Polyhydroxyalkanoate Materials Research Articles

Toward the production of block copolymers in microbial cells: achievements and perspectives.

The microbial production of polyhydroxyalkanoate (PHA) block copolymers has attracted research interests because they can be expected to exhibit excellent physical properties. Although post-polymerization conjugation and/or extension have been used for PHA block copolymer synthesis, the discovery of the first sequence-regulating PHA synthase, PhaCAR, enabled the direct synthesis of PHA-PHA type block copolymers in microbial cells. PhaCAR spontaneously synthesizes block copolymers from a mixture of substrates. To date, Escherichia coli and Ralstonia eutropha have been used as host strains, and therefore, sequence regulation is not a host-specific phenomenon. The monomer sequence greatly influences the physical properties of the polymer. For example, a random copolymer of 3-hydroxybutyrate and 2-hydroxybutyrate deforms plastically, while a block copolymer of approximately the same composition exhibits elastic deformation. The structure of the PHA block copolymer can be expanded by in vitro evolution of the sequence-regulating PHA synthase. An engineered variant of PhaCAR can synthesize poly(D-lactate) as a block copolymer component, which allows for greater flexibility in the molecular design of block copolymers. Therefore, creating sequence-regulating PHA synthases with a further broadened substrate range will expand the variety of properties of PHA materials. This review summarizes and discusses the sequence-regulating PHA synthase, analytical methods for verifying block sequence, properties of block copolymers, and mechanisms of sequence regulation. KEY POINTS: • Spontaneous monomer sequence regulation generates block copolymers • Poly(D-lactate) segment can be synthesized using a block copolymerization system • Block copolymers exhibit characteristic properties.

Synthesis and characterisation of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) multi-block copolymers comprising blocks of differing 3-hydroxyvalerate contents

Polyhydroxyalkanoates (PHAs) are attractive alternatives to commodity petroleum derived plastics, being bacterially derived, thermoprocessible and truly biodegradable under ambient conditions. However, the common short chain length PHAs - poly(3-hydroxybutyrate) (P3HB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) - are relatively stiff and brittle, and P3HB in particular has a narrow processing window. Block copolymer synthesis offers an opportunity to produce novel PHA materials comprised of different PHA phases, delivering a potential step-change improvement in PHA properties due to microphase separation. Therefore, this work demonstrates the synthesis and characterisation of novel high molecular weight PHBV-b-PHBV-b-PHBV block copolymers using isocyanate chemistry. High molecular weight, narrow compositional distribution, hydroxy-functionalised PHBV blocks of low (1 mol%) 3HV and high (67 mol%) 3HV contents were used as building blocks. The resulting block copolymers showed a very significant increase in elongation at break (to 76.0 %), as well as improved tensile toughness (to 15.0 MJ/m3) and tensile strength (to 18.5 MPa) compared to the starting materials or comparable blends. Based on the similarity in properties between the 1 mol% 3HV block copolymer products and their counterpart random copolymers, it is also shown that the introduction of urethane linkages had minimal effect on polymer properties. The morphologies of these novel PHBV materials were investigated, supporting the concept that the microphase separation of the different PHBV phases delivered these improved mechanical properties. This approach to PHA-b-PHA block copolymer synthesis offers an attractive generic platform for the production of novel biodegradable PHA materials for film and other applications.

Sustainable bioconversion of synthetic plastic wastes to polyhydroxyalkanoate (PHA) bioplastics: recent advances and challenges

Millions of tons of chemical plastics are accumulated annually worldwide in terrestrial and marine environments due to inadequate recycling plants and facilities and low circular use. Their continuous accumulation and contamination of soil and water pose a severe threat to the environment and to human, animal and plant health. There is therefore an urgent need to develop effective eco-environmental strategies to overcome the significant environmental impacts of traditional plastic waste management practises (incineration, landfilling, and recycling). In recent years, reports on microbial strains equipped with the potential of degrading plastic materials, which can further be converted into usable products such as PHA bioplastics have sprung up, and these offer a possibility to develop microbial and enzymatic technologies for plastic waste treatment and then progressing plastics circularity. In this chapter, an overview of the reported microbial and enzymatic degradations of petroleum-based synthetic plastics, specifically polyethylene, polystyrene, polypropylene, polyvinyl chloride, polyurethane and polyethylene terephthalate, is detailed. Furthermore, the harvesting of depolymerization products to produce new PHA materials with high added industrial value can be considered as an innovative solution, helping to increase synthetic plastic recycling rate and creating new circular economy opportunities. Finally, the challenge of ending plastic pollution is still difficult, but sustainable, renewable, bio-based and completely biodegradable, PHA will hold enormous promise for replacing plastics made from petrochemicals.

Recent Development of Polyhydroxyalkanoates (PHA)-Based Materials for Antibacterial Applications: A Review.

The diseases caused by microorganisms are innumerable existing on this planet. Nevertheless, increasing antimicrobial resistance has become an urgent global challenge. Thus, in recent decades, bactericidal materials have been considered promising candidates to combat bacterial pathogens. Recently, polyhydroxyalkanoates (PHAs) have been used as green and biodegradable materials in various promising alternative applications, especially in healthcare for antiviral or antiviral purposes. However, it lacks a systematic review of the recent application of this emerging material for antibacterial applications. Therefore, the ultimate goal of this review is to provide a critical review of the state of the art recent development of PHA biopolymers in terms of cutting-edge production technologies as well as promising application fields. In addition, special attention was given to collecting scientific information on antibacterial agents that can potentially be incorporated into PHA materials for biological and durable antimicrobial protection. Furthermore, the current research gaps are declared, and future research perspectives are proposed to better understand the properties of these biopolymers as well as their possible applications.

Combination of Hypotonic Lysis and Application of Detergent for Isolation of Polyhydroxyalkanoates from Extremophiles.

Production of polyhydroxyalkanoates (PHA), microbial biopolyesters, employing extremophilic microorganisms is a very promising concept relying on robustness of such organisms against microbial contamination, which provides numerous economic and technological benefits. In this work, we took advantage of the natural susceptibility of halophilic and thermophilic PHA producers to hypotonic lysis and we developed a simple and robust approach enabling effective isolation of PHA materials from microbial cells. The method is based on the exposition of microbial cells to hypotonic conditions induced by the diluted solution of sodium dodecyl sulfate (SDS) at elevated temperatures. Such conditions lead to disruption of the cells and release of PHA granules. Moreover, SDS, apart from its cell-disruptive function, also solubilizes hydrophobic components, which would otherwise contaminate PHA materials. The purity of obtained materials, as well as the yields of recovery, reach high values (values of purity higher than 99 wt.%, yields close to 1). Furthermore, we also focused on the removal of SDS from wastewater. The simple, inexpensive, and safe technique is based on the precipitation of SDS in the presence of KCl. The precipitate can be simply removed by decantation or centrifugation. Moreover, there is also the possibility to regenerate the SDS, which would substantially improve the economic feasibility of the process.

Production and Characterization of Polyhydroxyalkanoates from Wastewater via Mixed Microbial Cultures and Microalgae

In the context of circular economy and sustainable production of materials, this project investigated the feasibility of producing sustainable polyhydroxyalkanoates (PHA) from microalgae and sludge used in the treatment of municipal wastewater. The overall process was studied looking at the main steps: microalgae production, fermentation of the biomass, production and characterization of the PHAs. It was possible to obtain blends of hydroxybutyrate-hydroxyvalerate copolymers with high molecular weights and different compositions depending on the nature of the feedstock (mixed volatile fatty acids). In some cases, almost completely amorphous PHA materials were obtained, suggesting a potential diversification of uses and applications.

Enhancing the biological reductive dechlorination of trichloroethylene with PHA from mixed microbial cultures (MMC)

The biological reductive dechlorination (BRD) process is often limited by the lack of suitable electron donors, requiring the supply of long-lasting substrates to enhance the biological metabolism. Among the others, polyhydroxyalkanoates (PHA) are a particularly interesting slow-release source of electron-donors, being entirely biodegradable polyesters. Generally, industrial PHA production processes are based on pure culture fermentation with high related costs. In recent years, innovative and cost-effective PHA production from mixed microbial cultures (MMC) and waste feedstocks is attracting considerable attention. This research, for the first time, investigated the effect of distinctive types of PHA on the BRD process of trichloroethylene (TCE), in a continuous-flow lab-scale system using two PHA materials with different purity grade produced from MMC at pilot scale in comparison with a commercial PHA produced from pure culture. Promising results have been obtained with non-extracted MMC-PHA, a material consisting of both PHA (56%, w/w) and microbial cells, with constant production of acids over 110 days, which stimulated a nearly complete TCE dechlorination with the non-toxic ethene as the main byproduct (approximately 92%). Dehalococcoides mccartyi was the main microorganism responsible for TCE dechlorination process, representing ≥ 54.05% of the bacterial population, mainly carrying the reductive dehalogenase genes tceA and vcrA (≥9.17E+08 and ≥2.01E+07 gene copies/g of PHA or sand, respectively). This finding suggests the possibility to directly use PHA-rich biomass deriving from MMC production process, which does not require any polluting and expensive extraction procedures, as a novel material in the field of groundwater remediation with noticeable economic and environmental advantages.

Design of Polyhydroxyalkanoate (PHA) Microbeads with Tunable Functional Properties and High Biodegradability in Seawater

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) were used to prepare microbeads, with diameter ranging from 50 to 100 µm, by an emulsion-evaporation process. The emulsification-evaporation process enables the formation of spherical polyhydroxyalcanoate beads, with crystalline rates similar to the ones of the former polymer and with important surface roughness as compared to amorphous polylactic acid smooth beads. The mechanical properties of the different PHA beads are also found to be intimately linked with their crystalline content, with modulus varying between 1 and 7 GPa. The degradation behavior of these PHA microbeads was tested under marine environment and revealed a rapid degradation, similar to cellulose, and a degradation rate correlated with the crystalline content. These results emphasize the possibility and interest in developing PHA materials with tunable functions and degradation properties.

Reducing off-Flavour in Commercially Available Polyhydroxyalkanoate Materials by Autooxidation through Compounding with Organoclays.

Polyhydroxyalkanoates (PHAs) are nowadays considered competent candidates to replace traditional plastics in several market sectors. However, commercial PHA materials exhibit unsatisfactory smells that can negatively affect the quality of the final product. The cause of this typical rancid odour is attributed to oxidized cell membrane glycolipids, coming from Gram-negative production strains, which remain frequently attached to PHAs granules after the extraction stage. The aim of this research is the development of customised PHA bio-nano-composites for industrial applications containing organomodified nanoclays with high adsorbance properties able to capture volatile compounds responsible for the displeasing fragrance. To this end, a methodology for the detection and identification of the key volatiles released due to oxidative degradation of PHAs has been established using a headspace solid-phase microextraction technique. We report the development of nine bio-nano-composite materials based on three types of commercial PHA matrices loaded with three species of nanoclays which represent a different polar behaviour. It has been demonstrated that although the reached outcoming effect depends on the volatile nature, natural sepiolite might result in the most versatile candidate for any the PHA matrices selected.

Production of polyhydroxyalkanoates (PHA)-based renewable packaging materials using photonic energy: A bench and pilot-scale study

This work describes the utilization of a new photonic heat treatment technology in combination with polyhydroxyalkanoates (PHA) materials for the development of biofriendly packaging and coated substrate materials. This technology advances the use of aqueous PHA coatings as a replacement for less environmentally friendly extruded plastic and fluorotreated packaging papers and boards. The new technology utilizes short burst photonic energy to heat PHA latex coatings. Utilizing a rapid pulse of photonic energy enables PHA particles to melt and form a film within milliseconds, as compared to conventional equilibrium drying that takes several minutes. Coatings were applied to commercially produced papers in order to validate the combined use of photonic treatment technology with PHA materials for commercial applications. Photonic treatment for both bench-top and roll-toroll pilot-scale studies resulted in kit values of 12, and 2 min Cobb values of <2 g/m2. Repulpability studies showed the material to be completely repulpable, having 100% accepts after screening. The results demonstrate that the photonic treatment of PHA polymers could be used in current commercial settings to produce environmentally friendly packaging and coated products.

Review of Polyhydroxyalkanoates Materials and other Biopolymers for Medical Applications

Review of Polyhydroxyalkanoates Materials and other Biopolymers for Medical Applications

Binary polyhydroxyalkanoate systems for soft tissue engineering

Progress in tissue engineering is dependent on the availability of suitable biomaterials. In an effort to overcome the brittleness of poly(3-hydroxybutyrate), P(3HB), a natural biodegradable polyester, and widen its biomedical applications, plasticising of P(3HB) with oligomeric substances of related structure has been studied. A biosynthesised medium-chain-length polyhydroxyalkanoate (mcl-PHA) copolymer, the plasticiser precursor, was obtained using vegetable waste frying oil as a sole carbon source. The mcl-PHA was transformed into an oligomeric derivative by acid hydrolysis. The plasticising effect of the oligomeric mcl-PHA on P(3HB) was studied via characterisation of thermal and mechanical properties of the blends in the course of ageing at ambient conditions. Addition of oligomeric mcl-PHA to P(3HB) resulted in softer and more flexible materials based entirely on PHAs. It was shown that the oligomeric mcl-PHA transformed highly crystalline P(3HB) into materials with a dominant amorphous phase when the content of oligomeric mcl-PHA exceeded 10 wt%. In vitro biocompatibility studies of the new binary PHA materials showed high viability and proliferation of C2C12 myoblast cells. Thus, the proposed approach for P(3HB) plasticisation has the potential for the generation of more pliable biomaterials based on P(3HB) which can find application in unique soft tissue engineering applications where a balance between stiffness, tensile strength and ductility is required. Statement of SignificancePolyhydroxyalkanoates, a broad family of natural biodegradable and biocompatible polymers, have emerged as highly promising biomaterials both for bulk and biomedical applications. Here we describe an approach to tune the mechanical properties of stiff and brittle poly(3-hydroxybutyrate) and thereby to expand its potential biomedical applications. Plasticisation, a common practice in the plastic industry to modify polymer mechanical properties, has been used very cautiously for biomedical applications due to plasticiser toxicity and migration. We have developed a plasticiser for poly(3-hydroxybutyrate) based on a structurally related but softer and pliable medium chain length polyhydroxyalkanoate. Additives of oligomeric derivatives of this polymer improved ductility of poly(3-hydroxybutyrate), greatly widening the future applicability of this well-established biomaterial. In parallel, the binary polyhydroxyalkanoate materials also exhibited improved cell attachment and proliferation, a highly desirable outcome.

Polyhydroxyalkanoates (PHA) production in bacterial co‐culture using glucose and volatile fatty acids as carbon source

Mixed bacterial cultures are increasingly being used in the production of polyhydroxyalkanoates (PHAs), as they have the potential to be more cost effective than axenic pure cultures. The purpose of this study was to use pure cultures in combination to identify their potential of PHA production. In this work we used volatile fatty acids (VFAs) and glucose as carbon source to check the ability of selected strains ST2 (Pseudomonas sp.) and CS8 (Bacillus sp.) as co-culture. The production of PHA in pure co-cultures of bacteria was therefore investigated in order to understand the effect of combining cultures on PHA production parameters and material properties. Bacteria could use the feed in better way when mixed as compared to individual strain. In undertaking this analysis, model volatile fatty acids (i.e., acetic and propionic acids) were used alone and in combination with glucose as feedstock. The production by Pseudomonas was 34% while 24% by Bacillus. However when combined and mixed feed (glucose + propionic acid) was used, 35% PHA produced. Overall, it was found that the ability of the pure cultures to produce PHA was low but when selected cultures were mixed, their ability to produce PHA was enhanced. Copolymers were obtained instead of homopolymers with improved properties. This suggests that industrial wastewater rich in volatile fatty acids and carbohydrates can be a good carbon source for PHA production with variable properties.

Polyhydroxyalkanoate-based drug delivery systems

Microbial polyhydroxyalkanoates (PHAs) have been a subject of significant research interest in the past few decades. The recent development of novel functionalized PHAs has opened up new possibilities to combine the good biocompatibility of PHA-based drug delivery systems to, for example, improve drug loading and release properties, targeting or imaging functionalities. This mini-review presents some recent scientific developments in the preparation of functionalized PHAs, PHA–drug and PHA–protein conjugates, multifunctional PHA nanoparticles and micelles as well as biosynthetic PHA particles for drug delivery. These developments in combination with the generally excellent biocompatibility of PHA materials are expected to further expand the interest in PHA materials for drug delivery and other therapeutic applications. © 2016 Society of Chemical Industry

Class IV polyhydroxyalkanoate (PHA) synthases and PHA-producing Bacillus.

This review highlights the recent investigations of class IV polyhydroxyalkanoate (PHA) synthases, the newest classification of PHA synthases. Class IV synthases are prevalent in organisms of the Bacillus genus and are composed of a catalytic subunit PhaC (approximately 40kDa), which has a PhaC box sequence ([GS]-X-C-X-[GA]-G) at the active site, and a second subunit PhaR (approximately 20kDa). The representative PHA-producing Bacillus strains are Bacillus megaterium and Bacillus cereus; the nucleotide sequence of phaC and the genetic organization of the PHA biosynthesis gene locus are somewhat different between these two strains. It is generally considered that class IV synthases favor short-chain-length monomers such as 3-hydroxybutyrate (C4) and 3-hydroxyvalerate (C5) for polymerization, but can polymerize some unusual monomers as minor components. In Escherichia coli expressing PhaRC from B. cereus YB-4, the biosynthesized PHA undergoes synthase-catalyzed alcoholytic cleavage using endogenous and exogenous alcohols. This alcoholysis is thought to be shared among class IV synthases, and this reaction is useful not only for the regulation of PHA molecular weight but also for the modification of the PHA carboxy terminus. The novel properties of class IV synthases will open up the possibility for the design of new PHA materials.

On the use of different hydrocolloids as electrospun adhesive interlayers to enhance the barrier properties of polyhydroxyalkanoates of interest in fully renewable food packaging concepts

This paper compares the effect of adding different high barrier nanostructured interlayers with adhesive properties, made of electrospun whey protein isolate (WPI), pullulan, zein and zein blends with WPI and pullulan, to a polyhydroxyalkanoates material, specifically, polyhydroxybutyrate-co-valerate with a valerate content of 3% (PHBV3) in a multilayer form. The attained morphology, thickness and inherent permeability of the electrospun nanostructures played an important role on oxygen and water barrier properties of these multilayer systems, since both permeability values were only improved when fibrillar morphologies (pullulan and/or zein fibres) were obtained. More specifically, oxygen and water vapour permeability values were improved between 38–48% and 28–35%, respectively, in multilayer systems prepared by the addition of electrospun zein and/or pullulan nanofibres. However, the electrospun WPI nanostructures, exhibiting a beaded morphology, did not significantly decrease the water vapour and oxygen permeability values of the PHBV3-multilayer systems as did zein and pullulan (fibres). These results add new insight, to other previous studies, into the potential of this processing method for the development of biodegradable PHBV multilayer systems of significant interest in food packaging applications.

Nanostructured interlayers of zein to improve the barrier properties of high barrier polyhydroxyalkanoates and other polyesters

This paper compares the effect of incorporating high barrier self-adhesive nanostructured interlayers of zein between layers of other polyhydroxyalkanoate materials (a PHB homopolymer and a PHBV5 copolymer with 5% valerate content). Furthermore, similar reference multilayer structures based on both a polylactide (PLA) and a polyethylene terephthalate (PET) were also prepared, retaining high levels of transparency. The results indicate significant differences of the effect of the interlayer between different materials. Thus, while the oxygen and limonene barrier properties were significantly improved by the presence of a zein nanostructured interlayer, the water vapour permeability of these multilayer systems were seem to vary among materials since the zein interlayer was only efficient as a barrier element in the PHA materials as compared to PLA. Flexibility of PHB and PHBV multilayer structures were also greatly improved by the addition of zein interlayers whereas minor changes were observed in PLA and PET multilayer systems. These results provide complementary insight into the use of electrospun nanostructured zein to generate fully renewable solutions in multilayer form to enhance the barrier performance of polyhydroxyalcanoates.

Enhanced proliferation and differentiation of neural stem cells grown on PHA films coated with recombinant fusion proteins

Polyhydroxyalkanoates (PHAs) belong to a family of copolyesters with demonstrated biocompatibility. We hypothesize that genetically fusing evolutionarily preserved cell binding motifs, such as RGD or IKVAV, to the PHA-binding protein phasin (PhaP) for surface functionalization of PHA materials could better support the growth and differentiation of neural stem cells (NSCs). This hypothesis is tested on three polyester materials of the same aliphatic family: poly(L-lactic acid) (PLA) and two PHB copolymers, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate-co-3-hydroxyhexanoate) (PHBVHHx). Experimental results indicate that surface coating of the two fusion proteins, PhaP–RGD and PhaP–IKVAV, provides short-term advantages in promoting the adhesion, proliferation and neural differentiation of rat NSCs compared to the PhaP-coated or uncoated material. Among the tested samples, the combination of coating PhaP–IKVAV on an PHBVHHx surface yields the highest levels in cell adhesion and proliferation, while the PLA film coated with PhaP–IKVAV promotes better neural differentiation and neurite outgrowth in the early stage. Because both PhaP–RGD and PhaP–IKVAV could be produced in an inexpensive manner, our data suggest that PhaP–IKVAV is an ideal nonspecific coating agent to functionalize hydrophobic biomaterials in the application of neural tissue engineering.

Reaction of the Rat Tissues to Implantation of Polyhydroxyalkanoate Films and Ultrafine Fibers

The reaction of various tissues of rats to implantation of polyhydroxyalkanoate films and ultrafine fibers was studied by optic microscopy. Implantation of polyhydroxyalkanoate films into the abdominal cavity caused a peritoneal reaction, leading after 1 month to the formation of fibrous adhesions between polyhydroxyalkanoate and intestinal loops. Under the skin and in the muscle tissue polyhydroxyalkanoate films were encapsulated in a thick fibrous capsule. Implantation of polyhydroxyalkanoate ultrathin fibers led to formation of foreign body granulomas in all tissues with perifocal inflammation and sclerosis of the adjacent tissues. The polymer was fragmented in these granulomas and phagocytosed by macrophages with the formation of giant foreign body cells. Hence, polyhydroxyalkanoate materials implanted in vivo caused chronic granulomatous inflammatory reaction and were very slowly destroyed by macrophages.

The behaviour of neural stem cells on polyhydroxyalkanoate nanofiber scaffolds

Polyhydroxyalkanoates (PHA) have demonstrated their potentials as medical implant biomaterials. Neural stem cells (NSCs) grown on/in PHA scaffolds may be useful for repairing central nervous system (CNS) injury. To investigate this possibility, nanofiber matrices (scaffolds) prepared from several PHA via a novel phase separation process were studied to mimic natural extracellular matrix (ECM), and rat-derived NSCs grown in the PHA matrices were characterized regarding their in vitro differentiation behaviors. All three PHA materials including poly(3-hydroxybutyrate) (PHB), copolymer of 3-hydroxybutyrate and 4-hydroxybutyrate (P3HB4HB), and copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx) supported NSC growth and differentiation both on their 2D films and 3D matrices. Among three PHA nanofiber matrices, PHBHHx one showed the strongest potentials to promote NSC differentiation into neurons which is beneficial for CNS repair. Compared to the 2D films, 3D nanofiber matrices appeared to be more suitable for NSC attachment, synaptic outgrowth and synaptogenesis. It was suggested that PHBHHx nanofiber scaffolds (matrices) that promote NSC growth and differentiation, can be developed for treating central nervous system injury.