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The effect of pressure and temperature of supercritical carbon
dioxide on the biological activity of black yeast cells
Maja Čolnik1, Mateja Primožič1, Nina Gunde Cimerman2, Polona Zalar2, Maja Habulin1
Željko Knez*1,
1
University of Maribor, Faculty of Chemistry and Chemical Engineering,
Laboratory for Separation Processes and Product Design,
Smetanova ulica 17, 2000 Maribor, Slovenia
2
University of Ljubljana, Biotechnical Faculty, Department of Biology,
Večna pot 111, 1000 Ljubljana, Slovenia
Corresponding author: zeljko.knez@uni-mb.si; Phone: +386 2 2294 402; Fax: +386 2 2527 774
ABSTRACT
The suspension of black yeast was incubated in SC CO2 at different pressures and constant temperature. Culture
of the black yeast suspension was centrifuged before and after incubation in SC CO 2. The absorbance of the
supernatant was measured using a UV-Vis spectrophotometer at 595 nm for the α-amylase activity and at 340
nm for cellulase activity determination. The total protein concentration was assayed by the Bradford method at
the wavelength of 595 nm.
INTRODUCTION
Black yeasts represent an important form of microorganisms that are able to survive in extreme habitats such as
salt, hot and cold deserts and deep water. They have been isolated from a wide range of extreme environments,
oligotrophic stone base, highly saline habitats and Arctic glacier ice. Hortaea werneckii is the dominant black
yeast species which belongs to the order Dothideales [1]. H. werneckii is a very excellent adaptable fungi, it can
grow without salt and at extremely high concentration of salt. [1,2,3,4]. Black yeast contain many different
enzymes; free or bound to cellular structures (intracellular enzymes), and extracellular enzymes excrete in the
growth medium. From the technical and economical reasons, microorganisms are still the most important source
of enzymes. For these reasons, the suspension of H. werneckii was incubated in SC CO2 in order to use the
enzymes from these fungi for biotransformation.
In the SC CO2 inactivation treatments, different factors can affect the sensitivity of microorganisms to the
treatment, including the species and strain of the microorganism, the composition of the suspending medium, or
the pressure, temperature and duration of the process. The resistance of a microorganism to different stresses,
including high pressure processing, is known to be affected by the growth stage [5].
MATERIALS AND METHODS
H. werneckii was obtained from the Department of Biology, Biotechnical Faculty (Ljubljana, Slovenia). A small
amount of H. werneckii cells (Figure 1) were suspended in 10 mL of prepared meat extract medium. The mixture
was moved in high-pressure batch reactor. When the temperature reached the desired value (35 °C), CO2 was
pumped into the reactor. The experiments were carried out in the operative pressure range from 100 and 300 bar
and at temperature 35 °C. Afterwards, the reactor was slowly depressurized (3 bar/min). The suspension of H.
werneckii was immediately aseptically removed from the reactor and a sample was taken to determine the
survival of H. werneckii cells. Each sample of H. werneckii suspension was centrifuged. The supernatant was
used for subsequent analyses. The absorbance of the supernatant was measured using a UV-Vis for the α-
amylase activity and for cellulase activity determination. The total protein concentration was assayed by the
Bradford method [6].
Figure 1. MEA agar slant of the black yeast H. werneckii and H. werneckii cells under microscope (400x
magnification).
RESULTS AND DISCUSION
Protein concentration in H. werneckii suspension
H. werneckii suspension was centrifuged after incubation in SC CO2. The supernatant (without cells and debris)
was analyzed spectrophotometrically. The total protein concentration in the suspension of culture H. werneckii
was increased. High pressure led to permeabilization of cell walls and membranes, and consequently the
cytoplasmic substances had been extracted from H. werneckii cells in the suspension. The highest protein
concentration in suspension of H. werneckii was found after 24 h of incubation in SC CO 2 at 300 bar. These
results indicate that the protein concentration increases linearly with the SC CO2 treatment time at both
examined pressures (Figure 2).
1
0.9
0.8
c (mg/mL)
0.7
0.6
0.5
100 bar
300 bar
0.4
0.3
0.2
0.1
0
0
5
10
15
time (h)
20
25
30
Figure 2. Protein concentration in the H. werneckii cell suspension after SC CO2 treatment at 35 °C.
Cellulase and α-amylase activity in H. werneckii suspension
The impact of the SC CO2 on the activity of cellulase and α-amylase in the cell suspension of the culture of H.
werneckii were determined. Experiments were carried out at a constant temperature of 35 °C, at pressures 100
and 300 bar and at different times of treatment in SC CO 2. Cellulase and α-amylase activities in cell suspension
were measured before and after incubation in SC CO2. Incubation of H. werneckii cell suspension in SC CO2 led
to permeabilization of black yeast cells and therefore an increase of cellulase and α-amylase activities were
detected.
The activities of cellulase and α-amylase were determined with UV-Vis spectrophotometer before and after
incubation of the black yeast culture in SC CO2. Cellulase is an extracellular enzyme which activity was
determined in cell suspension of H. werneckii before incubation in SC CO2. The highest activity of cellulase in
cell suspension after incubation in SC CO2 was achieved for 1 h incubation time at 100 bar and 35 °C and
reached 0.391 U/mL of cell suspension. α-Amylase is in contrast to cellulase an intracellular enzyme. Low
activity of α-amylase before incubation in SC CO2 was detected. After incubation of the cells suspension of H.
werneckii at the pressure of 300 bar the highest α-amylase activity was observed at the incubation time of 1 h. At
these conditions, the activity of α-amylase was 0.0095 U/mL of cell suspension. With the increase of incubation
time, the activities of cellulase and α-amylase in cell suspension H. werneckii decreased. Deactivation of these
enzymes at both examined pressures (100 and 300 bar), can be seen from the Figures 3 and 4.
cellulase (U/mL of cell suspension H. werneckii)
0.45
0.4
0.35
0.3
0.25
100 bar
0.2
300 bar
0.15
0.1
0.05
0
0
5
10
15
time (h)
20
25
30
Figure 3. Experimental data of cellulase activity in cell suspension of H. werneckii exposed to SC CO2 at 100
and 300 bar as a function of treatment time.
α-amylase (U/mL of cell suspension H. werneckii
0.012
0.010
0.008
0.006
100 bar
300 bar
0.004
0.002
0.000
0
5
10
15
time (h)
20
25
30
Figure 4. Experimental data of α-amylase activity in cell suspension of H. werneckii exposed to SC CO2 at 100
and 300 bar as a function of treatment time.
CONCLUSION
This paper examined the effect of pressure and temperature of SC CO 2 on the biological activity of H. werneckii
cells. From the H. werneckii cells during the SC CO2 treatment a significantly greater amount of proteins was
released. It was found that the H. werneckii cells contain extracellular enzyme cellulase and intracellular enzyme
α-amylase. High pressure together with long incubation times in SC CO 2 caused a decreasing of cellulase and αamylase activity.
REFERENCES
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61, 2008, p. 67.
[3] GUNDE-CIMERMAN, N., OREN, A., PLEMENITAŠ, A. (eds), Springer, 2005.
[4] ABLIZ, P., FUKUSHIMA, K., TAKIZAWA K.., MIYAJI, M., NISHIMURA, K., Diagn. Microbiol. Infect.
Dis., 46, 2003, p. 89.
[5] ORTUÑO, C., MARTÍNEZ-PASTOR, M. T., MULET, A., BENEDITO, J., J. of Supercrit. Fluids, 63, 2012,
p. 8.
[6] BRADFORD M. M., Anal. Biochem., 72, 1976, p. 248.