A simple HPLC-MS method for the quantitative determination of the composition of bacterial medium chain-length polyhydroxyalkanoates (original) (raw)
Related papers
Journal of Chromatography A, 2007
Standard chromatographic methods for the quantification of bacterial poly(3-hydroxyalkanoate) (PHA) proved to be inappropriate for the analysis of medium-chain-length PHA (mcl-PHA). Transesterification catalyzed by protic acids is not quantitative for mcl-PHA under common conditions due to slow reaction kinetics and formation of side-products in case of functionalized side-chains. To circumvent these limitations, an improved method for the quantification of mcl-PHA by GC-FID was developed. Boron trifluoride in methanol was successfully applied to quantitatively methanolyse different mcl-PHA (recovery >94%). This novel method is well-suited for the analysis of purified mcl-PHA as well as for mcl-PHA in biomass.
Review of the Developments of Bacterial Medium-Chain-Length Polyhydroxyalkanoates (mcl-PHAs)
Bioengineering
Synthetic plastics derived from fossil fuels—such as polyethylene, polypropylene, polyvinyl chloride, and polystyrene—are non-degradable. A large amount of plastic waste enters landfills and pollutes the environment. Hence, there is an urgent need to produce biodegradable plastics such as polyhydroxyalkanoates (PHAs). PHAs have garnered increasing interest as replaceable materials to conventional plastics due to their broad applicability in various purposes such as food packaging, agriculture, tissue-engineering scaffolds, and drug delivery. Based on the chain length of 3-hydroxyalkanoate repeat units, there are three types PHAs, i.e., short-chain-length (scl-PHAs, 4 to 5 carbon atoms), medium-chain-length (mcl-PHAs, 6 to 14 carbon atoms), and long-chain-length (lcl-PHAs, more than 14 carbon atoms). Previous reviews discussed the recent developments in scl-PHAs, but there are limited reviews specifically focused on the developments of mcl-PHAs. Hence, this review focused on the mcl-...
Isolation and Screening of Polyhydroxyalkanoates 1
Isolation and Screening of Polyhydroxyalkanoates Producing Bacteria from Soils of Fruits, Food and Fermented Cassava Processing Sites., 2018
Polyhydroxyalkanoates(PHAs)are biodegradable polymers accumulated by bacteria under unbalanced growth conditions. They have chemical and mechanical properties comparable to synthetic polymers which make them candidate alternatives in plastic production. The objective of this work was to isolate and screen for bacteria with potential for PHAs production. Bacteria were isolated on nutrient agar using the pour-plate method and screened for PHAs production using the viable colony andSudan Black B staining methods. The colonial and cellular morphologies of the PHAs positive isolates were determined. Catalase, oxidase and starch hydrolysis tests were also done. DNA was extracted from each isolate and amplification of the PHAs synthase gene was achieved using four polymorphic primers. The detection of PCR amplification on agarose gel formed the basis of classification of each isolate. A total of 53 bacteria was isolated out of which 6 strongly PHAs positive were selected. The isolates produced orange, blue and purple fluorescence with Nile Red and Nile Blue A. Blue-back intracellular bodies were observed with Sudan Black B staining. Grams reaction revealed all isolates as Gram positive rods and cocci. Four isolates were positive for catalase, 5 for oxidase and all for starch hydrolysis tests. Analysis of the PCR amplicons revealed two isolates as class IV PHAs producing bacteria. Our results show that bacteria from soils of fruit, food and cassava processing sites have great potentials for PHAs production.
Bacterial polyhydroxyalkanoates: Opportunities, challenges, and prospects
Journal of Cleaner Production, 2020
Excessive utilization of synthetic plastics has led to a major detrimental impact on the environment. Plastic pollution and accumulation in water bodies have threatened the survival of marine life. Plastic pollution can be prevented by using biopolymers that are eco-friendly and can be naturally produced by certain living organisms. The biopolymers have environmental advantages over synthetic plastics, such as biodegradability and biocompatibility. In comparison to plants and other microbial systems, bacteria can accumulate a high amount of polyhydroxyalkanoates (PHAs). However, the major stumbling block in the production of bacterial PHAs is its low cost-effectiveness due to costs associated with fermentaion and down-stream processing. In consideration with the above properties, opportunities and challeges associated with bacterial PHAs, this review focuses on structural diversity of PHAs, biosynthesis mechanism in bacteria, biodegradation, life cycle analysis, and environmental impact of bioplastic production. It further enumerates the advanced tools and techniques for bacterial PHA production, along with various factors affecting the commercialization of bioplastics. Extraction methods, down-stream processing, and biomedical applications of PHAs are also discussed. The opportunities and challenges in the commercialization of bacterial PHAs along with future scenario and environmental sustainability are presented for the purpose of fostering sustainable development.
Methods for identification, quantification and characterization of polyhydroxyalkanoates
International Journal of Bioassays, 2016
Polyhydroxyalkanoates (PHAs) are the polymers of hydroxyalkanoates that accumulate as a carbon/energy storage material in various microorganisms. PHAs have been attracting considerable attention as biodegradable substitutes for conventional polymers, because of their similar material properties to conventional plastics. A number of review articles on the general features of PHAs, the physiology, genetics and molecular biology, the development of PHAs having novel monomer constituents, production processes, biodegradation of PHAs are available. Recently much effort has been devoted to develop a process for the economical production of PHAs. The isolation, analysis and characterization of PHAs are important factors for any process development. A number of methods have been developed for the analysis of PHAs. This paper is an effort to compile the methods available for the identification, quantitative estimation and characterization of PHAs. The methods described in this paper include-...
Gas-chromatographic analysis of poly(3-hydroxyalkanoates) in bacteria
Biotechnology Techniques, 1994
The accuracy and reproducibility of the gas-chromatographic method for the analysis of PHB and PHA in whole cells of Alcaligenes eutrophus H16 and Pseudomonas putida KT2442 were determined. It was found that for analysis of PHA the methanolysis time in the assay had to be increased to 4 h. Accuracy of the PHB and PHA assay were 0.018 mg and 0.304 mg respectively, based on estimation of the measurement error.
We synthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3-HB-co-3-HV)] copolymer having different contents of 3- hydroxyvalerate (3-HV) units (16.04, 16.3, 24.95, 25.62, and 16.52 mol % 3-HV) with different yields of polyhydroxyalkanoates (PHAs) by feeding with different cooking oils and with Alkaliphilus oremlandii OhILAs strain. The PHA production efficiency of the Alkaiphilus strain was compared with that of the control strain, Bacillus cereus. The synthesis of each PHA biopolymer was performed with different toxic spent oils as the sole carbon source in an oil-in-water-based microemulsion medium. We observed that the productivity of the poly(3- hydroxybutyrate) [P(3-HB)] copolymer from the Alkaliphilus strain was higher than those of the PHAs isolated from B. cereus and the Escherichia coli XL1B strain. The synthesized PHA copolymers were characterized by 1H-NMR and Fourier transform infrared (FTIR) spectroscopy. In the 1H-NMR spectra, a doublet resonance peak at 1.253 ppm of the/ methyl protons of the 3-hydroxybutyrate (3-HB) side group and one at 0.894 ppm due to the methyl protons of the 3-HV side group indicated the presence of 3-HB and 3-HV units in the copolymer. The chemical shift values at 1.25 and 2.2 ppm, due to the resonance absorption peaks of the methyl protons and methylene protons, confirmed the synthesis of the P(3-HB) homopolymer. From the FTIR spectra, a strong C@O stretching frequency in the range of 1745–1727 cm21, together with strong CAO stretching bands near 1200 cm21 and a strong band near 3400 cm21, confirmed the synthesis of P(3-HB-co-3-HV) and P(3-HB). Thus, waste cooking oil as a substrate provided an alternate route for the formation of P(3-HB-co-3- HV) and P(3-HB) by Alkaliphilus and E. coli strains, respectively
Journal of Chemical Technology and Biotechnology, 2007
BACKGROUND: Medium-chain-length polyhydroxyalkanoates (PHAs) are biodegradable polyesters accumulated intracellularly as energy resources by bacterial species such as Pseudomonas putida. The most popular method for PHA recovery is solvent extraction using trichloromethane (chloroform) and methyl alcohol (methanol). An alternative method is enzymatic treatment, which eliminates usage of these hazardous solvents. This research focuses on the characterization of PHAs recovered by enzymatic treatments and ultrafiltration. Comparisons are made with conventional solvent extracted PHA.RESULTS: The purity of PHA in water suspension recovered by enzymatic treatments as analyzed by gas chromatography was 92.6%. Enzymatically recovered PHA was comparable to conventional solvent-extracted PHA, which had a purity of 95.5%. PHA was further characterized for functional group analysis, structural composition analysis and molecular weight determination. It was found that the molecular weight of the PHA recovered by enzymatic treatment was less than solvent-extracted PHA, probably due to degradation of the lipopolysaccharide layer. However, functional group and structural composition analyses showed similar results for PHA recovered by both methods.CONCLUSION: PHAs recovered through enzymatic digestion treatment have good comparability with solvent-extracted PHAs. Thus enzymatic digestion has great potential as an alternative recovery method. Copyright © 2007 Society of Chemical Industry
Production of Polyhydroxyalkanoates, a bacterial biodegradable polymer
African Journal of Biotechnology, 2004
There has been considerable interest in the development and production of biodegradable polymer to solve the current problem of pollution caused by the continuous use of synthetic polymer of petroleum origin. Polyhydroxyalkanoates (PHAs) are known to be accumulated as intracellular inclusion in some bacteria. The materials properties exhibited by PHAs, ranging from stiff, brittle to rubber-like makes it a close substitute for the synthetic plastic. The high cost of PHAs production has restricted its applications. The possibility of producing this polymer commercially and at comparable cost has been the main focus in this area.
Proceedings of the National Academy of Sciences, India Section B: Biological Sciences, 2015
Municipal sewage triggers a stress prone environment to accumulate polyhydroxyalkanoates (PHAs) in the cytosol of bacteria. In view of that, different Bacillus species were isolated from municipal sewage and screened for evaluating their efficacy of PHA production. Growth parameters such as temperature, pH, glucose concentration and carbon nitrogen combinations were optimized with respect to higher biomass production as it is analogous to PHA accumulation. Under optimized conditions, the Bacillus species produced 3.09 g/L of PHAs which was estimated as a higher yield in comparison to other similar strains. Fourier transform infrared spectroscopic analysis of the extracted polyhydroxybutyrate confirmed the distinct peak corresponding to C=O group, whereas proton nuclear magnetic resonance (1 H NMR) and differential scanning colorimetric analysis exhibited detailed insight of its chemical structure and properties by reflecting monomeric unit. The high yielding bacterial isolate was identified by 16S rDNA sequencing and the sequence was confirmed as Bacillus subtilis with an accession no. KP172548 after submission to NCBI data base. The potential bacterium may be further exploited for cost effective and mass scale production of biopolymer.