Copolymers Of 3-hydroxybutyrate Research Articles

Modelling across Multiple Scales to Design Biopolymer Membranes for Sustainable Gas Separations: 2-Multiscale Approach

The majority of materials used for membrane-based separation of gas mixtures are non-renewable and non-biodegradable, and the assessment of alternative bio-based polymers requires expensive and time-consuming experimental campaigns. This effort can be reduced by adopting suitable modelling approaches. In this series of works, we propose various modelling approaches to assess the CO2/CH4 separation performance of eight different copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV) using a limited amount of experimental data for model calibration. In part 1, we adopted a fully atomistic approach based on Molecular Dynamics (MD), while, in this work, we propose a multiscale methodology where a molecular description of the polymers is bridged to a macroscopic prediction of its gas sorption behaviour. PHBV structures were simulated using MD to obtain pressure–volume–temperature data, which were used to parametrise the Sanchez–Lacombe Equation of State. This, in turn, allows for the evaluation of the CO2 and CH4 solubility in the copolymers at various pressures and compositions with little computational effort, enabling the estimate of the sorption-based selectivity. The gas separation performance obtained with this multiscale technique was compared to results obtained with a fully atomistic model and experimental data. The solubility–selectivity for the CO2/CH4 mixture is in reasonable agreement between the two models and the experimental data. The multiscale method presented is a time-efficient alternative to fully atomistic methods and detailed experimental campaigns and can accelerate the introduction of renewable materials in different applications.

Multi-stage process for mixed microbial culture production of polyhydroxyalkanoates from sugarcane stillage: Assessment of external nutrient supplementation

Sugarcane stillage is an abundant wastewater from ethanol production. It has drawn considerable interest as a potential feedstock for biotechnological processes aiming at the recovery of energy and value-added products. In this study we explored the use of stillage for the production of polyhydroxyalkanoates (PHA), which are microbial bioplastics with potential to replace conventional plastics in specific applications. As stillage is nutrient deficient, we investigated the impact of external nutrient supplementation on the selection of PHA-producing mixed cultures. Cultures selected under four different total-chemical-oxygen-demand-to-nitrogen ratios (COD.t/N) (15, 30, 40 and 80) were evaluated according to their PHA production performance and microbial composition. The experimental results demonstrated that the most favorable PHA production was achieved for the biomass selected under strict carbon-limiting conditions, with a COD.t/N ratio of 15. This resulted in an overall PHA volumetric productivity of 2.10 g PHA L−1 d−1, a maximum intracellular PHA content of 0.65 g PHA g VSS−1 and a PHA storage yield of 0.71 g CODPHA g CODH.Org−1. The resulting PHA was a copolymer of 3-hydroxybutyrate (73 %mol) and 3-hydroxyvalerate (27 %mol) monomers. The microbial consortium was enriched by members of the Brevundimonas genus, known PHA producers, with a relative abundance of 26.4 %.

Monodisperse polyhydroxyalkanoate nanoparticles as self-sticky and bio-resorbable tissue adhesives

Monodisperse nanoparticles of biodegradable polyhydroxyalkanoates (PHAs) polymers, copolymers of 3-hydroxybutyrate (3HB) and 4-hydroxybutyrate (4HB), are synthesized using a membrane-assisted emulsion encapsulation and evaporation process for biomedical resorbable adhesives. The precise control over the diameter of these PHA particles, ranging from 100 nm to 8 μm, is achieved by adjusting the diameter of emulsion or the PHA concentration. Mechanical properties of the particles can be tailored based on the 3HB to 4HB ratio and molecular weight, primarily influenced by the level of crystallinity. These monodisperse PHA particles in solution serve as adhesives for hydrogel systems, specifically those based on poly(N, N-dimethylacrylamide) (PDMA). Semi-crystalline PHA nanoparticles exhibit stronger adhesion energy than their amorphous counterparts. Due to their self-adhesiveness, adhesion energy increases even when those PHA nanoparticles form multilayers between hydrogels. Furthermore, as they degrade and are resorbed into the body, the PHA nanoparticles demonstrate efficacy in in vivo wound closure, underscoring their considerable impact on biomedical applications.

Modelling across Multiple Scales to Design Biopolymer Membranes for Sustainable Gas Separations: 1—Atomistic Approach

In this work, we assessed the CO2 and CH4 sorption and transport in copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), which showed good CO2 capture potential in our previous papers, thanks to their good solubility-selectivity, and are potential biodegradable alternatives to standard membrane-separation materials. Experimental tests were carried out on a commercial material containing 8% of 3-hydroxyvalerate (HV), while molecular modelling was used to screen the performance of the copolymers across the entire composition range by simulating structures with 0%, 8%, 60%, and 100% HV, with the aim to provide a guide for the selection of the membrane material. The polymers were simulated using molecular dynamics (MD) models and validated against experimental density, solubility parameters, and X-ray diffraction. The CO2/CH4 solubility-selectivity predicted by the Widom insertion method is in good agreement with experimental data, while the diffusivity-selectivity obtained via mean square displacement is somewhat overestimated. Overall, simulations indicate promising behaviour for the homopolymer containing 100% of HV. In part 2 of this series of papers, we will investigate the same biomaterials using a macroscopic model for polymers and compare the accuracy and performance of the two approaches.

Films Based on a Blend of PVC with Copolymer of 3-Hydroxybutyrate with 3-Hydroxyhexanoate.

Polymeric packaging materials are one of the factors of environmental pollution. Reducing the environmental burden is possible by increasing the environmental friendliness of packaging materials. In this work, we study polymer films based on polyvinyl chloride (PVC) with a copolymer of 3-hydroxybutyrate with 3-hydroxyhexanoate P (3-GB) (3-GG) with different component ratios. The process of processing blends in the process of obtaining a packaging film is considered. The optical characteristics of the obtained films are determined. Thermal analysis of the obtained films was carried out using the differential scanning calorimetry (DSC), TGA, and thermomechanical analysis (TMA) methods. The degree of gelling of the resulting mixture was determined. It is shown that PHB has miscibility with PVC.

Effects of the Organic Loading Rate on Polyhydroxyalkanoate Production from Sugarcane Stillage by Mixed Microbial Cultures.

Stillage is an abundant wastewater from the sugarcane ethanol industry. It is rich in fermentable substrates and presents low-nutrient content, constituting a promising substrate for polyhydroxyalkanoate (PHA) production by mixed microbial cultures (MMC). This work assessed the enrichment of a PHA-accumulating MMC from acidified sugarcane stillage in a sequencing batch reactor under increasing organic loading rates (OLR) and no external nutrient supplementation. The OLR was increased from 1.0 to 7.1kg COD m-3day-1 in four steps. A PHA-producing MMC with high storage response was selected in all experimental conditions. The volumetric biomass productivity and the maximal PHA storage capacity increased continuously with the OLR, reaching 0.061g VSS L-1h-1 and 0.49g PHA g VSS-1, respectively. The highest observed PHA storage yield (0.60g CODPHA g COD.t-1) and specific PHA storage rate (0.169g CODPHA g of CODX h-1) were obtained for the OLR of 4.5kg COD m-3day-1. The PHA produced was a co-polymer of 3-hydroxybutyrate (86-77%mol) and 3-hydroxyvalerate (14-23%mol). The performance of the biomass enrichment was comparable to those attained with other agro-industrial wastewaters, indicating the potential of acidified sugarcane stillage as a feedstock for MMC PHA production.

Effect of nitrogen limitation on polyhydroxyalkanoates production efficiency, properties and microbial dynamics using a soil-derived mixed continuous culture

ABSTRACTIn the present study, a soil-derived mixed microbial consortium was developed for polyhydroxyalkanoates (PHAs) production via nutrient limitation. The enhanced consortium was then cultured continuously in sequential batch and the effect of different nitrogen to carbon ratios (N:C) on the yields and properties of produced PHAs and on the changes of the microbial population were investigated. In all cases, the produced polymers were identified as blends or co-polymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) units, with 3HB being the dominant monomer. The degradation profile and the transition temperatures of the produced PHAs were further assessed and compared, as well as the molecular weights which ranged from 77.104 Da to 180.104 Da. In order to further investigate the effect of culturing conditions on the development and alteration of the microbial consortium, the Nile blue live staining was applied in the mixed cultures at the end of each operational period. Various fluorescent single colonies were selected based on differences in their morphology and were identified. Finally, the alterations in microbial diversity and community composition assessed via the RISA profiling method (rRNA Intergenic Spacer Analyses), resulted in the identification of a number of dominant organisms.

Engineering Pseudomonas entomophila for synthesis of copolymers with defined fractions of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates

Polyhydroxyalkanoates (PHA) composed of both short-chain-length (SCL) and medium-chain-length (MCL) monomers (SCL-co-MCL PHA) combine the advantages of high strength and elasticity provided by SCL PHA and MCL PHA, respectively. Synthesis of SCL-co-MCL PHA, namely, copolymers of 3-hydroxybutyrate (3HB) and MCL 3-hydroxyalkanoates (3HA) such as 3-hydroxydecanoate (3HD) and longer chain 3HA, has been a challenge for a long time. This study aims to engineer Pseudomonas entomophila for synthesizing P(3HB-co-MCL 3HA) via weakening its β-oxidation pathway combined with insertion of 3HB synthesis pathway consisting of β-ketothiolase (phaA) and acetoacetyl-CoA reductase (phaB). 3HB and MCL 3HA polymerization is catalyzed by a low specificity PHA synthase (phaC), namely, mutated PhaC61-3. The link between the fatty acid de novo synthesis and PHA synthesis was further blocked to increase the supply for SCL and MCL monomers in P. entomophila. The so-constructed P. entomophila was successfully used to synthesize novel PHA copolymers of P(3HB-co-3HD), P(3HB-co-3HDD) and P(3HB-co-3H9D) consisting of 3HB and 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD) and 3-hydroxy-9-decanent (3H9D), respectively. MCL 3HA compositions of P(3HB-co-3HD) and P(3HB-co-3HDD) can be adjusted from 0 to approximate 100 mol%. Results demonstrated that the engineered P. entomophila could be a platform for tailor-made P(3HB-co-MCL 3HA).

Polyhydroxyalkanoates (PHA) production by photoheterotrophic microbial consortia: Effect of culture conditions over microbial population and biopolymer yield and composition

Three microbial consortia (C2, C4, C5) grown under photoheterotrophic conditions assimilated acetate and butyrate, as individual and mixed substrates. By controlling the culture conditions, it was possible to manipulate the microbial population composition and thus the yield of polyhydroxyalkanoates (PHA) accumulation. Under limited ammonium conditions, pH control, and a sequential two-step process, C2 and C4 produced PHA. C4 showed the highest production of 44% of the cell dry mass (CDM), close to the theoretical value calculated with a stoichiometric balance. Analysis of the confocal microscopy images confirmed the accumulated biopolymer percentages produced by each consortium, and it was in close correlation with microbial distribution and substrate consumption pattern. 1H, 13C, NMR, and MALDI-TOF spectra identified the primary structure of the obtained biopolymers as copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). This composition allows for better mechanical properties compared to the PHB homopolymer. Microbial characterization showed a similar microbial population with different proportions for C2 and C4. The highest PHA production in C4 was associated with higher abundances of PHA producers, including Clostridium (29%), Pseudomonas (8%) and Rhodopseudomonas (5%). Both microbial consortia showed that a portion of their microbial populations were able to perform syntrophic reactions (Dysgonomonas and Clostridium).

Induced apoptosis of osteoblasts proliferating on polyhydroxyalkanoates

The mechanism study on behaviors of cells influenced by biomaterial surface properties can provide profound guidances for functional tissue engineering scaffolds design. In this study, regulation of integrin-mediated cell–substrate interactions using rat osteoblasts incubated on PHA films was investigated. Compared with tissue culture plate (TCP), poly-3-hydroxybutyrate (PHB), copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV) and copolymer of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx), osteoblasts inoculated on a terpolymer of 3-hydroxybutyrate, 3-hydroxyvalerate and 3-hydroxyhexanoate (PHBVHHx) were found to have higher apoptosis rates. Several integrin subunits in osteoblasts grown on PHBVHHx showed altered expressions. Simultaneously, extracellular matrics (ECM) were also remodeled on the material surface. Osteoblasts showed a higher expression of integrin subunit β3 and αv on PHBVHHx films compared with that on TCP. On the other hand, less vitronectin, osteopontin and fibronectin, the main ligands for integrin β3 were expressed and deposited in ECM. The unligated integrin β3 could recruit caspase-8 to the membrane and activate its downstream signaling which was proven by the caspase-8 activation assay. It was therefore concluded that the induced apoptosis of osteoblasts on PHBVHHx was regulated by recruitment of caspase-8 to the unligated integrin β3.

Characterization of polymeric microparticles based on resorbable polyesters of oxyalkanoic acids as a platform for deposition and delivery of drugs

The effect of the preparation technique (chemical composition of a polymer, type and method of emulsion mixing, and molecular mass of a drug) on the yield, structure, and size of microparticles obtained from resorbable polyesters of microbiological origin, polyhydroxyalkanoates, is studied. It is found that the concentration of the polymer solution and the method of emulsion mixing are the most significant factors affecting the diameter of microparticles based on polyhydroxyalkanoates; the surface structure of particles depends to a higher extent on the chemical composition of the polymer. The family of microparticles from 100–200 nm to 50–70 μm in diameter is synthesized. It is shown that the rate of drug release from microparticles in vitro into the medium is higher in the case of 3-hydroxybutyrate copolymers with 3-hydroxyvalerate than in the case of the homopolymer of 3-hydroxybutyrate. This parameter increases with the content of 3-hydroxyvalerate units in the copolymer and the porosity and mass fraction of the drug in particles with a decrease in their sizes. For in vitro systems containing a phosphate buffer, variation in the preparation parameters makes it possible to obtain microparticles with various characteristics suitable for deposition of drugs. For microparticles obtained from polyhydroxyalkanoates and having different diameters, the mathematical description of the kinetics of drug release from the polymer matrix is provided.

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.

Medical Application of Microbial Biopolyesters Polyhydroxyalkanoates

Polyhydroxyalkanoates (PHA) are a family of polyesters synthesized by microorganisms under unbalanced growth conditions. PHA including poly-3-hydroxybutyrate (PHB), copolymers of 3-hydroxybutyrate and 3-hydroxyvalerate (PHBV), poly-4-hydroxybutyrate (P4HB), copolymers of 3-hydroxybutyrate and 3-hydroxyhexanoate (PHBHHx), and poly-3-hydroxyoctanoate (PHO) are now available in sufficient quantity for tissue engineering medical application studies due to their reasonable biocompatibility, adjustable mechanical properties, and controllable biodegradability. This paper reviews many achievements based on PHA for medical devices development, tissue repair, artificial organ construction, drug delivery, and nutritional/therapeutic uses. Combined with the recent FDA approval for P4HB clinical application, one can expect a good prospect for PHA application in the medical fields.

Green technology for conversion of food scraps to biodegradable thermoplastic polyhydroxyalkanoates.

A new technology is developed and demonstrated that couples anaerobic digestion of food scraps with production of biodegradable thermoplastics, polyhydroxyalkanoates (PHAs). The food wastes were digested in an anaerobic reactor producing four major organic acids. The concentrations of acetic, propionic, butyric, and lactic acids reached 5.5, 1.8, 27.4, and 32.7 g/L, respectively. The fermentative acids were transferred through membranes via molecule diffusion into an air-bubbling reactor where the acids were utilized to produce PHAs in an enriched culture of Ralstonia eutropha. With a silicone rubber membrane, butyric acid and small amounts of acetic and propionic acids were transferred and used, producing a homopolymer PHA, poly(3-hydroxybutyrate). The dry cell weight and PHA content reached 11.3 g/L and 60.2% (w/w), respectively. With a dialysis membrane, the mass transfer rates of fermentative acids were enhanced, and the PHA production was significantly improved. The dry cell weight and its PHA content reached 22.7 g/L and 72.6% (w/w), respectively. The formed PHA was a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate (HV) with 2.8 mol % HV monomer unit. The polymer content (72.6% of dry cell mass) reported in this study is the highest one obtained from organic wastes and is comparable with the PHA content from pure glucose fermentation.

Purification of Aspergillus fumigatus (Pdf1) Poly(β-hydroxybutyrate) (PHB) Depolymerase Using a New, Single-Step Substrate Affinity Chromatography Method: Characterization of the PHB Depolymerase Exhibiting Novel Self-Aggregation Behavior

The extracellular poly(β-hydroxybutyrate) (PHB) depolymerase of Aspergillus fumigatus Pdf1 was purified by a new, simple, one-step affinity chromatography method using the substrate PHB. The purified enzyme was glycosylated, with the molecular mass of ≃40 KD, and exhibited a novel self-aggregation behavior by means of hydrophobic interaction that was resolved by Triton X-100 (TX-100) pretreatment of enzyme and also TX-100 incorporation in the native gel. The apparent Km value of purified enzyme for PHB was 119 μg/mL and 3-hydroxybutyrate was detected as the main endproduct of PHB hydrolysis. The depolymerase was insensitive to phenylmethyl sulfonyl fluoride (PMSF), sodium azide, ethylenediaminetetraacetic acid (EDTA), and para-chloromercuric benzoic acid (PCMB), but was inactivated by dithioerythritol (DTT) and showed specificity for short chain-length poly(β-hydroxyalkanoates) (PHAs) such as PHB, poly(hydroxyvalerate) (PHV), and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Medium-chain-length PHA failed to get hydrolyzed. The enzyme, however, exhibited strong cross reactivity with the Comamonas sp. PHB depolymerase antibodies, but not with PHV depolymerase antibodies of Pseudomonas lemoignei. Southern hybridization and dot blot analysis of A. fumigatus Pdf1 genomic DNA with alkaline phosphatase labeled probes of P. lemoignei PHB and PHV depolymerase genes revealed no homology, although the enzyme hydrolyzed both PHB and PHV.

Scanning electron microscopy of polyhydroxyalkanoate degradation by bacteria

Bacterial degradation of sheets of selected polyhydroxyalkanoates by Comamonas sp., Pseudomonas lemoignei and Pseudomonas fluorescens GK13 is reported. Five natural polyhydroxyalkanoates were used, namely poly(3-hydroxybutyrate), poly(3-hydroxyvalerate), a copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate, a copolymer of mainly 3-hydroxyoctanoate and minor amounts of 3-hydroxyhexanoate, and two rubber-like copolymers of saturated and unsaturated hydroxyalkanoic acids that had been modified by electron-beam-induced cross-linking. Each of these polymers was degraded by at least one bacterial strain, the rate of hydrolysis being dependent on the surface area of the polymer exposed to attack. Scanning electron microscopy of partially degraded samples showed that hydrolysis started at the surface and at physical lesions in the polymer and proceeded to the inner part of the material. No evidence for areas of non-degradable polymer was found for any of the polymers analysed, even if the polymer contained chemical cross-links.

Biodegradation of Poly (3 -hYdroxYalkanoates in Anaerobic Sludge and Characterization of a Poly (3 -hYdroxYalkanoates Degrading Anaerobic Bacterium

Summary The degradation of samples of poly (3-hydroxybutyrate) [P(3HB)], copolymers of 3-hydroxybutyrate and 10 and 20% 3-hydroxyvalerate [P(3HB-co-3HV)], and poly (3-hydroxyoctanoate-co-3-hydroxydecanoate) [P(3H0-co-3HD)] in anaerobic sludge was investigated by gravimetry, changes in molecular weight and mechanical properties. After 123 days, the mass of P(3HB) decreased by 15%. P(3HB-co-3HV) showed 7 and 11% mass loss, while P(3H0-co-3HD) showed no change in mass. The average molecular weight of all samples decreased with incubation time but this decrease was not correlated to the loss of mass, and is to be attributed to abiotic hydrolysis. The tensile strength of the P(3HB) and P(3HB-co-3HV) samples decreased with incubation time, but elongation remained practically unchanged. By enumeration, it was shown that anaerobic P(3HB) degrading bacteria were enriched during the incubation period. An anaerobic bacterium LMG 16094, able to degrade P(3HB) and P(3HB-co-19% 3HV) in pure culture, but not P(3HB-co-97%3HV), P(3H0-co-3HD) or poly (e-caprolactone), was isolated and characterized by fatty acid analysis. Its cells contained mainly straight chain, saturated and unsaturated fatty acids, with a high portion of fatty acids with 16 carbons, and did not contain hydroxylated or branched fatty acids. Its 16S rDNA gene sequence was found to be related to those of members of Clostridium group I ( Collins et al., 1994). In a simple in vitro test using strain LMG 16094 in pure culture, an extruded P(3HB-co-12% 3HV) film lost 10% of its initial mass within 21 days of anaerobic incubation.

Continuous production of poly(3-hydroxybutyrate-co-3-hyhroxyvalerate) byAlcaligenes eutrophus

Copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) were produced in a continuous culture ofAlcaligenes eutrophus with 17.5 gl −1 of fructose and 2.5gl −1 of pentanoic acid in the feed. The P(3HB-co-3HV) productivity was maximal at a dilution rate of 0.17h−1 and yielded 0.31gl −1h−1 under an ammonium-limited condition. The 3HV content in copolymers increased from 11 to 79 mol% as the dilution rate of cells was increased from 0.06 to 0.32h−1.

Pseudomonas lemoignei has five poly(hydroxyalkanoic acid) (PHA) depolymerase genes: A comparative study of bacterial and eukaryotic PHA depolymerases

Four polyhydroxyalkanoate (PHA) depolymerases were purified from the culture fluid ofPseudomonas lemoignei: poly(3-hydroxybutyrate) (PHB), depolymerase A (Mr, 55,000), and PHB depolymerase B (Mr, 67,000) were specific for PHB and copolymers of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) as substrates. The third depolymerase additionally hydrolyzed poly(3-hydroxyvalerate) (PHV) at high rates (PHV depolymerase;Mr, 54,000). The N-terminal amino acid sequences of the three purified proteins, of a fourth partially purified depolymerase (PHB depolymerase C), and of the PHB depolymerases ofComamonas sp. were determined. Four PHA depolymerase genes ofP. lemoignei (phaZ1,phaZ2,phaZ3, andphaZ4) have been cloned inEscherichia coli, and the nucleotide sequence ofphaZ1 has been determined recently (D. Jendrossek, B. Muller, and H. G. Schlegel,Eur. J. Biochem.218, 701–710, 1993). In this study the nucleotide sequences ofphaZ2 andphaZ3 were determined.PhaZ1,phaZ2, andphaZ4 were identified to encode PHB depolymerase C, PHB depolymerase B, and PHV depolymerase, respectively.PhaZ3 coded for a novel PHB depolymerase ofP. lemoignei, named PHB depolymerase D. None of the four genes harbored the PHB depolymerase A gene, which is predicted to be encoded by a fifth depolymerase gene ofP. lemoignei (phaZ5) and which has not been cloned yet. The deduced amino acid sequences ofphaZ1–phaZ3 revealed high homologies to each other (68–72%) and medium homologies to the PHB depolymerase gene ofAlcaligenes faecalis T1 (25–34%). Typical leader peptide amino acid sequences, lipase consensus sequences (Gly-Xaa-Ser-Xaa-Gly), and unusually high proportions of threonine near the C terminus were found in PhaZ1, PhaZ2, and PhaZ3. Considering the biochemical data of the purified proteins and the amino acid sequences, PHA depolymerases ofP. lemoignei are most probably serine hydrolases containing a catalytical triad of Asp, His, and Ser similar to that of lipases. A comparison of biochemical and genetic data of various eubacterial and one eukaryotic PHA depolymerases is provided also.

Formation of polyester blends by a recombinant strain ofPseudomonas oleovorans: Different poly(3-hydroxyalkanoates) are stored in separate granules

WhenPseudomonas oleovorans (GPo1) is grown on sodium octanoate under ammonium limiting conditions, it is able to accumulate a copolyester consisting of medium chain length 3-hydroxyalkanoic acids (PHAm). 3-Hydroxybutyrate is only incorporated in trace amounts. WhenP. oleovorans is equipped with the PHB biosynthetic genes ofAlcaligenes eutrophus (GPo1[pVK101::PP1]), it forms a polyester containing major amounts of 3-hydroxybutyrate. The resulting polymer however is a blend of PHAm and PHB, rather than a copolymer of 3-hydroxybutyrate and medium chain length 3-hydroxyalkanoic acids [11]. To establish whether PHAm and PHB molecules are stored in the same or separate granules by this recombinantP. oleovorans strain, we studied polymer forming cells by freeze-fracture electron microscopy. This approach is possible because previous freeze-fracture electron microscopy studies on PHAm and PHB accumulating strains have shown that PHAm and PHB granules can be distinguished from each other: PHAm granules from mushroom-like structures, whereas PHB granules from needle structures during freeze-fracturing. In this paper we show that stationary phase cells of GPo1[pVK101::PP1] contained both mushroom and needle-like structures, indicating that PHAm and PHB chains were stored in separate granules. To be able to determine whether the separation of PHAm and PHB is complete, the respective granules were separated on sucrose gradients. A total cell extract of GPo1[pVK101::PP1] which was subjected to sucrose gradient centrifugation revealed two white bands of different densities: the upper band with a density of 1.05 g/mL consisted exclusively of PHAm granules, while the lower band with a density of 1.19 g/mL consisted of PHB granules only. Thus, when bacteria synthesize both PHAm and PHB, the resulting polymer chains are segregated completely and stored in separate granules.