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Biological Sciences Division
Research Highlights

September 2005

NMR technologies developed for biofilm studies featured in recent journals

Researchers at PNNL have developed a unique capability to map metabolism in live, functioning biofilms. Biofilms are microbial communities that are attached to solid surfaces and to one another in an excreted polymer matrix. They are found in or on nearly every natural environment including humans, where they influence many environmental and health-related processes. The researchers have adapted a hybrid optical and nuclear magnetic resonance (NMR) microscope to provide detailed metabolic information for biofilms grown on microscope slides maintained under controlled growth conditions.

An article in the September 2005 issue of the Journal of Microbiological Methods describes the novel procedures and instrumentation, and demonstrates their use for the study of metabolism in Shewanella oneidensis strain MR-1 biofilms under various growth conditions. (MR-1 is a metal-reducing bacterium that has the potential for remediation of contaminated surface and ground water.) Developments include a perfused microscope sample chamber integrated into a flowing growth reactor, its integration with NMR microscopy hardware, and localized 1H NMR techniques to image biofilm growth and to measure the concentrations of hydrogen-containing metabolites (growth substrates and metabolic byproducts).

A second article in Water Science and Technology introduces a NMR spectroscopic-imaging method to map biofilm metabolism as a function of biofilm depth with 22-micron resolution. Microbe metabolism and phenotype vary rapidly with depth in a biofilms—this is responsible for biofilm properties such as the ability to induce surface corrosion and their resistance to antimicrobial agents.) Ex situ biofilm cultivation was employed, which yielded improved sample properties (uniform biofilm thickness and robust attachment to the glass microscope slide surface) suitable for depth-resolved measurements, and provided improved growth-environment control.

To date, studies have employed so called monoculture biofilms; that is, biofilms composed of microbes of a single bacterial species. PNNL researchers are currently adapting the NMR methods to study diverse, co-culture biofilms populated by different species. All natural biofilms are co-cultures.

This work was supported by PNNL's Biomolecular Systems Initiative. The research was performed in the W.R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by DOE's Office of Biological and Environmental Research and located at PNNL. The researchers include Paul D. Majors, Jeffrey S. McLean, Grigoriy E. Pinchuk, James K. Fredrickson, Yuri A. Gorby, Kevin R. Minard, and Robert A. Wind.


Illustration of the recirculation/dilution flow loop system for NMR microscopy of in situ biofilms. The inset shows an exploded view of the perfusable cell culture sample chamber containing a glass cover slip that serves as a biofilm growth surface at the measurement location. Full Image

References

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. DOI:10.1016/j.mimet.2005.04.017.

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.


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