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PubMed2004

Glutamic acid removal and PHB storage in the activated sludge process under dynamic conditions.

Glutamic acid removal in the activated sludge process is studied herein, primarily the formation of storage polymers under dynamic conditions. The activated sludge process was operated by using a sequencing batch reactor (sludge age of 6 d) fed with a synthetic mixture of readily available carbon sources, including glutamic acid. Removal of glutamic acid as the only carbon sources was studied in batch tests, along with oxygen consumption, ammonia uptake-release, and formation of storage polymers. It was found that poly-3-hydroxybutyrate (PHB) was stored and that the storage also occurred simultaneously to biomass growth. PHB storage accounted for 16% of the overall solids that were formed from glutamic acid, as the average value of nine batch tests. Neither other Polyhydroxyalkanoates nor polyglutamic acid were detected. Nuclear magnetic resonance analysis, performed on biomass extracts, allowed us to clarify the main metabolic pathways involved in glutamic acid removal and, in particular, the pathways involved in PHB storage. It was found that glutamic acid enters the Krebs cycle as alpha-ketoglutaric acid and exits to form pyruvic acid and then acetyl-CoA, which is the starting point of PHB production pathway.

Ammonia↗
PubMed2005

A mathematical model for regulating monomer composition of the microbially synthesized polyhydroxyalkanoate copolymers.

A mathematical model is proposed for predicting the copolymer composition of the microbially synthesized polyhydroxyalkanoate (PHA) copolymers. Based on the biochemical reactions involved in the precursor formation and polymerization pathways, the model correlates the copolymer composition with the cultivation conditions, the enzyme levels and selectivity, and the metabolic pathways. It suggests the following points: (1) in the case of a sole carbon source, the copolymer composition depends mainly on the topology of the metabolic pathways and the selectivity of both the enzymes involved in the precursor formation and the polymerization route; (2) the copolymer composition can be varied in a wide range via alteration of the flux ratio of different types of monomers channeled from two or more independent and simultaneous pathways; (3) the enzymes which should be over-expressed or inhibited to obtain the desired copolymer composition can be predicted. For example, inhibition of the beta-oxidation pathway will increase the content of the monomer units with longer chain length. To test the model, various experiments were envisaged by varying cultivation time, concentration and chain length of the sole carbon source, and molar ratio of the cosubstrates. The predictions from the model agree well with the experimental results. Therefore, the proposed model will be useful in predicting the PHA copolymer composition under different biochemical reaction conditions. In other words, it can provide a guide for the synthesis of desired PHA copolymers.

Computer Simulation↗
PubMed2006

Staining and quantification of poly-3-hydroxybutyrate in Saccharomyces cerevisiae and Cupriavidus necator cell populations using automated flow cytometry.

BACKGROUND: Poly [(R)-3-hydroxybutyric acid] (PHB) is a prokaryote storage material for carbon and energy that accumulates in cells under unbalanced growth conditions. Because this class of biopolymers has plastic-like properties, it has attracted considerable interest for biomedical applications and as a biodegradable commodity plastic. Current flow cytometric techniques to quantify intracellular PHB are based on Nile red. Here, an improved cytometric technique for cellular PHB quantification utilizing BODIPY 493/503 staining was developed. This technique was then automated using an automated flow cytometry system. MATERIALS: Using flow cytometry, the fluorescence of Saccharomyces cerevisiae and Cupriavidus necator with varying PHB content after staining with BODIPY 493/503 and Nile red was compared, and automated staining techniques were developed for both cultures. RESULTS: BODIPY 493/503 staining had less background staining, higher sensitivity and specificity to PHB, and higher saturation values than did Nile red staining. The developed automated staining procedure was capable of analyzing the PHB content of a bioreactor sample every 25 min and measured the average PHB content with accuracy comparable to offline GC analysis. CONCLUSION: BODIPY 493/503 produced an overall better staining for PHB than did Nile red. When combined with the automated system, this technique provides a new method for the online monitoring and control of bioreactors.

Boron Compounds↗
PubMed1995

Fibroblast seeding and culture in biodegradable porous substrates.

A natural poly(hydroxybutyrate-co-9% hydroxyvalerate) copolyester was processed into a three-dimensional porous foam structure by salt leaching/solvent casting with previously sieved sodium chloride salts. Laboratory-built P(HB-9% HV) foams and commercial collagen sponges were cut into small rectangular specimens, sterilized, and prewetted using ethanol, rinsed with Dulbecco's minimum essential medium + 10% serum culture media, and seeded with fibroblasts isolated from canine anterior cruciate ligaments. The fibroblast cultures into such porous substrates were performed from 0 to 35 days by incubation (5% CO2) at 37 degrees C. It demonstrated that the P(HB-HV) sustained a cell proliferation rate similar to that observed in collagen sponges, up to at least 35 days, with a maximal cell density on the day 28 in culture. On the other hand, the P(HB-HV) materials kept their structural integrity during the culture period while the collagen foams contracted greatly. Further, the total protein production after 4 weeks in culture was found to be twice as high (190 +/- 10%) in the P(HB-9% HV) foam than in the collagen foam. Porous P(HB-HV) materials appear to be adequate polymeric substrates for cell cultures. However, further evaluations are still required to confirm such preliminary results.

Animals↗
PubMed2002

Low pressure plasma treatment of poly(3-hydroxybutyrate): toward tailored polymer surfaces for tissue engineering scaffolds.

Thin films of poly(3-hydroxybutyrate) were modified by microwave ammonia plasma treatment. The results of the modification were studied by means of contact angle goniometry, ellipsometry, Fourier transform infrared-attenuated total reflection spectroscopy, atomic force microscopy, and X-ray photoelectron spectroscopy. To prove the presence of amino groups on the poly(3-hydroxybutyrate) surface, chemical labeling with 4-trifluoromethyl benzaldehyde was performed before X-ray photoelectron spectroscopy analysis. Under the applied plasma conditions, a hydrophilic surface with a good long-term stability was obtained.

Electron Probe Microanalysis↗
PubMed1993

Tissue response and in vivo degradation of selected polyhydroxyacids: polylactides (PLA), poly(3-hydroxybutyrate) (PHB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/VA).

The tissue response and in vivo molecular stability of injection-molded polyhydroxyacids--polylactides (PLA), poly(3-hydroxybutyrate) (PHB), and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHB/VA, 5-22% VA content)--were studied. Polymers were implanted subcutaneously in mice and extirpated at 1, 3, and 6 months in order to study tissue response and polymer degradation. All polymers were well tolerated by the tissue. No acute inflammation, abscess formation, or tissue necrosis was observed in tissues adjacent to the implanted materials. Furthermore, no tissue reactivity or cellular mobilization was evident remote from the implant site. Mononuclear macrophages, proliferating fibroblasts, and mature vascularized fibrous capsules were typical of the tissue response. Degradation of the polymers was accompanied by an increase in collagen deposition. For the polylactide series, the inflammatory response after 1 month of implantation was less for materials containing the D-unit in the polymer chain, whereas in the case of the polyhydroxybutyrate/valerates, the number of inflammatory cells increased with increasing content of the valerate unit in the polymer chain. Between 1-3 months, there was slightly more tissue response to the PHB and PHB/VA polymers than to PLA. This response is attributed to the presence of leachable impurities and a low molecular weight soluble component in the polyhydroxybutyrate/valerates. At 6 months, the extent of tissue reaction was similar for both types of polymers. All polylactides degraded significantly (56-99%) by 6 months. For a poly(L-lactide) series, degradation rate in vivo decreased with increasing initial molecular weight of the injection-molded polymer. Several samples showed pronounced bimodal molecular weight distributions (MWD), which may be due to differences in degradation rate, resulting from variability in distribution of crystalline and amorphous regions within the samples. This may also be the result of two different mechanisms, i.e., nonenzymatic and enzymatic, which are involved in the degradation process, the latter being more extensive at the later stage of partially hydrolyzed polymer. The PHB and PHB/VA polymers degraded less (15-43%) than the polylactides following 6 months of implantation. Generally, the polymer with higher valerate content (19%, 22%) degraded most. The decrease in molecular weight was accompanied by a narrowing of the MWD for PHB and copolymers; there was no evidence of a bimodal MWD, possibly indicating that the critical molecular weight that would permit enzyme/polymer interaction had not been reached. Weight loss during implantation ranged from 0-50% for the polylactides, whereas for the PHB polymers weight loss ranged from 0-1.6%.

Absorption↗
PubMed2005

Effects of surface modification of poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) on physicochemical properties and on interactions with MC3T3-E1 cells.

As a new member of the polyhydroxyalkanoate (PHA) family, poly (3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) has better mechanical and processible properties than poly (3-hydroxybutyrate) (PHB). Still, it is difficult to introduce functional groups to the polyester carbon chain of PHBHHx, which restricts the modification of PHBHHx for a wide range. In this study, a procedure for the modification of the surface of PHBHHx films under strongly alkaline conditions was described. Through this kind of modification, carboxyl and hydroxyl groups were introduced to the surface and the total surface free energy was increased, which was mainly due to the increased polar components. Meanwhile, this process makes the surface rougher, resulting in larger total surface areas. After mineralization in simulated body fluids (SBFs), the apatite nucleation and growth on the surface-hydrolyzed PHBHHx films were significantly faster than on the unmodified PHBHHx films. This phenomenon should have a close relationship with the increased carboxyl and hydroxyl groups. The physicochemical properties also influenced the cell response to PHBHHx films. Compared to unmodified PHBHHx, fibronectin adsorption, and MC3T3-E1 cell attachment and proliferation were significantly greater on surface-hydrolyzed PHBHHx, which may be due to the increased surface free energy and rougher surface. Therefore, surface hydrolysis makes PHBHHx more suitable for osteoblast cell response and for application in bone-tissue engineering.

3-Hydroxybutyric Acid↗
PubMed2006

Improvement in hydrophilicity of PHBV films by plasma treatment.

Poly(3-hydroxybutyrate-co-hydroxyvalerate) (PHBV) films were prepared by the solvent cast method. Oxygen and nitrogen plasma treatment were used to improve the hydrophilicity of the surface of PHBV films. The surface properties were characterized by contact angle measurement and X-ray photoelectron spectroscopy (XPS). The water contact angle of PHBV films decreased after plasma treatment, which suggested that the surfaces became more hydrophilic. The effects of exposure time, plasma generating power, and chamber pressure on water contact angle were investigated. Although the decrease of contact angle with both plasma treatments was similar, the change of PHBV surface structure was different. XPS analysis showed that the oxygen content and the ratio of O/C increased markedly after oxygen plasma treatment, while after nitrogen plasma treatment, the surface was enriched with nitrogen atoms. Furthermore, it was found that C-O and C-C bonds were broken with both plasma treatment, while different new bonds were formed, that is, COOH (for oxygen plasma), and C-N, C=N, and amide bonds (for nitrogen plasma) have been generated after plasma irradiation. Dog bone marrow stromal cell culture studies showed that cells grew better on the plasma treated surface. These results demonstrate potentiality of this method for grafting useful component in future tissue-engineering applications.

Animals↗
PubMed2007

In vitro biocompatibility evaluation of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) copolymer in fibroblast cells.

Among the various biomaterials available for tissue engineering and therapeutic applications, microbial polyhydroxyalkanoates offer the most diverse range of thermal and mechanical properties. In this study, the biocompatibility of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB); containing 50 mol % of 4-hydroxybutyrate] copolymer produced by Delftia acidovorans was evaluated. The cytotoxicity, mode of cell death, and genotoxicity of P(3HB-co-4HB) extract against V79 and L929 fibroblast cells were assessed using MTT assay, acridine orange/propidium iodide staining, and alkaline comet assay, respectively. Our results demonstrate that P(3HB-co-4HB) treated on both cell lines were comparable with clinically-used Polyglactin 910, where more than 60% of viable cells were observed following 72-h treatment at 200 mg/mL. Further morphological investigation on the mode of cell death showed an increase in apoptotic cells in a time-dependent manner in both cell lines. On the other hand, P(3HB-co-4HB) at 200 mg/mL showed no genotoxic effects as determined by alkaline comet assay following 72-h treatment. In conclusion, our study indicated that P(3HB-co-4HB) compounds showed good biocompatibility in fibroblast cells suggesting that it has potential to be used for future medical applications.

Animals↗
PubMed2007

Evaluation of the biological responses of osteoblast-like UMR-106 cells to the engineered porous PHBV matrix.

Poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) has been investigated for biomedical applications due to its many biologically favorable properties. However, to explore its application in bone tissue engineering, the poorly bioactive surface property of PHBV must be improved. To engineer PHBV to achieve a biologically active surface, in this study each porous PHBV matrix was prepared by solute leaching of salt/PHBV cast film and was treated with ozone followed by dip coating with type I collagen. The biological responses of osteoblast-like UMR-106 cells after being grown on the engineered PHBV matrix were evaluated. Confocal microscopy and the MTT assay were used to map and quantify the viable cell proliferation on the PHBV matrix, respectively. The cells were cultivated in osteogenic media containing beta-glycerophosphate and later stained with alizarin red to visualize mineralization of the matrix. RNA was extracted from the UMR-106 cells, and reverse transcriptase-polymerase chain reaction (RT-PCR) was applied to detect expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (a house keeping gene) and bone sialoprotein (BSP) (marker of the osteoblastic phenotype). The results showed that the UMR-106 cells after cultivation on the engineered PHBV matrix retained the osteoblastic phenotype characteristics, indicating that the porous PHBV matrix after ozone treatment and collagen dip coatings are a promising scaffold for bone tissue engineering applications.

Animals↗
PubMed2007

Characteristic comparison of bioactive scaffolds based on polyhydroxyalkoanate/bioceramic hybrids.

In this study, bioactive hybrid scaffolds were prepared by combining poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate) (PHBV) with three kinds of bioceramics, which are sol-gel bioglass (SGBG), tricalcium phosphate (TCP), and hydroxyapatite. The bioactivity and biodegradation of various scaffolds were analyzed with simulated experiments in vitro by immersing the scaffolds into simulated body fluid to evaluate their biomineralizing ability. The changes of the surface components and structures were determined by FTIR, XRD, and SEM analyses. Cell cultures and observation were made in vitro to assess the proliferation of osteoblast on these scaffolds. The results show that there might be ion exchanges and biodegradation reactions occurring between the hybrids and simulated body fluid solution, which result in the weight loss of the scaffolds and the pH changes in solution. An obvious biomineralized deposit layer was formed on the surface of the PHBV/SGBG and PHBV/TCP scaffolds, which was determined to be the bonelike crystalline hydroxyl-carbonate-apatite. The experiments on cell culture show that osteoblast could grow and proliferate on the bioactive hybrid scaffolds.

Animals↗
PubMed2007

Growing tissue-like constructs with Hep3B/HepG2 liver cells on PHBV microspheres of different sizes.

In this study, an oil-in-water emulsion solvent evaporation technique was used to fabricate poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV, 8% PHV), microspheres as scaffold, to guide liver cell growth. Human hepatoma cell lines, HepG2 and Hep3B, were cultured in vitro on both the microspheres and polymer films. SEM and optical microscope images showed that multilayer cells were formed among the microspheres to bridge them together and developed into cell-construct aggregates after 1 week of culture. MTT results showed that the cell proliferation on the microspheres was more than two times higher than that on the films after 12 days of culture. The cells seeded on microspheres secreted albumin 2-4 times more than that on the positive control after 1 week of culture, which indicated that this hepatic function was greatly improved by the aggregation of cells on microspheres. Although HepG2 failed to express P-450 activity, this hepatic function was preserved when Hep3B cultured on microspheres. All the results indicated that PHBV microspheres are appropriate scaffolds for liver tissue engineering.

Cell Culture Techniques↗
PubMed1992

Control of drug release with a combination of prodrug and polymer matrix: antitumor activity and release profiles of 2',3'-diacyl-5-fluoro-2'-deoxyuridine from poly(3-hydroxybutyrate) microspheres.

Drug release was controlled by a combination of prodrug and polymer matrix. Prodrugs of 5-fluoro-2'-deoxyuridine with different physicochemical properties were synthesized by esterification with aliphatic acids (propionate, n-butyrate, and n-pentanoate). Microspheres containing these ester prodrugs were prepared with poly(3-hydroxybutyrate) of three molecular weights (65,000, 135,000, and 450,000). The release rates from the spheres depended on both the lipophilicity of the prodrug and the molecular weight of the polymer. Regardless of the polymer, the relative release rates were propionate greater than butyrate greater than pentanoate. The release of butyrate and pentanoate from the spheres consisting of low-molecular-weight polymer (M(r), 65,000) was faster than that from the spheres of higher molecular weight (M(r), 135,000 or 450,000). A single intraperitoneal injection of spheres of the highest molecular weight polymer containing butyrate or pentanoate resulted in higher antitumor effects against P388 leukemia in mice than did free prodrugs given over a period of five consecutive days. The polymer sphere itself showed low toxicity to and good biocompatibility with mice and rats.

Animals↗
PubMed2004

In-situ atomic force microscopy observation of enzymatic degradation in poly(hydroxyalkanoic acid) thin films: normal and constrained conditions.

The enzymatic degradation of lamellar crystals in poly(hydroxyalkanoic acid) thin films has been visualized by using in-situ dynamic force mode (tapping mode) atomic force microscopy (AFM) in buffer solution. It was found that poly(hydroxybutyric acid) (PHB) depolymerase from Ralstonia pickettii T1 degraded the thin surface layers formed at room temperature first, and that lamellar crystals formed at the crystallization temperature (110 degrees C) were eroded from the crystallographic a-axis to show splintered morphologies at the tips of the crystals. In some cases, lamellar crystals were hydrolyzed from the crystallographic b-axis, resulting in the formation of small crevices. These results suggest that disordered molecular chain-packing regions exist in the crystal along the crystallographic a- and b-axes, and that enzymatic degradation predominantly occurs from these defective regions. In addition, cantilever-tip-induced enzymatic degradation was carried out in the presence of PHB depolymerase. A concave area was artificially formed on the stacked lamellar crystals by the AFM tip. In-situ AFM observation has revealed that enzymatic degradation proceeds along both the longitudinal and lateral directions of the lamellae. At the same time, the PHB depolymerase preferentially eroded the concave area along the crystallographic c-axis. These results demonstrated that the PHB depolymerase predominantly degrades the less-ordered molecular chain-packing regions in the crystals.

3-Hydroxybutyric Acid↗
PubMed2004

Biosynthesis and compositional regulation of poly[(3-hydroxybutyrate)-co-(3-hydroxyhexanoate)] in recombinant ralstonia eutropha expressing mutated polyhydroxyalkanoate synthase genes.

A new strategy for bacterial polyhydroxyalkanoate (PHA) production by recombinant Ralstonia eutropha PHB(-)4 harboring mutated PHA synthase genes (phaC(Ac)) from Aeromona caviae was investigated. The strain harboring wild-type phaC(Ac) gene produced a PHA copolymer consisting of (R)-3-hydroxybutyrate and (R)-3-hydroxyhexanoate [P(3HB-co-3HHx)] with 3.5 mol-% of 3HHx fraction from soybean oil. When the mutants of phaC(Ac) gene were applied to this production system, 3HHx fraction in copolymers was varied in the range of 0-5.1 mol-%. Thus, the regulation of PHA copolymer compositions has been achieved by the use of mutated PHA synthase genes.

3-Hydroxybutyric Acid↗
PubMed2004

Synthesis and properties of graft copolymers based on poly(3-hydroxybutyrate) macromonomers.

Graft copolymers of poly(methyl methacrylate) with poly(3-hydroxybutyrate), PHB, segments as long side chains were prepared by the macromonomer method. PHB macromonomers were prepared from the esterification of oligomers with 2-hydroxyethyl methacrylate at their carboxylic acid end. Esterification products displayed low polydispersity indices (ca. 1.2) and a functionality of over 83%, with a Mn of 2,020. Using free radical polymerization methods, the macromonomers were copolymerized with methyl methacrylate to yield graft (comb type) copolymers at different comonomer feed ratios. The graft copolymers contained from 0.5 to 14 mol-% of PHB blocks, with a glass transition temperature decreasing from 100 to 3 degrees C.

Free Radicals↗
PubMed2004

Metabolic engineering for the production of copolyesters consisting of 3-hydroxybutyrate and 3-hydroxyhexanoate by Aeromonas hydrophila.

Aeromonas hydrophila 4AK4 was able to synthesize copolyesters consisting of 3-hydroxybutyrate (3HB) and about 15 mol-% 3-hydroxyhexanoate (3HHx) (PHBHHx) when grown in long chain fatty acids such as dodecanoate regardless of growth conditions. To regulate the unit fraction in PHBHHx, phbA and phbB genes encoding beta-ketothiolase and acetoacetyl-CoA reductase in Ralstonia eutropha, were introduced into A. hydrophila 4AK4. When gluconate was used as cosubstrate of dodecanoate, the recombinant produced PHBHHx containing 3-12 mol-% 3HHx, depending on the gluconate concentration in media. Vitreoscilla hemoglobin gene, vgb, was also introduced into the above recombinant, resulting in improved PHBHHx content from 38 to 48 wt.-% in shake flask study. Fermentor studies also showed that increased gluconate concentration in medium containing dodecanoate promoted the recombinant strain harboring phbA and phbB genes to incorporate more 3HB unit into PHBHHx, resulting in reduced 3HHx fraction. Recombinant A. hydrophila harboring phbA, phbB and vgb genes demonstrated better PHBHHx productivity and higher conversion efficiency from dodecanoate to PHBHHx than those of the recombinant without vgb in fermentation study. Combined with the robust growth property and simple growth requirement, A. hydrophila 4AK4 appeared to be a useful organism for metabolic engineering.

3-Hydroxybutyric Acid↗
PubMed2004

Elastic properties of poly(hydroxybutyrate) molecules.

Elasticity of various poly(hydroxybutyrate) (PHB) molecules of regular and irregular conformational structure was examined by the molecular mechanics (MM) calculations. Force - distance functions and the Young's moduli E were computed by stretching of PHB molecules. Unwinding of the 2(1) helical conformation H is characterized at small deformations by the Young's modulus E = 1.8 GPa. The H form is transformed on stretching into the highly extended twisted form E, similar to the beta-structure observed earlier by X-ray fiber diffraction. The computations revealed that in contrast to paraffins, the planar all-trans structure of undeformed PHB is bent. Hence, a PHB molecule attains the maximum contour length in highly straightened, but slightly twisted conformations. A dependence of the single-chain moduli of regular and disordered conformations on the chain extension ratio x was found. The computed data were used to analyze elastic response of tie (bridging) molecules in the interlamellar (IL) region of a semi-crystalline PHB. A modification of the chain length distribution function of tie molecules tau(N) due to secondary crystallization of PHB was conjectured. The resulting narrow distribution tau(N) comprises the taut tie molecules of higher chain moduli prone to overstressing. The molecular model outlined is in line with the macroscopically observed increase in the modulus and brittleness of PHB with storage time.

Elasticity↗