Biosynthesis and characterization of poly(3-hydroxydodecanoate) by β-oxidation inhibited mutant of Pseudomonas entomophila L48

A medium-chain-length (MCL) polyhydroxyalkanoates (PHAs) producer Pseudomonas entomophila L48 was investigated for microbial production of 3-hydroxydodecanote homopolymer. Pseudomonas entomophila L48 was found to produce MCL PHA consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), and 3-hydroxydodecanoate (3HDD) from related carbon sources fatty acids. In this study, some of the genes encoding key enzymes in β-oxidation cycle of P. entomophila such as 3-hydroxyacyl-CoA dehydrogenase, 3-ketoacyl-CoA thiolase, and acetyl-CoA acetyltransferase were deleted to study the relationship between β-oxidation and PHA synthesis in P. entomophila. Among the mutants constructed, P. entomophila LAC26 accumulated over 90 wt % PHA consisting of 99 mol % 3HDD. A fed-batch fermentation process carried out in a 6 L automatic fermentor produced 7.3 g L(-1) PHA consisting of over 97 mol % 3HDD fraction. Properties of MCL PHA were significantly improved along with increasing 3HDD contents. P(2.1 mol % 3HD-co-97.9 mol % 3HDD) produced by P. entomophila LAC25 had the widest temperature range between T(g) and T(m), which were -49.3 and 82.4 °C, respectively, in all MCL PHA reported so far. The new type of PHA also represented high crystallinity caused by side-chain crystallization compared with short side chain PHA. For the first time, P(3HDD) homopolymers were obtained.     

Formation of blends of various poly(3-hydroxyalkanoic acids) by a recombinant strain of Pseudomonas oleovorans

Summary Recombinant strains of Pseudomonas oleovorans, which harbour the poly(3-hydroxybutyrate)-biosynthetic genes of Alcaligenes eutrophus, accumulated poly(hydroxyalkanoates), composed of 3-hydroxybutyrate(3HB), 3-hydroxyhexanoate (3HHx) and 3-hydroxyactanoate (3HO), up to 70% of the cell dry weight if the cells were cultivated with sodium octanoate as the carbon source. Physiological and chemical analysis revealed multiple evidence that this polymer is a blend of the homopolyester poly(3HB) and of the copolyester poly(3HHx-co-3HO) rather than a random or a block copolyester of 3HB, 3HHx and 3HO. The molar ratio between poly(3HHx-co-3HO) and poly(3HB) varied drastically during the process of fermentation. Whereas synthesis of poly(3HHx-co-3HO) started immediately after ammonium was exhausted in the medium, synthesis of poly(3HB) occurred only after a lag-phase. From freeze-dried cells poly(3HHx-co-3HO) was much more readily extracted with chloroform than was poly(3HB). The blend was fractionated into petrol-ether-insoluble poly(3HB) and petrol-ether-soluble poly(3HHx-co-3HO). The molecular weight values of these polyesters measured by gel permeation chromatography were 2.96 × 10⁶ and 0.35 × 10⁶ and were similar of those polymers accumulated by A. eutrophus or by wild-type P. oleovorans, respectively. Offprint requests to: A. Steinbüchel     

Microbial degradation of poly(3-hydroxybutyrate) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) in soils.

The microbial degradation of tensile test pieces made of poly(3-hydroxybutyrate) [P(3HB)] or a copolymer of 90% 3-hydroxybutyric acid and 10% 3-hydroxyvaleric acid was studied in soils incubated at a constant temperature of 15, 28, or 40 degrees C for up to 200 days. In addition, hydrolytic degradation in sterile buffer at temperatures ranging from 4 to 55 degrees C was monitored for 98 days. Degradation was measured through loss of weight (surface erosion), molecular weight, and mechanical strength. While no weight loss was recorded in sterile buffer, samples incubated in soils were degraded at an erosion rate of 0.03 to 0.64% weight loss per day, depending on the polymer, the soil, and the incubation temperature. The erosion rate was enhanced by incubation at higher temperatures, and in most cases the copolymer lost weight at a higher rate than the homopolymer. The molecular weights of samples incubated at 40 degrees C in soils and those incubated at 40 degrees C in sterile buffer decreased at similar rates, while the molecular weights of samples incubated at lower temperatures remained almost unaffected, indicating that molecular weight decrease is due to simple hydrolysis and not to the action of biodegrading microorganisms. The degradation resulted in loss of mechanical properties. From the samples used in the biodegradation studies, 295 dominant microbial strains capable of degrading P (3HB) and the poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymer in vitro were isolated and identified.(ABSTRACT TRUNCATED AT 250 WORDS)     

Engineering of Ralstonia eutropha for production of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) from fructose and solid-state properties of the copolymer

Recombinant Ralstonia eutropha capable of producing poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) copolymer [P(3HB-co-3HHx)] from fructose was engineered by introduction of genes for crotonyl-CoA reductase (CCR) from Streptomyces cinnamonensis (ccrSc) and for PHA synthase and (R)-specific enoyl-CoA hydratase from Aeromonas caviae (phaC-JAc). In this recombinant strain, C6-acyl-CoA intermediates were provided via beta-ketothiolase-mediated elongation of butyryl-CoA, which was generated from crotonyl-CoA by the function of CCR. The recombinant strain could accumulate the copolyester up to 48 wt % of dry cell weight with 1.5 mol % of 3HHx fraction from fructose, when the expression of ccrSc under the control of the PBAD promoter was induced with 0.01% L-arabinose. The absence of L-arabinose or the deletion of ccrSc from the plasmid resulted in accumulation of poly(3-hydroxybutyrate) homopolymer, indicating the critical role of CCR in the formation of the 3-hydroxyhexanoate unit. Higher CCR activity obtained by the addition of a larger amount of L-arabinose did not affect the composition but reduced the intracellular content of the copolyester. The P(3HB-co-1.5 mol % 3HHx) copolyester produced from fructose by the recombinant R. eutropha showed relatively lower melting temperatures (150 degrees C and 161 degrees C) and lower crystallinity (48 +/- 5%) compared to those (175 degrees C and 60 +/- 5%) of P(3HB) homopolymer. It has been found that the incorporation of a small amount (1.5 mol %) of 3HHx units into P(3HB) sequences leads to a remarkable change in the solid-state properties of P(3HB) crystals. The present study demonstrates the potential of the engineered pathway for the production of copolyesters having favorable characteristics from inexpensive carbon resources.     

Synthesis of polyhydroxyalkanoates from different short-chain fatty acids by mixed cultures submitted to aerobic dynamic feeding

The production of polyhydroxyalkanoates from acetate and propionate by two mixed cultures well adapted to each of these substrates was evaluated. Sludge fed with acetate (A), produced a homopolymer of hydroxybutyrate (HB), whereas sludge fed with propionate (P) produced a copolymer of HB and HV (hydoxyvalerate). Switching the substrate feeds, propionate to sludge A and acetate to culture P, a terpolymer of HB, HV and hydroxymethylvalerate (HMV) was obtained with culture A and a copolymer of P(HB/HV) by sludge P. Regardless of the population used, the polymer yield and productivity were much higher for acetate than for propionate. Feeding a mixture of acetate and propionate, in equal parts, to both cultures resulted in an increase of HV units produced per C mol of propionate consumed, relative to the situation where only propionate was used. The individual use of butyrate and valerate by culture A was also studied. Butyrate produced a homopolymer whereas valerate was stored as a terpolymer of P(HB/HV/HMV). The polymer yields on acetate and butyrate were higher than those on propionate and valerate. The polymer productivity was higher for acetate and propionate than for butyrate and valerate. Results showed that the polymer composition, and consequently the polymer properties, could be manipulated by varying the volatile fatty acid feed composition and/or the population.     

Production and characterization of homopolymer polyhydroxyheptanoate (P3HHp) by a fadBA knockout mutant Pseudomonas putida KTOY06 derived from P. putida KT2442

Pseudomonas putida KT2442 commonly produces medium-chain-length polyhydroxyalkanoates (PHA) consisting of 3-hydroxyhexanoate (C6) to 3-hydroxydodecanoate (C12) when grown in glucose or even number fatty acid. When two of the beta-oxidation genes fadBA were deleted, the P. pudida KT2442 mutant named KTOY06 accumulated a homopolymer of poly-3-hydroxyheptanoate (P3HHp) up to 71 wt% of its cell dry weight in the presence of heptanoate as a single carbon source. P3HHp contents in the cell dry weight were in direct proportional to Na-heptanoate concentration up to 10 g/L. In contrast, under the same cultivation conditions, the wild type P. putida KT2442 produced a copolymer consisting of 3-hydroxyheptanoate (3HHp) and 5.3–8.4 mol% 3-hydroxynonanoate (3HN). Gas chromatography (GC), nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), thermogravimetric analysis (TGA) and gel permeation chromatography (GPC) were used to characterize the homopolymer P3HHp, respectively. It was found that the P3HHp with an average molecular weight of 455 kDa was a completely amorphous homopolymer without crystallinity. P3HHp is thermo-degradable at around 250 °C.     

Biosynthesis of poly(3-hydroxydecanoate) and 3-hydroxydodecanoate dominating polyhydroxyalkanoates by β-oxidation pathway inhibited Pseudomonas putida

Pseudomonas putida KT2442 produces medium-chain-length polyhydroxyalkanoates consisting of 3-hydroxyhexanoate (3HHx), 3-hydroxyoctanoate (3HO), 3-hydroxydecanoate (3HD), 3-hydroxydodecanoate (3HDD) and 3-hydroxytetradecanoate (3HTD) from relevant fatty acids. P. puitda KT2442 was found to contain key fatty acid degradation enzymes encoded by genes PP2136, PP2137 (fadB and fadA) and PP2214, PP2215 (fadB2x and fadAx), respectively. In this study, the above enzymes and other important fatty acid degradation enzymes, including 3-hydroxyacyl-CoA dehydrogenase and acyl-CoA dehydrogenase encoded by genes PP2047 and PP2048, respectively, were studied for their effects on PHA structures. Mutant P. puitda KTQQ20 was constructed by knocking out the above six genes and also 3-hydroxyacyl-CoA-acyl carrier protein transferase encoded by PhaG, leading to a significant reduction of fatty acid β-oxidation activity. Therefore, P. puitda KTQQ20 synthesized homopolymer poly-3-hydroxydecanoate (PHD) or P(3HD-co-84mol% 3HDD), when grown on decanoic acid or dodecanoic acid. Melting temperatures of PHD and P(3HD-co-84mol% 3HDD) were 72 and 78 °C, respectively. Thermal and mechanical properties of PHD and P(3HD-co-84mol% 3HDD) were much better as compared with an mcl-PHA, consisting of lower content of C10 or C12 monomers. For the first time, it was shown that homopolymer PHD and 3HDD monomers dominating PHA could be synthesized by β-oxidation inhibiting P. putida grown on relevant carbon sources.     

Intracellular degradation of poly(3-hydroxybutyric acid) accumulated by Azotobacter chroococcum MAL-201

Azotobacter chroococcum MAL-201 accumulates poly(3-hydroxybutyric acid) [P(3HB)] accounting 69% of cell dry weight (CDW) from glucose during growth in nitrogen-free Stockdale medium. Degradation of the accumulated polymer by the organism was studied under carbon-free medium following two-step cultivation method. P(3HB) content of cells decreased rapidly from 69% to 4.8% of CDW after 35 h under carbon-deprived condition. Autodigestion of P(3HB) was evident from the estimation of intracellular P(3HB) depolymerase (i-depolymerase) activity in cell-free extract using artificial P(3HB) granules as substrate. Polymer content decreased rapidly along with the increase in i-depolymerase activity and rate of polymer degradation when medium was supplemented with (NH4)2SO4 at 0.1% (w/v) level. However, the effects were reverse when organic nitrogenous substrate, beef extract at similar concentration was present in the medium. The optimum temperature and pH for i-depolymerase activity were 35 degrees C and 7.7 respectively. The oxygen-limiting condition (culture volume per flask volume, 50%) decreased 10.7% activity of i-depolymerase over control resulting a slow P(3HB) degradation. The presence of NaCl (6 x 10(3) microg/ml) showed a positive effect on i-depolymerase whereas EDTA (40 microg/ml) resulted in 20% less activity. Furthermore, the intracellular degradation of P(3HB) decreased the intrinsic viscosity, molecular weight and tensile strength of the accumulated polymer.     

Biosynthesis of Poly(3-Hydroxyalkanoic Acid) Copolymer from CO(inf2) in Pseudomonas acidophila through Introduction of the DNA Fragment Responsible for Chemolithoautotrophic Growth of Alcaligenes hydrogenophilus

Pseudomonas acidophila is a bacterial strain producing a poly(3-hydroxyalkanoic acid) (PHA) copolymer from low-molecular-weight organic compounds such as formate and acetate. The genes responsible for PHA production were cloned in cosmid pIK7 containing a 14.8-kb HindIII fragment of P. acidophila DNA. With the aim of developing a means of producing a PHA copolymer from CO(inf2), cosmid pIK7 was introduced into a polymer-negative mutant of the chemolithoautotrophic bacterium Alcaligenes eutrophus PHB(sup-)4. However, the recombinant strain produced a homopolymer of 3-hydroxybutyric acid (polyhydroxybutyric acid) from CO(inf2). Since it was thought that the composition of the accumulated polymer might depend not on the PHA biosynthetic genes but on the metabolism of the host strain, a recombinant plasmid, pFUS, containing the genes for chemolithoautotrophic growth of the hydrogen-oxidizing bacterium A. hydrogenophilus was introduced into P. acidophila by conjugation. The recombinant plasmid pFUS was stably maintained in P. acidophila in the absence of chemolithoautotrophic or antibiotic selection. This pFUS-harboring strain possessed the ability to grow under a gas mixture of H(inf2), O(inf2), and CO(inf2) in a mineral salts medium, and PHA copolymer accumulation was confirmed by nuclear magnetic resonance spectral analysis. A gas chromatogram obtained by gas chromatography-mass spectrometry showed the composition of the polymer to be 52.8% 3-hydroxybutyrate, 41.1% 3-hydroxyoctanoate, and 6.1% 3-hydroxydecanoate. This is the first report of the production of a PHA copolymer from CO(inf2) as sole carbon source.     

Role of heme oxygenase-2 in pial arteriolar response to acetylcholine in mice with and without transfusion of cell-free hemoglobin polymers

Carbon monoxide derived from heme oxygenase (HO) may participate in cerebrovascular regulation under specific circumstances. Previous work has shown that HO contributes to feline pial arteriolar dilation to acetylcholine after transfusion of a cell-free polymeric hemoglobin oxygen carrier. The role of constitutive HO2 in the pial arteriolar dilatory response to acetylcholine was determined by using 1) HO2-null mice (HO2-/-), 2) the HO inhibitor tin protoporphyrin IX (SnPPIX), and 3) 4,5,6,7-tetrabromobenzotriazole (TBB), an inhibitor of casein kinase-2 (CK2)-dependent phosphorylation of HO2. In anesthetized mice, superfusion of a cranial window with SnPPIX decreased arteriolar dilation produced by 10 microM acetylcholine by 51%. After partial polymeric hemoglobin exchange transfusion, the acetylcholine response was normal but was reduced 72% by SnPPIX and 95% by TBB. In HO2-/- mice, the acetylcholine response was modestly reduced by 14% compared with control mice and was unaffected by SnPPIX. After hemoglobin transfusion in HO2-/- mice, acetylcholine responses were also unaffected by SnPPIX and TBB. In contrast, nitric oxide synthase inhibition completely blocked the acetylcholine responses in hemoglobin-transfused HO2-/- mice. We conclude 1) that HO2 activity partially contributes to acetylcholine-induced pial arteriolar dilation in mice, 2) that this contribution is augmented in the presence of a plasma-based hemoglobin polymer and appears to depend on a CK2 kinase mechanism, 3) that nitric oxide synthase activity rather than HO1 activity contributes to the acetylcholine reactivity in HO2-/- mice, and 4) that plasma-based polymeric hemoglobin does not scavenge all of the nitric oxide generated by cerebrovascular acetylcholine stimulation.     

Biosynthesis and characterization of polyhydroxyalkanoate block copolymer P3HB-b-P4HB

Polyhydroxyalkanoates (PHA) synthesis genes phbC and orfZ cloned from Ralstonia eutropha H16 were transformed into beta-oxidation weakened Pseudomonas putida KTOY08ΔGC, a mutant of P. putida KT2442. The recombinant P. putida strain termed KTHH06 was able to produce a short-chain-length PHA block copolymer consisting of poly(3-hydroxybutyrate) (P3HB) as one block and poly(4-hydroxybutyrate) (P4HB) as another block. One-dimensional and two-dimensional nuclear magnetic resonance (NMR) clearly indicated the polymer was a diblock copolymer consisting of 20 mol % P3HB as one block and 80 mol % P4HB as another one. Differential scanning calorimetric (DSC) showed that P3HB block melting temperatures (T(m)) in the block copolymer P3HB-b-P4HB was shift to low temperature compared with homopolymer P3HB and a blend of P3HB and P4HB. The block copolymer with a number average molecular weight of 50000 Da and a polydispersity of 3.1 demonstrated a better yield and tensile strength compared with that of its related random copolymer and blend of homopolymers of P3HB and P4HB.     

Degradation of poly(3-hydroxyoctanoic acid) [P(3HO)] by bacteria: purification and properties of a P(3HO) depolymerase from Pseudomonas fluorescens GK13.

Twenty-five gram-negative bacteria and one gram-positive bacterium capable of growing on poly(3-hydroxyoctanoic acid) [P(3HO)] as the sole source of carbon and energy were isolated from various soils, lake water, and activated sludge. Most of the isolates degraded only P(3HO) and copolymers of medium-chain-length (MCL) hydroxyalkanoic acids (HA). Except for the gram-positive strain, which was able to hydrolyze P(3HO) and poly(3-hydroxybutyric acid) [P(3HB)], no isolate was able to degrade polymers of short-chain-length HA, such as P(3HB) or poly(3-hydroxyvalerate) [P(3HV)]. All strains utilized a large variety of monomeric substrates for growth. All gram-negative strains, but not the gram-positive strain, accumulated poly(hydroxyalkanoic acids) (PHA), consisting of MCL HA, if they were cultivated under accumulation conditions. One strain, which was identified as Pseudomonas fluorescens GK13 (biovar V), was selected and the extracellular P(3HO) depolymerase of this strain was purified from the culture medium of P(3HO)-grown cells by chromatography with Octyl-Sepharose CL4B and by gel filtration with Superose 12. The relative molecular weights of the native and sodium dodecyl sulfate-treated enzymes were 48,000 and 25,000, respectively. The purified enzyme hydrolyzed P(3HO), copolymers of MCL HA, and para-nitrophenyl esters of fatty acids. P(3HB), P(3HV), and characteristic substrates for lipases, such as Tween 80 or triolein, were not hydrolyzed. The P(3HO) depolymerase of P. fluorescens GK13 was insensitive to phenylmethylsulfonyl fluoride and dithioerythritol, unlike other PHA depolymerases. The dimeric ester of 3-hydroxyoctanoic acid was identified as the main product of enzymatic hydrolysis of P(3HO).(ABSTRACT TRUNCATED AT 250 WORDS)     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid.

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

Intracellular degradation of two structurally different polyhydroxyalkanoic acids accumulated in Pseudomonas putida and Pseudomonas citronellolis from mixtures of octanoic acid and 5-phenylvaleric acid

From a set of mixed carbon sources, 5-phenylvaleric acid (PV) and octanoic acid (OA), polyhydroxyalkanoic acid (PHA) was separately accumulated in the two pseudomonads Pseudomonas putida BM01 and Pseudomonas citronellolis (ATCC 13674) to investigate any structural difference between the two PHA accumulated under a similar culture condition using one-step culture technique. The resulting polymers were isolated by chloroform solvent extraction and characterized by fractional precipitation and differential scanning calorimetry. The solvent fractionation analysis showed that the PHA synthesized by P. putida was separated into two fractions, 3-hydroxy-5-phenylvalerate (3HPV))-rich PHA fraction in the precipitate phase and 3-hydroxyoctanoate (3HO)-rich PHA fraction in the solution phase whereas the PHA produced by P. citronellolis exhibited a rather little compositional separation into the two phases. According to the thermal analysis, the P. putida PHA exhibited two glass transitions indicative of the PHA not being homogeneous whereas the P. citronellolis PHA exhibited only one glass transition. It was found that the structural heterogeneity of the P. putida PHA was caused by a significant difference in the assimilation rate between PV and OA. The structural heterogeneity present in the P. putida PHA was also confirmed by a first order degradation kinetics analysis of the PHA in the cells. The two different first-order degradation rate constants (k₁), 0.087 and 0.015/h for 3HO- and 3HPV-unit, respectively, were observed in a polymer system over the first 20 h of degradation. In the later degradation period, the disappearance rate of 3HO-unit was calculated to be 0.020 h. The k₁ value of 0.083/h, almost the same as for the 3HO-unit in the P. putida PHA, was obtained for the P(3HO) accumulated in P. putida BM01 grown on OA as the only carbon source. In addition, the k₁ value of 0.015/h for the 3HPV-unit in the P. putida PHA, was also close to 0.019/h for the P(3HPV) homopolymer accumulated in P. putida BM01 grown on PV plus butyric acid. On the contrary, the k₁ values for the P. citronellolis PHA were determined to be 0.035 and 0.029/h for 3HO- and 3HPV-unit, respectively, thus these two relatively close values implying a random copolymer nature of the P. citronellolis PHA. In addition, the faster degradation of P(3HO) than P(3HPV) by the intracellular P. putida PHA depolymerase indicates that the enzyme is more specific against the aliphatic PHA than the aromatic PHA.     

ENHANCED BIOSYNTHESIS OF POLY(3-HYDROXYBUTYRATE) FROM POTATO STARCH BY Bacillus cereus STRAIN 64-INS IN A LABORATORY-SCALE FERMENTER

To decrease the polyhydroxyalkanoate (PHA) production cost by supplying renewable carbon sources has been an important aspect in terms of commercializing this biodegradable polymer. The production of biodegradable poly(3-hydroxyalkanoates) (PHA) from raw potato starch by the Bacillus cereus 64-INS strain isolated from domestic sludge has been studied in a lab-scale fermenter. The bacterium was screened for the degradation of raw potato starch by a starch hydrolysis method and for PHA production by Nile blue A and Sudan black B staining. Shake-flask cultures of the bacterium with glucose [2% (w/v)] or raw potato starch [2% (w/v)] produced PHA of 64.35% and 34.68% of dry cell weight (DCW), respectively. PHA production was also carried out in a 5-L fermenter under control conditions that produced 2.78 g/L of PHA and PHA content of 60.53% after 21 hr of fermentation using potato starch as the sole carbon source. Gas chromatography–mass spectroscopy (GC-MS) analyses confirmed that the extracted PHA contained poly(3-hydroxybutyrate) (PHB) as its major constituent (>99.99%) irrespective of the carbon source used. The article describes, for what we believe to be the first time, PHB production being carried out without any enzymatic or chemical treatment of potato starch at higher levels by fermentation. More work is required to optimize the PHB yield with respect to starch feeding strategies.     

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%.     

Production of polyhydroxyalkanoates with high 3-hydroxydodecanoate monomer content by fadB and fadA knockout mutant of Pseudomonas putida KT2442

Pseudomonas putida KT2442 produces medium-chain-length (MCL) polyhydroxyalkanoates (PHA) consisting of 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO), 3-hydroxydecanoate (HD), and 3-hydroxydodecanoate (HDD) from a wide-range of carbon sources. In this study, fadA and fadB genes encoding 3-ketoacyl-CoA thiolase and 3-hydroxyacyl-CoA dehydrogenase in P. putida KT2442 were knocked out to weaken the beta-oxidation pathway. Two-step culture was proven as the optimal method for PHA production in the mutant termed P. putida KTOY06. In a shake-flask culture, when dodecanoate was used as a carbon source, P. putida KTOY06 accumulated 84 wt % PHA, much higher than 50 wt % PHA in its wild type KT2442. The PHA monomer composition was completely different: the HDD fraction in PHA produced by KTOY06 was 41 mol %, much higher compared with 7.5 mol % only in KT2442. The fermentor-scale culture indicated the HDD fraction in PHA decreased during the culture time from 35 to 25 mol % in a one-step fermentation process or from 75 to 49 mol % in a two-step fermentation process. It is for the first time that PHA with a dominant HDD fraction was produced. Thermal and mechanical properties assays indicated that this new type PHA with a high HDD fraction had higher crystallinity and tensile strength than PHA with a low HDD fraction did, demonstrating an improved application property.     

Production of polyhydroxyalkanoates (PHAs) with canola oil as carbon source

Wautersia eutropha was able to synthesize medium chain length polyhydroxyalkanoates (PHAs) when canola oil was used as carbon source. W. eutropha was cultivated using fructose and ammonium sulphate as carbon and nitrogen sources, respectively, for growth and inoculum development. The experiments were done in a laboratory scale bioreactor in three stages. Initially, the biomass was adapted in a batch culture. Secondly, a fed-batch was used to increase the cell dry weight and PHA concentration to 4.36 g L(-1) and 0.36 g L(-1), respectively. Finally, after the addition of canola oil as carbon source a final concentration of 18.27 g L(-1) PHA was obtained after 40 h of fermentation. With canola oil as carbon source, the polymer content of the cell dry matter was 90%. The polymer was purified from dried cells and analyzed by FTIR, NMR and DSC using PHB as reference. The polymer produced by W. eutropha from canola oil had four carbon monomers in the structure of the PHA and identified by 1H and 13C NMR analysis as 3-hydroxybutyrate (3HB), 3-hydroxyvalerate (3HV), 3-hydroxyoctanoate (3HO), and 3-hydroxydodecanoate (3HDD).     

Microbial production of medium-chain-length 3-hydroxyalkanoic acids by recombinant Pseudomonas putida KT2442 harboring genes fadL, fadD and phaZ

Monomers of microbial polyhydroxyalkanoates, mainly 3-hydroxyhexanoic acid (3HHx) and 3-hydroxyoctanoic acid (3HO), were produced by overexpressing polyhydroxyalkanoates depolymerase gene phaZ, together with putative long-chain fatty acid transport protein fadL of Pseudomonas putida KT2442 and acyl-CoA synthetase (fadD) of Escherichia coli MG1655 in P. putida KT2442. FadL(Pp), which is responsible for free fatty acid transportation from the extracellular environment to the cytoplasm, and FadD(Ec), which activates fatty acid to acyl-CoA, jointly reinforce the fatty acid beta-oxidation pathway. Pseudomonas putida KT2442 (pYZPst01) harboring polyhydroxyalkanoates depolymerase gene phaZ of Pseudomonas stutzeri 1317 produced 1.37 g L(-1) extracellular 3HHx and 3HO in shake flask studies after 48 h in the presence of sodium octanoate as a sole carbon source, while P. putida KT2442 (pYZPst06) harboring phaZ(Pst), fadD(Ec) and fadL(Pp) achieved 2.32 g L(-1) extracellular 3HHx and 3HO monomer production under the same conditions. In a 48-h fed-batch fermentation process conducted in a 6-L fermentor with 3 L sodium octanoate mineral medium, 5.8 g L(-1) extracellular 3HHx and 3HO were obtained in the fermentation broth. This is the first time that medium-chain-length 3-hydroxyalkanoic acids (mcl-3HA) were produced using fadL(Pp) and fadD(Ec) genes combined with the polyhydroxyalkanoates depolymerase gene phaZ.     

Formation of short chain length/medium chain length polyhydroxyalkanoate copolymers by fatty acid beta-oxidation inhibited Ralstonia eutropha

Ralstonia eutropha has been considered as a bacterium, incorporating hydroxyalkanoates of less than six carbons only into polyhydroxyalkanoates (PHAs). Cells of the wild type cultivated with sodium octanoate as the carbon source in the presence of the fatty acid beta-oxidation inhibitor sodium acrylate synthesized PHAs composed of the medium chain length hydroxyalkanoates (3HA(MCL)) 3-hydroxyhexanoate (3HHx) and 3-hydroxyoctanoate (3HO) as well as of 3-hydroxybutyrate and 3-hydroxyproprionate as revealed by gas chromatography, (1)H NMR spectroscopy, and mass spectroscopy. The characterization of the polymer as a tetrapolymer was confirmed by differential solvent extraction and measurement of melting and glass transition temperature depression in the purified polymer compared to PHB. These data suggested that the R. eutropha PHA synthase is capable of incorporating longer chain substrates than suggested by previous in vitro studies. Furthermore, expression of the class II PHA synthase gene phaC1 from P. aeruginosa in R. eutropha resulted in the accumulation of PHAs consisting of 3HA(MCL) contributing about 3-5% to cellular dry weight. These PHAs were composed of nearly equal molar fractions of 3HO and 3-hydroxydecanoate (3HD) with traces of 3HHx. These data indicated that 3HA(MCL)-CoA thioesters were diverted from the fatty acid beta-oxidation pathway towards PHA biosynthesis in recombinant R. eutropha.