Bacterial polyhydroxyalkanoates-eco-friendly next generation plastic: Production,biocompatibility, biodegradation,physical properties and applications

ABSTRACT

Polyhydroxyalkanoates (PHAs) are intracellular aliphatic polyesters synthesized as energy reserves, in the form of water-insoluble, nano-sized discrete and optically dense granules in cytoplasm by a diverse bacteria and some archae under conditions of limiting nutrients in the presence of excess carbon source. Bacteria synthesize different PHAs from coenzyme A thioesters of respective hydroxyalkanoic acid, and degrade intracellularly for reuse and extracellularly in natural environments by other microorganisms. In vivo, PHAs exist as amorphous mobile liquid and water-insoluble inclusions but in vitro, exhibit material and mechanical properties, ranging from stiff and brittle crystalline to elastomeric and molding, similar to petrochemical thermoplastics. Further, they are hydrophobic, isotactic, biocompatible and exhibit piezoelectric properties. But as commodity plastics their applications are limited by high production cost, low yield, in vivo degradation, complexity of technology and difficulty of extraction. Therefore, to replace the conventional plastic with PHAs, it is prerequisite to standardize the PHA production systems.     

Metabolic engineering for microbial production and applications of copolyesters consisting of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates

Poly(hydroxyalkanoate)s (PHAs) are a class of microbially synthesized polyesters that combine biological properties, such as biocompatibility and biodegradability, and non-bioproperties such as thermoprocessability, piezoelectricity, and nonlinear optical activity. PHA monomer structures and their contents strongly affect the PHA properties. Using metabolic engineering approaches, PHA structures and contents can be manipulated to achieve controllable monomer and PHA cellular contents. This paper focuses on metabolic engineering methods to produce PHA consisting of 3-hydroxybutyrate (3HB) and medium-chain-length 3-hydroxyalkanoates (3HA) in recombinant microbial systems. This type of copolyester has mechanical and thermal properties similar to conventional plastics such as poly(propylene) and poly(ethylene terephthalate) (PET). In addition, pathways containing engineered PHA synthases have proven to be useful for enhanced PHA production with adjustable PHA monomers and contents. The applications of PHA as implant biomaterials are briefly discussed here. In the very near term, metabolic engineering will help solve many problems in promoting PHA as a new type of plastic material for many applications.     

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by metabolically engineered Escherichia coli strains

Biosynthesis of polyhydroxyalkanoates (PHAs) consisting of 3-hydroxyalkanoates (3HAs) of 4 to 10 carbon atoms was examined in metabolically engineered Escherichia coli strains. When the fadA and/or fadB mutant E. coli strains harboring the plasmid containing the Pseudomonas sp. 61-3 phaC2 gene and the Ralstonia eutropha phaAB genes were cultured in Luria-Bertani (LB) medium supplemented with 2 g/L of sodium decanoate, all the recombinant E. coli strains synthesized PHAs consisting of C4, C6, C8, and C10 monomer units. The monomer composition of PHA was dependent on the E. coli strain used. When the fadA mutant E. coli was employed, PHA containing up to 63 mol% of 3-hydroyhexanoate was produced. In fadB and fadAB mutant E. coli strains, 3-hydroxybutyrate (3HB) was efficiently incorporated into PHA up to 86 mol%. Cultivation of recombinant fadA and/or fadB mutant E. coli strains in LB medium containing 10 g/L of sodium gluconate and 2 g/L of sodium decanoate resulted in the production of PHA copolymer containing a very high fraction of 3HB up to 95 mol%. Since the material properties of PHA copolymer consisting of a large fraction of 3HB and a small fraction of medium-chain-length 3HA are similar to those of low-density polyethylene, recombinant E. coli strains constructed in this study should be useful for the production of PHAs suitable for various commercial applications.     

Utilization of 2-alkenoic acids for biosynthesis of medium-chain-length polyhydroxyalkanoates in metabolically engineered Escherichia coli to construct a novel chemical recycling system

This study describes the biosynthesis and thermal degradation of medium-chain-length polyhydroxyalkanoate (PHA), focusing on 2-alkenoic acids as a recyclable carbon source. Using metabolically engineered Escherichia coli, PHA consisting of 3-hydroxydecanoate (3HD) was synthesized from 2-decenoic acid. Solvent cast film of poly(3HD) [P(3HD)] was transparent and showed thermal property similar to that of polycaprolactone. In addition, the use of various 2-alkenoic acids (C6-C12) resulted in production of PHAs with over 95 mol% of the corresponding single monomer units. The pyrolysis product of P(3HD) was dominantly 2-decenoic acid used for the P(3HD) biosynthesis. This demonstrates the feasibility of PHA recycling via 2-alkenoic acids, which act as pyrolysis products and raw materials for PHA biosynthesis.     

Biodegradable Polyhydroxyalkanoates by Stereoselective Copolymerization of Racemic Diolides: Stereocontrol and Polyolefin‐Like Properties

Bacterial polyhydroxyalkanoates (PHAs) are a unique class of biodegradable polymers because of their biodegradability in ambient environments and structural diversity enabled by side‐chain groups. However, the biosynthesis of PHAs is slow and expensive, limiting their broader applications as commodity plastics. To overcome such limitation, the catalyzed chemical synthesis of bacterial PHAs has been developed, using the metal‐catalyzed stereoselective ring‐opening (co)polymerization of racemic cyclic diolides (rac‐8DL^R, R=alkyl group). In this combined experimental and computational study, polymerization kinetics, stereocontrol, copolymerization characteristics, and the properties of the resulting PHAs have been examined. Most notably, stereoselective copolymerizations of rac‐8DL^Me with rac‐8DL^R (R=Et, Bu) have yielded high‐molecular‐weight, crystalline isotactic PHA copolymers that are hard, ductile, and tough plastics, and exhibit polyolefin‐like thermal and mechanical properties.     

Crystalline morphology and thermal properties for random copolyesters of (R)‐3‐hydroxybutyric acid with different hydroxyalkanoic groups

The solid‐state structures and thermal properties of melt‐crystallized films of random copolymers of (R)‐3‐hydroxybutyric acid (3HB) with different hydroxyalkanoic acids such as (R)‐3‐hydroxypentanoic acid (3HV), (R)‐3‐hydroxyhexanoic acid (3HH), medium‐chain‐length (R)‐3‐hydroxyalkanoic acids (mcl‐3HA; C8‐C12), 4‐hydroxybutyric acid (4HB), and 6‐hydroxyhexanoic acid (6HH) were characterized by means of small‐angle X‐ray scattering, differential scanning calorimetry, and optical microscopy. The randomly distributed second monomer units except for 3HV in copolyesters act as defects of P(3HB) crystal and are excluded from the P(3HB) crystalline lamellae. The lamellar thickness of copolymers decreased with an increase in either the main‐chain or the side‐chain carbon numbers of second monomer units. In addition, the growth rate of spherulites decreased with an increase in the carbon numbers of second monomer units for copolymers with an identical comonomer composition. These results indicate that the steric bulkiness of second monomer unit affects on the crystallization of 3HB segments in random copolyesters.     

Electrospinning nanofibers of microbial polyhydroxyalkanoates for applications in medical tissue engineering

Differently to most chemically synthesized medical materials, polyhydroxyalkanoates (PHAs) are intracellular carbon and energy storage granules, which is a family of natural bio-polymers synthesized by microorganism's materials. Due to excellent biocompatibility reasonable biodegradability and versatile material difference, PHAs are well medical biomaterials candidates for applications in tissue engineering and drug delivery, including commercial PHB, PHBV, PHBHHx, PHBVHHx, P34HB and few uncommercial PHAs. Electrospinning nanofibers with the size of 10–10,000 nm can improve the mechanical properties and decrease the crystallinity of PHA, meanwhile simulate the structure and function of native extracellular matrix of cells. Hence, PHAs electrospinning nanofibers as engineered scaffolds have been widely used for tissue engineering scaffolds in cardiovascular, vascular, nerve, bone, cartilage and skin; also, as carriers for application in drug delivery system. In this review, we highlight the extraction and properties of medical PHAs from natural or engineered microorganism, and microstructure, current manufacturing techniques and medical applications of electrospinning nanofibers of PHAs. Moreover, the current challenges and prospects of PHAs electrospinning nanofibers are discussed rationally, providing an insight into developing vibrant fields of PHAs electrospinning nanofibers-based biomedicine.     

Medical applications of biopolyesters polyhydroxyalkanoates

Microbial polyhydroxyalkanoates(PHAs) are a family of biopolyesters produced by many wild type and engineered bacteria.PHAs have diverse structures accompanied by flexible thermal and mechanical properties.Combined with their in vitro biodegradation,cell and tissue compatibility,PHAs have been studied for medical applications,especially medical implants applications,including heart valve tissue engineering,vascular tissue engineering,bone tissue engineering,cartilage tissue engineering,nerve conduit tissue engineering as well as esophagus tissue engineering.Most studies have been conducted in the authors’ lab in the past 20+ years.Recently,mechanism on PHA promoted tissue regeneration was revealed to relate to cell responses to PHA biodegradation products and cell-material interactions mediated by microRNA.Very importantly,PHA implants were found not to cause carcinogenesis during long-term implantation.Thus,PHAs should have a bright future in biomedical areas.     

Poly-3-Hydroxyalkanoates containing unsaturated repeating units produced by pseudomonas oleovorans

Poly-3-hydroxyalkanoates (PHAs) containing repeating units with terminal alkene substituents at the 3-position were produced by Pseudomonas oleovorans grown with either 7-octeneoic acid [OA(?)] alone, or 10-undeceneoic acid [UND(?)] alone, or mixtures of UND(?) and either nonanoic acid (NA) or octanoic acid (OA). For the latter, the biomass and PHA yields decreased as the fraction of UND(?) increased in the mixed carbon substrates. Essentially all of the repeating units in the PHA obtained from cells grown with UND(?) alone contained terminal alkene groups, including 3-hydroxy-10-undeceneoate, 3-hydroxy-8-noneneoate, and 3-hydroxy-6-hepteneoate units, but less than half of the units in the PHA from OA(?) had alkene substituents. The PHAs obtained from cells grown with various mixtures of UND(?) and either OA or NA were random copolymers, and the fraction of units with alkene substituents in these polymers increased in proportion to the fraction of UND(?) in the mixed carbon substrates. © 1995 John Wiley & Sons, Inc.     

Asymmetric ZnO Panel‐Like Hierarchical Architectures with Highly Interconnected Pathways for Free‐Electron Transport and Photovoltaic Improvements

Through a rapid and template‐free precipitation approach, we synthesized an asymmetric panel‐like ZnO hierarchical architecture (PHA) for photoanodes of dye‐sensitized solar cells (DSCs). The two sides of the PHA are constructed differently using densely interconnected, mono‐crystalline and ultrathin ZnO nanosheets. By mixing these PHAs with ZnO nanoparticles (NPs), we developed an effective and feasible strategy to improve the electrical transport and photovoltaic performance of the composite photoanodes of DSCs. The highly crystallized and interconnected ZnO nanosheets largely minimized the total grain boundaries within the composite photoanodes and thus served as direct pathways for the transport and effective collection of free electrons. Through low‐temperature (200 °C) annealing, these novel composite photoanodes achieved high conversion efficiencies of up to 5.59 % for ZnO‐based quasi‐solid DSCs.     

Novel routes to epoxy functionalization of PHA‐based electrospun scaffolds as ways to improve cell adhesion

Straightforward and versatile routes to functionalize the surface of poly(3‐hydroxyalkanoate) (PHA) electrospun fibers for improving cell compatibility are reported under relatively mild conditions. The modification of nanofibrous PHAs is implemented through two different methodologies to introduce epoxy groups on the fiber surface: (1) preliminary chemical conversion of double bonds of unsaturated PHAs into epoxy groups, followed by electrospinning of epoxy‐functionalized PHAs blended with nonfunctionalized PHAs, (2) electrospinning of nonfunctionalized PHAs, followed by glycidyl methacrylate grafting polymerization under UV irradiation. The latter approach offers the advantage to generate a higher density of epoxy groups on the fiber surface. The successful modification is confirmed by ATR‐FTIR, Raman spectroscopy, and TGA measurements. Further, epoxy groups are chemically modified via the attachment of a peptide sequence such as Arg‐Gly‐Asp (RGD), to obtain biomimetic scaffolds. Human mesenchymal stromal cells exhibit a better adhesion on the latter scaffolds than that on nonfunctionalized PHA mats. © 2013 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 816–824     

Microbial synthesis and characterization of polyhydroxyalkanoates with fluorinated phenoxy side groups from Pseudomonas putida

Microbial poly(3-hydroxyalkanoates) (PHAs) with fluorinated phenoxy side groups were produced by Pseudomonas putida when fluorophenoxyalkanoic acids were used as carbon sources. 11-(2-Fluorophenoxy)undecanoic acid (2FPUDA), 11-(3-fluorophenoxy)undecanoic acid (3FPUDA), 11-(4-fluorophenoxy)undecanoic acid (4FPUDA), 11-(2,4-difluorophenoxy)undecanoic acid (2,4DFPUDA), 11-(2,4,6-trifluorophenoxy)undecanoic acid (2,4,6TFPUDA), and 11-(2,3,4,5,6-pentaflurophenoxy)undecanoic acid (2,3,4,5,6PFPUDA) were used as carbon sources in the present study. When cells were grown with 2,4DFPUDA, the production of homo poly(3-hydroxy-5-(2,4-difluorophenoxy)pentanoate) was confirmed by NMR and GC/MS analyses. Fluorine atoms inserted into the side chain of the PHA dramatically affected its physical properties. In marked contrast to medium chain length (MCL) PHA, this fluorinated PHA was opaque, cream colored, and possessed greater crystallinity and a higher melting point (∼100 °C) than did the other MCL PHAs. Surface contact angle evaluation revealed that the PHA with two fluorine atoms possessed water-shedding properties. The number of substituted fluorine atoms in the carbon source affected cell growth and difluorine-substituted phenoxyalkanoic acids reduced cell growth, and polymer production compared to non-substituted phenoxyalkanoic acids. No polymeric materials were obtained using either 2,4,6TFPUDA or 2,3,4,5,6PFPUDA.     

Evaluation of the effects of crude glycerol on the production and properties of novel polyhydroxyalkanoate copolymers containing high 11‐hydroxyoctadecanoate by Cupriavidus necator IPT 029 and Bacillus megaterium IPT 429

Crude glycerol (CG), a by‐product from biodiesel production, is a carbon source with potential as feedstock for polyhydroxyalkanoate (PHA) production. PHAs are biological macromolecules synthesized by microorganisms as intracellular carbon and energy storage granules. PHA production and its properties were investigated using Cupriavidus necator IPT 029 and Bacillus megaterium IPT 429 cultivated with CGs from different origins. The highest PHA extraction percentage (71.56% [w/v]) occurred when C. necator IPT 029 metabolized CG 3 (from the processing of biodiesel from castor bean oil). The gas chromatography–mass spectrometry analyses revealed novel PHA constituents as building blocks of medium (3‐hydroxytetradecanoate) and long (11‐hydroxyoctadecanoate) chains. Molar mass distribution revealed range of 121–6900 kDa. The initial degradation temperature ranged from 181.83 to 287.50°C and the crystallinity ranged from 35.30 to 66.70%. The results obtained indicate that C. necator IPT 029 from CG 3 could produce copolymers with industrially applicable thermophysical properties. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthesis,recovery and possible application of medium-chain-length polyhydroxyalkanoates: A short overview

Bacterial polyhydroxyalkanoates (PHAs) are polyesters of 3-hydroxyacids produced as intracellular granules by a large variety of bacteria, currently receiving much attention because of their potential as renewable and biodegradable plastics. The monomer units in these microbial polyesters are all in the R-configuration due to the stereospecificity of biosynthetic enzymes. Pseudomonads synthesise mainly medium-chain-lenght PHAs, formed of monomers of 6 to 14 carbons. The PHA monomer composition is influenced by the substrate added to the growth media and determines the physical properties of the plastic material. The capability of Pseudomonads to incorporate many different functional groups into the PHAs does extend their physical properties and potential applications, and suggests various possibilities to produce tailor-made polymers. The mcl-PHAs are of major interest for specific uses, where chirality and elastomeric property of the polymers are important. In this report we will focus on the biotechnological production, recovery and possible applications of mcl-PHAs.     

FUNCTIONAL POLYHYDROXYALKANOATES SYNTHESIZED BY MICROORGANISMS*

Many bacteria have been found to synthesize a family of polyesters termed polyhydroxyalkanoate, abbreviated asPHA. Some interesting physical properties of PHAs such as piezoelectricity, non-linear optical activity, biocompatibility andbiodegradability offer promising applications in areas such as degradable packaging, tissue engineering and drag delivery.Over 90 PHAs with various structure variations have been reported and the number is still increasing. The mechanicalproperty of PHAs changes from brittle to flexible to elastic, depending on the side-chainlength of PHA. Many attempts havebeen made to produce PHAs as biodegradable plastics using various microorganisms obtained from screening naturalenvironments, genetic engineering and mutation. Due to the high production cost, PHAs still can not compete with the non-degradable plastics, such as polyethylene and polypropylene. Various processes have been developed using low cost rawmaterials for fermentation and an inorganic extraction process tbr PHA purification. However, a super PHA production strainmay play the most critical role for any large-scale PHA production. Our recent study showed that PHA synthesis is acommon phenomenon among bacteria inhabiting various locations, especially oil-contaminated soils. This is very importantfor finding a suitable bacterial strain for PHA production. In fact, PHA production strains capable of rapid growth and rapidPHA synthesis on cheap molasses substrate have been found on molasses contaminated soils. A combination of novelproperties and lower cost will allow easier commercialization of PHA for many applications.     

Synthesis and NMR Analysis in Solution of Oligo(3‐hydroxyalkanoic acid) Derivatives with the Side Chains of Alanine,Valine, and Leucine (β‐Depsides): Coming Full Circle from PHB to β‐Peptides to PHB

Oligomers of 3‐hydroxyalkanoic acids that contain two, three, and six residues with and without O‐terminal (tBu)Ph₂Si and C‐terminal PhCH₂ protection have been synthesized in such a way that the side chains on the oligoester backbone were those of the proteinogenic amino acids Ala (Me), Val (CHMe₂), and Leu (CH₂CHMe₂). The enantiomerically pure 3‐hydroxyalkanoates were obtained by Noyori hydrogenation of the corresponding 3‐oxo‐alkanoates with [Ru((R)‐binap)Cl₂](binap=2,2′bis(diphenylphosphanyl)‐1,1′‐binaphthalene)/H₂ (Scheme 1), and the coupling was achieved under the conditions (pyridine/(COCl)₂, CH₂Cl₂, −78°) previously employed for the synthesis of various oligo(3‐hydroxybutanoic acids) (Schemes 2 and 3). The Cotton effects in the CD spectra of the new oligoesters provided no hints about chiral conformation (cf. a helix) in MeOH, MeCN, octan‐1‐ol, or CF₃CH₂OH solutions (Figs. 1 and 2). Detailed NMR investigations in CDCl₃ solution (Figs. 3–6, and Tables 1–5) of the hexa(3‐hydroxyalkanoic acid) with the side chains of Val (HC), Ala (HB), Leu (HH), Val, Ala, Leu (from O‐ to C‐terminus; 3 ) gave, on the NMR time‐scale, no evidence for the presence of any significant amount of a 2₁‐ or a 3₁‐helical conformation, comparable to those identified in stretched fibers of poly[(R)‐3‐hydroxybutanoic acid], or in lamellar crystallites and in single crystals of linear and cyclic oligo[(R)‐3‐hydroxybutanoic acids], or in the corresponding β‐peptide(s) (the oligo(3‐aminoalkanoic acid) analogs; 1 – 3 ). Thus, the extremely high flexibility (averaged or ‘random‐coil' conformation) of the polyester chain (CO−O rotational barrier ca. 13 kcal/mol; no hydrogen bonding), as compared to polyamide chains (CO−NH barrier ca. 18 kcal/mol; hydrogen bonding) has been demonstrated once again. The possible importance of this structural flexibility, which goes along with amphiphilic properties, for the role of PHB in biology, in evolution, and in prebiotic chemistry is discussed. Structural similarities of natural potassium‐channeling proteins and complexes of oligo(3‐hydroxybutanoates) with Na⁺, K⁺, or Ba²⁺ are alluded to (Figs. 7–9).     

Microwave‐assisted synthesis and characterization of biodegradable block copolyesters based on poly(3‐hydroxyalkanoate)s and poly(D,L‐lactide)

Microwave (MW)‐assisted ring‐opening polymerization (ROP) provides a rapid and straightforward method for engineering a wide array of well‐defined poly(3‐hydroxyalkanoate)‐b‐poly(D,L ‐lactide) (PHA‐b‐PLA) diblock copolymers. On MW irradiation, the bulk ROP of D,L ‐lactide (LA) could be efficiently triggered by a series of monohydroxylated PHA‐based macroinitiators previously produced via acid‐catalyzed methanolysis of corresponding native PHAs, thus affording diblock copolyesters with tunable compositions. The dependence of LA polymerization on temperature, macroinitiator structure, irradiation time, and [LA]₀/[PHA]₀ molar ratio was carefully investigated. It turned out that initiator efficiency values close to 1 associated with conversions ranging from 50 to 85% were obtained only after 5 min at 115 °C. A kinetic investigation of the MW‐assisted ROP of LA gave evidence of its “living”/controlled character under the experimental conditions selected. Structural analyses and thermal properties of biodegradable diblock copolyesters were also performed. © 2012 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2012     

Synthesis of macromonomers bearing hydrolyzable segments derived from α‐hydroxyalkanoic acids

Methacrylic macromonomers bearing hydrolyzable oligoester segments are prepared by derivatization of oligo(α‐hydroxyalkanoic acids) (obtained by thermal polycondensation) with methacrylic acid and copolymerized with tert‐butyl acrylate.     

An Efficient Thiol‐Ene Chemistry for the Preparation of Amphiphilic PHA‐Based Graft Copolymers

We present a straightforward method to prepare amphiphilic graft copolymers consisting of hydrophobic poly(3‐hydroxyalkanoates) (PHAs) backbone and hydrophilic α‐amino‐ω‐methoxy poly(oxyethylene‐co‐oxypropylene) (Jeffamine®) units. Poly(3‐hydroxyoctanoate)‐co‐(3‐hydroxyundecenoate) (PHOU) was first methanolyzed to obtain the desired molar mass. The amino end groups of Jeffamine were converted into thiol by a reaction with N‐acetylhomocysteine thiolactone and subsequently photografted. This “one‐pot” functionalization prevents from arduous and time‐consuming functionalization of the hydrophilic precursor or tedious modifications of PHAs, thus simplifying the process. The amphiphilic nature of modified PHAs leads to water‐soluble copolymers exhibiting thermoresponsive behavior.     

Effect of C∶N molar ratio on monomer composition of polyhdroxyalk anoates produced by Pseudomonas mendocina 0806 and Pseudomonas pseudoalkaligenus YS1

Polyhydroxyalkanoates (PHAs) are biodegradable polymers produced by bacteria. In this study, the effect of C∶N molar ratio on the monomer composition of PHAs was investigated, including medium chain length PHA produced by Pseudomonas mendocina 0806 and PHA blends consisting of monomers of 3-hydroxybutyrate and medium chain length hydroxyalkan⇘te produced by Pseudomonas pseudoalkaligenus YS1. It was observed that there were some fixed ranges of C∶N molar ratio that affect the monomer composition of PHA independently of the substrate. For strain 0806, the ranges were C∶N <20, 20<C∶N<200, and C∶N>200. The monomer composition was constant among these ranges when using glucose and octanoate as the sole substrate. For strain YS1, the ranges were C∶N<20, 20<C∶N<45, and C∶N>45. These results are useful for controlling monomer composition in PHA production.