH₄ gas to bacterial communities creates problems, and there is a need to search for innovative strategies that can make the process easier and more successful (Kraakman et al., 2011). Mechanical stress of the bioreactor limits the growth of methanotrophs by hindering the agitation rate (Cantera et al., 2018b). For this reason, conventional bioreactors remain limited (Muñoz et al., 2007; Kraakman et al., 2011; Estrada et al., 2012). Furthermore, an increase in both investment and operating costs is influenced by a low gas-liquid concentration gradient (Estrada et al., 2012). For microbial conversion of waste gas streams (rich in CH₄) into valuable bio-products, suspended growth bioreactors are found suitable (Muñoz et al., 2007). Suspended growth membrane diffusion and pressurized bioreactors have been developed to enhance mass transport and require low-moderate energy for operation. Also, through internal gas recirculation, a high mass transfer rate of CH₄ can be achieved (Estrada et al., 2014). Conversely, some environmental variables, such as culture conditions, pH, temperature, dissolved oxygen (O₂) concentration, CH₄ and O₂ ratio, cultivation time, and the composition of cultivation media also play a major role in the CH₄ conversion process (Semaru et al., 2010; Liao et al., 2016). The addition of a high salt concentration with stable pH values avoids contamination of the whole process. Co-substrates addition to the CH₄ conversion process could be applied to tailor the quality of the byproducts. One study demonstrated dioxygenase enzyme for methane activation by oxidizing two methane molecules simultaneously. This approach could enhance process performance (Liao et al., 2016). In this context, “omics” technology will soon contribute to improving methanotroph-based biorefinery. Several companies, such as the Intrexon Corporation and Calysta are conducting more research and investment to find a single methanotroph capable of producing multiple value-added bioproducts (Cantera et al., 2018a; Cantera et al., 2018b). An advanced reactor set-up would certainly be helpful for enhanced bioavailability and conversion of gaseous C₁ substrates.

The generation of high value-added bioproducts could be achieved by fed-batch cultivation through a controlled nutrient feed strategy in mechanically stirred fermenters to gain high bacterial cell densities (Harding et al., 2007). Syngas contain CO, that has a high affinity towards metal-containing enzymes which makes the fermentation process toxic. To avoid such toxic substances, enzymatic or chemical extraction methods are used. Although the conversion of C₁ substrates to PHA seems lucrative, several limitations need to be overcome to enhance the economic status of PHA (Singh et al., 2017). An eco-friendly, cost-effective PHA recovery is required from biomass.

PHA polymers may possess more than 160 types of monomers that endow a wide range of physical properties (Choi et al., 2020a; Alcântara et al., 2020). Also, the biocompatibility and biodegradability qualities of PHAs have attracted more attention towards the medical field and therapeutic material industries (Figure 4). Therefore, applications of PHAs range from preparing commodity products to agricultural sectors. PHAs are more suitable agents for biomedical applications because the in vivo degradation of PHA is less acidic as compared to polylactic acid and poly (lactic-co-glycolic acid), which cause local necrosis and inflammation (Choi et al., 2020b). The biomedical use of PHAs started when surgical sutures were tested using P (3HB). Sutures made of PHAs were implanted in rats and were found to remain active for a long time without any adverse effects (Shishatskaya et al., 2004). Several co-polymers of PHAs were later exploited in biomedical field in the form of heart valve scaffolds, drug delivery agents, surgical sutures, bone marrow scaffolds, stents, cardiovascular patches, orthopedic pins, adhesion barriers, tendon repair devices, wound dressings, and (Gumel et al., 2013; Ray and Kalia, 2017c; Chen and Zhang, 2018). Owing to their biocompatible nature, PHA-based scaffolds such as P(3HB-co-3HV) used in the preparation of laser-perforated biodegradable scaffold films that repair damaged tissue and enable a higher rate of cell adhesion, growth, and migration (Ellis et al., 2011) (Figure 4). The fabrication of carbon nanotubes with PHBHHx nanocomposite film was reported to support osteogenesis process (Wang et al., 2011; Wu et al., 2013) and similarly, pre-osteoblast cells development was also found by the blending of P (3HB-co-3HHx) and polycaprolactone (Puppi et al., 2017). Other unique approaches have recently been developed, for example, PHB with poly-lactide-caprolactone was used for the preparation of electrospun nanofibrous scaffolds to accelerate progression in cell cycle and prevent necrosis (Daranarong et al., 2014), and PHA microspheres were used for proliferating stem cells and osteoblast regeneration (Wei et al., 2018). Conversely, the application of PHA is focused on the regeneration of new bone by employing bone tissue engineering. A blend of 8% 3HV with 30% hydroxyapatite composition had shown suitable mechanical compressive strength with lower inflammatory response and mineralization (Galego et al., 2000). Terpolyester of PHA scaffolds enhanced osteoblast attachment, propagation, proliferation, and differentiation in nerve cells (Li et al., 2005; Wang et al., 2010). Among various PHA polyesters, P(3HB-co-3HV) was shown to be the best for bone tissue regeneration (Yang et al., 2004). The degradative PHA (3-hydroxybutyrate) has been shown to promote the growth of murine osteoblast MC3T3-E1 cells in vitro. In addition, in vivo studies on ovariectomized rats suggested the role of 3HB in preventing osteoporosis, as revealed by a bone material density and bone biomechanics study (Zhao et al., 2007). A unique PHBHHx-based microgroove surface pattern (10 µm size) influences the interfacial behaviors of bone marrow mesenchymal stem cells and differentially expressed miRNAs in comparison to smooth-surfaced PHBHHx substrates (Wang et al., 2013). PHA also strengthens the skeleton, increases the rate of calcium deposition, and lowers the level of serum osteocalcin (Zhao et al., 2007; Guyot et al., 2020). PHB/chitosan nanofibers have been helpful in cartilage tissue engineering (Sadeghi et al., 2016). These studies demonstrate the beneficial biomaterial properties of PHAs in different medical applications.

A study reported that PHB film was used for cancer detection (Sabarinathan et al., 2018). Thus, the use of PHB films for cancer detection was time-saving and painless compared to biopsy (Sabarinathan et al., 2018). PHA can also be used as a drug carrier. PHA copolymers were used to release anticancer drug 5-fluorouracil (Choi et al., 2020a). Several periodontal disease bacteria were killed by P(3HB-co-3HV) microspheres and microcapsule containing tetracycline [151]. Electrospun fibrous meshes of PHB-polyethylene oxide were used as drug delivery agents for chlorhexidine against some pathogenic bacteria (Meng et al., 2013). Similarly, a blend of PHB-chitosan was used to deliver ketoprofen, which is an antipyretic (Lins et al., 2014). Also, 3HB derivatives of PHA polymers can be used as effective drugs against mitochondrial damage (Zhang et al., 2013). Methyl ester of the 3HB monomer has shown to be effective against Alzheimer’s and Parkinson’s diseases (Camberos-Luna et al., 2016; Camandola mattson, 2017). Hydroxy acid produced from PHA degradation has also been shown to play a major role in calcium ion stimulation in memory enhancers (Raza et al., 2020). 3HB also a degradative product of PHA has an anti-osteoporosis effect (Cao et al., 2014).

Applications of PHA have been further extended through various chemical functionalization approaches. In addition to medical use, PHAs are also suitable for food packaging, cosmetics, and agricultural applications. Produced from municipal biowastes, P (3HB-co-3HV) were electrospun to prepare bio papers for food packaging (Melendez-Rodriguez et al., 2020). Due to the high radical scavenging activity, high water vapor transmission, antioxidant and antibacterial property, PHB film can act as a biodegradable active packaging material. For example, PHB film is used in the wrapping of chilled salmon (Ma et al., 2018). PHA can be used to make straws and bottles. A degradable straw was prepared using Nodax PHA (Danimer Scientific, U.S.A.) in 2018 (Choi et al., 2020a). Recently, Nestle Co. with Danimer Scientific developed biopolymer-based bottles (Choi et al., 2020b). Moreover, several PHAs blended with different biobased materials are used in the cosmetic industry for the preparation of biobased beauty masks and sanitary napkins (Choi et al., 2020b). PHA microplastics can be used in face cleansers and toothpaste as an alternative to synthetic microplastics, which are harmful to live beings, including aquatic animals (Bouwmeester and Hollmanpeters, 2015). Recently, a sun protection product containing PHA micropowder was introduced (Unilever, U.K.) as containing the first biodegradable cosmetic ingredients derived from renewable biomass (Choi et al., 2020a). Several farmers use plastic materials for greenhouse films and protection nets, which are essential for increasing the quality of crops and protecting them from hazards. High-density polythene is a commonly used synthetic plastic material but owing to its non-biodegradability, PHA films are currently being considered. Large quantities of bags are used in the agricultural sector for fertilizers and seedlings, among others (Adane and Muleta, 2011). In this context, PHA bags have many advantages PHA polymer bags do not release any toxic chemicals into the soil.

PHAs can also be helpful in denitrification. Recently, a modified sequencing batch biofilm reactor was designed to treat landfill leachate. Here, ammonia-oxidizing bacteria and denitrifying bacteria are capable of transforming organic carbon compounds into intracellular PHA (Yin et al., 2018). In this reactor, the process of nitrification (removes NH₄ ⁺ –N to produce NO₂ ⁻ –N), simultaneous nitrification and denitrification (removes NO₂ ⁻ –N), and endogenous denitrification (removes residual NO₂ ⁻ –N) utilized PHA as a carbon source (Yin et al., 2018). PHA films can be used to prepare biodegradable waste bags, which can contribute to reducing landfills and making the composting process more efficient. PHA films can be used in agriculture and reduce disposal costs (Winnacker, 2019). Recent developments in these areas are promising, and will certainly increase the value of PHA polymers for their useful applications in daily life. PHAs are used in 3D printing implants for humans (Rydz et al., 2019). Chemically modified PHAs such as Polyhydroxyalkanoate-g-poly (N-isopropylacrylamide) has shown thermal responsive hydrophilicties and biocompatibilities at different temperature (Ma et al., 2016). Modified PHA with EU3⁺ and Tb3⁺ possesses intense photoluminescence properties with enhanced hydrophilicity and biocompatibility (Yu et al., 2019a, Yu et al., 2019b). PHAs can be also used as nano-vaccines in drug delivery (Parlane et al., 2012).

Various companies are known for the production of PHAs. TianAN is the largest PHAs production company. PhaBuilder, Medpha, COFCO, and Bluepha have been set up to explore Next-Generation Industrial Biotechnology for low-cost PHAs production. Metabolix company was sold to Korean company CJ. RWDC and Danimer have employed recombinant R. eutropha, which can utilize fatty acids for P (3HB-co-3HHx) production. New light and Full cycle companies utilize greenhouse gas, organic wastes respectively for the production of PHAs. Recently, a global organization is known as GO! PHA has been established to produce PHAs (Table 3.).

For the microbial production of several value added bio-products, C1 sources are considered as the most promising and Next-generation carbon sources. Significant efforts have been made on the production of PHAs from C1 sources as it is eco-friendly, economical feasible and it has closed carbon cycle. Several strategies such as processes and metabolic engineering have been evolved to enhance the production efficiency of PHAs and their co-polymers in native autotrophs and methylotrophs. These strategies have extended the utilization of C1 sources for the production of PHAs. Companies like Mango materials and Newlight Technologies have already achieved the goal in producing PHAs on commercial scale from C1 carbon substrates (Joo 2008). The real motive of PHA scale up production is to collaborate with waste fermentation facilities or power plant as they emit GHGs at a substantial rate. However, these waste gases might not contain the actual composition of C1 gases, but a potential still available for the development of a bioreactor. Thus, PHA production from C1 sources can be considered as the most effective solution for the generation of plastics (Joo 2008).

Several waste substrates have been exploited for the production of PHA. However, C₁ compounds are a promising feedstock for the biotechnological production of value-added chemicals. These compounds would help protect dwindling fossil fuel resources, reduce greenhouse gas emissions, and contribute to a cleaner climate. In the present scenario, it is easy to modify the microorganisms that contain the indigenous substrate pathways, for example, fixation of the CO₂, degradation pathway, and CH₄ oxidation. However, it is necessary to evaluate the theoretical limitations of the pathway. In this review, PHB production has studied from CO₂, but the yield have been relatively low as compared to other carbon sources. To address this issue, several strategies have been developed to enhance the yield with better physicochemical properties. C1- fermenting microbes need to be engineered for the production of PHB with high yield and to tolerate other toxic gases. In addition to this, metabolic engineering approaches should be developed for the production of PHA copolymers. Life cycle assessment of C1- fermentation based biopolymer production needs to access the potential of C 1 sources mitigation and the circular economy. Furthermore, to evaluate the economic feasibility technoeconomic analysis should be done. All the discussed parameters need to be studied to replace synthetic plastics over biopolymers.

To overcome such problems, genetic engineering can be an approach. Optimizing the discovery and design of novel enzymes and pathways, modifying microorganisms, and developing innovative and efficient operating technologies will contribute towards the ultimate success of sustainable biopolymer production by utilizing C₁ carbon sources. Synthetic biology and morphology engineering approaches will enhance the cell density and high volumetric productivity for process economics and suitable downstream development. To improve the thermal and mechanical properties of PHAs microorganisms are capable of utilizing various precursors through metabolic engineering approaches. Apart from this artificial intelligence helps to improve the industrial production of PHAs. To make PHAs production more economy, a co-production of PHAs with other chemicals such as ectoine, inositol, amino acids, hydroxy acids can be done. Next-Generation Industrial Biotechnology offers several opportunities for bioproduction of PHAs with fully automatic bioprocessing plants can be expected.