(516i) Investigation Into The Effect Of Bacterial Chemotaxis On In-Situ Bioremediation Rate

This research
investigates the effect of bacterial chemotaxis on degradation rate in an
experimental model for in situ bioremediation (ISB). The process of
sensing an attractant chemical by a motile bacterium and subsequent motion
towards that attractant, known as chemotaxis, occurs in Pseudomonas putida
G7,
Pseudomonas stutzeri KC, Rhizobium meliloti, and many other subsurface strains along
with Escherichia coli. To date, there have been no widely accepted
experimental studies that demonstrate whether bacterial chemotaxis can enhance
biodegradation of contaminants, which can be chemoattractants, in the
subsurface. This research presents novel experimental methodologies, developed
to investigate bacterial chemotaxis and migration, providing for the systematic
evaluation of the effect of the chemotaxis phenomenon in a saturated porous
medium. The hypothesis formulated is that bacteria undergoing chemotaxis in a
porous medium will be able to sense an attractant chemical, bias their motion
towards it, and subsequently degrade the attractant at a higher rate than
bacteria exhibiting non-chemotactic behavior by increasing the bacterial
density in the contaminated region.

This
experimental model has been developed to measure the degradation rate of
serine, a simulated contaminant and chemoattractant. E. coli RP437 was
used as a representative chemotactic in situ bacteria while E. coli
RP5700, a tsr- mutant strain of RP437 that lacks the serine
chemoreceptor, was used as the non-chemotactic control strain. RP5700 exhibits
random motility similar to RP437 in the presence and absence of serine
gradients. These two strains were highly characterized for this work, a process
which was rigorous and performed in more detail than in prior works.
Chemotactic ability of RP437 toward 1 mM serine was validated via capillary and
swarm plate assays. Swimming speeds, run lengths, and turn angles were compared
using a tracking microscope and were statistically similar. Serine uptake rates
in liquid media were also statistically similar: 861.94 ± 39 pmol/(min·10⁷
CFU) for RP437 and 882.84 ±
44 pmol/(min·10⁷ CFU) for RP5700. These results show that these strains are suitable for
investigating any enhancing effect of chemotaxis on biodegradation rate.

For ISB
experiments, a model aquifer has been designed to introduce RP437 and RP5700
bacteria to serine in saturated sand via a sharp gradient. The aquifer was used
to compare serine degradation rates and migration rates through sand. Degradation
rate results over a 21 hour period were 4.5 x 10^-3 ± 1.3 x 10^-3
mM/h for RP437 and 4.6 x 10^-3 ± 1.4 x 10^-3 mM/h for
RP5700. These results show that the degradation rate of serine was
statistically similar for both strains, indicating that enhancement was not
detected. The experimental parameters chosen for this study did not elucidate
degradation or migration enhancements due to chemotaxis. However, the
experimental methodologies developed to acquire these results represent novel
contributions to the field of chemotaxis analysis in porous media. These
methodologies can easily be extended for the variation of other sets of
parameters, such as particle size, cell densities, growth conditions, and
selection of chemoattractants. An additional test in the enhancement of
chemotaxis to complement these results was performed. Chemical enhancement of chemotaxis
was tested in a simple swarm plate study with growth where caffeine was used to
stimulate RP437 bacteria. With the addition of 1.0 µg/ml and 10 µg/ml
caffeine, swarms were 8.5% larger in diameter relative to a control with no
caffeine. This represents 16% more biomass growing and penetrating into the
agar. While degradation in the swarm plates was not measured, this result
demonstrates the potential for enhancement of migration and degradation in ISB
systems. A significant result of this work was the success of using the
RP437/RP5700 pair for chemotaxis studies, due to their high degree of
similarity. This similarity is likely due to the fact that RP437 is the parent
strain for RP5700. It is recommended that work with this pair continue in
studies comparing chemotactic and non-chemotactic behavior.