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In Situ Magnetic Resonance Investigations of Metabolism and Mass Transport in Biofilms

EMSL Project ID


Most microbes in nature attach to surfaces and to one another thus forming biofilms. Biofilms are diverse, robust microbiological communities that can serve beneficial (remedial ecological) or detrimental (invasive biological) functions. These characteristics are thought to arise from drastic changes in microbial phenotype and metabolism with biofilm depth. While time- and depth-resolved metabolism measurements are crucial for understanding and exploiting these biofilm physiological gradients, the required experimental methods were previously nonexistent.

We are developing methods to study biofilms under known and reproducible conditions using nuclear magnetic resonance (NMR) microscopy, spectroscopy and combined NMR/optical techniques. Objectives include: time and depth-resolved metabolism, metabolic pathways and flux rates, mass transport and their correlation with gene expression in bacterial films. These methods are being developed in conjunction with Shewanella oneidensis (strain MR-1) monoculture bacterial film studies, but are equally applicable to other biofilm systems of interest to DOE and NIH. Metabolic and transport process modeling of NMR results will help to establish these new research tools to study adherent cell metabolism.

Previous project results include:
1. Initial time- and depth-resolved metabolite concentrations and biofilm volumes and distributions were monitored for Shewanella oneidensis strain MR-1 biofilms under aerobic and anaerobic media conditions.
2. Spatially resolved metabolite measurements were used to demonstrate depth-varying metabolism for MR-1 biofilms.
3. An initial 13C-labeled lactate metabolic pathway study for MR-1 biofilms was performed (Majors et al., 2004).
4. Several microscopy protocol and hardware changes were made to improve biofilm characteristics, environmental control and imaging performance, and to facilitate future analytical modeling efforts.
5. Initial combined NMR / confocal fluorescence microscopy experiments were performed.
6. Peak assignments for most MR-1 metabolites observed with slow magic angle spinning (slow-MAS) in cells cultured on agar plates were completed by employing 2D-resolved NMR spectroscopy.
7. A high-field (11.7T) slow-MAS probe compatible with existing imaging (gradient) hardware was developed to improve spectral resolution and provide spatial information.

The expected outcomes are:
∑ The combined confocal and NMR microscope developed at PNNL (Wind et al 2002) will be used to correlate depth-resolved (NMR) metabolism and transport information with depth-resolved (optical) genomic (green fluorescent protein reporters), cell viability and cell / exopolymer information for Shewanella biofilms.
∑ Lactate metabolism by Shewanella will be characterized as a function of biofilm depth, cell density and oxygen availability using combined NMR/optical microscopy. Depth-resolved, proton-detected 13C NMR labeling studies will be correlated with depth-resolved biomass measurements and confocal assay of living/dead cells.
∑ A new high-field slow-MAS imaging probe will be evaluated and used to resolve key metabolites for Shewanella via 13C-labeled 2-D NMR studies.

7. Majors, P.D., J.S. McLean, G.E. Pinchuk, J.K. Fredrickson, Y.A. Gorby, K.R. Minard and R.A. Wind, “NMR methods for in-situ biofilm metabolism studies,” Journal of Microbiological Methods (submitted April 2004).

R.A. Wind, P.D. Majors, K.R. Minard, E.J. Ackerman, G.R. Holtom, B.D. Thrall, and T.J. Weber, “Combined Confocal and Magnetic Resonance Microscopy,” Appl. Magn. Reson., 22, 145-158 (2002).

Project Details

Project type
Capability Research
Start Date
End Date


Principal Investigator

Paul Majors
Washington State University

Team Members

Jeffrey Mclean
University of Washington

Jian-zhi Hu
Pacific Northwest National Laboratory

Robert Wind
Pacific Northwest National Laboratory

Related Publications

Hu JZ, DN Rommereim, KR Minard, A Woodstock, BJ Harrer, RA Wind, RP Phipps, and PJ Sime. 2008. "Metabolomics in Lung Inflammation: A High Resolution ¹H NMR Study of Mice Exposed to Silica Dust ." Toxicology Mechanisms and Methods 18(5):385-398. doi:10.1080/15376510701611032
Line narrowing in 1H MAS spectrum ofmesoporous silica by removing adsorbed H2O using N2
Majors PD, JS Mclean, GE Pinchuk, JK Fredrickson, YA Gorby, KR Minard, and RA Wind. 2005. "NMR methods for in-situ biofilm metabolism studies." Journal of Microbiological Methods 62(3):337-344.
Majors PD, JS Mclean, JK Fredrickson, and RA Wind. 2005. "NMR methods for in-situ biofilm metabolism studies: spatial and temporal resolved measurements." Water Science and Technology 52(7):7-12.
Wood BD, F Golfier, PD Majors, M Quintard, and S Whitaker. 2004. "Biofilms in Porous Media: Upscaling Conservation Equations and Measurement of Microbial Distributions via NMR." Biofilms 2004: Structure and Activity of Biofilms Conference, Las Vegas, NV.