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Microbial Biofilms

Developing biofilm of Shewanella oneidensis MR-1
Developing biofilm of Shewanella oneidensis MR-1. The three-dimensional structure of biofilms is revealed by live imaging using confocal microscopy. Red color indicates cellular DNA and green color indicates extracellular polysaccharides, which play a structural role in the biofilm and may actively and passively sequester metals and radionuclides from the environment. Each unit represents 40 microns. Enlarged View

What Is a Microbial Biofilm?

A biofilm is a collection of microbial communities enclosed by a matrix of extracellular polymeric substance (EPS) and separated by a network of open water channels. These communities adhere to manmade and natural surfaces, such as metals and teeth, typically at a liquid-solid interface (1). Their architecture is an optimal environment for cell-cell interactions, including the intercellular exchange of genetic material, communication signals, and metabolites, which enables diffusion of necessary nutrients to the biofilm community. The matrix in which microbes in a biofilm are embedded protects them from UV exposure, metal toxicity, acid exposure, dehydration and salinity, phagocytosis, antibiotics, and antimicrobial agents (1). The protective EPS and the unsurpassed metabolic versatility and phenotypic plasticity of microbes, likely explain how bacteria are able to persist in so many different types of environments, including those that are inhospitable to higher forms of life. By forming organized communities with other microbes, they can even further extend their ability to adapt and thrive in even the most hostile environments.

What Role Do Biofilms Play?

The majority of bacteria in natural, clinical, and industrial settings persist in association with living or abiotic surfaces. Some contributions by bacterial communities are positive. In nature, they play an important role in the synthesis and degradation of organic matter; the degradation of environmental pollutants; and the cycling of nitrogen, sulfur, and metals. These metabolic processes are complex and typically can only be conducted through the concerted effort of multiple metabolically distinct microbes. In industrial settings, biofilms are important in processing sewage, treatment of petroleum-contaminated groundwater, and nitrification.

However, biofilms also contribute to biocorrosion, are associated with tooth decay, and are responsible for infections of the human body. With regard to biocorrosion, sulfate-reducing bacteria (SRB), such as Desulfovibrio vulgaris, contribute to the corrosion of steel. The presence of Streptococcus mutans in dental plaque is a hallmark of dental caries. Also, biofilms account for more than 80% of all microbial infections of the human body. Examples of biofilm infections of the human body include device-related infections, such as intravenous catheters and joint prostheses; infective endocarditis; and cystic fibrosis pneumonia (1). The protective nature of the biofilm structure makes the bacteria embedded within them remarkably difficult to treat with antimicrobials; biofilms are resistant to doses of antimicrobials 100- to 1000-fold over the minimum lethal dose for microbes outside of biofilms (2). Also, biofilms are highly resistant to both immunological and non-specific defense mechanisms of the body.

The Complexities of Biofilms Research

Past microbiology research focused primarily on pure culture, planktonic (free-swimming) bacteria. While extremely useful for deconvoluting the functional components and the regulatory networks of a single cell, this research tells us very little about community behavior. However, there are many difficulties associated with biofilm research. Biofilms are more difficult to culture than planktonic bacteria. Culturing biofilms must take into account the transport kinetics of nutrients throughout the biofilm and fluid forces on the biofilm. Simple experiments, such as dose response measurements used in liquid cultures, are made difficult by the heterogeneous, complex structure and undefined transport kinetics of biofilms (1).

PNNL Is Developing New and Innovative Technologies for Biofilms Research

Scientists at Pacific Northwest National Laboratory (PNNL) are addressing the complexities associated with biofilm research. We are creating new and innovative ways to study microbial biofilms. Our Microbial Cell Dynamics Laboratory houses a variety of equipment for microbial research, including two biofilm reactors. New technologies are being developed at PNNL for biofilm research, which are nondestructive, quantitative, and capable of producing spatially resolved temporal measurements. These efforts address a major challenge in microbiology, and they complement an objective of the U.S Department of Energy's (DOE) Genomics: Genomes to Life (GTL) program to obtain a functional understanding of multi-species microbial communities.

PNNL's Biofilms Research Is Supported by a Variety of Projects

Controlled Cultivation, Molecular Biology, and Advanced Imaging of Microbial Biofilms - Researchers at PNNL are developing new and innovative approaches to analyze biofilms. We are designing and evaluating a fermentor to grow biofilms reproducibly; constructing genetically modified bacteria to study biofilm development; investigating single- and multiple-species interactions with reactive surfaces; and providing well-defined, controlled samples for developing biofilm research capabilities.

Noninvasive Biofilm Characterization Using Acoustic Microscopy New acoustic microscopy-based capabilities are being developed and demonstrated at PNNL to better understand biofilm community structure and dynamic space-time processes in three-dimensional, heterogeneous, living biofilms.

Experimental Metabolism Studies of Oral Biofilm Communities — Using nuclear magnetic resonance (NMR) methodologies developed at PNNL, metabolite production is being characterized in dental biofilms. Concurrently, stable isotope probing (SIP) will be used to identify the bacterial types that actively produce organic acids from the common sugar, glucose. This combined approach will pave the way for studying the metabolism of other complex microbial communities.

Proteome and Bioenergetic Analysis of Growth States in a Syntrophic Quad-Culture — Scientists at PNNL are developing a method to predict biochemical behavior in a simple, four-microorganism community based on analyses of transcriptome and macroscopic process data describing carbon and energy flow. Organisms of particular interest in this study are the sulfate-reducing and/or syntrophic Desulfovibrio vulgaris and Syntrophobacter fumaroxidans as well as the methanogenic bacterium Methanosarcina barkeri.

Dynamics and Spatial Expression of Signal Proteins in the Desulfovibrio vulgaris Biofilm and its Implication to Iron Corrosion — Anaerobic corrosion of iron is frequently linked to the activity of sulphate-reducing bacteria (SRB), which can form biofilms on the surface of steel. We are measuring the dynamic and spatial expression of green fluorescent protein-fused, two-component signal transduction system proteins in SRB, which may control biofilm formation.

References

(1) Hall-Stoodley L, JW Costerton, and P Stoodley. 2004. "Bacterial Biofilms: From the Natural Environment to Infectious Diseases." Nature Reviews: Microbiology. 2:95-108.

(2) Lewis K. 2004. "Persistor Cells and the Riddle of Biofilm Survival." Biochemistry (Moscow). 70(2):267-274.

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