(18a) Multi-Scale Approach to Fundamental Understanding of Biofilm-Mineral Interactions

Multi-scale
approach to fundamental understanding of biofilm-mineral interactions

Somayeh Ramezanian and Nehal Abu-Lail.

Voiland School of Chemical Engineering
& Bioengineering, Washington State University, Pullman, WA 99164-6515.

Somayeh.ramezanian@gmail.com, nehal@wsu.edu

Introduction. The search for
the development of sustainable applications which utilize biofilms for soil
treatment is ongoing. The
growth of bacterial biofilms in geo-media results in clogging of the porous
media and consequently reducing the permeability of fluids in soil. This is
important for many applications in soil including creating biobarriers to
prevent migration of toxic compounds from a contaminant plum to ground water or
an adjacent river. In addition, bacterial biofilms can produce a layer of
extracellular polymeric substances (EPS) and coat the minerals or accumulate in
pore spaces resulting in improved grain contact. Owing to EPS, biofilms usually
show a three-dimensional coiled structure which can mechanically resist an
external tensile stress. Biocoating of mineral grains with biofilms can
increase the shear stiffness and strength of soil and protect soil from erosion
or disruption.

Formation
of biofilms in subsurface sediment environment offers the potential to innovative
and sustainable solutions for many geotechnical problems. However lack of the
fundamental knowledge of how biofilms and minerals interact together blocks the
advancement towards these solutions. Furthermore, the effects of the environmental
conditions including pH and ionic strength of pore water on the particle-level
adhesion forces of biofilms to minerals is not currently well understood. Lack
of such knowledge is an important problem, hindering the effort toward the use
of microbial biofilms in geo-engineering.

Therefore,
the objective of this study is to investigate the interactions between biofilms
and mineral surfaces using nanoscale and macroscale approaches. Here, the
effects of environmental conditions including pH and ionic strength on the nanoscale
adhesion forces between bacterial surface biopolymers and a silicon nitride tip,
which mimics properties of sand, were studied using atomic force microscopy
(AFM). Also quantitative information on bacterial elasticity was obtained from
modeling the measured force-indentation data using the Hertz model of contact
mechanics. In
addition, a sand column was used to represent and study the transport of
bacteria in groundwater.

Materials
and methods.

Culture.
A
strain of Pseudomonas putida was kindly provided by Dr. James Harsh from
the Department of Crop and Soil Sciences of Washington State University.
Bacteria were precultured in tryptic soy broth (TSB) medium overnight. Overnight
cultures were diluted in 1:100 in TSB (10%) growth medium and incubated for
additional 12 h to reach to the late exponential growth phase. For sand column
experiments, a minimal growth medium (NaCl 1.3g/l, MgCl₂ 1 mM, Na₂HPO₄.2H₂O
1.5 g/l, KH₂PO₄ 0.75 g/l, (NH₄)₂SO₄
0.2 g/l, CaCl₂.2H₂O 0.4 mM, FeCl₃.6H₂O
0.01 mM, Glucose 2g/l) was used. Bacterial cells were harvested by
centrifugation at 7000 rpm for 10 min and washed once with saline (0.85% (w/v)
NaCl in water) solution.

AFM
sample preparation. Silicon slides were sonicated in ethanol and DI
water following by incubation in Piranha solution (H₂SO₄
75% and H₂O₂ 25%) for 1 hr. The silicon slides then were
washed and dried and immersed in 30% (v/v) 3-aminopropyltrimethoxysilane
(Sigma-Aldrich, St. Louis, MO) in methanol for 20 min. Cell pellets were
resuspended in the saline solution containing 3 mM
1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride (EDC) (Sigma-Aldrich
St. Louis, MO) and incubated for 15
min. Then 1.2 mM N-hydroxysulfosuccinimide (NHS) (Thermo Fisher
Scientific, Rockford, IL) was added and the solution incubated for additional 15
min. Modified silicon slides then were immersed in the bacterial suspension in
EDC/NHS solution and agitated at 70 rpm for 20 h.

Atomic
force microscopy (AFM). A PicoForce Scanning Probe Microscope with a
Nanoscope IIIa controller and extender module (Bruker AXS Inc.,) was used to
perform the force measurements on bacterial surface biopolymers. Cantilevers
were
calibrated before every experiment in water and the spring constant (~ 0.06 N/m)
and deflection sensitivity of the cantilevers were determined. Images of 6 µm ×
6 µm were captured in tapping mode to locate individual bacterial cells on
silicon slides. On average 15 force curves were collected on the top of each
cell and about 10 cells were investigated at each condition (pH 4 and 7).

Packed
bed sand column experiments. Macroscale experiments were performed
using sand columns that represent the transport of bacteria in ground water. In
these experiments, a bacterial suspension at a certain concentration (~ 1×10⁰⁸)
was allowed to pass through a sand column (1.9 cm in diameter and 10.6 cm in
height) at a given environmental condition (pH 4, 7 & 10). The
bacterial suspension was introduced to the column using a peristaltic pump at
an approach velocity of 0.069 ml/s and all experiments were performed in
triplicates. The
concentration of bacteria in the effluent was continuously sampled and
concentrations were read using a spectrophotometer at 600 nm. Finally
breakthrough curves were drawn. The one dimensional filtration theory model was
used to quantify the collision efficiency of bacterial attachment to sand
particles in different environmental conditions.

Results and discussion.
Our
results indicate that both the magnitude of interaction forces between
bacterial cells and the negatively charged silicon nitride tip, and the elastic
moduli of the Pseudomonas putida cells depend on the pH of the buffer. A
repulsive barrier to bacterial attachment to the AFM tip is seen at pH 7 while
small attraction is observed at pH 4. The larger adhesion force between the
cells and the negatively charged silicon nitride at pH 4 is probably due to the
fact that the functional groups on the bacteria surface biopolymers are less
negatively charged at the lower pH. Also Hertz model estimated higher rigidity
for bacterial cells at pH 4 compared to pH 7. This might be explained by the
fact that at lower pH the biopolymers are less negatively charged resulting in a
more collapsed brush layer on the cell which provide a more rigid surface for
the bacteria surface. While at higher pH the negative charge on the polymer
units increases the repulsive forces and hence causes the biopolymers to extend
in the solution. Our macroscale experiments demonstrated that about 75%, 65%
and 45 % of the initial bacteria population were retained in the sand column at
pH 4, 7 & 10, respectively. This indicates a higher attachment of the cells
to sand particles at pH 4 compared to pH 7 and 10.  Both macroscale and
nanoscale experiments predicted similar adhesion trends between bacteria and
soil mimicking surfaces under variable pH conditions.

Conclusions.
Collectively, our results indicate that at pH 4, cells are more
rigid and adherent to silicon nitride tip compared to cells exposed to pH 7.
Also our macroscale results are in good agreement with our nanoscale adhesion
forces which showed higher adhesion when cells were exposed to pH 4 compared to
pH 7.

Acknowledgements:
This study is supported financially by National Science Foundation grant #
1266366. We also would to thank Dr. Muhunthan for providing the sand which was
used in the macroscale experiments.