In vivo and Post-synthesis Strategies to Enhance the Properties of PHB-Based Materials: A Review

The transition toward “green” alternatives to petroleum-based plastics is driven by the need for “drop-in” replacement materials able to combine characteristics of existing plastics with biodegradability and renewability features. Promising alternatives are the polyhydroxyalkanoates (PHAs), microbial biodegradable polyesters produced by a wide range of microorganisms as carbon, energy, and redox storage material, displaying properties very close to fossil-fuel-derived polyolefins. Among PHAs, polyhydroxybutyrate (PHB) is by far the most well-studied polymer. PHB is a thermoplastic polyester, with very narrow processability window, due to very low resistance to thermal degradation. Since the melting temperature of PHB is around 170–180°C, the processing temperature should be at least 180–190°C. The thermal degradation of PHB at these temperatures proceeds very quickly, causing a rapid decrease in its molecular weight. Moreover, due to its high crystallinity, PHB is stiff and brittle resulting in very poor mechanical properties with low extension at break, which limits its range of application. A further limit to the effective exploitation of these polymers is related to their production costs, which is mostly affected by the costs of the starting feedstocks. Since the first identification of PHB, researchers have faced these issues, and several strategies to improve the processability and reduce brittleness of this polymer have been developed. These approaches range from the in vivo synthesis of PHA copolymers, to the enhancement of post-synthesis PHB-based material performances, thus the addition of additives and plasticizers, acting on the crystallization process as well as on polymer glass transition temperature. In addition, reactive polymer blending with other bio-based polymers represents a versatile approach to modulate polymer properties while preserving its biodegradability. This review examines the state of the art of PHA processing, shedding light on the green and cost-effective tailored strategies aimed at modulating and optimizing polymer performances. Pioneering examples in this field will be examined, and prospects and challenges for their exploitation will be presented. Furthermore, since the establishment of a PHA-based industry passes through the designing of cost-competitive production processes, this review will inspect reported examples assessing this economic aspect, examining the most recent progresses toward process sustainability.

Introducing the Newly Isolated Bacterium Aneurinibacillus sp. H1 as an Auspicious Thermophilic Producer of Various Polyhydroxyalkanoates (PHA) Copolymers–2. Material Study on the Produced Copolymers

Aneurinibacillus sp. H1 is a promising, moderately thermophilic, novel Gram-positive bacterium capable of the biosynthesis of polyhydroxyalkanoates (PHA) with tunable monomer composition. In particular, the strain is able to synthesize copolymers of 3-hydroxybutyrate (3HB), 4-hydroxybutyrate (4HB) and 3-hydroxyvalerate (3HV) with remarkably high 4HB and 3HV fractions. In this study we performed an in-depth material analysis of PHA polymers produced by Aneurinibacillus sp. H1 in order to describe how the monomer composition affects fundamental structural and physicochemical parameters of the materials in the form of solvent-casted films. Results of infrared spectroscopy, X-ray diffractometry and thermal analysis clearly show that controlling the monomer composition enables optimization of PHA crystallinity both qualitatively (the type of the crystalline lattice) and quantitatively (the overall degree of crystallinity). Furthermore, resistance of the films against thermal and/or enzymatic degradation can also be manipulated by the monomer composition. Results of this study hence confirm Aneurinibacillus sp. H1 as an auspicious candidate for thermophilic production of PHA polymers with material properties that can be tuned together with their chemical composition by the corresponding adjustment of the cultivation process.

Biosynthesis of Poly(3-Hydroxybutyrate-co-3-Hydroxyvalerate) by Cupriavidus necator B-10646 from Mixtures of Oleic Acid and 3-Hydroxyvalerate Precursors

Polyhydroxyalkanoates have attracted much attention as biodegradable alternative to petroleum-based synthetic plastics. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymer is one of the best characterized PHA copolymers because of its high commercial potential. However, commercial use of PHAs has been limited by their high price. One approach to reducing the cost of PHA production is to use inexpensive carbon sources (fatty acids, plant oils, etc.). The aim of this work was to study synthesis of P(3HB-co-3HV) by the Cupriavidus necator B-10646 bacterium grown on oleic acid and different biochemical precursors of 3HV. Bacterial cells were grown for 72 h at 30°C and 200 rpm on an incubator shaker. Salts of propionic or valeric acids were used as precursors of 3HV. The content and the composition of the polymer were determined by gas chromatography of fatty acid methyl esters. Lipids and polymer were extracted from biomass using the method of Folch. The addition of potassium propionate and valerate did not inhibit bacterial growth and polymer synthesis, the cell concentration and polymer content reaching 9.3-9.5 g/L and 80-83%, respectively. The addition of potassium valerate or propionate led to the synthesis of (P(3HB-co-3HV)) copolymer containing 21.2 and 14.3 mol% of 3HV, respectively. The number average molecular weight (Mn) of the polymer synthesized by the bacterium on oleic acid alone was 220 kDa; the polydispersity of the polymer was 3.5. The polymer synthesized in the presence of potassium valerate and propionate was characterized by a lower Mn (156-178 kDa) and a higher polydispersity of the polymer (4.4-4.9). The main fatty acids (FA) of intracellular lipids were oleic (33.26% of the total FA) and palmitic acid (27.48% of the total FA). The addition of potassium propionate or valerate did not cause any significant changes in the composition of the FA of intracellular lipids of the strain studied. This study demonstrates the ability of C. necator B-10646 to synthesize P(3HB-co-3HV) from mixtures of oleic acid and 3HV precursors. The data obtained can be used to develop and implement an economically feasible process of the P(3HB-co-3HV) production

Introducing the Newly Isolated Bacterium Aneurinibacillus sp. H1 as an Auspicious Thermophilic Producer of Various Polyhydroxyalkanoates (PHA) Copolymers–1. Isolation and Characterization of the Bacterium

Extremophilic microorganisms are considered being very promising candidates for biotechnological production of various products including polyhydroxyalkanoates (PHA). The aim of this work was to evaluate the PHA production potential of a novel PHA-producing thermophilic Gram-positive isolate Aneurinibacillus sp. H1. This organism was capable of efficient conversion of glycerol into poly(3-hydroxybutyrate) (P3HB), the homopolyester of 3-hydroxybutyrate (3HB). In flasks experiment, under optimal cultivation temperature of 45 °C, the P3HB content in biomass and P3HB titers reached 55.31% of cell dry mass and 2.03 g/L, respectively. Further, the isolate was capable of biosynthesis of PHA copolymers and terpolymers containing high molar fractions of 3-hydroxyvalerate (3HV) and 4-hydroxybutyrate (4HB). Especially 4HB contents in PHA were very high (up to 91 mol %) when 1,4-butanediol was used as a substrate. Based on these results, it can be stated that Aneurinibacillus sp. H1 is a very promising candidate for production of PHA with tailored material properties.

Production of Polyhydroxyalkanoates Copolymers by Recombinant Pseudomonas in Plasmid- and Antibiotic-Free Cultures

Three different polyhydroxyalkanoate (PHA) synthase genes (<i>Ralstonia eutropha</i> H16, <i>Aeromonas</i> sp. TSM81 or <i>Aeromonas hydrophila</i> ATCC7966 <i>phaC</i>) were introduced into the chromosome of two <i>Pseudomonas</i> strains: a native medium-chain-length 3-polyhydroxyalkanoate (PHA_MCL) producer (<i>Pseudomonas</i> sp. LFM046) and a UV-induced mutant strain unable to produce PHA (<i>Pseudomonas</i> sp. LFM461). We reported for the first time the insertion of a chromosomal copy of <i>phaC</i> using the transposon system mini-Tn<i>7</i>. Stable antibiotic marker-free and plasmid-free recombinants were obtained. Subsequently, P(3HB-<i>co</i>-3HA_MCL) was produced by these recombinants using glucose as the sole carbon source, without the need for co-substrates and under antibiotic-free conditions. A recombinant harboring <i>A. hydrophila phaC</i> produced a terpolyester composed of 84.2 mol% of 3-hydroxybutyrate, 6.3 mol% of 3-hydroxyhexanoate, and 9.5 mol% of 3-hydroxydecanoate from only glucose. Hence, we were successful in increasing the industrial potential of <i>Pseudomonas</i> sp. LFM461 strain by producing PHA copolymers containing 3HB and 3HA_MCL using an unrelated carbon source, for the first time in a plasmid- and antibiotic-free bioprocess.