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1. Introduction

Nowadays, plastic materials are widely used and essential components of product packaging, cars, household/office appliances, computer equipment, and medical devices [1]. Conventional plastics accumulate in the environment because they are not biologically degradable, producing a large spectrum of pollutants [1]. Circa 140 million tons of plastic are consumed/produced every year all around the world. Plastic production requires the processing of approximately 150 million tons of fossil fuels and a huge amount of waste is produced whose depolymerization can take thousands of years [2]. In May 2018, the European Commission proposed new European rules aimed at reducing plastic pollution in cities and oceans by banning several plastic products. EU officials noted that the single-use plastic objects and fishing gear accounts for 70 percent of trash in the ocean and that the move towards “innovative alternatives” could create around 30,000 jobs. EU First Vice President Frans Timmermans hailed the draft directive, saying: “Plastic waste is undeniably a big issue and Europeans need to act together to tackle this problem, because plastic waste ends up in our air, our soil, our oceans, and in our food” (http://europa.eu/rapid/press-release_IP-18-3927_en.htm).

The methods used for the disposal of plastic materials are challenging. Indeed, in landfills, the rates of degradation are tremendously low. Incineration generates toxic by-products. Recycling can be done, but it is a very time-consuming process and also causes changes in the properties of plastic materials [3].

To reduce world pollution, researchers have been focused on biodegradable plastics as alternative to conventional fossil-derived plastics. Plastics are biodegradable at a tunable rate depending on the surrounding environmental conditions (e.g., location or temperature) and on the material (http://www.european-bioplastics.org/). The biodegradable plastics can be classified into four categories, as reported in Table 1 [4].

Currently, 359 million tons of plastic are annually produced of which bioplastics represent about 1% of the whole market (http://www.european-bioplastics.org/). However, the bioplastic market is growing by about 20% to 100% per year to fulfill the rising demand and the request for more sophisticated materials, applications, and emerging products. According to the latest market data compiled by European Bioplastics in cooperation with the Research Institute Nova-Institute, the global bioplastics production capacity is set to increase from around 2.11 million tons in 2019 to approximately 2.43 million tons in 2024 (http://www.european-bioplastics.org/).

Biodegradable plastics altogether, including polylactic acid (PLA), polyhydroxyalkanoates (PHA), starch blends, and others, account for over 55.5 percent (over 1 million tons) of global bioplastics production capacities. The production of biodegradable plastics is expected to increase to 133 million in 2024, especially due to PHA’s significant growth rates. Indeed, in the current scenario of increasing environmental awareness, PHAs are becoming potential substitutes for non-biodegradable polymers [1]. Poly-β-hydroxybutyrate (PHB) is a well-known and characterized PHA found in bacteria. The production capacity of PHB has been growing steadily during the last few years.

The present review reports main issues regarding the PHB production and the challenging issues to be investigated/developed. The PHB structure as well as its formation/accumulation in the cyanobacteria is presented. The biotechnological route to produce the PHB is presented with the potential solution to improve the productivity and reduce the cost of the production. The effects of the PHB structure in the cell on the recovery performance is discussed. 2. Poly-β-hydroxybutyrate (PHB) Structure Polyhydroxyalkanoates (PHAs) are the main blocks of biodegradable plastics. They are polyesters produced by various microorganisms, such as cyanobacteria. Due to their chemical and physical features, PHAs are the most investigated biodegradable polymers.

The PHAs are divided into three groups: short-chain-length-PHAs (SCL-PHAs), medium-chain-length-PHAs (MCL-PHAs), and long-chain-length-PHA (LCL-PHA) [5,6,7]. The polyhydroxybutyrate (PHB) (Figure 1) is the most common of the SCL-PHAs. PHBs have been characterized with respect to the following interesting features: thermoplastic processability, hydrophobicity, biodegradability, biocompatibility, and optical purity [8]. Key physical features of PHBs are a melting temperature of about 177 °C, glass transition temperature of about 2 °C, crystallinity of about 60%, tensile strength of about 43 MPa, and extension to break of about 5% [9].

3. Commercial Status of PHB

The current bioplastic production plants (Figure 2) are characterized on a small scale when compared to plant petroleum-based plastics. One example is China’s TianAn PHA plant that produces approximately 2000 metric tons of PHA per year. This production is quite small if compared to the standards. However, since bioplastics represent an interesting product that can be used in various sectors, some of the producers became leaders in the biopolymer field production all over the world [4,10].

PHA bioplastic businesses are largely used in North America, Europe, Asia-Pacific, etc. [2]. Companies such as Metabolix Inc. (USA), Shenzhen Ecomann Technology Co. Ltd. (China), Tianjin GreenBio Materials Co. Ltd. (China), Meredian Inc. (USA), Mango Materials (USA), Newlight (USA), and Biomer (Germany) are the main producers of PHA [2] (Table 2).

In 2006, Metabolix and Archer Daniel Midland (ADM) developed a joint company called Telles that became the major PHA producer in the USA. Unfortunately, this company ended production in 2012 [11].

Another company in the USA called Meridian Inc. produces around 15,000 tons of PHA bioplastics per year obtained from the fermentation of bacteria grown on oils (fatty acids). The obtained products are used for films, non-woven fabrics, and food-contact packaging. The full capacity of the plant is able to produce more than 300,000 tons of PHA per year [2,11].

The Mango Materials, founded in the USA in 2010, focused its production on PHB-based bioplastic using a new technology based on intellectual property licensed from Stanford University. In this case, the PHB is produced by methanotrophs bacteria that grow using the methane contained in wastewaters. The PHB is extracted from the cellular biomass, transformed into bioplastic, and finally converted into products of interest [11].

The Dutch company DSM and the Chinese company Tianjin Green Bio-Science Co. created a joint venture to produce bioplastics. The company is now producing PHA resin (10,000 metric tons per year) [2,11].

Of note, the Japanese company Kaneka produced 50,000 metric tons annually of PHB in 2010 [2,11].

4. Conventional Production System of PHB

The commercial PHB production is based on cultures of microorganisms, such as Cupriavidus necator, Alcaligenes, Azotobacter, Bacillus, Nocardia, Pseudomonas, Rhizobium [12,13]. Under optimal conditions using fructose as carbon source, bacteria—e.g., Ralstonia eutropha (also known as Cupriavidus necator)—can produce PHB up to 80% of the cell dry weight [14]. Table 3 reports an overview of bacterial strains used to produce PHB, the feedstock used as carbon source, and the chemical structure of the produced copolymers.

The high cost of the raw materials (which represent 30–40% of the total production costs) for PHB production by bacterial fermentation makes this process not economically competitive (about 2.6 U.S.D./kg as reported by Lopez-Arenas et al. [39]). Therefore, low cost photosynthetic production systems have been proposed as alternative [40,41]. However, the use of transgenic plants compete directly with subsistence crops for agricultural acreage and have led to ethical concerns because the dissemination of transgenic plants could be difficult to regulate [2].

5. Cyanobacteria and PHB

Cyanobacteria display all the advantages of bacteria when compared to the chemoheterotrophs. In addition, cyanobacteria are characterized by the potential to exploit sunlight energy and to convert CO2-abundant and not competitive carbon-based feedstock into a product of interest, such as PHB. Two further advantages of the cyanobacterial system are that (i) they do not compete with the agro-food market for resources [42,43,44] and (ii) they contribute to capture and to use the CO2, decreasing the pressure of the greenhouse gas release into the atmosphere.

Cyanobacteria—or blue-green algae—are prokaryotic organisms that have proved to be interesting because of their potential contribution to the sustainable economy. They can perform oxygenic photosynthesis capturing free energy from (sun) light for the synthesis of ATP and NADPH [42]. Valuable products from CO2, sunlight, and water can be produced by photosynthetic metabolism [44].

The ancestors of cyanobacteria were responsible for the oxygen enrichment in the—originally anoxic—atmosphere of the earth more than 3.2 billion years ago. This event had an enormous impact on life on earth resulting in favorable conditions for aerobic organisms. Subsequently, the thylakoid membranes of the ‘primordial cyanobacteria’ emerged from oxygenic photosynthesis evolution [45] leading to the development of plants.

The cyanobacteria evolution succeeded due to their unique metabolic pathways that can carry out oxygenic photosynthesis and respiration simultaneously in the same cell compartment. Furthermore, many cyanobacterial species can fix nitrogen and survive under many environmental conditions [46].

Photosynthesis and respiration involve electron transport pathways that are catalyzed by protein complexes in membranes. The oxygenic photosynthesis is characterized by the conversion of CO2 and water to sugars fixing the energy from sunlight. This process is the reverse of respiration, which is the conversion of sugars to CO2 and water releasing energy. The respiratory electron transport chain is in the cytoplasmic membrane—the membrane that separates the cytoplasm and the periplasm—or can occur in thylakoids. Instead, the photosynthetic electron transport in most cyanobacteria occurs only in thylakoids. A schematic representation of the respiratory and photosynthetic electron transport chains in cyanobacterial thylakoid membranes is shown in Figure 3.

The spectrum of potential applications of cyanobacteria or of their components is very large. Cyanobacteria have been used as food and feed sources. Strains of Spirulina are rich in vitamins and proteins and are used as food supplement. One of the largest industrial applications of cyanobacteria is their capability of being effective fertilizers. Because cyanobacteria can fix nitrogen, they are able to provide support for plant growth in rice fields [47]. Cyanobacteria were also investigated to produce anti-fungal, anti-bacterial, and anti-cancer agents. The cyanophycin polymer, built from aspartate and arginine, can replace polyacrylate (which is derived from propylene and thus from petroleum) in paints and surface coatings. Cyanobacteria are capable to produce a protein–pigment complex called phycocyanin, which is used as a food colorant in cosmetics and diagnostics [48].

Under photoautotrophic conditions and minimal nutrient conditions, such as phosphate, nitrate, magnesium, and calcium, as well as macro-minerals, ferrous, manganese, zinc, cobalt, and copper [9], cyanobacteria may accumulate the homopolymer of PHB [49]. Indeed, PHB can be used by cyanobacteria as a specialized biomass for carbon and energy storages [50].

The cyanobacterial strain Synechocystis sp. PCC 6803 is the most used and well characterized strain because its growth is quite fast (minimal doubling time of less than seven hours) and it does not have specific nutritional demands [51].

6. Improvement of PHB Production in Cyanobacteria 6.1. Optimization of Growth Conditions

A deficiency or limitation of nutrients, such as nitrogen and phosphate, stimulates the accumulation of PHB in cyanobacteria under photoautotrophic growth conditions [52,53,54] (Table 4). A PHB production fraction in cyanobacteria as high as 71% (of dcw) may be produced in Nostoc muscorum Agardh cultures carried out in a medium supplemented with glucose, acetate, and valerate coupled with nitrogen and phosphate limitation [52]. The PHB pool in Spirulina sp. LEB 18 reached 31% (of dcw) under photoheterotrophic conditions in a medium supplemented with sodium bicarbonate and deficiency of nitrogen and phosphorus [55]. Aulosira fertilissima CCC 444 accumulated 77% of PHB—with respect to the dry cell weight—for growth in a medium that contained fructose and valerate and under phosphorus deficiency [53]. For Synechocystis sp. PCC 6803, PHB accumulation of 38% (of dcw) under nitrogen depletion with fructose and acetate supplementation was reported [56]. The same cyanobacterium accumulated PHB: (i) up to 4%—with respect to the dry cell weight—under photoautrophic growth conditions and nitrogen depletion [50]; and (ii) up to 8% under photoautrophic conditions—CO2 concentration in the gas stream 2% (v/v)—and nitrate concentration reduced to half of the optimal cell growth concentration (BG1/2 medium) [57]. Haase et al. [58] reported that Nostoc muscorum CCAP 1453/9 reached a PHB content of 22% under photoautotrophic conditions and deficiency of nitrogen. Calothrix scytonemicola TISTR 8095 accumulated 25% of PHB—with respect to the dry cell weight—under photoautotrophic growth conditions and deficiency of nitrogen.

The comments above and the data summarized in Table 4 highlight the necessity to grow cells under the unbalanced feeding of nitrogen and phosphorus in order to activate PHB production [2]. The high accumulation of PHB under limited nitrogen conditions can be rationalized by considering that when photosynthesis continued to fix CO2 the absence of nitrogen did not support the synthesis of proteins. Therefore, cyanobacteria accumulate the carbon energy as glycogen and/or PHB [59].

Bhati and Mallick [52], Panda and Mallick [56], and Samantary and Mallick [53] measured the maximum PHB content for cultures carried out under heterotrophic growth conditions. Although the PHB production may be very interesting, this PHB production requires the supply of a reduced carbon source (like a sugar). Coelho et al. [55], Haase et al. [58], and Kaewbai-ngam et al. [60] reported interesting results for cultures carried out under autotrophic conditions in shake flasks. They cultivated Calothrix scytonemicola TISTR 8095, Nostoc muscorum CCAP 1453/9, and Spirulina sp. LEB 18, respectively. However, these strains are biofilm forming strains [61] and their performance may be strongly impaired when cultivated in photobioreactors, the culture system that preserve the high quality of the product. Synechocystis PCC6803 was characterized by high PHB productivity under heterotrophic growth conditions [62]. However, its performance was drastically reduced when cells were grown under autotrophic conditions [50]. Indeed, the PHB fraction reported by Wu et al. [50] (4% of PHB) was half the value reported by Carpine et al. [57].

6.2. Genetic Engineering Approach

Genetic engineering of photosynthetic microbes—cyanobacteria—may enhance the conversion of CO2 into chemical commodities [44,69]. The development and application of engineered cyanobacteria is a very promising approach to develop renewable and carbon neutral processes to provide chemicals typically produced by fossil sources and alternative energy vectors [70].

Several cyanobacterial strains can be employed to transfer exogenous DNA across the cell membrane into the host organism by a yet unresolved mechanism [70] due to their natural competence. Synechocystis sp. PCC 6803, Synechococcus elongatus PCC 7942, and Synechococcus sp. PCC 7002 are naturally transformable and highly suitable for genetic engineering applications [71].

Cyanobacteria can be considered the cell factories of the future. For example, introducing an exogenous biosynthetic pathway in a cyanobacterial cell may improve a certain product fabrication (a chemical, a biofuel, or a precursor of a bioplastic) obtained from the conversion of CO2 (Figure 4) [72].

Cyanobacteria, genetically engineered with foreign genes, can produce two main products: (i) proteins and (ii) small-molecule chemicals. Depending on the targeted product, cyanobacteria can be engineered with the addition of a single gene or of an entire new pathway. The central carbon metabolism of cyanobacteria is a highly complex network which includes the CBB cycle, glycolysis, the pentose phosphate (PP) pathway and the TCA cycle [72].

Cyanobacteria may accumulate considerable amounts of PHB as product of the wild metabolism and as results of the genetic engineering of strains by heterologous transformation with genes involved in the PHB pathway of R. eutropha [73]. In many cyanobacteria, PHB is a native polymer that stores carbon, produced via the polyhydroxyalkanoate (PHA) biosynthetic pathway [74]. The pathway from acetyl-CoA to PHB includes the following three catalytic steps:

  • The condensation of two molecules of acetyl-CoA into acetoacetyl-CoA by PHA-specific beta-ketothiolase (phaA) [75];

  • The reduction of acetoacetyl-CoA to (R)-3-hydroxybutyryl-CoA by acetoacetyl-CoA reductase (phaB);

  • The (R)-3-hydroxybutyryl-CoA condensation into PHB by PHA synthase (phaC/phaE) [75] (Figure 5).

Genetic and metabolic engineering together with recombinant DNA technology have shown the ability to increase the PHB accumulation to produce a considerable amount of biomass [4]. Table 5 lists the numerous genetic engineering approaches described in the literature and that have been applied to increase the PHB content in cyanobacteria. The degree of success of those approaches depends on several issues. Several studies have evaluated the PHB accumulation under mixotrophic growth conditions [54,76,77] in mutants of Synechocystis and Synechococcus. The researchers have investigated the effect of supplementation of the growth medium with acetate. The acetate, which can be converted into acetyl-CoA via the activity of acetyl-CoA synthetase, may support many metabolic pathways. Those pathways are the TCA cycle for cell growth, glycogen synthesis, and fatty acid metabolism relevant for biofuel production (such as butanol, propanol, and isopropanol [72]) and PHB biosynthesis [75]. The heterologous expression of the PHA-synthesizing gene operon from the bacterium R. eutropha in Synechocystis sp. PCC 6803 has been reported to increase the PHB content from 7% to 11% of dcw when grown in a N-deprived medium supplemented with 10 mM acetate [76]. Similar results have been obtained for the cyanobacterium Synechococcus sp. PCC 7942 in which the entire A. eutrophus PHB-synthesizing apparatus was heterologously expressed. These recombinant Synechococcus cells accumulated 25% PHB of dcw [75] for cultures carried out supplementing 10 mM acetate and under N-starvation [53]. The PHB content of the phaAB-carrying strain obtained by Khetkorn et al. [75] increased to 35% of dcw for cultures carried out supplementing 4 mM acetate under N-starvation conditions. The accumulation was very high when compared with the 13% PHB of dcw measured for the wild type cultures carried out under the same operating conditions.

The PHB content reported for cultures carried out under photoautotrophic growth conditions using just CO2 as carbon source was lower [78,79] than that measured for cultures with other carbon sources. The overexpression of the native sigE gene (encoding of the minor sigma factors of the organism) provided the integration of the latter in the Synechocystis chromosome, and increased the PHB content from 0.6% to 1.4% [74] when cells were grown in a N-deprived medium. Hondo et al. [78] transformed Synechocystis cells with the vector pAM461c, harboring a PHA biosynthetic operon from Microcystis aeruginosa NIES-843, and reached a PHB content of about 7% in the N-deprived medium [74]. In a recent publication of our group, Carpine et al. [74] measured a PHB content larger than 12% (of dcw) by overexpression of phosphoketolase (xfpk), combined with the double deletion of phosphotransacetylase (pta) and acetyl-CoA hydrolase (ach) (under balanced growth conditions, i.e., using BG11 [80] as the growth medium). This result was almost two-fold higher than previously reported under photoautotrophic growth conditions [30].

6.3. Continuous PHB Production

A possible way to implement the process of PHB production by a genetically modified strain of Synechocystis PCC6803 is to carry out the process under a continuous mode. As comprehensively described in the literature, PHB production includes two distinct phases for most production strains. During the first phase, enough concentration of active biomass with low PHB accumulation is produced in a nutritionally balanced growth medium. During the second phase, a nutritional stress is induced by restricting the supply of a growth-essential nutrient (e.g., nitrogen, phosphate, etc.). This implies PHB accumulation due to the deviation of the carbon flux from biomass production. In this second phase, PHB accumulation is much higher than biomass formation [81]. A potential reactor configuration could be the serial arrangement of two sequential reactors: (i) reactor I for biomass production under optimal growth conditions; (ii) reactor II for PHB accumulation under nitrate-starvation conditions. The advantages of this configuration are

  • The possibility to separate the biomass growth phase from the PHB production phase. The two phases are characterized by different optimal operating conditions;

  • Minimization of equipment downtime and time loss due to the lag phase of the microbial cultures [82];

  • The continuous cultivation can guarantee the growth of microorganisms and their long-term genetic stability under defined nutrient limitations for prolonged time periods, resulting in both high productivities and constant product quality [82];

  • The possibility to harvest biomass at a desired PHB-mass fraction;

  • The applied dilution rate D may significantly influence the molar mass of PHB. The PHB productivity/concentration may be affected by the dilution; indeed, alteration of the dilution rate could affect both utilization of the substrate and the growth of cells [83].

6.4. Mathematical Modeling

The researchers and industries involved in the blue biotechnology have focused on the potential replacement of conventional plastics by PHBs. The improvement of large-scale productivity and biochemical/genetic properties of producing strains requires mathematical modeling and process/strain optimization procedures [84]. In any case, the development of industrial processes asks for mathematical models of cell growth and PHB production.

Models available in literature to describe cell growth and the PHB production rate depend on the PHB production modality. Three classes of PHB producing microorganisms can be identified:

  • Strains characterized by distinct phases: biomass growth phase and PHA production phase, typically induced by N or P limitation (prototype organisms: Pseudomonas sp. 2F, Methylmonas extorquens) [8];

  • Strains characterized by the accumulation of PHB during cell growth phase under balanced nutritional conditions and by the increase of the PHB fraction under the non-growth phase (usually induced by N or P limitation) (prototype organism: Cupriavidus necator) [85];

  • Strains characterized by remarkable PHB formation during the (exponential) growth phase without limitation of any essential growth components [86,87] (prototype organisms: Azohydromonas lata DSM 1122, or Pseudomonas putida GPo1 ATTC 29347).

The mathematical models reported in literature that describe the PHB production from microorganisms can be classified as follows: (i) models that are based on enzyme and/or microbial kinetics (kinetic models); (ii) models that are based on the characteristics of metabolic networks that are time invariant (dynamic models); models that are based on cellular metabolism (metabolic models); (iii) models that are based on microbial processes and on the metabolic regulation by the cybernetic framework (cybernetic models); and (iv) hybrid models (that combine at least two of the types of model properties mentioned before) [85].

A number of metabolic and bioreactor models have been proposed in literature for PHB production by bacteria (Table 6). Mozumder et al. [88] reported the autotrophic PHB production in Cupriavidus necator by using two-phase processes: biomass growth and subsequent PHB production. This study simulated the two distinct phases to achieve a high overall PHB production rate and PHB content. Faccin et al. [89] presented a model that aimed at the kinetics growth of Bacillus megaterium for the production of P(3HB). Khanna and Srivastava [90] proposed a structured unsegregated model for PHB accumulation in Ralstonia eutropha based on exhaustive investigations on substrate limitation and inhibition by the carbon, nitrogen, and phosphorus sources. Our group [57] proposed the first model to describe photoautotrophic PHB production by Synechocystys PCC6803 using a light/dark cycle (18 h light/6 h dark) for cyanobacterial growth. This model, presented in a publication by Carpine et al. [57], was based on mass balances. The effect of significant factors and nutrients on the cyanobacterial system was considered. In particular, the proposed model included the nitrate and phosphate utilization rate, the PHB production rate, the cellular growth and the rate of lysis of the cells. One of the innovations of this model was that the cells were considered to be in the cultures in two different statuses: the growing cells (X) that were able to grow, and the cells that were able to produce PHB (XPHB). The specific velocity (kT) of XPHB formation was considered dependent on the initial concentration of nitrate [57].

7. PHB Recovery

The market penetration of PHB has the limitation of high production cost. The economic bottleneck is the cost of the fermentation and the recovery/purification technologies. The recovery/purification technologies issue significantly affects the overall process economics. The recovery of PHB granules from bacterial cytoplasm significantly increases total processing costs [91]. The process characterized by the lowest recovery cost was with the sodium hydroxide and sulphuric acid treatments (1.02 € kg−1 and 1.11 € kg−1, respectively) [92]. However, an efficient, economical, and environmentally friendly extraction of PHB from cells is required for a sustainable process from a cost point of view and from a cost-effective industrial production point of view.

The PHB recovery requires more details regarding its formation/accumulation mechanism in the cell to address the more appropriate recovery technique. PHB is a lipid-like compound synthesized by many microorganisms as storage material. The PHB is accumulated intracellularly under the form of inclusion bodies (PHB granules). PHB granules are wrapped by a considerable number of proteins: the granule structure suggests that they are supramolecular complexes with specific functions rather than being only simple sacks rich in carbon and energy [93] (Figure 6). The formation of PHB granules in the cells is currently under intense discussion. Three models of PHB granule formation were developed based on theoretical considerations and experimental data obtained from Ralstonia eutropha cultures. The first model—the Micelle model—assumes that soluble (cytoplasmic) PHB synthase molecules (phaC) start to produce the hydrophobic PHB molecule only when the concentration of the substrate (3-hydroxybutyryl-CoA) is sufficiently high [94]. The initial polymer chains of PHB form aggregates characterized by hydrophobicity and low solubility in an aqueous environment. The aggregates form the micelle structures in the cytoplasm of the cells. As a consequence of the Micelle model, the PHB granules can be randomly localized in the cell [93,95]. The second model—the Budding model—assumes that the PHB synthase is located in the cytoplasmic membrane and that the newly formed PHB chains are realized into the bilayer of the membrane [96]. The third model—the Scaffold model—assumes that PHB synthase of nascent PHB granules is—or becomes—attached to a yet unknown scaffold molecule within the cell [93]. The granules-scaffold complexes are bound to the bacterial nucleoid and constitute the PHB granule initiation complexes. In this case, subcellular localization of PHB granules would depend on the nature and localization of the scaffold of the PHB-accumulating cell [93].

Whatever the PHB formation/accumulation mechanism, the recovery of the PHB granules requires the rupture of the bacterial cell and the removal of the protein layer wrapping the PHB granules. A possible alternative is the selective dissolution of the PHB granules by means of suitable solvents and successive precipitation of the dissolved PHB.

Solvent extraction is the most widely adopted method to recuperate PHB from the cellular biomass. This method is simple and rapid and allows the assessment of the PHB fraction accumulated in the cells [99]. The two main steps of the PHB extraction are the modification of permeability of the cell membrane to enhance the release and solubilization of PHB and the PHB non-solvent precipitation [100]. Extraction of PHB with solvents such as chlorinated hydrocarbons—e.g., chloroform, 1,2-dichloroethane [101]—and cyclic carbonates—e.g., ethylene carbonate and 1,2-propylene carbonate—is very common. Precipitation of PHB is commonly induced by a non-solvent, such as methanol and ethanol [101]. Solvent extraction has undoubtedly advantages over the other extraction methods of PHB in terms of efficiency. Furthermore, this method can remove bacterial endotoxin and cause insignificant degradation of the formed polymers [100]. Therefore, it is possible to obtain very pure PHB with a high molecular weight. Unfortunately, a large-scale application of solvent extraction is considered not environmentally friendly.

The digestion technique to recover the PHB requires the solubilization of the cellular material wrapping the PHB granules. The digestion techniques are well established approaches developed as an alternative to solvent extraction.

Table 7 reports information about the main PHB recovery techniques. Results regarding the PHB purity and the yield are listed as well as reported in the reviewed paper.

A simple, practical, efficient, and cost-effective system of PHB recovery using non-halogenated solvents should be developed. Different strategies for PHB recovery are reported hereinafter:

  • Recovery of PHB accumulated in cyanobacteria using ionic liquids to dissolve cyanobacteria and retain PHB [102]. Ionic liquids (ILs) have emerged as a potential solvent for several applications. ILs are characterized by a melting point below 100 °C. They are characterized by interesting properties—e.g., high ionic conductivity and high thermal stability—which are difficult to achieve in general organic solvents [102]. Furthermore, ILs are characterized by negligible vapor pressure. They are gaining attention as novel green solvents;

  • Spontaneous liberation of intracellular PHB granules using a genetic engineering approach. Jung et al. [103] manipulated the initial inoculum size and the composition of the medium and obtained an Escherichia coli strain that was able to produce PHB at a very high fraction [103]. After spontaneous cell lysis, PHB granules were released into the medium;

  • Extraction of PHB using non-chlorinated solvents (e.g., anisole) [104] or non-halogenated solvents (e.g., butyl acetate) [105];

  • Extraction of PHB with a solvent-free approach using enzyme digestion in an aqueous medium [106]. Martino et al. [106] used biomass of Cupriavidus necator DSM 428 grown on used cooking oil (UCO) for extraction of the PHB granules using sodium dodecyl sulphate (SDS), ethylenediaminetetraacetic acid (EDTA), and the enzyme alcalase in an aqueous medium. The recovered PHB granules showed >90% purity and no crystallization.

8. Conclusions Poly-β-hydroxybutyrate (PHB) is one of the best biodegradable polymers among those proposed as alternatives to conventional plastics. The photoautotrophic system of cyanobacteria is considered the system of choice for a low-cost production of PHB. One of the challenges is to render the PHB production by cyanobacteria, a competitive process, using various strategies described in this paper: (i) medium optimization; (ii) genetic engineering approach; (iii) tuning of mathematical model for cyanobacterial growth and coupled PHB production; (iv) PHB production using continuous operations. The synergic effect of the improvements achieved with these approaches will give the best chances to maximize PHB production by cyanobacteria. So far, the main drawback of the commercial production and application of PHB in the industry is its high production cost compared with conventional plastics. The recovery of PHB granules from cytoplasm significantly increases total processing costs. The other big challenge of the PHB production process from cyanobacteria is to find a low-cost and environmentally friendly strategy to recover it.

Figure 1. General structure of poly-β-hydroxybutyrate.

Figure 2. PHB production plants: (A) Bio-On in Italy that produces Minerva; (B) PHB industrial in Brazil that produces Biocycle; (C) Meredian in the USA; (D) Metabolix in the USA.

Figure 3. Schematic representation of the intersecting photosynthetic and respiratory electron transport pathways of the cyanobacterium Synechocystis sp. PCC 6803.

Figure 4. Schematic representation of a cyanobacterial cell factory. (A) A non-modified cyanobacterial cell is investing carbon (in form of CO2) and energy sources into growth and the generation of biomass. (B) A modified cyanobacterial (here: through the introduction of an alternative biosynthetic pathway) cell factory is investing the majority of the incoming carbon and energy sources into a (excreted) product (here: carbon containing products), allowing only the replacement of dying cells and the maintenance of the cells to be fueled by the native metabolism.

Figure 5. PHB biosynthetic pathway.

Figure 6. PHB granules inside the cyanobacteria cells: (A) Synechococcus sp. PCC7942 [27]; (B) Synechococcus sp. PCC7942 [97]; (C) Synechocystis PCC6803 [23]; (D) Cyanothece sp. strain PCC 7822 [98]; (E) Synechococcus sp. MA19 [72].

Types of Biodegradable Plastics Examples
Bioplastics that are biobased (biomass products).This group includes polysaccharides, such as starches or animal protein (e.g., casein, whey and gelatin).
Bioplastics that are biobased (from microorganisms).This group includes biodegradable biopolymer extracted from microorganisms (e.g., polyhydroxyalkanoates (PHA), polyhydroxybutyrate (PHB), poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV)).
Bioplastics that are biobased (from biotechnology).This group includes biodegradable biopolymer produced by a conventional synthesis of bio derived monomers (e.g., polylactic acid (PLA)).
Bioplastics that are fossil-based. This group includes bioplastics from petrolchemical products obtained from conventional synthesis of synthetic monomers (e.g., polycaprolactone (PCL), poly(butylene adipate-co-terephthalate) (PBAT)).

1 after Singh et al. [4].

Name of the Company City/Country Production (kt Year−1) Raw Material Brand Name
Biomer Schwalbach am Taunus/Germany-PHBBiomer
Biomatera Toronto/Canada-PHABiomatera
Bio-OnBologna/Italy10PHAMinerv
KanekaOsaka/Japan 10PHB
Tianjin Green-BioTaijin/China10PHAGreen Bio
Imperial Chemical Industries (ICI) London/UK0.3PHB-
Danimer ScientificGeorgia/USA0.3PHA-
PHB IndustrialSerrana/Brazil0.1PHBBiocycle
TEPHAMassachusetts/USA-PHATephaflex/TephElas
TinanZhejiang/China10PHBEnmat
SIRIMSelangor/Malaysia2PHA-
Shenzhen EcomannShandong/China5PHAAmBio

Bacterial Strain Carbon Source Polymer (s) PHB % References
Aeromonas hydrophila 4AK4 mutantLauric acid, oleic acidMCL-PHA 164[15]
Alcaligenes latusMalt, soy waste, milk wastePHB 270[16]
Azotobacter chroococcum H23Waste water from olive oil millsPHB 280[17]
Bacillus cereus UW85GlucosePHB 29[18]
Bacillus megaterium ATCC 6748Corn steep liquor and molassesPHB 243[19]
Bacillus spp. 87IGlucosePHB 267[20]
Burkholderia sacchari sp. IPT101Glucose PHB 2 PHBV 368[21]
Burkholderia cepacia IPT 048bagassePHB 262[22]
Caulobacter crescentus DSM 4727 GlucosePHB 218[23]
Cupriavidus necator DSM 545Corn syrupPHB 230[24]
Cupriavidus necator DSM 545waste glycerolPHB 262[25]
Enterobacter aerogenes 12BiwastewaterPHB 243[26]
Escherichia coli mutantsXylose PHB 227[27]
Halomonas boliviensis LC1Starch, hydolysate, maltose, maltotetraose, and maltohexaosePHB 256[28]
Legionella pneumophila 74/81Nutrient brothPHB 216[29]
Methylocystis sp. GB 25 DSM 7674MethanePHB 251[30]
Microlunatus phosphovorus DSM 10555Glucose, acetate PHB 230[31]
Mixed Microbial CultureLactatePHB 290[11]
Pandoraea sp.Crude glycerolPHB 263[32]
Pseudomonas aeruginosa NCIB 40045Agro-industrial oily wastesMCL-PHA 166[33]
Pseudomonas hydrogenovora DSM 1749Dairy wheyMCL-PHA 121[34]
Pseudomonas putida CA-3Petrochemical plastic wastePHB 230[35]
Pseudomonas fluorescens A2a5Sugar cane liquorPHB 270[36]
Rhodopseudomonas palustris SP5212AcetatePHB 234[37]
Ralstonia pickettii 61A6Sugar cane liquorPHB 210[38]

1 MCL-PHA: medium-chain-length-polyhydroxyalkanoates 2 PHB: polyhydroxybutyrate 3 PHBV: poly(3-hydroxybutyrate-co-3-hydroxyvalerate).

Cyanobacteria Strain Culture Conditions Polymer Polymer Content (%) References
Arthrospira (Spirulina) platensisPhotoautotrophic PHB6[63]
Anabaena cylindrica 10C PropionateP(3HB-co-3HV)2[64]
Aulosira fertilissima CCC 444Citrate + acetate and K2HPO4PHB85[65]
Aulosira fertilissima CCC 444Fructose + valerateP(3HB-co-3HV)77[26]
Gloeothece sp. PCC 6909AcetatePHB9[66]
Nostoc muscorum AgardhNitrogen deficiency + acetate + glucose + valerateP(3HB-co-3HV)71[25]
Nostoc muscorum CCAP 1453/9CO2PHB22[31]
Scytonema geitleri BharawajaAcetate 30 mMPHB7[67]
Synechococcus sp. PCC7942 Acetate + nitrogen deficiencyPHB26[27]
Synechocystis sp. PCC 6803CO2; Nitrate concentration is the half of the optimal concentrationPHB8[30]
Synechocystis sp. PCC 6803CO2; Nitrate deficiencyPHB4[23]
Synechocystis sp. PCC 6803 Phosphate-deficiency + gas exchange limitation + acetatePHB38[35]
Spirulina sp. LEB 18sodium bicarbonate + nitrogen and phosphorus deficiencyPHB31[28]
Spirulina platensis UMACC 161Acetate and CO2PHB10[68]

Cyanobacterial Strain Engineered Genes Carbon Source Culture Conditions Reactor Polymer Fraction (% dcw) References
Synechocystis sp. PCC 6803 mutant strainThe agp gene was inserted CO2 0.035%, Acetate 5 mMNitrogen deprivedflask18.6[43]
Synechococcus sp. PCC7942 mutant strainA PHB synthesizing enzyme from A. eutrophus was expressed from a plasmidCO2 5%, Acetate 10 mMNitrogen deprivedflask25[27]
Synechocystis sp. PCC 6803 mutant strainPHA synthase from C. necator was expressed from a plasmidCO2 1%, Acetate 10 mMNitrogen deprivedflask11[42]
Synechocystis sp. PCC 6803 mutant strainThe native sigE was expressed from the chromosomeCO2 1%Nitrogen deprivedflask1.4[45]
Synechocystis sp. PCC 6803 mutant strainA PHA biosynthetic operon from M. aeruginosa was expressed from a plasmidCO2 2%Nitrogen deprivedflask7[44]
Synechocystis sp. PCC 6803 mutant strainThe native phaAB was overexpressed from the chromosomeCO2 0.035%, Acetate 4 mMNitrogen deprivedflask35[41]
Synechocystis sp. PCC 6803 mutant strainxfpk from B. breve was expressed in a double pta and ach knock out.CO2 2%BG11PBR12[46]

Microorganism Growth Rate Production Rate dXdt dCdt dNdt dPdt dPHBdt References
Bacillus megateriumµ1=µmax·(CC+KSC)·(NN+KSN)µ2=µmax·(NN+KSN)µ3=µmax·(1−HHm)·(NN+KSN)µ4=µmax·(1+k1HH+k2H·H)·(NN+KSN)-(µ−kd)·X−(ɑ·µ+·)·X−µi·XYXN-K1·µi·Φ+k2·X [56]
Cupriavidus necatorµXS=µXSmax(H2KXH2+H2)·(O2KXO2+O2)·(CO2KXCO2+CO2)·(NKN+N+N2KIN)µpS=µPSmax(H2KpH2+H2)·(O2KpO2+O2+O22KPIO2)·(CO2KpCO2+CO2)·[1−(fPHBfPHBmax))β](KPINN+KPIN)µXS·XkL aCO2(CO2*−CO2)−(µXSYXCO2+µpSYpCO2)·XFN NFV−µXSYXN·XwhereFN=(1NF)(µXSYXN)XV-µpS·X [55]
Ralstonia eutrophaµ=[Cn1Cn1+KCn1·Nn2Nn2+KNn2]·[1−(C)ɑ1(Cm1)ɑ1]·[1−(N)ɑ2(Nm2)ɑ2]-µ·FV·X−(ɑ·µ+·)·X+F1V·C1−FV·C1−(µYXN+ms)·X+F2V·S2−FV·N−µ·XYXN−ms·X+F2V·N−FV·N-k1·dXdt+k2·X−FV·PHB [57]
Synechocystis PCC6803µ=µm·(CikxCi +Ci)·IavkSI+Iav·(1−qnQn)·(1−qpQp)rPHB=kPHB·CO2·XPHBµX−rT−rL2-YNNO3−·ρNO3−·X−YNX·µ·XwhereρNO3−=ρmNO3−·NO3−kρNO3−+NO3−YPPO43−·ρPO4−3·X−YPX·µ·XwhereρPO4−3=ρmPO4−3·PO4−3KρPO4−3+PO4−3rPHB [30]

Extraction Method Materials Strain Results References
Solvent extractionChloroformBacillus cereus SPVPurity: 92%; Yield: 31% [107]
ChloroformCupriavidus necator DSM 545Purity: 95%; Yield: 96%[108]
1,2-Propylene carbonate Cupriavidus necator DSM 545Purity: 84%; Yield: 95%[108]
Methyl tert-butyl etherPseudomonas putida KT2440Yield: 80–85%[109]
Butyl acetateC. necatorPurity: 99; Yield: 96[91]
Non halogenated acetone/ethanol/propylene carbonateC. necatorPurity: 93%; Yield: 92%[110]
AcetoneP. putida GPo1Yield: 94%[111]
Digestion Method
SurfactantSDSRecombinant Escherichia coliPurity: 99%; Yield: 89%[112]
Palmitoyl carnitineC. necator, Alcaligenes latusDegree of lysis: 56–78%[113]
Surfactant-sodium hypochloriteSDS-Sodium hypochloriteAzotobacter chroococcum G-3Purity: 98%; Yield: 87%[114]
Triton X-100-sodium hypochloriteC. necator DSM 545Purity: 98% [115]
Sodium hypochloriteSodium hypochloriteC. necator Recombinant Escherichia coliPurity: 86%; Purity: 93%[116]
Sodium hypochloriteStaphylococcus epidermidis [117]
Surfactant ChelateTriton X-100-EDTASinorhizobium melilotiPurity: 68%[118]
Betaine-EDTA disodium saltC. necator DSM 545Purity: >96%; Yield: 90%[119]
Dispersion of sodium hypochlorite and chloroformChloroform-sodium hypochloriteB. cereus SPV Purity: 95%; Yield: 30%[107]
Chloroform-sodium hypochloriteC. necator Recombinant Escherichia coliPurity: >98%[116]
Selective dissolution by protonsSulfuric acidC. necatorPurity: >97%; Yield: >95%[120]
Enzymatic digestionEnzyme combined with SDS-EDTAP. putidaPurity: 93%[121]
Alcalase combined with SDS and (EDTA),Cupriavidus necator DSM 428Purity: >90%; Yield: >90%[71]
Mechanical disruptionBead millA. latus [122]
High pressure homogenizationA. latus [122]
SDS-High pressure homogenizationMetylobacterium sp V49Purity: 95%; Yield: 98%[123]
Spontaneous liberation E. coliAutolysis of 80%[68]

Author Contributions

R.C.: conceptualization, writing-original draft, writing-review & editing. G.O.: conceptualization, writing-original draft, writing-review & editing. K.J.H.: writing-review & editing. A.P.: writing-review & editing. A.M.: writing-review & editing.

Funding

Part of the work of R.C. was supported by a grant from the Stichting tot Bevordering van het Wetenschappelijk Onderzoek in de Biochemie, Amsterdam, The Netherlands.

Conflicts of Interest

The authors report no conflict of interest.

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Roberta Carpine1,2,*, Giuseppe Olivieri1,3,*, Klaas J. Hellingwerf4, Antonino Pollio5 and Antonio Marzocchella11Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione Industriale, Università degli Studi di Napoli “Federico II”, P.le V. Tecchio 80, 80125 Napoli, Italy

2Department of Organic Chemistry, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland

3Bioprocess Engineering Group, Wageningen University & Research, Droevendaalsesteeg 1, 6708PB Wageningen, The Netherlands

4Molecular Microbial Physiology Group, Swammerdam Institute for Life Sciences—Science Park, 904 1098 XH Amsterdam, The Netherlands

5Biology Department, Università degli Studi di Napoli “Federico II”, Via Foria 223, 80139, Napoli, Italy

*Authors to whom correspondence should be addressed.

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© 2020. This work is licensed under http://creativecommons.org/licenses/by/3.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.

Abstract

The increasing impact of plastic materials on the environment is a growing global concern. In regards to this circumstance, it is a major challenge to find new sources for the production of bioplastics. Poly-β-hydroxybutyrate (PHB) is characterized by interesting features that draw attention for research and commercial ventures. Indeed, PHB is eco-friendly, biodegradable, and biocompatible. Bacterial fermentation processes are a known route to produce PHB. However, the production of PHB through the chemoheterotrophic bacterial system is very expensive due to the high costs of the carbon source for the growth of the organism. On the contrary, the production of PHB through the photoautotrophic cyanobacterium system is considered an attractive alternative for a low-cost PHB production because of the inexpensive feedstock (CO2 and light). This paper regards the evaluation of four independent strategies to improve the PHB production by cyanobacteria: (i) the design of the medium; (ii) the genetic engineering to improve the PHB accumulation; (iii) the development of robust models as a tool to identify the bottleneck(s) of the PHB production to maximize the production; and (iv) the continuous operation mode in a photobioreactor for PHB production. The synergic effect of these strategies could address the design of the optimal PHB production process by cyanobacteria. A further limitation for the commercial production of PHB via the biotechnological route are the high costs related to the recovery of PHB granules. Therefore, a further challenge is to select a low-cost and environmentally friendly process to recover PHB from cyanobacteria.

Details

Title
Industrial Production of Poly-β-hydroxybutyrate from CO2: Can Cyanobacteria Meet this Challenge?
Author
Carpine, Roberta; Olivieri, Giuseppe; Hellingwerf, Klaas J  VIAFID ORCID Logo  ; Pollio, Antonino  VIAFID ORCID Logo  ; Marzocchella, Antonio
First page
323
Publication year
2020
Publication date
2020
Publisher
MDPI AG
e-ISSN
22279717
Source type
Scholarly Journal
Language of publication
English
DOI
https://doi.org/10.3390/pr8030323
ProQuest document ID
2377004308
Copyright
© 2020. This work is licensed under http://creativecommons.org/licenses/by/3.0/ (the “License”). Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.