Yield of poly-D(-)-3-hydroxybutyrate from various carbon sources: a theoretical study

The theoretical yield of poly-D(-)-3-hydroxybutyrate (PHB) has been estimated from the biochemical pathway leading to PHB when a carbohydrate (glucose), a C(1) compound (methanol), a C(2) compound (acetic acid), or a C(4) compound (butyric acid) is used as a carbon source. In estimating the yield, recycling (or regeneration) of NADP(+)/ (NADPH + H(+)) and NAD(+) /(NADH + H(+)) have been taken into account. A special emphasis is made on te regeneration of NADPH, which is the coenzyme of acetoacetyl-CoA reductase, one of three key enzymes involved in the biosynthesis of PHB. As a NADPH-regenerating enzyme, glucose-6-phosphate dehydrogenase or isocitrate dehydrogenase is conceived. An equation which predicts the overall yield of PHB when non-PHB residual biomass is actually formed has been derived as a function of both the theoretical yield and PHB content of the dry cell mass. The ratio of the overall yield to the theoretical yield is roughly proportional to the PHB content. (c) 1993 John Wiley & Sons, Inc.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Degradation of poly (3-hydroxybutyrate) and its copolymer poly (3-hydroxybutyrate-co-3-hydroxyvalerate) by a marine Streptomyces sp. SNG9.

A marine Streptomyces sp. SNG9 was characterized by its ability to utilize poly(3-hydroxybutyrate) (PHB) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate P (3HB-co-HV). The bacterium grew efficiently in a simple mineral liquid medium enriched with 0.1% poly(3-hydroxybutyrate) powder as the sole carbon source. Cells excreted PHB depolymerase and degraded the polymer particles to complete clarity in 4 days. The degradation activity was detectable by the formation of a clear zone around the colony (petri plates) or a clear depth under the colony (test tubes). The expression of PHB depolymerase was repressed by the presence of simple soluble carbon sources. Bacterial degradation of the naturally occurring sheets of poly(3-hydroxybutyrate) and its copolymer poly(3-hydroxybutyrate-co-3-hydroxyvalerate) was observed by scanning electron microscopy (SEM). Morphological alterations of the polymers sheets were evidence for bacterial hydrolysis.     

Production of poly(3-hydroxybutyrate) by solid-state fermentation with <Emphasis Type="Italic">Ralstonia eutropha</Emphasis>

The use of solid-state fermentation is examined as a low-cost technology for the production of poly(hydroxyalkanoates) (PHAs) by Ralstonia eutropha. Two agroindustrial residues (babassu and soy cake) were evaluated as culture media. The maximum poly(hydroxybutyrate) (PHB) yield was 1.2 mg g^–1 medium on soy cake in 36 h, and 0.7 mg g^–1 medium on babassu cake in 84 h. Addition of 2.5% (w/w) sugar cane molasses to soy cake increased PHB production to 4.9 mg g^–1 medium in 60 h. Under these conditions, the PHB content of the dry biomass was 39% (w/w). The present results indicate that solid-state fermentation could be a promising alternative for producing biodegradable polymers at low cost.Revisions requested 31 August 2004; Revisions received 12 October 2004     

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.     

Production of poly(3-hydroxybutyrate) from inexpensive substrates

Two inexpensive substrates, starch and whey were used to produce poly(3-hydroxybutyrate) (PHB) in fed-batch cultures of Azotobacter chroococcum and recombinant Escherichia coli, respectively. Oxygen limitation increased PHB contents in both fermentations. In fed-batch culture of A. chroococcum, cell concentration of 54 g l⁻¹ with 46% PHB was obtained with oxygen limitation, whereas 71 g l⁻¹ of cell with 20% PHB was obtained without oxygen limitation. The timing of PHB biosynthesis in recombinant E. coli was controlled using the agitation speed of a stirred tank fermentor. A PHB content of 80% could be obtained with oxygen limitation by increasing the agitation speed up to only 500 rpm.     

Degradation of microbial polyester poly(3-hydroxybutyrate) in environmental samples and in culture

Poly(3-hydroxybutyrate) [P(3HB)] test-pieces prepared from the polymer produced by Azotobacter chroococcum were degraded in natural environments like soil, water, compost and sewage sludge incubated under laboratory conditions. Degradation in terms of % weight loss of the polymer was maximum (45%) in sewage sludge after 200 days of incubation at 30°C. The P(3HB)-degrading bacterial cultures (36) isolated from degraded test-pieces showed different degrees of degradation in polymer overlayer method. The extent of P(3HB) degradation increases up to 12 days of incubation and was maximum at 30°C for majority of the cultures. For most efficient cultures the optimum concentration of P(3HB) for degradation was 0.3% (w/v). Supplementation of soluble carbon sources like glucose, fructose and arabinose reduced the degradation while it was almost unaffected with lactose. Though the cultures degraded P(3HB) significantly, they were comparatively less efficient in utilizing copolymer of 3-hydroxybutyrate and 3-hydroxyvalerate [P(3HB-co-3HV)].     

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.     

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.     

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°C and in 250 g/l molasses although the optimal growth temperature was 35–37°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.     

Poly(3-hydroxybutyrate) (PHB) synthesis by recombinant Escherichia coli harbouring Streptomyces aureofaciens PHB biosynthesis genes: effect of various carbon and nitrogen sources

Recombinant Escherichia coli (ATCC:PTA-1579) harbouring poly(3-hydroxybutyrate) (PHB) synthesising genes from Streptomyces aureofaciens NRRL 2209 accumulates PHB. Effects of different carbon and nitrogen sources on PHB accumulation by recombinant E. coli were studied. Among the carbon sources used glycerol, glucose, palm oil and ethanol supported PHB accumulation. No PHB accumulated in recombinant cells when sucrose or molasses were used as carbon source. Yeast extract, peptone, a combination of yeast extract and peptone, and corn steep liquor were used as nitrogen sources. The maximum PHB accumulation (60% of cell dry weight) was measured after 48 h of cell growth at 37 degrees C in a medium with glycerol as the sole carbon source, and yeast extract and peptone as nitrogen sources. Scanning electron microscopy of the PHB granules isolated from recombinant E. coli revealed these to be spherical in shape with a diameter ranging from 0.11 to 0.35 pm with the mean value of 0.23 +/- 0.06 pm.     

Isolation of a Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterium from compost, and cloning and characterization of a gene encoding PHB depolymerase of Bacillus megaterium N-18-25-9

A Gram-positive poly(3-hydroxybutyrate) (PHB)-degrading bacterial strain was isolated from compost. This organism, identified as Bacillus megaterium N-18-25-9, produced a clearing zone on opaque NB-PHB agar, indicating the presence of extracellular PHB depolymerase. A PHB depolymerase gene, PhaZ(Bm), of B. megaterium N-18-25-9 was cloned and sequenced, and the recombinant gene product was purified from Escherichia coli. The N-terminal half region of PhaZ(Bm) shared significant homologies with a catalytic domain of other PHB depolymerases. Although the C-terminal half region of PhaZ(Bm) showed no significant similarity with those of other PHB depolymerases, that region was necessary for the PHB depolymerase activity. Therefore, this enzyme's domain structure is unique among extracellular PHB depolymerase domain structures. The addition of PHB to the medium led to a sixfold increase in PhaZ(Bm) mRNA, while the presence of glucose repressed PhaZ(Bm) expression. The maximum activity was observed at pH 9.0 at 65 degrees C.     

Structure of native poly(3-hydroxybutyrate) granules characterized by X-ray diffraction

The structure of native poly(3-hydroxybutyrate) (PHB) granules of Alcaligenes eutrophus was characterized in wet cells or wet granules by analysis of X-ray diffraction. The PHB granules in intact cells were completely amorphous, but became crystalline after treatment with alkali or sodium hypochlorite. The native PHB granules were isolated from the cells by treatment with enzymes and sonic oscillation. The isolated PHB granules remained amorphous in suspension. The PHB granules were crystallized by various treatments with aqueous acetone, alkaline solution (of either NaOH or sodium hypochlorite), and lipase in an aqueous environment. These results suggest that crystallization of PHB molecules is started by the removal of a lipid component from native granules by various treatments.     

Anaerobic degradation of poly-3-hydroxybutyrate and poly-3-hydroxybutyrate-co-3-hydroxyvalerate

The anaerobic degradation of the polyesterspoly-3-hydroxybutyrate (PHB) andpoly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) wasinvestigated with special regard to intermediateproducts, kinetics, and yields. During the degradationof PHBV acetate, propionate, n-butyrate, andn-valerate were detected. Additionally,3-hydroxybutyrate and 3-hydroxyvalerate and fourdimeric esters of these two molecules were identifiedby GC-MS measurements. Three different test systemsfor the anaerobic degradation of polyesters werestudied. It was not possible to get reproducibleresults by means of the Anaerobic Sturm-test, a simplesystem based on carbon dioxide measurement. Secondly,a system based on the GC measurement of accumulatedorganic acids was investigated. A degradation of 90%in two days was calculated by a carbon balance. Bestresults were reached with the third test system basedon the measurement of methane with a gas meter. Adegradation of 99% was observed within 30 days.     

Purification and Characterization of Fungal Poly(3-hydroxybutyrate) Depolymerase from Paecilomyces lilacinus F4-5 and Enzymatic Degradation of Poly(3-hydroxybutyrate-co-3-hydroxyvalerate) Film

Summary Poly(3-hydroxybutyrate) [P(3HB)] depolymerase was purified from a poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)]-degrading fungus, Paecilomyces lilacinus F4-5 by hydrophobic and ion exchange column chromatography, and showed a molecular mass of 45 kDa. The optimum temperature and pH of the P(3HB) depolymerase were 50 °C and 7.0, respectively. The enzyme was stable for at least 30 min at temperatures below 40 °C, while the activity abruptly decreased over 55 °C. Enzymatic P(3HB-co-3HV) degradation showed a similar degradation pattern to that of film overlaid by fungal hyphae. It reflects that the fungal degradation of P(3HB-co-3HV) in soil is mainly caused by extracellular depolymerases.     

Production of poly(3-hydroxybutyrate) from whey by cell recycle fed-batch culture of recombinant Escherichia coli

A new fermentation strategy using cell recycle membrane system was developed for the efficient production of poly(3-hydroxybutyrate) (PHB) from whey by recombinant Escherichia coli strain CGSC 4401 harboring the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes. By cell recycle, fed-batch cultivation employing an external membrane module, the working volume of fermentation could be constantly maintained at 2.3 l. The final cell concentration, PHB concentration and PHB content of 194 g l^–1, 168 g l^–1 and 87%, respectively, were obtained in 36.5 h by the pH-stat cell recycle fed-batch culture using whey solution concentrated to contain 280 g lactose l^–1 as a feeding solution, resulting in a high productivity of 4.6 g PHB l^–1 h^–1.     

Production of poly(3-hydroxybutyrate) from whey by fed-batch culture of recombinant Escherichia coli in a pilot-scale fermenter

Production of poly(3-hydroxybutyrate) [P(3HB)] from wheyby fed-batch culture of recombinant Escherichia coli CGSC 4401 harboring a plasmid containing the Alcaligenes latus polyhydroxyalkanoate (PHA) biosynthesis genes was examined in a 30 l fermenter supplying air only. With lactose below 2 g l^–1, cells grew to 12 g dry cell l^–1 with 9% (w/w) P(3HB) content. Accumulation of P(3HB) could be triggered by increasing lactose to 20 g l^–1. By employing this strategy, 51 g dry cell l^–1 was obtained with a 70% (w/w) P(3HB) content after 26 h. The productivity was 1.35 g P(3HB) l^–1 h^–1. The same fermentation strategy was used in a 300 l fermenter, and 30 g dry cell l^–1 with 67% (w/w) P(3HB) content was obtained in 20 h.     

Production of 3-hydroxypropionate homopolymer and poly(3-hydroxypropionate-co-4-hydroxybutyrate) copolymer by recombinant Escherichia coli

Conversion of 3-hydroxypropionate (3HP) from 1,3-propanediol (PDO) was improved by expressing dehydratase gene (dhaT) and aldehyde dehydrogenase gene (aldD) of Pseudomonas putida KT2442 under the promoter of phaCAB operon from Ralstonia eutropha H16. Expression of these genes in Aeromonas hydrophila 4AK4 produced up to 21 g/L 3HP in a fermentation process. To synthesize homopolymer poly(3-hydroxypropionate) (P3HP), and copolymer poly(3-hydroxypropionate-co-3-hydroxybutyrate) (P3HP4HB), dhaT and aldD were expressed in E. coli together with the phaC1 gene encoding polyhydroxyalkanoate (PHA) synthase gene of Ralstonia eutropha, and pcs' gene encoding the ACS domain of the tri-functional propionyl-CoA ligase (PCS) of Chloroflexus aurantiacus. Up to 92 wt% P3HP and 42 wt% P3HP4HB were produced by the recombinant Escherichia coli grown on PDO and a mixture of PDO+1,4-butanediol (BD), respectively.     

Production and characterization of homopolymer poly(3-hydroxyvalerate) (PHV) accumulated by wild type and recombinant Aeromonas hydrophila strain 4AK4

Aeromonas hydrophila 4AK4 normally produces copolyesters (PHBHHx) consisting of 3-hydroxybutyrate (C4) and 3-hydroxyhexanoate (C6). Wild type and recombinant A. hydrophila 4AK4 (pSXW02) expressing vgb and fadD genes encoding Vitreoscilla haemoglobin and Escherichia coli acyl-CoA synthase respectively, were found able to produce homopolyester poly(3-hydroxyvalerate) (PHV) (C5) on undecanoic acid as a single carbon source. The recombinant grew to 5.59 g/L cell dry weight (CDW) containing 47.74 wt% PHV in shake flasks when growth was conducted in LB medium and PHV production in undecanoic acid. The cells grew to 47.12 g/L CDW containing 60.08 wt% PHV in a 6 L fermentor study. Physical characterization of PHV produced by recombinant A. hydrophila 4AK4 (pSXW02) in fermentor showed a weight average molecular weight (M_w) of 230,000 Da, a polydispersity of 3.52, a melting temperature of 103 °C and a glass transition temperature of −15.8 °C. The degradation temperature at 5% weight loss of the PHV was around 258 °C.     

Biocompatibility of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolyesters produced byAlcaligenes sp. MT-16

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate), poly(3HB-co-3HV), copolyesters, with 3-hydroxyvalerate (3HV) contents ranging from 17 to 60 mol%, were produced byAlcaligenes sp. MT-16, and their biocompatibility evaluated by the growth of Chinese hamster ovary (CHO) cells and the adsorption of blood proteins and platelets onto their film surfaces. The number of CHO cells that adhered to and grew on these films was higher with increasing 3HV content. In contrast, the tendency for blood proteins and platelets to adhere to the copolyester surfaces significantly decreased with increasing 3HV content. Examination of the surface morphology using atomic force microscopy revealed that the surface roughness was an important factor in determining the biocompatibility of theses copolyesters. The results obtained in this study suggest that poly(3HB-co-3HV) copolyesters, with >30 mol% 3HV, may be useful in biocompatible biomedical applications.