Poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) production from biodiesel by‐product and propionic acid by mutant strains of Pandoraea sp.

Pandoraea sp. MA03 wild type strain was subjected to UV mutation to obtain mutants unable to grow on propionic acid (PA) but still able to produce poly(3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) [P(3HB‐co‐3HV)] from glycerol and PA at high 3HV yields. In shake flask experiments, mutant prp25 was selected from 52 mutants affected in the propionate metabolism exhibiting a conversion rate of PA into 3HV units of 0.78 g g^?1. The use of crude glycerol (CG) plus PA or valeric acid resulted in a copolymer with 3HV contents varying from 21.9 to 30 mol% and 22.2 to 36.7 mol%, respectively. Fed‐batch fermentations were performed using CG and PA and reached a 3HV yield of 1.16 g g^?1, which is 86% of the maximum theoretical yield. Nitrogen limitation was a key parameter for polymer accumulation reaching up to 63.7% content and 18.1 mol% of 3HV. Henceforth, mutant prp25 is revealed as an additional alternative to minimize costs and support the P(3HB‐co‐3HV) production from biodiesel by‐products. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1077–1084, 2017     

A study of synthesis and properties of poly‐3‐hydroxybutyrate/diethylene glycol copolymers

This study investigates synthesis of poly(3‐hydroxybutyrate)/diethylene glycol copolymers (P3HB/DEG) by Cupriavidus eutrophus B‐10646 cells as related to DEG concentration in the medium and the time when it is added to the culture of cells synthesizing P3HB. The study determines the limits of physiological effect of DEG on C. eutrophus cells, showing that at DEG concentrations above 30 g/L, it inhibits cell growth, decreasing cell concentration and total P3HB/DEG yield and inducing an increase in the degree of saturation of fatty acids in lipids of cell cytoplasmic membrane. A series of copolymers containing different molar fractions of DEG (between 0.13 and 3.0 mol%) have been synthesized and their physicochemical, physical/mechanical, and biological properties have been investigated as related to the chemical composition and proportions of DEG monomers of the polymers. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1017–1028, 2016     

Metabolic engineering of Escherichia coli for the production of polylactic acid and its copolymers

Polylactic acid (PLA) is a promising biomass‐derived polymer, but is currently synthesized by a two‐step process: fermentative production of lactic acid followed by chemical polymerization. Here we report production of PLA homopolymer and its copolymer, poly(3‐hydroxybutyrate‐co‐lactate), P(3HB‐co‐LA), by direct fermentation of metabolically engineered Escherichia coli. As shown in an accompanying paper, introduction of the heterologous metabolic pathways involving engineered propionate CoA‐transferase and polyhydroxyalkanoate (PHA) synthase for the efficient generation of lactyl‐CoA and incorporation of lactyl‐CoA into the polymer, respectively, allowed synthesis of PLA and P(3HB‐co‐LA) in E. coli, but at relatively low efficiency. In this study, the metabolic pathways of E. coli were further engineered by knocking out the ackA, ppc, and adhE genes and by replacing the promoters of the ldhA and acs genes with the trc promoter based on in silico genome‐scale metabolic flux analysis in addition to rational approach. Using this engineered strain, PLA homopolymer could be produced up to 11 wt% from glucose. Also, P(3HB‐co‐LA) copolymers containing 55–86 mol% lactate could be produced up to 56 wt% from glucose and 3HB. P(3HB‐co‐LA) copolymers containing up to 70 mol% lactate could be produced to 46 wt% from glucose alone by introducing the Cupriavidus necator β‐ketothiolase and acetoacetyl‐CoA reductase genes. Thus, the strategy of combined metabolic engineering and enzyme engineering allowed efficient bio‐based one‐step production of PLA and its copolymers. This strategy should be generally useful for developing other engineered organisms capable of producing various unnatural polymers by direct fermentation from renewable resources. Biotechnol. Bioeng. 2010; 105: 161–171. © 2009 Wiley Periodicals, Inc.     

Regulation of poly-(R)-(3-hydroxybutyrate-co-3-hydroxyvalerate) biosynthesis by the AtoSCDAEB regulon in phaCAB + Escherichia coli

AtoSC two-component system (TCS) upregulates the high-molecular weight poly-(R)-3-hydroxybutyrate (PHB) biosynthesis in recombinant phaCAB ⁺ Escherichia coli strains, with the Cupriavidus necator phaCAB operon. We report here that AtoSC upregulates also the copolymer P(3HB-co-3HV) biosynthesis in phaCAB ⁺ E. coli. Acetoacetate-induced AtoSC maximized P(3HB-co-3HV) to 1.27 g/l with a 3HV fraction of 25.5 % wt. and biopolymer content of 75 % w/w in a time-dependent process. The atoSC locus deletion in the ?atoSC strains resulted in 4.5-fold P(3HB-co-3HV) reduction, while the 3HV fraction of the copolymer was restricted to only 6.4 % wt. The ?atoSC phenotype was restored by extrachromosomal introduction of AtoSC. Deletion of the atoDAEB operon triggered a significant decrease in P(3HB-co-3HV) synthesis and 3HV content in ?atoDAEB strains. However, the acetoacetate-induced AtoSC in those strains increased P(3HB-co-3HV) to 0.8 g/l with 21 % 3HV, while AtoC or AtoS expression increased P(3HB-co-3HV) synthesis 3.6- or 2.4-fold, respectively, upon acetoacetate. Complementation of the ?atoDAEB phenotype was achieved by the extrachromosomal introduction of the atoSCDAEB regulon. Individual inhibition of β-oxidation and mainly fatty acid biosynthesis pathways by acrylic acid or cerulenin, respectively, reduced P(3HB-co-3HV) biosynthesis. Under those conditions, introduction of atoSC or atoSCDAEB regulon was capable of upregulating biopolymer accumulation. Concurrent inhibition of both the fatty acid metabolic pathways eliminated P(3HB-co-3HV) production. P(3HB-co-3HV) upregulation in phaCAB ⁺ E. coli by AtoSC signaling through atoDAEB operon and its participation in the fatty acids metabolism interplay provide additional perceptions of AtoSC critical involvement in E. coli regulatory processes towards biotechnologically improved polyhydroxyalkanoates biosynthesis.     

Biosynthesis of polylactic acid and its copolymers using evolved propionate CoA transferase and PHA synthase

For the synthesis of polylactic acid (PLA) and its copolymers by one‐step fermentation process, heterologous pathways involving Clostridium propionicum propionate CoA transferase (Pct_Cp) and Pseudomonas sp. MBEL 6‐19 polyhydroxyalkanoate (PHA) synthase 1 (PhaC1_Ps6‐19) were introduced into Escherichia coli for the generation of lactyl‐CoA endogenously and incorporation of lactyl‐CoA into the polymer, respectively. Since the wild‐type PhaC1_Ps6‐19 did not efficiently accept lactyl‐CoA as a substrate, site directed mutagenesis as well as saturation mutagenesis were performed to improve the enzyme. The wild‐type Pct_Cp was not able to efficiently convert lactate to lactyl‐CoA and was found to exert inhibitory effect on cell growth, random mutagenesis by error‐prone PCR was carried out. By employing engineered PhaC1_Ps6‐19 and Pct_Cp, poly(3‐hydroxybutyrate‐co‐lactate), P(3HB‐co‐LA), containing 20–49 mol% lactate could be produced up to 62 wt% from glucose and 3HB. By controlling the 3HB concentration in the medium, PLA homopolymer and P(3HB‐co‐LA) containing lactate as a major monomer unit could be synthesized. Also, P(3HB‐co‐LA) copolymers containing various lactate fractions could be produced from glucose alone by introducing the Cupriavidus necator β‐ketothiolase and acetoacetyl‐CoA reductase genes. Fed‐batch cultures were performed to produce P(3HB‐co‐LA) copolymers having 9–64 mol% of lactate, and their molecular weights, thermal properties, and melt flow properties were determined. Biotechnol. Bioeng. 2010; 105: 150–160. © 2009 Wiley Periodicals, Inc.     

En route to economical eco‐friendly solvent system in enhancing sustainable recovery of poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate) copolymer

Separation of poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate) [P(3HB‐co‐4HB)] from bacterial cell matter is a critical step in the downstream process with respect to material quality and eco‐balance as P(3HB‐co‐4HB) is widely used for biomedical applications. Therefore, an efficient and eco‐based extraction of P(3HB‐co‐4HB) using a combination of NaOH and Lysol in digesting the non‐polymeric cell material (NPCM) digestion is developed. The NaOH and Lysol show synergistic influence on the copolymer extraction at a high purity and recovery of 97 and 98 wt% respectively. The optimized cell digestion method was found applicable to a vast batch of cells containing copolymers from various 4HB monomer compositions. At the largest extraction volume of 100 L, P(3HB‐co‐4HB) with a purity of 89 wt% was extracted with a maximum recovery of 90 wt%. The method developed has also eliminated the cell pretreatment step. The extraction method developed in this research has not only produced an economic and efficient copolymer recovery but has also retained the copolymer quality, in term of its molecular weight and thermal properties. It demonstrates a practical and promising downstream processing method in recovering the copolymer effectively from the bacterial biomass.     

Production of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by <Emphasis Type="Italic">Rhodopseudomonas palustris</Emphasis> SP5212

Summary Production of poly(3-hydroxybutyric acid) [P(3HB)] by Rhodopseudomonas palustris SP5212 isolated in this laboratory has been optimized under phototrophic microaerophilic conditions. Cells grown in malate medium accumulated 7.7% (w/w) P(3HB) of cellular dry weight at the early stationary phase of growth. The accumulated P(3HB) however, attained 15% (w/w) of cellular dry weight when acetate (1.0%, w/v) was used as the sole carbon source under nitrogen-limiting conditions. Synthesis and accumulation of polymer was favoured by sulphate-free conditions and at a phosphate concentration sub-optimal for growth. The polymer content of cells was increased drastically (34% of cellular dry weight) when the acetate containing medium was supplemented with n-alkanoic acids. Compositional analysis by H¹ NMR revealed that these accumulated polymers were composed of 3-hydroxybutyric acid and 3-hydroxyvaleric acid (3HV). The contents of 3HV in these copolymers ranged from 14 to 38 mol%.     

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.     

Biosynthesis of novel polyhydroxyalkanoate containing 3-hydroxy-4-methylvalerate by Chromobacterium sp. USM2

Aims: Polyhydroxyalkanoate (PHA) with enhanced physicochemical properties will be ideal for a wide range of practical applications. The incorporation of 3‐hydroxy‐4‐methylvalerate (3H4MV) into the polymer backbone is known to improve the overall properties of the resulting polymer. However, the most suitable micro‐organism and PHA synthase that can synthesize this monomer efficiently still remain unknown at present. Therefore, we evaluated the abilities of a locally isolated Chromobacterium sp. USM2 to produce PHA containing 3H4MV. Methods and Results: The ability of Chromobacterium sp. USM2 to synthesize poly(3‐hydroxybutyrate‐co‐3‐hydroxy‐4‐methylvalerate) [P(3HB‐co‐3H4MV)] was evaluated under different culture conditions. It was found that Chromobacterium sp. USM2 can synthesize P(3HB‐co‐3H4MV) when glucose and isocaproic acid were fed as carbon source. However, the highest molar fraction of 3H4MV, 22 mol% was detected in Chromobacterium sp. USM2 when isocaproic acid was provided as the sole carbon source. In addition, aeration was identified as a crucial factor in initiating the accumulation of high 3H4MV molar fractions. Conclusions: Chromobacterium sp. USM2 was able to synthesize broad comonomer compositional distribution of P(3HB‐co‐3H4MV). Significance and Impact of the Study: Compared with Cupriavidus necator and Burkholderia sp., Chromobacterium sp. USM2 was found to have better ability to bioconvert isocaproic acid to form 3H4MV unit.     

Application of random mutagenesis to enhance the production of polyhydroxyalkanoates by Cupriavidus necator H16 on waste frying oil

Using random chemical mutagenesis we obtained the mutant of Cupriavidus necator H16 which was capable of improved (about 35 %) production of poly(3-hydroxybuytrate) (PHB) compared to the wild-type strain. The mutant exhibited significantly enhanced specific activities of enzymes involved in oxidative stress response such as malic enzyme, NADP-dependent isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase and glutamate dehydrogenase. Probably, due to the activation of these enzymes, we also observed an increase of NADPH/NADP⁺ ratio. It is likely that as a side effect of the increase of NADPH/NADP⁺ ratio the activity of PHB biosynthetic pathway was enhanced, which supported the accumulation of PHB. Furthermore, the mutant was also able to incorporate propionate into copolymer poly(3-hydroxybuytyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] more efficiently than the wild-type strain (Y3HV/prec = 0.17 and 0.29 for the wild-type strain and the mutant, respectively)). We assume that it may be caused by lower availability of oxaloacetate for the utilization of propionyl-CoA in 2-methylcitrate cycle due to increased action of malic enzyme. Therefore, propionyl-CoA was incorporated into copolymer rather than transformed to pyruvate via 2-methylcitrate cycle. Thus, the mutant was capable of the utilization of waste frying oils and the production of P(3HB-co-3HV) with better yields and improved content of 3HV resulting in better mechanical properties of copolymer than the wild-type strain. The results of this work may be used for the development of innovative fermentation strategies for the production of PHA and also it might help to define novel targets for the genetic manipulations of PHA producing bacteria.     

Estimation of the Number of Polyhydroxyalkanoate (PHA)-Degraders in Soil and Isolation of Degraders Based on the Method of Most Probable Number (MPN) Using PHA-Film

A new method to estimate the number of polyhydroxyalkanoates (PHA)-degraders in soil and to isolate degraders, called the film-MPN method, is proposed. The incubation time was measured by the first order reaction (FOR) model. This method was used to estimate numbers of poly (3-hydroxybutyrate-co-3-hydroxyvalerate)[P(3HB-co-3HV)]- and poly(3-hydroxyvalerate-co-4-hydroxybutyrate)[P(3HB-co-4HB)]-degraders in garden soil (4.30 × 10⁵ and 2.15 × 10⁵ aerobic degraders per gram of dry soil, respectively). The number of P(3HB-co-3HV)-degraders in paddy field soil was 5.06 × 10⁵ aerobic degraders per gram dry soil. Also, several P(3HB-co-3HV)-degraders were isolated directly from positive-growth tubes of high dilution.     

Purification and properties of poly(3-hydroxyvaleric acid) depolymerase from Pseudomonas lemoignei

During growth on poly(3-hydroxyvaleric acid), P(3HV), or valerate Pseudomonas lemoignei secretes a P(3HV) depolymerase. This P(3HV) depolymerase was purified from the culture medium of valerate-grown cells by ammonium sulphate precipitation, chromatography on DEAe-sephacel and CM-Sepharose CL 6B. The relative molecular masses of the native as well as the sodium dodecyl sulphate (SDS)-treated enzyme were 53 000 or 54 000, respectively. In contrast to the poly(3-hydroxybutyric acid), P(3HB), depolymerase of Comamonas sp. and P(3HB) depolymerases A and B of P. lemoignei, which are specific for the hydrolysis of P(3HB), the purified P(3HV) depolymerase hydrolysed P(3HB), P(3HV) and co-polymers of 3-hydroxybutyric acid and 3-hydroxyvaleric acid at similar rates. Poly(hydroxyalkanoic acids), consisting of monomers with six and more carbon atoms or substrates characteristic for lipases such as Tween 80 or triolein were not hydrolysed. Maximum activities were measured in 50mm TRIS-HCl buffer, pH 8.0, at 55° C. The apparent K _m values of the purified P(3HV) depolymerase for P(3HB) and P(3HV) were 77 and 65 g polyester/ml, respectively. As the main product of enzymatic hydrolysis of P(3HV), 3-hydroxyvalerate was identified. The depolymerase was insensitive to p-hydroxymercuribenzoate but sensitive to dithioerythritol and phenylmethylsulphonyl fluoride, indicating the absence of active reduced sulphur groups and the presence of essential disulphide bonds and serine residues. Correspondence to: D. Jendrossek     

Mole Fraction Control of Poly([R]‐3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHB/HV) Synthesized by Paracoccus denitrificans

Different substrate mixtures of acetic acid and valeric acid were used to synthesize copolymers of poly([R]‐3‐hydroxybutyrate‐co‐3‐hydroxyvalerate) (PHB/HV) with Paracoccus denitrificans under N‐limited conditions. A correlation between the substrate ratio and HV content was found in batch experiments, which seemed to be suitable to produce a number of defined copolymers. In fed‐batch fermentation, such correlation could only be found with carbon substrate mixtures of very restricted composition. Due to the individual substrate consumption rates with this technique, a polymer with 16.5 mol.‐% HV content [w/w] could be reproducibly synthesized. However, under N‐limited chemostatic cultivation conditions it was possible to produce a spectrum of definitely composed copolymers (3.0 %–46.3 mol.‐% HV) from different mixtures of acetic acid and valeric acid.     

Polyhydroxyalkanoate biosynthesis and simultaneous remotion of organic inhibitors from sugarcane bagasse hydrolysate by Burkholderia sp.

Burkholderia sp. F24, originally isolated from soil, was capable of growth on xylose and removed organic inhibitors present in a hemicellulosic hydrolysate and simultaneously produced poly-3-hydroxybutyrate (P3HB). Using non-detoxified hydrolysate, Burkholderia sp. F24 reached a cell dry weight (CDW) of 6.8 g L^?1, containing 48 % of P3HB and exhibited a volumetric productivity (P_P3HB) of 0.10 g L^?1 h^?1. Poly-3-hydroxybutyrate-co-3-hydroxyvalerate copolymers (P3HB-co-3HV) were produced using xylose and levulinic acid (LA) as carbon sources. In shake flask cultures, the 3HV content in the copolymer increased from 9 to 43 mol% by adding LA from 1.0 to 5.0 g L^?1. In high cell density cultivation using concentrated hemicellulosic hydrolysate F24 reached 25.04 g L^?1 of CDW containing 49 % of P3HB and P_P3HB of 0.28 g L^?1 h^?1. Based on these findings, second-generation ethanol and bioplastics from sugarcane bagasse is proposed.     

Comparative evaluation of physico-chemical characteristics of biopolyesters P(3HB) and P(3HB-co-3HV) produced by endophytic <Emphasis Type="Italic">Bacillus cereus</Emphasis> RCL 02

Background

Bacteria endogenously residing within the plant tissues have attracted significant attention for production of biopolyester, polyhydroxyalkanoates (PHAs). Bacillus cereus RCL 02 (MCC 3436), a leaf endophyte of oleaginous plant Ricinus communis L. accumulates 81% poly(3-hydroxybutyrate) [P(3HB)] of its cell dry biomass when grown in mineral salts (MS) medium.

Methods

The copolymer production efficiency of B. cereus RCL 02 was evaluated in valeric acid supplemented MS medium under biphasic cultivation condition. The copolymer so produced has been compared with the P(3HB) isolated from RCL 02 in terms of thermal, mechanical and chemical properties.

Results

Valeric acid supplementation as co-substrate in the medium has led to the production of copolymer of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) [P(3HB-co-3HV)] with 14.6 mol% 3HV. The identity of the polymers has been confirmed by X-ray diffraction (XRD) analysis, Fourier transform infrared (FTIR) and nuclear magnetic resonance (NMR) spectroscopic studies. Thermogravimetric analysis (TGA) revealed that P(3HB) and P(3HB-co-3HV) films degraded at 278.66°C and 273.49°C, respectively. The P(3HB-co-3HV) showed lower melting temperature (165.03°C) compared to P (3HB) (170.74°C) according to differential scanning calorimetry (DSC). Incorporation of 3HV monomers decreased the tensile strength (21.52 MPa), tensile modulus (0.93 GPa), storage modulus (E′) (0.99 GPa) and increased % elongation at break (12.2%) of the copolyester. However, P(3HB) showed better barrier properties with lower water vapor transmission rate (WVTR) of 0.55 g-mil/100 in²/24 h.

Conclusion

These findings emphasized exploration of endophytic bacterial strain (RCL 02) to produce biodegradable polyesters which might have significant potential for industrial application.

     

Synthesis of poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate)/chitosan/silver nanocomposite material with enhanced antimicrobial activity

This work aims to shed light in the fabrication of poly(3‐hydroxybutyrate‐co‐44%‐4‐hydroxybutyrate)[P(3HB‐co‐44%4HB)]/chitosan‐based silver nanocomposite material using different contents of silver nanoparticle (SNP); 1–9 wt%. Two approaches were applied in the fabrication; namely solvent casting and chemical crosslinking via glutaraldehyde (GA). A detailed characterization was conducted in order to yield information regarding the nanocomposite material. X‐ray diffraction analysis exhibited the nature of the three components that exist in the nanocomposite films: P(3HB‐co‐4HB), chitosan, and SNP. In term of mechanical properties, tensile strength, and elongation at break were significantly improved up to 125% and 22%, respectively with the impregnation of the SNP. The melting temperature of the nanocomposite materials was increased whereas their thermal stability was slightly changed. Scanning electron microscopy images revealed that incorporation of 9 wt% of SNP caused agglomeration but the surface roughness of the material was significantly improved with the loading. Staphylococcus aureus and Escherichia coli were completely inhibited by the nanocomposite films with 7 and 9 wt% of SNP, respectively. On the other hand, degradation of the nanocomposite materials outweighed the degradation of the pure copolymer. These bioactive and biodegradable materials stand a good chance to serve the vast need of biomedical applications namely management and care of wound as wound dressing. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:1469–1479, 2014     

Biosynthesis of polyhydroxyalkanoate copolymers from mixtures of plant oils and 3-hydroxyvalerate precursors

The combination of plant oils and 3-hydroxyvalerate (3HV) precursors were evaluated for the biosynthesis of polyhydroxyalkanoate (PHA) copolymers containing 3HV monomers by Cupriavidus necator H16. Among various mixtures of plant oils and 3HV-precursors, the mixture of palm kernel oil and sodium propionate was suitable for the biosynthesis of high concentration of PHA (6.8gL(-1)) containing 7mol% of 3HV. The 3HV monomer composition can be regulated in the range of 0-23mol% by changing culture parameters such as the initial pH, and the nitrogen source and its concentration. PHA copolymers with high weight-average molecular weights (Mw) ranging from 1,400,000 to 3,100,000Da were successfully produced from mixtures of plant oils and 3HV-precursors. The mixture of plant oils and sodium propionate resulted in PHA copolymers with higher M(w) compared to the mixture of plant oils and sodium valerate. DSC analysis on the PHA containing 3HV monomers showed the presence of two distinct melting temperature (Tm), which indicated that the PHA synthesized might be a blend of P(3HB) and P(3HB-co-3HV). Sodium propionate appears to be the better precursor of 3HV than sodium valerate.     

Physico-chemical properties of polyhydroxyalkanoate produced by mixed-culture nitrogen-fixing bacteria

Ultra-high molecular weight polyhydroxyalkanoates (PHAs) with low polydispersity index (PDI = 1.3) were produced in a novel, pilot scale application of mixed cultures of nitrogen-fixing bacteria. The number average molecular weight (M _n) of the poly(3-hydroxybutyrate) (P(3HB)) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (P(3HB-co-3HV)) was determined to be 2.4 × 10⁶ and 2.5 × 10⁶ g mol⁻¹, respectively. Using two types of carbon sources, biomass contents of the P(3HB) and P(3HB-co-3HV) were 18% and 30% (PHA in dry biomass), respectively. The extracted polymers were analysed for their physical properties using analytical techniques such as nuclear magnetic resonance (NMR) spectroscopy, differential scanning calorimetry (DSC) and gel permeation chromatography (GPC). NMR confirmed the formation of homopolymer and copolymer. DSC showed a single melting endotherm peak for both polymers, with enthalpies that indicated crystallinity indices of 44% and 37% for P(3HB) and P(3HB-co-3HV), respectively. GPC showed a sharp unimodal trace for both polymers, reflecting the homogeneity of the polymer chains. The work described here emphasises the potential of mixed colony nitrogen-fixing bacteria cultures for producing biodegradable polymers which have properties that are very similar to those from their pure-culture counterparts and therefore making a more economically viable route for obtaining biopolyesters.     

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.