The effect of high pressure on viability of Botryococcus braunii cells

The effect of high pressure on viability of Botryococcus braunii cells

The effect of high pressure on
viability of Botryococcus braunii
cells under aseptic conditions
Ece Yildiz-Ozturka, Joao Gouveiab, Ozlem Yesil-Celiktasa,*
aDepartment
bWageningen
of Bioengineering, Faculty of Engineering, Ege University,
35100 Bornova-Izmir, Turkey
UR Food & Biobased Research. Wageningen, Netherlands
Historical background
In June 2016, the scientific community will celebrate
the 39th anniversary of the discovery of volcanic hot
vents at the Galapagos Rift at the bottom of the
Pacific Ocean.
Ecosystem at these extreme conditions
(1200 bar, 2-100 °C, no sunlight, scarce supply of
organic nutrients)
High pressure applications
The invention of thermophilic and
piezophilic organisms and their
enzymes speeded up the research
Biological applications of high hydrostatic
pressure
• Food industry
•
• Proteins from extremophiles as
•
stable tools for biotechnological
•
applications
•
• High pressure effects on allergenicity •
and digestibility
•
• Disinfection of biomaterials
•
• Modulation of enzymatic activities
•
• Stabilization of protein intermediates
N. Rivalain et al., 2010, Biotechnology Advances 28,659-672.
Dissociation of protein complexes
Protein-DNA interactions
Vaccine development
Preparation of viral vectors
Genetic transformation of cells
Cell extraction
Pressure-assisted cryopreservation
Applications in oncology
High hydrostatic pressure in biosciences
Pressure effects on various components of biological systems
 Pressure effects on proteins
 Pressure effects on lipids and biomembranes
 Pressure effects on nucleic acids
Pressure effects on more complex living systems
 Pressure effects on mammalian cells
- for pressures around 200 MPa, cell death is the result of apoptosis,
- for pressures higher than 300 MPa, cell death occurs through a necrotic-like pathway
 Pressure effects on pathogens
Bacteria, Bacterial spores
Viruses, Parasites, Yeasts and molds
High pressure processing for inactivation
J.P.P.M. Smelt, 1998, Trends in Food Science & Technology, 9, 152 -158.
Inactivation vs activation?
High pressure inactivates
enzymes and microorganisms
How about stabilization and
activation?
Cell viability experiments: effect of hydrostatic high
pressure on B. braunii
 B. braunii for B6 strains were treated with high
pressure (50-100-150-200-250 bar) under aceptic
conditions by using subcritical water in order to
investigate the effect of high pressure on cells.
MB uptake
MB uptake/normalized
% Cell Viability
Fresh
4.339
0.281
90.83
50 bar
6.836
0.443
67.71
100 bar
6.865
0.445
67.43
150 bar
9.243
0.599
45.41
200 bar
7.608
0.493
60.55
250 bar
8.559
0.555
51.74
Dead
15.425
1.000
0.00
High pressure equipment 100 ml
max : 350 bar – 500 OC
 The cell viability was carried out by
using methylene blue uptake method.
Visualization of microscopic images subsequent
to hydrostatic pressure treatment
Control (fresh culture)
After 50 bar (100X)
After 100 bar (40X)
After 150 bar (40X)
After 200 bar (40X)
After 250 bar (40X)
Can B. braunii be cultivated subsequent to high
pressure treatment?
Bacteria cocktail (3 ml) was
inoculated into high pressure
treated B. braunii cultures.
The images of B. braunii cultures, after high pressure treatments (1. Day)
The images of B. braunii cultures after bacteria coctail (3 ml) was inoculated into pressure treated B. braunii cultures, (12. Day)
Supercritical CO2 extraction in SFE 100 System (Izmir)
 Supercritical CO2 extraction was carried out
using SFE 100 System (Thar Instruments,
Inc., UK, 2006).
 The extractor volume was 100 ml, thus it
was filled with about 10 g of lyophilized B.
braunii.
 The independent variables were pressure
(120, 160, 200 bar) and CO2 flow rate (5, 7,
9 g/min). Temperature was kept at 40oC.
High pressure equipment
100 ml (max : 300 bar)
Hydrocarbon contents of supercritical CO2 extracts for
lyophilized AC761 strain
Extraction Conditions
Squalene equivalent
hydrocarbon
concentration (mg/L)
Hydrocarbon Amount in
Total Extract (mg)
mg hydrocarbon
(squalene based) /
g biomass
160 bar, 7 g/min, 40OC
8301.83
1283.27
128.33
120 bar, 7 g/min, 40OC
9848.15
1177.57
117.76
160 bar, 9 g/min, 40OC
8770.07
1303.40
130.34
200 bar, 7 g/min, 40OC
8105.48
1422.72
142.27
160 bar, 7 g/min, 40OC
8209.76
1239.86
123.99
160 bar, 7 g/min, 40OC
7242.37
937.79
93.78
200 bar, 5 g/min, 40OC
8100.92
1379.68
137.97
120 bar, 5 g/min, 40OC
6819.20
759.49
75.95
200 bar, 9 g/min, 40OC
8716.36
1407.61
140.76
160 bar, 5 g/min, 40OC
7837.16
1150.68
115.07
160 bar, 7 g/min, 40OC
9201.93
1315.07
131.51
160 bar, 7 g/min, 40OC
8034.59
1169.51
116.95
120 bar, 9 g/min, 40OC
4418.31
491.74
49.17
Solvent extraction (1:30 g/ml, 40 oC, 1 h)
Solvent
Extraction
Squalene equivalent
hydrocarbon
concentration (mg/L)
Hydrocarbon
Amount in Total
Extract (mg)
mg hydrocarbon
(squalene based) /
g biomass
Hexane
2277.91
36.45
36.45
Dichloromethane
2048.70
43.02
43.02
Ethylacetate
1585.84
38.06
38.06
Chloroform/MeOH
1815.66
32.68
32.68
Supercritical CO2 extraction in Hastelloy vessel (Wageningen)
High pressure equipment - Hastelloy vessel, 250 ml (max : 300 bar)
 High pressure treatments were carried out by using hastelloy vessel that has 250 ml extractor
volume. The volume of broth was 100 ml.
 The independent variables were pressure (100-175-250 bar) and time (1-1.5-2 hour) for
extraction of hydrocarbons from B. braunii.
 Temperature was kept at 40oC and the CO2 flow rate was kept at 4 ml/min.
Hydrocarbon contents of supercritical CO2 extracts for
fresh algal broth (5 g/L)
Squalene equivalent
Hydrocarbon
Extraction Conditions
hydrocarbon
amount in extract
(mg)
concentration (mg/L)
mg hydrocarbon
(squalene based) / g
biomass
100 bar, 4 ml/min, 1 h
586.35
1.76
3.52
175 bar, 4 ml/min, 1h
860.27
2.58
5.16
250 bar, 4 ml/min, 1 h
2329.02
6.99
13.97
The microscopic images of fresh broth (control)
The microscopic images of broth treated at 40oC
Visualization of microscopic images
Control (fresh culture)
After 50 bar, 25oC, 1h
After 50 bar, 40oC, 1h
After 100 bar, 40oC, 1h
After 175 bar, 40oC, 1h
After 250 bar, 40oC, 1h
After 250 bar, 40oC, 1.5 h
After 250 bar, 40oC, 2h
Recultivation of Botryococcus braunii after supercritical CO2
treatments
The image of recultivation experiment
of pressure treated broths
(100, 175, 250bar)
a
b
c
The microscopic images of cultivated broths a) 100 bar, b) 175 bar, c) 250bar
Cell structure of B. braunii
(A) Color differential interference contrast (DIC)
microscopy image of a partial B. braunii colony
(B) Model of the B. braunii cell
and its surrounding extracellular matrices
 Portions of the hydrocarbon ECM
around the cell edge and the upper left quadrant
are drawn with the liquid
hydrocarbons removed to show the underlying
structure of these regions
T.L. Weiss et al., 2012, Eukaryotic Cell Journals, Vol: 11, Number:12, p. 1424–1440.
Effect of CO2 concentrations on the algal growth
 The growth did not change significantly in the range of 0.2–5% CO2
 The growth decreased over the 5% CO2 and stopped at 50% CO2
 As CO2 concentration in the bubbling air affects the pH of the medium, the pH
declined with increasing CO2 cconcentrations
 Optimum growth was observed in pH 5.44–6.65, and the growth fell to zero in pH
4.54
T. Yoshimura et al., 2013, Bioresource Technology 133 (2013) 232–239.
R. Rao, A., 2007, J. Microbiol. Biotechnol. 17(3), 414–419.
Conclusions
 The impact of pressurization and depressurization on the cellulose
membrane of algae
 Saturation of the broth with CO2 subsequent to the high pressure
extraction
 CO2 as a stress factor on the algae cell due to significant pH
difference
 Supercritical CO2 extract had a hydrocarbon content of 142.27 mg/g
biomass, whereas hydrocarbon content was between 32 and 43
mg/g in solvent extraction by different solvents
 Lower yields for directly treated broth at 5g/L concentration
NOVEL FLUIDIC TECHNOLOGIES GROUP
Research areas
 Life science applications of supercritical fluids (pharmaceutical compounds,
enzymes, scaffolds, sol-gel monoliths, microchip sterilization)
 Design of drug delivery systems (nanoparticle synthesis by
sol-gel, encapsulation, vesicular systems, topical formulations)
Microfluidic applications (cells, macromolecules)
Collaborators & Funding Agencies
•
Bilkent University, Dept. of Mechanical Engineering,
Microfluidics & Lab-on-a-chip Research Group
•
Izmir Institute of Technology, Molecular Biology and
Genetics
•
Kocaeli University, Biotechnology
•
Oklahoma State University, Dept. of Biosystems and
Agricultural Engineering
•
Technical University of Hamburg-Harburg, Institute of
Microsystems
•
University of Bremen
•
Wageningen University, Biobased Research Group
Acknowledgement
Arnoud Togtema for the support at supercritical CO2 experiments
at Wageningen University, Biobased Research Group