Towards understanding inter-strain attachment variations of Escherichia coli during transport in saturated quartz sand - Transport of Multiple Escherichia Coli Transport in Saturated Porous Media - page 30

3.2 Materials and Methods
3.2.1 Bacteria strains.
We used 54 E. coli strains. Of those, 48 strains were obtained from the Zoo in Rotterdam, The
Netherlands; six other strains we used in previous research (Lutterodt et al., 2009; UCFL strains
in Table 3. 2 ) . The Zoo strains were isolated during the last 5-10 years from various animals
living in the Zoo, and from various sources (liver, kidney, feces, lungs, etc.). All Zoo strains
were previously serotyped by the National Institute of Public Health and the Environment of the
Netherlands. Strains were grown in 50mL of nutrient broth (Oxoid CM001) for 24 h at 37 °C.
Bacteria were washed and centrifuged (14000xg) three times in Artificial Ground Water (AGW).
AGW was prepared by dissolving 526 mg/L CaCl 2 .2H 2 O and 184 mg/L MgSO 4 .7H 2 O, and
buffering 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. In this way, we
hoped to create an environment inside the column with a low repulsive double layer energy
barrier, in order to enhance attachment of cells.
3.2.2 Porous media
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 in our column: assuming a bacteria equivalent spherical diameter of 1.5 m, the ratio
of colloid and grain diameter was 0.004, which 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 de-mineralized water
until the electrical conductivity was close to zero (Li et al., 2004).
3.2.3 Column experiment
Column experiments were conducted in borosilicate glass columns with an inner diameter of 2.5
cm (Omnifit, Cambridge, U.K.) with polyethylene frits (25 m pore diameter) and one adjustable
endpiece. The column was packed wet with the quartz sand with vibration to minimize any
layering or air entrapment. Column sediment length was 7 cm. All column experiments were
conducted in artificial groundwater (AGW) at a velocity of 0.25 PV per minute (fluid approach
velocity = 10 -4 m/s). To eliminate E. coli retention variations resulting from variations in packing
of the sand, all experiments were carried out in one column, packed at the start of the entire set
of column experiments. Prior to each experiment, and in order to remove retained cells of the
previous experiment, the column was rinsed with 1 PV of 1.9 M HCl, immediately followed by a
pulse of 1.5 M NaOH to restore pH. Then, the column was equilibrated 50-60 pore volumes with
AGW in order to restore pH and EC. Usually, a suspension of E. coli with a concentration of ~
10 8 cells/mL was flushed through the column for 4 minutes (approximately equal to one pore
volume) followed by a flush of E. coli -free AGW. The E. coli concentration was determined