Introduction: The current plastic scenario
Humanity has gone through several significant epochs during its evolution. Important examples in the past were were the stone age and the diverse metal ages; currently, we not only live in the silicon age, moreover, we are also in the middle of the “plastic age”. Each of these epochs was or is, respectively, marked by quantum leaps in the use and evolution of novel materials that have been permanently integrated in response to the ever-growing demands of an intellectually and organizationally exponentially advancing society. Based on the multifarious beneficial properties of plastics, they are among the most widely produced and applied materials in our today´s world; regarding their low density, convenient processability, stability and flexibility, plastics have undisputed countless advantages over materials used in previous centuries, such as metals or minerals, but they also carry a lot of environmental and health risks (1). One could say that today, plastics emerged to an indispensable evil (2). As a matter of fact, the growing amounts of petrol-based plastics used for various purposes of our daily life are among the fundamental ecological grievances of our modern time. In this context, we are currently threatened by headwords dominating the media like “growing dumps of plastic garbage”, “marine ecosystems contaminated by plastics”, “microplastic”, and, very recently, political initiatives to “ban all-day plastic commodities” such as wide spread plastic shopping bags, cotton swabs with plastic rods, plastic cutlery, or plastic beakers (3).
Indeed, 330 megatons (Mt) of petrochemical plastics are currently produced globally per year. Producing them depletes limited available fossil raw materials; moreover, after being used, these petrochemical “full-carbon-backbone plastics” must be supplied to appropriate disposal strategies due to their high recalcitrance towards biodegradation. Until now (2018), an estimated quantity of 150 Mt of plastic waste has already entered the oceans; as a shocking estimate, this marine plastic waste is supposed to cause the death of about 100,000 marine mammals and about one million birds annually (4). Recently, the medially disseminated death of a sperm whale, which carried an incredible quantity of around eighty plastic bags in its digestive tract, which made it impossible for this marine giant to absorb organic nutrients, had upset the general community (5). In November 2018, another perished whale was found at the Indonesian coast; in the stomach of this animal, a total of 6 kg of plastics was found, inter alia 115 plastic cups, 25 plastic bags and more than 1000 other plastic parts (6). Especially microplastics have also direct effects on security of human nutrition; such plastics particles of a size of less than 5 mm easily pass through the whole food supply chain until they finally reach it´s end, namely the human body (7, 8, 9). In this context, food production often resorts to equipment made of plastic parts, which causes microplastic formation by abrasion, while food storage in plastic containers and bottles is another direct source of food contamination by microplastics (10). Only by now, the scientific community starts to mechanistically understand the harmful effects of microplastics on the human organism, and to estimate these effects qualitatively and quantitatively. Just the other day (autumn 2018), a total of nine different petrochemical plastics, predominantly polypropylene (PP) and polyterephtalate (PET) with particle sizes between 50 and 500 μm were for the first time identified by an Austrian research team in the human intestinal system; it is suspected that these microparticles can potentially be forwarded from the intestine to the blood and the lymphatic system and to various organs (11). Apart from plastics themselves, other products associated to polymer production and processing cause harmful effects to the human metabolism, such as crude oil-based o-phthalate or diverse other plasticizers; also these additive need replacement by biological alternatives, as manifested by very recent studies (12).
Regarding the problem of landfilling for final disposal of plastic, it should be noted that in Europe not even a third of plastic waste undergoes recycling; the rest ends up in landfills, pollutes terrestrial and aquatic environments or the forests, or is simply combusted in waste incineration plants (3). In the context of recycling, which is often extolled as a universal remedy to treat plastic waste, it should be mentioned that this strategy rather delays than avoids its final disposal in landfills or incineration. On the other hand, considerable parts of landfilled plastic discards arrive via detours, such as wind or river systems, into the oceans, where they cause the above described fatal effects on living beings (11). In the EU alone, this plastic migration from landfills to the sea applies to annually about 500,000 t of plastic rejects. Needless to say, this leads to a significant burden on the entire global ecosystem and human health. Also from a resource-political point of view, the exuberant production and use of plastic is more than shortsighted, unsustainable, and actually inadmissible. This does not just mean that plastic manufacturing leads to the exploitation of fossil resources; in addition, one has to keep in mind that most of the plastic items used today are for single use only. This situation acts as an as an amplifier of the ecological misery insofar as many plastic objects can, on the one hand, easily be produced, and, on the other hand, they are very cheap, which hardly promotes any change in the mental attitude of broad sections of the population towards not using them (4).
In order to change this situation, rational and ecological pricing of conventional plastic materials is required, which not only takes into account the benefits of such polymers, but also accounts for the environmental burden caused by their high ecological footprint. As a result, plastic would no longer be disposed of en masse. In addition, fair pricing, wherever possible and reasonable, would encourage the development and production of bio-inspired, sustainable alternatives.
Biological alternatives to established plastics
To an increasing extent, biopolymers with plastic-like properties are considered potential alternatives to replace well-established petrochemical plastics on the market; among these bio-alternatives, thermoplastic starch, poly(lactic acid), or, as the topic of this review, microbial polyhydroxyalkanoates (PHA), are in the spotlight of current R&D endeavors (13). Especially PHA, a family of microbial biopolyoxoesters produced as intracellular storage compounds by numerous bacteria and a range of extremophilic haloarchaea, are nowadays in the focus of microbiologists, polymer scientists, process engineers, and, more recently, scientists involved in synthetic biology. What makes PHA that fascinating are the tunable material features, which enables their implementation in numerous sectors of the plastics market; here, they have the potential to displace diverse currently dominating petrol-based plastomers and technomers (14,15). Most of all, PHA are the only family of so called “green plastics” sensu stricto, because they are biobased (biosynthesis starting from renewable resources), biosynthesized (intracellular products of the secondary metabolism of prokaryotic microbes), biodegradable, compostable, and fermentable to products like water, CO2, or CH4; moreover, they are biocompatible, which makes them attractive as materials of choice for biomedical applications. Production of these biological plastics does not deplete fossil resources because of being derived from renewable resources. Their entire life span is embedded into the closed carbon cycle of nature, which implies that their degradation, in contrast to degradation of petro-based plastics, does not cause an increase of the atmospheric CO2 level; hence, it does not amplify or accelerate climate change (Fig. 1) (16). Feedstocks typically used to manufacture PHA and other “green plastics” are renewable resources like sugars, high-molecular carbohydrates or fats and oils, which are natural products and substrates of the carbon cycle of living beings. It should be visualized that, in contrast to fossil resources used to produce established plastics, these renewable feedstocks were not captured within Earth´s interior since eons. After the use of biopolymers, they undergo biodegradation and composting by the metabolism of living organisms, which generates biomass and CO2; CO2, in turn, gets fixed again by the photosynthetic performance of green plants to rebuild the renewable feedstocks originally used for biopolymer synthesis, which closes the cycle of anabolism and catabolism of biopolymer feedstocks. This differs drastically from the life-cycle processes of petrochemical plastics, the thermal degradation of which results in a misbalance of CO2 through combustion; excess carbon that has been removed from the natural carbon cycle over millions of years is suddenly released into the atmosphere as surplus CO2 (Fig. 2). It would take millions of years to recover fossil fuels by means of photosynthetic fixation and fossilization of the resulting biomass (16).
In fact, this sounds promising, so why don´t we just switch to these biopolymers? Indeed, we have to consider ethical, economic and sustainability aspects especially when selecting the adequate raw material for biopolymer production, such as for biosynthesis of PHA. It is exhaustively reported that using nutritionally important carbohydrates like sugars or edible oils for PHA production results in high product formation rates, with numerous fermentation protocols being available for diverse production strain–substrate combinations; this goes as well for laboratory scale experiments as well as for (semi)industrial PHA production processes. However, applying such edible substrates for bioplastic production on a larger scale cannot be accepted based on the high number of people suffering from starvation on this planet; using such edible resources to feed PHA-producing microbes fuels the current “plate-versus-plastics” conflict, as does using converting food resources for biofuel production, which provokes the “plate-versus-tank” controversy (14). This makes clear that simply replacing established plastics by biobased alternatives cannot be considered the one and only panacea for the plastic problem, when the reflection of the entire process chain and biopolymer life cycle is not warranted (17). A sustainable biopolymer production process has to take aspects of cost-efficiency, environmental protection, optimized engineering, and ethics into consideration (14). As a more and more acknowledged exit strategy out of this predicament, one could link food industry with biopolymer industry in a synergistic way. Companies involved in food and feed production and processing produce huge quantities of carbon-rich waste streams, which could be used at zero costs as feedstocks by biopolymer producing companies (18). To underline the feasibility and importance of this still new paradigm, one should keep in mind that an estimated annual quantity of about 1012 kg of food is not consumed by humans, but simply rejected (19). Substituting purified carbon feedstocks by such waste streams of the food-industrial sector on the one hand safes about 50% of the entire costs of the biopolymer production chain, and, on the other hands, provides (agro)industry a tool to upgrade their disposal products in a value-generating manner (20). Examples of (agro)industrial waste used as PHA-feedstock during the last years, albeit not yet beyond the laboratory scale, encompass molasses from sugar industry (21, 22), hydrolysates of lignocellulose materials (23) like bagasse (24), wheat straw (25), rice straw (26), or wood (27, 28), sugars obtained by hydrolysis of green grass (29) or saccharified ryegrass (30), empty oil palm fruit bunches (31), cellulose-digested corn stover (32), steamed soybean wastewater (33), waste glycerol from biodiesel production (34, 35, 36, 37), saturated biodiesel fractions of animal origin (38, 39), waste office paper hydrolysate (40), or spent sulfite liquor obtained by chemical pulping during the paper production process (41). From gastronomy, hydrolyzed spent coffee grounds (42) or waste cooking oil (43) are available as PHA feedstocks at ample quantity. Only recently, even petrochemical plastic waste (polyethylene) was successfully converted to PHA (44). On an almost pilot scale of some 100 L, lactose-rich surplus whey from dairy industry was used for production of PHA copolyesters by the haloarchael production strain Haloferax mediterranei (45).
In contrast to the use of pure, defined feedstocks, it is pivotal to understand the composition of such complex carbon sources, and the impact of eventually present inhibiting compounds on growth and biopolymer production kinetics of a selected microbial production strain. Therefore, it is important to develop proper upstream processing methods to convert a waste stream into a biotechnological feedstock. In this context, it is often needed to hydrolyze di-, oligo-, or polymeric compounds into smaller substrates, which are easily convertible by the microbial production strain (46). Moreover, such complex mixtures of hydrolysis products frequently contain inhibitors, which require removal for detoxification (28, 47). Beside the use of food and agricultural waste as biotechnological substrates to cultivate microbial production strains, it is an emerging R&D trend to apply a range of surplus materials from agriculture and food production as filler materials to generate new biocomposites. This encompasses, e.g., lignocellulosics like bagasse and straw, wood dust, or cellulose (nano)fibers. Properties of such new biopolymer composite materials can be fine-tuned by triggering the ratio of biopolymer (PHA, PLA, thermoplastic starch, etc.), the filler material, and eventual additional plasticizers like glycerol or special plant oils; this way, one can achieve unprecedented, customized material properties and high performance in terms of, e.g., gas permeability, crystallinity, compression behavior, elongation-at-break, and others. Hence, smart bioplastic composite materials to be used in defined fields of the plastic market can be developed, fine-tuned to a special purpose, such as food packaging, automotive parts, and many more (48, 49, 50).
Now, it´s already more than nine decades ago, since Maurice Lemoigne described for the first time the microscopic observation of PHA granules as light-refractive inclusions in Bacillus megaterium cells (reviewed by 51). Typically, PHA synthesis in microbes is boosted when the cells are exposed to conditions of limited availability of a growth-essential nutrient, in most cases nitrogen or phosphate, in parallel to sufficient supply with exogenous carbon source (substrate). As their principal biological role as carbon- and energy reserve compounds, presence of PHA enables microbial cells to better endure periods of carbon starvation. In its role as “electron and NADH sink”, PHA regenerates reducing equivalents under conditions when central metabolic pathways, mainly the tricarboxylic acid cycle (TCC), are shut down, as it is the case when culturing cells under nitrogen limited conditions. Here, PHA biosynthesis is a “pseudofermentative” strategy applied by microbes to keep their metabolism running (52). More recently, the scientific community revealed the significant protective role of PHA against a number of stress factors, which are harmful or even lethal for PHA-free microbial cells (53). In this context, it was demonstrated that presence of PHA exerts a protective effect against the impact of elevated temperature (54), attenuates the destructive impact of excessive UV-irradiation by scattering light (55), helps to repair damage caused by repeated freezing-and-thawing cycles (56) or sudden osmotic imbalances (57), and elevates the burdens caused by chemical oxidants in general (58), and by sudden generation of reactive oxygen species (ROS) in particular (59). Regarding elevated salt concentrations (hyperosmotic conditions), presence of PHA granules in living microbes turned out to support the cells to better survive the harmful effects of osmotic up-shock; this also clarifies why PHA granules are found in so many haloarchaeal species isolated up to date from extremely saline ecological niches (57).
Microbiology and genetic engineering to support PHA biosynthesis and production
PHA bioproduction can be carried out either using established wild type strains, new mesophilic (60, 61) or extremophilic (62, 63) microbial isolates, or, to an increasing extend, by using genetically modified strains. Such genetic modifications (“genome editing”) of PHA-accumulating microbes have originally been carried out to understand the principal biochemical, enzymatic, genetic, and metabolic features of PHA biosynthesis and intracellular degradation of these biopolyesters. Only since the last couple of years, we witness a switch from genetic manipulation of PHA producers for academic interest towards the design of new powerful cellular platforms for high-throughput biosynthesis of tailor-made PHA, preferably from inexpensive feedstocks. These activities are embedded in the topical field of “Next Generation Industrial Biotechnology” (NGIB), which describes the use of robust, genetically tailored production strains in large-scale industrial bioprocesses, often operated under restricted sterility precautions (64).
In the meanwhile, genetic manipulation was already tested to improve all the stages of the PHA production process (65), from the optimization of substrate uptake and utilization (prime example: insertion of β-galactosidase genes in PHA producers not able to utilize lactose as substrate) (66)), the optimization of oxygen availability to enhance biomass growth by expression of bacterial hemoglobin (67), boosted PHA productivity under oxygen-limited conditions (68), CRISPR/Cas9 genome editing to enhance the pathways leading to poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBHV) biosynthesis (69)), the pre-determination of the monomeric PHA composition by knocking out specific pathways (prime example: designing β-oxidation weakened mutants to generate PHA homopolyesters consisting of medium-chain-length building blocks (70), or to generate PHA with pending aromatic side chains under controlled conditions (71)). Moreover, synthetic biology approaches, hence, the construction of artificial metabolic pathways in production strains, enable the production of high-quality PHA copolyesters from simple, unrelated carbon sources, as demonstrated by the poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) production from glucose as sole carbon source by genetically engineered Pseudomonas stutzeri (72), or by poly(4-hydroxybutyrate) (P4HB) hyperproduction from glucose by recombinant Escherichia coli (73). Even for downstream processing (prime example: insertion of genes encoding nuclease, which, after cell breakdown, facilitates separation of released PHA granules from the liquid phase), genome editing was reported as a tool for process improvement (74).
From the economic point of view, the development of engineered microbes able to convert inexpensive substrates (waste streams) into PHA is of particular significance (75). As a matter of fact, many natural microorganisms can synthesize PHAs with wide-ranging monomeric composition, which impacts the material properties of PHA. However, incorporation of diverse building blocks in growing PHA chains in most cases requires co-feeding bacteria with structurally related precursor substrates, which are frequently expensive, water insoluble, and inhibiting already at low concentration levels (76). This makes the production of PHA copolyesters of fine-tuned composition problematic especially on industrial scale. Therefore, a current R&D direction follows the exploitation of such microbial wild type or genetically engineered strains able to convert inexpensive renewable carbon sources towards desired PHA building blocks without the need to supplement established precursors; a prime example in this context is the above reported production of P4HB from glucose (73). In addition to economic considerations related to the substrate(s) used, particular attention must be dedicated to the microbial aspects of the process. Most of all, substrate-to-product (PHA) conversion yields need to be improved, either by optimizing the cultivation conditions (e.g., triggered oxygen supply to avoid excess CO2 and byproduct formation), or, to an increasing extent, by genetic engineering in order to divert the metabolic carbon flux predominately towards PHA biosynthesis instead of towards production of side-products like organic acids or CO2.
Bioengineering/Fermentation for advanced PHA production
PHA production under controlled conditions, especially on a larger scale, is typically carried out in bioreactors (77, 78); such bioreactors (previously also known as “fermenters”) can be operated in different modes, such as batch (substrate feed only at the beginning, product harvest only at the end of the process), fed-batch (repeated substrate supply according to substrate analysis, harvest only at the end), and continuous (permanent supply of substrates and products harvest) setups. In this context, PHA biosynthesis has to be understood as a multiphase process, because product (PHA) accumulation as a intracellular secondary metabolite is typically not coupled to the preceding phase of microbial growth (formation of active biomass). Therefore, both feeding strategy and bioreactor operation mode need to be carefully adapted to the selected production strain, substrate, and kinetics of growth and product formation (79). Traditional PHA production setups operated in batch (80), repeated batch (“fill-and-empty” or “drain-and-fill” cultivation) (81), fed-batch (82), or cyclic fed-batch mode (83) often show unsatisfactory control of PHA composition, and are often characterized by modest volumetric productivity (77, 78).
The above discussed use of (agro)industrial waste streams as feed streams, which often contain the carbon sources in a considerably diluted form, makes the operation in batch- or fed-batch mode impracticable; each re-feed drastically increases the volume of the bioreactor content (78). In such cases, fedbatch feeding processes coupled to cell recycling were recently developed, where a membrane module is directly coupled with the bioreactor due retain active biomass, but to discard substrate-poor fermentation broth; promising volumetric PHA productivities are reported for such setups, which also avoids an accumulation of inhibiting compounds in the fermentation broth (84).
A detailed understanding of the kinetics of microbial growth and PHA biosynthesis is needed for advanced continuous cultivation processes, which can be carried out in single-, two-, and multi-stage bioreactor arrangements. Optimizing such continuous processes implies matching the kinetics of microbial growth and PHA accumulation with the operation mode (dilution rate, etc.). Such continuous processes not only allow for high volumetric productivity, but also for a better control of the monomeric composition of the products. Moreover, continuous cultivations allow using substrates toxic already at low concentration, such as precursors of special PHA building blocks. While one-stage continuous processes are rather the method of choice for production of compounds of the primary metabolism, but not for secondary products synthesized by multistage processes, as it is the case for PHA, it is beneficial to produce catalytically active biomass in a first continuous stage at high cell densities; in a second stage, this biomass accumulates PHA under nutritionally challenged conditions (e.g., limitation if nitrogen source) at high productivity. In general, such continuous processes offer the possibility to feed the carbon source exactly at the rate as it is consumed by the cells, which causes an almost zero concentration of substrate in spent fermentation broth, thus saving substrate expenses (85, 86). Moreover, multistage continuous processes (“bioreactor cascades”), which, with increasing number of individual reactors connected in series, approach the characteristics of tubular plug flow reactors, allow even higher volumetric productivity; the highest volumetric PHA productivity in continuous processes ever of was reported for a 5-stage continuous bioreactor cascade (87). Moreover, such cascades allow for fine-tuning the cultivation conditions in each individual process stage (bioreactor). This offers the advantage of triggering the substrate feed in each stage, which makes it possible to generate blocky structured PHA of pre-defined composition and properties (87, 88, 89). In addition, it is possible to study the morphology of cells and PHA granule formation under different defined conditions; the latter is of importance for the final downstream processing, where it is beneficial to have large PHA granules in cells instead of small inclusions, which are more complicated to separate after cell disintegration (90, 91).
Gaseous substrates, especially those not or hardly water soluble, require different bioengineering approaches than easily water-soluble heterotrophic substrates. In this context, we currently observe the development of an increasing number of CO2-based PHA production processes, which use cyanobacteria (formerly “blue-green algae”) as photoautotrophic cell factories (92, 93, 94). Such light-dependent processes resort to discontinuously or continuously operated photobioreactors similar to those used for farming eukaryotic microalgae. In an optimized scenario, the needed substrate CO2 stems from industrial effluent gas; this strategy combines CO2-mitigation with formation of valued bioproducts. To enhance cyanobacterial cultivation, the photobioreactor arrangement needs to be optimized in terms of illumination regime, geometric characteristics, mixing behavior, and gassing/degassing performance. Only recently, a comprehensive review summarized the types of photobioreactors already tested for cultivation of cyanobacteria; it was shown that the optimal photobioreactor design strongly depends on the nutritional and illumination requirements of the individual organisms (95). Recent pilot scale results suggest that tubular photobioreactors might be best suitable for PHA production by cyanobacteria like Synechocytis sp. (96). Apart from cyanobacteria, several hydrogen-oxidizing species such as Idonella sp. are also reported to produce PHA from CO2 (97, 98).
In parallel to the gaseous substrate CO2, there are also other C1-compounds available as substrates for PHA production (99). In this context, methane (main product of natural gas and biogas plants) (100), methanol (101), and CO-rich syngas are other emerging substrate options for bioplastic production. In the case of methane, a major greenhouse gas, methanotrophic production strains like Methylocystis sp. were reported to produce PHA at yields exceeding 0.5 g PHA per g CH4, which is superior to the theoretical value of heterotrophic PHA production from sugars, which amounts to 0.48 g/g (reviewed by 102). Syngas, obtained by gasification of organic waste, can be converted towards PHA by anaerobic photosynthetic bacteria like Rhodospirillum rubrum (103); also here, tools of genetic engineering were already implemented for process improvement, such as the insertion of genes encoding for mcl-PHA biosynthesis to produce rubber-like PHA from syngas as sole carbon source (104). Of course, the industrial-scale production of PHA from methane or syngas needs bioreactor systems with optimized gas transfer, such as bubble columns or advanced airlift bioreactors. In the case of methane, a recent review describes the use of loop bioreactors for PHA production based on natural gas; here, it is shown how simulation software tools (computational fluid dynamics and mathematical modelling) help to better understand such biochemical processes. Gas holdup, liquid velocity vectors, shear stress and gas transfer coefficients of the bioreactor need to be studied in order to understand the correlations between the process parameters and product formation, which strongly facilitates the process development and upscaling (105). In the case of syngas, Karmann and colleagues addressed the fact that working with this substrate poses safety risks; therefore, the authors presented a syngas fermentation platform with advanced safety installations and process analytical technology (PAT). Besides measuring devices to control gas supply and consumption, this platform contained an in‐line flow cytometer to determine the biomass concentration and intracellular PHA fraction. The platform was used in fed‐ batch and continuous syngas-based PHA production processes with R. rubrum (106).
Advanced downstream processing for sustainable and efficient PHA recovery from biomass
After biosynthesis, the development of sustainable and inexpensive technologies to recover intracellular PHA from microbial biomass is the final crucial step in the entire PHA production chain. Several comprehensive reviews summarize reported methods for PHA recovery; in principle, such methods resort to the use of practicable PHA-solvents to extract PHA from the cells, the chemical or enzymatic digestion of the microbial biomass accommodating PHA granules, or the mechanical disintegration of the non-PHA cell mass (107, 108, 109). Importantly, well-established methods for PHA extraction, which result in best recovery yields and highest product purity, resort to toxic halogenated solvents, predominately chloroform, which should not take part in a sustainable production chain. Therefore, alternative methods for PHA production are currently in status of development, which resort to fundamentally different approaches:
Marudkla and colleagues reported an optimized chloroform-free PHA recovery approach using sodium dodecyl sulfate (SDS) and sodium hypochlorite (NaOCl); optimized by an experimental Taguchi-design, the authors described the optimal conditions for the maximum PHA recovery (78.7%) when using 0.5% w/v SDS combined with 6% v/v NaOCl. Apart from this chemical method, a new enzymatic approach for digestion of non-PHA biomass was developed by Kachrimanidiou et al., who produced crude enzyme by solid state fermentation of the fungus Aspergillus oryzae. This enzyme cocktail was used to lyse C. necator cells to recover PHA. Temperature and pH-value were optimized for highest C. necator lysis, which amounted to about 90%; PHA recovery yield and purity amounted to 98% and 97%, respectively (110).
Among new solvent-based methods for PHA extraction, the use of the green solvent dimethyl carbonate (DMC) is described. DMC is completely biodegradable and less harmful to humans and the environment than the established “gold-standard” solvent chloroform. This process can be applied to extract PHA from dry biomass, or even directly to concentrated bacterial fermentation slurries; it results in expediently high polymer recovery rates of more than 85% and outstanding product purity of more than 95%. In both cases (dry biomass or slurry), the extraction does not cause any degradation of the polymer´s molecular mass. Another technique developed in the same study applies fatty acid carboxylates; these surfactants disrupt cell membranes, which sets PHA granules free at excellent polymer recovery rates of more than 99% and high purity exceeding 90%. Among these surfactants, ammonium laurate can be efficaciously used and simply recycled (111). Two other non-chlorinated solvents, cyclohexanone and γ-butyrolactone (GBL), were recently examined by Jiang and colleagues to extract PHA from microbial biomass. The biopolyester used in this study was produced by the bacterium Cupriavidus necator H16 by conversion of vegetable oil as the sole carbon source. Using cyclohexanone as solvent at 120°C, 95% of PHA was extracted from the cells within 3 min; the product had a purity similar to that obtained using chloroform. GBL revealed considerably lower recovery yields at the same temperature. For both solvents, the molecular mass and dispersity of PHA were similar to chloroform-extracted products, while the contamination of the polymers by nitrogen residues was marginally higher when extracted using the two new solvents than in the reference case with chloroform. Especially cyclohexanone turned out as a proficient candidate for new, sustainable processes for PHA recovery (112). Technically more advanced approaches for solvent-based PHA extraction resort to special equipment, which allows extracting PHA under extreme process conditions with benign solvents; in this context, Koller et al. described an extraction apparatus, which was successfully used to extract PHA from microbial biomass under elevated temperature and pressure by using acetone as solvent, which does not dissolve crystalline PHA like poly(3-hydroxybutyrate) (PHB) or PHBHV under ambient conditions. Under conditions of about 120°C and 7 bar, it was possible to extract PHA at recovery yields and purity levels even exceeding those of parallel setups with chloroform. Cooling down the solution of PHA in acetone after extraction results in convenient precipitation of highly pure product, while residual biomass is retarded in an integrated filter system (113).
A completely different, entirely biological method of recovering PHA from bacterial biomass was developed by Ong and colleagues; these authors used larvae of the mealworm beetle Tenebrio molitor as PHA-recovery engine. These insects were fed with PHA-rich biomass of C. necator, whereby they digested the non-PHA part of biomass. The remaining white fecal pellets were simply washed with alkaline water. Purity of PHA obtained by this unique process amounted to 94%; moreover, the product did not show any reduction of molecular mass (114).
Major fields of application for PHA and follow-up products
Apart from the most prominent field of application of “bioplastics”, namely replacing plastics as packaging materials, PHA enter to an increasing extent the therapeutic, surgical and tissue engineering realm. In this context, recent reviews summarize current strategies to produce PHA at purity levels required for in vivo use (115, 116, 117, 118, 119, 120). Especially low endotoxin (lipopolysaccharides produced in the cell wall of Gram-negative microbes) levels are required for biopolymers used in vivo in order to prevent inflammatory reactions, which puts Gram-positive PHA production strains (no endotoxin producers) like Bacillus sp. or Streptomyces sp. in a new limelight (121). Such in vivo applications of PHA of varying compositions and their advanced follow-up products encompass carriers for triggered in vivo release of antibiotics or anti-cancer drugs, production of enhanced biodegradable sutures, surgical threads, implants or meshes, scaffolds for tissue engineering, or guidance conduits for nerve repair. To an increasing extend, especially PHA micro- and nanoparticles are used as carriers of active compounds, as demonstrated, e.g., by the retarded release of insulin from phospholipid-coated PHBHHx nano-spheres (122). In the field of cancer therapy, the cytostatic drug docetaxel can be delivered from nano-sized PHBHHx micelles to efficiently treat melanoma (123).
Particularly for the biomedical use, traditional processing techniques for production of PHA-based items, such as melt-spinning, melt extrusion, or solvent evaporation, get more and more outperformed by new emerging processing techniques such as additive manufacturing (3D-printing) to produce scaffolds for tissue engineering (124) or bone development (125), scaffold design by electrospinning (126) or computer-aided wet-spinning (127), or laser perforation (128) and plasma treatment (129) to enhance adhesion and attachment of stem cells and other cells to PHA surfaces. In vivo applicability of PHA can also be enhanced by post-synthetic functionalization of the biopolyesters; only recently, it was shown by Bhatia et al. that PHA functionalized with ascorbic acid provides a new biomaterial with decreased crystallinity, enhanced hydrophilicity, increased thermal degradation temperature, and superior biodegradability; these authors proposed this new product as powerful in vivo radical scavenger (130). In addition to the biomedical use of PHA in its polymeric form, it should be emphasized that the monomeric building blocks obtained by hydrolysis of microbial PHA reveal pharmaceutical activity, as demonstrated by the mitochondria protection mechanism exerted by 3-hydroxybutyrate (3HB), which makes this monomer active against the Alzheimer´s disease (131).
Apart from the biomedical field, another important direction of development is the design of PHA-based composite materials to be used for smart food packaging (132). Recent reviews on this topic underline especially the importance of nanotechnology to improve the properties of PHA-based packaging materials (133, 134, 135). In general, the inherent shortcomings of some biodegradable polymers like PHA such as modest mechanical properties, thermo-oxidative stability, narrow processing windows, and insufficient electrical and thermal properties can be overcome by producing PHA-based composites reinforced with various nanofillers (136). For food packaging, high gas barrier properties are of special importance. In this context, Kovalcik et al. combined PHA with optimized amounts of lignin by thermoforming to design new composite materials for packaging. The impact of the lignin fraction on melting and crystallization behavior, mechanical and viscoelastic properties, thermo-oxidative stability, and gas permeability was studied. Lignin turned out to be highly compatible with the biopolyester, and acted as active additive by reinforcing the polymer. Gas permeability decreased by 77% for oxygen and by 91% for CO2, respectively, when combining PHA with 1 wt.-% lignin. Also thermo-oxidation stability was drastically increased for the materials containing lignin. The authors suggested the use of this new class of PHA-based materials for packaging especially of easily perishable goods like food (137).
Barrier properties of biopolymers can also be improved by developing multi-layer biocomposites, as demonstrated by Fabra et al., who designed multi-layer composites for potential food packaging applications; in this study, layers obtained by melt blending of thermoplastic starch and bacterial nanocellulose whiskers were combined with layers of hybrid electrospun PHA reinforced with bacterial nanocellulose. These composites exerted excellent adhesion between the layers, and superior barriers for oxygen and water, as desired for the purpose of food packaging (138). Apart from organic nanofillers, also inorganic materials can be used to improve PHA properties as packaging material (139). Only recently, Akin and Tihminlioglu investigated the impact of nano-clay particles on water permeability and mechanical properties of PHA homo- and copolyesters. It turned out that, provided an optimal distribution of the nanoparticles in the polymer matrix, water vapor permeability can be drastically reduced, while both tensile strength and strain at break can be significantly increased in PHA samples loaded with 3% nano-clay (140).
Holistic life cycle considerations of the entire PHA production chain
As mentioned before, the motivation to gradually switch towards polymers originated from renewable resources is their hypothetical benefit regarding their environmental impact when compared with their petrol-based challengers. This assumption is generally due to the belief that materials based on renewable resources hold an intrinsic environmental benefit over those based on fossil resources. However, this statement has to be validated as the case arises by assessing all the ecological impacts caused by the production of a given material along its entire life cycle, which spans the bridge from production of the raw material to the fate of the final product after its use. Only systematic and complete life cycle assessments (LCA) are appropriate tools to deliver solid, quantifiable and comprehensive data about the ecological footprint of products; this is the conditio sine qua non needed to reliably answer the question if a given “bioplastic” like PHA and its follow-up products are that superior to outperform established petrol-based plastics in terms of their environmental performance.
As recently reviewed, the current discourse about environmental performance of PHA is exhaustively and controversially discussed in the present scientific literature (17). Based on these studies, it becomes clear that all of the above discussed stages of the PHA production chain need to be considered to identify the most important factors, which decide about the ecological performance of PHA and other biopolymers, and, most of all, to recognize the major fields for further process improvement that, without any doubt, are still present in the PHA production chain. However, comparing these studies makes it clear that PHA-based plastic products can indeed excel fossil competitors regarding their ecological performance, especially if their production is based on the use of (agro)industrial waste and by-products, the use of energy from clean sources, the switch to sustainable techniques for PHA recovery, an optimized bioengineering to run cultivation processes at maximum efficiency in terms of substrate-and energy demand, and the closing of the material cycles. Comprehensive LCA studies for PHA production based on (surplus) products from sugar industry (141), cheese making industry (45), animal processing industry (142, 143, 144), or soybean oil manufacturing (145) clearly substantiate this paradigm. Most of all, these studies underline that, in order to safe transportation costs, efficient PHA production needs to be carried out in-house, hence, in close vicinity to the (agro) industrial production plant, where the substrates used for PHA production accrues as waste stream. Biorefinery concepts, such as those presented in the case of PHA production from animal processing waste or from lignocelluloses, are currently considered those solutions with highest potential for large-scale realization (146). These biorefinery concepts increasingly resort to the implementation of microbial cell factories engineered by the tools of synthetic biology, and especially to robust, often extremophilic production strains, which saves costs for precursor substrates, energy, and quality requirements set for the bioreactor.
By the “Next Generation Industrial Biotechnology” (NGIB) approach described in this review article, PHA and follow-up products can become key players in the emerging global bio-economy game, which is nowadays in the focus of decision makers and opinion leaders from politics and the industrial sector in many global regions (147). As a final summary, Fig. 3 provides a schematic sketch of the entire PHA production chain; in this illustration, the individual key stages of the process are illustrated as planets surrounding the holistic NGIB concept illustrated as central star. The moons circling around the planets illustrate the particular tasks assigned to the process stages, which have to be optimized and considered to develop an economically, ethically and environmentally sustainable PHA production process.
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