Characterization of partially transesterified poly(beta-hydroxyalkanoate)s by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used for the characterization of a partially transesterified poly(beta-hydroxyalkanoate) (PHA), a polymer produced by the bacterial strain Alcaligenes eutrophus with saponified vegetable oils as the sole carbon sources. The transesterification was carried out separately under acidic and basic conditions to obtain PHA oligomers weighing <10 kDa. The intact oligomers were detected in their cationized forms, [M + Na]+ and [M + K]+, by MALDI-TOFMS. A composition analysis, using the MALDI-TOF spectra, indicated that the oligomers obtained via acid catalysis contained a methyl 3-hydroxybutyrate end group, and those obtained by base catalysis had a methyl crotonate (olefinic) end group. In addition to hydroxybutyrate (HB), the oligomers were found to contain a small percentage of hydroxyvalerate, which was independently confirmed by gas chromatography/mass spectrometry. In comparison, analysis of a commercial PHA polymer, transesterified under identical conditions, showed only the presence of HB, i.e., a pure poly(HB) homopolymer.     

Determination of the fatty acid composition of saponified vegetable oils using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) for the determination of the fatty acid composition of vegetable oils is described and illustrated with the analysis of palm kernel oil, palm oil, olive oil, canola oil, soybean oil, vernonia oil, and castor oil. Solutions of the saponified oils, mixed with the matrix, meso-tetrakis(pentafluorophenyl)porphyrin, provided reproducible MALDI-TOF spectra in which the ions were dominated by sodiated sodium carboxylates [RCOONa + Na]+. Thus, palm kernel oil was found to contain capric acid, lauric acid, myristic acid, palmitic acid, oleic acid, and stearic acid. Palm oil had a fatty acid profile including palmitic, linoleic, oleic, and stearic. The relative percentages of the fatty acids in olive oil were palmitoleic (1.2 +/- 0.5), palmitic (10.9 +/- 0.8), linoleic (0.6 +/- 0.1), linoleic (16.5 +/- 0.8), and oleic (70.5 +/- 1.2). For soybean oil, the relative percentages were: palmitoleic (0.4 +/- 0.4), palmitic (6.0 +/- 1.3), linolenic (14.5 +/- 1.8), linoleic (50.1 +/- 4.0), oleic (26.1 +/- 1.2), and stearic (2.2 +/- 0.7). This method was also applied to the analysis of two commercial soap formulations. The first soap gave a fatty acid profile that included: lauric (19.4% +/- 0.8), myristic (9.6% +/- 0.5), palmitoleic (1.9% +/- 0.3), palmitic (16.3% +/- 0.9), linoleic (5.6% +/- 0.4), oleic (37.1% +/- 0.8), and stearic (10.1% +/- 0.7) and that of the second soap was: lauric (9.3% +/- 0.3), myristic (3.8% +/- 0.5), palmitoleic (3.1% +/- 0.8), palmitic (19.4% +/- 0.8), linoleic (4.9% +/- 0.7), oleic (49.5% +/- 1.1), and stearic (10.0% +/- 0.9). The MALDI-TOFMS method described in this communication is simpler and less time-consuming than the established transesterification method that is coupled with analysis by gas chromatography/mass spectrometry (GC/MS). The new method could be used routinely to determine the qualitative fatty acid composition of vegetable oils, and, when fully validated by comparison with standard analytical methodologies, should provide a relatively fast quantitative measurement of fatty acid mixtures and/or soap formulations that contain saturated and unsaturated hydrocarbon moieties.     

Determination of acrolein in french fries by solid-phase microextraction gas chromatography and mass spectrometry

The frying of foods in the home can be a cause of indoor pollution due to the formation of acrolein. The emission of acrolein formed during frying in soybean, corn, canola, sunflower and palm oils was studied. A GC/MS method has been developed to determine acrolein in French fries using SPME as the sampling technique after derivatization with 2,4-dinitrophenylhydrazine (DNPH). Optimum SPME conditions included desorption at 250°C for 2min after an adsorption time of 10min at room temperature. The method presented good resolution, repeatability, detection and quantification limits, and linearity of response. French fries were prepared in five different oils with four frying steps. The results showed that changes in acrolein concentration occurred after frying potatoes in different types of oil and at different frying cycles. Potatoes fried in soybean oil contained the lowest concentration of acrolein. Shoestring potatoes contained a lower concentration of acrolein than potato chips and French fries, respectively, because of the higher surface/volume ratio.     

Microbial synthesis and characterization of physiochemical properties of polyhydroxyalkanoates (PHAs) produced by bacteria isolated from activated sludge obtained from the municipal wastewater works in Hong Kong

The first objective of this study was the measurement of physical properties of P(3HB-co-3HV) copolymers with different (hydroxybutyrate) HB to (hydroxyvalerate) HV ratios produced by Bacillus cereus (TRY2) isolated from activated sludge. The 3HV PHBV copolymers were 0.05, 22.6, 39.2, 54.1, and 69.1 mol%, respectively. The second objective was to study possible wastewater treatment and production of PHAs at the same time by B. cereus (TRY2) and Pseudomonas spp. (TOB17) (both were isolated from activated sludge), recombinant Bacillus DH5α, and a combination of the above three bacteria. The results were satisfactory; the maximum COD and TOC of the sewage sludge reduced were 53.5% and 67.5%, respectively.     

Effect of gamma irradiation on cell lysis and polyhydroxyalkanoate produced by Bacillus flexus

Bacillus flexus cultivated on sucrose and sucrose with plant oil such as castor oil produced polyhydroxybutyrate (PHB), a homopolymer of polyhydroxyalkanoate (PHA) and PHA copolymer (containing hydroxybutyrate and hexanoate), respectively. Gamma irradiation of these cells (5–40 kGy) resulted in cell damage and aided in the isolation of 45% and 54% PHA on biomass weight, correspondingly. Molecular weight of PHB increased from 1.5×10⁵ to 1.9×10⁵ after irradiation (10 kGy), with marginal increase of tensile strength from 18 to 20 MPa. At the same irradiation dosage, PHA copolymer showed higher molecular weight increase from 1.7×10⁵ to 2.3×10 ⁵ and tensile strength from 20 to 35 MPa. GC, GC–MS, FTIR and ¹H NMR were used for the characterization of PHA. Gamma irradiation seems to be a novel technique, to induce cross-linking and molecular weight increase of PHA copolymer and aid in easy extractability of intracellular PHA, simultaneously.     

Compatibilizing effect of a graft copolymer on bacterial poly(3‐hydroxybutyrate)/poly(methyl methacrylate) blends

Blends of isotactic (natural) poly(3‐hydroxybutyrate) (PHB) and poly(methyl methacrylate) (PMMA) are partially miscible, and PHB in excess of 20 wt % segregates as a partially crystalline pure phase. Copolymers containing atactic PHB chains grafted onto a PMMA backbone are used to compatibilize phase‐separated PHB/PMMA blends. Two poly(methyl methacrylate‐g‐hydroxybutyrate) [P(MMA‐g‐HB)] copolymers with different grafting densities and the same length of the grafted chain have been investigated. The copolymer with higher grafting density, containing 67 mol % hydroxybutyrate units, has a beneficial effect on the mechanical properties of PHB/PMMA blends with 30–50% PHB content, which show a remarkable increase in ductility. The main effect of copolymer addition is the inhibition of PHB crystallization. No compatibilizing effect on PHB/PMMA blends with PHB contents higher than 50% is observed with various amounts of P(MMA‐g‐HB) copolymer. In these blends, the graft copolymer is not able to prevent PHB crystallization, and the ternary PHB/PMMA/P(MMA‐g‐HB) blends remain crystalline and brittle. © 2002 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 40: 1390–1399, 2002     

Influence of feeding strategies of mixed microbial cultures on the chemical composition and microstructure of copolyesters P(3HB‐co‐3HV) analyzed by NMR and statistical analysis

NMR spectroscopy was applied for quantitative and qualitative characterization of the chemical composition and microstructure of a series of poly(3‐hydroxybutyrate‐co‐3‐hydoxyvalerate) copolymers, P(3HB‐co‐3HV), synthesized by mixed microbial cultures at several different feeding strategies. The monomer sequence distribution of the bacterially synthesized P(3HB‐co‐3HV) was defined by analysis of their high‐resolution 1D ¹³C NMR and 2D ¹H/¹³C HSQC and ¹H/¹³C HMBC NMR spectra. The results were verified by employment of statistical methods and suggest a block copolymer microstructure of the P(3HB‐co‐3HV) copolymers studied. Definitive distinction between block copolymers or a mixture of random copolymers could not be achieved. NMR spectral analysis indicates that the chemical composition and microstructure of the copolymers can be tuned by choosing a correct feeding strategy. Copyright © 2009 John Wiley & Sons, Ltd.     

Biosurfactant Production by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Grown in Residual Soybean Oil

Pseudomonas aeruginosa PACL strain, isolated from oil-contaminated soil taken from a lagoon, was used to investigate the efficiency and magnitude of biosurfactant production, using different waste frying soybean oils, by submerged fermentation in stirred tank reactors of 6 and 10 l capacities. A complete factorial experimental design was used, with the goal of optimizing the aeration rate (0.5, 1.0, and 1.5 vvm) and agitation speed (300, 550, and 800 rpm). Aeration was identified as the primary variable affecting the process, with a maximum rhamnose concentration occurring at an aeration rate of 0.5 vvm. At optimum levels, a maximum rhamnose concentration of 3.3 g/l, an emulsification index of 100%, and a minimum surface tension of 26.0 dynes/cm were achieved. Under these conditions, the biosurfactant production derived from using a mixture of waste frying soybean oil (WFSO) as a carbon source was compared to production when non-used soybean oil (NUSO), or waste soybean oils used to fry specific foods, were used. NUSO produced the highest level of rhamnolipids, although the waste soybean oils also resulted in biosurfactant production of 75–90% of the maximum value. Under ideal conditions, the kinetic behavior and the modeling of the rhamnose production, nutrient consumption, and cellular growth were established. The resulting model predicted data points that corresponded well to the empirical information.     

Degradation of medium-chain-length polyhydroxyalkanoates in tropical forest and mangrove soils

Bacterial polyhydroxyalkanoates (PHAs) are perceived to be a suitable alternative to petrochemical plastics because they have similar material properties, are environmentally degradable, and are produced from renewable resources. In this study, the in situ degradation of medium-chain-length PHA (PHA_MCL) films in tropical forest and mangrove soils was assessed. The PHA_MCL was produced by Pseudomonas putida PGA1 using saponified palm kernel oil (SPKO) as the carbon source. After 112 d of burial, there was 16.7% reduction in gross weight of the films buried in acidic forest soil (FS), 3.0% in the ones buried in alkaline forest soil by the side of a stream (FSst) and 4.5% in those buried in mangrove soil (MS). There was a slight decrease in molecular weight for the films buried in FS but not for the films buried in FSst and in MS. However, no changes were observed for the melting temperature, glass transition temperature, monomer compositions, structure, and functional group analyses of the films from any of the burial sites during the test period. This means that the integral properties of the films were maintained during that period and degradation was by surface erosion. Scanning electron microscopy of the films from the three sites revealed holes on the film surfaces which could be attributed to attack by microorganisms and bigger organisms such as detritivores. For comparison purposes, films of polyhydroxybutyrate (PHB), a short-chain-length PHA, and polyethylene (PE) were buried together with the PHA_MCL films in all three sites. The PHB films disintegrated completely in MS and lost 73.5% of their initial weight in FSst, but only 4.6% in FS suggesting that water movement played a major role in breaking up the brittle PHB films. The PE films did not register any weight loss in any of the test sites.     

多环芳烃指纹用于渤海采油平台原油的鉴别

采用气相色谱/质谱方法,对渤海海上4个不同区块、5个平台的6口油井原油进行了烷基化多环芳烃系列化合物和美国环保署（EPA）优先控制多环芳烃系列化合物的准确定性定量分析。通过多环芳烃原始指纹谱图、多环芳烃组分分布模式和特征比值的比较对上述原油进行鉴别。结果证明不同区块的原油中多环芳烃指纹信息不尽相同,即使在同一平台不同油井中所产的原油其指纹也存在一定差异。为确保原油鉴别的准确性,分析过程中必须在仪器的稳定性和样品前处理方面实施严格的质量控制措施。     

Tracking juniper berry content in oils and distillates by spectral deconvolution of gas chromatography/mass spectrometry data

The complex nature of botanicals and essential oils makes it difficult to identify all of the constituents by gas chromatography/mass spectrometry (GC/MS) alone. In this paper, automated sequential, multidimensional gas chromatography/mass spectrometry (GC-GC/MS) was used to obtain a matrix-specific, retention time/mass spectrometry library of 190 juniper berry oil compounds. GC/MS analysis on stationary phases with different polarities confirmed the identities of each compound when spectral deconvolution software was used to analyze the oil. Also analyzed were distillates of juniper berry and its oil as well as gin from four different manufacturers. Findings showed the chemical content of juniper berry can be traced from starting material to final product and can be used to authenticate and differentiate brands.     

Analysis of poly-beta-hydroxybutyrate in environmental samples by GC-MS/MS

Application of gas chromatography-mass spectrometry (GC-MS) can significantly improve trace analyses of compounds in complex matrices from natural environments compared to gas chromatography only. A GC-MS/MS technique for determination of poly-beta-hydroxybutyrate (PHB), a bacterial storage compound, has been developed and used for analysis of two soils stored for up to 319 d, fresh samples of sewage sludge, as well as a pure culture of Bacillus megaterium. Specific derivatization of beta-hydroxybutyrate (3-OH C4:0) PHB monomer units by N-tert-butyl-dimethylsilyl-N-methyltrifluoracetamide (MTBSTFA) improved chromatographic and mass spectrometric properties of the analyte. The diagnostic fragmentation scheme of the derivates tert-butyldimethylsilyl ester and ether of beta-hydroxybutyric acid (MTBSTFA-HB) essential for the PHB identification was shown. The ion trap MS was used, therefore the scan gave the best sensitivity and with MS/MS the noise decreased, so the S/N was better and also with second fragmentation the amount of ions increased compared to SIM. The detection limit for MTBSTFA-HB by GC-MS/MS was about 10(-13) g microL(-1) of injected volume, while by GC (FID) and GC-MS (scan) it was around 10(-10) g microL(-1) of injected volume. Sensitivity of GC-MS/MS measurements of PHB in arable soil and activated sludge samples was down to 10 pg of PHB g(-1) dry matter. Comparison of MTBSTFA-HB detection in natural soil sample by GC (FID), GC-MS (scan) and by GC-MS/MS demonstrated potentials and limitations of the individual measurement techniques.     

Simultaneous kinetic determination of 3-hydroxybutyrate and 3-hydroxyvalerate in biopolymer degradation processes

A new kinetic method is proposed for the simultaneous determination of 3-hydroxybutyrate (3HB) and 3-hydroxyvalerate (3HV) based on the different rate of the 3-hydroxybutyrate dehydrogenase-catalysed reactions of these compounds with coenzyme NAD⁺. A flow injection system with two reactors of immobilised 3-hydroxybutyrate dehydrogenase and dual detection is used. The concentrations of NADH produced after two different reaction times are measured by fluorometry or spectrophotometry and multivariate linear calibration is applied for quantification. Concentrations of 3HB and 3HV between 1 × 10⁻⁶ and 1 × 10⁻⁴ M can be determined at an average sampling frequency of 20 h⁻¹. In contrast to usual methods, the proposed here makes possible the discrimination of 3HB and 3HV without previous separation so that usual extraction with chlorinated solvents and/or chromatographic separation is not required. The method is of interest in a wide variety of fields concerning PHAs, as it can provide information on the degradation rate and mechanism, composition and structure of these polymers. Its applicability has been proved through the determination of 3HB and 3HV in the digests of some chemically degraded commercial PHAs.     

Use of headspace solid-phase microextraction for the quantification of poly(3-hydroxybutyrate) in microbial cells

Biopolymers such as poly(hydroxyalkanoates) (PHAs) have received much attention due to their physico-chemical properties, biodegradability, and biocompatibility that make them good candidates for industrial and medical applications. Produced by some microorganisms PHAs accumulate within the cells of these organisms. The optimization of microbial processes to produce PHAs at a lower cost requires rapid and accurate techniques for quantification of the biopolymer in biomass. The present study describes a method based on solid-phase microextraction (SPME) coupled to gas chromatography (GC) for the determination of poly(3-hydroxybutyrate) (PHB) in Alcaligenes latus cells. First PHB was depolymerized by either methanolic or hydrolytic digestion into methyl 3-hydroxybutyrate (Me-3-HB) or crotonic acid (CA), respectively. The resulting analytes were then subjected to analysis by headspace SPME/GC with flame ionization detection (FID). The two depolymerization/SPME/GC-FID methods were optimized and applied to the analysis of PHB in bacterial biomass harvested from a fermentation process that uses A. latus. Results were compared with those obtained using GC-FID analysis of MeOH/CHCl(3) digested samples. Excellent agreement was found between the three methods but the two SPME-based methods were environmentally friendly and easier to perform.     

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.     

Tetrahydrogestrinone: discovery, synthesis, and detection in urine

Tetrahydrogestrinone (18a-homo-pregna-4,9,11-trien-17beta-ol-3-one or THG) was identified in the residue of a spent syringe that had allegedly contained an anabolic steroid undetectable by sport doping control urine tests. THG was synthesized by hydrogenation of gestrinone and characterized by mass spectrometry and NMR spectroscopy. We developed and evaluated sensitive and specific methods for rapid screening of urine samples by liquid chromatography/tandem mass spectrometry (LC/MS/MS) of underivatized THG (using transitions m/z 313 to 241 and 313 to 159) and gas chromatography/high-resolution mass spectrometry (GC/HRMS) analysis of the combination trimethylsilyl ether-oxime derivative of THG (using fragments m/z 240.14, 254.15, 267.16, and 294.19). A baboon administration study showed that THG is excreted in urine.     

聚β－羟基烷酸酯热分解行为的直接裂解质谱研究

用ＤＰＭＳ、ＴＧ、ＤＴＡ等方法研究了聚β－羟基丁酸酯（ＰＨＢ）及其共聚物Ｐ（ＨＢ－ｃｏ－ＨＶ）的热分解行为和某些结构性质．能提供多至７个链节重复单元的裂解碎片．可较好地反应共聚物的组成和分布．结果表明：聚β－羟基烷酸酯的热分解具有较高的选择性，通过β－Ｈ转移反应形成由羧基和烯烃结尾的齐聚物．齐聚物准分子离子可进一步脱去一分子水．     

Biomedical Investigations of Biodegradable PHAs

This work is a review of the results of biomedical studies of polymer devices (films, fibers, microparticles, 3D implants) made from resorbable PHAs synthesized by the bacterium Wautersia (Ralstonia) eutropha B5786, using the technology developed at the Institute of Biophysics of the Siberian Branch of the Russian Academy of Sciences. Two types of PHAs – polyhydroxybutyrate (PHB) and a hydroxybutyrate/hydroxyvalerate copolymer (PHB/PHV) – have been proven to be biocompatible in vitro in cultures of fibroblasts, endothelial cells, hepatocytes, and osteoblasts, and in short- and long-duration experiments on animals. Polymer films and membranes have been found to be usable as scaffolds for functioning cells and monofilament suture fibers – for stitching muscular-fascial wounds and in abdominal surgery. Ectopic bone formation assay and experiments with the model of segmental osteotomy showed that 3D PHB and PHB/HA implants can be used for reparative osteogenesis. The paper reports beneficial results of using polymers to repair bone defects in oral surgery.     

Modeling mechanical properties of polyhydroxyalkanoate during degradation in animal tissue

Polyhydroxyalkanoates (PHAs) are a family of biodegradable and biocompatible polymers produced by several species microorganisms that possess favorable mechanical properties (e.g. strength and elongation properties). Different types of PHA polymers have been used in medical applications. However, in order to better understand the use of this polymer in the different applications, a thorough understanding of the kinetics of in vivo degradation is one of the major requirements. In this study, poly(3‐hydroxybutyrate) (PHB) was subcutaneously implanted in mice and incubated for 2, 4, 8, or 16 weeks. After removal from the animal, the strength, elongation, mass loss, and enthalpy of the PHB were tested for each time point. From these data, a mathematical model was generated by Rayleigh's method of dimensional analysis, where polymer strength over tissue contact time could be predicted. To prove the model, previous data obtained by our group were used: poly(3‐hydroxybutyrate‐co‐3‐hydroxyhexanoate) [P(HB‐co‐HHx)] incubation in the presence of human embryonic kidney cells (HEK). It was found that the developed model was aligned with experimental results, could predict the strength of the polymer when in contact with cells, and the predicted strength follows the trend of the experimental data. Also, the dimensionless constant (K) value associated with the model is different for both experiments, where this constant, produced via experimental data, is used for construction of a homogeneous equation. Copyright © 2017 John Wiley & Sons, Ltd.