Biology Reference
In-Depth Information
2.2.3 Column experiments
To study the effects of the surface properties of the
E. coli
strains on their transport in the
subsurface, column experiments were conducted in demineralised (DI) water and in artificial
groundwater (AGW). The latter was prepared by dissolving 526 mg/L CaCl
2
.2H
2
O and 184
mg/L MgSO
4
.7H
2
O in DI water, and buffering it with 8.5 mg/L KH
2
PO
4
, 21.75 mg/L K
2
HPO
4
and 17.7 mg/L Na
2
HPO
4
. The final pH-value ranged from 6.6 to 6.8 and the EC-value ranged
from 1025 to 1054 S/cm. The porous media comprised of 99.1% pure quartz sand (Kristall-
quartz sand, Dorsilit, Germany) with sizes ranging from 180 to 500 m, while the median of the
grain size weight distribution was 356 m. With this grain size, we excluded straining as a
possible retention mechanism is our column: assuming a bacteria equivalent spherical diameter
of 1.5 m, the ratio of cell and grain diameter was 0.004. This was well below the ratio (0.007)
for which straining was observed by Bradford et al. (2007) for carboxyl latex microspheres with
a diameter of 1.1 mm suspended in solutions with ionic strengths up to 31mM (the ionic strength
of the solutions we used was 4.7 mmol/L only). Total porosity was determined gravimetrically to
be 0.40. Prior to the experiments, to remove impurities, the sand was rinsed sequentially with
acetone, hexane and concentrated HCl, followed by repeated rinsing with DI water until the
electrical conductivity was close to zero (Li et al., 2004).
The column consisted of a 5 m transparent acrylic glass (Perspex) tube with an inner diameter of
10 cm, and with seven sampling ports placed at 10-50 cm intervals along the tube. A stainless
steel grid for supporting the sand was placed at the bottom of the tube. The column was gently
filled with the clean quartz sand under saturated conditions, while the sides of the column were
continuously tapped during filling, to avoid layering or trapping of air. The column was
connected both at the funnel shaped effluent end and influent end with two Masterflex pumps
(Console Drive Barnant Company Barrington Illinois, USA) via teflon tubes, and the pumps
were adjusted to a mean fluid approach velocity of 1.16x10
-4
m/s, coinciding with flushing the
column with 1 pore volume (PV) per working day. We considered this fluid approach velocity
fast enough to minimize the effects of die-off of
E. coli
on the measured bacteria concentrations
at the various sampling ports. Prior to a column experiment, the column was flushed for two days
with either DI or AGW to arrive at stable fluid conditions inside the column. Bacteria influent
suspensions were prepared by washing and centrifuging at 3000 rpm for 10 minutes (90.7 g-
force ) three times in either DI or AGW, and then diluting 1000 times to arrive at bacteria cell
concentrations of approximately 10
5
cells/mL. Experiments were conducted by applying a pulse
of 0.15 PV (approximately 2.2L) of bacteria influent suspension to the column, followed by
bacteria free DI or AGW. Samples were taken at 7 distances from the column inlet, and
immediately plated (0.1 mL) in duplicate on Chromocult agar (Merck). Bacteria inactivation was
assessed in all experiments by plating samples of the influent at an hourly frequency during the
entire experiment. All plates were incubated at 37 °C for (at least) 18 hours.
After each experiment, to clean the sand in the column, and to prepare for the next experiment, a
pulse of 5 L 1.9 M HCl followed by a pulse of 5 L 1.5 M NaOH was flushed through the column,
followed by flushing with DI water until the electrical conductivity of the effluent was well
below 3 S/cm.
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