Microscopical investigation of poly(3-hydroxybutyrate) granule formation in Azotobacter vinelandii

Poly(3-hydroxybutyrate) (PHB) granule formation in Azotobacter vinelandii was investigated by laser scanning fluorescence microscopy after staining the cells with Nilered and Baclight. Cells that had been starved for a carbon source for > or =3 days were almost free of PHB granules. Formation of visible PHB granules started within 1-2 h after transfer of the cells to a medium permissive for PHB accumulation. Fluorescent PHB granules at the early stages of formation were exclusively found in the cell periphery of the 2-3 mum ovoid-shaped cells. After 3 h of PHB accumulation or later, PHB granules were also found to be detached from the cell periphery. Our results indicate that PHB granule formation apparently begins at the inner site of the cytoplasmic membrane. This finding is different from previous assumptions that PHB granule formation occurs randomly in the cytoplasm of PHB-accumulating bacteria.     

Influence of oxygen transfer rate on the accumulation of poly(3-hydroxybutyrate) by Bacillus megaterium

Poly(3-hydroxybutyrate)—P(3HB)—is a natural biodegradable polyester synthesized by several bacteria, produced from renewable resources. The effects of oxygen transfer rate on the intracellular accumulation of P(3HB) was evaluated, aiming at increasing P(3HB) synthesized by Bacillus megaterium DSM 32^T in bioreactor batch cultures. Bench-scale bioreactor cultivations were performed under different volumetric oxygen mass transfer coefficients, k_La, setting stirrer speed on specified values. The results of this work show that oxygen transfer is a key factor on P(3HB) accumulation by B. megaterium, increasing the P(3HB) intracellular mass fraction from 39% to 62% of CDW at k_La condition of 0.006 s^?1.     

A mutation impairing alginate production increased accumulation of poly-β-hydroxybutyrate in Azotobacter vinelandii

An algK::Tn 5mutation impairing alginate production in Azotobacter vinelandiiled to a 50% increase in poly--hydroxy-butyrate production to 75% of the dry cell wt.     

Poly(3-hydroxybutyrate) synthesis genes in Azotobacter sp. strain FA8.

Genes responsible for the synthesis of poly(3-hydroxybutyrate) (PHB) in Azotobacter sp. FA8 were cloned and analyzed. A PHB polymerase gene (phbC) was found downstream from genes coding for beta-ketothiolase (phbA) and acetoacetyl-coenzyme A reductase (phbB). A PHB synthase mutant was obtained by gene inactivation and used for genetic studies. The phbC gene from this strain was introduced into Ralstonia eutropha PHB-4 (phbC-negative mutant), and the recombinant accumulated PHB when either glucose or octanoate was used as a source of carbon, indicating that this PHB synthase cannot incorporate medium-chain-length hydroxyalkanoates into PHB.     

Poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate)-based biopolymer systems

Biodegradable biopolymers attract much attention in biology and medicine due to its wide application. The present review considers a biodegradable and biocompatible polymer of bacterial origin, poly(3-hydroxybutyrate), which has wide perspectives in medicine and pharmaceutics. It highlights basic properties of biopolymer (biodegradability and biocompatibility) and also biopolymer systems: various materials, devices and compositions based on the biopolymer. Application of poly(3-hydroxybutyrate)-based biopolymer systems in medicine as surgical implants, in bioengineering as cell culture scaffolds, and in pharmacy as novel drug dosage forms and drug systems are also considered.     

Production of poly (3-hydroxybutyrate) from starch by Azotobacter chroococcum

Production of poly (3-hydroxybutyrate) (PHB) from starch was investigated in flask, batch, and fed-batch cultures of Azotobacter chroococcum. In flask culture, PHB content increased up to 74% of dry cell wt with increasing culture volume. In batch culture, PHB content increased to 44% with O2 limitation. In fed-batch culture, cell concentration of 71 g/l with 20% PHB was obtained without O2 limitation, whereas cell concentration of 54 g/l with 46% PHB was obtained with O2 limitation.     

The oxygen transfer rate influences the molecular mass of the alginate produced by Azotobacter vinelandii

The influence of oxygen transfer rate (OTR) on the molecular mass of alginate was studied. In batch cultures without dissolved oxygen tension (DOT) control and at different agitation rates, the DOT was nearly zero and the OTR was constant during biomass growth, hence the cultures were oxygen-limited. The OTR reached different maximum levels (OTR_max) and enabled to establish various relative respiration rates. Overall, the findings showed that OTR influences alginate molecular mass. The mean molecular mass (MMM) of the alginate increased as OTR_max decreased. The molecular mass obtained at 3.0 mmol l⁻¹ h⁻¹ was 7.0 times higher (1,560 kDa) than at 9.0 mmol l⁻¹ h⁻¹ (220 kDa). An increase in molecular mass can be a bacterial response to adverse nutritional conditions such as oxygen limitation.     

Poly(3-hydroxybutyrate) granules ofBacillus megaterium

Cells ofBacillus megaterium contain 35–45% of poly(3-hydroxybutyrate) (PHB) at the beginning of the stationary phase. This amount is only slightly affected by the medium composition. The PHB granules are spherical with the mean diameter of 1.15 μm.     

Two-stage fermentation process for alginate production by Azotobacter vinelandii mutant altered in poly-β-hydroxybutyrate (PHB) synthesis

Aims:  A two-stage fermentation strategy, based on batch cultures conducted first under non-oxygen-limited conditions, and later under oxygen-limited conditions, was used to improve alginate production by Azotobacter vinelandii (AT6), a strain impaired in poly-β-hydroxybutyrate (PHB) production.
Methods and Results:  The use of sucrose as carbon source, as well as a high oxygen concentration (10%), allowed to obtain a maximum biomass concentration of 7·5 g l⁻¹ in the first stage of cultivation. In the second stage, the cultures were limited by oxygen (oxygen close to 0%) and fed with a sucrose solution at high concentration. Under those conditions, the growth rate decreased considerably and the cells used the carbon source mainly for alginate biosynthesis, obtaining a maximum concentration of 9·5 g l⁻¹, after 50 h of cultivation.
Conclusion:  Alginate concentration obtained from the AT6 strain was two times higher than that obtained using the wild-type strain (ATCC 9046) and was the highest reported in the literature. However, the mean molecular mass of the alginate produced in the second stage of the process by the mutant AT6 was lower (400 kDa) than the polymer molecular mass obtained from the cultures developed with the parental strain (950 kDa).
Significance and Impact of the Study:  The use of a mutant of A. vinelandii impaired in the PHB production in combination with a two-stage fermentation process could be a feasible strategy for the production of alginate at industrial level.     

Formation of poly(hydroxybutyrate-co-hydroxyvalerate) by Azotobacter vinelandii UWD.

Azotobacter vinelandii UWD formed polyhydroxyalkanoate (PHA) copolymers containing beta-hydroxybutyrate and beta-hydroxyvalerate (HV) when grown in a medium containing glucose as the primary C source and valerate (pentanoate) as a precursor. Copolymer was not formed when propionate was added to the glucose medium but was formed when heptanoate, nonanoate, or trans-2-pentenoate was present. Optimal levels of HV were formed when valerate was added at the time of maximum PHA synthesis, although HV incorporation was not dependent on glucose catabolism. HV content in the polymer was increased from 17 to 24 mol% by adding 10 to 40 mM valerate to glucose medium, but HV insertion into the polymer occurred at a fixed rate. Similarly, the addition of valerate to a fed-batch culture of strain UWD in beet molasses in a fermentor produced 19 to 22 g of polymer per liter, containing 8.5 to 23 mol% HV after 38 to 40 h. The synthesis of HV in these cultures also occurred at a fixed rate (2.3 to 2.8 mol% h-1), while the maximum PHA production rate was 1.1 g liter-1 h-1. During synthesis of copolymer in batch or fed-batch culture, the yield from conversion of glucose into PHA (YP/S) remained at maximum theoretical efficiency (greater than or equal to 0.33 g of PHA per g of glucose consumed). Up to 45 mol% C source, but the PHA produced amounted to less than 1 g/liter. The combination of 30 mM valerate as a sole C source and 0.5 mM 4-pentenoate increased the HV content in the polymer to 52 mol%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Formation of poly(hydroxybutyrate-co-hydroxyvalerate) by Azotobacter vinelandii UWD.

Azotobacter vinelandii UWD formed polyhydroxyalkanoate (PHA) copolymers containing beta-hydroxybutyrate and beta-hydroxyvalerate (HV) when grown in a medium containing glucose as the primary C source and valerate (pentanoate) as a precursor. Copolymer was not formed when propionate was added to the glucose medium but was formed when heptanoate, nonanoate, or trans-2-pentenoate was present. Optimal levels of HV were formed when valerate was added at the time of maximum PHA synthesis, although HV incorporation was not dependent on glucose catabolism. HV content in the polymer was increased from 17 to 24 mol% by adding 10 to 40 mM valerate to glucose medium, but HV insertion into the polymer occurred at a fixed rate. Similarly, the addition of valerate to a fed-batch culture of strain UWD in beet molasses in a fermentor produced 19 to 22 g of polymer per liter, containing 8.5 to 23 mol% HV after 38 to 40 h. The synthesis of HV in these cultures also occurred at a fixed rate (2.3 to 2.8 mol% h-1), while the maximum PHA production rate was 1.1 g liter-1 h-1. During synthesis of copolymer in batch or fed-batch culture, the yield from conversion of glucose into PHA (YP/S) remained at maximum theoretical efficiency (greater than or equal to 0.33 g of PHA per g of glucose consumed). Up to 45 mol% C source, but the PHA produced amounted to less than 1 g/liter. The combination of 30 mM valerate as a sole C source and 0.5 mM 4-pentenoate increased the HV content in the polymer to 52 mol%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Poly(3-hydroxybutyrate) production by Cupriavidus necator using waste glycerol

Polyhydroxyalkanoates (PHAs) have been recognized as good substitutes for the non-biodegradable petrochemically produced polymers. However, their high (real or estimated) current production cost limits their industrial applications. This work exploits two strategies to enhance PHAs substitution potential: the increase in PHA volumetric productivity in high density cultures and the use of waste glycerol (GRP), a by-product from the biodiesel industry, as primary carbon source for cell growth and polymer synthesis. Cupriavidus necator DSM 545 was used to accumulate poly(3-hydroxybutyrate) (P(3HB)) from GRP and from commercial glycerol (PG) as control substrate. On PG, productivities between 0.6 g_PHB L^?1 h^?1 and 1.5 g_PHB L^?1 h^?1 were attained. The maximum cell DW was 82.5 g_DW L^?1, the P(3HB) content being 62%. When GRP was used, 68.8 g_DW L^?1 with a P(3HB) accumulation of 38% resulting in a final productivity of 0.84 g_PHB L^?1 h^?1 was obtained. By decreasing the biomass concentration at which accumulation was triggered, a productivity of 1.1 g_PHB L^?1 h^?1 (50% P(3HB), w/w) was attained using GRP. P(3HB) molecular weights (M_w) ranged from 7.9 × 10⁵ to 9.6 × 10⁵ Da.     

Poly(3-hydroxybutyrate) production from xylose by recombinant Escherichia coli

Several recombinant Escherichia coli strains harboring the Alcaligenes eutrophus polyhydroxyalkanoate biosynthesis genes were used to produce poly(3-hydroxybutyrate), PHB, from xylose. By flask culture of TG1 (pSYL107) in a defined medium containing 20?g/l xylose, PHB concentration of 1.7?g/l was obtained. Supplementation of a small amount of cotton seed hydrolysate or soybean hydrolysate could enhance PHB production by more than two fold. The PHB concentration, PHB content, and PHB yield on xylose obtained by supplementing soybean hydrolysate were 4.4?g/l, 73.9%, and 0.226?g PHB/g xylose, respectively.     

Poly(3-hydroxybutyrate) synthesis in fed-batch culture of Ralstonia eutropha with phosphate limitation under different glucose concentrations

Poly(3-hydroxybutyrate) (PHB) was produced by fed-batch cultures of Ralstonia eutropha with phosphate limitation under different glucose concentrations. When glucose was kept at 2.5 g l^–1, cell growth and PHB synthesis were limited due to the shortage of carbon source but a higher PHB content occurred in the cell-growth stage. This shows that a low glucose concentration is favorable for PHB accumulation in R. eutropha. PHB obtained with glucose at 9 g l^–1 is 1.6 times that obtained with 40 g l^–1. When glucose was in the range of 9 to 40 g l^–1, PHB concentration and productivity decreased significantly with the increase of glucose concentration. The highest PHB productivity was obtained with glucose at 9 g l^–1.