Biofilm – more than just slime.

The word biofilm is frequently mentioned by water treatment companies and Legionella consultants. The description of slime on the side of pipes is what is generally used to describe a biofilm. It is widely acknowledged as the source of problems with Legionella contamination and the failure of disinfection in building water systems.

In reality there is much more complexity to these structures than mere slime and it is worth understanding some more detail to recognise why biofilm can have such a negative effect on water quality in buildings, and can be so hard to control.

So what is a biofilm?

The word biofilm is often associated with J W (Bill) Costerton who with his colleagues pioneered research in this area. It was first used in scientific literature in the 1970’s. The history of the discovery of the influence of biofilms is traceable to Claude Zobell a marine microbiologist. Zobell became interested in the microbial load in sea water while working in the 1930’s in San Francisco,.

Summarising his work, he observed that a litre of seawater was likely to have a fairly constant microbial population in relation to the available nutrients. However the microbial population could increase in concentration by up to 1000 times with the same level of nutrients if the same volume of seawater was filled with glass beads. Increasing the surface area for microbial attachment increased the capacity of the water to support microbial growth. This was the first lesson in biofilms: increase the surface area of a system and the population will increase regardless of nutrient levels.

From these earlier experiments and understanding of biofilms and more intense research work in the 1970’s a definition emerged:

‘a physiologically co-ordinated community of microorganisms held in a polysaccharide glycocalyx with entrained organic and inorganic nutrients’

Quite a mouthful and a lot to get your head around! So let’s look at it in sections.

Biofilm Establishment

Biofilms are not chance meetings of microorganisms that settle in one place. The process of biofilm formation usually depends on ‘pioneer organisms’. Usually bacteria, these are the first organisms to attach to a surface. The natural organic material in the water supply binds to surfaces and protrudes like molecular hairs in to the surrounding water. Some bacteria will associate and bind to these organic molecules. This gives off the signal to produce sticky sugars (exopolysaccharides) that attach the cells more closely to the surface. An interesting twist in this process is often bacteria attach best to surfaces they are able to degrade; a good (or perhaps bad) example would be the bacteria that cause tooth decay.

This exopolysaccharide layer is the ‘slime’ base of the biofilm. Once attached and slime coated other bacteria, including Legionella, can associate with the slime and join in the pioneer colony. This process will depend on how well the different species ‘get on’ and sometimes a particular biofilm will prevent colonisation by certain microorganisms. Different microorganisms will excrete different nutrients into the slime which will be beneficial to and select for other members. The production of more slime increases the capacity for new members.

 

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Biofilm formation

Mature Biofilm

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A mature biofilm

 

At some point the predators move in: protozoa, some fungi and simple worms (nematodes) will graze the biofilms. Many will only graze the surface because they cannot penetrate the slime, others can burrow into the matrix. The relationship between predators and hosts is complex. Biofilms that have predators are often healthier and grow better.

The analogy is that Lions ensure the ‘survival of the fittest’ in herds of wildebeest. The composition of the biofilm will also attract specific predators looking for specific organisms to graze. Sometimes the bacteria they graze parasitize the predators. A growing number of other bacteria are being shown to possess have this ability including Legionella, Mycobacteria, Pseudomonas.

 

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Protozoa grazing biofilm in waste water

 

The nature of the surface (metal /plastic /stone), temperature, acidity, alkalinity and nutrient concentrations all play a role in selecting for the biofilm population that will colonise the surface. Some bacteria have clear preferences for some materials as opposed to others.

These processes combine to ensure that the biofilm is a co-ordinated community not just a chance occurrence. The slime also has other properties.

A slimy cap

Glycocalyx literally means ‘slimy cap’. Some microorganisms are particularly good at producing slime, and in response to low nutrients levels will be even more productive. The slime acts firstly as a glue that binds very strongly to the surface and secondly as a coating that protects the members of the community form the external environment. The glycocalyx is hydrophobic, that is it pushes water away from the surface allowing its attachment. This hydrophobic nature also helps to limit the diffusion of chemicals in the water into the slime. This is an important step in preventing disinfectants penetrating the biofilm.

 

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Glycocalyx limits diffusion into and out of the biofilm. This results in a range of environmental conditions quite different from the bathing medium.

A Nutrient Trap

Often slime carries a weak negative surface charge. This charge is what initially allows the bacteria to bind to the surface material. Organisms preferentially bind by attraction to a surface with an opposite charge or a surface with the same charge repels them. Organic material such as proteins and even DNA tend to be ‘sticky’. A sticky slime surface will soon become a trap for free organic matter and debris in the water system. The slime becomes a ‘nutrient net’ concentrating the nutrients at surfaces in the system.

A negative charge will attract oppositely molecules such as nitrate and phosphate and so acts as a nutrient trap collecting valuable nutrients from the surrounding water, and in turn metal ions. The charge will also repel like charged molecules such as negatively charged detergents and disinfectants. In this way the biofilm entrains inorganic nutrients.

Survival Advantages

Costerton and his colleagues also noted that biofilms exhibit ‘phenotypic plasticity’. What does this mean? It means a change in physical behaviour. A simple analogy is that you probably behave differently in different situations. Compare your behaviour at work on a Monday morning to yourself at a football match! The same person; same DNA – but a different behaviour. Biofilms exhibit a couple of different behaviours that assist their survival. Firstly, association with a suitable surface switches on slime production and attachment of the cell. Secondly development of the colony triggers the inhabitants to slow down their metabolism and grow and reproduce more slowly.

The development of a well attached and protected community are the benefits of these two behaviours. Slowing down the metabolism makes nutrient sharing work better for all the inhabitants. It also means that toxic chemicals such as disinfectants take longer to be effective, so they either need to be at higher concentrations or present for longer. These two behaviours are the major (but not only) reasons for the resistance of biofilms to disinfection.

Multi Storey Car Parks

By considering car parks we can undertsand how Claude Zobell observed the huge increase in microbial load when he added glass beads. Like biofilms car parks can be a single layer (car lot) or multi-storey. Like a multi-storey carpark biofilms can fit a lot more cars into the same area as a car lot. More storeys mean more capacity within the same area. Also like multi-storey car parks biofilms afford some protection to the occupants – but also allow the restricted entry of the surrounding environmental conditions. Wind, dust, rain, leaves and light all enter the car park. Biofilms grow in columns with arches and tunnels that allow the flow of water through them bringing oxygen and nutrients.

Pieces of biofilm ‘blow away’ with water turbulence or physical disruptions (like building works). Unlike a carpark where a tornado or an earthquake would destroy the building, the pieces of detached biofilm move on with the water flow to colonise other areas in the system or end up mixed into the water and leaving through an outlet.

Stagnation

Stagnation of the water can lead to a lack of nutrient supply. This causes sections of the biofilm to starve. Once water starts to move again these starved sections detach easily and then pass through the system. So the notion that stagnation improves biofilm growth is probably not strictly true. Stagnation starves and loosens established biofilm causing its detachment.

An example of stagnation and biofilm detachment is a naval practice for removing biofilm on ships hulls. In this case the biofilm includes seaweeds, molluscs and various other creatures. A heavily biofilm laden ship can lose up to 30% of its engine capacity in dragging biofilm around the ocean. If this appears to be a problem the ship can anchor for 3 days or so. This allows stagnation and starvation of the biofilm as there is no flow of water and nutrients through the biofilm, causing it to loosen. If the ship then ‘takes off’ as rapidly as possible a significant proportion of biofilm will detach improving engine efficiency. This is the same principle as flushing outlets that are not in use for several days to control Legionella. The sudden turbulent flow flushes away the loosened biofilm.

More than just slime…

Biofilms are co-ordinated structures that permit microorganisms to exploit surface materials increasing in numbers, trap nutrients, and resist predators and disinfectants. A lot more complex than mere slime! To tackle them in building water systems requires a comprehensive risk management plan and water treatment program.

The content and diagrams remain the intellectual property of Hindmarsh Plumbing Services Pty Ltd and may not be copied, or used without their prior written approval, photos supplied by Dr Richard Bentham. Use of any material within this document if allowed for replication or use must be noted and accredited to Hindmarsh Water Treatment – contact us on 1300 730 462 or at mailto:legionella@hindmarshplumbing.com.au