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PubMed2002

The synthesis of short- and medium-chain-length poly(hydroxyalkanoate) mixtures from glucose- or alkanoic acid-grown Pseudomonas oleovorans.

Pseudomonas oleovorans NRRL B-778 accumulated mixtures of poly-3-hydroxybutyrate (PHB) and medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs) when grown on glucose, octanoic acid or oleic acid, whereas growth on nonanoic acid or undecanoic acid resulted in copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-co-HV). Acetone fractionation verified the presence of PHB/mcl-PHA mixtures. The acetone-insoluble (AIS) fractions of the polymers derived from glucose (PHA-glucose), octanoic acid (PHA-octanoic) and oleic acid (PHA-oleic) were exclusively PHB while the acetone-soluble (AS) fractions contained mcl-PHA composed of differing ratios of 3-hydroxy-acid monomer units, which ranged in chain length from 6 to 14 carbon atoms. In contrast, both the AIS and AS fractions from the polymers derived from nonanoic acid (PHA-nonanoic) and undecanoic acid (PHA-undecanoic) were composed of comparable ratios of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). The unfractionated PHA-glucose, PHA-octanoic and PHA-oleic polymers had melting temperatures (Tm) between 177 and 179 degrees C, enthalpies of fusion (AHf) of 20 cal/g and glasstransition temperatures (Tg) of 3-4 degrees C. This was due to the large PHB content in the polymer mixtures. On the other hand, the PHA-nonanoic and PHA-undecanoic polymers had thermal properties that supported their copolymer nature. In both cases, the Tm values were 161 degrees C, deltaHf values were 7 cal/g and Tg values were - 3 degrees C.

Fermentation↗
PubMed2002

Alginate production by Azotobacter vinelandii mutants altered in poly-beta-hydroxybutyrate and alginate biosynthesis.

Mutant AT268 of Azotobacter vinelandii - showing diminished production of poly-beta-hydroxybutyrate (PHB) due to a mutation in phbR (the gene coding for the transcriptional activator of the phbBAC biosynthetic operon); mutant CNT26, containing a mutation (muc26) that increases the transcription of gene algD (encoding GDP mannose dehydrogenase, the key enzyme in alginate biosynthesis); and mutant DM, carrying both phbR and muc26 mutations - were characterised in terms of alginate production, broth viscosity, and molecular weight of the alginate. All the mutants evaluated produced 25% less alginate with respect to that produced by the wild type. Unexpectedly and with no apparent relation to the phbR and muc26 mutations, mutant DM exhibited the highest molecular weight ever reported for a bacterial alginate (up to 4 x 10(6) Da), with a very low polydispersity index (1.3). Acetyl content in the alginate produced by this strain was low (1.4-2.6%). These characteristics make this mutant a very valuable source for producing alginates with improved properties.

Alginates↗
PubMed2001

Biosynthesis of PHB tercopolymer by Bacillus cereus UW85.

AIMS: The study was attempted to determine the ability of a Gram-positive Bacillus cereus UW85 strain to biosynthesize poly (3-hydroxybutyrate) copolymers when epsilon-caprolactone, or epsilon-caprolactone and glucose, were used as carbon sources. METHODS AND RESULTS: Bacillus cereus was grown for 24 h under nitrogen-limited conditions in a mineral salts medium. Growth was monitored by measurement of turbidity. Glucose level was determined by the colorimetric anthrone METHOD: The epsilon-caprolactone concentration was determined by gas chromatography. The bacterial biopolymers were extracted with chloroform in a Soxhlet extractor and then characterized by nuclear magnetic resonance and gel permeation chromatography. When epsilon-caprolactone was used as a carbon substrate, the bacterial strain produced tercopolymer with 3-hydroxybutyrate, 3-hydroxyvalerate and 6-hydroxyhexanoate units. However, when caprolactone and glucose were supplied together, only homopolymer of poly (3-hydroxybutyrate) was produced. CONCLUSION: All tercopolymers isolated from B. cereus UW85 cells were obtained with yields up to 9% (w/w) and low number-average molecular weight compared with the homopolymer PHB. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus cereus UW85 produced tercopolymer with a low molecular weight from one substrate (epsilon-caprolactone) used as a carbon source. The results are significant for the potential future application of Bacillus biopolymers to bioplastics production.

Bacillus cereus↗
PubMed2001

Synthesis of intracellular storage polymers by Amaricoccus kaplicensis, a tetrad forming bacterium present in activated sludge.

AIMS: The study investigated the physiology of Amaricoccus kaplicensis to determine whether it could outcompete polyphosphate accumulating bacteria in activated sludge systems removing phosphorus, by preferentially assimilating substrates in the anaerobic stages of these processes. METHODS AND RESULTS: The storage processes were investigated under anaerobic, anoxic and aerobic conditions in both batch and periodically fed cultures in an aerobic sequencing batch reactor (SBR). Amaricoccus kaplicensis showed a high capacity for storing aerobically large amounts of acetate as poly beta-hydroxybutyrate (PHB) at high rates. However, no acetate assimilation under anaerobic conditions and very slow assimilation under anoxic conditions could be detected. CONCLUSION: Amaricoccus kaplicensis in pure culture does not behave as polyphosphate accumulating bacteria competitor; therefore it is difficult to understand why anaerobic/aerobic systems often contain such large numbers of Amaricoccus cells. SIGNIFICANCE AND IMPACT OF THE STUDY: Amaricoccus kaplicensis is probably not responsible for the failure of activated sludge systems removing phosphorus, and other organisms capable of anaerobic substrate assimilation should be sought.

Acetates↗
PubMed2002

The influence of nutritional and environmental conditions on the accumulation of poly-beta-hydroxybutyrate in Bacillus mycoides RLJ B-017.

AIMS: To optimize the nutritional and environmental conditions for growth of and poly-beta-hydroxybutyrate (PHB) accumulation in Bacillus mycoides RLJ B-017. METHODS AND RESULTS: An isolate, identified as B. mycoides, was grown on different sources of carbon and nitrogen. Among these, sucrose, beef extract and di-ammonium sulphate were found to be the most suitable for growth and PHB accumulation. The overall maximum value of PHB (%) in cells, PHB yield (Yp/s) and productivities (Qp and qp) were 69.4 +/- 0.4% dry cell weight (DCW), 0.21 gp gS(-1), 0.104 +/- 0.012 gp l(-1) h(-1) and 0.03 gp gx(-1) h(-1), respectively when grown in a medium containing 20 gs l(-1) sucrose, supplemented with di-ammonium sulphate. The addition of beef extract increased the value of PHB (%) in cells, PHB yield and productivities by 17.58 +/- 0,3, 23.8, 19.23 +/- 0.3 and 13.8 +/- 0.2% , respectively. The overall maximum values of PHB (% DCW), PHB yield and productivities were obtained at pH 7.0 +/- 0 .1, temperature 30 +/- 0.5 degrees C, agitation 650 rev min(-1) and oxygen transfer rate 3.8 mmol O(2) l(-1) h(-1). CONCLUSIONS: Sucrose, glucose and fructose were found to be more suitable for cell growth and PHB accumulation, but sucrose was less expensive than glucose. Among the nitrogen sources, beef extract and di-ammonium sulphate promoted PHB synthesis. The accumulation of PHB was observed to be growth associated. SIGNIFICANCE AND IMPACT OF THE STUDY: Gram-positive bacteria have not been reported to accumulate large amounts of polyhydroxyalkanoate and hence have not been considered as potent candidates for industrial production. A number of Bacillus spp. have been reported to accumulate 9-44.5% DCW PHB. By comparison, Bacillus RLJ B-017 contained 69.4 +/- 0.4% DCW PHB. Therefore, this strain has been considered as a potent organism for industrial interest. A relatively high yield of PHB was obtained in this wild strain and PHB synthesis was independent of nutrient limitation. The conditions for the higher PHB yield and productivity will be optimized in the next phase using fed-batch culture.

Bacillus↗
PubMed2003

Identification of polyhydroxyalkanoate (PHA)-producing Bacillus spp. using the polymerase chain reaction (PCR).

AIMS: The aim of the work was to develop efficient method to identify polyhydroxyalkanoate (PHA)-producing species of Bacillus from numerous soil isolates of bacteria. Identification of the isolates and characterization of the PHA produced by strains positive on the polymerase chain reaction (PCR) was envisaged. METHODS AND RESULTS: Different bacteria isolated from soil were screened by PCR using two sets of primers designed for Bacillus megaterium. Amongst 23 isolates examined, the DNA of 12 isolates reacted positively with the primers giving amplicons identical in size to that obtained from B. megaterium. The isolates which were identified as strains of B. sphaericus, B. circulans, B. brevis and B. licheniformis, produced 11- 41% of PHA in biomass, in sucrose-containing medium, over a growth period of 24-72 h. The nature of the PHA thus produced was analyzed by Fourier transform infrared spectroscopy, gas chromatography and by nuclear magnetic resonance (NMR) and found to contain polyhydroxy butyrate and polyhydroxyvalerate. CONCLUSIONS: The results indicate that most of our isolates from different species contained the B. megaterium type of PHA synthase. Bacillus licheniformis appeared to belong to another group as it did not react with both sets of primers. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the universality of the B. megaterium type of PHA synthase in soil isolates of Bacillus. Some variations were also found.

Acyltransferases↗
PubMed2003

A composite poly-hydroxybutyrate-glial growth factor conduit for long nerve gap repairs.

There is considerable evidence that peripheral nerves have the potential to regenerate in an appropriate microenvironment. We have developed a novel artificial nerve guide composed of poly 3-hydroxybutyrate (PHB) filled with glial growth factor (GGF) suspended in alginate hydrogel. Gaps of 2-4 cm in rabbit common peroneal nerve were bridged using a PHB conduit containing either GGF in alginate hydrogel (GGF) or alginate alone (Alginate), or with an empty PHB conduit (Empty). Tissues were harvested 21, 42 and 63 days post-operatively. Schwann cell and axonal regeneration were assessed using quantitative immunohistochemistry. At 21 days, addition of GGF increased significantly the distance of axonal and Schwann cells regeneration in comparison with that observed in Alginate and Empty conduits for both gap lengths. The axons bridged the 2-cm GGF conduits gap by 63 days, with a comparable rate of regeneration seen in 4-cm conduits. Schwann cells and axonal regeneration quantity was similar for both gap lengths in each group. However, at all time points the quantity of axonal and Schwann cells regeneration in GGF grafts was significantly greater than in both Alginate and Empty conduits, the latter showing better regeneration than Alginate conduits. The results indicate an inhibitory effect of alginate on regeneration, which is partially reversed by the addition of GGF to the conduits. In conclusion, GGF stimulates a progressive and sustainable regeneration increase in long nerve gap conduits.

Alginates↗
PubMed1998

Production of poly-3-hydroxybutyrate from lactose and whey by Methylobacterium sp. ZP24.

Methylobacterium sp. ZP24, isolated from a local pond, is able to grow in a medium containing 12 g l-1 lactose as a sole source of carbon, giving 5.25 g l-1 biomass yield and poly-3-hydroxybutyrate (PHB) up to 59% of its dry weight in 40 h. The isolate was also able to utilize cheese whey and produce 1.1 g l-1 PHB. Addition of ammonium sulphate increased the production of PHB from whey 2.5-fold. The potential of Methylobacterium sp. ZP24 in PHB production from cheese whey is discussed.

Ammonium Sulfate↗
PubMed2003

Poly-beta-hydroxyalkanoates consumption during degradation of 2,4,6-trichlorophenol by Sphingopyxis chilensis S37.

AIMS: To analyse the possible effect of poly-beta-hydroxyalkanoate (PHA) consumption on 2,4,6-trichlorophenol (2,4,6-TCP) degradation during starvation by Sphingopyxis chilensis S37 strain, which stores PHAs and degrades 2,4,6-TCP. METHODS AND RESULTS: The strain was inoculated in saline solution supplemented with 2,4,6-TCP (25-400 microm). Chlorophenol degradation was followed both spectrophotometrically and by chlorine released; viable bacterial counts were also determined. Cells starved for 24, 48 or 72 h were incubated with 25 microm of 2,4,6-TCP and PHA in cells investigated by spectrofluorimetric and flow cytometry. Results demonstrated that starvation decreased the ability to degrade 2,4,6-TCP. After 72 h of starvation, degradation of 2,4,6-TCP decreased to less than 10% and the relative PHA content diminished to ca 50% during the first 24 h. CONCLUSION: Utilization of PHA may be an important factor for the degradation of toxic compounds, such as 2,4,6-TCP, in bacterial strains unable to use this toxic compound as carbon and energy source. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study describing a relationship between intracellular PHA consumption and 2,4,6-TCP degradation. Therefore, PHAs provides an endogenous carbon and energy source under starvation and can play a significant role in the degradation of toxic compounds.

Alphaproteobacteria↗
PubMed1999

A resorbable nerve conduit as an alternative to nerve autograft in nerve gap repair.

Poly-3-hydroxybutyrate (PHB) occurs within bacterial cytoplasm as granules and is available as bioabsorbable sheets. Previously, the advantage of PHB in primary repair has been investigated while in this study the same material has been used to bridge an irreducible gap. The aim was to assess the level of regeneration in PHB conduits compared to nerve autografts. The rat sciatic nerve was exposed, a 10 mm nerve segment was resected and bridged with either an autologous nerve graft or a PHB conduit. The grafted segments were harvested up to 30 days. Immunohistochemical staining was performed and computerised quantification of penetration distance and volume of axonal regeneration was estimated by protein gene product (PGP) immunostaining and calcitonin gene-related peptide (CGRP) positive fibres. Penetration and proliferation density of Schwann cells into the conduit was measured by quantifying S-100 staining. The inflammatory response was quantified with ED-1 staining for macrophages. Antibodies to vWf provided an assessment of angiogenesis and capillary infiltration. Percentage immunostaining for PGP in autograft and PHB groups showed a progressive increase up to 30 days with a significant linear trend with time and an increase in the volume of axonal regeneration. A similar pattern of progressive increase with time was observed with CGRP immunostaining for both groups and with S-100 in the PHB group. Good angiogenesis was present at the nerve ends and through the walls of the conduit. The results demonstrate good nerve regeneration in PHB conduits in comparison with nerve grafts.

Animals↗
PubMed2002

Poly-3-hydroxybutyrate (PHB): a resorbable conduit for long-gap repair in peripheral nerves.

The repair of peripheral nerve injuries with an autologous nerve graft is still the treatment of choice, although it is widely recognised that the method is associated with donor-site morbidity and a suboptimal functional outcome. An alternative approach is to use a nerve conduit to provide a guidance channel for the regenerating nerve. This study investigates the potential of poly-3-hydroxybutyrate (PHB) nerve conduits to bridge long nerve gaps (up to 4 cm) in a rabbit common-peroneal-nerve injury model. Regeneration was assessed up to 63 days postoperatively, and compared with that achieved using nerve autografts. By 42 days, regenerating axons had bridged nerve gaps of all lengths in groups with nerve autografts and in those with PHB conduits. The regeneration distance increased with time but did not vary with gap length (P </= 0.009, 14 versus 21 days, PHB tube 2 cm, 3 cm and 4 cm, Mann-Whitney U -test). At 42 days, the area of immunostained regenerating fibres in the PHB group was greater than that in the nerve autograft group (P < 0.001, PHB versus autograft, 21 and 42 days, three-way analysis of variance (ANOVA)). These results suggest that PHB conduits support peripheral nerve regeneration up to 63 days, and that they are suitable for long-gap nerve injury repair.

Analysis of Variance↗
PubMed1999

A new resorbable wrap-around implant as an alternative nerve repair technique.

Poly-3-hydroxybutyrate (PHB), a bacterial storage product, is available as bioabsorbable sheets and has been used in this study for primary nerve repair. The aim was to assess axonal regeneration following such repair and determine the inflammatory response to PHB. In 20 adult cats, the transected superficial radial nerve was wrapped in PHB sheets, while primary epineural repair was carried out in the contralateral limb. At 6 and 12 months, the repair sites were assessed immunohistochemically for macrophage infiltration and myelinated axons were counted in the distal nerve. Mean macrophage counts across the whole width of the nerve in both groups at 6 and 12 months showed no statistically significant difference. Nor was there any significant difference between the two groups at both time-points in axon counts, axon diameter, myelin thickness and g-ratio. There was a statistically significant increase in fibre diameters at 12 months, indicating that fibres were undergoing continuous maturation.

Animals↗
PubMed2002

[Poly-beta-hydroxybutyric acid (PHB) films and plates in defect covering of the osseus skull in a rabbit model].

BACKGROUND: Titan plates have proven a success in the operative assistance of middle face fractures for stabile osteosynthesis. Also bioresorbant materials are being used increasingly. METHOD: Films and plates made from poly-beta-hydroxybutyric acid (PHB) with two holes and bolts are tested subperiostally on the osseus skull of 15 rabbits or respectively on cut trough zygomatic arches. The test bodies and the surrounding tissue are explantated after a defined period and are clinically and histologically prepared. RESULTS: All implants healed well. There were no macroscopic or microscopic signs of inflammation. A very slow, clinically not recordable decomposition followed. A "leap" in the decomposition evidently took place between the 20th and 25th month. Only initial signs of resorbation were to be found microfocally on the surface of the implant until 20 months after implantation but no more test body could be detected after 25 months. CONCLUSION: PHB is suitable for defect covery of the osseus skull or respectively as osteosynthesis material for fractures of the visceral cranium.

Animals↗
PubMed2003

[In vitro culture of cells from respiratory mucosa on foils of collagen, poly-L-lactide (PLLA) and poly-3-hydroxy-butyrate (PHB)].

BACKGROUND: The replacement of respiratory mucosa after surgical resections at different locations in the ENT-field (trachea, nasal septum, sinus maxillaris) seems to be essential or at least of great benefit for patients. Presently a satisfying solution for this challenge does not exist. Therefore we have analysed the growth of cells from respiratory mucosa on different matrices. METHODS: Cell cultures are initiated mainly by growing out cells from tissue pieces but also by seeding of cells after enzymatic dissociation (sequential trypsinization) of mucosa. Cells were cultured with serum-containing and serum-free media on 3 different foils: made of collagen, of Poly-L-lactic acid (PLLA) and Poly-hydroxybutyric acid (PHB). Culturing time was on average about 4 - 5 weeks. RESULTS: On all tested materials fibroblasts and epithelial cells have grown in principle. PLLA was the material with best properties concerning the aim of this study whereas culturing of cells on PHB only happens after surface modification by amino-functionalisation with plasma treatment. Visualisation of cells on materials of collagen could not realised by light-microscopy due to the 3-dimensional structure and the optical properties of this material. Here the cultures were analysed by cell membrane staining and by REM. We could not find a differentiation of epithelial cells with beating cilia 6 weeks after starting the culture. However 20 days after starting the culture on PLLA (good conditions for observation with light-microscope) cells with beating cilia could be observed, in our opinion resulting from dissociated cells from tissue pieces and not from proliferated cells. CONCLUSIONS: In principle the analysed materials are useful for the culturing of cells from respiratory mucosa. However, for obtaining differentiated epithelial cells the culture conditions have to be modified.

Cell Adhesion↗
PubMed1998

Pericardial substitution after cardiopulmonary bypass surgery: a trial of an absorbable patch.

Primary closure of the pericardium affords some protection against adhesion formation and the consequent hazards of resternotomy. However, its completion may be impractical and hazardous, and therefore the pursuit of an ideal pericardial substitute has prompted much research. Twenty calves were divided into 3 groups for the study. All animals underwent right posterolateral thoracotomy. The test group (group X), consisting of 6 animals, received a poly-beta-hydroxybutyrate patch (PHB) to close the pericardium following cardiopulmonary bypass (CPB). In group Y (9 animals) the pericardium was left open following CPB. Group Z (5 animals) also had their pericardium left open but did not undergo CPB (non-CPB). The plasminogen activating activity (PAA) of homogenates of pericardial tissue samples were measured in 5 animals in group X, and 5 in group Z. Samples were taken at three time points from the time of pericardiotomy, and at reoperation 4 weeks later. In group X (CPB) there was a significant reduction in the PAA during the operation with some recovery at reoperation. The reduction in the pericardial PAA of group Z (non-CPB) animals did not reach significance. For both group X and group Z the progress of mesothelial damage, compared with that at zero time, showed a significant increase. In addition, their pericardial inflammatory features became more apparent in the later samples but more significantly in group Z. This study demonstrated no significant short-term differences in adhesion formation or postoperative coronary anatomy visibility between any of the groups. At reoperation the patch material contained pronounced macrophage activity but no regenerative mesothelium. There were no infective episodes in any of the animals studied. Furthermore, this study suggests that CPB in comparison to non-CPB has a significant affect on pericardial PAA.

Animals↗
PubMed1995

Enzyme-catalyzed synthesis of poly[(R)-(-)-3-hydroxybutyrate]: formation of macroscopic granules in vitro.

A combined chemical and enzymatic procedure has been developed to synthesize macroscopic poly[(R)-(-)-3-hydroxybutyrate] (PHB) granules in vitro. The granules form in a matter of minutes when purified polyhydroxyalkanoate (PHA) synthase from Alcaligenes eutrophus is exposed to synthetically prepared (R)-3-hydroxybutyryl coenzyme A, thereby establishing the minimal requirements for PHB granule formation. The artificial granules are spherical with diameters of up to 3 microns and significantly larger than their native counterparts (0.5 micron). The isolated PHB was characterized by 1H and 13C NMR, gel-permeation chromatography, and chemical analysis. The in vitro polymerization system yields PHB with a molecular mass > 10 x 10(6) Da, exceeding by an order of magnitude the mass of PHAs typically extracted from microorganisms. We also demonstrate that the molecular mass of the polymer can be controlled by the initial PHA synthase concentration. Preliminary kinetic analysis of de novo granule formation confirms earlier findings of a lag time for the enzyme but suggests the involvement of an additional granule assembly step. Minimal requirements for substrate recognition were investigated. Since substrate analogs lacking the adenosine 3',5'-bisphosphate moiety of (R)-3-hydroxybutyryl coenzyme A were not accepted by the PHA synthase, we provide evidence that this structural element of the substrate is essential for catalysis.

Acyl Coenzyme A↗