Microstructure of bacterially synthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Kamiya, Noriho; Yamamoto, Yasuhiko; Inoue, Yoshio; Chûjô, Riichirǒ; Doi, Yoshiharu

doi:10.1021/ma00194a030

Cited by 182 publications

(204 citation statements)

References 2 publications

(2 reference statements)

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“…The compositions and sequence distributions of P(3HB-co-4HB) copolymers were determined by analysis of 1 Hand 13 C NMR spectra [11], which were recorded on a JEOL GSX-270 spectrometer. The 1 H NMR spectra were recorded at 808C for a CDCl 3 solution of P(3HB-co-4HB) (10 mg ml 21 ) with 4.7 ms pulse width, 5 s pulse repetition, 4000 Hz spectral width, 16K data points and 32 accumulations.…”

Section: Analytical Proceduresmentioning

confidence: 99%

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Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) produced by Comamonas acidovorans

Mitomo

Hsieh

Nishiwaki

et al. 2001

Polymer

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Poly(3-hydoxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] was biosynthesized by Comamonas acidovorans with mixed carbon sources of n-butyric acid and 1,4-butanediol. P(3HB) and P(3HB-co-94 mol% 4HB) were obtained by fermentation with sole carbon source of n-butyric acid and 1,4-butanediol, respectively. The content of 4HB component in P(3HB-co-4HB) increased as the composition of 1,4-butanediol in mixed carbon sources increased. These biosynthesized copolymers are not homogeneous, but mixtures of copolymers having different 4HB contents. Fractionation was carried out to obtain fractionated copolymers having a narrower distribution of composition. The change in structural and physical properties of the fractionated copolymers were investigated against the 4HB contents. Crystallinities of P(3HB-co-4HB)s were estimated using density values and the heat of fusion of P(3HB) measured was 125 J g 21 and that of P(4HB) was 76 J g 21 . q

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Section: Analytical Proceduresmentioning

confidence: 99%

“…The composition of both components could be also estimated using areas of peak 3 (CH of 3HB) and peak 8 (CH 2 of 4HB), though it was usually estimated from 1 H NMR spectrum. The spectra of carbonyl The other expression of randomness in sequence distribution is as follows [11]:…”

Section: Microbial Synthesis Of P(3hb-co-4hb)mentioning

confidence: 99%

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) produced by Comamonas acidovorans

Mitomo

Hsieh

Nishiwaki

et al. 2001

Polymer

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“…High resolution NMR spectroscopy has been used as a powerful tool to determine the microstructural heterogeneity (specifically, heterogeneity in local sequences) of the microbial copolyesters (2,4,5). It is possible to assign the 13 C resonances present in local dyad, triad, and tetrad sequences.…”

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confidence: 99%

“…1 (the steady-state method, see text for the details). The suggested microstructural heterogeneity in the 3HB-4HB copolymer was detected in detail at a molecular level by using high resolution 13 C NMR spectroscopy, in which each resonance peak is usually shifted by the presence of a different neighboring monomer unit, and thus every shifted resonance has information on the local monomer sequence of the copolyester (2,4,5). For the copolymers composed of 3HB and 4HB monomer units, the sequences of dyad, triad, and tetrad were assigned assuming a first order Markovian random copolymerization in cells (4).…”

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confidence: 99%

Local Sequence Dependence of Polyhydroxyalkanoic Acid Degradation in Hydrogenophaga pseudoflava

Yoon

Choi

1999

Journal of Biological Chemistry

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The first order intracellular degradation of various polyhydroxyalkanoic acid (PHA) inclusions in Hydrogenophaga pseudoflava cells was investigated by analyzing the compositional and microstructural changes of the PHA using gas chromatography, 13 C NMR spectroscopy, and differential scanning calorimetry. Two types of PHA, copolymers and blend-type polymers, were separately accumulated in cells for comparison. The constituent monomers were 3-hydroxybutyric acid (3HB), 4-hydroxybutyric acid (4HB), and 3-hydroxyvaleric acid (3HV). It was found that the 3HB-4HB copolymer was degraded only when the polymer contained a minimal level of 3HB units. With the cells containing a 3HB/4HB blend-type polymer, only poly(3HB) was degraded, whereas poly(4HB) was not degraded, indicating the totally inactive nature of the intracellular depolymerase against poly(4HB). On the basis of the magnitude of the first order degradation rate constants, the relative substrate specificity of the depolymerase toward the constituting monomer units was determined to decrease in the order 3HB > 3HV > 4HB. 13 C NMR resonances of the tetrad, triad, and dyad sequences were analyzed for the samples isolated before and after degradation experiments. The results showed that the intracellular degradation depended on the local monomer sequence of the copolymers. The relative substrate specificity of the depolymerase determined from the NMR local sequence analysis agreed well with that obtained from the kinetics analysis. It is suggested that, without isolation and purification of the intracellular PHA depolymerase and "native" PHA substrates, the relative specificity of the enzyme as well as the microstructural heterogeneity of the PHA could be determined by measuring in situ the first order degradation rate constants of the PHA in cells.

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“…NMR spectra were used to study composition as well as study sequence monomer distribution of the produced PHA using two parameters: D and R, as reported by Kamiya et al (Kamiya et al 1989) and Žagar et al (Žagar et al 2006) The main factors impacting the extraction are: concentration of biomass in solvent, the biomass particle size, cell-wall structure, the length of the extraction, and the temperature (Kshirsagar et al 2013). A first set of extraction experiments using 2-butanol at temperatures ranging from 70 °C to 150 °C in 10 °C increments was made using PHA-rich biomass.…”

Section: Nmr)mentioning

confidence: 99%

Composition of mixed culture PHA biopolymers and implications for downstream processing

Montano-Herrera¹

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Polyhydroxyalkanoates (PHAs) are biopolymers synthesised and accumulated by most bacteria for carbon and energy storage. They have properties and applications comparable to petrochemical thermoplastics. Although PHAs are produced at high yields using pure biological cultures, the use of mixed cultures can significantly reduce the production costs and make use of waste streams to produce environmentally sustainable materials. In order to produce mixed culture PHAs of relevance for broader industry applications it is necessary to develop the ability to tailor polymers with diversified mechanical properties through understanding and controlling the monomer composition and compositional distribution.Diverse and complex PHA polymer structures have been achieved in mixed microbial cultures using time-based feeding strategies. However, PHA monomer composition and compositional distribution in PHA random and blocky copolymers are sensitive to substrate feeding history, making more complex the prediction of final PHA content and composition.In this sense, a better understanding of the changing cell physiologies that develop in response to different feeding strategies and substrates is necessary to design optimised feeding strategies and process control algorithms for PHA production.This thesis presents for the first time a comprehensive flux characterisation of monomer development during mixed culture PHA accumulation concurrent with biomass growth using Metabolic Flux Analysis (MFA). A dynamic trend in active biomass growth and in polymer composition was observed and was consistent over replicate accumulations. Monomer (3-hydroxybutyrate and 3-hydroxyvalerate) incorporation into poly(3-hydroxybutyate-co-3-hydroxyvalerate) copolymers in a pilot scale production system was evaluated based on published models that describe polymer production during PHA accumulation. However, it was found that these existing models could not describe the fluctuations in the proportion of Additionally, thermal degradation of mixed culture PHAs during melt processing was assessed by Near-Infrared (NIR) spectroscopy coupled to Multivariate Data Analysis. It was shown that, with correct pretreatment, a copolymeric product that was much more stable to extrusion processing than commercially available PHA was produced.Overall, this thesis explores both the fundamental and applied aspects of PHA production by mixed microbial cultures with concurrent active biomass growth. The use of computational iii tools to explore and help understand the underlying metabolic processes was explored.Polymer composition seems to follow a very complex regulation processes which can be described through the incorporation of more detailed reactions in current metabolic models.Furthermore, this thesis gives further insight into the fundamental properties of the materials produced and an assessment of their potential for degradation during processing.iv

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Microstructure of bacterially synthesized poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

Cited by 182 publications

References 2 publications

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) produced by Comamonas acidovorans

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) produced by Comamonas acidovorans

Local Sequence Dependence of Polyhydroxyalkanoic Acid Degradation in Hydrogenophaga pseudoflava

Composition of mixed culture PHA biopolymers and implications for downstream processing

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