Microbial Poly Research Articles

Crystallization and Evaluation of Physical Properties of Microbial Poly(3-hydroxybutyrate-co-4-hydroxybutyrate).

The bulk crystallization behavior of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] were studied using polarized light microscopy, differential scanning calorimetry, and wide- and small-angle X-ray diffraction. Under the isothermal crystallization condition, the linear growth rates of the P(3HB-co-4HB) spherulites were measured at various temperatures and the influence of mutual disturbance for both comonomer components was clarified. The long period of P(3HB-co-4HB) increased much less with an increase in the crystallization temperature, compared to that of P(3HB). These results imply that the P(3HB) crystal excluded the 4HB component when, for example, the content of 4HB component in the copolymer was 5 mol%.

Synthesis of microbial poly(beta-hydroxybutyrate) modified with oligo(pentaerythritol ethoxylate) by Ralstonia eutropha.

Poly(beta-hydroxybutyrate) (PHB) modified with different amounts of pentaerythritol ethoxylate (PEE) has been synthesized using Ralstonia eutropha. The growth kinetics and the synthesis of PHB in the presence of PEE were modeled using appropriate differential equations for the mass balance of the two-stage process. The influence of PEE addition on the morphology of PHB was studied by various microscopic and scattering techniques. Light microscopic and wide-angle X-ray measurements indicated that the addition of PEE had a nucleating effect on the crystallization of PHB. The spherulite growth rate was widely independent of the PEE addition. The lamellae of PHB became more disordered when PEE was added as demonstrated by atomic force microscopy. Furthermore, small-angle X-ray data indicated a decrease in the long period with increasing PEE content of the modified PHB.

Molecular dynamics of the spin probes in dry and wet poly(3-hydroxybutyrate) films with different morphology

The molecular mobilities of the spin probes TEMPO and TEMPOL in microbial poly(3-hydroxybutyrate) (PHB) have been studied by the ESR method in a range of temperatures from 293 to 363K. The spectra of the dry and wet films with isotropic and textured morphology are analyzed as a spectral superposition of the “fast-” and “slow-rotating” fractions. Nonpolar TEMPO is sensitive to structural heterogeneity and practically nonsensitive to water sorption. The polar TEMPOL can interact with the carbonyl groups of PHB and the ESR spectra are affected by water molecules. The differences of spectral characteristics between the textured and isotropic PHB films are manifested in the rotation mobility (rotation correlation times) and the mole fractions of the “fast-” and “slow-rotating” ESR probes.Comparison of ESR data with water diffusivities in the textured and isotropic samples reveals the relationship between transport phenomena and morphology of the novel biopolymer material.

Biological effects of poly(ethylene glycol) on the microbial poly(β-hydroxyalkanoates) produced by pseudomonads microorganisms

Poly(ethylene glycol) of Mn 200 g/mol (PEG-200) was added to cultivation media of Pseudomonas oleovorans and Pseudomonas putida during fermentation. The carbon source and the medium used were sodium octanoate and medium E*, respectively. It was discovered that PEG-200 can control product molecular weight as well as cell productivity for both microorganisms. However, PEG-200 had no significant effect on the polymer compositions. The introduction of PEG-200 in the second-stage culture for 24 hr did not result in any notable cellular toxicity-measured in terms of colony forming unit/mL. Increasing the PEG-200 concentration to 8%, however, caused a decrease in the total polymer yield, the cell productivity as the well as Mn values. Gas chromatography analysis of products indicated that the major repeat units were β-hydroxycaproate, β-hydroxyoctanoate, and β-hydroxydeca noate. Thus, this work initiates new research opportunities for the control of product molecular weight for in-vivo microbial polymer synthetic reactions.

Efficient and economical recovery of poly(3-hydroxybutyrate) from recombinant Escherichia coli by simple digestion with chemicals.

A simple method for the recovery of microbial poly(3-hydroxybutyrate) [P(3HB)] from recombinant Escherichia coli harboring the Ralstonia eutropha PHA biosynthesis genes was developed. Various acids (HCl, H2SO4), alkalies (NaOH, KOH, and NH4OH), and surfactants (dioctylsulfosuccinate sodium salt [AOT], hexadecyltrimethylammonium bromide [CTAB], sodium dodecylsulfate [SDS], polyoxyethylene-p-tert-octylphenol [Triton X-100], and polyoxyethylene(20)sorbitan monolaurate [Tween 20]) were examined for their ability to digest non-P(3HB) cellular materials (NPCM). Even though SDS was an efficient chemical for P(3HB) recovery from recombinant E. coli, it is expensive and has waste disposal problem. NaOH and KOH were also efficient and economical for the recovery of P(3HB), and therefore, were used to optimize digestion condition. When 50 g DCW/L of recombinant E. coli cells having the P(3HB) content of 77% was treated with 0.2 N NaOH at 30 degrees C for 1 h, P(3HB) was recovered with purity of 98.5%. Using this simple recovery method, the effect of recovery method on the final production cost of P(3HB) was examined. Processes for the production of P(3HB) by recombinant E. coli from glucose with two different recovery methods, surfactant-hypochlorite digestion and simple digestion with NaOH, were designed and analyzed. By employing the fermentation process that resulted in P(3HB) concentration, P(3HB) content and P(3HB) productivity of 157 g/L, 77%, and 3.2 P(3HB) g/L-h, respectively, coupled with the recovery method of NaOH digestion, the production cost of P(3HB) was US$ 3.66/kg P(3HB), which was 25% less than that obtained by employing the surfactant-hypochlorite digestion method.

Thermal properties and crystallization behavior of poly(3-hydroxybutyric acid) in blends with chitin and chitosan

Films of blends of microbial poly(3-hydroxybutyric acid) (PHB) with α-chitin and chitosan were prepared as completely biodegradable polyester-polysaccharide composites. DSC revealed that the crystallization of PHB in these blends is suppressed when the proportion of the polysaccharide is increased. The same tendency was evident from the FT-IR band intensity of the carbonyl stretching absorption from PHB. Chitosan was found to have a stronger ability to suppress the crystallization of PHB than α-chitin. PHB in the blends was found by 13C NMR spectroscopy to be trapped in the `glassy' environment of the polysaccharide. Upon blending with PHB, the chitosan resonances are significantly broadend as compared to the chitin resonances. Hydrogen bonds are suggested to be formed between the carbonyl groups of PHB and the amide NH groups of chitin and chitosan.

Hydrolytic degradation and mechanical properties of hydrogels prepared from microbial poly(amino acid)s

The thermal hydrolytic degradability of hydrogels prepared from microbial poly(γ-glutamic acid) (PGA) and poly(ɛ-lysine) (PL) by γ-irradiation were measured in excess deionized water. PGA and PL hydrogels were slightly degraded near room temperature and degraded over 60 °C. It was found that PGA and PL hydrogels were hydrodegradable. In addition, the mechanical properties of PGA and PL hydrogels were measured by compression. PGA and PL hydrogels were not very strong and were the same as agar hydrogel (2%).

Poly(?-glutamic acid) hydrogel prepared from microbial poly(?-glutamic acid) and alkanediamine with water-soluble carbodiimide

Poly( y-glutamic acid) (PGA) hydrogels have been prepared from microbial PGA produced by Bacillus subtilis F-02-1, water-soluble carbodiimide (WSC), and alkanediamines such as 1,3-propanediamine (1,3-PD), 1,4-butanediamine (1,4-BD), and 1,6-hexanediamine (1,6-HD) in aqueous medium. The carboxyl groups of PGA were activated by the addition of WSC in deionized water, and the PGA-WSC adduct was produced. PGA hydrogels could be produced after the mixing of PGA-WSC and alkanediamine in deionized water. This alkanediamine to which both amino groups reacted with the carboxyl groups of PGA plays the role of a crosslinking point. When the amount of PGA was 100 mg, WSC was 50 mg or more, and 1,3-PD was 25 μL or more in 2 mL of deionized water, PGA hydrogels could be produced. Specific water contents (weight of absorbed water/weight of dry gel) ranged from 300 to 1,993 g/g in the case of 1,3-PD. If the PGA-WSC adduct was freeze-dried, the yield of the PGA hydrogel became higher than that when PGA-WSC was not freeze-dried. The highest yield of the PGA hydrogel from 100 mg of PGA, 100 mg of WSC, and 100 μL of 1,3-PD in 2 mL of deionized water using the freeze-dry method was 39.9 mg of dry PGA hydrogel with a 650 g/g specific water content. The order of yield was 1,6-HD > 1,4-BD > 1,3-PD from 100 mg of PGA-100 mg of WSC in 2 mL of deionized water. The order of the specific water content was 1,3-PD (462 g/g) > 1,4-BD (234 g/g) > 1,6-HD (199 g/g). This order may be due to the higher reaction probability between the activated carboxyl groups in the PGA-WSC and both amino groups in the alkanediamine with longer methylene chains, indicating that the crosslinking density of the PGA hydrogel is higher and the specific water content is lower.

Preparation conditions and swelling equilibria of biodegradable hydrogels prepared from microbial poly(γ-glutamic acid) and poly(ε-lysine)

Biodegradable hydrogels prepared by γ-irradiation from microbial poly(amino acid)s are reviewed. pH-sensitive hydrogels were prepared by means of γ-irradiation of poly(γ-glutamic acid) (PGA) produced byBacillus subtilis IFO3335 and poly(ε-lysine) (PL) produced byStreptomyces albulus in aqueous solutions. The preparation conditions, swelling equilibria, hydrolytic degradation, and enzymatic degradation of these hydrogels were studied. A hydrogel with a wide variety of swelling behaviors has been produced by γ-irradiation from a mixture solution of PGA and PL.

Separation and Structural Characterization of Cyclic and Open Chain Oligomers Produced in the Partial Pyrolysis of Microbial Poly(hydroxybutyrates)

To explore the possibility of using the partial pyrolysis of microbial poly(hydroxybutyrates) (PHBs) for obtaining single 3HB, 4HB, and mixed 3HB/4HB oligomers, the pyrolysis of P(3HB), P(4HB), and P(3HB-co-4HB) was performed and the products obtained were analyzed by HPLC and fast atom bombardment mass spectrometry (FAB-MS). Our results show that the partial pyrolysis of P(3HB) and P(4HB), followed by HPLC separation of the products, affords a suitable route for the synthesis of linear 3HB oligomers and of 4HB oligolides, respectively. The thermal degradation of P(3HB), P(4HB), and P(3HB-co-4HB) copolymers containing 3-34% of 3HB units was investigated by using several techniques: monitoring the time-dependent changes in molecular weight of melt samples in the range 180-200 °C ; direct pyrolysis mass spectrometry ; preparative pyrolysis and subsequent IR, NMR, HPLC, and FAB-MS analysis of the pyrolysate. Contrary to the claim that the rate constant (K d ) of chain scission is not influenced by the composition of the copolyesters, our results show that the rate of chain scission is influenced by the composition of copolyesters and that a mixture of cyclic and open chain oligomers is formed in the pyrolysis process. These findings indicate that P(3HB-co-4HB) copolymers undergo thermal degradation by two competitive random chain scission mechanisms : a β-CH hydrogen transfer at the 3HB units and an ester exchange cyclization at the 4HB units.

Synthesis and characterization of pH‐sensitive and biodegradable hydrogels prepared by γ irradiation using microbial poly(γ‐glutamic acid) and poly(ϵ‐lysine)

AbstractPoly(γ‐glutamic acid) (PGA) and poly(ϵ‐lysine) (PL) solutions were used as components to prepare mixed hydrogels by γ irradiation. A PGA and PL mixed solution was crosslinked to form a hydrogel with specific water content (weight of absorbed water/weight of dry gel) of 10–100 when the 5 wt % solution of mixed polymer was exposed to γ radiation of 87 kGy dosage under N2 atmosphere. The specific water content increased with increasing PGA content of the PGA/PL mixed gel. The influence of pH and salt concentration on equilibrium swelling was studied. A characteristic pH‐sensitive swelling behavior was obtained using compositional changes of PGA and PL in the gel. PGA/PL 50/50 wt % mixed gel swelling in acid (pH < 4.0) and alkaline (pH > 6.0) conditions and was deswelled between pH 4.0 and 6.0 due to the ionic composition changes of the gel network. With an increase in the ratio of PGA to PL, the hydrogels showed increasing sensitivity to salt solutions (NaCl, Na2SO4, and CaCl2). In addition, degradation of PGA/PL gel by protease produced from Aspergillus oryzae was investigated at 40°C and pH 7.0. PL gel was degraded completely within 2 days. An increase in the ratio of PAG in the PGA/PL mixed gel led to a decrease in the degree of degradation as expected. Some subtle degradation changes were found in the 50/50 and 80/20 wt % (PGA/PL) gels that were degraded by only 3.5 and 3.8% by protease, respectively. © 1995 John Wiley & Sons, Inc.

Properties of biodegradable hydrogels prepared by γ irradiation of microbial poly(ϵ‐lysine) aqueous solutions

AbstractPoly(ϵ‐lysine) (PL) hydrogels have been prepared by means of γ irradiation of PL produced by Streptomyces albulus in aqueous solutions. When the dosage of γ irradiation was 70 kGy or more and the concentration of PL in water was 1–7 wt %, transparent hydrogels (opaque hydrogels for 1–3 wt % PL concentration) could be produced. In the case of 70 kGy of γ irradiation and 5 wt % PL concentration, the specific water content (wt of absorbed water/wt of dry hydrogel) of the PL hydrogel was approximately 160. Specific water contents of PL hydrogels decreased markedly with an increase in the dosage of γ irradiation. The specific water contents were increased with an increase in PL concentration in the irradiated solution. This result indicates the presence of a radical scavenger in the PL solution. Swelling equilibria of PL hydrogels were measured in water or in aqueous solutions of various pHs or concentrations of NaCl, Na2SO4, and CaCl2. Under acid conditions, the PL hydrogel swelled due to the ionic repulsion of the protonated amino groups in the PL molecules. The degree of deswelling in electrolyte solution was smaller than that of other ionic hydrogels [poly(γ‐glutamic acid), poly(acrylic acid) etc.]. In addition, the enzymatic degradations of PL hydrogel were studied at 40°C and pH 7.0 in an aqueous solution of the neutral protease [Protease A (Amano)] produced from Aspergillus oryzae. The rate of enzymatic degradation of the respective PL hydrogels was much faster than the rate of simple hydrolytic degradation. The rate of enzymatic degradation decreased with the increase in γ‐irradiation dose during preparation of the PL hydrogel. © 1995 John Wiley & Sons, Inc.

Preparation conditions and swelling equilibria of hydrogel prepared by γ-irradiation from microbial poly(γ-glutamic acid)

Crosslinked microbial poly(γ-glutamic acid) (PGA) hydrogels were prepared by γ-irradiation, and their preparation conditions such as irradiation dose and PGA concentration were studied. When PGA solution was exposed to γ-radiation, PGA was crosslinked to yield high water sorption materials with specific water content of 100–1400 times their dry volume depending on the prepara tion conditions [e.g. irradiation dose (>20 kGy), PGA concentration (2–10 wt%)]. With an increase of γ-irradiation dosage, the gel content of PGA gels was inclined to increase but the specific water content decreased markedly. The equilibrium swelling behaviour in various aqueous solutions was investigated as a function of pH and concentration of electrolyte (NaCl, Na 2SO 4, CaCl 2). These PGA hydrogels exhibited reduced swelling when exposed to low pH or electrolyte solutions. The ionic contribution to swelling thus seems to be the main factor governing the swelling of these gels. In addition, the thermal hydrolytic degradability was measured in excess deionized water. PGA gels were almost completely degraded after about 1 h at 100°C.

Hydrolytic Degradation and Biodegradation of Hydrogel Prepared from Microbial Poly(.EPSILON.-lysine).

Hydrolytic and enzymatic degradation processes of hydrogels prepared by γ-irradiation from microbial poly (e-lysine) (PL) were studied. Hydrolysis studies on PL hydrogels were carried out in deionized water at 40, 75, and 95°C. PL hydrogel, which was prepared with 100 kGy γ-irradiation dose and 5 wt% PL concentration. was degraded by only 2% at 40°C for 17 hours, and degraded by 14% at 75°C for 13.5 hours. As the heating temperature was increased, the degradation rate was increased. The specific water content (wt. of absorbed water/wt. of dry gel) was increased with the progress of hydrolytic degradation, The rate of hydrolytic degradation of heterogeneous opaque PL hydrogel (≤ 3 wt% PL concentration) was slower than that of homogeneous transparent PL hydrogel. The thermal stability of PL hydrogel was found to be dependent on the structure of the gel. The enzymatic degradations of PL hydrogels were studied at 40°C and pH 7.0 in the 25 mM phosphate buffer with Protease A produced from Aspergillus oryzae. PL hydrogels were degraded by 20-60% for 17 hours by Protease A. The rate of enzymatic degradation of PL hydrogel was much faster than the rate of simple hydrolytic degradation. The enzymatic degradation occurred at the surface of the PL hydrogel. The surface erosion by Protease A was confirmed by monitoring the time-dependent changes in the specific water content of the PL hydrogel.

Effect of Nucleating Agents on Crystallization of Poly(3-hydroxybutyrate) and Its Copolymer.

Crystallization and nucleation behavior of microbial poly (3-hydroxybutyrate) and poly (3-hydroxybutyrate-co-3-hydroxyvalerate) were investigated by differential scanning calorimetry. The polymers mixed with talc as a nucleating agent showed promotion of crystallization of the polymers, while the addition of saccharin hardly promoted crystallization. However, melting points and long periods of these polymers crystallized isothermally after mixing with talc and saccharin, decreased considerably. The depression in melting points and long periods of the polymers mixed with saccharin was larger than that mixed with talc, implying that saccharin was included more easily within the polymer crystal playing the role of diluent. The concentration of around 4% is most appropriate to reveal typical effect of nucleating agents without severe deterioration of mechanical properties. Large spherulites were formed in plain polymers and those mixed with saccharin, whilst extremely small spherulites were formed in the polymers mixed with talc.

Synthesis and Characterization of Poly[(R,S)-3-hydroxybutyrate-b-6-hydroxyhexanoate] as a Compatibilizer for a Biodegradable Blend of Poly[(R)-3-hydroxybutyrate] and Poly(6-hydroxyhexanoate)

A block copolymer (P[R,S)-3HB-b-6HH]) of atactic poly[(R,S)-3-hydroxybutyrate] (P[(R,S)-3HB]) and poly(6-hydroxyhexanoate) (P(6HH)) was prepared by ring-opening polymerization of (R,S)-β-butyrolactone followed by the subsequent polymerization of e-caprolactone in the presence of Zn(C 2 H 5 ) 2 /H 2 O catalyst. The block copolymer was an A-B diblock copolymer comprising atactic P[(R,S)-3HB] (A) and P(6HH) (B) segments. The block copolymer was used as a compatibilizer for the immiscible blend of microbial poly[(R)-3-hydroxybutyrate] (P[(R)-3HB]) and P(6HH). The addition of a small amount of the block copolymer into the P[(R)-3HB]/P(6HH) blend reduced the size of the P(6HH) dispersed domains

Optimization of microbial poly(3‐hydroxybutyrate) recover using dispersions of sodium hypochlorite solution and chloroform

Optimization was carried out for the recovery of microbiol poly(3-hydroxybutyrate) (PHB) from Alcaligenes eutrophus. This process involved the use of a dispersion made of sodium hypochlorite solution and chloroform. The dispersion enabled us to take advantage of both differential digestion by hypochlorite and solvent extraction by chloroform. The PHB recovery (%) from cell powder was maximized using a 30% hypochlorite concentration, a 90-min treatment time, and a 1:1 (v/v) chloroform-to-aqueous-phase ratio. Under these optimal conditions, the recovery was about 91% and the purity of recovered PHB was higher than 97%. The number average molecular weight, M(n) of recovered PHB was about 300,000 and the weight average molecular weight M(w) was about 1,020,000, compared to the original M(n) of 530,000 and M(w) of 1,272,000. The moderate decrease in both M(n) and M(w) might be ascribed to the shielding effect of chloroform. In addition, the relatively small decrease in M(w) probably resulted from the loss of short PHB chains which might be water soluble. The crystallinity of recovered PHB was in the range of 60 to 65%although a slightly higher crystallinity was observed when the dispersion was used. (c) 1994 John Wiley & Sons, Inc.

Hydrolytic degradation of blends of poly(3-hydroxybutyrate) with poly(3-hydroxybutyrate- co-3-hydroxyvalerate)

The hydrolytic degradation behaviour and some other properties were analysed for blends of microbial poly(3-hydroxybutyrate) [P(3HB)] with microbial poly(3-hydroxybutyrate- co-3-hydroxyvalerate) [P(3HB- co-3HV); 3HV content, 22.3 mol%]. From the analysis of differential scanning calorimetry (d.s.c.) curves, the main component of the crystalline phase in the blend films was found to be P(3HB). The crystallinity of P(3HB) decreases on blending with P(3HB- co-3HV). The hydrolytic degradation of the blend films was carried out on the solution-cast films in phosphate buffer at 55°C and pH 7.4. The rate of hydrolytic degradation of the blend films was accelerated by blending with P(3HB- co-3HV), and showed linear correlation with crystallinity. Therefore, the degradation rate of P(3HB) can be controlled by blending with P(3HB- co-3HV). The lower the initial crystallinity of the blend films, the higher the rate of hydrolytic degradation is seen to be.

Thermal degradation of microbial poly(4-hydroxybutyrate)

An almost pure poly(4-hydroxybutyrate) (P(4HB)) sample (i.e., a P(3HB-co-4HB) copolymer containing only 3% of 3HB units) was obtained by microbial synthesis. The thermal degradation of this material was investigated using thermogravimetry, direct pyrolysis mass spectrometry and abo by preparative pyrolysis and subsequent NMR analysis of the pyrolyzate. Previous studies on P(3HB-co-4HB) copolyesters have reported that the thermal degradation process occurs by a β-CH hydrogen transfer, similar to the well-known case of P(3HB). However, the latter work did not distinguish between the thermal degradation fate of 3HB and 4HB units. Our indings indicate that the thermal

Physical Properties and Biodegradability of Microbial Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

Physical Properties and Biodegradability of Microbial Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)