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PubMed2006

In vitro biocompatibility of electrospun poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) fiber mats.

In the present contribution, the potential for use of the ultrafine electrospun fiber mats of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) as scaffolding materials for skin and nerve regeneration was evaluated in vitro using mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) as reference cell lines. Comparison was made with PHB and PHBV films that were prepared by solution-casting technique. Indirect cytotoxicity assessment of the as-spun PHB and PHBV fiber mats with mouse fibroblasts (L929) and Schwann cells (RT4-D6P2T) indicated that the materials were acceptable to both types of cells. The attachment of L929 on all of the fibrous scaffolds was significantly better than that on both the film scaffolds and tissue-culture polystyrene plate (TCPS), while RT4-D6P2T appeared to attach on the flat surfaces of TCPS and the film scaffolds much better than on the rough surfaces of the fibrous scaffolds. For L929, all of the fibrous scaffolds were superior in supporting the cell proliferation to the film counterparts, but inferior to TCPS at days 3 and 5, while, for RT4-D6P2T, the rough surfaces of the fibrous scaffolds appeared to be very poor in supporting the cell proliferation when comparing with the smooth surfaces of TCPS and the film scaffolds. Scanning electron microscopy was also used to observe the behavior of both types of cells that were cultured on both the fibrous and the film scaffolds and glass substrate for 24 h.

Animals↗

PubMed2006

Biosynthesis and native granule characteristics of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in Delftia acidovorans.

The ability of Delftia acidovorans to incorporate a broad range of 3-hydroxyvalerate (3HV) monomers into polyhydroxyalkanoate (PHA) copolymers was evaluated in this study. Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] containing 0-90 mol% of 3HV was obtained when a mixture of sodium 3-hydroxybutyrate and sodium valerate was used as the carbon sources. Transmission electron microscopy analysis revealed an interesting aspect of the P(3HB-co-3HV) granules containing high molar ratios of 3HV whereby, the copolymer granules were generally larger than those of poly(3-hydroxybutyrate) [P(3HB)] granules, despite having almost the same cellular PHA contents. The large number of P(3HB-co-3HV) granules occupying almost the entire cell volume did not correspond to a higher amount of polymer by weight. This indicated that the granules of P(3HB-co-3HV) contain polymer chains that are loosely packed and therefore have lower density than P(3HB) granules. It was also interesting to note that a decrease in the length of the side chain from 3HV to 4-hydroxybutyrate (4HB) corresponded to an increase in the density of the respective PHA granules. The presence of longer side chain monomers (3HV) in the PHA structure seem to exhibit steric effects that prevent the polymer chains in the granules from being closely packed. The results reported here have important implications on the maximum ability of bacterial cells to accumulate PHA containing monomers with longer side chain length.

Centrifugation, Density Gradient↗

PubMed2006

Supercritical antisolvent precipitation of PHBV microparticles.

The micronization of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) from organic solutions using supercritical antisolvent (SAS) technique has been successfully achieved. SAS experiments were carried out at different operational conditions and microspheres with mean diameters ranging from 3 to 9 microm were obtained. The effect of CO(2) and liquid flow, temperature and pressure on particle size and particle size distribution was evaluated. The microspheres were precipitated from a dichloromethane (DCM) solution. The best process conditions for this mixture were, according to our study, 40 degrees C, 100 bar, 1 mL min(-1) liquid flow and 10 L min(-1) carbon dioxide flow. Experiments with polymers containing different HV percentages were carried out. The powders obtained became more spherical as the HV content decreased.

Calorimetry, Differential Scanning↗

PubMed2005

Possibilities for controlling a PHB accumulation process using various analytical methods.

Poly-beta-hydroxybutyrate (PHB) and other polyesters can be produced by various species of bacteria. Of the possible carbon sources, methane could prove to be one of the most suitable substrates for the manufacture of PHB. The methanotrophic strain Methylocystis sp. GB 25 DSM 7674 was applied in order to accumulate PHB in a rapid, non-sterile process. Cultivation was performed in two stages: a continuous growth phase (dilution rate 0.17 h(-1)) and a PHB accumulation phase under deficiency conditions of an essential nutrient (e.g. phosphorus) in batch culture. The PHB content of the biomass was as high as 51%; efficiency was the highest during the first 5 h of the product formation process. The PHB produced is of very high quality, having a high molecular mass of up to 2.5 x 10(6) Da. In order to monitor and control the process, a rapid analysis method based upon turbidimetry in the visible range (438 nm) was applied. Moreover, the PHB content of the biomass was determined using an FT-IR-spectroscopic method with ATR sampling and multivariate calibration. We achieved a value of 1.4% as the best standard error of cross validation. The nitrogen content of the PHB final product (a product quality parameter) was estimated by spectroscopic method in the visible range.

Biomass↗

PubMed2005

Genetic network driven control of PHBV copolymer composition.

We developed a detailed mathematical model describing the coupling between the molecular weight distribution dynamics of poly(3-hydroxybutyrate-co-3hydroxyvalerate) (PHBV) copolymer chains with those of hydroxybutyrate (HB) and hydroxyvalerate (HV) monomer formation. Sensitivity analysis of the model revealed that both the monomer composition and the molecular weight distribution of the copolymer chains are strongly affected by the ratio between the rates at which the two-monomer units are incorporated into the chains. This ratio depends on the relative HB and HV availability, which in turn is a function of the expression levels of genes encoding enzymes that catalyze monomer formation. Regulation of gene expression was accomplished through the aid of an artificial genetic network, the patterns of expression of which can be controlled by appropriately tuning the concentration of an extracellular inducer. Extensive simulations were used to study the effects of operating conditions and parameter uncertainties on the range of achievable copolymer compositions. Since the predicted conditions fell in the range of feasible bioprocessing manipulations, it is expected that such strategy could be successfully employed. Thus, the presented model constitutes a powerful tool for designing genetic networks that can drive the formation of PHBV copolymer structures with desirable characteristics.

Bacteria↗

PubMed2006

Synthesis of PHBV block copolymers driven by an oscillatory genetic network.

Artificial genetic networks constitute a powerful tool to achieve various biotechnological objectives. In this work, we propose the modification of an oscillatory genetic network, known as the repressilator, to drive synthesis of poly(3hydroxybutyrate-co-3hydroxyvalerate) (PHBV) block copolymer chains in recombinant Escherichia coli cells. To study the feasibility of this idea, we developed a detailed mathematical model describing the dynamics of the genetic network, which drive the formation of monomer units that are subsequently incorporated into actively growing block copolymer chains. Extensive simulation studies have shown that appropriate choice of the molecular characteristics of the network and manipulation of extracelllular conditions lead to tight control of both the micro- and macro-structures of the resulting block copolymer chains. Thus, the model can guide network design aiming at producing block copolymer structures with desirable characteristics.

Biological Clocks↗

PubMed2005

Preparation, characterization and in vitro release of gentamicin from PHBV/wollastonite composite microspheres.

Composite microspheres have been prepared from bioactive wollastonite (W) and biodegradable poly (hydroxybutyrate-polyhydroxyvalerate) (PHBV) in the present study. Gentamicin was encapsulated into the microspheres by the absorption method and the in vitro release of the gentamicin from the microspheres was performed in distilled water, modified simulated body fluid (SBF) and phosphate buffered saline (PBS) at 37 degrees C for 22 days, respectively. The results showed that the release behavior of gentamicin from PHBV/W composite microspheres was similar to that from the pure PHBV microspheres when the experiment was performed in distilled water. However, in the PBS and SBF solutions, gentamicin released from the PHBV/W composite microspheres at a relatively lower rate as compared to that of the pure PHBV microspheres and 90% of the total amount of gentamicin released from the composite microspheres after soaking for 22 days, which was much longer than that for the release of the same amount gentamicin from the pure PHBV microspheres (8 days). Scanning electron microscopy (SEM) and energy-dispersive spectrometer (EDS) analysis on the microspheres after release in SBF and PBS revealed that a microporous apatite layer was formed on the composite microspheres surface, which resulted in a controlled release behavior of the gentamicin from the PHBV/W composite microspheres. All of these results provided the possibility that the PHBV/W composite microspheres could be applied as alternative drug controlled release systems, especially as bone fillings for bone repair due to their advantages of controlled releasing antibiotics and apatite-formation ability, through which the implanted microspheres could chemically bond to the surrounding tissue in vivo.

Algorithms↗

PubMed2006

Determination of doxorubicin in rabbit ocular tissues and pharmacokinetics after intravitreal injection of a single dose of doxorubicin-loaded poly-beta-hydroxybutyrate microspheres.

A validated HPLC method was developed for the quantification of doxorubicin in rabbit ocular tissues using solid phase extraction and ultraviolet detection. Chromatographic separation of doxorubicin in various ocular tissues was performed on a C18 column. The mobile phase was composed of 0.2 M KH2PO4 buffer solution, acetonitrile and triethylamine in volumetric ratio of 70/30/0.2, adjusted to pH 4.0 with orthophosphoric acid. The calibration curve was linear over the range of 0.03-10, 0.03-10, 0.05-10 and 0.05-10 microg/ml in vitreous body, iris, retina/choroids and sclera, respectively. The intra-day and inter-day precisions in all ocular tissues were smaller than 4.95% and 5.73%, and the accuracies were about 100%. The extraction recoveries of doxorubicin in all of the ocular tissues were between 83.47% and 96.33%. After intravitreal administration of doxorubicin-loaded poly-beta-hydroxybutyrate microspheres, doxorubicin level in ocular tissues was much lower than that for administration of free doxorubicin, which was helpful to reduce the associated toxicity to surrounding tissues. Doxorubicin was detectable even after tens of days in the studied ocular tissues.

Animals↗

PubMed2005

Poly-beta-hydroxybutyrate accumulation in Nostoc muscorum and Spirulina platensis under phosphate limitation.

Nostoc muscorum and Spirulina platensis were grown under phosphate deficiency in order to investigate the role of internal phosphate pool and activity of alkaline phosphatase on poly-beta-hydroxybutyrate (PHB) accumulation. PHB accumulation in N. muscorum increased to 22.7% of dry weight (dw) after 4 day of phosphate deficiency, while the internal phosphate pool reduced to the level of 0.02 microM mg dw(-1) at a maximum APase activity of 2.57nM PNP mg dw(-1) h(-1). In contrary, S. platensis depicted maxima of 1.39nM PNP mg dw(-1) h(-1) on day 30 of incubation, which was about 2 fold lower than the observed value of N. muscorum. PHB content in S. platensis remained low even after prolonged phosphate starvation, and a rise only up to 3.5% of dw was recorded on day 60 of phosphate deficiency. Supplementation of NADPH exogenously to S. platensis cultures grown under phosphate deficiency favoured PHB accumulation in 10, 20 and 30 days old cultures, but not in the cultures grown under phosphate deficiency for 60 days. The possible role of phosphate limitation on PHB accumulation is discussed.

Alkaline Phosphatase↗

PubMed2006

Poly-beta-hydroxybutyrate accumulation in Nostoc muscorum: effects of metabolic inhibitors.

Poly-beta-hydroxybutyrate (PHB) accumulation in Nostoc muscorum was studied in presence of various metabolic inhibitors. Supplementation of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) was found to suppress PHB accumulation in phosphate-limited N. muscorum under photoautotrophic growth condition. PHB accumulation increased up to 21% and 17% from an initial PHB content of 8.5% of dry weight, respectively, under carbonylcyanide m-chlorophenylhydrazone (CCCP) and dicyclohexylcarbodiimide (DCCD) treatment, whereas 2,4 -dinitrophenol (DNP) supplementation depicted insignificant effect on PHB pool of the test cyanobacterium. Supplementation of l-methionine-dl-sulfoximine (MSX) and azaserine was also found to increase PHB accumulation in N(2) -fixing and NH(4)(+) -grown N. muscorum, but not in NO(3)(-) -grown cells. The stimulatory action of monofluoroacetate on PHB accumulation was suppressed in presence of alpha-ketoglutarate and DCMU. Interestingly, 2,3 -butanedione supplementation was not only found inhibitory for accumulation of PHB in P-deficient, N-deficient and chemoheterotrophically grown N. muscorum but suppression of PHB synthesis was also evident in control cultures in presence of 2,3 -butanedione. The possible mechanisms are discussed.

Azaserine↗

PubMed2005

NMR relaxometry and imaging of water absorbed in biodegradable polymer scaffolds.

Porous substrates made of poly(3-hydroxybutyrate-3-hydroxyvalerate) (PHBHV) were prepared by a particulate leaching method. After removing the salt by extraction in water, proton nuclear magnetic resonance (NMR) relaxometry and imaging were performed on sets of PHBHV substrates immersed in phosphate-buffered solution during 3 months at different time points. Polarized optical microscopy studies were performed on thin sections, 25 and 5 mum, of the PHBHV samples. The results of NMR relaxometry showed two (1)H nuclei populations, well distinguishable on the free induction decay (FID), due to the different decay time constants, a factor of 10(2) apart. Thus, it was possible to separate the two populations, giving separate distributions of T(1) relaxation times. One population could be associated with water protons in the pores and the other to macromolecular protons. The distributions of T(1) and T(2) of the water proton shifted to lower values with increasing immersion time to a constant value after 30 days. The results obtained by NMR imaging showed an initial increase in the apparent porosity, reaching a plateau after 25 days of immersion. This increase is attributed mainly to the absorption of water in the microporosity as supported by the results of the relaxometry measurements and shown by scanning electron microscopy. The average porosity measured by NMR imaging at the plateau, 78+/-3%, is slightly higher than that determined by optical microscopy, 73+/-9%, which may be due to the fact that the latter method did not resolve the microporosity. Overall, the results suggest that at early stages after immersing the scaffolds in the aqueous medium, first 30 days approximately, NMR imaging could underestimate the porosity of the substrate.

Absorption↗

PubMed2005

Melting behavior of poly(3-hydroxybutyrate) investigated by two-dimensional infrared correlation spectroscopy.

The melting behavior of a bacterially synthesized biodegradable polymer, poly(3-hydroxybutyrate) (PHB), was investigated by using generalized two-dimensional infrared (2D IR) correlation spectroscopy. Temperature-dependent spectral variations in the regions of the C-H stretching (3100-2850 cm(-1)), C=O stretching (1800-1680 cm(-1)), and C-O-C stretching (1320-1120 cm(-1)) bands were monitored during the melting process. The asynchronous 2D correlation spectrum for the C=O stretching band region resolved two crystalline bands at 1731 and 1723 cm(-1). The intense band at 1723 cm(-1) may be due to the highly ordered crystalline part of PHB, and the weak band at 1731 cm(-1) possibly arises from the crystalline part with a less ordered structure. These crystalline bands at 1731 and 1723 cm(-1) share asynchronous cross peaks with a band at around 1740 cm(-1) assignable to the C=O band due to the amorphous component. This observation indicates that the decreases in the crystalline components do not proceed simultaneously with the increase in the amorphous component. In the 3020-2915 cm(-1) region where bands due to the asymmetric CH3 stretching and antisymmetric CH2 stretching modes are expected to appear, eight bands are identified at 3007, 2995, 2985, 2975, 2967, 2938, 2934, and 2929 cm(-1). The bands at 2985 and 2938 cm(-1) are ascribed to the amorphous part while the rest come from crystal field splitting, which is a characteristic of polymers with a helical structure.

Carbon↗

PubMed2005

Denitrification on poly-beta-hydroxybutyrate in microbial granular sludge sequencing batch reactor.

Microbial granules were successfully cultivated in an alternating aerobic-anaerobic sequencing batch reactor (SBR) for removing organic carbon and nitrogen. It was found that almost all input ammonium was converted to nitrite and nitrate in the aerobic phase, while the efficiency of denitrification was highly related to the availability of external carbon source in the anaerobic phase. Complete denitrification was achieved with sufficient supply of external carbon, while only partial denitrification was observed with no addition of external carbon. Results showed that in the absence of external carbon source, pre-accumulated poly-beta-hydroxybutyric acid (PHB) in microbial granules could be utilized for cell maintenance and denitrification. With supply of external carbon but no addition of nitrate, PHB accumulation accounted for the main mechanism of the organic removal. Under balanced growth conditions (with organic carbon and nitrogen supply), external organic carbon was consumed simultaneously for denitrification, PHB storage as well as for cell functions. It was revealed that the potential role of PHB for denitrification by microbial granules was very limited, i.e. less than 28 mg nitrate-nitrogen l(-1) was found to be denitrified with internally accumulated PHB. This study for the first time shows the limiting capacity of PHB as reducing power for denitrification by microbial granules.

Bacteria, Aerobic↗

PubMed2005

Long-term aeration management for improved N-removal via SND in a sequencing batch reactor.

Management of the aeration length in a sequencing batch reactor (SBR) can improve N-removal by minimising the amount of organic substrate that is oxidised aerobically. This study investigates the long-term effect of aeration control on N-removal via simultaneous nitrification and denitrification (SND) by a mixed culture in a 2L acetate-fed SBR, using PHB as the electron donor for denitrification. The reactor was operated continuously with automated termination of the aerobic phase after ammonium depletion, using the specific oxygen uptake rate (SOUR) as the control parameter. This resulted in an increase of the organic loading rate (OLR) from 0.33 to 0.59 g BOD g(-1)d(-1). Over the first 12 cycles of operation, the PHB content of the biomass increased three-fold and resulted in a progressively increasing SOUR, which allowed an increased amount of nitrogen removal via SND from 34% to 52%. After one month of continuous operation with controlled aeration, the settling efficiency of the biomass had significantly improved (SVI 70 mL g(-1) X). Long-term oxygen management resulted in biomass with a higher capacity for N-removal via SND and improved settling characteristics. Our results may help to explain long-term historical effects of N-removal capabilities in WWTPs and assist design engineers in choosing an appropriate aeration length and OLR.

Aerobiosis↗

PubMed2006

The fractional contributions of elementary modes to the metabolism of Escherichia coli and their estimation from reaction entropies.

The metabolism of a cell can be viewed as a weighted sum of elementary modes. Due to the multiplicity of modes the identification of the individual weights represents a non-trivial problem. To enable the determination of weighting factors we have identified and implemented two gene deletions in combination with defined growth conditions that limit the metabolism from 4374 original elementary modes to 24 elementary modes for a non-PHB synthesizing control and 40 modes for a PHB synthesizing strain. These remaining modes can be further grouped into five families that have the same overall stoichiometry. Thus, the complexity of the problem is significantly reduced, and weighting factors for each family of modes could be determined from the measurement of accumulation rates of metabolites. Moreover, it is shown that individual weights are inversely correlated with the entropy generated by the operation of the used pathways defined in elementary modes. This suggests that evolution developed cellular regulatory patterns that permit diversity of pathways while favoring efficient pathways with low entropy generation. Furthermore, such correlation provides a rational way of estimating metabolic fluxes based on the thermodynamic properties of elementary modes. This is demonstrated with an example in which experimentally determined, intracellular fluxes are shown to be highly correlated with fluxes computed based on elementary modes and reaction entropies. The analysis suggests that the set of elementary modes can be interpreted analogous to a metabolic ensemble of quantum states of a macroscopic system.

Anaerobiosis↗

PubMed2003

Escherichia coli lacking the AcrAB multidrug efflux pump also lacks nonproteinaceous, PHB-polyphosphate Ca2+ channels in the membrane.

PHB(polyP) complexes bind calcium and form calcium channels in the cytoplasmic membrane in Escherichia coli and are likely to be important in Ca(2+) homeostasis in this organism. E. coli N43, which lacks the AcrA component of a major multidrug resistance pump, was shown to be defective in calcium handling, with an inability to maintain submicromolar levels of free Ca(2+) in the cytoplasm. Therefore, using an N-phenyl-1-napthylamine (NPN)-dependent fluorescence assay, we measured temperature-dependent phase transitions in the membranes of intact cells. These transitions specifically depend on the presence of PHB(Ca(2+)polyP) complexes. PHB(Ca(2+)polyP) channel complexes, particularly in stationary phase cultures, were detected in wild-type strains; however, in contrast, isogenic acrA(-) strains had greatly reduced amounts of the complexes. This indicates that the AcrAB transporter may have a novel, hitherto undetected physiological role, either directly in the membrane assembly of the PHB complexes or the transport of a component of the membrane, which is essential for assembly of the complexes into the membrane. In other experiments, we showed that the particular defective calcium handling detected in N43 was not due to the absence of AcrA but to other unknown factors in this strain.

Calcium↗

PubMed1995

Poly-3-hydroxybutyrate/polyphosphate complexes form voltage-activated Ca2+ channels in the plasma membranes of Escherichia coli.

The lipidic polymer, poly-3-hydroxybutyrate (PHB), is found in the plasma membranes of Escherichia col complexed to calcium polyphosphate (CaPPi). The composition, location, and putative structure of the polymer salt complexes led Reusch and Sadoff (1988) to propose that the complexes function as Ca2+ channels. Here we use bilayer patch-clamp techniques to demonstrate that voltage-activated Ca2+ channels composed of PHB and CaPPi are in the plasma membranes of E. coli. Single channel calcium currents were observed in vesicles of plasma membranes incorporated into planar bilayers of synthetic 1-palmitoyl, 2-oleoyl phosphatidylcholine. The channels were extracted from cells and incorporated into bilayers, where they displayed many of the signal characteristics of protein Ca2+ channels: voltage-activated selective for divalent over monovalent cations, permeant to Ca2+, manner by La3+, Co2+, Cd2+, and Mg2+, in that order. The channel-active extract, purified by size exclusion chromatography, was found to contain only PHB and CaPPi. This composition was confirmed by the observation of comparable single channel currents with complexes reconstituted from synthetic CaPPi and PHB, isolated from E. coli. This is the first report of a biological non-proteinaceous calcium channel. We suggest that poly-3-hydroxybutyrate/calcium polyphosphate complexes are evolutionary antecedents of protein Ca2+ channels.

Calcium Channels↗

PubMed2003

Exogenous leukaemia inhibitory factor enhances nerve regeneration after late secondary repair using a bioartificial nerve conduit.

The clinical outcome of peripheral nerve injuries remains disappointing, even in the ideal situation of a primary repair performed with optimal microsurgical techniques. Primary repair is appropriate for only about 85% of injuries, and outcome is worse following secondary nerve repair, partly owing to the reduced regenerative potential of chronically axotomised neurons. Leukaemia inhibitory factor (LIF) is a gp-130 neurocytokine that is thought to act as an 'injury factor', triggering the early-injury phenotype within neurons and potentially boosting their regenerative potential after secondary nerve repair. At 2-4 months after sciatic nerve axotomy in the rat, 1 cm gaps were repaired using either nerve isografts or poly-3-hydroxybutyrate conduits containing a calcium alginate and fibronectin hydrogel. Regeneration was determined by quantitative immunohistochemistry 6 weeks after repair, and the effect of incorporating recombinant LIF (100 ng/ml) into the conduits was assessed. LIF increased the regeneration distance in repairs performed after both 2 months (69%, P=0.019) and 4 months (123%, P=0.021), and was statistically comparable to nerve graft. The total area of axonal immunostaining increased by 21% (P>0.05) and 63% (P>0.05), respectively. Percentage immunostaining area was not increased in the 2 months group, but increased by 93% in the repairs performed 4 months after axotomy. Exogenous LIF, therefore, has a potential role in promoting peripheral nerve regeneration after secondary repair, and can be effectively delivered within poly-3-hydroxybutyrate bioartificial conduits used for nerve repair.

Animals↗