Final Report: NMR Imaging of Biofilm Growth in Porous Media

EPA Grant Number: R821268
Title: NMR Imaging of Biofilm Growth in Porous Media
Investigators: Sharma, Mukul M. , Georgiou, George , Majors, Paul D.
Institution: The University of Texas at Austin
EPA Project Officer: Hiscock, Michael
Project Period: September 1, 1995 through August 1, 1999
Project Amount: $449,760
RFA: Exploratory Research - Chemistry and Physics of Water (1995) RFA Text |  Recipients Lists
Research Category: Water , Land and Waste Management , Engineering and Environmental Chemistry


The transport and growth of microorganisms in the subsurface is of relevance to microbial ecology in aquifers and sediments and to the in-situ biodegradation of organic contaminants. In many instances, biofilm growth is the dominant mechanism by which cell populations colonize subsurface environments. There is currently no direct method for monitoring in-situ such bacteria populations.

In this project we introduce the use of nuclear magnetic resonance imaging (NMRI) techniques as a methodology for the detection and visualization of biofilms. Ultimately the techniques are to be applied to biofilm growth in porous media.

The objective of the first part of this work was to verify optically that NMRI techniques could be used to detect biofilms. A sorted relaxation-resolved NMRI technique is introduced for the non-destructive, quantitative spatial resolution of biofilm in a bioreactor. T1 and T2 relaxation NMR imaging data were used to resolve spatial maps of biofilm. Experiments with a transparent, open-flow and glass bead-packed parallel-plate bioreactor, yielded NMR biofilm images comparable to photographs of the bioreactor. Sorting of the relaxation data improves NMRI by using a threshold biofilm relaxation time value from relaxation time histograms. Ranges of relaxation times for biofilm were determined to be between 0.3 and 2.2 seconds for T1, and 0.07 and 0.11 seconds for T2.

The spatial resolution of the NMR technique was also investigated by running experiments with bacterial colonies on agar, centrifuged cells in test tubes and varying free cell concentration. The spatial resolution experiments showed that E. coli colonies on agar with diameters as small as 1 mm and heights as small as 0.2 mm could be detected and measured with reasonable accuracy using the NMRI technique. These limits correspond with the resolution of the NMRI apparatus used in this study, and are, therefore, not inherent.

Dilute concentrations of free E. coli cells (as opposed to cells within a biofilm) could not be detected easily, indicating that the NMR method might not be a suitable method for detectingfreecellsofthisparticularstrain. NMR velocity images of flow in bioreactors were also conducted. NMR velocity profiles compare very well with calculated velocities. The evolution of biofilm growth patterns with time were different with fluid fiow when compared to no flow conditions.

The second part of this work involved the investigation of the effect of different substrates and flow velocities on bacterial adhesion and biofilm growth. These studies show that biofilms develop sooner and cover a greater percentage of the flow area when the nutrient flow rate is low, thus indicating that it is easier for the cells to a&ere when the shear forces are small. The same trend was detected for both Plexiglas ? and albumen coated Plexiglas. By adding albumen the surface charge density is reduced, and the surface also becomes more hydrophilic. Both factors should, and do, reduce bacterial adhesion.

T1 and T2-resolved NMRI of biofilm in a bead-packed bioreactor are generally comparable with corresponding optical images. The T1-resolved images agreed with the optical image on 80% of the pixels with a net error of 4%. The T1-weighted image agreed with the optical image on 80% of the pixels with a 9% net error. The range of T1 values for biofilm are roughly the same as for the open flow bioreactor. They can be expected to be between 0.3 and 2.2 seconds. The T2-resolved image agreed with the optical image 82% of the pixels with a net error of 2%. The T2 values for biofilm are quite similar to those in the open flow experiment with a range of 0.08 to 0.11 seconds.

Overall resolution is not as high for the bead-packed experiment because of the lower signal-to-noise ratio generated from the smaller biofilm volume in the bioreactor. The lower resolution only affected the NMRI where there were small colonies of biofilm.

Biofilm growth experiments were also conducted in a sandstone sample. NMRI did not satisfactorily indicate the presence of biofilm in the core. There is some evidence of bacteria/biofilm in a T1-weighted cross-section. The vertical line graph that indicates differences in signal intensity between NMRI obtained two weeks apart, shows an increase in signal intensity. This increased signal intensity means that bacteria are in the rock sample at that position. However, resolution was unsatisfactory due to the presence of clays and paramagnetic elements (such as iron) in the core. This indicates that NMR imaging of biofilms in-situ is possible only in relatively clean (clay free) porous media.

We have developed a technique using Atomic Force Microscopy (AFM) that can be employed as an exquisitely sensitive and versatile tool for quantifying the interaction between bacteria and surfaces in physiological solutions. The forces of interaction between an AFM cantilever tip and a uniform lawn of bacteria immobilized on glass were determined. By comparing the interactions of cantilever tips with lawns of isogenic E. coli strains carrying genetic lesions that alter their cell surface composition, it was possible to evaluate the effect of macromolecules such as lipopolysaccharide and capsular polysaccharide on the adhesion process. Mutations that result in the synthesis of truncated lipopolysaccharide or in the overproduction of the negatively charged capsular polysaccharide colanic acid render the interaction of the bacteria with the AFM tip unfavorable due to increased electrostatic repulsion. Furthermore, AFM could be used to evaluate the adhesion of bacteria onto subsurface minerals and commercially relevant biomaterials. In one approach, micron-size polystyrene beads were attached to AFM tips which were then used to measure forces. Unfortunately, this approach is limited by the meager number of materials manufactured as beads of a size suitable for AFM measurements. As an alternative approach, AFM cantilever tips were coated with a confluent layer of bacteria and used to probe planar surfaces. In this configuration, AFM could be employed to measure the force of interaction between virtually any bacterium and surface of interest.

We have measured interactions between hydrophilic and hydrophobic surfaces in an aqueous medium at various pH and ionic strengths as well as in some organic solvents using atomic force microscopy and analyzed them in terms of adhesion and detachment from surfaces. In hydrophilic systems the forces observed were found to be well described by DLVO theory at large separation distances. Very long-range hydrophobic forces were not observed in hydrophilic-hydrophobic systems. Nevertheless the jump into contact was found to occur at distances greater that those predicted by just van-der-Waals attraction. The interaction between two hydrophobic surfaces was dominated by the long-range attraction due to hydrophobic forces. This interaction was found to be sensitive to the type of substrate as well as to the pH and electrolyte concentration.

Measured pull-off forces showed poor reproducibility. However, average values showed clear trends and were used to estimate interfacial energies or work of adhesion. These values were compared to those calculated by the surface tension component theory using the acid-base approach. Good qualitative agreement was obtained giving support for the usefulness of this approach in estimating interfacial energies between surfaces in liquid media. A comparison of the measured adhesion force with hydrodynamic detachment experiments showed good qualitative agreement.

Escherichia coli cells were immobilized onto the tip of a standard AFM cantilever and force measurements were performed by approaching the modified cantilever onto mica, hydrophilic glass, hydrophobic glass, polystyrene and Teflon. Consistent with prior qualitative observations, we show that bacterial adhesion is indeed enhanced by the surface hydrophobicity of the substrate. The forces of interaction measured with the AFM are compared to model predictions based on an extended-DLVO approach. In this model, short-range acid-base and steric interactions are included with the conventional van der Waals attraction and electrostatic components. The theoretical predictions agree well with experimental data for E. coli D21f2, a strain whose outer surface consists of lipopolysaccharide molecules with severely truncated carbohydrate chains. However the adhesive behavior of E. coli strains with more complex cell surface structures was found to be more difficult to model due to the possible involvement of steric and bridging effects or specific receptor-ligand interactions that remain to be resolved.

We have conducted A preliminary investigation into the ability of synthetic surfactants to reduce or eliminate the force of adhesion between Escherichia coli (E. coli ) and both hydrophobic and hydrophilic surfaces. The two surfactants investigated were the block copolymers PEG-lysine dendron and Pluronic F127. The PEG-lysine dendron consists of a polyethylene glycol (PEG) chain coupled to a lysine dendron consisting of 32 positively charged amino acid residues. The lysine dendron serves as the anchor by adsorbing onto anionic surfaces while the PEG chain acts as a buoy and radiates from the surface. Reversible adsorption of PEG-lysine dendron onto anionic polymer surfaces reduced the interaction of eukaryotic cells and proteins with biomaterial surfaces. Another surfactant believed to prevent bacterial adhesion is Pluronic F127 which is both water-soluble and non-toxic. This block copolymer consists of a central hydrophobic segment of poly(propylene oxide) flanked by two hydrophilic segments of poly(ethylene oxide).

The force of interaction between bacteria and planar surfaces was measured using the Atomic Force Microscope (AFM) as described by Razatos et al. (1998a). Bacterial adhesion to surfaces in the presence or absence of surfactant was also examined visually in a flow-cell chamber with a fluorescence microscope. Results indicate that the both the F127 and the PEG-lysine dendron were effective at reducing bacterial adhesion. The effect was most pronounced on hydrophobic substrates.

It was found that the adhesion between particle and substrate increases with the hydrophobicity of the surfaces in aqueous media as predicted by acid-base theory. Good agreement was also obtained for non-aqueous systems. In general the acid-base approach was found to provide reasonable agreement with experiments if long-range electrostatic repulsion is properly accounted for. Results for a range of particles and substrates of varying hydrophobicity and for a range of solvents can be consistently explained using this approach.

Journal Articles on this Report : 5 Displayed | Download in RIS Format

Other project views: All 6 publications 5 publications in selected types All 5 journal articles
Type Citation Project Document Sources
Journal Article Freitas AM, Sharma MM. Effect of surface hydrophobicity on the hydrodynamic detachment of particles from surfaces. Langmuir 1999;15(7):2466-2476. R821268 (Final)
  • Abstract: Langmuir
  • Journal Article Hoskins BC, Fevang L, Majors PD, Sharma MM, Georgiou G. Selective imaging of biofilms in porous media by NMR relaxation. Journal of Magnetic Resonance 1999;139(1):67-73. R821268 (Final)
  • Abstract from PubMed
  • Abstract: Science Direct
  • Journal Article Ong Y-L, Razatos A, Georgiou G, Sharma MM. Adhesion forces between E. coli bacteria and biomaterial surfaces. Langmuir 1999;15(8):2719-2725. R821268 (Final)
  • Abstract: Langmuir
  • Journal Article Razatos A, Ong Y-L, Sharma MM, Georgiou G. Molecular determinants of bacterial adhesion monitored by atomic force microscopy. Proceedings of the National Academy of Sciences of the United States of America 1998;95(19):11059-11064. R821268 (Final)
  • Full-text from PubMed
  • Abstract from PubMed
  • Journal Article Razatos A, Ong Y-L, Sharma MM, Georgiou G. Evaluating the interaction of bacteria with biomaterials using atomic force microscopy. Journal of Biomaterials Science. Polymer Edition 1998;9(12):1361-1373. R821268 (Final)
  • Abstract from PubMed
  • Supplemental Keywords:

    RFA, Scientific Discipline, Waste, Water, Nutrients, Contaminated Sediments, Physics, Environmental Chemistry, Chemistry, chemical mixtures, Bioremediation, Engineering, Engineering, Chemistry, & Physics, NMR spectroscopy, biodegradability, hazardous waste treatment, nuclear magnetic resonance, bioremediation model, decontamination of soil and water, microbial degradation, nutrient concentrations, subsurface ecology, hydrocarbon, aquifer sediments, biodegradation, geophysical imaging, contaminated sediment, PCBs, sediment, subsurface imaging, subsurface systems, aromatic substrates, bioremediation of soils, contaminants in soil, hazardous waste cleanup, aquifer remediation design, soil contaminants, biochemistry, bioacummulation, contaminated aquifers, nuclear magnetc resonance, bioaugmentation, NMR , organic contaminants

    Progress and Final Reports:

    Original Abstract
  • 1996
  • 1997
  • 1998