The synthesis of short- and medium-chain-length poly(hydroxyalkanoate) mixtures from glucose- or alkanoic acid-grown <Emphasis Type="Italic">Pseudomonas oleovorans</Emphasis>

Pseudomonas oleovorans NRRL B-778 accumulated mixtures of poly-3-hydroxybutyrate (PHB) and medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs) when grown on glucose, octanoic acid or oleic acid, whereas growth on nonanoic acid or undecanoic acid resulted in copolymers of poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHB-co-HV). Acetone fractionation verified the presence of PHB/mcl-PHA mixtures. The acetone-insoluble (AIS) fractions of the polymers derived from glucose (PHA-glucose), octanoic acid (PHA-octanoic) and oleic acid (PHA-oleic) were exclusively PHB while the acetone-soluble (AS) fractions contained mcl-PHA composed of differing ratios of 3-hydroxy-acid monomer units, which ranged in chain length from 6 to 14 carbon atoms. In contrast, both the AIS and AS fractions from the polymers derived from nonanoic acid (PHA-nonanoic) and undecanoic acid (PHA-undecanoic) were composed of comparable ratios of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV). The unfractionated PHA-glucose, PHA-octanoic and PHA-oleic polymers had melting temperatures (T _m) between 177 and 179°C, enthalpies of fusion (ΔH _f) of 20 cal/g and glass transition temperatures (T _g) of 3–4°C. This was due to the large PHB content in the polymer mixtures. On the other hand, the PHA-nonanoic and PHA-undecanoic polymers had thermal properties that supported their copolymer nature. In both cases, the T _m values were 161°C, ΔH _f values were 7cal/g and T _g values were −3°C. Journal of Industrial Microbiology & Biotechnology (2002) 28, 147–153 DOI: 10.1038/sj/jim/7000231 Received 30 July 2001/ Accepted in revised form 04 November 2001     

Hyperproduction of polyesters consisting of medium-chain-length hydroxyalkanoate monomers by strain Pseudomonas stutzeri 1317

Pseudomonas stutzeri strain 1317 was found to grow on various fatty acids, alcohols, diols, as well as glucose and gluconate for the synthesis of polyhydroxyalkanoates (PHA) with various monomer units. The PHA monomer structures were dependent on the type of fatty acids and alcohols, as well as the diols in the culture media. Only even number monomers, such as 3-hydroxyhexanoate (HHx), 3-hydroxyoctanoate (HO) and 3-hydroxydecanoate (HD), were accumulated when even numbered fatty acids, alcohols, glucose and gluconate, as well as diol were used as carbon sources. Odd numbered fatty acids and odd numbered alcohols led to the formation of odd numbered monomers, such as 3-hydroxyvalerate (HV), 3-hydroxyheptanoate (HHp), 3-hydroxynonanoate (HN) and 3-hydroxyundecanoate (HU). The strain tolerated up to 1.5% of ethanol and made 8.3% of PHA when growth was conducted in 1.2% of ethanol. PHA formed up to 77% of cell dry weight when the strain was grown in tridecanoate. PHA synthesis was highly dependent on the nitrogen source. A depletion in nitrogen supply immediately resulted in PHA accumulation in cells grown in the glucose mineral medium.     

In situ copolyesters containing poly(L-lactide) and poly(hydroxyalkanoate) units

In situ copolyesters containing polylactide (PLA) and polyhydroxyalkanoate (PHA) segments were obtained via ring-opening polymerization of L-lactide using PHA as a macroinitiator with stannous octoate as catalyst. Incorporation of PHA (20 wt %) into PLA affords a novel copolymer with Mn values ranging from 25 to 50 KDa and low polydispersities of 1.8-2.3. DSC analysis of the copolymer indicates well-defined crystallization and melting transitions different from the homopolymers and corresponding blend. The polymers were characterized by FT-IR, GPC, DSC, optical microscopy, NMR, and TGA. The results show successful reactivity of PHA as a macroinitiator for the ring-opening polymerization of lactide.     

Methyl-branched poly(hydroxyalkanoate) biosynthesis from 13-methyltetradecanoic acid and mixed isostearic acid isomer substrates

Nonylphenol, the most abundant environmental pollutant with endocrine disrupting activity, is also toxic to plants and microorganisms, but its actual impact in the field is unknown. In this study, diversity of culturable soil microfungal and plant communities was assessed in a disused industrial estate, at three sites featuring different nonylphenol pollution. Although soil microfungal assemblages varied widely among the sites, no significant correlation was found with point pollutant concentrations, thus suggesting indirect effects of soil contamination on microfungal assemblages. The potential of indigenous fungi and plants to remove nonylphenol was assessed in mesocosm experiments. Poplar plants and a fungal consortium consisting of the most abundant strains in the nonylphenol-polluted soil samples were tested alone or in combination for their ability to reduce, under greenhouse conditions, nonylphenol levels either in a sterile, artificially contaminated sand substrate, or in two non-sterile soils from the original industrial area. Introduction of indigenous fungi consistently reduced nonylphenol levels in all substrates, up to ca. 70% depletion, whereas introduction of the plant proved to be effective only with high initial pollutant levels. In native non-sterile soil, nonylphenol depletion following fungal inoculation correlated with biostimulation of indigenous fungi, suggesting positive interactions between introduced and resident fungi. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Mass production of medium-chain-length poly(3-hydroxyalkanoates) from hydrolyzed corn oil by fed-batch culture of Pseudomonas putida

A novel biopolyester, Medium-Chain-Length Polyhydroxyalkanoates (MCL-PHAs), was made from the corn oil hydrolysate, an inexpensive and renewable carbon source, by a fed-batch culture of Pseudomonas putida with a phosphate limitation. The cell growth, MCL-PHAs accumulation and feeding strategies of corn oil hydrolysate in the cultures of P. putida were investigated in 5 l and 30 l fermentors respectively. In the optimal fermentation, the final cell and MCL-PHAs concentrations reached 103 and 28 g/l, which represents a MCL-PHAs productivity of 0.61 g/l h. It was confirmed by its NMR spectrum that this MCL-PHAs from corn oil hydrolysate contained 4 saturated and 3 unsaturated monomers with a chain length of 6–14 carbon atoms.     

Improved film properties of radiation-treated medium-chain-length poly(hydroxyalkanoates)

Medium-chain-length poly(hydroxyalkanoates) (mcl-PHAs) were synthesized from coconut oil (PHA-C), tallow (PHA-T), and soybean oil (PHA-S) by bacterial fermentation using Pseudomonas resinovorans as the producer strain. Films were solution-cast and subjected to 50 kGy of -irradiation. This resulted in crosslink formation based on the number of olefinic groups present in the polymer side-chains. In each case, radiation improved the tensile strength (104% and 63%), percent elongation (49% and 13%), and Young's modulus (30% and 76%) of PHA-C and PHA-T films, respectively. The greatest effect was on PHA-S, which was converted from an amorphous, liquid-like material to a solid elastomeric film. © Rapid Science Ltd. 1998     

Synthesis of multiarm star poly(glycerol)-block-poly(2-hydroxyethyl methacrylate)

Well-defined multiarm star block copolymers poly(glycerol)-b-poly(2-hydroxyethyl methacrylate) (PG-b-PHEMA) with an average of 56, 66, and 90 PHEMA arms, respectively, have been prepared by atom transfer radical polymerization (ATRP) of HEMA in methanol by a core-first strategy. The hyperbranched macroinitiators employed were prepared on the basis of well-defined hyperbranched polyglycerol by esterification with 2-bromoisobutyryl bromide. Polydispersites M(w)/M(n) of the new multiarm stars were in the range of 1.11-1.82. Unexpectedly, with the combination of CuCl/CuBr(2)/2,2'-bipyridyl as catalyst, the polymerization conversion can be driven to maximum values of 79%. The control of CuCl catalyst concentration is also very important to achieve high conversion and narrow polydispersity. The absolute M(n) values of the obtained multiarm star polymers were in good agreement with the calculated ones, and the highest M(n) values of the multiarm star copolymer is around 10(6) g/mol. Kinetic analysis shows that an induction period exists in the polymerization of HEMA. After this induction period, a linear dependence of ln ([M](0)/[M](t)()) on time was observed. Due to the star architecture, the viscosity of the obtained multiarm star PHEMA is much lower than that of linear PHEMA.     

Miscibility of bioerodible polyphosphazene/poly(lactide-co-glycolide) blends

We have previously demonstrated the feasibility of blending bioerodible polyphosphazenes with poly(lactide-co-glycolide) (PLGA) to form versatile polymeric materials with altered bioerosion properties. These studies demonstrated the effective neutralization of the acidic degradation products of PLGA by the polyphosphazene hydrolysis products. In the present study, five new polymers of dipeptide polyphosphazenes poly[(ethyl glycinato)x(glycyl-ethyl glycinato)yphosphazene] and novel blends of these polyphosphazenes with poly(lactide-co-glycolide) (PLGA) were synthesized and fabricated. The miscibility was analyzed using differential scanning calorimetry and scanning electron microscopy. Hydrogen bonding within the blends was assessed by attenuated total reflectance infrared spectroscopy. The phosphazene component of the blend contained varying ratios of the glycyl-glycine ethyl ester to the glycine ethyl ester. Poly[(ethyl glycinato)0.5(glycine ethyl glycinato)1.5phosphazene formed completely miscible blends with PLGA (50:50) and PLGA (85:15). This is ascribed to the multiple hydrogen-bonding sites within the side groups of the polyphosphazene. The components of the blend act as plasticizers for each other because a glass transition temperature for each blend was detected at a lower temperature than for each individual polymer. A hydrolysis study showed that unblended solid poly[(ethyl glycinato)0.5(glycyl ethyl glycinato)1.5phosphazene] hydrolyzed in less than 1 week. However, the blends degraded at a slower rate than both parent polymers. This is attributed to the buffering capacity of the polyphosphazene hydrolysis products, which increases the pH of the degradation media from 2.5 to 4, thereby slowing the degradation rate of PLGA.     

Rapid genetic characterization of poly(hydroxyalkanoate) synthase and its applications

Microorganisms containing short-chain-length (scl-) or medium-chain-length (mcl-) poly(hydroxyalkanoates) (PHAs) are commonly screened by applying rapid staining methods using lipophilic reagents. These methods provide powerful means for general screening of organisms actively producing and accumulating PHAs. The Southern blot hybridization method additionally allows the identification of potential PHA-producing microorganisms. Polymerase chain reaction (PCR)-based detection methods further afford rapid and sensitive means to screen for PHA biosynthesis genes. Specific PCR assays had been developed for the simultaneous or individual detection of the class II mcl-PHA synthase genes of Pseudomonas. The amplicons (approximately 0.54 kb) can be directly sequenced or used as probes for hybridization studies. The sequence information can further be used to initiate chromosome walking for an eventual cloning of the complete PHA biosynthesis operon. In addition, the amplification pattern and sequence data can be used to differentiate subgroups of organisms, as demonstrated for P. corrugata and P. mediterranea. Other researchers reported PCR methods for the detection of scl-PHA synthase genes and those of Bacillus spp., thus greatly expanding the types of PHA synthase gene and the organisms that can be characterized by this approach. The vast sequence information obtainable through PCR-based studies of various PHA synthase operons should facilitate the identification or construction of new PHA synthases capable of synthesizing novel PHAs.     

Biosynthesis of medium-chain-length poly(hydroxyalkanoates) with altered composition by mutant hybrid PHA synthases

Pseudomonas resinovorans harbors two isogenic poly(hydroxyalkanoates) (PHAs) synthase genes (phaC1 _Pre , phaC2 _Pre ) responsible for the production of intracellular medium-chain-length (mcl-)PHAs. Sequence analysis showed that the putative gene-products of these genes contain a conserved α/β-hydrolase fold in the carboxy-terminal half of the proteins. Hybrid genes pha7 and pha8 were constructed by exchanging the α/β-hydrolase-fold coding portions of phaC1 _Pre and phaC2 _Pre at the 3′ terminal. When grown with decanoate as carbon source, the pha7- or pha8-transformed Escherichia coli LS1298 produced PHAs containing 73–75% β-hydroxydecanoate (β-HD) and 25–27% β-hydroxyoctanoate (β-HO). Deletion mutants, Δpha7 and Δpha8, were isolated during the PCR-based construction of pha7 and pha8, respectively. Cells harboring these mutants produced PHAs containing 55–60 mol% β-HD and 40–45 mol% β-HO. These results demonstrate the feasibility of generating active hybrid mcl-PHA synthase genes and their mutants with the potential of producing polymers having a varied repeat-unit composition.     

Lamellar thickening and cocrystallization of poly(hydroxyalkanoate)s on annealing.

Lamellar thickening behavior of microbial polyesters, poly(3-hydroxybutyrate) [P(3HB)], poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] and poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] annealed at various temperatures was investigated to make sure of the occurrence of cocrystallization of both components. All the copolymers showed steep increases in melting points accompanied by partial melting as the annealing temperature increased up to just below the melting points. In contrast, long periods of P(3HB-co-7mol% 3HV) increased to twice, similar to those of P(3HB), with increasing annealing temperature up to just below the melting point, while long periods of P(3HB-co-7mol% 4HB) and P(3HB-co-92mol% 3HV) only increased up to one and a half times. Lattice indices of unit cell of the former crystal were increased slightly, while those of the latter crystal remained unchanged. These results imply that the P(3HB) crystal can occlude the 3HV component to some extent, but hardly includes the 4HB component, and P(3HV) crystal also excludes the 3HB component.     

Study of biodegradable polylactide/poly(butylene adipate-co-terephthalate) blends

Both polylactide (PLA) and poly(butylene adipate-co-terephthalate) (PBAT) are biodegradable polymers. They are thermoplastics which can be processed using most conventional polymer processing methods. PLA is high in strength and modulus (63 MPa and 3.4 GPa, respectively) but brittle (strain at break 3.8%) while PBAT is flexible and tough (strain at break approximately 710%). In view of their complementary properties, blending PLA with PBAT becomes a natural choice to improve PLA properties without compromising its biodegradability. In this study, PLA and PBAT were melt blended using a twin screw extruder. Melt elasticity and viscosity of the blends increased with the concentration of PBAT. Crystallization of the PLA component, phase morphology of the blend, mechanical properties, and toughening mechanism were investigated. The blend comprised an immiscible, two-phase system with the PBAT evenly dispersed in the form of approximately 300 nm domains within the PLA matrix. The PBAT component accelerated the crystallization rate of PLA but had little effect on its final degree of crystallinity. With the increase in PBAT content (5-20 wt %), the blend showed decreased tensile strength and modulus; however, elongation and toughness were dramatically increased. With the addition of PBAT, the failure mode changed from brittle fracture of the neat PLA to ductile fracture of the blend as demonstrated by tensile test and scanning electron microcopy (SEM) micrographs. Debonding between the PLA and PBAT domains induced large plastic deformation in PLA matrix ligaments.     

Sulfate reduction and anaerobic glycerol degradation by a mixed microbial culture

Anaerobic glycerol degradation by a mixed microbial culture from a fermenter fed with industrial alcohol distillation waste water, was investigated in the absence or presence of sulfate, at 37°C and at a constant pH of 7.2. In the absence of sulfate, glycerol utilization was found to be characterized by the transient formation of 1,3-propanediol prior to propionate and acetate accumulation. In the presence of sulfate, 1,3-propanediol production was minor, and the carbon balance reflected a considerable accumulation of intermediate(s). A study of the role of sulfate reduction and methanogenesis on anaerobic 1,3-propanediol degradation showed that consumption of this substrate by the mixed microbial culture required a terminal electron acceptor. The number of fermentative and sulfate-reducing bacteria with glycerol or 1,3-propanediol as carbon and energy source revealed that sulfate-reducing bacteria outcompete fermentative bacteria for these substrates. The possible ecological role of sulfate-reducing bacteria in the metabolism of these reduced substrates is discussed.     

Extracellular poly(hydroxyalkanoate) depolymerases and their inhibitor from Pseudomonas lemoignei.

Enzymatic degradation processes of microbial copolyesters, poly(3-hydroxybutyrate-co-3-hydroxyvalerate): P(3HB-co-3HV) and poly(3-hydroxybutyrate-co-4-hydroxybutyrate): P(3HB-co-4HB), were studied by the weight loss (erosion) of copolyester films. These studies employed three extracellular depolymerases which degrade poly(3-hydroxybutyrate): P(3HB). Two enzymes were purified from the culture supernatant of Pseudomonas lemoignei and one from Alcaligenes faecalis T1. The rate of enzymatic degradation of microbial copolyester films with various compositions showed an almost similar tendency to three different P(3HB) depolymerases, and decreased in the following order: P(3HB-co-4HB) greater than P(3HB) greater than P(3HB-co-3HV). An inhibitory protein of P(3HB) depolymerases in the succinate culture medium of P. lemoignei was isolated and characterized. The molecular weight of P(3HB) depolymerase inhibitor was 35,000 as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This inhibitor of a single polypeptide chain may reversibly bind the serine residues at the active site of P(3HB) depolymerase. This inhibitory protein was not induced in the culture medium when P. lemoignei was grown on P(3HB) as the sole carbon source.     

Compatible ternary blends of chitosan/poly(vinyl alcohol)/poly(lactic acid) produced by oil-in-water emulsion processing

Ternary compatible blends of chitosan, poly(vinyl alcohol), and poly(lactic acid) were prepared by an oil-in-water (O/W) emulsion process. Solutions of chitosan in aqueous acetic acid, poly(vinyl alcohol) (PVA) in water, and poly(lactic acid) (PLA) in chloroform were blended with a high-shear mixer. PVA was used as an emulsifier to stabilize the emulsion and to reduce the interfacial tension between the solid polymers in the blends produced. It proved to work very well because the emulsions were stable for periods of days or weeks and compatible blends were obtained when PVA was added. This effect was attributed to a synergistic effect of PVA and chitosan because the binary blends PVA/PLA and chitosan/PLA were completely incompatible. The blends were characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), thermal mechanical analysis (TMA), stress-strain tests, and Fourier transform infrared spectroscopy (FTIR). The results indicated that despite the fact that the system contained distinct phases some degree of molecular miscibility occurred when the three components were present in the blend.     

Efficient production of medium-chain-length poly(3-hydroxyalkanoates) from octane by Pseudomonas oleovorans: economic considerations

Poly(3-hydroxyalkanoates) (PHA) have the potential to become a biodegradable alternative for conventional plastics. In order to produce PHA at competitive costs in comparison with commonly used plastics, efficient PHA production systems will have to be developed. Poly(3-hydroxybutyrate) fermentations are well developed and in actual use on an industrial scale; medium-chain-length PHA (mcl-PHA) production is less well described, although the vast majority of all PHA known today are mcl-PHA. This paper compares and describes mcl-PHA production systems with respect to the volumetric productivity, the cellular PHA content and the polymer yield on carbon substrates. Nitrogen was shown to be the most effective limitation to trigger PHA formation in P. oleovorans after different nutrient limitations had been compared. By using an economic model for the calculation of PHA production costs, we show that it should be possible to produce octane-based mcl-PHA on a large scale (more than 1000 tonnes/year) at costs below U.S. $ 10 kg⁻¹. Received: 4 April 1997 / Accepted: 20 May 1997     

Two-stage continuous process development for the production of medium-chain-length poly(3-hydroxyalkanoates)

Pseudomonas oleovorans forms medium-chain-length poly(3-hydroxyalkanoate) (PHA) most effectively at growth rates below the maximum specific growth rate. Under adequate conditions, PHA accumulates in inclusion bodies in cells up to levels higher than half of the cell mass, which is a time-consuming process. For PHA production, a two-stage continuous cultivation system with two fermentors connected in series is a potentially useful system. It offers production of cells at a specific growth rate in a first compartment at conditions that lead cells to generate PHA at higher rates in a second compartment, with a relatively long residence time. In such a system, dilution rates of 0.21 h(-1) in the first fermentor (D(1)) and 0.16 h(-1) in the second fermentor (D(2)) were found to yield the highest volumetric PHA productivity. Transient-state experiments allowed investigation of D(1) and D(2) over a wide dilution rate range at high resolution in time-saving experiments. Furthermore, the influence of temperature, pH, nutrient limitation, and carbon source on PHA productivity was investigated and results similar to optimum conditions in single-stage chemostat cultivations of P. oleovorans were found. With all culture parameters optimized, a volumetric PHA productivity of 1.06 g L(-1) h(-1) was determined. Under these conditions, P. oleovorans cells contained 63% (dry weight) PHA in the effluent of the second fermentor. This is the highest PHA productivity and PHA content reported thus far for P. oleovorans cultures grown on alkanes.     

Characterization of medium-chain-length polyhydroxyalkanoate biosynthesis by Pseudomonas mosselii TO7 using crude glycerol

Polyhydroxyalkanoates (PHAs) are biopolyesters produced by microorganisms that are environmentally friendly. PHAs can be used to replace traditional plastic to reduce environmental pollution in various fields. PHA production costs are high because PHA must be produced from a carbon substrate. The purpose of this study was to find the strain that can used the BDF by-product as the sole carbon source to produce high amounts of medium-chain-length PHA. Three isolates were evaluated for potential PHA production by using biodiesel-derived crude glycerol as the sole carbon source. Among them, Pseudomonas mosselii TO7 yielded high PHA content. The PHA produced from P. mosselii TO7 were medium-chain-length-PHAs. The PHA content of 48% cell dry weight in 48 h with a maximum PHA productivity of 13.16 mg PHAs L^?1 h^?1. The narrow polydispersity index value of 1.3 reflected the homogeneity of the polymer chain, which was conducive to industrial applications.     

Preparation of poly(l-lactide) blends and biodegradation by Lentzea waywayandensis

As a biodegradable polyester, polylactide (PLA) has applications as a packaging material, in biomedical fields and tissue engineering. With the dual aim of improving its properties and biodegradability, PLA was blended with other polymers such as gum arabic, thermoplastic starch, microcrystalline cellulose, polyethylene glycol and polyhydroxy butyrate in 1:1 (w/w) by melt-blending technique. The thermal properties of the blends were compared with that of unblended PLA by thermo-gravimetric analysis. Biodegradation using Lentzea waywayandensis was in the order of PLA–gum arabic?>?PLA–thermoplastic starch?>?PLA(virgin)?>?PLA–microcrystalline cellulose?>?PLA–polyethylene glycol?>?PLA–polyhydroxy butyrate. Weight loss of 99?% (w/w) was noted within 4?days for PLA–thermoplastic starch and PLA-gum arabic blends.