Applications brief: Chloroform induces outstanding crystallization of poly(hydroxybutyrate) (PHB) vesicles within bacteria

Key points

  • AFM-IR is used to determine the crystallinity degrees of PHB in various environments
  • Using nanoscale IR spectroscopy, it was determined that PHB crystallized extremely well when exposed to chloroform

Key words

AFM-IR | Chemical Map | Degree of Crystallinity | Bacterial poly(hydroxybutyrate) | Nanoscale Infrared Spectrsoscopy


R. Rebois, D. Onidas, C. Marcott, I. Noda, A. Dazzi


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.

Image: Spectrum of PHB within a bacterium recorded via AFM-IR. The amide I band is detected at 1658 cm-1 and the ester carbonyl band, characteristic of PHB presence, is revealed at 1739 cm-1.

AFM-IR spectra (a–d) recorded at four different positions (points 1, 2, 3, and 4 in e) of the AFM tip on the same sample. Positions 1, 2, 3, and 4 are located on topographic image of bacteria (e) and correspond to PHB vesicles inside four different bacteria. The black and red curves represent the spectra recorded, respectively, before and after chloroform exposure. Spectra are normalized to the amide I band intensity.

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