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.     

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.     

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.     

The Methane Monooxygenase Intrinsic Activity of Kinds of Methanotrophs

Methanotrophs have promising applications in the epoxidation of some alkenes and some chlorinated hydrocarbons and in the production of a biopolymer, poly-β-hydroxybutyrate (poly-3-hydroxybutyrate; PHB). In contrast with methane monooxygenase (MMO) activity and ability of PHB synthesis of four kinds of methanotrophic bacteria Methylosinus trichosporium OB3b, M. trichosporium IMV3011, Methylococcus capsulatus HD6T, Methylomonas sp. GYJ3, and the mixture of the four kinds of strains, M. trichosporium OB3b is the highest of the four in the activity of propene epoxidation (10.72 nmol/min mg dry weight of cell [dwc]), the activity of naphthalene oxidation (22.7 mmol/mg dwc), and ability in synthesis of PHB(11% PHB content in per gram dry weight of cell in 84 h). It could be feasible to improve the MMO activity by mixing four kinds of methanotrophs. The MMO activity dramatically decreased when the cellular PHB accumulated in the second stage. The reason for this may be the dilution of the MMO system in the cells with increasing PHB contents. It has been found that the PHB contents at the level of 1–5% are beneficial to the cells for maintenance of MMO epoxidation activity when enough PHB have been accumulated. Moreover, it was also found that high particulate methane monooxygenase activity may contribute to the synthesis of PHB in the cell, which could be used to improve the yield of PHB in methanotrophs.     

Improvement of substrate conversion to molecular hydrogen by three-stage cultivation of a photosynthetic bacterium,Rhodovulum sulfidophilum

In photosynthetic bacteria, after transition to light-anaerobic and nitrogen-deficient conditions, hydrogen evolution starts with expression of nitrogenase activity. Until the expression of enough activity,Rhodovulum sulfidophilum consumed substrates and converted them to poly(3-hydrox-ybutyrate) (PHB), resulting in a decrease in the proportion of substrate converted into hydrogen gas. To prevent conversion to PHB during the period when nitrogenase activity is derepressed, the authors employed a cultivation method consisting of three stages: cell growth, nitrogenase derepression, and hydrogen production. Cells cultivated by this method exhibited no lag time before the commencement of hydrogen evolution and gave an improved yield of hydrogen from the algal fermentative products.     

Poly(β-hydroxyalkanoates): Biorefinery polymers in search of applications

Poly(β-hydroxyalkanoates) are nature's high molecular weight thermoplastic polyesters. They occur as storage granules in a variety of bacteria. From a plastics, film, and fibres perspective, the statistically random copolyesters based on β-hydroxybutyrate (HB) and β-hydroxyvalerate (HV) repeating units have high potential for commercial exploitation because of their biodegradability. They provide a range of melting points from 180°C down to 80°C and all compositions exhibit high crystallinity due to isodimorphism. The latter is due to the similar crystalline conformations of poly(β-hydroxybutyrate) (PHB) and poly(β-hydroxyvalerate) (PHV). The nascent granules of a 21 mole % HV copolyester sample in freeze-dried bacterial cells were examined by ¹³C solid-state NMR. A disorder in the HV ethyl side group was noted but backbone carbons for HB and HV units showed evidence of crystalline order which was confirmed by x-ray diffraction. In keeping with the isodimorphous properties of this system, electron diffraction of copolyester single crystals for compositions up to 21 mole % HV confirmed a lattice expansion previously observed for bulk crystallized P(HB-co-HV). Solution or melt crystallized films showed decreasing rates of crystallization with increasing HV content. Homogeneous blends of PHB with P(HB-co-HV) could be formed which showed a single melting peak by differential scanning calorimetry. The potential of P(HB-co-HV) as a source of value-added small molecules is discussed. Depending on the method of degradation (i.e., chemical or pyrolytic) chiral synthons or vinylic small molecules are obtainable in nearly quantitative yields. Because their physical properties resemble those of polyolefins this family of chiral thermoplastics will probably find wide use in biomedical applications where compatibility and absorbability are essential features.     

Aerobic and anaerobic microbial degradation of poly-beta-hydroxybutyrate produced by Azotobacter chroococcum

Food industry wastewater served as a carbon source for the synthesis of poly-β-hydroxybutyrate (PHB) by Azotobacter chroococcum. The content of polymer in bacterial cells grown on the raw materials reached 75%. PHB films were degraded under aerobic, microaerobic, and anaerobic conditions in the presence and absence of nitrate by microbial populations of soil, sludges from anaerobic and nitrifying/denitrifying reactors, and sediment from a sludge deposit site. Changes in molecular mass, crystallinity, and mechanical properties of PHB were studied. Anaerobic degradation was accompanied by acetate formation, which was the main intermediate utilized by denitrifying bacteria or methanogenic archaea. On a decrease in temperature from 20 to 5° C in the presence of nitrate, the rate of PHB degradation was 7.3 times lower. Under anaerobic conditions and in the absence of nitrate, no PHB degradation was observed, even at 11°C. The enrichment cultures of denitrifying bacteria obtained from soil and anaerobic sludge degraded PHB films for a short time (3–7 d). The dominant species in the enrichment culture from soil were Pseudomonas fluorescens and Pseudomonas stutzeri. The rate of PHB degradation by the enrichment cultures depended on the polymer molecular weight, which reduced with time during biodegradation.     

Biosynthesis and biocompatibility of biodegradable poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

Poly(3-hydroxybutyrate), PHB, and poly(3-hydroxybutyrate-co-4-hydroxybutyrate), P(3HB-co-4HB), consisting of 0-94% mole fraction of 4HB content, were produced in high content by Cupriavidus necator strain A-04. The carbon sources used for PHB production included sugars made locally in Thailand: refined sugarcane, brown sugarcane, rock sugar, toddy palm sugar and coconut palm sugar. The switching of the ratios of carbon to nitrogen, together with the ratios of fructose to 1,4-butanediol, were applied to P(3HB-co-4HB) production in fed-batch cultures. Optimal P(3HB-co-4HB) production was achieved with 112 g biomass and 73 g P(3HB-co-4HB) with 38% mole fraction of 4HB content. Next, P(3HB-co-4HB) with a 0, 5, 24, 38 and 64% mole fraction of 4HB content were purified and prepared as plastic films. The mechanical properties and biocompatibility of these films were tested and compared with commercial PHB, polystyrene (PS) and polyvinylchloride (PVC) prepared without additives. The results demonstrated that PHB had thermal and mechanical properties similar to those of commercial PHB. The P(3HB-co-4HB) polymers possessed melting temperature and glass transition temperature values higher than those reported previously. The mechanical properties were compared with those of PS and PVC. The in vitro biocompatibility was assessed using L929, human dermal fibroblast and Saos-2 human osteosarcoma cells. The cytotoxicity results and scanning electron micrographs showed that P(3HB-co-4HB) films have good surface characteristics and can promote cell attachment, proliferation and differentiation. Combined with their good mechanical properties, P(3HB-co-4HB) polymers possess potential usefulness for biomaterial applications in artificial skin tissue support and orthopedic support.     

有机酸对甲烷利用菌Methylosinus trichosporium IMV3011生物合成聚-3-羟基丁酸酯的影响

为了降低聚-3-羟基丁酸酯(PHB)的生产成本,利用甲烷和甲醇生长的甲烷利用菌是具有研究价值的一类菌种.文章针对如何克服甲烷利用菌IMV3011积累PHB过程中受到制约的因素进行了考察,以改善此菌种生产PHB的能力.研究发现,通过添加适量的三羧酸(TCA)循环的抑制剂——苹果酸可以达到很高PHB产量的目的.在细胞进行两阶段培养过程中,营养平衡和营养受限培养各进行24h,即加入不同浓度的苹果酸(前者控制在5.7×10-4g/L,后者控制在0.03g/L),可以使PHB的积累量达到55%(未添加只能达到40%).实验还通过对PHB合成中所需的酶的活性研究,从一定程度上证明了适当的抑制三羧酸循环对保持PHB合成的酶系的活性将更有利于PHB的合成.     

甲烷氧化细菌生物催化合成聚-β-羟基丁酸酯的分子量调控

在甲烷氧化细菌Methylosinus trichosporium IMV3011细胞内生物催化合成聚-β-羟基丁酸酯(PHB)的过程中,对影响聚合物分子量的各种因素进行了研究.发现碳源、培养基组分NH4+,NO3-,HPO24-,Mg2+,某些导向PHB合成的关键中间产物以及PHB的提取方法均会对PHB的分子量产生影响.同时,通过对胞内PHB合成酶系中关键作用酶的活性变化进行研究,发现β-酮硫解酶催化着控制进入PHB循环入口的关键反应,而PHB分子量的变化则主要取决于PHB合成酶和PHB降解酶的协同作用.     

Assessment of PHB with varying hydroxyvalerate content for potential packaging applications

Poly-(hydroxybutyrate) (PHB) is biodegradable aliphatic polyester that is produced by a wide range of microorganisms. Basic PHB has relatively high glass transition and melting temperatures. To improve flexibility for potential packaging applications, PHB is synthesized with various co-polymers such as poly-(3-hydroxyvalerate) (HV) leading to a decrease of the glass transitions and melting temperatures. In addition, the HV broadens the processing window since there is improved melt stability at lower processing temperatures. In this study, PHB synthesized with different valerate contents (5%, 12%, and 20%) and varying in molecular weights were characterized. All PHBV materials displayed a glass transition between −10 and 20 °C. The two melting transitions found for Aldrich 5%, 12%, and Tianan 20%, resulted from crystals formed during cooling of the samples. The complex viscosity decreased with increasing temperature due to a decrease in molecular weights of the samples. These results suggest that processing the co-polymer below 160 °C would be beneficial with low screw speed. The mechanical results indicate all PHBV materials had high elastic modulus and flexural strength with low tensile strength and elongation at break. The WVTR results indicated the polymer to be very hydrophilic, resulting in higher water transmission rates.     

On-line fermentation gas analysis: Error analysis and application of mass spectrometry

On-line fermentation gas analysis is of general interest because it permits the determination of metabolic rates in almost any biological process using living organisms. The consumption and production of gases (O₂, CO₂, CH₄, etc.) and volatile compounds may be determined without causing any risk of infection. Elemental balancing permits the determination of other metabolic rates if the stoichiometry is known. This was studied with the production of poly-β-hydroxybutyrate (PHB) by Alcaligenes latus. Estimations were based on the measurement of gas partial pressure and flow-rates, pH and alkali consumption rate. Experiments with a small quadrupole mass spectrometer showed unacceptable error propagation. Therefore, dynamic error propagation for all rates was studied using simulation. It was found that, for example, a 1% relative offset-calibration error for oxygen can result in an error in PHB estimation of > 50%. It is suggested that this culture is used in combination with elemental balancing for thorough tests of the accuracy of on-line gas analysis equipment. An on-line process gas analyser based on a quadrupole mass spectrometer (Balzers PGM 407) gave the following precision values (abs. vol.?%) during cultivation of Bacillus subtilis: nitrogen (m/z 14), 0.024; oxygen (m/z 32), 0.020; argon (m/z 40), 0.0011; and carbon dioxide (m/z 44), 0.0034. These values, combined with automatic recalibration, would be sufficient for reasonable estimation of PHB, biomass and substrates.     

Biodegradation of Polyhydroxyalkanoate Films in Natural Environments

Summary: Biodegradation of film specimens from polyhydroxyalkanoates (PHAs) of two types – poly-3-hydroxybutyrate (PHB) and poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV) – was analysed in different environments: tropical sea waters of the South China Sea (Nha Trang, Vietnam) and soils in the environs of Hanoi (Vietnam), Nha Trang (Vietnam) and Krasnoyarsk (Siberia, Russia). In seawater, the mass loss of the specimens of both types was almost equal. However, in tropical soils, PHB degraded quicker than PHBV. In the Siberian soil, the degradation rate of the PHBV was generally higher than that of PHBV. Analysis of molecular mass of PHA specimens showed its decreasing during biodegradation. In the tropical sea conditions, PHA degrading microorganisms were represented by bacteria of Enterobacter, Bacillus and Gracilibacillus genera. Among PHA degrading bacteria, Burkholderia, Alcaligenes, Bacillus, Mycobacterium and Streptomyces genera were identified in Vietnamese soils, and Variovorax, Stenotrophomonas, Acinetobacter, Pseudomonas, Bacillus and Xanthomonas genera in Siberian soils. Micromycetes of Gongronella, Paecilomyces, Penicillium and Trichoderma genera exhibited PHA degrading activity in Vietnamese soils, and Paecilomyces, Penicillium, Acremonium, Verticillium and Zygosporium genera – in Siberian soils.     

Biosynthesis of poly-β-hydroxybutyrate and exopolysaccharides on azotobacter chroococcum strain 6B utilizing simple and complex carbon sources

Coproduction of poly-β-hydroxybutyrate (PHB) and exopolysaccharides (EPS) was investigated with Azotobacter chroococcum strain 6B isolated from soil samples. The bacterium was cultured using various carbon sources solely or with 0.1 g/L of ammonium sulfate. Ammonium addition resulted in reduced PHB and EPS production with glucose, fructose, and sucrose media, but cellular mass remained constant except for sucrose. Protein was nearly twofold higher in ammonium-grown cultures. Glucose and fructose alone biosynthesized high amounts of EPS (maximum 2.1 and 1.1 g/L, respectively, at 72 h), whereas PHB was accumulated only in glucose-grown cells. Sucrose almost did not produce EPS. Conversely, PHB content was the highest obtained from all experimented conditions (1.1 g/L at 48 h, 40% cell dry wt). When a complex carbon source such as sugar cane molasses was utilized, PHB was accumulated concomitant with EPS production from the initial time to 48 h (0.75 g/L, 37% cell dry wt and 0.6 g/L, respectively), and then PHB decayed at 72 h (0.2 g/L). On the other hand, EPS continued to be biosynthesized (1.1 g/L, 72 h). PHB fractions of total intra- and extracellular biopolymers were calculated. Sucrose-modified Burk’s medium without ammonium addition is suggested as a medium capable of diverting the carbon source for the production of intracellular PHB rather than EPS with A. chroococcum 6B.     

Mass spectrometry feedback control for synthesis of polyhydroxyalkanoate granule microstructures in Ralstonia eutropha

Polyhydroxyalkanoate (PHA) granules with core-shell layered microstructure were synthesized in Ralstonia eutropha using periodic feeding of valeric acid into a growth medium containing excess fructose. The O2 consumption and CO2 evolution rates, determined by off-gas mass spectrometry, have been used as sensitive measures to indicate the type of nutrients utilized by R. eutropha during PHA synthesis. Domains of poly-3-hydroxybutyrate (PHB) were formed during polymer storage conditions when only fructose was present. Feeding of valeric acid (pentanoic acid) resulted in the synthesis of hydroxyvalerate (HV) monomers, forming a poly-3-hydroxybutyrate-co-valerate (PHBV) copolymer. The synthesis of desired polymer microstructures was monitored and controlled using online mass spectrometry (MS). The respiratory quotient (RQ) was unique to the type of polymer being synthesized due to increased O2 consumption during PHBV synthesis. MS data was used as the control signal for nutrient feeding strategies in the bioreactor. The core-shell structures synthesized were verified in cells using transmission electron microscopy after thin sectioning and staining with RuO4. It was demonstrated that the synthesis of core-shell microstructures can be precisely controlled utilizing a MS feedback control system.     

Polymerization and copolymerization of β-butyrolactone and benzyl-β-malolactonate by aluminoxane catalysts

High molecular weight poly-β-hydroxybutyrate (PHB) and poly (β-hydroxybutyrate-co-β-benzyl malate) [P (HB? BM)], were prepared by ring-opening polymerization reactions of racemic β-butyrolactone (BL) and racemic β-benzyl malolactonate (BM) using two types of oligomeric aluminoxane catalysts prepared by the reaction of water with either triethyl-aluminum (EAO) or triisobutylaluminum (IBAO). The stereoregularities, crystallinities, and molecular weights were determined for both the PHB homopolymers and the P (HB? BM) copolymers by nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), and gel permeation chromatography (GPC). All homopolymers and copolymers obtained could be separated into acetone-soluble and acetone-insoluble fractions. In every case the latter had higher degrees of crystallinity, higher molecular weights and higher degrees of stereoregularity (84–87% isotactic dyads) than the former. Hence all of the polymers obtained from both types of catalysts apparently had stereoblock isotactic structures. Copolymer compositions and monomer dyad sequence distributions were determined by NMR spectroscopy.     

Evaluation of the transport properties of Poly(3-hydroxybutyrate) and its 3-hydroxyvalerate copolymers for packaging applications

Poly(3-hydroxybutyrate) (PHB) and ist 3-hydroxyvalerate containing copolymers form a family of fully biodegradable polyesters with many potential applications. In this work, the results obtained in our laboratory concerning carbon dioxide, water and organic solvent transport through PHB and three low 3-hydroxyvalerate copolymers are reviewed. Comparison established between the former results and some data taken from the literature, have revealed that PHB and the above mentioned copolymers show transport properties similar to other common thermoplastics such as PVC and PET, particularly in the case of carbon dioxide and water. Consequently, PHB and copolymers can be catalogued as good barrier materials against these penetrants. On the other hand, these biopolymers show a low barrier character against organic compound permeation. All these features conform a good balance of physicochemical properties for these polyhydroxyalkanoates, which may take them suitable for several applications, including its use in biodegradable packaging materials.     

Potential of various archae- and eubacterial strains as industrial polyhydroxyalkanoate producers from whey

Three different microbial wild-type strains are compared with respect to their potential as industrial scale polyhydroxyalkanoate (PHA) producers from the feed stock whey lactose. The halophilic archaeon Haloferax mediterranei as well as two eubacterial strains (Pseudomonas hydrogenovora and Hydrogenophaga pseudoflava) are investigated. H. mediterranei accumulated 50 wt.-% of poly-3-(hydroxybutyrate-co-8%-hydroxyvalerate) from hydrolyzed whey without addition of 3-hydroxyvalerate (3HV) precursors (specific productivity q(p): 9.1 mg x g(-1) x h(-1)). Using P. hydrogenovora, the final percentage of poly-3-hydroxybutyrate (PHB) amounted to 12 wt.-% (q(p): 2.9 mg x g(-1) x h(-1)). With H. pseudoflava, it was possible to reach 40 wt.-% P-3(HB-co-5%-HV) on non-hydrolyzed whey lactose plus addition of valeric acid as 3HV precursor (q(p): 12.5 mg x g(-1) x h(-1)). A detailed characterization of the isolated biopolyesters and an evaluation with regard to the economic feasibility completes the study.     

Crystallization behaviors and morphology of biodegradable poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

Crystallization behaviors and spherulitic morphology of poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] with different 4-hydroxybutyrate (4HB) molar fraction were investigated by differential scanning calorimetry and polarized optical microscopy. Crystallization behaviors of P(3HB-co-4HB) are significantly affected by 4HB molar fraction. The melting temperature (T _m), glass transition temperature (T _g), and crystallinity (X _c) decrease with the increase of 4HB molar fraction. Banded spherulites are observed in poly (3-hydroxybutyrate) (PHB) and P(3HB-co-4HB) copolymers. The band spacing decreases with the increase of 4HB molar fraction. The morphology and growth rate of the spherulites strongly depend on 4HB molar fraction and the crystallization temperatures. The introduction of 4HB unit can inhibit the emergence of cracks in PHB spherulites.     

Evaluation of mass-transfer and kinetic parameters forRhodospirillum rubrum in a continuous stirred tank reactor

The photosynthetic bacteriumRhodospirillum rubrum has been evaluated for its ability to produce hydrogen from carbon monoxide and water in a continuous stirred tank reactor according to the watergas shift reaction. An assessment of mass-transfer parameters and reaction kinetics was made for this sparingly soluble substrate system. Experiments were conducted in a nonsteady-state fashion with continuous liquid and gas flow, which allowed for separation of the mass-transfer and kinetic-limited regions. Based on the data obtained, mass-transfer coefficients for the system were determined, and a mathematical expression for the reaction kinetics was formulated. The results showed that the hydrogen production was inhibited by elevated levels of dissolved carbon monoxide in the liquid.