ABSTRACTS – ORAL PRESENTATIONS - AMCA, spol. s r.o.

technological properties PHB is considered as an alternative to petrochemical polymers
such as polyethylene or polypropylene. Moreover, unlike synthetic polymers, PHB can
be produced from renewable or waste substrates and it is also fully biodegradable and
biocompatible (Kessler and Wilholt 2001).
Analysis of intracellular PHB content of the bacterial cells is an important challenge
connected with biotechnological production of PHB. The most common technique to
quantify PHB is based on Gas Chromatography (GC), however, this method does not
provide rapid tool to analyze PHB content of cells in bioreactor due to labor intensive and
time-consuming sample preparation. From this point of view, flow cytometry analysis of
Nile Red stained cells seems to be superior to GC due to its simplicity and rapidity (Vidal-
Mas et al. 2001).
The aim of this work was to develop method for analysis of intracellular PHB content
by using Nile Red staining and Flow Cytometry. Cupriavidus necator H16 (formerly
Alcaligenes eutrophus, Wautersia eutropha and Ralstonia eutropha) was cultivated in
5 l bioreactor (Biostat B plus, Sartorius Biotech, Germany) using mineral salt medium
and waste frying oil as a carbon source (Obruca et al. 2010). Samples were withdrawn at
regular intervals; cells were pelleted, washed with PBS buffer and fixed with cold ethanol
(30%, 15 min). After that, the cell suspension (1 ml, approx. 10 6 of cells/ml) was stained
with 1 ml of Nile Red (1 mg/ml of DMSO). Analysis of cell population was performed
using Apoggee A50 (Apogee, GB) (excitation at 488 nm, emission was analyzed using
orange channel). PHB none-producing mutant strain Cupriavidus necator H16/PHB - 4 was
used as a negative control.
According to our results, Nile Red staining and Flow Cytometry analysis of PHB cell
content provides rapid and reliable tool to monitor process of PHB production. The
interval between sampling and result analysis does not exceed 45 min. which enables to
control the process of PHB production.
Furthermore, we developed protocol to stain the bacterial cells without application of
ethanol fixation. When the cells were stained under slightly elevated temperature (40-
45°C), the PHA positive cells were stained, but; unlike in case of ethanol fixation, the
cells remained viable and cultivable. This protocol may be useful for selection of PHAoverproducing
strains or mutants by Fluorescence Activated Cell Sorting (FACS).
Acknowledgement
This work was supported by project “Centre for Materials Research at FCH BUT” No.
CZ.1.05/2.1.00/01.0012 from ERDF and by the project „Excellent young researcher at
BUT“ No. CZ.1.07./2.3.00/30.0039.
References
Kessler B, Wilholt B (2001) Factors involved in the regulatory of polyhydroxyalkanoate
metabolism. J Biotech 86: 97–104.
Vidal-Mas J, Resina-Pelfort O, Haba E, Comas J, Maresa A, Vives-Rego J (2001) Rapid flow
cytometry – Nile red assessment of PHA cellular content and heterogenitity in cultures
of Pseudomonas aeruginosa 4ZT2 (NCIB 40044) grown in waste frying oil. Antonie van
Leeuwenhoek 80: 57-63.
Obruca, S., Marova, I., Snajdar, O., Mravcova, L. and Svoboda, Z. (2010a) Production of
poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Cupriavidus necator from waste rapeseed
oil using propanol as a precursor of 3-hydroxyvalerate. Biotech Lett 39: 1925-1932.
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