Bacterial Polyhydroalkanoate in a Sustainable Future

Version	Summary	Created by	Modification	Content Size	Created at	Operation
1		Diogo Proença	--	3818	2023-02-17 15:13:47	\|
2	format	Jason Zhu	Meta information modification	3818	2023-02-20 02:56:43	\|

This entry is adapted from the peer-reviewed paper 10.3390/ijerph20042959

Environmental challenges related to the mismanagement of plastic waste became even more evident during the COVID-19 pandemic. The need for new solutions regarding the use of plastics came to the forefront again. Polyhydroxyalkanoates (PHA) have demonstrated their ability to replace conventional plastics, especially in packaging. Its biodegradability and biocompatibility makes this material a sustainable solution. The cost of PHA production and some weak physical properties compared to synthetic polymers remain as the main barriers to its implementation in the industry. The scientific community has been trying to solve these disadvantages associated with PHA.

polyhydroxyalkanoate bacteria sustainability

1. Polyhydroxyalkanoates (PHA) and Bacterial Producers

PHA are polyesters produced by many Gram-positive and Gram-negative bacteria ^[1]. The first report of PHA production was in 1926 and was detected in Bacillus megaterium ^[2]. These natural polymers are accumulated in the form of water-insoluble granules of 0.2–0.5 µm diameter inside the cells ^[3] and serve as a storage material for carbon and energy ^[1]. Stressors such as fluctuations in temperature, osmolarity, pH, elevated pressure, or the presence of microbial growth inhibitors were reported to affect PHA production ^[4]. Most known PHA bacterial producers show better polymer production under nutrient limitation (e.g., nitrogen, phosphorus, oxygen, and magnesium) and excess carbon source. Still, some bacterial groups do not need nutrient limitations to produce PHA ^[5]. Bacteria such as Alcaligenes lactus, a mutant strain of Azotobacter vinelandii, and recombinant Escherichia coli can produce and store PHAs during the growth phase ^[5]^[6]. To date, about 92 bacterial genera can produce PHA under both anaerobic and aerobic conditions and more than 150 monomers of PHA are known. Studies show that PHA constitutes a biological advantage for producers when exposed to freezing ^[7] and other stress conditions such as oxidative and osmotic pressure ^[8]. Ralstonia eutropha H16 is considered the model organism for the study of PHA production ^[8]. Pseudomonas species are also widely studied for PHA production due to their versatility and ability to produce polymers from various carbon sources ^[9].

PHAs are increasingly arousing the interest of the scientific community and industry. This offers the possibility to replace synthetic plastics, reaching the so-desired circular economy. The aim is to find new forms of this polymer that can fulfil certain properties and functions ^[10]. The PHA polymers are composed of (R)-hydroxyalkanoic acid (HA) monomers. These monomers can vary in the alkyl side chain (R), which differs in the number of carbons, and this is responsible for the wide variety of PHAs ^[11]^[12]. The molecular mass of PHA can vary between 50 kDa and 100 kDa depending on the PHA producer ^[13].

Depending on their chain length, PHAs can be divided into three main groups: short-chain length (scl), medium-chain length (mcl), and long-chain length (lcl). Depending on their monomeric constitution, PHAs can be homopolymers, i.e., constituted by equal monomers, or heteropolymers, constituted by different monomers ^[5]. Scl-PHAs have 3–5 carbon atoms in their structure. PHB, the most well-studied PHA type, belongs to this group, and it is a homopolymer. Many bacteria such as Bacillus megaterium, Burkholderia cepacia, Cupriavidus necator, and others can produce scl-PHAs ^[14]. Mcl-PHAs are polymers with 6–14 carbons. Some examples of these polymers are poly(3-hydroxyhexanoate) P(3HHx) and poly(3-hydroxyoctanoate) P(3HO) ^[5]. Some bacteria such as Pseudomonas aeruginosa, P. oleovorans, and P. corrugata can produce mcl-PHAs ^[14]. Lcl-PHAs have more than 14 carbon atoms and are rarely produced by microorganisms ^[3]. The physical properties of polymers are distinct in these groups, with scl-PHAs being known to be brittle, while mcl-PHAs have elastomeric properties ^[13]^[15].

PHB is the most studied polymer belonging to the PHA class due to its properties similar to the synthetic polymer polypropylene (PP). The composition of the carbon sources used by the microbial producers affects the properties of PHB ^[16]. However, when compared to synthetic polymers, PHB has disadvantages, namely in its physical properties. The structure of PHA can be altered by physical, chemical, or biological methods to improve its properties. Currently, several PHB copolymers (polymers constituted by 3-hydroxybutyrate and other monomers) are known, which present diversity in their structures and properties, thus being able to be used in a wide range of applications. Examples of PHB copolymers, their properties, and applications are reviewed by Raza et al. (2019) ^[17]. One of the most used examples of copolymers is poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)), which can be produced by several bacteria ^[18]. This copolymer has greater flexibility and other characteristics different from PHB. Terpolymers have more than one secondary monomer in their constitution and are also currently being explored as an alternative to copolymers. Examples of these polymers produced by bacteria are P(3HB-co-3HV-co-3HHx) and P(3HB-co-3HV-co-4HB). Recently, a genetically engineered Ralstonia strain was able to produce P(3HB-co-3HV-co-HHx) by using tung oil, obtained from the nut seed of the tung tree, as a substrate ^[19]. The production of copolymers and terpolymers allows for a multitude of polymers with different characteristics, which can be adjusted to achieve the desired properties. As research progresses, new forms of polymers and new types of bonds are being discovered, allowing the spectrum of properties and applications achieved by these polymers to increase. This versatility allows PHA polymers to become an increasingly effective alternative to synthetic polymers in various applications.

2. Feedstocks for PHA Production

PHA can be produced from a diversity of substrates. In the industry, the majority of PHAs are produced from sucrose, sugar corn, and vegetable oils ^[20]. The use of these substrates has two main disadvantages: the competition for substrates with the food industry ^[20] and the cost of production ^[21]. Despite its numerous advantages, PHA production is not well established in the industry due to its high production and recovery costs ^[5]^[22]. Currently, researchers are trying to use cheaper substrates for PHA production, such as waste feedstocks (WF), lignocellulosic feedstocks (LF), dairy industry waste, oil industry waste, municipal wastes, biodiesel industrial waste, and waste syngas ^[23]. Some of the recent developments of feedstocks, strains, and associated process developments for PHA production are reviewed in Li and Wilkins (2020). The use of LF for PHA production involves pre-treatment and hydrolysis processes, which allow more availability of carbon sources for microbial fermentation and PHA production ^[24]. Currently, this is still a process that needs further investigation due to low production efficiencies. Nevertheless, Argiz et al. (2021) studied the use of fish oil waste as a carbon source for PHA production in a two-stage process (culture selection and accumulation of intracellular compounds) taking advantage of the use of mixed microbial cultures (MMC). The substrate is hydrolysed in two phases without the need for prior treatment by bacteria belonging to MMC, releasing soluble free fatty acids (FFA). Subsequently, soluble FFA can enter cells, functioning as a substrate for various metabolic pathways ^[25]. Volatile fatty acids (VFA) have been used as a carbon source to produce PHA. Different bacterial strains were reported to reach PHA accumulations up to 80% using this carbon source and, therefore, this application is considered an alternative for the reduction of fermentation costs. Recently, Vu et al. (2022) showed the effect of different concentrations of VFAs as a sole carbon source for the biosynthesis of PHAs using Cupriavidus necator, formerly known as Ralstonia eutropha ^[26]. Crude glycerol has also been explored for PHA production. This is a by-product produced by the biodiesel industry. Crude glycerol is one of the few waste sources that can be used directly as the substrate for PHA production, therefore, it is considered a promising alternative substrate in reducing production cost ^[27]. Wen et al. (2020) investigated PHA production dynamics with the different changing directions of the crude glycerol gradient. Reverse glycerol gradient was demonstrated to be more effective in accumulation batch assays using MMC with origins in wastewater treatment tanks ^[28]. Future studies will be important for optimizing the WF and LF valorisation processes in the PHA production process to make it increasingly sustainable and implemented in the industry.

3. Metabolic Pathways Involved in PHA Synthesis

Diverse genomic and metabolic studies allow more understanding about PHA biosynthesis and degradation. The production of PHA can be divided into two main steps: generation of hydroxy acyl-CoA and polymerization of hydroxy acyl-CoA into PHA ^[1]. In the first step, three main metabolic pathways allow the production of PHA, which are acetoacetyl-CoA synthesis, de novo fatty acid synthesis, and fatty acid β-oxidation ^[10]. Acetyl-CoA and acyl-CoA are common intermediates in all three pathways ^[3] and have an important role in the regulation of its production ^[22].

Bacteria can produce PHA through sugar or fatty acid sources, varying the metabolic pathway used. When the main carbon source is sugar, bacteria can produce through two metabolic pathways: acetoacetyl-CoA synthesis (pathway I), whose reactions and enzymes required for the process will be discussed in the following section, and fatty acid biosynthesis (pathway II). During the reactions of the latter metabolic pathway, intermediates are formed that can be used as precursors in PHA synthesis, typically mcl-PHAs ^[29]. One of the compounds formed during the synthesis of fatty acids is (R)-3-hydroxyacyl-ACP, which can be converted to (R)-3-hydroxyacyl-CoA, via the acyl-ACP: CoA transacylase, encoded by the phaG gene ^[30]. The (R)-3-hydroxyacyl-CoA is an intermediate in the PHA formation process ^[29]. When PHA production originates from fatty acids as the main carbon source, the most common metabolic pathway is fatty acid β-oxidation. This pathway gives rise to several intermediates that can be converted into (R)-3-hydroxyacyl-CoA through the action of different enzymes, such as hydratases, epimerases, or reductases ^[29]. This allows an immense diversity in the production of mcl-PHAs. Using this pathway, it is also possible to produce scl-PHAs through the action of the enzyme enoyl-CoA hydratase (PhaJ) which converts enoyl-CoA intermediates to (R)-3-hydroxyacyl-CoA precursors ^[30]. All metabolic pathways end with the polymerization reaction through the enzyme PHA synthase (PhaC).

PHA metabolism of bacterial cells is controlled at multiple levels, via nutrient imbalances or stressors, and by specific regulators present in the pha gene cluster ^[31]. Different bacterial cell growth phases have been studied. Most bacteria that are PHA producers were shown to produce this polymer at the stationary growth phase. Some of them have been shown to form granules of crystalline PHB at those conditions ^[32]. To control the so-called PHA metabolic cycle, genome editing tools have been addressed to improve PHA production ^[33].

4. Bacterial Genes and Enzymes Involved in PHA Metabolism

In bacteria, PHA polymer is produced in the form of hydrophobic granules. These granules have several enzymes on their surfaces that are involved in polymer production, stabilization, mobilization, and degradation. Examples of some enzymes are phasins (PhaP, PhaI, PhaF, and others), which have a role in stabilizing the polymer, regulating proteins (PHA synthesis repressor-PhaR and PHB-responsive repressor-PhaQ) like the PHA synthase activating protein (PhaM) and an enzyme that promotes PHA degradation (PhaZ and PhaY) ^[1]. Phasins are not essential in the polymerization of PHA, however, they play an important role in the regulation of the size, number, and surface-to-volume ratio of PHA granules ^[22].

Several enzymes participate in PHA production, PhaC being considered the key enzyme of this process ^[34]. This enzyme is present in all PHA-producing bacteria and there are four main classes, according to their substrate preference and their primary structure. Type I and type III PHA synthases are responsible for the production of scl-PHAs, while type II normally produces mcl-PHAs ^[12]. Type I and type II PHA synthases are characterized by an enzyme with only one subunit with 60–70 kDa and can be found in R. eutropha and P. aeruginosa, respectively ^[12]^[35]. Type III is present in C. vinosum and has two subunits (PhaC and PhaE), which together are responsible for polymer production ^[12]^[35]. Type IV is present in Bacillus species and comprises two subunits (PhaC and PhaR) that are essential for PHA production ^[1].

The two main enzymes responsible for PHA degradation are PHA hydrolase (PhaY) and PHA depolymerase (PhaZ). The degradation of PHA depends on a few factors such as the chemical composition of the polymer, polymeric chain length, crystallinity, and complexity ^[5]. Biodegradation of PHA can happen in aerobic or anaerobic conditions, resulting in different products. In the first case, PHA degradation results in carbon dioxide and water, and in the second, the products of PHA degradation are carbon dioxide and methane ^[13].

Oftentimes, the genes involved in PHA metabolism are inserted in gene clusters ^[3]. The most studied one is the phaCBA gene cluster which can be found in R. eutropha H16 ^[22]. The three genes present in this gene cluster (phaA, phaB, and phaC) encoded three proteins that are responsible for PHA production. This production is divided into three phases. Firstly, PhaA-β-ketothiolase (encoded by the phaA gene) catalyses the synthesis of acetoacetyl-CoA from acetyl-CoA. Then, PhaB-acetoacetyl-CoA reductase (encoded by the phaB gene) is responsible for the reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA, which is incorporated into the growing polymer by PhaC-PHA synthase (encoded by phaC gene) in the final step. Kutralam-Muniasamy et al. (2017) demonstrated the presence of other cluster groups in PHA producers.

5. Strategies for Sustainable Production of PHA

5.1. Upstream Processes in PHA Production

In the upstream procedures, research is essentially related to the discovery of new strains that produce PHA, or to the improvement of the production of PHA by new or existing strains already described through optimization of culture conditions and feeding strategies ^[1]. The fact that most current commercial PHB producers are heterotrophic ^[36] and that the production process is not economically sustainable leads to the search for other potential producers of the polymer. An example is the exploration of cyanobacteria as potential PHA producers. Some genera of cyanobacteria are already described as PHA producers, e.g., Synechococcus, Nostoc, Spirulina, etc. These bacteria have the main advantage that they do not require exogenous sugar for growth because they use CO₂ for this purpose. The increase in CO₂ in the last years has had negative impacts on the environment, namely the aggravation of global warming. Green technologies for the conversion and utilization of CO₂ in a sustainable manner is a goal of today’s society in order to decrease its concentration and impacts on the environment; the use of cyanobacteria in PHA production is an example. Optimization of production parameters is also a useful tool for process sustainability and is the focus of several recent studies. In Chmelová et al. (2021), the effects of various culture medium components were studied through statistical design on the biomass production of P. oleovorans. Since 50% of PHA production cost is due to the cost of the carbon sources used in this process, some research focuses on investing in cheaper substrates including waste materials such as whey, starch, sugar-cane molasses waste, vegetable oils, and wastewater for the production of this polymer ^[37]. The use of waste in PHA production not only reduces production costs but also allows waste reduction, thereby reducing environmental pollution. In a recent study, it has been possible to verify a coupling between the two approaches mentioned above, the exploitation of cyanobacterial species for PHA production and the use of waste as a substrate for fermentation ^[38]. In that research, N. muscorum accumulated 31.3% CDW of P(HB-co-HV) from unrectified glycerol substrate derived from biodiesel industry waste.

Another approach to lower PHA production costs is the use of mixed microbial cultures (MMC) using WF as a substrate. The use of MMC has advantages such as cheaper carbon sources and no need for sterility control. However, when using MMC in PHA production, lower values of PHA content are obtained than those obtained by pure cultures ^[39]. In the last years, several studies have tried to optimize PHA production by MMC. The selection of bacterial strains and the culture conditions are extremely important for the success of this technique. Volatile fatty acids seem to be the best precursors for PHA production in MMC ^[28]. Shen et al. (2022) showed that the highest PHA productivity values to date were achieved using MMC with wastewater. The addition of pyruvate was the determining factor for the increased yield ^[40]. Recently, Vermeer et al. (2022) demonstrated the production of poly(3-hydroxyisobutyrate) (PHiB) using microbial enrichment cultures ^[41].

Due to the great evolution in the field of molecular biology in the last decades, it is possible to manipulate bacteria at the genetic and metabolic levels to obtain desired products efficiently. Transcriptome and metabolome analysis allow people to understand the metabolic changes inherent in the production of PHA, thus facilitating better-targeted genetic manipulation to make the production process more efficient ^[1]. One of the most adopted ways to increase the production of the desired product is to hyper-activate the pathway that leads to the formation of this product and inhibit or eliminate competitive metabolic pathways. Some techniques, such as gene mutation, editing CRISP/Cas9 genes, and using strong promoters for essential genes for PHA production are used in an attempt to increase PHA production ^[3]. In Xiong et al. (2018), a genome editing method, the electroporation-based CRISPR-Cas9 technique, was developed to increase the efficiency of genetic manipulation of R. eutropha. Five putative restriction endonuclease genes were disrupted, and Cas9 expression was optimized enabling genome editing via homologous recombination based on CRISPR-Cas9 in R. eutropha ^[42]. Later, Jung et al. (2019) used the same technique in recombinant E. coli to improve PHA production. They deleted genes of competitive pathways and overexpressed a gene which catalyses the interconversion of NADH and NADPH, leading to a more efficient PHA production ^[43]. This technique may be used in the future by several producers to manipulate the carbon flux into PHA production.

Another widely used technique is genetic recombination which allows non-PHA-producing bacteria to produce the polymer ^[3], creating microbial cell factories. Typically, these bacteria are genetically well-studied, which facilitates genetic manipulation to increase PHA production. Recombinant organisms allow for solving some limitations of natural PHA producers such as long generation time, relatively low optimal growth temperature, and being hard to lyse. Another advantage is that recombinants usually lack the PHA degradation pathway, thus allowing for higher PHA yielding. One of the most commonly used non-PHA-producing bacteria in these types of studies is E. coli. This bacterium is considered a powerful microbial cell factory for PHA production on a commercial scale. By using different designs of recombinant E. coli strains, different types of PHA can be obtained. A novel PHA copolymer, P(3HB-co-3H2MB), was synthesized by constructing an artificial metabolic pathway in E. coli L55218. The copolymer was synthesized with various monomer compositions and their thermal properties were investigated. Last year, Goto et al. (2022) studied the effect of chaperones on PHA production in recombinant E. coli expressing PHA synthase from B. cereus YB-4. The results showed that chaperones have a positive action on the activation of PHA synthases and PHA productivity ^[41]. Recent studies also seek to find ways to optimize the conditions for PHA production by recombinant E. coli ^[6]^[44]^[45].

Pilot platforms for PHA production have been implemented in Treviso (northeast of Italy), Carbonera (northeast of Italy), and Lisbon to produce PHAs by open MMCs. These pilots exploited different organic biowastes as raw materials for biodegradable polymers, characterized by thermal and chemical properties comparable to commercial plastics ^[46]. A pilot for production of PHA from paper industry wastewater was also investigated. The pilot plant was designed as a three-step process comprising anaerobic fermentation for maximization of the volatile fatty acid (VFA) concentration, enrichment of PHA-producing biomass, and accumulation for maximization of the PHA content of the biomass. They identified VFA production and pH control as major bottlenecks ^[47].

5.2. Downstream Processes

Several processes are applied in PHA extraction and recovery from biomass after its production in bacteria. Problems related to downstream processes, such as high recovery costs and low PHA contents obtained, need to be overcome. The excessive use of environmentally harmful solvents is also a worrying factor that has been considered in current research. Usually, the extraction is carried out when the growth peak of the bacterial culture is reached to optimize the polymer yield ^[48]. The initial step usually consists of separating the biomass from the culture medium. This can be done by techniques such as centrifugation, filtration, and sedimentation, among others. The next step is cell disruption, extraction, and purification of the polymer. Depending on the technique chosen, the PHA is extracted by solubilizing it or by solubilizing the biomass together with the polymer. Then, the technique used for polymer purification will be dependent on the future application of the polymer. The solvent extraction method is one of the most widely used due to its simplicity and the high purity of the polymer obtained. This process has the advantage of eliminating impurities such as bacterial toxins ^[5]. In this process, the cells are pre-treated to facilitate the access and solubilisation of the polymer, usually in chloroform ^[49]. Then, the polymer is precipitated by the action of alcohols, such as methanol or ethanol. Despite being widely used at the laboratory level, its use is not recommended when high amounts of polymer are produced due to the vast number of solvents used and its toxic effects on humans and the environment ^[1]. Then, the need arose to find more eco-friendly ways to extract the polymer. In these methods, the cells can be digested through mechanical, enzymatic and/or chemical action and the PHA isolated through centrifugation or filtration ^[1]. Mechanical methods, such as high-pressure homogenization (HPH), are widely used in PHA extraction ^[50]. In this technique, cell lysis is based on the passage of biomass through a valve with a narrow orifice, followed by depressurization and a large increase in flow velocity, with consequent cavitation and high shear stress causing deformation/rupture of the cells in suspension ^[51]. The chemical digestion principle involves the solubilisation of non-PHA components using various surfactants, such as sodium dodecyl sulphate (SDS), Triton X-100, sodium hypochlorite, etc., after cell lysis ^[52]. In enzymatic digestion, enzymes such as lysozyme are used, allowing components such as proteins to dissolve. The PHA beads are separated by filtration or another method ^[52].

In large-scale production of PHA, halogenated extraction continues to be one of the most used methods that, in turn, results in higher production costs due to chemicals and the risk to operators and environmental contamination ^[53]. In addition to these methods, green solvents have been explored to substitute chlorinate solvents in PHA extraction, such as DMC. DMC is an acyclic alkyl carbonate that is currently produced in the industry ^[53]. Its versatility and non-toxicity are two properties that have increased interest in this solvent. In Samorì et al. (2015), C. necator freeze-dried biomass containing 74 ± 2 wt% of P(3HB) was extracted with DMC. This method was successful in the extraction of PHA from both freeze-dried biomass and highly concentrated microbial slurry without any pre-treatment before the extraction. The recovery and purity of the polymer were high, conserving the thermophysical characteristics of the polymers ^[53]. This reagent is more efficient in extracting and recovering PHA from MMC compared to more traditional methods ^[39].

Through this analysis, it is possible to see that there are several techniques currently used both upstream and downstream of the PHA production process. Their use will depend on the desired goals, and the right combination of the various techniques can lead to the desired PHA yield. Nowadays, researchers not only seek processes that result in higher PHA production but also processes that are more eco-friendly. Cost reduction, use of non-toxic reagents and solvents, and reuse of materials are important factors in developing industrially applied processes.

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