Effects of surface characteristics on the transport of multiple Escherichia coli isolates in large scale columns of quartz sand - Transport of Multiple Escherichia Coli Transport in Saturated Porous Media - page 12

that, under environmentally relevant growth conditions, the majority of E. coli isolates (88%)
encoding Ag43 formed thick biofilms, while the majority of E. coli isolates not encoding Ag43
(75%) formed thin biofilms. Thus, Ag43 was involved in the attachment of E. coli cells.
Although studies have been conducted to determine the influence of LPS, bacteria growth stage
and evolution of cell surface macro-molecules on cell adhesion (Walker et al., 2004 and 2005),
the effect of bacteria properties on bacteria transport over longer travel distances (> 0.5 m) has
not been systematically studied. In addition, the range of environmentally realistic sticking
efficiencies, that determines bacteria travel distances in aquifers, are not known.
The objective of this chapter is to study the effects of a number of bacteria properties (Ag43
expression, motility, hydrophobicity, outer surface potential, and cell sphericity) of six E. coli
strains on their transport behavior over environmentally realistic transport distances, up to 5 m.
2.2 Materials and methods
2.2.1 Extraction of manure and bacteria growth
We used six Escherichia coli ( E. coli ) strains (UCFL-71, UCFL-94, UCFL-131, UCFL-167,
UCFL-263 and UCFL-348) obtained from a soil in a cattle grazing field(Yang et al., 2004). To
mimic environmental conditions, E. coli isolates were grown in an extract of cow manure (Yang
et al., 2006). Thereto, fresh cow manure was collected from a farm (cow ranch 'Ackersdijk',
Delft, The Netherlands), and stored at -20 °C in batches of 50 g. Prior to each experiment, one
batch of 50 g cow manure was defrosted and mixed with demineralized (DI) water at a 1:20 ratio
(EPA - 1312 Leach Method). To facilitate extraction, the mixture was acidified to a pH of
5±0.05 with concentrated sulphuric acid and nitric acid at 60/40 weight percent mixture, and
extraction was performed for 2 hours. The mixture was then centrifuged (IEC Centra GP 8- rotar
218/18cm) for 10 min at 4600 rpm (1185 g-force), and then at 9000 rpm for 10 min (816.5 g-
force) (MSE high speed 18). The supernatant was sequentially filtered through a 0.45 m and a
0.2 m mesh size cellulose acetate membrane filter (47 mm diameter) . E. coli isolates were
activated from a glass test tube (pepton agar stock) and grown in Luria Bertoni (LB) broth
(DifcoTM LB Broth, Miller) for 6 hours at 37 °C while shaking at 120 rpm on an orbital shaker.
The inoculum was then diluted 10 5 fold in the cow manure extract and incubated, while shaking
on the orbital shaker at 120 rpm, for 72 hours at 21 °C until a stationary growth phase was
reached, resulting in a concentration of ~10 8 cells/ml.
2.2.2 Characterization of cell properties
We determined hydrophobicity, motility, outer surface potential of the E. coli cells, cell width
and cell length, while data on Ag43 expression were obtained from Yang et al. (2006).They
detected the presence of Ag43 using rabbit anti-Ag43Α and FITC-labeled goat anti-rabbit serum
and evaluated coagulation of cells using phase-contrast and epiflourescence micrographs. For a
more detailed description we refer to Yang (2005).
Hydrophobicity was determined with the Microbial Adhesion To Hydrocarbons (MATH) method
(Pembrey et al., 1999; Walker et al., 2005), where percentage partitioning of cells into dodecane