Chapter 11 Lab Exercises
Section 15 Biofilms as Biobarriers
Page 3 Student
Copyright © Alfred B. Cunningham, John E. Lennox, and Rockford J. Ross, Eds. 2001-2010
Biofilms as Biobarriers1
Under certain circumstances, biofilms can be used to retard
the spread of organic and inorganic contaminants in ground
water by filling the pores of the soil with EPS (extracellular
polymeric substances). The EPS fills the pore spaces of the
soil and reduces the mass transport of water. In this exercise
you will create a biofilm in a sand column and measure the rate
of flow of water through the column as the biofilm grows.
||sand column (See Figure 1)
||overnight culture of Pseudomonas
fluorescens (or other BSL-1 organism)
||Tubes containing 20 ml aliquots of molasses
||150 ml beaker
||sterile 25 ml pipette
||container for the disposal of waste medium
Safety note: During this exercise
you should wear latex or other protective gloves. The column
should be mounted securely in a ringstand and the entire
apparatus should be placed on a tray to collect any spillage in
the event of an accident.
Staff, Center for Biofilm Engineering, Montana State University, Bozeman
Figure 1. Column for demonstrating biobarrier
- Obtain a culture of Pseudomonas fluorescens
from your instructor. This is a soil bacterium noted for
its production of EPS. It has been grown up in Molasses
medium, the same medium that you will be using to
“feed” the column. The sand in the column is
intended to represent soil, a rather porous medium.
- Using a sterile 25 ml pipette, add enough of the
culture at the top of the column to bring the medium to the
level of the sand. At this point, all the pores in the sand
column should be filled with culture.
- Using the same pipette, now add an additional 15 ml of
the culture to the column. Carefully mark the 15 ml level
on the column with a magic marker or a piece of lab
- While holding the Tygon™ tubing at the base of
the column closed, remove the clamp.
- Release the Tygon™ tubing and with a stopwatch,
record the time taken for the culture to just reach
the surface of the packing material. Record this
value as the pre-biofilm drainage time. Re-attach the clamp
and incubate the column at room temperature until the next
laboratory period. Make sure the medium is just at or
slightly above the packing material surface. If you miss
recording the timing for any reason, not to worry, just add
more medium to the 15 ml line and try again.
- On the desk you will find tubes of sterile molasses
medium. Add sterile molasses medium to the column to the 15
ml mark. As before, remove the clamp while holding the
Tygon™ tubing closed. Release the tubing and record
the time taken for the medium to just reach the surface of
the packing material.
- Record the time it takes to drain 15 ml of medium in a
table indicating time and number of days after inoculation
of the column.
- Note any evidence of microbial growth in the pores of
the column seen through the wall of the column.
- On a graph, plot drainage time vs day post inoculation
in seconds. You maay find that recording ml/sec a more
useful measure of biobarrier formation. (15 ml/drainage
time in sec = ml/sec).
Subsequent lab periods:
- Continue to take readings on the flow rate of the
column as long as any significant change is noted. One
might pool class data and graph the results.
- At the end of the exercise, how much (percentage) has
the flow rate been reduced by the presence of the
- Sometime during the growth of the biofilm in the
column, the flow rate will temporarily increase. Based on
your understanding of biofilm growth, can you account for
- Describe a scenario or write a news report in which
this phenomenon might be used to reduce the effects of
Subsurface biofilm barriers for the containment
and remediation of contaminated groundwater
Cunningham AB, Sharp RR, Hiebert R, James G
Bioremediation Journal, 2003; 7:151-164
Educational Program Curricula and Teaching
Developed in collaboration with Dr. John Lennox,
Penn State University-Altoona
©1999-2008 Center for Biofilm Engineering,