Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Analysis of Biofilms Kendrick B. Turner Analytical/Radio/Nuclear ChemistrySeminar March 24, 2006 Overview Introduction What is a biofilm? Biofilm Formation Where are biofilms found? Industrial applications of biofilms Detection/Characterization Methods Indirect methods Direct methods 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. 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 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 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 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 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 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 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 GC/MS Isolated Growth ICP, HPLC, TOC (total organic carbon) analysis of solid or liquid growing media for changes in concentration of metals and organic components with time. 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. 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. 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. http://www.olympusconfocal.com/theory/LSCMIntro.pdf 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 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 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. 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 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 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. 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 Reviews 15 (2002): 155-166. Gromly, S., Adams, V., Marchand, E. “Physical Simulation for Low-Energy Astrobiology Environmental Scenarios.” Astrobiology 3 (2003): 761-770 Kuehn, M., et al. “Automated Confocal Laser Scanning Microscopy and Semiautomated Image Processing for Analysis of Biofilms.” Applied and Environmental Microbiology 64 (1998): 4115-4127. Kral, T., Bekkum, C., McKay, C. “Growth of Methanogens on a Mars Soil Simulant.” Origins of Life and Evolution of the Biosphere 34 (2004): 615-626 LaPaglia, C., Hartzell, P. “Stress-Induced Production of Biofilm in the Hyperthermophile Archeioglobus fulgidus.” Applied and Environmental Microbiology 63 (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