Analysis of Biofilms
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Analysis of Biofilms
Kendrick B. TurnerAnalytical/Radio/Nuclear ChemistrySeminar
March 24, 2006
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Overview
Introduction What is a biofilm? Biofilm Formation Where are biofilms found? Industrial applications of biofilms
Detection/Characterization Methods Indirect methods Direct methods
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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.
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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
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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
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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
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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
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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
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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
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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.
Indirect Detection Methods
Isolated Growth
GC/MS
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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.
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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.
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Direct Detection Methods
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
Laser Scanning Confocal Microscopy
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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
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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
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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.
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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
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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
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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.
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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