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Infos - Biofilm

Simply put, biofilms are a collection of microorganisms surrounded by the slime they secrete, attached to either an inert or living surface. You are already familiar with some biofilms: the plaque on your teeth, the slippery slime on river stones, and the gel-like film on the inside of a vase which held flowers for a week. Biofilm exists wherever surfaces contact water.

More than 99 percent of all bacteria live in biofilm communities. Some are beneficial. Sewage treatment plants, for instance, rely on biofilms to remove contaminants from water. But biofilms can also cause problems by corroding pipes, clogging water filters, causing rejection of medical implants, and harboring bacteria that contaminate drinking water.

As in any water system, 99 percent of the bacteria in an automated watering system is likely to be in biofilms attached to internal surfaces. Biofilms are the source of much of the free-floating bacteria in drinking water, some of which can cause infection and disease in laboratory animals. One common biofilm bacteria, Pseudomonas aeruginosa, is a secondary pathogen which can infect animals with suppressed immune systems. Besides being a reservoir of bacteria which can affect animal health, biofilms can also cause corrosion in stainless steel piping systems. In order to design and operate automated watering systems that deliver the bacterial quality required by our customers, we should understand how biofilms develop, some of the problems they can cause, and how they can be controlled.

Understanding bacteria in biofilms is one step in preparing for the future. We are currently meeting the most demanding microbiological water quality requirements by supplying chlorinated reverse osmosis water and by maintaining water quality through flushing and sanitization. But, what if chlorine use in animal drinking water is prohibited?

Of course, you might just want to learn about biofilms to marvel at the ability of bacteria to adapt to their environment and to evade our attempts to eliminate them.

Steps in Biofilm Development
The instant a clean pipe is filled with water, a biofilm begins to form. The development of the biofilm occurs in the following steps:

Step 1.
Surface conditioning
The first substances associated with the surface are not bacteria but trace organics. Almost immediately after the clean pipe surface comes into contact with water, an organic layer deposits on the water/solid interface (Mittelman 1985). These organics are said to form a "conditioning layer" which neutralizes excessive surface charge and surface free energy which may prevent a bacteria cell from approaching near enough to initiate attachment. In addition, the adsorbed organic molecules often serve as a nutrient source for bacteria.

Biofilm01

Figure 1.

Adsorption of organic molecules on a clean surface forms a conditioning film.

(Characklis 1990)

Step 2.
Adhesion of ‘pioneer’ bacteria
In a pipe of flowing water, some of the planktonic (free-floating) bacteria will approach the pipe wall and become entrained within the boundary layer, the quiescent zone at the pipe wall where flow velocity falls to zero. Some of these cells will strike and adsorb to the surface for some finite time, and then desorb. This is called reversible adsorption. This initial attachment is based on electrostatic attraction and physical forces, not any chemical attachments. Some of the reversibly adsorbed cells begin to make preparations for a lengthy stay by forming structures which may permanently adhere the cell to the surface. These cells become irreversibly adsorbed.

Biofilm02

Figure 2.

Transport of bacteria cells to the conditioned surface, adsorption, desorption, and irreversible adsorption.

(Characklis 1990)

Step 3.
Glycocalyx or ‘slime’ formation
Biofilm bacteria excrete extracellular polymeric substances, or sticky polymers, which hold the biofilm together and cement it to the pipe wall. In addition, these polymer strands trap scarce nutrients and protect bacteria from biocides. According to Mittelman (1985), "Attachment is mediated by extracellular polymers that extend outward from the bacterial cell wall (much like the structure of a spider’s web). This polymeric material, or glycocalyx, consists of charged and neutral polysaccharides groups that not only facilitate attachment but also act as an ion-exchange system for trapping and concentrating trace nutrients from the overlying water. The glycocalyx also acts as a protective coating for the attached cells which mitigates the effects of biocides and other toxic substances."

Biofilm03

Figure 3.

Wild bacteria are "hairy" cells with extracellular polymers which stick to surfaces.

(Mittelman 1985)

As nutrients accumulate, the pioneer cells proceed to reproduce. The daughter cells then produce their own glycocalyx, greatly increasing the volume of ion exchange surface. Pretty soon a thriving colony of bacteria is established. (Mayette 1992)

Biofilm04

Figure 4.

Biofilm is made up microbes and a "spiders web" of extracellular polymers.

In a mature biofilm, more of the volume is occupied by the loosely organized glycocalyx matrix (75-95%) than by bacterial cells (5-25%).

(Geesey 1994)

Because the glycocalyx matrix holds a lot of water, a biofilm-covered surface is gelatinous and slippery.

Step 4.
Secondary Colonizers
As well as trapping nutrient molecules, the glycocalyx net also snares other types of microbial cells through physical restraint and electrostatic interaction. These secondary colonizers metabolize wastes from the primary colonizers as well as produce their own waste which other cells then use in turn. According to Borenstein (1994), these "other bacteria and fungi become associated with the surface following colonization by the pioneering species over a matter of days."

Step 5.
Fully Functioning Biofilm  A cooperative "consortia" of species
The mature, fully functioning biofilm is like a living tissue on the pipe surface. It is a complex, metabolically cooperative community made up of different species each living in a customized microniche. Biofilms are even considered to have primitive circulatory systems. Mature biofilms are imaginatively described in the article "Slime City":

"Different species live cheek-by-jowl in slime cities, helping each other to exploit food supplies and to resist antibiotics through neighborly interactions. Toxic waste produced by one species might be hungrily devoured by its neighbor. And by pooling their biochemical resources to build a communal slime city, several species of bacteria, each armed with different enzymes, can break down food supplies that no single species could digest alone." "The biofilms are permeated at all levels by a network of channels through which water, bacterial garbage, nutrients, enzymes, metabolites and oxygen travel to and fro. Gradients of chemicals and ions between microzones provide the power to shunt the substances around the biofilm." (Coghlan 1996)

Biofilms grow and spread
A biofilm can spread at its own rate by ordinary cell division and it will also periodically release new ‘pioneer’ cells to colonize downstream sections of piping. As the film grows to a thickness that allows it to extend through the boundary layer into zones of greater velocity and more turbulent flow, some cells will be sloughed off. According to Mayette (1992), "These later pioneer cells have a somewhat easier time of it than their upstream predecessors since the parent film will release wastes into the stream which may serve as either the initial organic coating for uncolonized pipe sections down stream or as nutrient substances for other cell types."

Biofilm05

Figure 5.

Bacteria and other microorganisms develop cooperative colonies or "consortia" within the biofilm.

An anaerobic biofilm may develop underneath the aerobic layer.

The biofilm thickness will reach an equilibrium as flowing water detaches cells extending out into turbulent flow.

(Borenstein 1994)

How fast does biofilm develop?
According to Mittelman (1985), the development of a mature biofilm may take several hours to several weeks, depending on the system. Pseudomonas aeruginosa is a common ‘pioneer’ bacteria and is used in a lot of biofilm research. In one experiment (Vanhaecke 1990) researchers found that Pseudomonas cells adhere to stainless steel, even to electropolished surfaces, within 30 seconds of exposure.