Biofilms

Much of the material in this section on biofilms was abstracted from Costerton & Stewart - Scientific American July 2001 and the American Society for Microbiology - Education Website

Some other typical biofilm locations and types:

The pristine lake shown in this picture is in the northern Rocky Mountains of Montana. Biofilm communities such as that shown below, form here and are composed of a range of different types of organisms, both autotrophic and heterotrophic. Algae derive their energy from photosynthesis and their carbon from dissolved carbon dioxide. Bacteria, which are generally heterotrophic, obtain their energy from organic matter produced by the algae or from organic matter washing into the lake from the surrounding terrestrial habitat.

This is one kind of biofilm from a pristine aquatic alpine ecosystem as seen through a conventional microscope. The larger, roughly spherical cells that appear green to brown are algae while the smaller dark cells are associated bacteria. Both types of cells produce a polymeric extracellular slime layer which encloses the cells. This complex aggregate of cells and polysaccharide is the biofilm community.

Biofilms are also commonly associated with living organisms, both plant and animal. Tissue surfaces such as teeth and intestinal mucosa which are constantly bathed in a rich aqueous medium rapidly develop a complex aggregation of microorganisms enveloped in an extracellular polysaccharide they themselves produce.

Here, human dental plaque has been exposed to 5 % sucrose for 5 minutes, after which Gram's iodine (0.33% Iodine in 0.66% KI) was applied. The sucrose solution was applied to the left central incisor (which appears on the right) while the right central incisor served as a control.

A scanning electron micrograph of co-adhering oral microorganisms in dental plaque, showing so-called corncob structures. Bacteria in the photograph have a typical corncob structure. Each kernel is a bacterium, and what one sees is an aggregate of organisms stacked on top of each other. Scale bar = 10 µm.

Rolf Bos, H. J. Busscher, W. L. Jongebloed, and H. C. van der Mei, Laboratory for Materia Technica, University of Groningen, Groningen, The Netherlands

Humans have made considerable use of microbial biofilms, primarily in the area of habitat remediation. Water treatment plants, waste water treatment plants and septic systems associated with private homes remove pathogens and reduce the amount of organic matter in the water or waste water through interaction with biofilms.

This image is a scanning electron micrograph of the naturally occurring biofilm on sand grains in the clog mat of a septic system infiltration mound.

Scale Bar= 150 micrometers.

How do biofilms form?

Typically, within minutes, an organic monolayer adsorbs to the surface of the slide substrate. This changes the chemical and physical properties of the glass slide or other substrate. These organic compounds are found to be polysaccharides or glycoproteins. These adsorbed materials condition the surface of the slide and appear to increase the probability of the attachment of planktonic bacteria.

Free floating or planktonic bacteria encounter the conditioned surface and form a reversible, sometimes transient attachment often within minutes.

This attachment called adsorption is influenced by electrical charges carried on the bacteria, by Van der Waals forces and by electrostatic attraction although the precise nature of the interaction is still a matter of intense debate. In some instances, as for example, in the association between a pathogen and the receptor sites of cells of its host there may be a stereospecificity which though still reversible is stronger than that achieved strictly by ionic or electrostatic forces.

If the association between the bacterium and its substrate persists long enough, other types of chemical and physical structures may form which transform the reversible adsorption to a permanent and essentially irreversible attachment.

The final stage in the irreversible adhesion of a cell to an environmental surface is associated with the production of extracellular polymer substances or EPS. Most of the EPS of biofilms are polymers containing sugars such as glucose, galactose, mannose, fructose, rhamnose, N-acetylglucosamine and others.

This layer of EPS and bacteria can now entrap particulate materials such as clay, organic materials, dead cells and precipitated minerals adding to the bulk and diversity of the biofilm habitat. This growing biofilm can now serve as the focus for the attachment and growth of other organisms increasing the biological diversity of the community.

Colonization and adsorption to a surface are followed by the matrix production and development of the water channels.

A mature biofilm in a flowing environment may lose bacteria to the surrounding water.

The biofilm may exhibit “streamers” where these cells are being lost together with some of the matrix materials

Scanning electron micrograph (SEM) of a Pseudomonas aeruginosa PANO67 biofilm that was grown in a square glass tubing flow cell. The flow cell was 3 x 3 mm across and 20 cm in length. The biofilm was grown under high shear, turbulent, flow with a flow velocity of 1 m/s (a corresponding flow rate of 540 ml/min). The arrows indicate the direction of flow in the flow cell. The flow cell was positioned in a recirculating loop attached to a chemostat. Nutrients (a minimal salts with glucose as the sole carbon) were delivered by peristaltic pump and the recycle flow rate was controlled with a vane head pump.

Enlarged view of “A” above showing details of “streamers”

By:

Paul Stoodley, Center for Biofilm Engineering, Montana State University, Bozeman, Montana; Frieda Jørgensen, Food Microbiology Research Unit, Public Health Laboratory, Exeter, UK; Hilary M. Lappin-Scott, Environmental Microbiology Research Group, Exeter University, School of Biology, Exeter, UK

Biofilms can show surprising variation in environmental conditions within very short internal distances.

Large oxygen variations occur within a few hundredths of a millimeter and significant diffusion gradients of nutrients can also be established if they are used by the bacteria in the biofilm. Another effect is that of protection of the bacteria deeper in the biofilm against toxic chemicals.

These images below are micrographs of biofilm cross-sections composed of two bacterial species (Klebsiella pneumoniae and Pseudomonas aeruginosa) with progressive exposure to disinfectant. Untreated biofilm samples (control) and those following exposure to a low level (4 mg/L) of chloramine were stained with two fluorogenic compounds, frozen and cut into thin (5 µm) sections that were observed by fluorescence microscopy and photographed. The base of the biofilm that rests on the substratum is at the bottom of each image; the biofilm surface that is exposed to the overlying bulk fluid is the upper aspect of each picture. A combination of 4'6-diamido-2-phenylindole (DAPI) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) was used to stain the bacterial cells. This combination of stains distinguishes individual cells with active respiration (red-gold) from those that are non-respiring (green).

	Figure 1 shows the untreated control biofilm which is predominantly composed of respiring bacteria. .
	Figure 2 shows the biofilm which is predominantly composed of respiring bacteria, after 30 min. exposure to disinfectant
	Figure 3 shows the biofilm after 60 min. exposure to disinfectant. More bacteria have lost respiratory activity and the biofilm has become thinner.
	Figure 4 shows the biofilm after 90 min. exposure to disinfectant Gordon McFeters, Center for Biofilm Engineering, Department of Microbiology, Montana State University, Bozeman, Mont., USA

Another example is the protection of the deeper cells against the activity of chlorine bleach

Biofilms may also play a role in the biodegradation of resistant chemicals since they can consist of stable aggregations of many different organisms. An example might be that of PCB degradation (below) carried out by a consortium of different microorganisms

Perchloroethylene (PCE), used as a dry-cleaning agent throughout the world, is one of the most commonly encountered groundwater contaminants in the United States. PCE is a priority pollutant regulated by the Environmental Protection Agency. Scientists in the laboratory have discovered that certain bacteria can use PCE for food in the absence of oxygen. Attempts to expand this biodegradation process for the clean up of contaminated soil and groundwaters have met with difficulty. In the real world, conditions cannot be controlled as easily as in the laboratory. Degradation or bioremediation may take place in either bioreactors or in the subsurface environment. However, the microorganisms responsible for the degradation or consumption of perchloroethylene must be able to withstand shocks such as high concentrations of chemicals, mixtures of chemicals, high or low temperatures and extremes of pH. In addition the bacteria cultivated in the laboratory must be able to compete for limiting resources with other microorganisms present if they are introduced into the subsurface.

Jennifer Bower and Ralph Mitchell, Laboratory of Applied Microbiology, Division of Applied Sciences, Harvard University, Cambridge, Mass., USA

Groups of bacteria (consortia) grown on surfaces (biofilms) have been shown to be shock-resistant relative to cultures of a single type of bacteria. Growth on a surface is advantageous when compared to that in liquid because it increases the local density of the organisms, may facilitate the concentration of nutrients (especially important in low nutrient environments such as contaminated subsurface waters) and reduce exposure to shear stresses. In addition, consortia have diverse metabolic capabilities simply as a result of the genetic diversity present within the biofilm conferring to them a selective advantage over individual organisms within the environment.

Biofilms are present in groundwater and may play a role in microbial activities there.

Visualizing bacteria in environments dominated by nonbiological particles is very difficult. When the field is stained with acridine orange, a stain that reacts with nucleic acids, and viewed through a fluorescent microscope, the bacteria are clearly visible as yellow/green rods (Fig. 2).

William Ghiorse, Section of Microbiology, Cornell University, Ithaca, N.Y., USA

Biochemistry and Interactions in Biofilms:

Some recent observations:

Bacteria such as Pseudomonas aeruginosa have genes that are turned on in about 15 mins after the attach to a surface. - one gene is algC and is needed to make alginate - one of the components of the polysaccharide matrix material.

Many biofilm bacterial cells typically make dozens or hundreds of proteins not found in "free-floating" cells.

The cells signal to each other as the approach the "quorum" or number required to initiate biofilm formation. It seems as if a certain number of cells are needed to produce enough of the signal molecules to "switch over" the cells to matrix production - this is the "quorum".

In Pseudomonas aeruginosa and similar cells, the signal molecule is known - they are acylated homoserine lactones. If the gene for these compounds are missing - no biofilms are formed.

Some red algae produce compounds called substituted furanones - they have almost no biofilm on their fronds in sea water. It seems that they block the signal transmissions due to the acylated homoserine lactones since they bind to the receptor sites normally used for signaling.