Analysis of biofilms
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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

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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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