Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids.

Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid [P(HB-co-HV)] copolymer when supplied with glucose (or sucrose in the case of A. latus) and propionic acid under nitrogen-limited conditions. A fed-batch culture of A. eutrophus produced 24 g of poly-beta-hydroxybutyric acid (PHB) liter-1 under ammonium limitation conditions. When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-1 was produced. The P(HB-co-HV) contained 5.0 mol% beta-hydroxyvaleric acid (HV). Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-1 in a chemostat culture of A. eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass. In comparison, A. latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate. When propionic acid was added to the first stage of a two-stage chemostat, A. latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV. In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate. The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed. Poly-beta-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid.     

Production of poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acids

Alcaligenes latus, Alcaligenes eutrophus, Bacillus cereus, Pseudomonas pseudoflava, Pseudomonas cepacia, and Micrococcus halodenitrificans were found to accumulate poly-(beta-hydroxybutyric-co-beta-hydroxyvaleric) acid [P(HB-co-HV)] copolymer when supplied with glucose (or sucrose in the case of A. latus) and propionic acid under nitrogen-limited conditions. A fed-batch culture of A. eutrophus produced 24 g of poly-beta-hydroxybutyric acid (PHB) liter-1 under ammonium limitation conditions. When the glucose feed was replaced with glucose and propionic acid during the polymer accumulation phase, 17 g of P(HB-co-HV) liter-1 was produced. The P(HB-co-HV) contained 5.0 mol% beta-hydroxyvaleric acid (HV). Varying the carbon-to-nitrogen ratio at a dilution rate of 0.15 h-1 in a chemostat culture of A. eutrophus resulted in a maximum value of 33% (wt/wt) PHB in the biomass. In comparison, A. latus accumulated about 40% (wt/wt) PHB in chemostat culture under nitrogen-limited conditions at the same dilution rate. When propionic acid was added to the first stage of a two-stage chemostat, A. latus produced 43% (wt/wt) P(HB-co-HV) containing 18.5 mol% HV. In the second stage, the P(HB-co-HV) increased to 58% (wt/wt) with an HV content of 11 mol% without further addition of carbon substrate. The HV composition in P(HB-co-HV) was controlled by regulating the concentration of propionic acid in the feed. Poly-beta-hydroxyalkanoates containing a higher percentage of HV were produced when pentanoic acid replaced propionic acid.     

利用糖蜜发酵生产生物降解塑料PHB的研究

由Ａｌｃａｌｉｇｅｎｅｓｌａｔｕｓ经单菌落分离,从中筛选到一株高效利用糖蜜产聚羟基丁酸（ＰＨＢ）的优良菌株１０１８。在６Ｌ台式发酵罐（Ｎ.Ｂ.Ｓ）中,进行了该菌利用甜菜糖蜜和甘蔗糖蜜积累ＰＨＢ的分批补料培养的研究。结果表明,培养５４ｈ左右发酵液中细胞干重达７０～８５ｇ／Ｌ,ＰＨＢ含量占细胞干重的６０％～７０％,生产强度１．０ｇＰＨＢ／Ｌ／ｈ以上;发酵液经非有机溶剂提取制得ＰＨＢ产品,纯度９５％,提取收率８０％以上。     

Recovery and characterization of poly(3-hydroxybutyric acid) synthesized in Alcaligenes eutrophus and recombinant Escherichia coli.

We studied recovery of poly(3-hydroxybutyric acid) (PHB) from Alcaligenes eutrophus and a recombinant Escherichia coli strain harboring the A. eutrophus poly(3-hydroxyalkanoic acid) biosynthesis genes. The amount of PHB degraded to a lower-molecular-weight compound in A. eutrophus during the recovery process was significant when sodium hypochlorite was used, but the amount degraded in the recombinant E. coli strain was negligible. However, there was no difference between the two microorganisms in the patterns of molecular weight change when PHB was recovered by using dispersions of a sodium hypochlorite solution and chloroform. To understand these findings, we examined purified PHB and lyophilized cells containing PHB by using a differential scanning calorimeter, a thermogravimetric analyzer, and nuclear magnetic resonance. The results of our analysis of lyophilized whole cells containing PHB with the differential scanning calorimeter suggested that the PHB granules in the recombinant E. coli strain were crystalline, while most of the PHB in A. eutrophus was in a mobile amorphous state. The stability of the native PHB in the recombinant E. coli strain during sodium hypochlorite treatment seemed to be due to its crystalline morphology. In addition, as determined by the thermogravimetric analyzer study, lyophilized cell powder of the recombinant E. coli strain containing PHB exhibited greater thermal stability than purified PHB obtained by chloroform extraction. The PHB preparations extracted from the two microorganisms had identical polymer properties.     

Optimization of microbial poly(3-hydroxybutyrate) recover using dispersions of sodium hypochlorite solution and chloroform

Optimization was carried out for the recovery of microbiol poly(3-hydroxybutyrate) (PHB) from Alcaligenes eutrophus. This process involved the use of a dispersion made of sodium hypochlorite solution and chloroform. The dispersion enabled us to take advantage of both differential digestion by hypochlorite and solvent extraction by chloroform. The PHB recovery (%) from cell powder was maximized using a 30% hypochlorite concentration, a 90-min treatment time, and a 1:1 (v/v) chloroform-to-aqueous-phase ratio. Under these optimal conditions, the recovery was about 91% and the purity of recovered PHB was higher than 97%. The number average molecular weight, M(n) of recovered PHB was about 300,000 and the weight average molecular weight M(w) was about 1,020,000, compared to the original M(n) of 530,000 and M(w) of 1,272,000. The moderate decrease in both M(n) and M(w) might be ascribed to the shielding effect of chloroform. In addition, the relatively small decrease in M(w) probably resulted from the loss of short PHB chains which might be water soluble. The crystallinity of recovered PHB was in the range of 60 to 65%although a slightly higher crystallinity was observed when the dispersion was used. (c) 1994 John Wiley & Sons, Inc.     

Biodeuteration of poly(beta-hydroxybutyrate).

The formation of poly(beta-hydroxybutyrate), PHB, by Rhodobacter sphaeroides and Alcaligenes eutrophus was studied using the following carbon sources and solvents: (1), acetate in H2O; (2), D3-acetate in H2O; (3), acetate in 90 to 92% D2O; and (4), D3-acetate in 90 to 92% D2O. The growth of Rb. sphaeroides cultured under condition (2) showed no apparent deuterium isotope effect, while considerably slowed growth in the presence of D2O was observed under conditions (3) and (4). In all cases, the PHB produced under deuterium enriched conditions was of high molecular weight. Interestingly, comparatively high volumetric formation of partially deuterated PHB was obtained using culture condition (4) for A. eutrophus. Fourier transform infrared spectroscopy (FT-i.r.), pyrolysis gas chromatography mass spectrometry (PGC-m.s.), and nuclear magnetic resonance (n.m.r.) were used to establish the extent and distribution of deuterium in the PHB samples produced. Partially deuterated PHB was obtained in each case, using a deuterium enriched culture. Considerable differences in the extent and distribution of deuterium were found between micro-organisms and culture conditions.     

Cloning and expression in Escherichia coli of the Alcaligenes eutrophus H16 poly-beta-hydroxybutyrate biosynthetic pathway.

The poly-beta-hydroxybutyrate (PHB) biosynthetic pathway from Alcaligenes eutrophus H16 has been cloned and expressed in Escherichia coli. Initially, an A. eutrophus H16 genomic library was constructed by using cosmid pVK102, and cosmid clones that encoded the PHB biosynthetic pathway were sought by assaying for the first enzyme of the pathway, beta-ketothiolase. Six enzyme-positive clones were identified. Three of these clones manifested acetoacetyl coenzyme A reductase activity, the second enzyme of the biosynthetic pathway, and accumulated PHB. PHB was produced in the cosmid clones at approximately 50% of the level found in A. eutrophus. One cosmid clone was subjected to subcloning experiments, and the PHB biosynthetic pathway was isolated on a 5.2-kilobase KpnI-EcoRI fragment. This fragment, when cloned into small multicopy vectors, can direct the synthesis of PHB in E. coli to levels approaching 80% of the bacterial cell dry weight.     

Increased PHB productivity by high-cell-density fed-batch culture of Alcaligenes latus, a growth-associated PHB producer

Alcaligenes latus, a growth-associated PHB producer, was cultivated by a pH-stat modal fed-batch culture technique to attain high PHB productivity. Both sucrose solution and inorganic medium were fed in conjunction with the supply of ammonia solution which serves as a nitrogen source and as a means of pH control. Compositions of the inorganic medium were formulated by elemental analysis of A. latus cell mass. The effect on inoculum size was examined to reduce culture time. High concentrations of cell (142 g/L) and PHB (68.4 g/L) were obtained in a short culture time (18 h) with an inoculum size of 13.7 g/L. The PHB content and the PHB productivity at the end of the fed-batch culture were 50% of dry cell weight and 4.0 g PHB/(L . h), respectively. (c) 1996 John Wiley & Sons, Inc.     

Production of poly(3-hydroxybutyric acid) by fed-batch culture of Alcaligenes eutrophus with glucose concentration control

Alcaligenes eutrophus NCIMB 11599 was cultivated to produce poly(3-hydroxybutyric acid) (PHB) from glucose by the automatic fed-batch culture technique. The glucose concentration of the culture broth was controlled at 10 to 20 g/L by two methods: using exit gas data obtained from a mass spectrometer and using an on-line glucose analyzer. The effect of ammonium limitation on PHB synthesis at different culture phases was studied. The final cell concentration, PHB concentration, and PHB productivity increased as ammonia feeding was stopped at a higher cell concentration. High concentrations of PHB (121 g/L) and total cells (164 g/L) were obtained in 50 h when ammonia feeding was stopped at the cell concentration of 70 g/L. The maximum PHB content reached 76% of dry cell weight and the productivity was 2.42 g/L h with the yield of 0.3 g PHB/g glucose.     

Turnover of poly(3-hydroxybutyrate) (PHB) and its influence on the molecular mass of the polymer accumulated by Alcaligenes eutrophus during batch culture

Abstract Radiolabelled glucose was added to a batch culture of Alcaligenes eutrophus during the accumulation of poly(3-hydroxybutyrate) (PHB) to label newly synthesized polymer. The specific radioactivity of the polymer continued to increase, by approximately 30%, after the cessation of PHB accumulation, indicating that turnover of PHB was occurring. Fractionation of PHB showed that high molecular mass polymer was gradually replaced by PHB of lower molecular mass. Turnover of PHB is the cause of the slow decline in the molecular mass of PHB following the cessation of polymer accumulation but is unlikely to be the sole reason for the more rapid decrease in the molecular mass of PHB during the accumulation phase.     

Cloning of poly(3-hydroxybutyric acid) synthase genes of Rhodobacter sphaeroides and Rhodospirillum rubrum and heterologous expression in Alcaligenes eutrophus

From genomic libraries of the purple non-sulfur bacteria Rhodospirillum rubrum Ha and Rhodobacter sphaeroides ATCC 17023 in the broad-host range cosmid pVK100, we cloned a 15- and a 14-kbp HindIII restriction fragment, respectively. Each of these fragments restored the ability to accumulate poly(3-hydroxybutyrate) (PHB), in the PHB-negative mutant Alcaligenes eutrophus PHB-4. These hybrid cosmids also complemented PHB-negative mutants derived from wild-type R. rubrum or R. sphaeroides. Both fragments hybridized with the PHB synthase structural gene of A. eutrophus H16 and conferred the ability to express PHB synthase activity. Only the 15-kbp HindIII fragment from R. rubrum conferred on the mutant PHB-4 the ability to form large PHB granules (length up to 3.5 microns).     

Production of poly(3-hydroxybutyrate) by high cell density fed-batch culture of Alcaligenes eutrophus with phospate limitation

High cell density fed-batch fermentation of Alcaligenes eutrophus was carried out for the production of poly(3-hydroxybutyrate) (PHB) in a 60-L fermentor. During the fermentation, pH was controlled with NH(4)OH solution and PHB accumulation was induced by phosphate limitation instead of nitrogen limitation. The glucose feeding was controlled by monitoring dissolved oxygen (DO) concentration and glucose concentration in the culture broth. The glucose concentration fluctuated within the range of 0-20 g/L. We have investigated the effect of initial phosphate concentration on the PHB production when the initial volume was fixed. Using an initial phosphate concentration of 5.5 g/L, the fed-batch fermentation resulted in a final cell concentration of 281 g/L, a PHB concentration of 232 g/L, and a PHB productivity of 3.14 g/L . h, which are the highest values ever reported to date. In this case, PHB content, cell yield from glucose, and PHB yield from glucose were 80, 0.46, and 0.38% (w/w), respectively. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 28-32, 1997.     

Physiology and molecular genetics of poly(β-hydroxyalkanoic acid) synthesis in Alcaligenes eutrophus

The Alcaligenes eutrophus genes for beta-ketothiolase, NADPH-dependent acetoacetyl-CoA reductase and poly(beta-hydroxybutyric acid) synthase (PHB synthase) which comprise the three-step PHB-biosynthetic pathway, were cloned. Molecular studies revealed that these genes are organized in a single operon. The A. eutrophus PHB-biosynthetic genes are readily expressed in other bacteria, and DNA fragments harbouring the operon can be used as a cartridge to confer to other bacteria the ability to synthesize PHB from acetyl-CoA. The biochemical and physiological capabilities of A. eutrophus for the synthesis of a wide variety of polyhydroxyalkanoates are discussed.     

Experimental optimization of fed-batch culture for poly-beta-hydroxybutyric acid production

The optimal feed rate profiles of glucose and ammonium hydroxide were calculated using a proposed model, and implemented for the production of poly-beta-hydroxybutyric acid (PHB) by Alcaligenes eutrophus. By implementing these optimal feed rates with a high glucose feed concentration of 700 g/L and an ammonium hydroxide concentration of 7%(w/w), it was possible to achieve a high final cell concentration of 141 g/L and a high PHB concentration of 105 g/L in 40 h of fed-batch operation. The PHB productivity was as high as 2.63 g/(L hr). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 697-705, 1997.     

Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M

Abstract Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zooglea ramigera I-16-M were examined. The release of d (−)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of d (−)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing d (−)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.     

Intracellular degradation of poly(3-hydroxybutyrate) granules of Zoogloea ramigera I-16-M.

Intracellular degradation of poly(3-hydroxybutyrate) (PHB) in bacteria is not yet clear. The properties of the autodigestion of native PHB granules from Zoogloea ramigera I-16-M were examined. The release of D(-)-3-hydroxybutyrate was observed only at pH values higher than about 8.5 and at relatively high ionic strength (optimal concentration 200 mM NaCl). Triton X-100 and diisopropylfluorophosphate inhibited this reaction. Addition of the supernatant fraction of Z. ramigera did not increase the release of D(-)-3-hydroxybutyrate from the native PHB granules. On the other hand, using the protease-treated PHB granules from Alcaligenes eutrophus as a substrate, PHB depolymerase activity was detected in the supernatant fraction of Z. ramigera cells. The soluble PHB depolymerase showed similar properties to the enzyme in the PHB granules. Since PHB depolymerase activity was found in fractions containing D(-)-3-hydroxybutyrate oligomer hydrolase activity, which were separated by DEAE-Toyopearl or by Sephacryl S-100, it is possible that the intracellular PHB depolymerase is identical to the oligomer hydrolase which has been purified already.     

High cell density culture of metabolically engineered Escherichia coli for the production of poly(3-hydroxybutyrate) in a defined medium

A recombinant Escherichia coli strain XL1-Blue harboring a stable high-copy-number plasmid pSYL107 containing the Alcaligenes eutrophus polyhydroxyalkanoate biosynthesis genes and the Escherichia coli ftsZ gene was employed for the production of poly(3-hydroxybutyrate) (PHB) by fed-batch culture in a defined medium. Suppression of filamentation by overexpressing the cell division protein FtsZ allowed production of PHB to a high concentration (77 g/L) with high productivity (2 g/L/h) in a defined medium, which was not possible with the recombinant E. coli that underwent filamentation. Further optimization of fed-batch culture condition resulted in PHB concentration of 104 g/L in a defined medium, which was the highest value reported to date by employing recombinant E. coli.     

Production of Poly(3-hydroxybutyrate) by fed-batch culture of recombinant Escherichia coli with a highly concentrated whey solution

Fermentation strategies for the production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes were developed. The pH-stat fed-batch cultures of E. coli CGSC 4401 harboring pJC4, a stable plasmid containing the A. latus PHA biosynthesis genes, were carried out with a concentrated whey solution containing 280 g of lactose equivalent per liter. Final cell and PHB concentrations of 119.5 and 96.2 g/liter, respectively, were obtained in 37.5 h, which resulted in PHB productivity of 2.57 g/liter/h.     

Poly(3-Hydroxybutyrate) Production with High Productivity and High Polymer Content by a Fed-Batch Culture of Alcaligenes latus under Nitrogen Limitation

Alcaligenes latus has been known to produce poly(3-hydroxybutyrate) (PHB) in a growth-associated manner even under nutrient-sufficient conditions. However, the PHB content obtained by fed-batch culture was always low, at ca. 50%, which makes the recovery process inefficient. In this study, the effect of applying nitrogen limitation on the production of PHB by A. latus was examined. In flask and batch cultures, the PHB synthesis rate could be increased considerably by applying nitrogen limitation. The PHB content could be increased to 87% by applying nitrogen limitation in batch culture, which was considerably higher than that typically obtainable (50%) under nitrogen-sufficient conditions. In fed-batch culture, cells were first cultured by the DO-stat feeding strategy without applying nitrogen limitation. Nitrogen limitation was applied at a cell concentration of 76 g (dry cell weight)/liter, and the sucrose concentration was maintained within 5 to 20 g/liter. After 8 h of nitrogen limitation, the cell concentration, PHB concentration, and PHB content reached 111.7 g (dry cell weight)/liter, 98.7 g/liter, and 88%, respectively, resulting in a productivity of 4.94 g of PHB/liter/h. The highest PHB productivity, 5.13 g/liter/h, was obtained after 16 h.     

Effect of overexpression of a soluble pyridine nucleotide transhydrogenase (UdhA) on the production of poly(3-hydroxybutyrate) in Escherichia coli

A soluble pyridine nucleotide transhydrogenase (UdhA) has been used to increase the productivity and yield of PHB in vivo. By inducing a high level of UdhA, which can transfer reducing equivalents between NAD and NADP, we have increased NADPH availability, resulting in high yield and productivity of PHB in Escherichia coli. Coexpression of the phb operon from Alcaligenes eutrophus H16 and the native udhA from E. coli from high copy plasmids resulted in an increase in PHB yield from 49 to 66% g of PHB per gram of total cell dry weight and an increase in final concentration from 3.52 to 6.42 g/L; the PHB concentration of the udhA carrying strain is almost twice that of the control strain expressing only the phb operon. The results of this study demonstrate the effectiveness of cofactor manipulation and its application as a tool in metabolic engineering.