Microbial and Natural Macromolecules: Synthesis and Applications

Microbial and Natural Macromolecules: Synthesis and Applications brings together active scientists and academicians in the field who share updated information and research outcomes from global experts. Microbial macromolecular diversity, molecular composure, genetics, usability of advanced molecular tools and techniques for their study as well as their applicability are discussed with detailed research perspectives.

• Illustrates fundamental discoveries and methodological advancements
• Discusses novel functional attributes of macromolecules
• Updates progress on microbial macromolecular research

• Language: English
• Publisher: Academic Press
• Release date: Sep 15, 2020
• ISBN: 9780128200858

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macromolecules

Chapter 1: Production, properties, and processing of microbial polyhydroxyalkanoate (PHA) biopolyesters

Martin Kollera,b    a Office Research Management and Service, c/o Institute of Chemistry, NAWI Graz, University of Graz, Graz, Austria

b ARENA—Association for Resource Efficient and Sustainable Technologies, Graz, Austria

Abstract

Polyhydroxyalkanoates (PHA) are reserve materials produced by various prokaryotic microorganisms. Biologically, PHA primarily provides microbial cells with advantages to endure challenging environmental conditions like nutrient deprivation or environmental stress. Technologically, PHA constitute plastic-like basic materials with characteristics similar to those of currently widely applied plastics produced by petrochemistry, and are expected to sooner or later compete these established petrochemistry-derived plastics. Material properties of these intriguing biopolyesters, such as degradability, composting performance, thermal melting and decomposition behavior, crystallinity, strength, or elasticity are predefined during their biosynthesis in living cells. This high versatility of PHA allows their use in sectors like biomedicine, packaging, electronics, and many more. Current efforts to make PHA production efficient and sustainable in terms of economics and environmental impact are mainly related to the applied microbial production strains, advanced bioreactor types, continuous or discontinuous cultivation processes, sustainable and efficient techniques for product recovery, and, in particular, the choice of raw materials. Only recently, also the scientific disciplines of genetic engineering, metabolomics, bioinformatics, and nanotechnology consolidate their role in the field of PHA research.

Keywords

Bacteria; Bioplastic; Bioreactor; Downstream processing; Feedstocks; Fermentation; Haloarchaea; Polyhydroxyalkanoates; Polymer composites and blends; Product recovery

1: Introduction

Polyhydroxyalkanoates (PHA), as a versatile class of microbial produced biopolyesters, are currently in the scientific focus of material scientists, process engineers, microbiologists, and more and more, of systems- and synthetic biologists. This interest originates from PHA’s versatile material characteristics, making it attractive to be used in numerous areas of the plastics market, which is currently dominated by diverse technomers and plastomers of petrochemical origin (Koller, Maršálek, Miranda de Sousa Dias, & Braunegg, 2017; Kourmentza et al., 2017).

PHA was for the first time detected by light microscopic observation more than 90 years ago as light-refractive inclusions in cells of the bacterium Bacillus megaterium (reviewed by Lenz & Marchessault, 2005). Fig. 1 shows electron-microscopic pictures of PHA-rich cells of the strain Cupriavidus necator DSM 545 cultivated in a cascade of bioreactors. The cells were supplied with glucose as a sole carbon substrate for growth and PHA accumulation.

Fig. 1 C. necator cells with a PHA biopolyester mass fraction of 0.65 g/g. Magnification: 70,000  ×. Picture kindly provided by E. Ingolić, FELMI-ZFE, Graz, Austria.

PHA display all features characterizing green plastics; they are biobased, biosynthesized, biodegradable, compostable, and biocompatible. Hence, they can be considered the only group of real green plastics sensu stricto. Moreover, PHA are water-insoluble, heat resistant (at least the highly crystalline representatives), and have an attractive surface structure. Importantly, PHA pellets can be processed on standard machines used for processing of petrochemistry-derived plastics. PHA melt behaves like liquid crystalline polymers, which allows molding thin-walled or complex structures, which is of significance to produce scaffolds for biomedical use (Rodríguez-Contreras, 2019).

Typically, high PHA synthesis rates are observed for cultures exposed to the limitation of an essential nutrient medium component, while at the same time an exogenous carbon source needs to be present in excess. As their primary biological function, PHA serve the microbial production strains as carbon-rich reserve compounds to better survive periods of starvation, and as pseudo-fermentative electron sinks serving for the regeneration of reducing equivalent in situations when central metabolic pathways (e.g., the tricarboxylic acid cycle, TCC, or nucleic acid biosynthesis) are blocked (Braunegg, Lefebvre, & Genser, 1998).

A range of further functions of PHA was more recently revealed, such as their protective role against stress factors (Obruca, Sedlacek, Koller, Kucera, & Pernicova, 2018) like elevated temperature (Obruca, Sedlacek, Mravec, Samek, & Marova, 2016), UV-irradiation (Slaninova et al., 2018), challenging freezing and thawing cycles (Obruca et al., 2016), or oxidative stress (Obruca, Marova, Stankova, Mravcova, & Svoboda, 2010). Hence, in the context of halophilicity, the adaptation of organisms to high salinity, presence of PHA in living cells was shown to support them to overcome the detrimental effects of osmotic up-shock (Obruca et al., 2017) or sudden osmotic imbalances in general (Sedlacek et al., 2019), which explains their frequent occurrence in haloarchaea typically thriving in extremely saline habitats (Koller, 2019).

2: Different types of PHA

2.1: PHA homo- and heteropolyesters

Depending on the number of distinct building blocks, monomers present in a PHA polyester, it is possible to distinguish PHA homopolyesters from PHA heteropolyesters. Homopolyesters contain only one single monomer type, with poly(3-hydroxybutyrate) (PHB), an isotactic, linear, thermoplastic polyester, being the most prominent and by far best-studied example. Heteropolyesters encompass copolyesters with two different monomers (poly(3-hydroxybutyrate- co-3-hydroxyvalerate) (PHBHV) being the best-studied example), and terpolyesters (three different building blocks) (Koller, 2018a).

2.2: scl-, mcl-, and lcl-PHA and their characteristics

The group of short-chain-length-PHA (scl-PHA) encompasses all PHA monomers with three to five carbon atoms, with the chiral monomers (R)-3-hydroxybutyrate (3HB) and (R)-3-hydroxyvalerate (3HV), and the achiral monomers 4-hydroxybutyrate (4HB) being the most important representatives. In contrast, medium-chain-length and long-chain-length-PHA (mcl-PHA and lcl-PHA, respectively) building blocks have 6 to 14 (mcl-PHA) or more than 14 (lcl-PHA) carbon atoms. Scl-PHA are typically crystalline, rather brittle thermoplastic materials with narrow melting temperature ranges and low flexibility; the homopolyester of 4HB and 4HB-rich copolyesters of 3HB and 4HB are exceptions by having high flexibility and decreased crystallinity. Mcl- and lcl-PHA, in contrast, are latex-like resins, hence, they have the characteristics of elastomers with a low glass transition temperature (Tg), low degree of crystallinity (Xc), broad melting temperature ranges, and lower molecular masses than scl-PHA (Zinn, 2010).

2.2.1: scl-PHA

In the case of scl-PHA, high Tg (typically at least 0°C), sharp melting points of even more than 180°C for highly crystalline PHB, and high molecular mass are characteristic material features of scl-PHA (Troschl, Meixner, & Drosg, 2017). Generally, lower polydispersity (Ði), describing molecular mass distribution in a PHA sample, is found in case of mcl-PHA than for scl-PHA, which means that scl-PHA have a narrower distribution of PHA chain length in an investigated sample (Zinn, 2010).

PHB, the homopolyester of 3HB, is by far the best-studied member scl-PHA and the entire PHA family. PHB was discovered about 90 years ago (Lemoigne, 1923), and, until 1974, it was believed to be the only natural PHA accumulated by microorganisms. Since PHB is highly brittle, has a high melting point (up to 180°C), and high crystallinity (Xc; typically 60%–70%), its application, especially for flexible packaging, is not feasible. In 1974, Wallen and Rohwedder isolated microorganisms from samples of sewage water, and noticed that the characteristics of the PHA accumulated by these species were substantially different than known for the homopolyester PHB; especially the low Tm of <  100°C was unexpected. It was revealed that the PHA contained not only 3HB, but also 3HV monomers. For the production of PHBHV copolyesters instead of PHB homopolyester, most described scl-PHA production strains, e.g., C. necator, need to be supplied with precursor co-substrates structurally related to 3HV. This was first demonstrated by simultaneously supplying A. eutrophus (today: C. necator) with butyric acid (3HB precursor) plus valeric acid as 3HV precursor. Feeding valeric acid as the only carbon source resulted PHA consisting of up to 90% of 3HV (Wallen & Rohwedder, 1974).

4HB is the only thoroughly studied achiral building block in microbial PHA. 4HB-containing PHA was first detected when supplying C. necator butyric acid and the 4HB-precursor compounds 4-hydroxybutyric acid or 4-chlorobutyric acid, while supplying butyric acid as sole carbon source resulted in PHB homopolyester biosynthesis. Especially a drastically decreased crystallinity was observed when increasing the 4HB fraction in poly(3HB-co-4HB) (Kunioka, Kawaguchi, & Doi, 1989). The effect of varied 4HB fractions in poly(3HB-co-4HB) on Tm, Tg, and storage modulus was also studied, revealing that all these parameters decreased when increasing the 4HB share. While yield stress and breaking stress only slightly decrease with increasing 4HB fraction, the elongation at break strongly increases, making these materials highly flexible and elastic. Material features of the homopolyester poly(4HB) are entirely different to those of other scl-PHA like PHB or poly(3HB-co-3HV); poly(4HB) has an enormous elongation at break (up to 1000%), is highly flexible and stretchable, and of interest for surgical applications (Saito, Nakamura, Hiramitsu, & Doi, 1996). In this context, the company Tepha, Inc., USA, produces and commercializes various PHA-based biomedical products like sutures or bioresorbable surgical threads; TephaFLEX sutures consisting of poly(4HB) are approved by the US Food and Drug Administration (FDA) (Brigham & Sinskey, 2012).

2.2.2: mcl-PHA

Mcl-PHA contain monomers with 6–14 carbon atoms and are less crystalline than scl-PHA. Some mcl-PHA even contain monomers with functional (e.g., olefinic) groups; this paves the way for post-synthetic modification as a tool to adapt PHA’s properties. Mcl-PHA samples often look like rubbers or biological latexes; since their Tg values are remarkably low, they are highly amorphous materials with structures which do not easily crystallize and do not become brittle even far below the freezing point. Pseudomonas putida (formerly Pseudomonas oleovorans) is the most frequently used mcl-PHA production strain; it incorporates also functionalized (epoxy-group harboring, halogenated, unsaturated, etc.) building blocks into mcl-PHA when supplied with appropriate functionalized precursor compounds (Gopi, Kontopoulou, Ramsay, & Ramsay, 2018; Zinn, 2010).

Mcl-PHA synthesis occurs via fatty acid β-oxidation of substrates, generating a mixture of different (S)-acyl-CoAs, which are isomerized to the (R)-isomers; these are polymerized towards mcl-PHA copolyesters (De Waard, Van der Wal, Huijberts, & Eggink, 1993). Alternatively, mcl-PHA production also occurs via fatty acid de novo synthesis from structurally unrelated substrates like sugars or glycerol (Huijberts, Eggink, De Waard, Huisman, & Witholt, 1992). Different from β-oxidation, fatty acid de novo synthesis directly provides the (R)-isomers of acyl-CoAs. In this context, it was only recently reported by Sathiyanarayanan et al. that the psychrophilic species Pseudomonas sp. PAMC 28620, isolated from Arctic glacier fore-field soil, accumulates the mcl-PHA poly(3HTD-co-3HDD-co-3HD-co-3HO) with high mcl-PHA contents in biomass of more than 50 wt% when using glycerol as a sole carbon source (Sathiyanarayanan et al., 2017).

2.2.3: lcl-PHA

Reports on lcl-PHA, polyesters containing building blocks with more than 14 carbon atoms are still scarcely described. Barbuzzi et al. reported the first study on a 3-hydroxypentadecanoate (3HPD)-containing PHA by detecting low 3HPD fractions of 2% in PHA produced by Pseudomonas aeruginosa ATCC 27853 grown on pentadecanoic acid as a sole carbon source (Barbuzzi et al., 2004). The up-to-date longest PHA building block found in microbial PHA, 3-hydroxyoctadecanoate (3HOD), was detected by Ray and Kalia, who produced PHA copolyesters containing 3HD, 3HHD, and 3HOD by co-feeding Bacillus thuringiensis EGU45 with glucose, CGP, and propionate (Ray & Kalia, 2017). A more recent report describes lcl-PHA production when cultivating C. necator IPT027 on the inexpensive carbon source crude palm oil rich in unsaturated lipids. This lcl-PHA contained mainly 3HD beside several other saturated and unsaturated monomers with 18 carbon atoms; it was highly amorphous with high thermal stability and low polydispersity (Guo et al., 2013).

Fig. 2 provides the chemical structures of selected PHA homo- and heteropolyesters discussed in this chapter.

Fig. 2 Chemical structures of selected types of PHA. (A–D) Short-chain length PHA ( scl -PHA), (E–H) medium-chain length PHA ( mcl -PHA). (A–H) Polyoxoesters; (I) PHA with thioester groups. (a) Poly(3-hydroxybuytrate) (PHB); (b) poly(3-hydroxybutyrate- co -3-hydroxyvalerate) (PHBHV); (c) poly(3-hydroxybutyrate- co -4-hydroxybutyrate); (d) poly(3-hydroxybutyrate- co -3-hydroxyvalerate- co -4-hydroxybutyrate) (PHBHV4HB); (e) poly(3-hydroxyoctanoate- co -3-hydroxydecanoate- co -3-hydroxydodecanoate); (f) poly(3-hydroxydodecanoate); Fig. 2, Cont’d (g) poly(3-hydroxynon-8-enoate- co -3-hydroxydecanoate- co -3-hydroxyundec-10-enoate); (h) poly(3-hydroxyhexanoate- co -3-hydroxydec-9-enoate- co -3-hydroxy-5-phenylvalerate); (i) poly(3-hydroxybuytrate- co -3-mercaptobutyrate). * indicates chiral centers in the monomers; x : degree of polymerization; (m) molar fraction of 3-hydroxybutyrate (3HB); (n) molar fraction of 3-hydroxyvalerate (3HV); (o) molar fraction of 4-hydroxybutyrate (4HB); (p) molar fraction of 3-hydroxydecanoate; (q) molar fraction of 3-hydroxydodecanoate; (r) molar fraction of 3-hydroxyoctanoate; (s) molar fraction of 3-hydroxynon-8-enoate; (t) molar fraction of 3-hydroxyundec-10-enoate; (u) molar fraction of 3-hydroxyhexanoate; (v) molar fraction of 3-hydroxydec-9-enoate; (w) molar fraction of 3-hydroxy-5-phenylvalerate; (y) molar fraction of 3-mercaptobutyrate. Reproduced with permission from Koller, M., Maršálek, L., Miranda de Sousa Dias, M., Braunegg, G. (2017). Producing microbial polyhydroxyalkanoate (PHA) biopolyesters in a sustainable manner, New Biotechnology 37 (A), 24-38. by Elsevier.

2.3: Microstructure of PHA heteropolyester

Besides the type of monomers present in PHA, one can also differentiate diverse types of PHA regarding their microstructure. In this context, intracellular polymer blends display mixtures of different homo- or heteropolyesters present in microbial cells; such blends can often be fractionated by enhanced solation techniques, and typically show multiple melting endotherms in DSC-thermograms. The blends can consist of fractions of heteropolyesters with different fractions of given monomers, and/or of fractions of PHA with strongly different molecular mass (reviewed by Koller, 2018a).

In addition, heteropolyesters can either display random distribution of the building blocks, or consist of different homo- or heteropolyester blocks (blocky structured PHA—b-PHA) (Fig. 3). Chains of b-PHA comprise a minimum of two distinctive polymer sections (blocks), which are covalently linked. Properties of each block contribute to the properties of the entire polyester, which paves the way towards novel polymer properties, which are not reachable by simply blending polymers with the composition of the individual bocks. Diblocks like [3HB]x-[3HV]y, triblocks like [3HB]x-[3HV]y-[4HB]z, or repeated multiblocks like [3HB-3HV]n can be differentiated (Impallomeni et al., 2018). Recently, b-PHA production increasingly fascinates polymer scientists, firstly, because of the outstanding controllability of polymer properties during their biosynthesis, and, secondly, since the processes and properties are better reproducible if compared to PHA of random distribution. In addition, b-PHA are not so prone to polymer aging by progressing crystallization, as shown for the diblock copolymer poly(3HB-b-3HHx). Moreover, novel di-block copolymers of PHB, PHBV, or PHO and atactic PHB (chemically produced) were developed and appeared promising for use as blend compatibilizers for cardiovascular engineering (Pederson, McChalicher, & Srienc, 2006).

Fig. 3 Schematic representation of different PHA microstructures. Black spheres: 3HB; gray spheres: 3HV; textured spheres: 4HB; white, thick black edged spheres: 3HHx; light gray spheres: 3HO; white, black edged spheres with insert DIC: linker diisocyanate. (A) PHB homopolyester; (B) copolyesters with random distribution ( left : poly(3HB- co -3HV), right : Nodax-type poly(3HB- co -3HHx)); (C) poly(3HB- co -3HV- co -4HB) terpolyester with random distribution; (D) poly(3HB- co -3HV- co -4HB- co -3HHx) quarterpolyester with random distribution; (E) diblock b -PHA poly(PHB- b -PHV); (F) triblock b -PHA poly(PHB- b -PHV- b -P4HB); (G) polyesterurethane (PEU) with hard PHB and soft PHO blocks linked by diisocyanate; (H) PHA blend of 3HV-rich and 3HV-poor poly(3HB- co -3HV) randomly distributed copolyesters. Reproduced in adapted form with permission from Koller, M. (2018a). Chemical and biochemical engineering approaches in manufacturing Polyhydroxyalkanoate (PHA) biopolyesters of tailored structure with focus on the diversity of building blocks. Chemical and Biochemical Engineering Quarterly 32(4), 413–438.

Practically, b-PHA are produced by living cells when supplying them subsequently with one type of carbon substrate after depletion of another, both acting as precursor compounds for the monomers building the individual PHA blocks (Impallomeni et al., 2018). Especially dual nutrient-limited conditions mentioned before, where both carbon and nitrogen sources are supplied at low concentrations, which enables their immediate uptake by the cells, are convenient to generate b-PHA (Zinn, Witholt, & Egli, 2004). Such processes can be efficiently performed in continuous bioreactors; in case it is desired to generate a b-PHA composed of a series of different blocks, a continuously operated multistage cascade of bioreactor should be the most appropriate engineering setup to generate such tailor-made PHA by strict sequence regulation (idea described in Atlić et al. (2011)).

3: Challenges in PHA biopolyesters production

3.1: Microbial PHA production strains

3.1.1: Gram-negative Eubacteria as PHA producers

The predominant groups of well-described PHA production strains are Gram-negative eubacteria isolated from soil (e.g., C. necator, Burkholderia sacchari, Azohydromonas australica, Azotobacter vindelandii, Pseudomonas putida, etc.) or from aquatic environments (e.g., Halomonas sp., Bacillus megaterium uyuni S29, etc.). Such organism generally displays high substrate-to-product yields due to the lack of substrate-demanding sporulation, and, in many cases, high intracellular PHA loads on a PHA-in total biomass-basis, and high specific PHA productivity. However, PHA production by Gram-negative strains is accompanied by the formation of endotoxins, a group of lipopolysaccharides (LPS) produced in the bacterial cell wall. Such LPS need to be removed during PHA recovery and purification because of the inflammatory reactions caused by them when PHA are used in vivo (Zinn, Witholt, & Egli, 2001).

Typically, PHA biosynthesis by Gram-positive eubacteria is based on chemoheterotrophic cultivations, where organic substrates serve as both energy- and carbon source. The cultivation of Gram-negative bacteria on simple organic substrates like sugars or lipids in bioreactors, which do not require any illumination, is still the most frequently described process for PHA production. Comparing the use of sugars to lipids as chemo-organic substrates for PHA production, there are typically fundamental differences regarding the substrate-to-product conversion yields. In Gram-negative bacteria, substrates like glucose or glycerol are mainly catabolized aerobically via the KDPG pathway and, to a lower extent, the glycolysis pathway (also known in eukaryotic organisms); this, however, routes a considerable part of the carbon source towards reparative CO2 formation, resulting in a theoretical maximum conversion yield of only 0.48 g/g. When lipids like long-chain fatty acids or their derivatives are converted, degradation occurs via the β-oxidation pathway, which generates high quantities of acetyl-CoA to be converted into biomass or, under growth-limiting conditions, to PHA. For such processes, lipid-to-PHA exceeded yield of 0.6 g/g is typically reported (Koller & Braunegg, 2015b). As a typical example, a conversion yield of only 0.29 g/g was reported for PHA production by C. necator when using crude glycerol phase as substrate (Koller & Braunegg, 2015b); a similar yield (0.33 g/g) was obtained when using glucose as a carbon source (Haas, El-Najjar, Virgolini, Smerilli, & Neureiter, 2017). In contrast, 0.60 g/g was achieved with the same strain when using saturated fatty acid methyl esters (SFAME; low-quality biodiesel) as substrate. This high conversion yield was also substantiated when using SFAE for mcl-PHA production by other organisms; for Pseudomonas citronellolis, 0.59 g/g were obtained (Muhr et al., 2013b), while even 0.62 g/g when using Pseudomonas chlororaphis (Muhr et al., 2013a). Using sugar-rich apple-pulp waste for mcl-PHA production by P. citronellolis, the yield dropped to only 0.12 g/g (Rebocho et al., 2019).

However, there are alternatives to chemoorganotrophic PHA production, which involves the use of CO2, CO, or CH4 as a carbon source by Gram-negative organisms like cyanobacteria, knallgasbacteria, or purple bacteria. These aspects are described in detail in Section 3.2.3.

3.1.2: Gram-positive Eubacteria as PHA producers

Studies reporting PHA biosynthesis by Gram-positive microorganisms are rather scarcely found in the literature in comparison to the vast variety of reports about Gram-negative PHA producers. However, it is often forgotten that the first microorganism ever described as a PHA producer, in fact, was a Gram-positive strain, namely Bacillus megaterium, which was studied by Lemoigne already in the 1920s (Lemoigne, 1923). In the subsequent decades, PHA production by Bacilli and other Gram-positives was rather considered a shenanigan by the global scientific community. Only about 10 years ago, this situation suddenly changed by detecting some PHA producing representatives of the genus Bacillus, which are characterized by possessing Class IV PHA synthases (Tsuge, Hyakutake, & Mizuno, 2015). Examples are Bacillus sp. JMa5, an organism with outstanding PHA production capacity from molasses, a side stream of sugar industry (Wu et al., 2001), Bacillus cereus CFR06, a bacterium, which accumulates intracellular PHA pools of more than half of its biomass (Halami, 2008), Bacillus cereus YB-4 (Mizuno, Ohta, Hyakutake, Ichinomiya, & Tsuge, 2010), or Bacillus sp. IPCB-403, a strain with PHA accumulation exceeding even 0.7 g per g biomass (Dave, Ramakrishna, & Desai, 1996). Finally, PHA production on a larger scale was carried out using the new Gram-positive isolate Bacillus cereus SPV (Valappil et al., 2007; Valappil, Rai, Bucke, & Roy, 2008). Importantly, in contrast to Gram-negative strains, Gram-positive bacteria do not synthesize unwanted lipopolysaccharides (LPS), a group of inflammatory active endotoxins, in their cell wall. When PHA produced by Gram-negative biomass is recovered, LPS get co-isolated together with PHA, which drastically hampers using PHA from Gram-negative bacteria in vivo, hence, in the medical field, which encompasses the production of surgical sutures, implants, and others (Koller, 2018b; Luef, Stelzer, & Wiesbrock, 2015; Zinn et al., 2001).

In this context, especially the halotolerant B. megaterium uyuni S29, an organism isolated from a Bolivian salt lake, constitutes a robust and definitely promising and PHA production strain; this organism was shown to accumulate LPS-free PHA biopolyesters displaying a quality according to the requirements allowing their biomedical application. For experiments in laboratory bioreactors with this strain, Rodríguez-Contreras et al. reported a PHB concentration of 8.5 g/L, a PHB fraction in biomass of 0.30 g/g, and volumetric productivity for PHB of 0.25 g/(L h), which, according to the current state of knowledge, constitute the highest values for PHA production by a Gram-positive microorganism (Rodríguez‐Contreras et al., 2013). Later, inexpensive sugar beet molasses with a sucrose content of about 150 g/L, an abundant waste stream, was supplied as a carbon source for PHA production by B. megaterium uyuni S29. Under phosphate-limited cultivation conditions, a biomass concentration, PHA fraction in biomass and volumetric PHA productivity of 16.7 g/L, 0.60 g/g, 0.42 g/(L h), respectively, were obtained after 24 h of batch cultivation. Notably, this strain immediately hydrolyses sucrose to glucose and fructose by excreting extracellular invertase (Schmid et al., 2019).

3.1.3: Haloarchaea as PHA producers

Haloarchaea, the extremely halophilic branch of the Archaea domain, encompass species which accumulate polyhydroxyalkanoate biopolyesters in their cytoplasm. Such ancient organisms, which thrive in highly challenging, often hostile habitats characterized by salinities between 100 and 300 g/L NaCl, have the potential to outperform established PHA production strains. This optimism presents multifarious reasons, including cultivation setups at extreme salinities can be performed at minimized sterility precautions by excluding the growth of microbial contaminants; the high inner-osmotic pressure in haloarchaea cells facilitates the recovery of intracellular biopolyester granules by cell disintegration in hypo-osmotic media; many haloarchaea utilize carbon-rich waste streams as main substrates for growth and PHA biosynthesis, which allows coupling polyhydroxyalkanoate production with bio-economic waste management. Finally, in many cases, haloarchaea are reported to produce copolyesters from structurally unrelated inexpensive substrates, and PHA biosynthesis often occurs in parallel to the production of additional marketable bio-products such as pigments or technologically useful polysaccharides. In any case, the haloarchaeon Haloferax mediterranei, an organism originally isolated from highly saline water samples taken at the coast near Alicante in Spain, is the prototype strain for haloarchaea accumulating PHA at reasonable quantities (reviewed by Koller, 2019).

It was only during the last decade when profound information about the enzymatic and genomic particularities of Hfx. mediterranei, with special emphasis dedicated to the mechanisms involved in PHA biosynthesis, was elaborated (Xiang, 2016). This covers studies on the special Hfx. mediterranei PHA synthase enzymes (Lu, Han, Zhou, Zhou, & Xiang, 2008), haloarchaeal phasins as enzymes essential for PHA granule formation (Cai et al., 2012), the identification and mapping of the phaB genes encoding PHA biosynthetic enzymes in Hfx. mediterranei (Feng et al., 2010), the multiple pathways generating the 3HV-precursor propionyl-CoA (Han et al., 2013), or patatin, the first haloarchaeal enzyme identified to serve the in vivo mobilization (depolymerization) of native Hfx. mediterranei granules (Liu et al., 2015). A range of diverse inexpensive carbon-rich food and agro-industrial waste and side products have already been tested as feedstocks for PHA production by Hfx. mediterranei. This encompasses surplus whey from cheese and the dairy industry (Koller et al., 2007a, 2007b; Pais, Serafim, Freitas, & Reis, 2016), crude glycerol phase (CGP) as the main by-product of biodiesel production (Hermann-Krauss et al., 2013), extruded corn starch (Chen, Don, & Yen, 2006), extruded rice bran (Huang, Duan, Huang, & Chen, 2006a), stillage from bioethanol manufacturing (Bhattacharyya et al., 2014, 2015), molasses wastewater form sugar industry (Cui, Zhang, Ji, & Wang, 2017), olive mills wastewater (Alsafadi & Al-Mashaqbeh, 2017; Pozo, Martınez-Toledo, Rodelas, & Gonzalez-Lopez, 2002; Ribera, Monteoliva-Sanchez, & Ramos-Cormenzana, 2001), vinasse from molasses-based ethanol production (Bhattacharyya et al., 2012), or macroalgae (seaweeds) hydrolyzed by advanced techniques (Ghosh et al., 2019). Importantly, Hfx. mediterranei produces high-quality PHBHV copolyesters from unrelated, inexpensive carbon sources like glycerol or sugars without the need to supplement precursors structurally related with 3HV. A cost assessment for PHBHV production by Hfx. mediterranei in hydrolyzed whey lactose, based on experiments carried out on a 300-L bioreactor scale, estimated a production price per kg PHBHV of <  3 € (Koller, Sandholzer, Salerno, Braunegg, & Narodoslawsky, 2013). Further, it was shown that saline waste streams of the production process, namely spent fermentation broth and PHA-free cell debris can be reutilized in next fermentation batches to safe material and disposal costs (Koller, 2015).

Besides Hfx. mediterranei, several other haloarchaea, mainly from the genera Haloarcula, Halobiforma, Haloterrigena, Halopiger, Halococcus, Halogranum, Halorhabdus, Haloquadratum, or Natrinema, were positively tested for PHA bioproduction; however, PHA production by these strains was tested only on a shaking flask scale (exception: the extremely halophilic and at the same time thermophilic strain Haloterringena hispanica, which was cultivated under controlled conditions in bioreactors), and productivities and PHA contents in cell mass were typically low (reviewed by Obruca et al., 2014). Only during the last years, the strain Halogemometricum borinquense turned out as a potential candidate for large-scale PHA production in the future; based on inexpensive waste streams like cane sugar bagasse (Salgaonkar & Bragança, 2017) or cassava bagasse (Salgaonkar, Mani, & Bragança, 2019), this organism accumulates PHA at a quantity amounting to more than half of its cell dry mass. Similar to Hfx. mediterranei, also Hgm. borinquense produces PHBHV copolyesters from unrelated carbon sources (Salgaonkar & Bragança, 2015). Table 1 provides a section of diverse genera encompassing PHA-accumulating species.

Table 1

Superscript c indicates cyanobacteria.

3.1.4: Mixed cultures for PHA production

As an alternative to the use of monoseptic, pure microbial cultures for biotechnology, considerable efforts are currently dedicated to the use of mixed microbial cultures (often also referred to as mixed microbial consortia (MMC)). Generally, MMC cultivations originate from the natural approach of selection and competition, also termed evolutionary engineering, which is a fundamentally different approach to genetic or metabolic engineering strategies to tailor microbial whole cell biocatalysts for a given target, e.g., enhanced substrate uptake, improved productivity, etc. Such evolutionary engineering is carried out by exerting selective pressure on a microbial consortium/culture by adapting feeding- and cultivation conditions in the cultivation environment (bioreactor); this means that it is the ecosystem which gets engineered, not the microorganisms. Since PHA are storage compounds biosynthesized under dynamic environmental conditions, cyclic feast-and-famine cultivation regimes can be expediently applied to exert effective selective pressure on a PHA-producing microbial consortium. These cyclic feast-and-famine regimes involve repetitively alternating availability (feast phase) and lack (famine phase) of substrate. The majority of described relevant studies run such systems as repeated batch cultivations (Reis et al., 2003; Serafim, Lemos, Oliveira, & Reis, 2004).

3.2: Feedstocks for PHA production

3.2.1: How to select appropriate carbon sources for PHA production

High production costs of PHA in comparison to their petrochemical counterparts still hamper PHA’s broad market penetration. In the first instance, the price of the carbon source defines the final production price; commonly used carbon sources like pure sugars, edible oils, or fatty acids typically account for about 50% of the entire PHA production costs (Choi & Lee, 1999). Hence, the use of inexpensive raw materials as carbon sources is necessary, especially for industrial-scale PHA production (Kaur & Roy, 2015). Besides the cost aspects, one has to consider the holistic aspects of ethics and sustainability when choosing adequate raw material for biopolymer production. In addition, such waste materials need to be available at sufficient quantity all over the year; off-season availability of storable substrates of constant composition, and resistivity towards microbial degradation needs to be warranted, which is the case for well-storable lignocellulosic materials like bagasse. Alternatively, two-step processes first ferment perishable raw materials via anaerobic bioprocesses, generating conveniently storable fermentation products like lactic acid and various volatile fatty acids, which are used as substrates for aerobic PHA production of PHA in a subsequent bioprocess with PHA-accumulating microbes. Briefly, a sustainable process for biopolymer production needs to encompass aspects of economics, environmental protection, optimized engineering, and ethics (Brigham & Riedel, 2018; Koller et al., 2017).

3.2.2: Examples of viable biogenic organic carbon sources for PHA production

Waste streams from rendering and animal processing industry, namely low-quality (highly saturated) biodiesel fractions, crude glycerol originating from transesterification of animal-based surplus lipids (SFAME), and offal materials were successfully used as substrates for PHA biosynthesis by different production strains (Titz et al., 2012). The low-quality biodiesel fractions are excellent substrates for scl-PHA production by C. necator (Hejazi, Vasheghani‐Farahani, & Yamini, 2003), and for mcl-PHA production using strains from the Pseudomonas genus, such as P. citronellolis (Muhr et al., 2013b) or P. chlororaphis (Muhr et al., 2013a). Techno-economic assessment of PHA production from slaughtering waste, encompassing SFAME, crude glycerol, offal materials, and meat-and-bone meal as substrates, calculated PHA production cost varying from 1.41 to 1.64 €/kg, depending on fluctuating market prices for offal materials, biodiesel, and meat and bone meal (Shahzad et al., 2017). Crude glycerol was successfully used as sole carbon source for PHA biosynthesis by various strains such as Hfx. mediterranei (Hermann-Krauss et al., 2013), Burkholderia glumae MA13 (de Paula et al., 2019), or C. necator (Gahlawat & Kumar Soni, 2019; Koller & Braunegg, 2015b).

Among lipophilic surplus materials, waste cooking and frying oil from gastronomy is an abundantly available carbon source for scl-PHA production by mesophilic (Kourmentza et al., 2018; Obruca, Snajdar, Svoboda, & Marova, 2013) and, as described only recently, also by halophilic (Kumar & Kim, 2019) microbes. For the use of C. necator it was shown by Benesova and colleagues that the addition of chicken feather hydrolysate considerably enhances both biomass and PHA yields when using waste frying oil as a main carbon source (Benesova, Kucera, Marova, & Obruca, 2017). In addition, mcl-PHA production from waste cooking oil was reported for using the strain Pseudomonas putida KTT2440 (Ruiz, Kenny, Narancic, Babu, & O’Connor, 2019).

Another gastronomy waste stream accruing in huge quantities in the various global region is spent coffee ground; after hexane-extraction, the oil fraction of this waste can be employed to produce scl-PHA by the well-established production strain C. necator with PHA contents in biomass up to 0.9 g/g (Obruca et al., 2014), while the residual lignocellulose fraction, after hydrolysis to fermentable sugars, can undergo conversion towards scl-PHA by the superior lignocellulose converter Burkholderia sacchari or Bacillus megaterium (Obruca, Benesova, Kucera, Petrik, & Marova, 2015). Oil extraction from spent coffee ground for PHA biosynthesis was later optimized by employing sCO2 (Cruz et al., 2014), while Kovacik et al. demonstrated that toxic compounds present in acidic hydrolysates of the lignocellulose fraction (phenolics, etc.) can conveniently be removed by adsorption on styrene-divinylbenzene-based resins; this way, hydrolyzed spent coffee ground fibers can also be used as substrates for PHA production using more sensitive organisms like Halomonas halophila (Kovalcik et al., 2018).

Other biorefinery projects combine PHA production with the conversion of abundantly available lignocellulose-rich waste, consisting of lignin with a complex polyphenolic structure that is strongly linked to cellulosse and hemicellulose fibers; lignocellulosics constitute the most abundant renewable resources on earth, with 60% of all plant biomass consisting of lignocellulose. The composition of lignocellulosic biomass differs in terms of the shares of lignin (10%–25%), cellulose (30%–60%), and hemicellulose (25%–35%) (Kumar, Singh, & Singh, 2008; Peters, 2006; Tengerdy & Szakacs, 2003). In most cases, lignocellulose waste originates from agriculture and forestry. Examples for tehir use as PHA-feedstocks encompass desugarized sugar beet molasses (using Bacillus megaterium uyuni S29) (Schmid et al., 2019), sugar cane bagasse (B. sacchari) (Silva et al., 2004), tequila bagasse (Saccharophagus degradans) (Alva Munoz & Riley, 2008; González-García, Grieve, Meza-Contreras, Clifton-García, & Silva-Guzmán, 2019), wood straw (C. necator) (Saratale & Oh, 2015), softwood hydrolysates (Paraburkholderia sacchari IPT 101) (Dietrich, Dumont, Orsat, & Del Rio, 2019), or rice husks (B. sacchari) (Heng, Hatti‐Kaul, Adam, Fukui, & Sudesh, 2017).

Other carbohydrate-rich waste materials successfully tested as feedstocks for PHA biosynthesis are waste of pulp and paper industry (B. sacchari) (Al-Battashi et al., 2019), residual juices from cashew nut production (Arumugam et al., 2019), carbon-rich municipal wastewater (Pittmann & Steinmetz, 2017), wastewater streams of the olive processing industry (alpechìn) (Alsafadi & Al-Mashaqbeh, 2017; Pozo et al., 2002; Ribera et al., 2001), or chicory roots (Haas et al., 2015).

As one of the most important surplus materials for biotechnological production of PHA, lactose-rich surplus whey from cheese and dairy industry needs to be highlighted. Dependent on the production strain, lactose, the main carbon source present in full whey must be either hydrolyzed into the monomers glucose and galactose (examples: Hfx. mediterannei, or Paraburkholderia funghorum [former Pseudomonas hydrogenovora]), or, when the strain per se has sufficient activity of β-galactosidase, can be supplied in a nonhydrolyzed form (e.g., Bacillus megaterium, recombinant E. coli, Hydrogenophaga pseudoflava, or Ensifer meliloti). In principle, processing of whey to a substrate for PHA production starts with a skimming step to remove lipids. After that, skimmed whey gets separated into a lactose-rich (ca. 200 g/L) permeate fraction, and a proteinaceous retentate fraction, via ultrafiltration. The permeate fraction, either hydrolyzed or intact, gets sterilized and is supplied as a carbon feedstock for the bioprocess (Koller, Marsalek, & Braunegg, 2016). The utilization of simply deproteinized, not ultrafiltrated whey as feedstock for PHA production was reported by Obruca et al., who used Bacillus megaterium CCM 2037 as production strain. These authors reached about 1.5 g/L PHA when adding a small amount of the metabolic stress factor ethanol at the beginning of the stationary phase (Obruca, Marova, Melusova, & Mravcova, 2011).

Such biorefinery concepts are characterized by value-added utilization of as many constituents of raw material as possible; moreover, on-site running of all the process steps is a characteristic feature of such biorefineries for safe transportation costs. As a prime example, an integrated biorefinery concept was realized by the Brazilian company PHB Industrial S/A (PHBISA). This process utilizes in-house waste streams of sugar and biofuel manufacturing for the production of PHA biopolyesters. Starting from sugar cane, saccharose is produced, and, by fermentative conversion of remaining molasses, the biofuel ethanol. Waste streams from sugar (bagasse) and ethanol (fusel alcohols) production are integrated into the PHA production process and make it economically feasible. PHB homopolyester and, by co-feeding the 3HV precursor propionic acid together with hydrolyzed sucrose during the PHA accumulation phase, PHBHV copolyesters are produced by this process at a reported annual quantity of 10,000 tons, and commercialized under the trademark BIOCYCLE. In this process, about 3 kg sucrose yields 1 kg PHB, when using the production strain C. necator. High-pressure steam stemming from bagasse combustion is used for generation of electrical power to run the plant. In addition, low-pressure steam for heating and sterilization is also generated by bagasse combustion. The process is integrated into an ethanol production plant, which generates fusel alcohols like iso-pentanol at the distillation step (Nonato, Mantelatto, & Rossell, 2001).

Molasses, another side stream of the sugarcane industry, remain after removing sucrose crystals from the liquor; they can be sold at up to half of the price of purified sugars. In 1989, Page used Azotobacter vinelandii strain UWD for PHA production. While more than 2 g/L PHB was produced during the exponential growth phase in defined media containing ammonium acetate and 10 g/L of glucose, fructose, sucrose, or maltose, more than 1.5 g/L PHB were produced with 10 g/L sodium gluconate or glycerol. After acetate depletion, PHB content in biomass soon amounted to 53%–70 wt%. More than 2 g/L PHB were also obtained when A. vinelandii was grown with 20 g/L of complex substrates like cane molasses, beet molasses, corn syrup, or malt extract. Especially beet molasses stimulated bacterial growth, which resulted in higher PHB productivity. Importantly, this study demonstrated that not purified (unrefined) carbon sources favor PHB biosynthesis, resulting in PHB yields similar to or even better than when suing refined sugars (Page, 1989).

Starch as another abundantly available carbon source has a lower price than glucose. Already in 1999, Choi and Lee had calculated that PHB production costs on a production scale of 100,000 annual tons should decrease from 5 US$ per kg below 4 US$ when replacing glucose (0.49 US$ per kg) by hydrolyzed corn starch (0.22 US$ per kg) (Choi & Lee, 1999). In many cases, starch-based PHA production needs enzymatic or chemical starch hydrolysis to convertible substrates (glucose, maltose) (Chen, Trong-Ming, & Hsiao-Feng, 2006; Huang, Duan, Huang, & Chen, 2006b). Moreover, PHBHV production by H. mediterranei on starch co-extruded with α-amylases was reported by Chen and colleagues. The product contained approximately 10 mol% 3HV and showed excellent thermomechanical features. Working with the same strain and a medium containing extruded rice bran and corn starch, Huang et al. reported a PHA concentration of almost 80 g/L, 55.6 wt% PHA in biomass and high volumetric productivity of 0.71 g/(L h) (Chen, Trong-Ming, & Hsiao-Feng, 2006). Later, Haas et al. also studied saccharified waste potato starch as an inexpensive carbon source for PHB production by C. necator NCIMB 11599 under conditions of phosphate limitation. High concentrations of 179 g/L biomass and 94 g/L PHB were obtained, and the starch-to-biomass and starch-to-PHB yields amounted to 0.46 and 0.22 g/g, respectively; volumetric PHB productivity was reported with 1.47 g/(L h) (Haas, Jin, & Zepf, 2008).

A different approach is provided by running the two phases of a PHA-production process (growth phase for biomass production, followed by nutrient-limited phase for PHA accumulation) using two different substrates. This makes sense in case of abundantly available raw materials, which can be used by the production strain to generate a high density of catalytically active biomass but are not converted by the strain to PHA in the second step. This was demonstrated on a 100-L pilot scale by Follonier et al., who used the sugar-rich extract of pomace of Gewüztraminer grapes for growth of P. putida KT2440 in a batch-feeding mode for the production of about 8.0 g/L residual (catalytically active) biomass. After the onset of the PHA-accumulation phase by nitrogen limitation, the substrate feed was exchanged by an equimolar mixture of octanoate and undecenoate, which was converted by the cells to a mcl-PHA consisting of a total of six identified building blocks, among them three saturated (3HHx, 3HO, and 3HD), and three unsaturated monomers (3-hydroxy-10-heptenoate, 3-hydroxynonenoate, 3-hydroxydecenoate). Conversion yield of fatty acids to mcl-PHA amounted to excellent 0.79 g/g, and the final mcl-PHA fraction in total biomass was about 0.4 g/g (Follonier, Riesen, & Zinn, 2015). A similar approach was recently reported by Pernicova and colleagues, who cultivated P. putida KT2440 on nontreated chicken feathers. The bacteria excreted the enzyme keratinase to utilize the material for biomass growth, without converting it into PHA. After the growth, the almost PHA-free biomass was separated and re-suspended in a nitrogen-limited cultivation medium containing waste frying oil as sole carbon source. On this substrate, the cells produced a PHA-copolyester consisting of 3HHx (27.2%) and 3HO (72.8%) with a mass fraction of 0.61 g/g in biomass. In addition, the extracellular keratinase from the first cultivation stage can be isolated and commercialized (Pernicova, Enev, Marova, & Obruca, 2019).

3.2.3: Examples of C1-compounds as viable carbon sources for PHA production

Cyanobacteria, a group of phototrophic organisms were formerly classified as blue algae in an incorrect way; they encompass species, which show more or less pronounced PHA bioaccumulation when cultivated under illumination and CO2-supply. This opens the door for converting CO2-rich exhaust gases from industrial plants, such as coal power plants, and coupling CO2-mitigation with biopolyesters synthesis. Together with PHA, cyanobacteria are well known to produce other marketable products, such as bioactive compounds of therapeutic significance, or pigments to be used in the food industry or for immunofluorescence labeling. For some cyanobacteria, it turned out reasonable for enhanced PHA productivity to adapt the cultivation regime in a way that dark/light cycles are alternating; in addition, it was shown that mixotrophic feeding, hence, the parallel supply of CO2 and organic substrates like acetate or sugar leads to higher product output. In any case, it is still a long road to make PHA production by cyanobacteria profitable. Factors like the long-term stability of large-scale process (Troschl et al., 2017), the adequate photobioreactor systems (Kamravamanesh, Lackner, & Herwig, 2018; Singh & Mallick, 2017), higher biomass concentration (Costa et al., 2018), appropriate downstream processing (Roja, Sudhakar, Anto, & Mathimani, 2019), and others still need to be addressed and optimized.

Apart from phototrophs, also bacteria typically known as chemoheterotrophic PHA producers were originally described as chemolithoautotrophic strains. This is especially valid for C. necator (originally names Alcaligenes eutrophus H16), which can be used as knallgas bacterium, which converts CO2 and H2 to biomass (single cell protein) and PHA (Heinzle & Lafferty, 1989). This synthesis pathway fell into oblivion more and more, until, during the last years, the concept of using knallgas bacteria as working horses for CO2-based biosynthesis is experiencing a revival, inspired by the current endeavors to mitigate greenhouse gas and to link this mitigation with generation of valued bioproducts (Milker, Kunze, Sydow, Kroner, & Holtmann, 2018).

Syngas, mostly containing H2, CO, and CO2, constitutes another volatile raw material for the production of various bioproducts, which can be obtained by gasification, hence, thermochemical conversion of biomass (Phillips, Huhnke, & Atiyeh, 2017). Even the microwave-mediated pyrolysis of household waste to syngas for PHA production was demonstrated (Revelles et al., 2017). The purple nonsulfur photosynthetic bacterium Rhodospirillum rubrum is able to produce, besides H2, PHA by syngas fermentation; especially carboxylases present in this organism enable the conversion of CO2 in syngas to PHA (Revelles, Tarazona, García, & Prieto, 2016). This enables the design of a biorefinery concept, where, starting from gasification of abundant raw materials like switchgrass or household waste, PHA, and H2 can be produced as vendible products by R. rubrum. Depending on the market price of H2, a PHA production price of below 2€ per kg can be achieved in such biorefinery plants (Choi, Chipman, Bents, & Brown, 2010). Only recently, it was shown that the inhibitory effect of syngas for R. rubrum can be overcome by diluting the gas feed with N2; in addition, also co-feeding of acetate turned out to be beneficial for PHA biosynthesis by this purple nonsulfur bacterium (Karmann, Panke, & Zinn, 2019).

Anaerobic degradation of organic material, also organic waste, results in a CH4 yield regarding the carbon balance of approximately 50%. This gas is often just flared or released into the atmosphere due to the high costs of storage and transport. For illustration, about 7 million tons of CH4 were released in the atmosphere just regarding the USA in the year 2008 (reviewed by Khosravi-Darani, Mokhtari, Amai, & Tanaka, 2013). Methylotrophs are able to produce PHA; in particular, these organisms produce PHA starting from methane, which occurs, e.g., in natural gas and biogas. Importantly, this approach converts the severe greenhouse gas methane into the environmentally friendly and valued bioproduct PHA. In this context, natural gas contains about 85%–90% of methane, and it is also formed by methanogens during biological degradation of organic materials. Stoichiometrically, the theoretically maximum methane-to-PHA conversion yield amounts to 0.67 g/g (Asenjo & Suk, 1986), while typically about 0.50–0.55 g/g are achieved in praxi (Wendlandt, Jechorek, Helm, & Stottmeister, 2001), which is still considerably higher than aerobic sugar-to-PHA conversion yields. Moreover, methane constitutes a chemically stable compound, which is in contrast to complex, inexpensive feedstocks of varying composition, which can influence PHA quality. Table 2 provides an overview of different industrial waste streams to be used as feedstocks for PHA biosynthesis.

Table 2

3.3: Bioprocess and cultivation regime

3.3.1: Batch mode for PHA production

Simple cultivation setups are operated in batch mode, were all substrates are added only at the beginning of the process; such cultivations typically result in low productivity and low PHA contents in biomass. Mathematical modeling of typical batch processes for PHA production by a wild type strain and its mutant was carried out by Torres-Cerna et al., showing the interrelation between substrate consumption and PHA productivity during the different stages of the cultivations (Torres-Cerna, Alanis, Poblete-Castro, & Hernandez-Vargas, 2017). An advanced version of batch cultivations is the so-called repeated batch technique, where, as soon as a predefined concentration of biomass is reached, the major part of cultivation broth is removed and processed, while the remaining volume acts as inoculum for fresh nutrient medium. After each removal, the inoculum gets more active, which results in the shorter time needed for each subsequent cycle. In addition, such repeated batch cultivation avoids nonproductive time (dead time, revamping time) required for cleaning, sterilization, and refilling of the bioreactor after each batch, which increases the overall volumetric productivity and thus the industrial feasibility of the process. Such simple fill-and-empty strategy was shown by Gahlawat et al. These authors removed three times (after 27, 48, and 60 h) 20% (v/v) of the culture broth and replaced it by the same volume of the fresh nutrient medium; the strain Azohydromonas australica was used as whole-cell biocatalyst. Compared to simple batch cultivation, a more than threefold increase in PHB concentration (from about 6 g/L to more than 20 g/L), and almost a duplication in volumetric productivity from 0.17 to 0.29 g/(L h) was achieved by this approach (Gahlawat & Srivastava, 2017).

3.3.2: Fed-batch mode for PHA production

PHA production processes are typically carried out in fed-batch mode, where especially the carbon source is added when needed according to substrate analysis. Moreover, substrate refed can be done as a direct reaction on dropping substrate concentration (online or offline substrate analysis needed), or indirectly according to parameters influenced by actual substrate consumption, e.g., dissolved oxygen (DO) concentration (DO-stat processes), CO2 concentration in exhaust air (CO2-stat processes), or pH-value (pH-stat processes) (Koller, 2018c).

Fed-batch mode helps to avoid excessive actual substrate concentrations, which might be inhibiting for microbial growth and/or PHA biosynthesis. In this context, five different strategies were studied by Hrnčiřík and colleagues for optimized fed-batch cultivation of Ps. putida KT2442 in a 7-L bioreactor on the substrate octanoate, which displays inhibiting effect on the production strain already at rather low concentration levels. These feeding strategies employed (i) continuous substrate supply proportional to the CO2 production rate (applicable primary during the exponential phase of growth), (ii) continuous substrate supply proportional to the O2 uptake rate (also applicable mainly during the logarithmic growth phase), (iii) continuous feeding according to a linear feeding profile (appropriate after completion of the initial batch phase), (iv) semicontinuous pulse-feeding according to dissolved oxygen (DO) concentration, (v) continuous feeding according to the DO signal (PID controlled DO-stat). These feeding strategies aimed at developing a fully automated dosage of the substrate to obtain high mcl-PHA productivity; in particular, the authors evaluated the proper sequence of and switching between the different strategies. In addition, the optimal ratio between the added carbon source and produced CO2 and consumed DO was evaluated. By this optimized sequence of feeding strategies, the octanoate feeding rate can be calculated from process variables mirroring the current state of the bacterial culture. Most importantly, the proposed DO- and CO2-based continuous strategies for substrate feeding during the exponential growth phase constitutes a viable alternative to frequently used pulse-feed pH-stat and DO-stat feeding, specifically in cases where permanent substrate feeding is required (Hrnčiřík, Náhlík, & Mareš, 2017).

Repeated fed-batch cultivations are advanced fed-batch approaches, where substrate pulses are supplied after certain time intervals, while parts of the fermentation broth are periodically replaced by fresh medium. As demonstrated by Biglari and colleagues, such systems can result in strongly increased productivity. These authors compared two different repeated fed-batch cultivations with a C. necator mutant strain using urea as nitrogen, and glucose as carbon source, respectively. In an incremental repeated fed-batch fill-and-draw process, higher volumes of fermentation broth (10%) were replaced after regular intervals in the accumulation phase by increasing quantities of glucose solution, while in the decrement mode, only half the volumes of fermentation broth (5%) were regularly replaced by fresh medium, but lower quantities of glucose were added with each refed cycle. As a major outcome, the incremental approach increased PHA (110 g/L) and biomass concentration; PHA productivity was more than two times higher than in the decremental mode. For both cases, high PHA fractions in biomass, by far exceeding 0.8 g/g, were reported. Surprisingly, the authors reported high glucose-to-PHA yields of 0.50 g/g (Biglari, Orita, Fukui, & Sudesh, 2020).

However, especially when using highly diluted substrates like typically the case for by-products with low carbon source content (e.g., whey, lignocellulosic hydrolysates, etc.), it easily comes to high dilution of the cultivation broth in the bioreactor; this can technically be overcome by cell recycling bioreactor systems, where spent (substrate-poor) cultivation supernatant is removed via a filtration unit, while active biomass is retained and gets resuspended in the culture medium. Such approach was successfully demonstrated by Haas and colleagues, who used an external microfiltration module in a model fermentation with a continuous feed of C. necator with a diluted (50 g/L) glucose feed solution mimicking a dilutes substrate stream of an inexpensive waste carbon source; glucose was almost immediately consumed by the cells, nearly glucose-free cultivation broth was permanently removed via the microfiltration unit, and biomass concentration was continuously increased up to 148 g/L, with a PHB mass fraction of 0.78 g/g. The achieved volumetric productivity amounted to 3.1 g/(L·h) (Haas et al., 2017).

3.3.3: Continuous mode for PHA production

Continuous cultivation processes are described for various different bioproducts, and diverse challenges and biological and engineering risks and means to handle them were comprehensively reviewed before (Koller, 2018c). Continuous cultivation process for PHA bioproduction can be operated in single-, two-, or multistage mode. All these continuous processes are typically operated in chemostat (chemical environment is constant) mode. In the steady state of such processes, hence, in the constant phase of a continuous bioprocess, concentration of products and substrates do not change; hence, they are parallel to the time axis when plotting concentrations against the cultivation time. Mathematically, the steady state of a chemostat bioprocess is characterized by the dilution rate D [1/h], which expresses the quotient of flow rate to bioreactor volume, equaling the specific growth rate μ. This allows forcing a biological system to react to a process engineering parameter, hence, μ can be triggered by adapting D. However, caution must be exercised in that one should not overburden the biological system; as soon as μ exceeds the value of D, there is a continuous dilution of the fermentation broth, the so-called washout of the biomass.

Such continuous processes offer the advantage of running the cultivations at constant product output, both in terms of product quantity and product quality, for extended periods without the need for laborious and time-consuming revamping of the bioreactors, which enables high volumetric productivity, and reduced demand for process support by staff. Moreover, the continuous harvest of cultivation broth also enables to process it in a continuous way (e.g., continuously operated centrifuges) for product recovery; this is in contrast to large batch setups, where a huge quantity of biomass needs to be processed after harvest (Koller & Braunegg, 2015a).

Single-stage PHA production processes, although being the simplest among such chemostat techniques, have inherent disadvantages, which originate from the kinetic particularities of PHA biosynthesis: as a product of the secondary metabolism, biomass growth, and PHA biosynthesis are temporarily decoupled (biomass growth is typically followed by the phase of predominant PHA formation, provoked by limitation of a growth-essential nutrient). In a single-stage continuous (chemostat) process, all nutrients needed for growth (carbon-, nitrogen-, and phosphate source plus minerals) need to be continuously supplied to the cells. Hence, it is not possible to design the composition of the feed stream in a way that both high biomass density and high PHA fractions in biomass can be reached at the same time. If growth essential components like the nitrogen and/or phosphate source are supplied in high concentration compared to the carbon source, it will be possible to reach a high density of active biomass, but at the expense of intracellular PHA content and hence at lower overall productivity. On the contrary, providing carbon source in high excess will result in higher PHA fractions in biomass, but in lower cell density, which again prevents higher PHA productivity. This dilemma becomes even worse when planning a process where medium components are almost depleted in the product stream leaving the bioreactor in order to save substrate costs. To sum up, single-stage continuous processes are no method of choice to tap the full biotechnological potential of production of secondary metabolites like PHA. As an exception, some strains are described to accumulate PHA at high rates already during balanced growth; in such cases, it becomes reasonable to run a continuous production process in single-stage mode (Koller & Muhr, 2014).

Two-stage continuous chemostat processes display considerable advantage in case of secondary metabolites like PHA; here, a first stage is dedicated to the production of high concentration of catalytically active biomass by continuously supplying all components needed for balanced cellular growth. Concentration of all components, as well as feeding rate, should be adapted in a way allowing maximum cell growth (dilution rate close to the biological maximum of the strain’s specific growth rate at given environmental conditions), and to avoid significant concentrations of the growth-limiting component (typically the nitrogen source) to enter the second stage of the process. Hence, the aim of this first stage is to continuously produce active, PHA-poor biomass in its logarithmic phase of growth (maximum specific growth rate μmax.). In the second stage, only substrates needed for PHA biosynthesis (main carbon source and eventually precursor substrates for special PHA building blocks) are continuously provided. Considering the fact that specific PHA accumulation rates are typically lower than specific growth rates for a given strain/substrate combination, the dilution rate D in the second stage will be lower to increase the residence time τ, which enables more complete substrate-to-product conversion (desirably no discarded substrate in the stream leaving the second stage), and higher intracellular PHA mass fraction; also here, the aim should be to operate the second stage at a D close to the biological maximum of the specific product formation rate qP max. Both μmax. and qP max. need to be estimated before in discontinuous processes. Again, care has to be taken that D does by no way exceed μmax. or qP max. in order to avoid washout of cells. Moreover, the acceptable actual substrate concentration depends on the microorganism’s substrate affinity constant Ks and on μ (or D at steady-state conditions, respectively) (reviews by Koller & Braunegg, 2015a and Koller & Muhr, 2014). Fig. 4 shows a schematic of the discussed cultivation regimes used for PHA production.

Fig. 4 Schematic illustrations of different cultivation regimes used for PHA production. Reproduced with kind permission from Koller, M. (2018c). A review on established and emerging fermentation schemes for microbial production of Polyhydroxyalkanoate (PHA) biopolyesters, Fermentation 4(2), 30, by MDPI publishers, Basel, Switzerland.

3.4: PHA separation from residual microbial biomass

3.4.1: Downstream processing in PHA production

After the bioprocess, downstream processing is needed to separate PHA-rich biomass from the spent fermentation broth, hence, the supernatant of fermentation broth. These are typically accomplished by means of sedimentation or centrifugation and to a lower extend by filtration. In case of continuous cultivations, biomass separation can also be accomplished by continuous operated separators. After this step, biomass is typically dried, and enhanced techniques are needed to recover PHA from dry microbial biomass at high yields, in a short time, without negative impact on the polymer quality, and by environmentally benign means. In a nutshell, solvent-based extraction methods and cell disruption by enzymatic, chemical, or mechanical techniques, or combinations thereof, are