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PubMed2003

Reduced mouse fibroblast cell growth by increased hydrophilicity of microbial polyhydroxyalkanoates via hyaluronan coating.

The mouse fibroblast cell line L929 was inoculated on 3D scaffolds of microbial polyesters, namely polyhydroxybutyrate (PHB) and poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) to evaluate their in vitro biocompatibility. It was found that both polyhydroxyalkanoates (PHA) subjected to lipase treatment and hyaluronan (HA) coating decreased the contact angle of water to the material surface approximately 30%, meaning an increased hydrophilicity on the PHA surface. At the same time, both the lipase treatment and the HA coating smoothened the PHA surface. After the lipase treatment or HA coating, the ratio of PHA hydrophilic groups including hydroxyl and carboxyl to carbonyl of PHA was approximately 1:1 or 2:1. Cells grown on scaffolds treated with lipase were approximately 4 x 10(5)/ml, twice in number of the control. However, PHA scaffolds coated with HA were observed with a 40% decrease in cell growth compared with that of the control. HA coating reduced the cell attachment and proliferation on PHA although the materials had increased hydrophilicity. In comparison, lipase treatment promoted the cell growth on PHA although the treatment did not lead to better hydrophilicity compared with HA coating. It appeared that an appropriate combination of hydrophilicity and hydrophobicity was important for the biocompatibility of PHBHHx, especially for the growth of L929 cells on the surface of this material. This may have instructive significance for biomaterial selection and design.

Animals↗
PubMed2001

Production of PHA from starchy wastewater via organic acids.

Polyhydroxyalkanoate (PHA) was produced from a starchy wastewater in a two-step process of microbial acidogenesis and acid polymerization. The starchy organic waste was first digested in a thermophilic upflow anaerobic sludge blanket (UASB) reactor to form acetic (60-80%), propionic (10-30%) and butyric (5-40%) acids. The total volatile fatty acids reached 4000 mg l(-1) at a chemical oxygen demand (COD) loading rate of 25-35 g l(-1) day(-1). A carbon balance indicates that up to 43% of the organic carbon in the starchy waste went to the organic acids and the rest to biogas, volatile suspended solids and residual sludge accumulated in the reactor. The acid composition profile was affected by COD loading rate: a medium rate around 9 g l(-1) day(-1) gave a high propionic acid content (29% wt) and a high rate around 26 g l(-1) day(-1) led to a high butyric acid content (34% wt). The acids in the effluent solution after microfiltration were utilized and polymerized into PHA by bacterium Alcaligenes eutrophus in a second reactor. Fifty grams of PHA was produced from 100 g total organic carbon (TOC) utilized, a yield of 28% based on TOC, which is comparable with 55 g PHA per 100 g TOC of pure butyric and propionic acids used. PHA formation from individual acids was further investigated in a semi-batch reactor with three acid feeding rates. With a limited nitrogen source (80-100 mg NH(3) per liter), the active biomass of A. eutrophus, not including the accumulated PHA in cells, was maintained at a constant level (8-9 g l(-1)) while PHA content in the cell mass increased continuously in 45 h; 48% PHA with butyric acid and 53% PHA with propionic acid, respectively. Polyhydroxybutyrate was formed from butyric acid and poly(hydroxybutyrate-hydroxyvalerate) formed from propionic acid with 38% hydroxyvalerate.

Alcaligenes↗
PubMed2001

Versatility of biodegradable biopolymers: degradability and an in vivo application.

Biodegradable materials have various important applications in the biomedical field. There are basically two groups of polyesters which have significant importance in this field. These are polylactides and polyhydroxybutyrates. Both groups degrade via hydrolysis with the rates of degradation depending on medium properties such as pH, temperature, solvent and presence of biocatalysts, as well as on chemical compositions. In order for these biomaterials to be suitable for use in load bearing applications without deformation or warping their strengths and their capability to maintain their form must be improved. To insure dimensional stability during degradation and to match modulus and strength to that of bone, introduction of a reinforcing structure for those applications to plate fixation through the creation of an interpenetrating network might be a feasible approach. In this study, poly(lactide-co-glycolide) (PLGA), was the major structural element to be strengthened by a three-dimensional network or "scaffold" of another biodegradable polymer, poly(propylene fumarate) (PPF). PPF would be crosslinked with a biocompatible vinyl monomer, vinylpyrrolidone (VP). Three different approaches were tested to create dimensionally stable bone plates. First, via in situ crosslinking of PPF in the presence of PLGA. Secondly, by blending of precrosslinked PPF with PLGA. Finally, by simultaneous crosslinking and molding of the PLGA, PPF and VP. These were compared against extruded or compression molded PLGA controls. Results showed that compression molding at room temperature followed by crosslinking under pressure at elevated temperature and subsequently by gamma-irradiation appeared to yield the most favorable product as judged by swelling, hardness and flexural strength data. The composition of the implant material, PLGA(3):PPF(1):VP(0.7), appeared to be suitable and formed the compositional and procedural basis for in vivo biocompatibility studies.

Absorbable Implants↗
PubMed2004

The activator of the Rhodospirillum rubrum PHB depolymerase is a polypeptide that is extremely resistant to high temperature (121 degrees C) and other physical or chemical stresses.

Hydrolysis of native (amorphous) polyhydroxybutyrate (nPHB) granules isolated from different sources by soluble PHB depolymerase of Rhodospirillum rubrum in vitro requires the presence of a heat-stable compound (activator). The activator was purified and was resistant against various physical and chemical stresses such as heat (up to 130 degrees C), pH 1-12, dryness, oxidation by H2O2, reducing and denaturing compounds (2-mercaptoethanol, 5 M guanidinium-HCl) and many solvents including phenol/chloroform. The activator coding gene was identified by N-terminal sequencing of the purified protein, and the deduced protein showed significant homology to magnetosome-associated protein (Mms16) of magnetotactic bacteria. Analysis of the activation process in vitro showed that the activator acts on nPHB granules but not on the depolymerase. The effect of the activator could be mimicked by pretreatment of nPHB granules with trypsin or other proteases but protease activity of the purified activator was not detected. Evidence is shown that different mechanisms were responsible for activation of nPHB by trypsin and activator, respectively. PHB granule-associated protein (PhaP) of Ralstonia eutropha nPHB granules were cleaved by trypsin but no cleavage occurred after activator treatment. Hydrolysis of artificial protein-free PHB granules coated with negatively charged detergents (sodium dodecyl sulfate (SDS), cholate but not cetyltrimethyl-ammonium bromide (CTAB)) did not require activation and confirmed that surface layer proteins of nPHB granules are the targets of the activator rather than lipids. All experimental data are in agreement with the assumption that trypsin and the activator enable the PHB depolymerase to find and to bind to the polymer surface: trypsin by removing a portion of proteins from the polymer surface, the activator by modifying the surface structure in a not yet understood manner presumably by interaction with phasins of the proteinous surface layer of nPHB.

Carboxylic Ester Hydrolases↗
PubMed1999

Enrichment, phylogenetic analysis and detection of a bacterium that performs enhanced biological phosphate removal in activated sludge.

Activated sludge communities which performed enhanced biological phosphate removal (EBPR) were phylogenetically analyzed by 16S rRNA-targeted molecular methods. Two anaerobic-aerobic sequencing batch reactors were operated with two different carbon sources (acetate vs. a complex mixture) for three years and showed anaerobic-aerobic cycles of polyhydroxybutyrate- (PHB) and phosphate-accumulation characteristic for EBPR-systems. In situ hybridization showed that the reactor fed with the acetate medium was dominated by bacteria phylogenetically related to the Rhodocyclus-group within the beta-Proteobacteria (81% of DAPI-stained cells). The reactor with the complex medium was also predominated by this phylogenetic group albeit at a lesser extent (23% of DAPI-stained cells). More detailed taxonomic information on the dominant bacteria in the acetate-reactor was obtained by constructing clone libraries of 16S rDNA fragments. Two different types of Rhodocyclus-like clones (R1 and R6) were retrieved. Type-specific in situ hybridization and direct rRNA-sequencing revealed that R6 was the type of the dominant bacteria. Staining of intracellular polyphosphate- and PHB-granules confirmed that the R6-type bacterium accumulates PHB and polyphosphate just as predicted by the metabolic models for EBPR. High similarities to 16S rDNA fragments from other EBPR-sludges suggest that R6-type organisms were present and may play an important role in EBPR in general. Although the R6-type bacterium is closely related to the genus Rhodocyclus, it did not grow phototrophically. Therefore, we propose a provisional new genus and species Candidatus Accumulibacter phosphatis.

Aerobiosis↗
PubMed1999

Genotypic diversity of Acidovorax strains isolated from activated sludge and description of Acidovorax defluvii sp. nov.

Fluorescence in situ hybridization of activated sludge samples from a municipal wastewater treatment plant using oligonucleotide probes specific for Acidovorax demonstrated that these bacteria are highly abundant in this environment. For the targeted cultivation of representatives belonging to this genus, isolates grown on agar plates after serial dilution were screened by whole-cell hybridization with specific probes. The obtained strains clustered in two phylogenetic groups as determined by 16S rRNA gene sequence analyses. The isolates of one cluster were phylogenetically and genotypically closely related to A. delafieldii. In contrast, the strains of the other cluster were genotypically and phenotypically distinct from the hitherto known Acidovorax species. Therefore, a new species, Acidovorax defluvii sp. nov., was proposed for these strains. The main characteristics of the newly defined species are as follows: Gram-negative, motile or non-motile rods with rounded ends, often with large polyhydroxybutyrate granules. In broth cultures flocs are formed. Test for cytochrome oxidase is positive with all strains. The majority of strains is catalase positive and reduces nitrate. All strains are metabolically inactive against most carbohydrates and organic acids. Fatty acid patterns are typical for the genus Acidovorax. The guanine-plus-cytosine content of DNAs varies between 62 and 64 mol%. The type strain of A. defluvii is BSB411T (DSM 12644). A new 16S rRNA-targeted oligonucleotide probe reacting by in situ hybridization with all known Acidovorax species, including A. defluvii sp. nov., was designed.

Base Sequence↗
PubMed2002

Multigene engineering: dawn of an exciting new era in biotechnology.

Development of a rice variety enriched in provitamin A, the accumulation of polyhydroxybutyrate polyester in Arabidopsis nuclear transgenic plants (with enzymes targeted to chloroplasts in both), and the expression of bacterial operons via the chloroplast genome are recent landmark achievements in multigene engineering. Hyper-expression of transgenes has resulted in the formation of insecticidal protein crystals or inclusion bodies of pharmaceutical proteins in transgenic chloroplasts, achieving the highest level of transgene expression ever reported in transgenic plants. These achievements illustrate the potential of multigene engineering to realize benefits of the post-genomic revolution.

Biopharmaceutics↗
PubMed2005

Detection of intermediates from the polymerization reaction catalyzed by a D302A mutant of class III polyhydroxyalkanoate (PHA) synthase.

Polyhydroxybutyrate (PHB) synthases catalyze the polymerization of (R)-3-hydroxybutyryl-CoA (HB-CoA) into high molecular weight PHB, biodegradable polymers. The class III PHB synthase from Allochromatium vinosum is composed of a 1:1 mixture of two approximately 40 kDa proteins: PhaC and PhaE. Previous studies using site-directed mutagenesis and a saturated trimer of hydroxybutyryl-CoA have suggested the importance of C149 (in covalent catalysis), H331 (in activation of C149), and D302 (in hydroxyl group activation for ester bond formation) in the polymerization process. All three residues are located on PhaC. We now report that incubation of D302A-PhaCPhaE with [14C]-HB-CoA results in detection, for the first time, of oligomeric HBs covalently bound to PhaC. The reaction products have been analyzed by SDS-PAGE, Westerns with PhaCPhaE antibodies, and autoradiography. Different migratory properties of D302A-PhaC on SDS-PAGE have been observed at [14C]-HB-CoA to enzyme (S/E) ratios between 5 and 100. Trypsin digestion and HPLC analysis of the D302A-PhaCPhaE (from a reaction with a S/E ratio of 5) allowed isolation of multiple radiolabeled peptides. N-Terminal sequencing, MALDI-TOF, and ESI mass spectrometric analysis of these peptides revealed that all of the peptides were identical but were modified by (HB)n ranging in size from n = 3 to n = 10. The in vitro results support the role of D302 in elongation rather than in activating the active site cysteine for acylation. This proposal has been further supported by our in vivo studies on a Wautersia eutropha strain in which the class I synthase gene has been replaced with the D302A-PhaCPhaE gene and the organism examined under PHB production conditions by transmission electron microscopy. Very small granules (<0.05 microm) were observed in contrast to the 0.2-0.5 microm granules observed with the wt strain. Use of the D302A synthase has allowed successful interrogation of the initiation and elongation steps catalyzed by the class III synthase.

Acyltransferases↗
PubMedDate not supplied

Biodegradation of aliphatic homopolyesters and aliphatic-aromatic copolyesters by anaerobic microorganisms.

The anaerobic degradability of natural and synthetic polyesters is investigated applying microbial consortia (3 sludges, 1 sediment) as well as individual strains isolated for this purpose. In contrast to aerobic conditions, the natural homopolyester polyhydroxybutyrate (PHB) degrades faster than the copolyester poly(hydroxybutyrate-co-hydroxyvalerate) (PHBV). For the synthetic polyester poly(epsilon-caroplacton) (PCL), microbial degradation in the absence of oxygen could be clearly demonstrated; however, the degradation rate is significantly lower than for PHB and PHBV. Other synthetic polyesters such as poly(trimethylene adipate) (SP3/6), poly(tetramethylene adipate) (SP4/6), and aliphatic-aromatic copolyesters from 1,4-butanediol, terephthalic acid, and adipic acid (BTA-copolymers) exhibit only very low anaerobic microbial susceptibility. A copolyester with high amount of terephthalic acid (BTA 40:60) resisted the anaerobic breakdown even under thermophilic conditions and/or when blended with starch. For the synthetic polymers, a number of individual anaerobic strain could be isolated which are able to depolymerize the polymers and selected strains where identified as new species of the genus Clostridium or Propionispora. Their distinguished degradation patterns point to the involvement of different degrading enzymes which are specialized to depolymerize either the natural polyhydroxyalkanoates (e.g., PHB), the synthetic polyester PCL, or other synthetic aliphatic polyesters such as SP3/6. It can be supposed that these enzymes exhibit comparable characteristics as those described to be responsible for aerobic polyester degradation (lipases, cutinases, and PHB-depolymerases).

Bacteria, Anaerobic↗
PubMed2002

The cobalt-catalyzed alternating copolymerization of epoxides and carbon monoxide: a novel approach to polyesters.

The metal-catalyzed synthesis of polyolefins, polyketones, and polycarbonates is well-known in academia and is already successfully applied in industrial processes. Still missing, however, is the metal-catalyzed synthesis of aliphatic polyesters, as one of the most important biodegradeable polymer families. We report here on the cobalt-catalyzed alternating copolymerization of propylene oxide and carbon monoxide, affording atactic and isotactic polyhydroxybutyrates (PHB). The postulated mechanism is supported by online ATR-IR analytics.

Journal Article↗
PubMed1997

Metabolism of micro-organisms responsible for enhanced biological phosphorus removal from wastewater. Use of dynamic enrichment cultures.

The removal of phosphorus from wastewater is already widely applied. In many cases use is made of micro organisms capable of accumulating phosphorus as polyphosphate inside the cell. The main characteristic providing the competitive advantage to these polyphosphate accumulating bacteria is the capability to use polyphosphate, in the absence of external electron acceptors, as energy source for the uptake and storage of acetic acid in the form of polyhydroxybutyrate (PHB). The reduction equivalents for the formation of PHB are derived from the conversion of glycogen to PHB. Despite the widespread use and study of enhanced biological phosphorus removal no pure culture, having the above mentioned characteristics, has been isolated yet. All ecophysiological studies on these type of cultures have therefore been performed by enrichment cultures. This paper reviews the research on these type of organisms, and shows that it is possible to understand a complex microbial process on a metabolic level, both stoichiometrically and kinetically, without the availability of a pure culture.

Acinetobacter↗
PubMed1999

Production of polyhydroxyalkanoates by Pseudomonas nitroreducens.

A strain coded AS 1.2343 was isolated from oil-contaminated soil in an oil-field in North China Tianjian City and it was identified as Pseudomonas nitroreducens. The strain demonstrated some unusual ability to synthesize polyhydroxybutyrate (PHB) homopolymer from medium-chain-length (mcl) fatty acids including hexanoate and octanoate. While polyhydroxyalkanoates (PHA) consisting of mcl hydroxyalkanoate (HA) monomers such as hydroxyoctanoate (HO) and hydroxydecanoate (HD) were the major compositions when butyrate, decanoate, lauric acid and tetradecanoic acid were used as substrates for the cell growth, respectively. PHA was accumulated up to 77% of the cell dry weight when growth was conducted in lauric acid, it appeared that the HA contents in the PHA would not be much affected by the changing of the lauric acid concentration. Varying the concentration ratio of butyrate to octanoate could change the composition of PHA accumulated by the strain. Yet PHB homopolymer was always the only polyester synthesized by the strain, regardless of the octanoate concentration change. Additionally, the ratio of carbon to nitrogen (C/N) in butyrate media was found to have effects on the PHA monomer content, as C/N increased from 2 to 100, content of HB decreased from 100% to 7%. PHA polyester synthesized by cells of Pseudomonas nitroreducens AS 1.2343 was a blend polymers consisting of acetone-insoluble HB and acetone-soluble mcl HA monomers.

Caprylates↗
PubMed1998

The healing process of palatal tissues after palatal surgery with and without implantation of membranes: an experimental study in dogs.

The aim of this study was to evaluate the wound-healing process clinically and histologically in growing beagle dogs after palatal repair according to von Langenbeck, with and without implantation of membranes of a copolymer of polyhydroxybutyrate 80%-hydroxyvalerate 20% (=PHB-co-HV 80/20). Von Langenbeck's repair was performed in 12 dogs (age 12 wk), while von Langenbeck's repair followed by implantation of PHV-co-HV membranes was carried out in 11 dogs (age 12 wk). Four dogs (age 12 wk) served as unoperated controls. Standardized intra-oral slides of the palate were taken and measurements of the wound surface areas were carried out. Histological sections were prepared at three different ages. The animals were studied until the age of 25 wk. It was found that wound closure after the von Langenbeck's procedure took about 3 wk, while the use of PHB-co-HV membranes after von Langenbeck's repair resulted in complete wound closure after approximately 7 wk after the membranes had sequestered. At the age of 25 wk, the histologic results after the von Langenbeck procedure showed that the entire scar tissue covering the former denuded bony areas was attached to the bone by means of Sharpey's fibres, while after implantation of the membranes only local scar tissue attachment by means of Sharpey's fibres was found. Further research is necessary to develop a membrane which allows wound closure without sequestration of it.

Journal Article↗
PubMed2001

Production of poly-3-hydroxybutyrate by Bacillus sp. JMa5 cultivated in molasses media.

A strain of Bacillus sp. coded JMa5 was isolated from molasses contaminated soil. The strain was able to grow at a temperature as high as 45 degrees C and in 250 g/l molasses although the optimal growth temperature was 35-37 degrees C. Cell density reached 30 g/l 8 h after inoculation in a batch culture with an initial concentration of 210 g/l molasses. Under fed-batch conditions, the cells grew to a dry weight of 70 g/l after 30 h of fermentation. The strain accumulated 25-35%, (w/w) polyhydroxybutyrate (PHB) during fermentation. PHB accumulation was a growth-associated process. Factors that normally promote PHB production include high ratios of carbon to nitrogen, and carbon to phosphorus in growth media. Low dissolved oxygen supply resulted in sporulation, which reduced PHB contents and dry weights of the cells. It seems that sporulation induced by reduced supply of nutrients is the reason that PHB content is generally low in the Bacillus strain.

3-Hydroxybutyric Acid↗
PubMedDate not supplied

Manufacture and evaluation of bioactive and biodegradable materials and scaffolds for tissue engineering.

For tissue regeneration and tissue engineering applications, a number of bioactive and biodegradable composites, either porous or non-porous, were fabricated. The newly developed materials included tricalcium phosphate reinforced polyhydroxybutyrate and its copolymer, poorly crystallized hydroxyapatite reinforced chitin, and plasma sprayed hydroxyapatite reinforced poly(L-lactic acid). It was shown that these new materials could be successfully produced using the manufacturing techniques adopted. In vitro experiments revealed that the incorporation of bioceramic particles in biodegradable polymers rendered the composites bioactive and significantly improved the ability of composites to induce the formation of bone-like apatite on their surfaces. Degradation of composite scaffolds in simulated body fluid was observed and could be due to the simultaneous degradation of polymer matrix and dissolution of bioceramic particles.

Journal Article↗
PubMed2002

Effect of lipase treatment on the biocompatibility of microbial polyhydroxyalkanoates.

Films made from microbial polyesters polyhydroxybutyrate (PHB) and poly(hydroxybutyrate-co-hydroxyhexanoate) (PHBHHx) were treated by lipases and NaOH solution. The change of the polyester biocompatibility was evaluated by inoculating mouse fibroblast cell line L929 on films of PHB, PHBHHx and their blends. Polylactic acid (PLA) was used as a control. It was found that untreated PHB and PLA films gave a poor support to the growth of L929 cells, viable cell density ranged from 0.1x10(4}) to 0.7x10(4) per ml only. While films of pure PHBHHx and PHB blended with PHBHHx showed improved biocompatibility, viable cell density observed increased from 9.6x10(2) to 6x10(4) on blended films of PHB/PHBHHx in ratios of 0.9/0.1 to 0/1, respectively. This result showed PHBHHx has a better biocompatibility compared with PHB. Films of PHB, PLA and the blends treated with lipases and 1 N NaOH, respectively, showed an improved ability to support cell growth. Biocompatibility of PHB was approximately the same as PLA after the treatment, while PHBHHx and its dominant blends showed improved biocompatibility compared with PLA. The sensitivity of the treatments was reduced when PHBHHx content increased in the PHB/PHBHHx blends. All three lipase treatments demonstrated more biocompatibility increase on all the films compared with the results of NaOH treatment. Scanning electron microscopy showed that PHB films changed its surface from multi-porous to rough non-porous after the lipase or NaOH treatment. While PHBHHx films showed little change after these treatments. The results showed that the polyester surface morphology played an important role in affecting cell attachment and growth on these materials.

Journal Article↗
PubMed1995

A biodegradable patch used as a pericardial substitute after cardiac surgery: 6- and 24-month evaluation with CT.

Biodegradable patches made of polyhydroxybutyrate (PHB) were recently shown to limit postoperative pericardial adhesions when inserted into the pericardium in sheep. The present study was carried out to evaluate the presence of post-operative retrosternal adhesions after cardiac surgery in patients operated on with the PHB patch compared with those operated on without the insertion of such a patch. 50 patients admitted for CABG and/or valvular replacement were randomised to treatment either with pericardial closure with a patch or with the pericardium left open. In 39 of these (19 with and 20 without a PHB patch) computerised tomography was carried out six and twenty-four months postoperatively. Computerised tomography indicated a lower incidence (p < 0.05) of postoperative adhesions between the patch and the cardiac surface in the patch group, compared with the corresponding area in the non-patch group. A reduction of patch size in 27% (p = 0.003) was also seen.

Aged↗
PubMed1995

Inorganic polyphosphates in the acquisition of competence in Escherichia coli.

A complex of polyhydroxybutyrate (PHB), Ca2+, and inorganic polyphosphate (polyP) was proposed as the membrane component responsible for competence for DNA entry in Escherichia coli (Reusch, R. N., and Sadoff, H. L. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 4176-4180). While chemical and immunological assays and 1H NMR have unequivocally established the identity and content of PHB in the complex, comparable methods were not available for polyP. With specific enzyme assays developed for polyP, we have identified, in chloroform extracts of competent cell membranes, a novel form of polyP of about 60 to 70 residues in a stoichiometric ratio of PHB to polyP of 2:1. In E. coli mutants, incapable of synthesizing the predominant, thousand-long polyP chains, appearance of this short polyP and its inclusion in membranes can account for their capacity to develop competence and indicates an auxiliary pathway for polyP synthesis. A variety of fluorescent lipid probes demonstrate the appearance of extensive rigid domains in membranes of competent cells. We propose that the PHB.Ca2+.polyP complex perturbs the conformation of the lipid matrix, making it more permeable to charged molecules and thus allowing the entry of DNA.

Cell Membrane↗