Development of a closed-loop control system for production of medium-chain-length poly(3-hydroxyalkanoates) (mcl-PHAs) from bacteria

Large scale availability of bacterial polyhydroxyalkanoates (PHAs) is still limited to a few types of short-chain-length PHAs, namely poly(3-hydroxybutyrate) (PHB) and its copolymer Biopol™, consisting of 3-hydroxybutyrate and 3-hydroxyvalerate repeating units. In order to increase the number of available medium-chain-length PHA (mcl-PHA) copolymers a flexible high-cell-density fed-batch process was developed. Continuous process monitoring and substrate control were achieved by coupling on-line gaschromatography (on-line GC) to a software-based Proportional Integral (PI) substrate controller. System development time and continuous system upgrading were considerably shortened by using LABView™, a powerful graphical programming environment. The control of octanoic acid and 10-undecenoic acid at 1.5 and 0.5 gL⁻¹ respectively, enabled the production of high levels of biomass (30 gL⁻¹) and mcl-PHA (10.5 gL⁻¹) by avoiding substrate limitations or toxicities. The resulting mcl-PHA was an amorphous copolyester consisting of 37 mol% unsaturated monomers. The present system represents a valuable tool for the production of tailor-made mcl-PHAs, where the desired monomer composition is determined by the ratio of added cosubstrates.     

Rapid microwave assisted esterification method for the analysis of poly-3-hydroxybutyrate in Alcaligenes latus by gas chromatography

In the present study, we used microwave energy instead of conventional heating to transform poly-3-hydroxybutyrate (PHB) into methyl 3-hydroxybutyrate (Me-3HB) in acidified methanol (H2SO4, 10%, v/v) mixture in less than 4 min at 10% microwave power. The microwave assisted method was then applied to analyze PHB produced by Alcaligenes latus. The PHB content in the biomass determined using microwave heating was comparable to the amount found by conventional heating. Moreover, the new esterification method was at least 50 times faster than the conventional method, affording a significant saving of time and energy.     

Gamma Irradiation Effects on Mechanical and Thermal Properties and Biodegradation of Poly(3-hydroxybutyrate) Based Films

Summary: Their biodegradable properties make polyhydroxyalkanoates (PHAs) ideal candidates for innovative applications. Many studies have been primarily oriented to poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxybutyrate-co-3-valerate) (PHBV) and afterwards to blends of PHAs with synthetic biodegradable polymers, such as poly(ε-caprolactone) (PCL). Medical and pharmaceutical devices require sterilization and γ irradiation could provide a proper alternative since it assures storage stability and microbiological safety. This contribution presents the effect of γ irradiation on the mechanical and thermal properties and on the biodegradation of PHB, PHBV and a commercial PHB/PCL blend. Samples, prepared by compression moulding, were irradiated in air at a constant dose rate of 10 kGy/h, from 10 to 179 kGy. Polymer chain scission was assessed by changes in the molecular weight, thermal properties and tensile behaviour. The correlation between absorbed dose and changes in the mechanical properties and biodegradation is discussed in detail. The optimum dose to guarantee microbiological sterilization without damage of the structure or meaningful loss of the mechanical properties is also reported.     

Novel block copolymers of atactic PHB with natural PHA for cardiovascular engineering: Synthesis and characterization

Low-molecular weight macroinitiators derived from natural poly(3-hydroxyalkanoates) (PHAs), which contain olefinic and activated by 18-crown-6 ether carboxylic end groups, were used in anionic ring opening polymerization (ROP) of racemic β-butyrolactone and new diblock copolymers of selected PHAs (PHB, PHBV, PHO) with atactic poly[(R,S)-3-hydroxybutyrate] (a-PHB) were obtained. These novel copolymers were characterized using ¹H NMR, GPC and DSC. Hydrolytic degradation studies of selected copolymers were also performed. Finally, the suitability of these polymeric materials for cardiovascular engineering and as blend compatibilizers was demonstrated.     

Characterization of copolymer hydroxybutyrate/hydroxyvalerate from saponified vernonia,soybean, and "spent" frying oils

Poly(beta-hydroxyalkanoate)s (PHAs) were biosynthesized by Ralstonia eutropha (formerly known as Alcaligenes eutrophus) by using saponified soybean, vernonia, and "spent" frying oils. These PHAs were isolated and characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS), gas chromatography/mass spectrometry (GC/MS), proton nuclear magnetic resonance spectrometry (1H NMR), and 2-dimensional homonuclear (1H-1H) correlation spectroscopy (COSY). The analytical results revealed that the PHAs produced from saponified vernonia and soybean oils were copolymers of hydroxybutyrate (HB) and hydroxyvalerate (HV), that is, P(HB/HV)s, whereas the saponified "spent" frying oil produced only poly(beta- hydroxybutyrate) (PHB) homopolymer. MALDI-MS, GC/MS, and NMR independently confirmed the composition of the PHAs. Saponified soybean oil and vernonia oil PHAs contained approximately 4 and 1% HV units, respectively. For comparison, commercial PHB and P(HB/HV), produced by R. eutropha by using glucose and a cosubstrate of glucose and propionic acid, respectively, as carbon sources, were similarly characterized.     

Effect of poly(vinyl acetate) (PVAc) on thermal behavior and mechanical properties of poly(3-hydroxybutyrate)/poly(propylene carbonate) (PHB/PPC) blends

To assess the compatibility of blends of synthetic poly(propylene carbonate) (PPC), with a natural bacterial poly(3-hydroxybutyrate) (PHB), a simple casting procedure of blend was used. poly(3-hydroxybutyrate)/poly(propylene carbonate) blends are found to be incompatible according to DSC and DMA analysis. In order to improve the compatibility and mechanical properties of PHB/PPC blends, poly(vinyl acetate) (PVAc) was added as a compatibilizer. The effects of PVAc on the thermal behavior, morphology, and mechanical properties of 70PHB/30PPC blend were investigated. The results show that the melting point and the crystallization temperature of PHB in blends decrease with the increase of PVAc content in blends, the loss factor changes from two separate peaks of 70PHB/30PPC blend to one peak of 70PHB/30PPC/12PVAc blend. It is also found that adding PVAc into 70PHB/30PPC blend can decrease the size of dispersed phase from morphology analysis. The result of tensile properties shows that PVAc can increase the tensile strength and Young’s modulus of 70PHB/30PPC blend, and both the elongation at break and the tensile toughness increase significantly with PVAc added into 70PHB/30PPC.     

Brush Type Copolymers of Poly(3-hydroxybutyrate) and Poly(3-hydroxyoctanoate) with Same Vinyl Monomers via “Grafting From” Technique by Using Atom Transfer Radical Polymerization Method

Brush type graft copolymers of poly(3-hydroxybutyrate) (PHB) and poly(3-hydroxyoctanoate) (PHO) with methylmethacrylate, (MMA), styrene, (S), and n-butylmethacrylate, (n-BuMA) were obtained by using Atom Transfer Radical Polymerization Method, (ATRP), via “grafting from” technique. Firstly PHB and PHO were chlorinated by passing chlorine gas through their solution in CHCl₃/CCl₄ (75/25 v/v) mixture and CCl₄, respectively, in order to prepare chlorinated PHB, PHB-Cl, and chlorinated PHO, PHO-Cl, with different chlorine contents. The determination of the chlorine content in chlorinated poly(3-hydroxyalkanoate) (PHA-Cl) was performed by the Volhard Method. Then ATRP of vinyl monomers was initiated by using PHA-Cl as macroinitiators in the presence of cuprous chloride (CuCl)/2,2′-bipyridine complex as catalyst, at 90 °C in order to obtain brushes containing PHAs. The polymer brushes were fractionated by fractional precipitation methods and the γ values calculated from the ratio of the volume of nonsolvent (methanol) and the volume of solvent (chloroform) of brushes varied between 0.82 and 6.50 depending on the composition of brushes. The polymer products were characterized by gel permeation chromatography (GPC), ¹H NMR, FTIR, thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC) techniques.     

Computer simulation studies of the miscibility of poly(3-hydroxybutyrate)-based blends

By means of the molecular dynamics simulation method, the miscibility of poly(3-hydroxybutyrate)/polyethylene (PHB/PE) blend has been investigated. Two glass transition temperatures of the PHB/PE are found by scrutinizing its volume-temperature curve, and this result is qualitatively in agreement with the experimental results. To further analyze the miscibility of poly(3-hydroxybutyrate)-based blends, the Flory-Huggins parameters of PHB/PE, poly(3-hydroxybutyrate)/poly(ethylene oxide) (PHB/PEO), poly(ethylene oxide)/polyethylene (PEO/PE) have been calculated via a Monte Carlo scheme, and the morphology of the PHB/PEO and the PHB/PE blend has been simulated using dissipative particle dynamics method. The time evolution of dividing interface for PHB/PEO/PE blend shows a dynamic phase separation process. All these results indicate that PHB and PEO tend to mix together, whereas PE aggregates to form PE-rich domains in the PHB/PE and PHB/PEO/PE blends.     

Study of enzymatic degradation of microbial copolyesters consisting of 3-hydroxybutyrate and medium-chain-length 3-hydroxyalkanoates

Enzymatic degradation of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) (PHBA) biopolyester consisting of 3-hydroxybutyrate (HB) and 15 mol% medium-chain-length 3-hydroxyalkanoates (HA) was studied using a polyhydroxyalkanoates (PHA) depolymerase produced by Ralstonia pickettii T1. It was found that PHBA films did not lose their weight after 25 h of depolymerase treatment. In contrast, three commercially available PHAs including poly-3-hydroxybutyrate (PHB), poly(3-hydroxybutyrate-19 mol% 3-hydroxyvalerate) (PHBV) and poly(3-hydroxybutyrate-19 mol% 3-hydroxyhexanoate) (PHBHHx) lost 75%, 94% and 39% of their original weights. Slow degradation of PHBA was also confirmed by the absence of HA monomers, dimers or trimers as degradation products in their depolymerase solution compared with abundance of degradation products released by the other three PHAs under the same condition. Surface erosion of PHBA was only observed after 48 h of enzymatic treatment compared with those of PHB, PHBV and PHBHHx which already had obvious surface changes after 7.5 h of same treatment. Although the crystallinities of PHB, PHBV, PHBHHx and PHBA were in the order PHB > PHBV > PHBHHx > PHBA valued at 55.8%, 47.8%, 45.9% and 40.9%, respectively, the order of degradability was PHBV > PHB > PHBHHx > PHBA. It can be proposed that PHA enzymatic degradation using this depolymerase was structure related: longer side-chain PHA including PHBHHx and PHBA was less favorable for the depolymerase degradation, longer the side chain, less the biodegradation.     

Non-isothermal crystallization kinetics of poly(3-hydroxybutyrate)/EVA 80 blends enhanced by NH4Cl as a nucleating agent

Journal of Thermal Analysis and Calorimetry - The crystallization kinetics of poly(3-hydroxybutyrate) (PHB), PHB/NH4Cl composites, PHB/ethylene-vinyl acetate (EVA 80) blends and PHB/EVA 80/1 mass%...     

Production of poly-3-hydroxyalkanoates from CO and H2 by a novel photosynthetic bacterium

A novel process is described to efficiently photoconvert low-grade organic materials such as waste biomass into natural biological plastics. When heterogeneous forms of dry biomass are thermally gasified, relatively homogeneous synthesis gas mixtures composed primarily of carbon monoxide and hydrogen are produced. Unique strains of photosynthetic bacteria were isolated that nearly quantitatively photoassimilate the carbon monoxide and hydrogen components of synthesis gas into new cell mass. Under unbalanced culture conditions when cellular growth is limited by shortages of nitrogen, calcium, magnesium, iron, or essential vitamins, up to 28% of the new cell mass is found as granules of poly-3-hydroxyalkanoate (PHA), a highmolecular-weight thermoplastic that can be solvent-extracted. The dominant monomeric unit of PHAs is 3-hydroxybutyrate (3HB), which is polymerized into the homopolymeric poly-3-hydroxybutyrate (PHB). PHB is marketed as a biodegradable plastic with physical properties similar to polystyrene. When a green alga was cocultured with the photosynthetic bacterium in light-dark (day-night) cycles, the bacteria synthesized a polymer of poly-3-hydroxybutyrate-3-hydroxyvalerate (PHB-V) with a composition of 70% 3HB and 30% 3-hydroxyvalerate (3HV) to an extent of 18% of the new cell mass. PHB-V is commercially marketed as Biopol and has physical properties similar to polypropylene or polyethylene. Our results demonstrate that a strain of photosynthetic bacteria capable of photoassimilating synthesis gas or producer gas is a potential candidate for large-scale production of biological polyesters.     

Characterization of poly[(R)-3-hydroxybutyrate-co-epsilon-caprolactone] copolymers by matrix-assisted laser desorption/ionization time-of-flight and electrospray ionization mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) of narrowly dispersed molecular weight gel permeation chromatography (GPC) fractions was used to characterize random and microblock poly[(R)-3-hydroxybutyrate-co-epsilon-caprolactone] [P(HB-co-CL)] copolymers obtained via the acid-catalyzed transesterification of the corresponding homopolymers, poly([(R)-3-hydroxybutyrate] (PHB) and poly(epsilon-caprolactone) (PCL). High-quality mass spectra were obtained, which made it possible to establish the nature of the polymer end groups. Besides the carboxylic termination, two other moieties were found: alcoholic and tosyl end groups. MALDI mass spectra of CL-rich samples possessed mostly tosyl end groups, while HB-rich samples possessed mostly alcoholic end groups, showing that the tosyl moiety is linked prevalently to CL terminal units. The higher resolution of electrospray ionization (ESI) mass spectra of lower molecular weight GPC fractions permitted the identification of the different oligomer species hypothesized in the assignment of the corresponding MALDI mass spectra. Partial methanolysis of these copolymers was explored as a method of producing mixed HB-CL oligomers to be utilized as new synthons, possessing a minor number of chiral centers from those obtained from hydrolysis of biotechnologically synthesized poly(hydroxyalkanoates) (PHAs).     

Synthesis and properties of graft copolymers based on poly(3-hydroxybutyrate) macromonomers

Graft copolymers of poly(methyl methacrylate) with poly(3-hydroxybutyrate), PHB, segments as long side chains were prepared by the macromonomer method. PHB macromonomers were prepared from the esterification of oligomers with 2-hydroxyethyl methacrylate at their carboxylic acid end. Esterification products displayed low polydispersity indices (ca. 1.2) and a functionality of over 83%, with a Mn of 2,020. Using free radical polymerization methods, the macromonomers were copolymerized with methyl methacrylate to yield graft (comb type) copolymers at different comonomer feed ratios. The graft copolymers contained from 0.5 to 14 mol-% of PHB blocks, with a glass transition temperature decreasing from 100 to 3 degrees C.     

Influence of poly (ethylene glycol) on the thermal, mechanical, morphological, physical-chemical and biodegradation properties of poly (3-hydroxybutyrate)

Blends of poly (3-hydroxybutyrate) (PHB) with poly (ethylene glycol) (PEG), (PHB/PEG), in different proportions of 100/0, 98/2, 95/5, 90/10, 80/20 and 60/40 wt%, respectively, were investigated for their thermal properties (using differential scanning calorimetry and thermogravimetric analysis), tensile properties, water vapor transmission rate, enzymatic biodegradation (using light microscopy) and mass retention. The addition of plasticizer did not alter the thermal stability of the blends, although an increase in the PEG content reduced the tensile strength and increased the elongation at break of pure PHB.     

Mixtures poly((<Emphasis Type="Italic">R</Emphasis>)-3-hydroxybutyrate) and poly(<Emphasis Type="SmallCaps">l</Emphasis>-lactic acid) subjected to DSC

Blends of bacterial poly((R)-3-hydroxybutyrate) (PHB) and poly(l-lactic acid) (PLLA) synthesized by polycondensation of l-lactic acid or by ring-opening polymerization of l-lactide were studied. Miscibility was investigated through both conventional differential scanning calorimetry (DSC) and temperature-modulated DSC (TMDSC). PHB and low-molar mass PLLA were miscible in a whole concentration range, and a single glass transition temperature was observed. On the other hand, PHB/high-molar mass PLLA mixtures phase separate, giving rise to two glass transition temperatures corresponding to PHB and PLLA. A treatment of blends at 190 °C leads to formation of block/multiblock/random copolymers, and blends become miscible.     

Melt and cold crystallization in a poly(3-hydroxybutyrate) poly(butylene adipate-co-terephthalate) blend

Journal of Thermal Analysis and Calorimetry - Melt and cold crystallization characteristics of poly(3-hydroxybutyrate) (PHB) and poly(butylene adipate-co-terephthalate) (PBAT), two biodegradable...     

以葡萄糖为碳源合成生物降解性聚酯的研究

利用从油田土壤中筛选的菌种ＤＧ１７ 以葡萄糖为碳源通过微生物发酵法合成了具有不同结构单元的新型生物可降解性聚合物———聚羟基脂肪酸酯（ＰＨＡｓ） ．初步研究了ＤＧ１７ 以葡萄糖为碳源的生物合成规律,并借助ＧＣ、ＮＭＲ 等分析手段对合成的聚合物进行了结构的分析表征,另外还研究了ＰＨＡｓ 的活性污泥降解情况．研究表明,在限氮条件下,只有碳氮比高于５后,ＤＧ１７ 才能在其体内合成ＰＨＡｓ．在过量碳源的存在下,氮磷比低,得到的聚合物是一种具长侧链的聚（ 羟基辛酸 ｃｏ 羟基癸酸） 的共聚物,为一种热塑性弹性体．在硫酸铵浓度为０－５ｇ／Ｌ,碳氮比为２０ 条件下合成的Ｐ（ＨＯ ｃｏ ＨＤ） 热塑性弹性体的数均分子量为１－１６ ×１０ － ５ ,分子量分散指数为２－４３ ．其玻璃化温度及熔融温度分别为Ｔｇ ＝ － ５２ ℃,Ｔｍ ＝ ５０ ℃．氮磷比高,则合成热塑性塑料ＰＨＢ．结果表明培养基中氮源与磷酸盐的相对浓度是影响ＤＧ１７ 生物合成路径的重要条件．     

Non-isothermal kinetic applied to thermal decomposition of commercial alkyd varnish

Summary PHB polyester poly(3-hydroxybutyrate) is an interesting biodegradable polymer and intensively investigated as cast and sheet films with applications in food industry or in medicine. The films obtained are typically brittle and many scientists have attempted to reduce this brittleness by blending with other polymers. PHB from Usina da Pedra was blended with PEG poly(ethyleneglycol) 300 resulting in blend 1 and blend 2. The two mixtures were melted at 200 and quenched at 0&deg;C. TG curves showed that the thermal stability of the blends and the PHB are identical. For these blends the crystallization temperature decreased compared to the pure PHB, which is probably due to the lower nucleation density.     

Nonisothermal crystallization and melting behavior of poly(3-hydroxybutyrate) and maleated poly(3-hydroxybutyrate)

Nonisothermal crystallization and melting behavior of poly(3-hydroxybutyrate) (PHB) and maleated PHB were investigated by differential scanning calorimetry using various cooling rates. The results show that the crystallization behavior of maleated PHB from the melt greatly depends on cooling rates and its degree of grafting. With the increase in cooling rate, the crystallization process for PHB and maleated PHB begins at lower temperature. For maleated PHB, the introduction of maleic anhydride group hinders its crystallization, causing crystallization and nucleation rates to decrease, and crystallite size distribution becomes wider. The Avrami analysis, modified by Jeziorny, was used to describe the nonisothermal crystallization of PHB and maleated PHB. Double melting peaks for maleated PHB were observed, which was caused by recrystallization during the heating process.     

Purification and Properties of an Extracellular Polyhydroxybutyrate Depolymerase from Pseudomonas mendocina DSWY0601

An extracellular polyhydroxybutyrate(PHB) depolymerase was purified to homogeneity from the culture supernatant of a PHB-degrading bacterium, Pseudomonas mendocina DSWY0601, which was isolated from brewery sewage for the ability to form clear zones on the PHB mineral agar plates. The molecular weight of the purified PHB depolymerase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) was approximately 59800 at the optimal temperature and pH value being 50 ℃ and 8.5, respectively. PHB depolymerase was stable in a temperature range of 20―50 ℃ and sensitive to pH value within a pH range of 8.0―9.5. PHB depolymerase degraded poly-3-hydroxybutyrate-co-4-hydroxybutyrate(P3/4HB) and poly-3-hydroxybutyrate-co-3- hydroxyvalerate(PHBV) but did not degrade poly(lactic acid)(PLA), poly(butylene succinate)(PBS) or poly- (caprolactone)(PCL). PHB depolymerase was sensitive to phenylmethylsulfonyl fluoride(PMSF), H₂O₂ and SDS. The main product after enzymatic degradation of PHB was indentified as 3-hydroxbutyrate monomer(3HB) by mass spectrometric analysis, suggesting that PHB depolymerase acted as an exo-type hydrolase. Analysis of phaZ_pm gene reveals that PHB depolymerase is a typical denatured short-chain-length PHA(dPHA_SCL, PHA=polyhydroxyalkanoate) depolymerase containing catalytic domain, linker and substrate-binding domain.