Hypochlorite digestion method for efficient recovery of PHB from <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis>

We reported the optimum amount of PHB accumulated by Alcaligenes faecalis during its 24 h growth under nitrogen deficient conditions. After 24 h incubation decrease in the amount of PHB was recorded. Hypochlorite digestion of biomass of organism followed by extraction with a solvent system consisting of 1:1 mixture of ethanol and acetone resulted in efficient recovery of PHB vis-à-vis earlier methods. This solvent system gave a high recovery yield, i.e. 5.6 gL⁻¹ vis-à-vis earlier reported yield, 1.34 gL⁻¹ (by same method), 0.63 gL⁻¹ (by chloroform extraction method) and 1.1 gL⁻¹ (by dispersion method).     

A rapid method for detecting bacterial polyhydroxyalkanoates in intact cells by Fourier transform infrared spectroscopy

Polyhydroxyalkanoates (PHA) are synthesized by many bacteria as inclusion bodies, and their biodegradability and structural diversity have been studied with a view to their potential application as biodegradable materials. In this paper, Fourier-transform infrared spectroscopy (FT-IR) was used to carry out rapid qualitative analysis of PHA in intact bacterial cells. The FT-IR spectra of pure PHA containing short-chain-length monomers, such as hydroxybutyrate (HB), medium-chain-length hydroxyalkanoate (mclHA) monomers including hydroxyoctanoate (HO) and hydroxydecanoate (HD), or both HB and mclHA monomers, showed their strong characteristic band at 1728 cm⁻¹, 1740 cm⁻¹ or 1732 cm⁻¹ respectively. Other accompanying bands near 1280 cm⁻¹ and 1165 cm⁻¹ helped identify the types of PHA. The intensity of the methylene band near 2925 cm⁻¹ provided additional information for PHA characterization. In comparison, bacterial cells accumulating the above PHA also showed strong marker bands at 1732 cm⁻¹, 1744 cm⁻¹ or 1739 cm⁻¹, corresponding to intracellular PHB, mclPHA and P(HB + mclHA) respectively. The accompanying bands visible in pure PHA were also observable in the intact cells. The FT-IR results were further confirmed by gas chromatography analysis. Received: 14 October 1998 / Received revision: 29 December 1998 / Accepted: 30 December 1998     

Influence of electron acceptor,carbon, nitrogen,and phosphorus on polyhydroxyalkanoate (PHA) production by <Emphasis Type="Italic">Brachymonas</Emphasis> sp. P12

Polyhydroxyalkanoates (PHAs), intracellular carbon and energy reserve compounds in many bacteria, have been used extensively in biodegradable plastics. PHA formation is influenced by nutrient limitations and growth conditions. To characterize the PHA accumulation in a new denitrifying phosphorus-removing bacterium Brachymonas sp. P12, batch experiments were conducted in which the electron acceptor (oxygen or nitrate) was varied and different concentrations of carbon (acetate), nitrogen (NH₄Cl), and phosphorus (KH₂PO₄) were used. Polyhydroxybutyrate (PHB) was the dominant product during PHA formation when acetate was the sole carbon source. The PHB content of aerobically growing cells increased from 431 to 636 mg PHB g⁻¹ biomass, but the PHB concentration of an anoxic culture decreased (−218 mg PHB g⁻¹ biomass), when PHB was utilized simultaneously with acetate as an electron donor for anoxic denitrification. The specific PHB production rate of the carbon-limited batch, 158.2 mg PHB g⁻¹ biomass h⁻¹, was much greater than that of batches with normal or excess carbon. The effects of phosphorus and nitrogen concentrations on PHB accumulation were clearly less than the effect of carbon concentration. According to the correlation between the specific PHB production rate and the specific cell growth rate, PHB accumulation by Brachymonas sp. P12 is enhanced by nutrient limitation, is growth-associated, and provides additional energy for the biosynthesis of non-PHB cell constituents to increase the cell growth rate beyond the usual level.     

Poly-(3-hydroxybutyrate) production from whey by high-density cultivation of recombinant Escherichia coli

Recombinant Escherichia coli strain GCSC 6576, harboring a high-copy-number plasmid containing the Ralstonia eutropha genes for polyhydroxyalkanoate (PHA) synthesis and the E. coli ftsZ gene, was employed to produce poly-(3-hydroxybutyrate) (PHB) from whey. pH-stat fed-batch fermentation, using whey powder as the nutrient feed, produced cellular dry weight and PHB concentrations of 109 g l⁻¹ and 50 g l⁻¹ respectively in 47 h. When concentrated whey solution containing 210 g l⁻¹ lactose was used as the nutrient feed, cellular dry weight and PHB concentrations of 87 g l⁻¹ and 69 g l⁻¹ respectively could be obtained in 49 h by pH-stat fed-batch culture. The PHB content was as high as 80% of the cellular dry weight. These results suggest that cost-effective production of PHB is possible by fed-batch culture of recombinant E. coli using concentrated whey solution as a substrate. Received: 19 December 1997 / Received revision: 17 March 1998 / Accepted: 20 March 1998     

Molecular characterization of Pseudomonas sp. LDC-5 involved in accumulation of poly 3-hydroxybutyrate and medium-chain-length poly 3-hydroxyalkanoates

Polyhydroxyalkanoates (PHAs) are biological polyesters, of which, Short-Chain-Length-Medium-Chain-Length (SCL-MCL) PHA copolymers are important because of their wide range of applications. The present study focused on molecular characterization of Pseudomonas sp. LDC-5 that is identified as SCL-MCL producer. Phase contrast, fluorescent and electron microscopic observation confirmed the presence of PHA granules in Pseudomonas sp. LDC-5. PCR analysis indicated the presence of expected amplicon for SCL phaC gene (∼500 bp), MCL phaC1 with phaZ (∼1.3), and phaC2 with phaZ (∼1.5 kb). Sequence analysis of the PHA synthase gene of Pseudomonas sp. LDC-5 revealed significant differences in phaC1 and phaC2 which were further confirmed by recombinant studies. Recombinant Escherichia coli harboring the partial phaC1 gene was able to accumulate PHA, whereas E. coli with phaC2 did not accumulate PHA as verified by fold analysis, immunoblotting, Gas Chromatography (GC), Differential scanning calorimetry (DSC), and FTIR studies. The predicted theoretical three-dimensional structure revealed that PhaC1 is consistent with α/β hydrolase fold. Monomer composition showed the presence of monomer ranging from C4 to C12: 1 when glucose and sodium octanoate fed as the carbon source. DSC revealed melting temperature peak at 153.12°C and glass transition (T_g) peaks at −0.37°C. Thermogravimetric analysis revealed that the polymer was stable up to 276°C. Fourier Transform Infrared Spectroscopy (FT-IR) spectral analysis showed the PHA specific wave number at 1,739.67 and 1,161.07 cm⁻¹. The potential of Pseudomonas sp. LDC-5 and its properties are discussed.     

Relationship between Poly- β-hydroxybutyrate Production and δ-endotoxin for Bacillus thuringiensis var. kurstaki

A linear relationship between total solid concentration (TSC), δ-endotoxin production [Cry = 0.2795(TSC)−0.2472, R² = 0.8644] and poly-β-hydroxybutyrate (PHB) accumulation [PHB = 0.1327(TSC) + 0.3974, R² = 0.9877] in Bacillus thuringiensis var. kurstaki HD-73 was observed. A similar correlation between δ-endotoxin and PHB accumulation [Cry = 2.1573(PHB)−1.1248, R² = 0.9181] was found. A minimum PHB accumulation of 0.52 mg l⁻¹ was required before the onset of δ-endotoxin production. Revisions requested 28 September 2005 and 4 November 2005; Revisions received 28 October 2005 and 1 February 2006     

Formation of new polyhydroxyalkanoate containing 3-hydroxy-4-methylvalerate monomer in Burkholderia sp

Burkholderia sp. synthase has been shown to polymerize 3-hydroxybutyrate (3HB), 3-hydroxyvalerate, and 3-hydroxy-4-pentenoic acid monomers. This study was carried out to evaluate the ability of Burkholderia sp. USM (JCM 15050) and its transformant harboring the polyhydroxyalkanoate (PHA) synthase gene of Aeromonas caviae to incorporate the newly reported 3-hydroxy-4-methylvalerate (3H4MV) monomer. Various culture parameters such as concentrations of nutrient rich medium, fructose and 4-methylvaleric acid as well as harvesting time were manipulated to produce P(3HB-co-3H4MV) with different 3H4MV compositions. The structural properties of PHA containing 3H4MV monomer were investigated by using nuclear magnetic resonance and Fourier transform infrared spectroscopy (FTIR). The relative intensities of the bands at 1,183 and 1,228 cm⁻¹ in the FTIR spectra enabled the rapid detection and differentiation of P(3HB-co-3H4MV) from other types of PHA. In addition, the presence of 3H4MV units in the copolymer was found to considerably lower the melting temperature and enthalpy of fusion values compared with poly(3-hydroxybutyrate) (P(3HB)). The copolymer exhibited higher thermo-degradation temperature but similar molecular weight and polydispersity compared with P(3HB).     

Heterologous expression of polyhydroxyalkanoate depolymerase from <Emphasis Type="Italic">Thermobifida</Emphasis> sp. in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and catalytic analysis by surface plasmon resonance

A polyhydroxyalkanote depolymerase gene from Thermobifida sp. isolate BCC23166 was cloned and expressed as a C-terminal His₆-tagged fusion in Pichia pastoris. Primary structure analysis revealed that the enzyme PhaZ-Th is a member of a proposed new subgroup of SCL-PHA depolymerase containing a proline–serine repeat linker. PhaZ-Th was expressed as two glycosylated forms with apparent molecular weights of 61 and 70 kDa, respectively. The enzyme showed esterase activity toward p-nitrophenyl alkanotes with V _max and K _m of 3.63 ± 0.16 μmol min⁻¹ mg⁻¹ and 0.79 ± 0.12 mM, respectively, on p-nitrophenyl butyrate with optimal activity at 50–55°C and pH 7–8. Surface plasmon resonance (SPR) analysis demonstrated that PhaZ-Th catalyzed the degradation of poly-[(R)-3-hydroxybutyrate] (PHB) films, which was accelerated in (R)-3-hydroxyvalerate copolymers with a maximum degradation rate of 882 ng cm⁻² h⁻¹ for poly[(R)-3-hydroxybutyrate-co-3-hydroxyvalerate] (12 mol% V). Surface deterioration, especially on the amorphous regions of PHB films was observed after exposure to PhaZ-Th by atomic force microscopy. The use of P. pastoris as an alternative recombinant system for bioplastic degrading enzymes in secreted form and a sensitive SPR analytical technique will be of utility for further study of bioplastic degradation.     

Repeated Batch Cultivation of Ralstonia eutropha for Poly (β-hydroxybutyrate) Production

Batch cultivation of Ralstonia eutropha NRRL B14690 attained 21 g biomass l⁻¹ and 9.4 g poly(β-hydroxybutyrate) l⁻¹ (0.45 g PHB g⁻¹ dry wt⁻¹) in 60 h. Repeated batch operation (empty-and-fill protocol) to remove 20% (v/v) of the culture broth and to supplement an equal volume of fresh media resulted in 49 g biomass l⁻¹ and 25 g PHB l⁻¹ (0.51 g PHB g⁻¹ dry wt⁻¹) with an overall productivity of 0.42 g PHB l⁻¹ h⁻¹ in 67 h. In the two cycles of repeated batch fermentation there was a 3-fold increase in productivity as compared to batch.     

Co-inoculation of Urea and DAP Tolerant <Emphasis Type="Italic">Sinorhizobium meliloti</Emphasis> and <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> as Integrated Approach for Growth Enhancement of <Emphasis Type="Italic">Brassica juncea</Emphasis>

Two plant growth promoting rhizobacteria––Sinorhizobium meliloti RMP1 and Pseudomonas aeruginosa GRC₂ were studied for integrated nutrient management to obtain improved yield of Brassica juncea. Low concentrations of urea and diammonium phosphate (DAP) stimulated the growth of both S. meliloti RMP1 and P. aeruginosa GRC₂. 1 M of urea and 0.35 M of DAP was found lethal for RMP1, while 1.3 M and 0.37 M concentrations of urea and DAP proved to be toxic for GRC₂. Lc₅₀ was observed as 0.49 M of urea and 0.15 M of DAP for RMP1, and 0.66 M urea and 0.18 M of DAP for GRC₂. Urea and DAP adaptive variants of RMP1 and GRC₂ was isolated. Adaptive bacterial variants had better growth rates at sub-lethal (Lc₅₀) concentrations of urea and DAP as compared to non-adaptive variants. They also retained plant growth promoting attributes similar to non adaptive variants. GRC₂ and RMP1 did not affect the growth of each other and were chemotactically active for DAP, urea as well as root exudates of B. juncea. Both the isolates colonized well in the rhizosphere of B. juncea, as their populations were recorded ≈5 log₁₀ cfu g⁻¹ after 120 days. Interestingly, the colonization ability was found even better when both strains were co-inoculated, as their population was recorded in the range of ≈6 log₁₀ cfu g⁻¹ after 120 days. In field trials, application of RMP1 and GRC₂ resulted in significant increase in biomass and yield of B. juncea as compared to control. However, yield was better with application of half dose and full dose of recommended fertilizers. Interestingly, the biomass as well as yield improved further when both isolates were applied together along with half dose of recommended fertilizers.     

A lower specificity PhaC2 synthase from Pseudomonas stutzeri catalyses the production of copolyesters consisting of short-chain-length and medium-chain-length 3-hydroxyalkanoates

A polyhydroxyalkanoate (PHA) synthase gene phaC2 _Ps from Pseudomonas stutzeri strain 1317 was introduced into a PHA synthase gene phbC _Re negative mutant, Ralstonia eutropha PHB⁻4. It conferred on the host strain the ability to synthesize PHA, the monomer compositions of which varied widely when grown on different carbon sources. During cultivation on gluconate, the presence of phaC2 _Ps in R. eutropha PHB⁻4 led to the accumulation of polyhydroxybutyrate (PHB) homopolymer in an amount of 40.9 wt% in dry cells. With fatty acids, the recombinant successfully produced PHA copolyesters containing both short-chain-length and medium-chain-length 3-hydroxyalkanoate (3HA) of 4–12 carbon atoms in length. When cultivated on a mixture of gluconate and fatty acid, the monomer composition of accumulated PHA was greatly affected and the monomer content was easily regulated by the addition of fatty acids in the cultivation medium. After the (R)-3-hydroxydecanol-ACP:CoA transacylase gene phaG _Pp from Pseudomonas putida was introduced into phaC2 _Ps-containing R. eutropha PHB⁻4, poly(3HB-co-3HA) copolyester with a very high 3-hydroxybutyrate (3HB) fraction (97.3 mol%) was produced from gluconate and the monomer compositions of PHA synthesized from fatty acids were also altered. This study clearly demonstrated that PhaC2_Ps cloned from P. stutzeri 1317 has extraordinarily low substrate specificity in vivo, though it has only 54% identity in comparison to a previously described low-substrate-specificity PHA synthase PhaC1_Ps from Pseudomonas sp. 61–3. This study also indicated that the monomer composition and content of the synthesized PHA can be effectively modulated by controlling the addition of carbon sources or by modifying metabolic pathways in the hosts.     

Production of PHB and P (3HB-co-3HV) biopolymers by <Emphasis Type="Italic">Bacillus megaterium</Emphasis> strain OU303A isolated from municipal sewage sludge

Bacillus megaterium strain OU303A isolated from municipal sewage sludge was selected for the study of biosynthesis of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-hydroxyvalerate P (HB-co-HV) copolymer. The strain yielded a maximum of 62.43% DCW polymer in the medium containing glycerol as carbon source, which was followed by 58.63% DCW polymer in glucose containing medium. We found that this strain was capable of producing 2.5% hydroxyvalerate copolymer from a single carbon substrate, glucose. The strain showed an increase in the amount of HV monomer content, when the precursor for the copolymer was included in the fermentation medium. The characterization of the biopolymers was carried out using FTIR, GC-MS, H¹ NMR and DSC. This is the first report of B. megaterium strain producing HV copolymer, without the addition of any precursor in the fermentation medium.     

Poly(3-hydroxybutyrate) synthesis from glycerol by a recombinant Escherichia coli arcA mutant in fed-batch microaerobic cultures

Poly(3-hydroxybutyrate) (PHB) synthesis was analyzed under microaerobic conditions in a recombinant Escherichia coli arcA mutant using glycerol as the main carbon source. The effect of several additives was assessed in a semi-synthetic medium by the ‘one-factor-at-a-time’ technique. Casein amino acids (CAS) concentration was an important factor influencing both growth and PHB accumulation. Three factors exerting a statistically significant influence on PHB synthesis were selected by using a Plackett–Burman screening design [glycerol, CAS, and initial cell dry weight (CDW) concentrations] and then optimized through a Box–Wilson design. Under such optimized conditions (22.02 g l⁻¹ glycerol, 1.78 g l⁻¹ CAS, and 1.83 g l⁻¹ inoculum) microaerobic batch cultures gave rise to 8.37 g l⁻¹ CDW and 3.52 g l⁻¹ PHB in 48 h (PHB content of 42%) in a benchtop bioreactor. Further improvements in microaerobic PHB accumulation were obtained in fed-batch cultures, in which glycerol was added to maintain its concentration above 5 g l⁻¹. After 60 h, CDW and PHB concentration reached 21.17 and 10.81 g l⁻¹, respectively, which results in a PHB content of 51%. Microaerobic fed-batch cultures allowed a 2.57-fold increase in volumetric productivity when compared with batch cultures. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. An erratum to this article can be found at     

FTIR spectroscopy as a potential tool to analyse structural modifications during morphogenesis of Candida albicans

Candida albicans is a polymorphic organism that grows under certain conditions as blastospores, hyphae or pseudohyphae. The potentials of FTIR spectroscopy for assessing structural differences in C. albicans blastospores and hyphae were investigated. The main observed differences were localised in the polysaccharide (950–1,185 cm⁻¹), protein (1,480–1,720 cm⁻¹), and the fatty acids (2,840–3,000 cm⁻¹) regions. Quantitative evaluation of differences between hyphae and blastospores by curve-fitting of these regions indicate that these modifications could be due to both changes in structure and content of components of the cell wall such as β-glucans, mannoproteins, and lipids. Furthermore, glycogen consumption could be involved during hyphae elongation. Thus, FTIR spectroscopy can be an interesting tool to investigate differences in structure and in content between blastospores and hyphae. We also demonstrate through this study that differentiation of C. albicans clinical strains using hyphae is feasible, as this has been previously shown with blastospores. This preliminary work on identification of C. albicans using hyphae is a prelude to a larger clinical study for early typing within 7 h from a pure culture.     

Production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Cupriavidus necator from waste rapeseed oil using propanol as a precursor of 3-hydroxyvalerate

Waste rapeseed oil is a useful substrate for polyhydroxyalkanoates (PHA) production employing Cupriavidus necator H16. In fed-batch mode, we obtained biomass and PHA yields of 138 and 105 g l⁻¹, respectively. Yield coefficient and volumetric productivity were 0.83 g PHA per g oil and 1.46 g l⁻¹ h⁻¹, respectively. Propanol at 1% (v/v) enhanced both PHA and biomass formation significantly and, furthermore, resulted in incorporation of 3-hydroxyvalerate units into PHA structure. Thus, propanol can be used as an effective precursor of 3-hydroxyvalarete for production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer. During the fed-batch cultivation, propanol concentration was maintained at 1% which resulted in 8% content of 3-hydroxyvalerate in copolymer.     

Incorporation of polyethylene glycol in polyhydroxyalkanoic acids accumulated by Azotobacter chroococcum MAL-201

Azotobacter chroococcum MAL-201 (MTCC 3853), a free-living nitrogen-fixing bacterium accumulates poly(3-hydroxybutyric acid) [PHB, 69% of cell dry weight (CDW)] when grown on glucose and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [PHBV with 19.2 mol% 3HV] when grown on glucose and valerate. Use of ethylene glycol (EG) and/or polyethylene glycols (PEGs) of low molecular weight as sole carbon source were detrimental to A. chroococcum growth and polymer yields. PEG-200, however, in the presence of glucose was incorporated into the polyhydroxyalkanoate (PHA) polymer. Addition of PEG-200 (150 mM) to culture medium during mid-log phase growth favored increased incorporation of EG units (12.48 mol%) into the PHB polymer. In two-step culture experiments, where valerate and PEG simultaneously were used in fresh medium, EG was incorporated most effectively in the absence of glucose, leading to the formation of a copolymer containing 18.05 mol% 3HV and 14.78 mol% EG. The physico-mechanical properties of PEG-containing copolymer (PHBV–PEG) were compared with those of the PHB homopolymer and the PHBV copolymer. The PHBV–PEG copolymer appeared to have less crystallinity and greater flexibility than the short-chain-length (SCL) PHA polymers.     

Production of Exo-polysaccharide by <Emphasis Type="Italic">Rhizobium</Emphasis> sp.

Two fold increase in the yield of glucose and maltose containing exo-polysaccharide (EPS) by Rhizobium sp. was observed during its growth in modified YEMB. EPS production, plant growth promotion activity and root colonization of Rhizobium sp. studies showed enhanced EPS synthesis, more seed germination and over all improvement in plant growth over control and R. meliloti treatment. Groundnut seeds bacterized with Rhizobium sp. resulted in 69.75% more root length, 49.51% more shoot height, 13.75% more number of branches and 13.60% more number of pods over the control and R. meliloti treatment. Bacterization of wheat seeds increased the dry matter yield of roots (1.7-fold), and roots adhering soil (RAS) (1.5) and shoot mass (1.9-fold). Rhizobium sp. inoculation also increased the population density of EPS-producing bacteria on the rhizoplane. Roots of plants inoculated with Rhizobium sp. maintained a higher K⁺/Na⁺ ratio and K⁺–Na⁺ selectivity.     

FTIR spectroscopy of the reaction center of Chloroflexus aurantiacus: Photooxidation of the primary electron donor

Photochemical oxidation of the primary electron donor P in reaction centers (RCs) of the filamentous anoxygenic phototrophic bacterium Chloroflexus (C.) aurantiacus was examined by light-induced Fourier transform infrared (FTIR) difference spectroscopy at 95 K in the spectral range of 4000–1200 cm⁻¹. The light-induced P⁺Q_A⁻/PQ_A IR spectrum of C. aurantiacus RCs is compared to the well-characterized FTIR difference spectrum of P photooxidation in the purple bacterium Rhodobacter (R.) sphaeroides R-26 RCs. The presence in the P⁺Q_A⁻/PQ_A FTIR spectrum of C. aurantiacus RCs of specific low-energy electronic transitions at ∼2650 and ∼2200 cm⁻¹, as well as of associated vibrational (phase-phonon) bands at 1567, 1481, and 1294–1285 cm⁻¹, indicates that the radical cation P⁺ in these RCs has dimeric structure, with the positive charge distributed between the two coupled bacteriochlorophyll a molecules. The intensity of the P⁺ absorbance band at ∼1250 nm (upon chemical oxidation of P at room temperature) in C. aurantiacus RCs is approximately 1.5 times lower than that in R. sphaeroides R-26 RCs. This fact, together with the decreased intensity of the absorbance band at ∼2650 cm⁻¹, is interpreted in terms of the weaker coupling of bacteriochlorophylls in the P⁺ dimer in C. aurantiacus compared to R. sphaeroides R-26. In accordance with the previous (pre)resonance Raman data, FTIR measurements in the carbonyl stretching region show that in C. aurantiacus RCs (i) the 13¹-keto C=O groups of P_A and P_B molecules constituting the P dimer are not involved in hydrogen bonding in either neutral or photooxidized state of P and (ii) the 3¹-acetyl C=O group of P_B forms a hydrogen bond (probably with tyrosine M187) absorbing at 1635 cm⁻¹. Differential signals at 1757(+)/1749(−) and 1741(+)/1733(−) cm⁻¹ in the FTIR spectrum of C. aurantiacus RCs are attributed to the 13³-ester C=O groups of P in different environments.     

Microbial production of polyhydroxyalkanoate block copolymer by recombinant Pseudomonas putida

Polyhydroxyalkanoate (PHA) synthesis genes phaPCJ _Ac cloned from Aeromonas caviae were transformed into Pseudomonas putida KTOY06ΔC, a mutant of P. putida KT2442, resulting in the ability of the recombinant P. putida KTOY06ΔC (phaPCJ _A.c ) to produce a short-chain-length and medium-chain-length PHA block copolymer consisting of poly-3-hydroxybutyrate (PHB) as one block and random copolymer of 3-hydroxyvalerate (3HV) and 3-hydroxyheptanoate (3HHp) as another block. The novel block polymer was studied by differential scanning calorimetry (DSC), nuclear magnetic resonance, and rheology measurements. DSC studies showed the polymer to possess two glass transition temperatures (T _g), one melting temperature (T _m) and one cool crystallization temperature (T _c). Rheology studies clearly indicated a polymer chain re-arrangement in the copolymer; these studies confirmed the polymer to be a block copolymer, with over 70 mol% homopolymer (PHB) of 3-hydroxybutyrate (3HB) as one block and around 30 mol% random copolymers of 3HV and 3HHp as the second block. The block copolymer was shown to have the highest tensile strength and Young’s modulus compared with a random copolymer with similar ratio and a blend of homopolymers PHB and PHVHHp with similar ratio. Compared with other commercially available PHA including PHB, PHBV, PHBHHx, and P3HB4HB, the short-chain- and medium-chain-length block copolymer PHB-b-PHVHHp showed differences in terms of mechanical properties and should draw more attentions from the PHA research community.     

Carbon-limited fed-batch production of medium-chain-length polyhydroxyalkanoates from nonanoic acid by <Emphasis Type="Italic">Pseudomonas putida</Emphasis> KT2440

Pseudomonas putida KT2440 grew on glucose at a specific rate of 0.48 h⁻¹ but accumulated almost no poly-3-hydroxyalkanoates (PHA). Subsequent nitrogen limitation on nonanoic acid resulted in the accumulation of only 27% medium-chain-length PHA (MCL-PHA). In contrast, exponential nonanoic acid-limited growth (μ = 0.15 h⁻¹) produced 70 g l⁻¹ biomass containing 75% PHA. At a higher exponential feed rate (μ = 0.25 h⁻¹), the overall productivity was increased but less biomass (56 g l⁻¹) was produced due to higher oxygen demand, and the biomass contained less PHA (67%). It was concluded that carbon-limited exponential feeding of nonanoic acid or related substrates to cultures of P. putida KT2440 is a simple and highly effective method of producing MCL-PHA. Nitrogen limitation is unnecessary.