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using optical density measurements (at 410 nm) with a 1 cm flow-trough glass cuvette and a
spectrophotometer (Cecil 1021, Cecil Instruments Inc., Cambridge, England). Absolute cell
numbers were deduced after calibration with plate counts on Chromocult agar (Merck,
Whitehouse Station, NJ). To check whether the flush with HCl followed by NaOH had indeed
removed all bacterial cells, at the beginning of each experiment, effluent samples were plated in
triplicate. All plates of all experiments were negative, indicating that, after the previous
experiment, all viable bacterial cells had indeed been removed from the column. To check for
consistency of the methodology, at the end of the entire set of experiments, 14 experiments with
varying breakthrough were repeated in random order. Breakthrough curves of all duplicate
column experiments were nearly identical, and the two group mean attachment efficiencies were
identical. From this, we concluded that the methodology we used was consistent and yielded
results that could be reproduced. In the absence of straining, we assumed that retention was
predominantly characterized by attachment of
E. coli
cells to the quartz grain surfaces in the
column. Bacteria attachment to the sand was quantified by computing the attachment efficiency (
Α
) as (Lutterodt et al., 2009a; Kretzschmar et al., 1997; Abudalo et al., 2005)
M
d
2
eff
Α
= −
c
ln
(3.1)
3 (1
−
Θ
)
L
Η
M
0
inf
where
d
is the minimum of the grain size weight distribution (m),
Η
is the single collector
contact efficiency (-) ,
Θ
is the total porosity of the sand (-),
L
is the travel distance (m),
is
M
inf
the total number of cells in the influent and
M
is the total number of cells in the effluent (-)
eff
obtained as (Kretzschzmar et al., 1997)
t
=
∫
M
q C t dt
( )
(3.2)
eff
0
where
q
is the volumetric flow rate (mL/min),
C
is the cell suspension ( # cells/mL) and
t
is
time (min). The TE correlation equation (Tufenkji and Elimelech, 2004a) was used to compute
Η
. For this, we assumed that the bacteria density was 1055 kg/m
3
, and the Hamaker constant
was estimated to 6.5×10
-21
J (Walker et al., 2004).
3.2.4 Cell characterization
To determine
width
and
length
of the cells, a light microscope (Olympus BX51) in phase contrast
mode, with a camera (Olympus DP2) mounted on top and connected to a computer, was used to
take images of cells. Averages of 50 images were imported into an image processing program
(DP-Soft 2) and the average cell width and cell length were measured. The equivalent spherical
diameter was determined as the geometric mean of average length and width (Rijnaarts et al.,
1993), while the cell sphericity was obtained from the ratio of average width to average length
(Weiss et al., 1995).
To determine
motility
, a 2 mL fresh culture was centrifuged (14000 xg) and washed three times
in AGW, and by means of a sterile toothpick, cells were picked from the remaining pellet in the
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