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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

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analysis of biofilms

Analysis of Biofilms

Kendrick B. Turner

Analytical/Radio/Nuclear ChemistrySeminar

March 24, 2006

overview
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
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
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
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
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 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
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
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 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

indirect detection methods1
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
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 methods1

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.
direct detection methods2
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 methods3
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
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 life1
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 life2
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
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
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
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