Abstract
Poly[(R)-3-hydroxyalkanoate]s or PHAs are aliphatic polyesters produced by numerous microorganisms. They are accumulated as energy and carbon reserve in the form of small intracellular vesicles. Poly[(R)-3-hydroxybutyrate] (PHB) is the most ubiquitous and simplest PHA. An atomic force microscope coupled with a tunable infrared laser (AFM-IR) was used to record highly spatially resolved infrared spectra of commercial purified PHB and native PHB within bacteria. For the first time, the crystallinity degree of native PHB within vesicle has been directly evaluated in situ without alteration due to the measure or extraction and purification steps of the polymer: native PHB is in crystalline state at 15% whereas crystallinity degree reaches 57% in commercial PHB. Chloroform addition on native PHB induces crystallization of the polymer within bacteria up to 60%. This possibility of probing and changing the physical state of polymer in situ could open alternative ways of production for PHB and others biopolymers.
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Nigmatullin R, Thomas P, Lukasiewicz B, Puthussery H, Roy I. Polyhydroxyalkanoates, a family of natural polymers, and their applications in drug delivery. J Chem Technol Biotechnol. 2015;90:1209–21.
Chen GQ, Hajnal I, Wu H, Lv L, Ye J. Engineering biosynthesis mechanisms for diversifying polyhydroxyalkanoates. Trends Biotechnol. 2015;33:565–74.
Lemoigne M. Produits de déshydration et de polymérisation de l’acide ß-oxobutyrique. Bull Soc Chim Biol. 1926;8:770–82.
Lenz RW, Marchessault RH. Bacterial polyesters: biosynthesis, biodegradable plastics and biotechnology. Biomacromolecules. 2005;6:1–8.
Luengo JM, Garcıa B, Sandoval A, Naharro G, Olivera ER. Bioplastics from microorganisms. Curr Opin Microbiol. 2003;6:251–60.
Thomson N, Summers D, Sivaniah E. Synthesis, properties and uses of bacterial storage lipid granules as naturally occurring nanoparticles. Soft Matter. 2010;6:4045–57.
Steinbüchel A, Valentin HE. Diversity of bacterial polyhydroxyalkanoic acids. FEMS Microbiol Lett. 1995;128:219–28.
Noda I, Green PR, Satkowski MM, Schechtman LA. Preparation and properties of a novel class of polyhydroxyalkanoate copolymers. Biomacromolecules. 2005;6:580–6.
Barnard GN, Sanders JKM. Observation of mobile poly(β-hydroxybutyrate) in the storage granules of Methylobacterium AM1 by in vivo 13C-NMR spectroscopy. FEBS Lett. 1988;231:16–8.
Barnard GN, Sanders JKM. The poly-β-hydroxybutyrate granule in vivo. A new insight based on NMR spectroscopy of whole cells. J Biol Chem. 1989;264:3286–91.
Kawaguchi Y, Doi Y. Structure of native poly(3-hydroxybutyrate) granules characterized by X-ray diffraction. FEMS Microbiol Lett. 1990;70:151–5.
de Koning GJM, Lemstra PJ. The amorphous state of bacterial poly[(R)-3-hydroxyalkanoate] in vivo. Polymer. 1992;33:3292–4.
Barham PJ. Nucleation behavior of poly-3-hydroxybutyrate. J Mater Sci. 1984;19:3826–34.
Horowitz DM, Sanders JKM. Amorphous, biomimetic granules of polyhydroxybutyrate: preparation, characterization, and biological implications. J Am Chem Soc. 1994;116:2695–702.
Bourbonnais R, Marchessault RH. Application of polyhydroxyalkanoate granules for sizing of paper. Biomacromolecules. 2010;11:989–93.
Lauzier C, Marchessault RH, Smith P, Chanzy H. Structural study of isolated poly(β-hydroxybutyrate) granules. Polymer. 1992;33:823–7.
Dazzi A, Prazeres R, Glotin F, Ortega JM. Local infrared microspectroscopy with subwavelength spatial resolution with an atomic force microscope tip used as a photothermal sensor. Opt Lett. 2005;30:2388–90.
Mayet C, Dazzi A, Prazeres R, Ortega JM, Jaillard D. In situ identification and imaging of bacterial polymer nanogranules by infrared nanospectroscopy. Analyst. 2010;135:2540–5.
Dazzi A, Prater CB, Hu Q, Chase DB, Rabolt JF, Marcott C. AFM-IR: combining atomic force microscopy and infrared spectroscopy for nanoscale chemical characterization. Appl Spectrosc. 2012;66:1365–84.
Deniset-Besseau A, Prater CB, Virolle MJ, Dazzi A. Monitoring TriAcylGlycerols accumulation by atomic force microscopy based infrared spectroscopy in Streptomyces species for biodiesel applications. J Phys Chem Lett. 2014;5:654–8.
Dazzi A, Glotin F, Carminati R. Theory of infrared nanospectroscopy by photothermal induced resonance. J Appl Phys. 2010;107:124519.
Xu J, Guo BH, Yang R, Wu Q, Chen GQ, Zhang ZM. In situ FTIR study on melting and crystallization of polyhydroxyalkanoates. Polymer. 2002;43:6893–9.
Padermshoke A, Katsumoto Y, Sato H, Ekgasit S, Noda I, Ozaki Y. Surface melting and crystallization behavior of polyhydroxyalkanoates studied by attenuated total reflection infrared spectroscopy. Polymer. 2004;45:6547–54.
Padermshoke A, Katsumoto Y, Sato H, Ekgasit S, Noda I, Ozaki Y. Melting behavior of poly(3-hydroxybutyrate) investigated by two-dimensional infrared correlation spectroscopy. Spectrochim Acta A Mol Biomol Spectrosc. 2005;61:541–50.
Sato H, Dybal J, Murakami R, Noda I, Ozaki Y. Infrared and Raman spectroscopy and quantum chemistry calculation studies of C–H–O hydrogen bondings and thermal behavior of biodegradable polyhydroxyalkanoate. J Mol Struct. 2005;744–747:35–46.
Chaijamrus S, Udpuay N. Production and characterization of polyhydroxybutyrate from molasses and corn steep liquor produced by Bacillus megaterium ATCC 6748. Agricultural Engineering International: the CIGR Ejournal. 2008;10:Manuscript FP 07 030.
Hahn SK, Chang YK, Lee SY. Recovery and characterization of poly(3-hydroxybutyric acid) synthesized in Alcaligenes eutrophus and recombinant Escherichia coli. Appl Environ Microbiol. 1995;61:34–9.
Porter MM, Yu J. Crystallization kinetics of poly(3-hydroxybutyrate) granules in different environmental conditions. J Biomater Nanobiotechnol. 2011;2:301–10.
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We gratefully acknowledge the Digital Surf team for their help and for supporting us with MountainsMap software which was used to post-process each nanoIR image.
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Rebois, R., Onidas, D., Marcott, C. et al. Chloroform induces outstanding crystallization of poly(hydroxybutyrate) (PHB) vesicles within bacteria. Anal Bioanal Chem 409, 2353–2361 (2017). https://doi.org/10.1007/s00216-017-0181-5
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DOI: https://doi.org/10.1007/s00216-017-0181-5