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 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

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

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

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