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PubMed2004

Attachment, proliferation and differentiation of osteoblasts on random biopolyester poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) scaffolds.

Rabbit bone marrow cells were inoculated on 3D scaffolds of poly(lactic acid) (PLA), poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBHHx) to evaluate their in vitro biocompatibilities. It was found that PHBHHx had the best performance on attachment, proliferation of bone marrow cells. The cells on PHBHHx scaffolds presented typical osteoblast phenotypes: round cell shape, high alkaline phosphotase (ALP) activity, strong calcium deposition, and fibrillar collagen synthesis. After incubation for 10 days, cells grown on PHBHHx scaffolds were approximately 2x10(5)ml(-1), 40% more than that on PHB scaffolds and 60% more than that on PLA scaffolds. ALP activity of the cells grown on PHBHHx scaffolds was up to about 65U/g scaffolds, 50% higher than that of PHB and PLA, respectively. The scanning electronic microscopy (SEM) results showed that PHBHHx scaffolds had the appropriate roughness for osteoblast attachment and proliferation comparing with PHB and PLA. All these indicated that PHBHHx was a suitable biomaterial for osteoblast attachment, proliferation and differentiation from bone marrow cells.

3-Hydroxybutyric Acid↗
PubMed2002

Polyhydroxyalknoate synthesis in plants as a tool for biotechnology and basic studies of lipid metabolism.

Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyacids naturally synthesized in bacteria as a carbon reserve. PHAs have properties of biodegradable thermoplastics and elastomers and their synthesis in crop plants is seen as an attractive system for the sustained production of large amounts of polymers at low cost. A variety of PHAs having different physical properties have now been synthesized in a number of transgenic plants, including Arabidopsis thaliana, rape and corn. This has been accomplished through the creation of novel metabolic pathways either in the cytoplasm, plastid or peroxisome of plant cells. Beyond its impact in biotechnology, PHA production in plants can also be used to study some fundamental aspects of plant metabolism. Synthesis of PHA can be used both as an indicator and a modulator of the carbon flux to pathways competing for common substrates, such as acetyl-coenzyme A in fatty acid biosynthesis or 3-hydroxyacyl-coenzyme A in fatty acid degradation. Synthesis of PHAs in plant peroxisome has been used to demonstrate changes in the flux of fatty acids to the beta-oxidation cycle in transgenic plants and mutants affected in lipid biosynthesis, as well as to study the pathway of degradation of unusual fatty acids.

Biotechnology↗
PubMed1999

Determination of polyhydroxyalkanoates in activated sludge by ion chromatographic and enzymatic methods.

Two new detection methods for the determination of poly-beta-hydroxybutyrate (PHB) and -valerate (PHV) are described. Both methods are based on depolymerization of PHB/PHV to 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). Depolymerization was achieved by either propanolic or hydrolytic digestion. Propanolic digestion transformed commercial PHB/PHV stoichiometrically into 3HB/3HV and yielded apparently complete recoveries of bacterial PHB/PHV from activated sludge. Hydrolytic digestion was suitable only for PHB determination. For quantification of 3HB and 3HV directly from digested sludge, a method based on ion-exchange chromatography and conductivity detection was developed (IC-method). Alternatively, the total of 3HB and 3HV was quantified using a commercial enzymatic test kit and colorimetric detection (enzyme method). Both detection methods are easier to perform than previous methods and are suitable for complex matrices such as activated sludge. The IC-method is recommended for high sample throughputs or if distinction between PHB and PHV is essential. Enzymatic detection is recommended if a few samples per day have to be measured immediately or if an ion chromatograph is unavailable.

1-Propanol↗
PubMed1998

Bacterial and other biological systems for polyester production.

Poly(3-hydroxybutyric acid) and other structurally related aliphatic polyesters from bacteria, referred to as polyhydroxyalkanoic acids, form biodegradable thermoplastics and elastomers that are currently in use, or being considered for use, in industry, medicine, pharmacy and agriculture. At present, they are produced by microbial fermentations; in the future, production will also be possible by in vitro methods or by agriculture using transgenic plants. Representatives from this highly diverse class of polyesters might be produced as commodity chemicals for bulk applications, and others as fine chemicals for special applications.

Archaea↗
PubMed2001

Factors involved in the regulatory network of polyhydroxyalkanoate metabolism.

Polyhydroxyalkanoates (PHAs) are bacterial storage materials which are accumulated by various bacteria under unbalanced growth conditions. Although PHAs are produced in larger amounts and are studied because of their plastic material properties, not much is known about the regulation of PHA accumulation and the regulatory interactions with the general cell metabolism. In this report, we point out the diversity of regulatory mechanisms involved in PHA metabolism, and present examples for factors acting at the transcription or enzymatic level.

Bacteria↗
PubMed2001

Degradation of natural and synthetic polyesters under anaerobic conditions.

Often, degradability under anaerobic conditions is desirable for plastics claimed to be biodegradable, e.g. in anaerobic biowaste treatment plants, landfills and in natural anaerobic sediments. The biodegradation of the natural polyesters poly(beta-hydroxybutyrate) (PHB), poly(beta-hydroxybutyrate-co-11.6%-beta-hydroxyvalerate) (PHBV) and the synthetic polyester poly(epsilon-caprolactone) (PCL) was studied in two anaerobic sludges and individual polyester degrading anaerobic strains were isolated, characterized and used for degradation experiments under controlled laboratory conditions. Incubation of PHB and PHBV films in two anaerobic sludges exhibited significant degradation in a time scale of 6-10 weeks monitored by weight loss and biogas formation. In contrast to aerobic conditions, PHB was degraded anaerobically more rapidly than the copolyester PHBV, when tested with either mixed cultures or a single strained isolate. PCL tends to degrade slower than the natural polyesters PHB and PHBV. Four PHB and PCL degrading isolates were taxonomically identified and are obviously new species belonging to the genus Clostridium group I. The depolymerizing enzyme systems of PHB and PCL degrading isolates are supposed to be different. Using one isolated strain in an optimized laboratory degradation test with PHB powder, the degradation time was drastically reduced compared to the degradation in sludges (2 days vs. 6-10 weeks).

Anaerobiosis↗
PubMed2001

Producing poly-3-hydroxybutyrate with a high molecular mass from methane.

Poly-3-hydroxybutyrate (PHB) and other polyesters can be produced by various species of bacteria. Of the possible carbon sources, methane could provide a suitable substrate for the production of PHB. Methane is cheap and plentiful - not only as natural gas, but also as biogas. The methanotrophic strain Methylocystis sp. GB 25 DSMZ 7674 is able to accumulate PHB in a brief non-sterile process. The studies were carried out using a 7-l and a 70-l pressure bioreactor. 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 (ammonium, phosphorus or magnesium) in batch culture. The PHB content of biomass was as high as 51%; efficiency was highest during the first 5 h of the product formation process. The maximum PHB yield relative to the methane consumed was estimated to be 0.55 g g(-1). The PHB produced is of very high quality, having a high molecular mass of up to 2.5x10(6) Da.

Alphaproteobacteria↗
PubMed2001

Continuous production of poly-3-hydroxybutyrate by Ralstonia eutropha in a two-stage culture system.

Poly-3-hydroxybutyrate (PHB) is mainly accumulated by Ralstonia eutropha under unbalanced growth conditions, which limits its production in batch or fed-batch modes. The continuous production of PHB was investigated in a two-stage continuous culture system. The first-stage produced cell mass giving the maximal cell dry weight of 27.1 g l(-1) at 0.21 h(-1) of dilution rate. High specific cell growth rate results in the decrease of PHB synthesis under glucose-limited and nitrogen-rich conditions in the first-stage. The second-stage produced PHB giving the maximal PHB concentration of 47.6 g l(-1) at 0.14 h(-1) of dilution rate. Specific PHB synthetic rate reached highest value at low dilution rate under nitrogen-limited condition in the second-stage, and decreased with the increase of ammonium concentration in the culture. In the continuous culture system, the maximal PHB productivity could reach 1.43 g l(-1) h(-1) at a dilution rate of 0.12 h(-1), but with relatively low PHB content of 47.6%. Maximal yield of PHB on glucose could reach 0.36 g g(-1) glucose at 0.075 h(-1) of dilution rate with relatively high PHB productivity of 1.23 g l(-1) h(-1) and PHB content of 72.1%, respectively.

Cupriavidus necator↗
PubMed2002

Model predictive controller for biodegradable polyhydroxyalkanoate production in fed-batch culture.

The aim of this study is to develop a model predictive controller (MPC) accompanied with a metabolic reaction model controller for controlling ethanol and n-pentanol concentrations and the mole fraction of monomer units in the production of poly(beta-hydroxybutyrate-co-beta-hydroxyvalerate), P(HB-co-HV), a biodegradable copolyester. The controller consists of two parts: one is for alcohol concentration control and the other one is for mole fraction control, and is based on the concept of metabolic flux distribution control. For control of alcohol concentration, conventional proportional and integral (PI) controller and feedforward/feedback controller did not function sufficiently because the large sampling interval of the biosensor led to a severe overshoot of concentration. A single-input and single-output (SISO) MPC is constructed for control of ethanol concentration in the growth phase, whereas a multi-input and multi-output (MIMO) MPC is constructed for control of both alcohol concentrations in the production phase. Specific ethanol consumption rate was estimated by the MPC using the past time series data of ethanol concentration. By means of simulations and experiments, the weighting parameters of the noise filters in the MPC were well adjusted. Ethanol and n-pentanol concentrations were well controlled by the MPC, compared with PI controller and feedforward/feedback controller. As a result, P(HB-co-HV) production was maximized with a given value of mole fraction of 3HV units at the end of cultivation.

Biodegradation, Environmental↗
PubMed2002

Effects of culture conditions on the production of polyhydroxyalkanoates by Azotobacter chroococcum H23 in media containing a high concentration of alpechín (wastewater from olive oil mills) as primary carbon source.

Large amounts of homopolymers containing beta-hydroxybutyrate (PHB) and copolymers containing beta-hydroxyvalerate (P[HB-co-HV]) are produced by Azotobacter chroococcum strain H23 when growing in culture media amended with alpechín (wastewater from olive oil mills) as the sole carbon source. Copolymer was formed when valerate (pentanoate) was added as a precursor to the alpechín medium, but it was not formed with the addition of propionate as a precursor. A. chroococcum formed homo- and copolymers of polyhydroxyalkanoates (PHAs) up to 80% of the cell dry weight, when grown on NH(4)(+)-medium supplemented with 60% (v/v) alpechín, after 48 h of incubation at 100 rev min(-1) and 30 degrees C. Production of PHAs by strain H23 using alpechín looks promising, as the use of a cheap substrate for the production of these materials is essential if bioplastics are to become competitive products.

Acetates↗
PubMed1997

Production of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) in recombinant Escherichia coli grown on glucose.

A recombinant Escherichia coli strain has been developed that produces poly(3-hydroxybutyrate-co-4-hydroxybutyrate) when grown in complex medium containing glucose. This has been accomplished by introducing into E. coli DH5 alpha separate plasmids harboring the polyhydroxyalkanoate (PHA) biosynthesis genes from Ralstonia eutropha (formerly named Alcaligenes eutrophus) and the succinate degradation genes from Clostridium kluyveri, respectively. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) levels reached 50% of the cell dry weight and contained up to 2.8 mol.% 4-hydroxybutyrate. The molecular weight of the polymer was 1.8 x 10(6).

Acyltransferases↗
PubMed1998

Formation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by PHA synthase from Ralstonia eutropha.

The acetoacetyl-CoA reductase and the polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha (formerly Alcaligenes eutrophus) were expressed in Escherichia coli, Klebsiella aerogenes, and PHA-negative mutants of R. eutropha and Pseudomonas putida. While expression in E. coli strains resulted in the accumulation of poly(3-hydroxybutyrate) [PHB], strains of R. eutropha, P. putida and K. aerogenes accumulated poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [poly(3HB-co-3HHx)] when even chain fatty acids were provided as carbon source, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)] when odd chain fatty acids were provided as carbon source. This suggests that fatty acid degradation can be directly accessed employing only the acetoacetyl-CoA reductase and the PHA synthase. This is also the first proof that the PHA synthase from R. eutropha can incorporate 3-hydroxyhexanoate (3HHx) into PHA and has, therefore, a broader substrate specificity than previously described.

3-Hydroxybutyric Acid↗
PubMed1998

Aqueous release and purification of poly(beta-hydroxybutyrate) from Escherichia coli.

The poly(beta-hydroxybutyrate) (PHB) biosynthetic genes of Ralstonia eutropha that are organized in a single operon (phaCAB) have been cloned in Escherichia coli, where the expression of the genes in the wild-type pha operon from plasmid pTZ18U-PHB leads to the formation of 50-80% PHB/celldry mass when the cells are grown in Luria-Bertani medium supplemented with 1% glucose (w/v). In combination with the phaCAB genes, expression of cloned lysis gene E of bacteriophage PhiX174 from plasmid pSH2 has been used to release PHB granules produced in E. coli. It was shown that small PHB granules in a semiliquid stage are squeezed out of the cells through the E-lysis tunnel structure which is characterized by a small opening in the envelope with borders of fused inner and outer membranes. All envelope components remain intact after E-lysis and can be removed from the mixture of released PHB granules by density gradient centrifugation. In addition, a modified E-lysis procedure is described which enables the release of PHB from cell pellets in pure water or low ionic strength buffer. PHB granules in aqueous solution can be aggregated by divalent cations. Addition of glassmilk speeds up the agglomeration of PHB granules and binding to glass beads can either be used for collection or further purification of PHB in aqueous solutions.

Bacteriolysis↗
PubMed1999

Dynamics and modeling on fermentative production of poly (beta-hydroxybutyric acid) from sugars via lactate by a mixed culture of Lactobacillus delbrueckii and Alcaligenes eutrophus.

The mixed culture system was considered in the present research where sugars such as glucose were converted to lactate by Lactobacillus delbrueckii and the lactate was converted to poly beta-hydroxybutyrate (PHB) by Alcaligenes eutrophus in one fermentor. For the modeling of the effect of NH3 concentration on the cell growth of A. eutrophus and PHB production rates, metabolic flux distributions were computed at two culture phases of cell growth and PHB production periods. It was found that the NADPH, generated through isocitrate dehydrogenate in TCA cycle, was predominantly utilized for the reaction from alpha-ketoglutalate to glutamate when NH3 was abundant, while it tended to be utilized for the PHB production through acetoacetyl CoA reductase as NH3 concentration decreased. This phenomenon was reflected in the development of mathematical model. In the mixed culture experiments, the two phases were observed, namely the lactate production phase due to L. delbrueckii and the lactate consumption phase due to A. eutrophus. The lactate concentration could be estimated on-line by the amount of NaOH solution and HCl solution supplied to keep the culture pH at constant level. Several mixed culture experiments were conducted to see the dynamics of the system. Finally, a mathematical model which can describe the dynamic behavior of the present mixed culture was developed and the model parameters were tuned for fitting the experimental data. The model may be used for several purposes such as control, optimization, and understanding process dynamics etc.

Alcaligenes↗
PubMed2000

Propionic acid metabolism and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (P3HB-co-3HV) production by Burkholderia sp.

Mutants of Burkholderia sp. that are unable to grow on propionic acid (prp) but still accumulate P3HB-co-3HV from carbohydrate and propionic acid were studied. In shaken flask tests, yields of 3HV from propionic acid (Y(3HV/Prop)) increased from 0.10 g g(-1) in the wild type to c.a. 0.35 g g(-1) in mutants affected in alpha-oxidation pathway or to 0.80 g g(-1) in mutants not affected in that pathway. In bioreactor tests, mutant IPT 189 showed Y(3HV/Prop) = 1.20 g g(-1), a yield very close to the theoretical maximum of 1.35 g g(-1). Accumulation of 3HV units from unrelated carbon sources was undetectable in these mutants indicating that 3HV units are produced directly from propionic acid. Thus, the industrial use of those mutants to produce the copolymer from sucrose and propionic acid could significantly reduce the production costs. The results strongly suggest the existence of at least two pathways that are involved in the oxidation of propionic acid in Burkholderia sp. Their rates would be modulated by the availability of propionic acid.

Bioreactors↗
PubMed2000

Calorimetrically recognized maximum yield of poly-3-hydroxybutyrate (PHB) continuously synthesized from toxic substrates.

The broader usage of poly-beta-hydroxybutyrate (PHB), for instance as bulk plastics, calls for cheap raw materials and greater overall process efficiency. The bacterial synthesis is generally induced and promoted by the limitation of growth via nitrogen, oxygen or phosphate depletion with the simultaneous excess and higher concentration of the carbon substrate. Consequently, toxic substrates have been considered unsuitable for PHB synthesis. Nevertheless, a single-stage continuous process for producing PHB from toxic substrates using microorganisms was developed and is reported here. The maximum heat flux during continuous growth and the maximum yield of PHB versus the substrate consumption rate were found to coincide. This suggests the possibility of controlling the conversion of a growth-inhibiting substrate into PHB and maximizing the process efficiency. The observed correlation occurred irrespective of the substrates investigated (phenol or sodium benzoate), the PHB-producing strain (Ralstonia eutropha JMP 134 or Variovorax paradoxus JMP 116), or the type of limitation imposed. The maximum PHB yields obtained comprised up to 50% of cell dry mass.

Ammonia↗
PubMed1999

Antibiotic release from biodegradable PHBV microparticles.

For the treatment of periodontal diseases, design of a controlled release system seemed very appropriate for an effective, long term result. In this study a novel, biodegradable microbial polyester, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), PHBV of various valerate contents containing a well established antibiotic, tetracycline, known to be effective against many of the periodontal disease related microorganisms, was used in the construction of a controlled release system. Tetracycline was loaded in the PHBV microspheres and microcapsules both in its acidic (TC) and in neutral form (TCN). Microcapsules of PHBV were prepared under different conditions using w/o/w double emulsion and their properties such as encapsulation efficiency, loading, release characteristics, and morphological properties were investigated. It was found that concentration of emulsifiers polyvinyl alcohol (PVA) and gelatin (varied between 0-4%) influenced the encapsulation efficiency appreciably. In order to increase encapsulation efficiency (from the obtained range of 18.1-30.1%) and slow down the release of the highly soluble tetracycline.HCl, it was neutralized with NaOH. Encapsulation efficiency of neutralized tetracycline was much higher (51.9-65.3%) due to the insoluble form of the drug used during encapsulation. The release behaviour of neither of the drugs was found to be of zero order. Rather the trends fitted reasonably well to Higuchi's approach for release from spherical micropheres. Biodegradability was not an appreciable parameter in the release from microcapsules because release was complete before any signs of degradation were observed.

Bacteria↗
PubMed2001

Occurrence, synthesis and medical application of bacterial polyhydroxyalkanoate.

Polyhydroxyalkanoates (PHAs) comprise a large class of polyesters that are synthesized by many bacteria as an intracellular carbon and energy compound. Analysis of isolated PHAs reveal interesting properties such as biodegradability and biocompatibility. Research was focused only recently on the application of PHA in implants, scaffolds in tissue engineering, or as drug carriers. Such applications require that PHA be produced at a constant and reproducible quality. To date this can be achieved best through bacterial production in continuous culture where growth conditions are kept constant (chemostat). Recently, it was found that PHA producing bacteria are able to grow simultaneously limited by carbon and nitrogen substrates. Thus, it became possible to produce PHA at high yields on toxic substrate and also control its composition accurately (tailor-made synthesis). Finally, applications of PHA in medicine are discussed.

Acyltransferases↗