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
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 Jupiters 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 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