Analysis of Biofilms

1. Analysis of Biofilms Kendrick B. Turner Analytical/Radio/Nuclear ChemistrySeminar March 24, 2006

2. Overview • Introduction • What is a biofilm? • Biofilm Formation • Where are biofilms found? • Industrial applications of biofilms • Detection/Characterization Methods • Indirect methods • Direct methods

3. What is a Biofilm? • A structured community of bacterial, algal, or other types of cells enclosed in a self-produced polymeric matrix and adherent to an inert or living surface • Bacteria prefer a sessile (surface-bound), community existence when possible, as this provides several advantages over a planktonic (free-floating) lifestyle.

4. Biofilm Pros and Cons • Advantages • Nutrients tend to concentrate at surfaces • Protection against predation and external environment • Pooling of resources (enzymes) from varying bacterial species in biofilm • Advantages • Waste can accumulate to toxic levels inside biofilm • Access to oxygen and water can become limited

5. Biofilm Formation • Steps in Biofilm Formation: • Adhesion to surface • Excretion of glycocalyx (glue-like, self-produced polymeric matrix) • Growth of bacteria within glycocalyx, expansion of bioflim

6. Where are Biofilms Found? • Biofilms are EVERYWHERE! • Tooth plaque • Ships hulls • Medical Implants (leading cause of rejection) • Contact lenses • Dairy/Petroleum pipelines • Rock surfaces in streams/geysers • Clogged drains

7. Biofilms in Extreme Environments • Biofilms most commonly form as a result of some stress. Therefore, biofilms are found in many extreme environments • Polar Regions • Acid Mine Drainage • High Saline Environments • Toxic/Polluted Locations • Hot Springs

8. Industrial Applications of Biofilms • Bioremediation: Bacterial degradation of polluted environments • Biofiltration: Selective removal of chemical species from solution • Biobarriers: Protection of objects using extremely rugged glycocalyx produced by biofilms • Bioreactors: Production of compounds using engineered biofilms

9. Detection/Characterzation Methods • Analytical techniques for monitoring biofilms follow two main strategies: • Indirect dection of organisms by analysis of waste and/or metabolism byproducts • Isolated growth, followed by analysis of headspace gas or growing media by a variety of methods (GC/MS, ICP, HPLC, etc.) • Direct detection of organisms • Microscopy techniques • Detection of proteins or DNA

10. Indirect Detection Methods • Indirect Detection of microorganism is accomplished by growth in an isolated environment followed by analysis: • GC/MS analysis of headspace gas for metabolic waste • ICP, HPLC, TOC (total organic carbon) analysis of solid or liquid growing media for changes in concentration of metals and organic components with time. GC/MS Isolated Growth

11. Indirect Detection Methods • Methane levels of a selection of methanobacteria on a Mars soil simulant • Bacteria innoculated on media with differing volumes of oxygen-free buffer, methane levels monitored in headspace.

12. Direct Detection Methods • Microscopy Techniques • Provides the best direct evidence of biofilm formation by imaging actual cells. • Most common microscopy technique is confocal laser scanning microscopy • Can produce blur-free images of thick specimens at various depths (up to 100µm) and then combine to form a 3D image.

13. http://www.olympusconfocal.com/theory/LSCMIntro.pdf Direct Detection Methods Laser Scanning Confocal Microscopy • A laser source (red line) is focused onto the sample by the objective lens. • The dye-labeled sample emits fluorescence (blue line), which is separated by the beam splitter from the source radiation and focused on a detector. • Fluorescence data from different layers in the sample is processed by a computer to reconstruct a 3D image of the sample.

14. Direct Detection Methods • Confocal Microscopy Image: • This image was taken of a biofilm consisting of a colonization of P. fluorescens at depths of 0, 1, 2, and 3µm. • Image at 1µm shows exopolymer surface of film. • Deeper images show population of cell inside biofilm

15. Direct Detection Methods • Isolation of nucleic acids (DNA/RNA) and proteins provides evidence of biological materials. • Isolation of nucleic acids or protein from a sample is carried out by lysis of cells and precipitation of nucleic acids and proteins. • Nucleic acids and proteins can be fluorescently labeled and detected/quantified

16. Detection as Biomarker for Extraterrestrial Life • It has been shown that biofilms exist in many extreme environments on Earth: • Extreme pH, temperature, salt concentrations • Presence of toxic compounds • It has been shown that biofilms made of methanobacteria can grow on a simulated Martian soil with simulated growing conditions.

17. Detection as Biomarker for Extraterrestrial Life • Application of current detection and characterization methods of biofilms require methods with several characteristics: • Automated, unmanned for robotic applications • Low power consumption • Small size/mass requirements • Simple or no sample prep • Operation in hostile environments

18. Detection as Biomarker for Extraterrestrial Life • Candidates for study: • Eurpoa: One of Jupiter’s moons believed to have liquid water beneath icy surface. • Mars: Bacteria shown to grow on simulated Mars soil and environmental conditions. http://nssdc.gsfc.nasa.gov/image/planetary/jupiter/europa_close.jpg http://antwrp.gsfc.nasa.gov/apod/ap010718.html

19. Conclusions • Bacteria have been shown to exist in virtually all environments on earth. • When induced by stress, bacteria tend to form biofilms. • Several methods exist for quantifying and characterizing biofilms. • Biofilms may be present in extreme extraterrestrial environments. • Methods for detection in these environments are needed which meet criteria for cost-effective, unmanned robotic missions.

20. References • Bond, P., Smriga, S., Banfield, J. “Phylogeny of Microorganisms Populating a Thick, Subaerial, Predominantly Lithotrophic Biofilm at an Extreme Acid Mine Drainage Site.” Applied and Environmental Microbiology 66 (2000): 3842-3849. • Dunne, W. “Bacterial Adhesion: Seen Any Good Biofilms Lately?” Clinical Microbiology Reviews15 (2002): 155-166. • Gromly, S., Adams, V., Marchand, E. “Physical Simulation for Low-Energy Astrobiology Environmental Scenarios.” Astrobiology3 (2003): 761-770 • Kuehn, M., et al. “Automated Confocal Laser Scanning Microscopy and Semiautomated Image Processing for Analysis of Biofilms.” Applied and Environmental Microbiology64 (1998): 4115-4127. • Kral, T., Bekkum, C., McKay, C. “Growth of Methanogens on a Mars Soil Simulant.” Origins of Life and Evolution of the Biosphere34 (2004): 615-626 • LaPaglia, C., Hartzell, P. “Stress-Induced Production of Biofilm in the Hyperthermophile Archeioglobus fulgidus.” Applied and Environmental Microbiology63 (1997): 3158-3163 • Prieto, B., Silva, B., Lantes, O. “Biofilm Quantification on Stone Sufaces: Comparison of Various Methods.” Science of the Total Environment 333 (2004): 1-7