Effects of NAPL Presence and Growth Stage
on the Movement of Pseudomonas
saccharophilia P15 through Soil Columns
Benjamin Durfee1 and Dr. Stefan Grimberg2
Civil and Environmental Engineering
Previous research has been conducted to model the transport of bacteria through soil
columns with non-aqueous phase liquids (NAPLs) present. The desire of this research was to
discover if NAPL-bacteria adhesion would affect the transport of Pseudomonas saccharophilia
P15 through a porous material. Pseudomonas saccharophilia P15 is a bacterial strain that has
been shown to digest polycyclic aromatic hydrocarbons, such as naphthalene, and to interact with
NAPLs, such as coal tars, which a by-product of industry and a contaminant of groundwater. The
objectives of this research were to determine how bacterial adhesion to hexadecane retarded
Pseudomonas transport, and to find the affects of varying growth conditions, including carbon
source, on this adhesion. For bacteria incubated on peptone as a carbon source it was shown that
adhesion did indeed retard the bacterial transport. However, for bacteria cultured on naphthalene,
lower adhesion failed to produce any discernable changes in transport. This is possibly due to the
relatively short length of the soil columns, with such short distances a small change in the
transport rate would not have time to become exaggerated to a noticeable point. For this reason, I
am repeating the naphthalene research with longer columns. The adhesion between the NAPL
and the Pseudomonas should hinder the transport of the bacteria through the soil column, and the
longer column should allow this change to be observable.
The setup will consist of a series of short-pulse column experiments varying the growth
stage and carbon source of the bacteria. These will be run through quartz sand columns with and
without NAPL presence. Hexadecane was used as a model NAPL for this experiment. A circuit
is used in which .01 M sodium “groundwater” is pumped through 16x2.5 cm diameter soil
columns filled with either hexadecane treated or untreated sand at the rate of .34 mL per hour.
When treating the columns with hexadecane, it is imperative to form a very thin film around the
sand particles so that the matrix is not blocked by the NAPL, interfering with fluid flow due to a
physical barrier. This setup is identical to that previously used with the exception of the longer
soil columns.
Analysis is performed using an UV absorbance meter set at 420 nm which is attached at
1 Class of 2008, Department of Engineering Studies, Clarkson University, Honors Researcher
2 Project Mentor, Department of Civil and Environmental Engineering, Clarkson University
2
the end of the loop to record the absorbance values as a function of time. When more particles
pass through this value will increase, these values can be graphed as a breakthrough curve. The
breakthrough curve can then be described by the following equation:
C(x, t)  no e
x
- kt
2 t 3 D
e

( x  vt ) 2
4 Dt
where C is the concentration, x is the distance, t is the time, k is the deposition rate coefficient, D
is the dispersion coefficient of the particles, and v is the velocity of the particles. The
normalization constant, No is determined by integrating the breakthrough curve of a bypass loop
for the volume of injected culture. The values of V and D are acquired by fitting the tracer
breakthrough curve to the equation, since the non-conservative tracer has a k value of 0. These
values can then be put in to fit the bacterial breakthrough curve to the equation, yielding the value
of k. This value k is what we are trying to find because it numerically expresses the adhesion
between the bacterial particles and the hexadecane.
So far I have results for the tracer test runs and have started analyzing them. The tracer
tests have confirmed that the columns are of similar hydrodynamic properties, as the
breakthrough curves for them have the very similar shapes and peak times (see fig. 1). The tracer
curves have also verified that the tracer is conserved, that is that none of it remains trapped within
the columns. This can be concluded because the integrals of the columns and the bypass loop
curves are the within a few percentage points of each other (see fig. 2). What remains to be done
are the tests with the bacteria, and the subsequent analysis of these tests. If these experiments
with the longer columns work we will know the extent of adhesion between naphthalene cultured
Pseudomonas and hexadecane. We will also know the effects of growth stage on said adhesion.
3
[fig. 1]
Concentration (mV)
Concentration as a Function of Time for Tracer
Runs
1200
1000
800
600
400
200
0
-200 0
Bypass Loop
Hexadecane
Treated Column
Untreated Column
50
100
150
Time (Minutes)
[Fig 2]
Tracer Run
Area Under Curve
Bypass Loop
753788295
Untreated Column
753374608
Hexadecane Column
735322224