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Chris on Koi

Filtration Facts and Fiction You MUST Know - Slime City

INTRODUCTION

When we enter the hobby of koi keeping we soak up as much information as possible. Much of this information has proven to be not entirely correct. Some of the information we are given has been misinterpreted and distorted. As science progresses and new discoveries are made so we must recognise them and adapt then to our hobby.

In previous articles in this series we have looked at some interesting facts and concepts surrounding filtration in Koi ponds. In this essay we turn our attention to one of the most important parts of the whole pond system - the media.

Whilst bacteria grow on all surfaces of the pond, the purpose of a biofilter or bioconverter is to provide an extra locale that will increase the surface area for nitrifying bacteria to grow on. We do this because the stocking densities in our ponds exceed that of a natural environment. Interestingly, in nature, the nitrifying bacteria will seek out areas where there is a food source concentration (ammonia) and colonise this area. By creating a chamber (or chambers) packed with a media of some sort (extended surface area) and by pumping the pond water through this area, we are, in reality, concentrating the ammonia etc. into this space. These are exactly the things found naturally in nature where nitrifying bacteria flourish - an increased surface area for bacterial colonisation and a feeding area of easily available ammonia for the bacteria.

Placing a media into a chamber or chambers, drawing the water through or pumping the water through them achieves flow or movement of the water, bacteria grow and we have a bioconverter.

The media can be used for both biological activity and in the mechanical part of the filter system. (More of this in the second part). When a media is used for the biological part of the filter – the bioconverter – we need to turn our attention to the bacteria that do such an important function in the system – the conversion of toxic ammonia to nitrite to nitrate to free nitrogen thus completing the nitrogen cycle. There are also many other bacterial species and microorganisms found in the system such as the heterotrophic bacteria. These also play an important role in the system and must not be ignored.

This essay on media is will be split into two parts. This first part will concentrate on fascinating aspects of the bacteria and the second part will discuss the media itself and what you have to do to get it to work for you.

We have learned that the nitrifying bacteria that convert ammonia to nitrite to nitrate are found on every surface in the pond. This is correct. What we were never told is a remarkable fact – that the majority of bacteria in the water are found within a biofilm and are not free floating. Some 98 - 99% percent of bacteria found in an aquatic environment survive within a biofilm surrounding.

This is significant as it affects many facts and myths regarding filtration on koi ponds. The biofilm houses not only nitrifying bacteria but also a host of other types of bacteria and microorganisms. The nitrifying bacteria do not seem to be in the majority within these biofilms.

Whilst we are on the subject of nitrifying bacteria we should consider a slight misnaming of a part of our filter systems. There is no such thing as a biofilter! Sorry guys and gals, after all these years we do not have biofilters on our ponds. The reaction between the nitrifying bacteria and the ammonia is a chemical reaction, not a filtering process. In other words the ammonia is converted by a chemical reaction to nitrite to nitrate. The solids are filtered from the water in the filter, the ammonia is chemically changed to less toxic substances in the bioconverter. Therefore, the part of the system that does this should be called the bioconverter or bioreactor but not a biofilter.

WHAT ARE BIOFILMS?

“Microbiologists have traditionally focused on free-floating bacteria growing in laboratory cultures; yet they have recently come to realize that in the natural world most bacteria aggregate as biofilms, a form in which they behave very differently. As a result, biofilms are now one of the hottest topics in microbiology." (Potera 1996)

Biofilms moved to the forefront of microbiology following a 1994 case that involved the infection of hundreds of asthmatics. It was found that all the asthmatics used the same inhalant contaminated with a bacterium known as Pseudomonas aeruginosa. This bacterium was able to survive the routine disinfection of the inhalant during manufacturing by forming a biofilm comprised of many colonies. The contaminated inhalers contained pieces of the biofilm that were transported directly to the lung tissue by the asthmatics. In the lung tissue the Pseudomonas biofilm was able to flourish. One hundred people died from the biofilm infection.

Scientists are studying the ways bacterial colonies form these slimy layers, which can be resistant to antibiotics, chlorine and disinfectants. Simply put, biofilms are a collection of microorganisms surrounded by the slime they secrete, attached to either an inert or living surface. Biofilms exists wherever surfaces contact water. Also researchers have now shown that a bacterium which attaches to a surface "turns on" a whole different set of genes, which makes it effectively a significantly different organism to deal with.

Dr. Andy Coghlan in New Scientist, August 31, 1996, made one of the best descriptions I have come across of a biofilm -
“In the past few years, scientists have learned how to observe the inner structures of biofilms or mucilages, which are built and populated by plain, humble bacteria such as E. Coli, salmonella and others. New findings about the genetics and biochemistry of biofilms are reported.”

“The mature, fully functioning biofilm is like a living tissue on the pipe or wall surface. It is a complex, metabolically cooperative community made up of different species each living in a customized micro-niche. Biofilms are even considered to have primitive circulatory systems.“

"Different species live cheek-by-jowl in slime cities, helping each other to exploit food supplies and to resist antibiotics through neighbourly interactions. Toxic waste produced by one species might be hungrily devoured by its neighbour. 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).
This biofilm structure was described by Andy Coghlan as a “Slime City”.

Andy Coghlan goes on to describe Slime City - "In most cases, the base of the biofilm is a bed of dense, opaque slime 5 to 10 micrometers (197-394 microinch) thick. It is a sticky mix of polysacharides, other polymeric substances and water, all produced by the bacteria. Soaring 100 to 200 micrometers (3940-7870 microinch) upwards are colonies of bacteria shaped like mushrooms or cones. Above street level comes more slime, this time of a more watery makeup and variable consistency with a network of channels through which water, bacterial garbage, nutrients, enzymes, metabolites and oxygen travel. "Slime City" (Coghlan 1996)

In a mature biofilm, more of the volume is occupied by the loosely organized slime or 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.

Biofilms are even found on the slime coat of fish. (Does account for the fact that we often cannot cure certain aliments and diseases associated with our koi?)

Slime City sets in motion a whole new set of ideas that makes us reconsider what we have learned in koi keeping.

STAGES OF BIOFILM DEVELOPMENT

In a new pond trace organics are the first molecules to be deposited on all solid surface/water interfaces. These trace organics become a conditioning layer for the bacterial cells for them to adhere to the new surface. This occurs almost immediately after the pumps are turned on. i.e. there is circulation of the water. (Mittelman 1985). The initial deposits of organics form a layer that starts to neutralize excessive surface charge and free energy. This free energy tends to push the bacterial cells away preventing them getting close enough to the surface to adhere to it so the initial organic deposit is a very good thing.

Once there is an organic deposit on the surfaces electrostatic attraction assists the bacteria cells to attach to the new surfaces. Some cells may initially break away but simply re-adhere elsewhere further along the flow. However, the cells that do stick to surfaces immediately begin forming structures and building colonies. These structures become extremely strong and resistant to external chemical treatments and forces trying to remove the bacteria. By forming the colonies the long-term survival of the bacterial colony is ensured. This is called a biofilm.

Each bacterium has a surrounding polymer or slime that looks something like a spiders web. It facilitates the attachment to a surface. Not only this but the polymer acts as an ion-exchange system for trapping and concentrating trace nutrients. (Mittleman 1985).  This polymer or glycocalyx (sticky substance) acts as a protective coating for the bacteria.

This slime is produced in considerable quantities and is responsible for the clogging up of mechanical filters and the system in general. It is produced all day and every day.

Stage 1 of Biofilm Development

The initial stage of development of a biofilm is easily damaged. The individual cells are attaching and detaching from the surfaces. The protective “slime’ has yet to form. The individual cells are exposed to the passing water and anything that we have added into it. This occurs within the first few hours of start up.

Stage 2

The second stage of biofilm development follows when the bacteria become fixed to the surfaces in groups. They immediately begin excreting their protective slime layer to ensure the survival of the individual and the colony. The bacteria begin to reproduce and the biofilm develops in size and complexity.

Stage 3

The mature biofilm. This structure is considered by some scientists to be virtually indestructible. If you want to remove the mature biofilm then you will have to resort to extremely powerful chemicals and possibly sand blasting the surface.

The mature biofilm is home to a host of microorganism and a wide variety of bacteria. Some 98 – 99% of the bacteria found in the pond are found within these biofilms.

As science investigates biofilms they have discovered that biofilm bacterial behaviour is much more complex than suspended cell behaviour. The bacteria within biofilms live in communities and appear to influence each other. According to Professor Gill Geesey of the Montana State University - “there are significant differences in the level of expression of genes involved in nutrient cycling among members of a single species bacterial population exposed to the same apparent conditions. Within these populations, there appears to be "division of labor" whereby some cells utilize available energy to turn on metabolic pathways that effect partial degradation of dead particulate matter, while other adjacent cells of the same population utilize the degradation products to produce new cells that are dispersed in the environment.”

SOME BIOFILM FACTS RELATIVE TO OUR HOBBY

1. Resistance to Chlorine

Researchers have demonstrated that biofilm associated bacteria may be 150-3000 times more resistant to free chlorine and 2-100 times more resistant to monochloramine than free-floating bacteria.
Rinsing filters with tap water may not be as damaging as we presumed.

2. Resistance to Antibiotics

Antibiotic doses that kill suspended bacterial cells need to be increased as much as 1,000 x to kill biofilm cells. As the antibiotics react with the watery slime they are used up. Unless the dosage of antibiotics is very strong it will not penetrate the protective slime biofilm. The levels of antibiotics needed to kill biofilms will kill the koi first.

3. Adhesion to Smooth Surfaces

According to Mayette (1992), "piping material that microorganisms cannot adhere to has yet to be discovered. Studies have shown that microbes will adhere to stainless steel, Teflon, PVC and PVDF (Kynar) with nearly equal enthusiasm."

Our perception that bacteria need a rough surface to adhere to seems to be antiquated. This would explain why plastic works as well as stone etc. Smooth surfaces do take longer to have biofilms form on them but will inevitably have biofilm development. Smoothness does not appear to significantly effect the total amount of biofilm that will attach to a surface. According to Meltzer (1993), "no surfaces have been found that are exempt from biofouling. Surface structure does appear to influence the rate of fouling, but only initially. In general, smooth surfaces foul at a slower initial rate than do rough ones, but biofilm formation after a period of days is inevitable."

4. Biofilm recovery (Regrowth)

Bacteria associated with biofilms are much more difficult to kill and remove from surfaces than planktonic organisms. According to Characklis (1990), numerous investigators and plant operators have observed "a rapid resumption of biofouling immediately following chlorine treatment. Incomplete removal of the biofilm will allow it to quickly return to its equilibrium state, causing a rebound in total plate counts following sanitization.”

If we do damage a mature biofilm it recovers rapidly. The time immediately after suspected damage must be treated with caution. Think of this time as a new pond – ease back on feeding and watch the water quality parameters. Do not be afraid to increase water changes to stabilise water quality until the biofilm has recovered.

In one researchers tests – “re-growth of the biofilm started after 2 days and was back up to equilibrium levels after 20 days”.

5. Water Velocity and Biofilms

The question inevitably arises whether back-washing filters will wash off the bacteria that have grown. We know that bacteria grow on all surfaces – including the inside of the pipes. The velocity of the water inside the pipes is far greater than will be experienced in backwashing a filter.

According to Mittelman (1985), "at higher flow rates, a denser, somewhat more tenacious biofilm is formed. As a result, these surfaces often appear to be free from foulants, since they are not slimy to the touch."

In our systems where we have high velocity in the pipes the biofilms will be denser and not quite as high as in the other parts of the system where the flow rates are slower. When the filters (slow flow rates compared to the pipes) are flushed the excess bacteria/bioflilms are sloughed off. This results in random ‘particle showers’ of bacteria in the pond system which can explain day-to-day fluctuations seen in total bacteria count results. If a plate count test is low, one shouldn’t assume that good or bad bacteria are not present in the watering system. They are housed quite comfortably in the protective layers of the biofilm.

6. Secondary Colonizers

Biofilms are a composition of billions of bacteria cells, fungi, algae and other living microbes as well as the organic slime they produce.

The biofilm traps nutrient molecules and also snares other types of microbial cells through physical restraint and electrostatic interaction. These secondary colonizers metabolise wastes from the primary colonizers as well as produce their own waste that other cells then use in turn.

7. Can Biofilms Survive Out of Water?

Biofilms can stay active for days and even weeks when out of the water. They can remain active for longer periods if they are kept damp. If you are ever contemplating building a new pond or extending you existing one then save the media. There are valuable bacterial colonies on your media that will seed your new filter. This natural seeding will start up the new system remarkable quickly.

8. The Down Side of Biofilms

Bacteria we need and bacteria that cause disease problems survive and are protected within biofilms. This explains the difficulty we sometimes experience in eradicating bacterial problems with our koi. 

Summary

Bacteria constitute a very successful life form. In their evolution, they have developed successful strategies for survival that include attachment to surfaces and development of protective biofilms where they behave very differently than free-floating bacteria.

Chris Neaves

References
Characklis, W.G.; Marshall, K.C. eds. Biofilms, John Wiley & Sons, Inc., New York (1990).

Coghlan, A. “Slime City”, New Scientist 15(2045), pp. 32-36 (August 31, 1996).

Costerton, J.W.; Lewandowski, Z.; Caldwell, D.E.; Korber, D.R.; Lappin-Scott, H.M. “Microbial Biofilms”, Annual Reviews of Microbiology 49, pp. 711-745 (1995).

Gillis, R.J.; Gillis, J.R. “A Comparative Study of Bacterial Attachment to High-Purity Water System Surfaces”, Ultrapure Water 13(6), (September 1996).

Editors: Allison D.S.; Gilbert P.; Lappin-Scott H. M. & Wilson M.; “Community Structure and Co-operation in Biofilms”, Society of General Microbiology  (2000)

Last Updated on Saturday, 19 September 2009 21:32