Geology Reference
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The cultures were harvested by centrifugation at
7000 rpm for 10 min at 20 8C, washed with 0.001 M
EDTA and three times washed with 0.1 M NaNO
3
.
The cells were centrifuged under the same con-
ditions listed above. The cells were finally batched
and re-suspended in the 0.1 M NaNO
3
to a
minimum of concentration of around 0.2 g L
21
of
bacteria. Our preliminary study showed that this
protocol is the most efficient in terms of EPS quan-
tities. The polysaccharides present in the cultures
were extracted with phenol according to the conven-
tional procedures for extracting bacterial polysac-
charides. These samples, consisting of cells, were
placed in a 2.0 mL Eppendorf-cap and centrifuged
for one minute at 13000 rpm. After discarding the
supernatant, another 2 mL of the sample was
added into Eppendorf-caps and again centrifuged.
In order to elute salts, the pellets were washed
with Phosphate-Buffer (pH 7), centrifuged again
and the supernatant was discarded.
To induce separation, the pellet was thoroughly
mixed with 0.5 mL Phosphate-Buffer and 0.5 mL
Phenol (80%) and incubated in heated water bath
for 20 minutes (Blaschek 1991). After every
5 minutes of heating the samples were thoroughly
mixed and put on ice for one minute. After
20 minutes of heat exposure the samples were
stored on ice for 5 minutes and centrifuged for
10 minutes at 5000 rpm. The supernatant was care-
fully transferred into a dialysis membrane (Sigma)
and closed with labelled clamps. The membrane
was stored overnight in nanopure water at þ4 8C.
The next day the samples were transferred into
1.5 mL Eppendorf caps and dried in a freeze dryer
overnight (Blaschek 1991). Finally, the dried
samples were dissolved in 50-250 mL of sterile
nanopure water and stored at 220 8Cin12mL
Eppendorf tubes.
strains in a background electrolyte of 0.01 M
NaNO
3
. All titrations were performed in a glass
vessel with a lid as part of a Metrohm GP 736
Titrino unit interfaced by Titrino software TITRI-
NET to a personal computer. Two separate buret
exchange units (20 mL and 10 mL) were used, one
for the acid and one for the base. We also used a
Metrohm titrator vessel lid. The temperature was
recorded with a temperature sensor; the error of
the temperature probe was 0.1 8C. The pH electrode
was three-point calibrated with buffers (pH 4, 7
and 10) before each experiment, and the slope was
constant at 99% of the Nernst value.
The Titrino unit was programmed with a
dynamic mode (DYN) for the titration, which adds
the variable amount of titrant according to the pH
changes: the smaller volume of titrant was added
at the slope of the pH curve. The successive titrant
additions were only made when the signal drift
reached 10 mV min
21
.
Potentiometric titration
The titrator electrode was first calibrated as men-
tioned above. The optical density of the bacterial
suspension in the NaNO
3
electrolyte solution was
measured prior to each extraction and titration run.
In order to determine the concentration of bacteria
(mg L
21
) and the bacterial cell numbers (cells
L
21
), the measured absorbance was compared to a
prepared calibration curve. The dry weight of bac-
teria was defined by drying at 65 8C until a constant
weight was attained. The absorbance used is ranged
between 0.41-0.81, which corresponds to 0.063-
0.122 g of bacteria L
21
. A known amount of suspen-
sion, c. 50 mL, was then transferred to the titration
vessel, which was immediately attached to the lid
setup connected with the N
2
gas line. A magnetic
stir bar was also added to the vessel. The whole
system was then degassed for 30-40 min to
exclude atmospheric CO
2
. Following the degassing
procedure a positive pressure of N
2
was maintained
by allowing a gentle flow of N
2
into the headspace
during the titration.
The EPS aliquot was then titrated quickly to
pH ¼ 2.9 with 0.1 M HCl. The buret exchange
unit was subsequently changed and the titration of
the aliquot (with NaOH up to pH ¼ 10) began.
The total time for each titration was c. 40 min.
Some titrations were reversed by conducting an
acid titration, immediately after the base titration.
The results of reversed titrations were not signifi-
cantly different from the forward titrations
suggesting reversibility of the proton adsorption-
desorption reactions.
The titration data were analyzed using the linear
programming method (LPM) or the so-called pK
a
spectrum method as proposed by Brassard and
Potentiometric titration and data analysis
Titration solutions
The solutions were degassed with N
2
for 20 minutes
to dissipate O
2
and CO
2
. The concentration of
NaNO
3
used in this experiment was 0.1 M. The
NaOH solution was prepared according to the fol-
lowing method: c. 0.1 M of solution was prepared
from NaOH using degassed 18 MV water. The
exact NaOH concentration was determined prior to
the titration experiment with a relative standard
deviation of 1% (Dittrich & Sibler 2005).
Titrator setting
Deprotonation constants and surface site concen-
trations were determined from acid-base titrations
of extracted polysaccharides from the three bacterial
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