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.