Transcript Analysis of Biofilms - University of Kentucky

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