Dear Pieter
I think that this is appropriate to discuss here, seeing as it involves the start up of a new
pond.... and I do not think it is spoiling the thread.... most people are here to learn and share experiences.....I think that is what the forums are all about...the sharing of knowledge... everyone is entitled to their own opinions at the end of the day, but if we fail to share knowledge we have gained over the years then I do feel that we fail every koi keeper out there who is trying to learn and become better koi / water keepers....... personally I would like to share my knowledge...I feel that some of the things I have learned are valuable to all...... please do not get hit up on the pee issue....as I have already stated that is just a bonus.... it is important to remove the oily films and here is the scientific explanations behind it........ I hope that this information assists others.....
Do we agree that biofilm colonises EVERY available surface area in a
pond.... including pipework, side walls, floor - everywhere.....
If you do not - then click on the link below ...... and read further
[You must be registered and logged in to see this link.]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.
"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)
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.
SOME BIOFILM FACTS REVELANT 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 biofilms. 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." Smooth surfaces do take longer to have biofilms form on them but will inevitably have biofilm development. 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 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 researcher’s tests -"re-growth of the biofilm started after 2 days and was back up to equilibrium levels after 20 days".
5. 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.
6. 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.
7. The downside 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.
OK - so we agree now that biofilms exist on every tiny inch of surface area in a
pond.... AND we can agree that they will eventually colonise even the smoothest of surfaces...... they will just take longer.....
Ok- here is where the PP trick comes in.......Koi Carp July 2006....
Healthy bio film or activated sludge?
"This journey started three and a half years ago when we were comparing Japanese matting with Kaldnes k1 moving bed technology, in combination with the Answer. The test set-up was three identical ponds with the same amount of koi that were getting exactly the same amount of food every day. The biological results were quite surprising for the Kaldnes moving bed – they were actually too good to be true. The nitrate removal rate was very high compared with the Japanese matting, and this lead to many discussions because the total surface area was the same. Also, sometimes the koi needed to be treated and all the ponds were given exactly the
same dose. We saw that often, the treatment was given a biological boost by the Kaldnes moving bed. After some thorough investigation, we understood why this difference was occurring. It surprised us, but in the end it was logical.
Bio Film Development
The discussion lead us to bio film development and how to generate it. So we had to go back to the basic science of bio film generation. In diagram 1 you can see a schematic diagram build-up of a bio film. The most important layer is the organic macro molecules that act as a primer layer for the Extracellulair Polymeric Substances (EPS), or in other words, the bio film. Without the macro molecules you cannot bind the bio film to the media. In picture 2 you can see a macro molecule on Japanese matting. As you can see, there aren't many macro molecules on the surface of the Japanese matting, so we were asked ourselves, 'why this is happening?'. We used some special techniques to find out... As you can see in pictures 3 and 4, the Japanese matting is glued together to create a stable matting. We were interested to find out what kind of glue they used. To find out, we had to prepare the media for a Scanning Electronic Microscope where
we could use an ESD technique to find out what the glue was made of. ESD is a materials analysing method that gives a chemical combination, by percentage, of the glue used.
In picture 5, by point A, we measured the glue and you can see the result in diagram 2 – this was interesting to us. In this diagram you can see many chlorine combinations, which are the reason why the macro molecules don't like to grow on the Japanese matting. In picture 6 you can see there is no bio film on the Japanese matting. So we analysed a piece of Japanese matting that had been treated with a lot of chemicals in a filter for many years. We used a confocale microscope where the sample is immersed in water – in this picture 7 you could see clearly that the glue was 'blown up' and would release chlorine combinations into the water. We showed the results to several microbiologists who concluded this specific Japanese matting could not hold or create a bio film, and is actually a mechanical filter that holds activated sludge as you can see in picture 8. This was quite shocking but was the correct explanation why in our test the Kaldnes moving bed was performing much better than the Japanese matting.
There was biological performance in the Japanese matting filter, but it was actually activated sludge. This means that in the waste the bacteria would do their job for us, but this could also harbour sulphate reducing bacteria (SRB)that could make our koi ill. We even suspected this could be one of the reasons why koi develop Hikkui in older systems that have Japanese matting as their core filtration. However, we had to check another type of Japanese matting to find out if it was using a glue too – this was not the case as you can see in diagram 3. But there was not much bio film development there either. So, we believe that Japanese matting is a mechanical filter that holds activated sludge that will do the biological action, however, if you clean the media, you would loose much of your bacteria.
Primer Layer
As we could see in the Japanese matting, it was very difficult to get the first layer as shown in diagram 1. The first layer is organic macro molecules that act as a primer layer for bacteria to grown on. Without this it is impossible for them to attach to the bio media. This primer could be generated in just one minute or it could take longer if the surface of the media is not clean.
However, each
pond's water is unique and has a different chemical balance – this could mean a long waiting time to generate this primer. So, the question was, 'how quickly we can generate the primer layer?' Some people used caustic soda to clean the K1 but the results were no better than the non-treated K1 and some dealers had high nitrite values, which you would not expect when using this method of cleaning. The other values where fine. It proved a complicated question because, although
this was not common, some people also found very high pH values. So we asked dealers to use live bacteria cultures to generate a bio film more quickly. It produced some better results and the nitrite problem was reduced, but not to a level we were happy with. The only
reasonable explanation was the primer was not 'working' yet and this is when we thought of using potassium permanganate to create the primer layer quickly on the bio media surface.
The PP Test
We encouraged people to do a blind test using potassium permanganate. In this test we used 40 gallons of tap water with 50 litres Kaldnes K1 media and one teaspoon of potassium permanganate crystals and aerated it all. And after this it was rinsed twice with clean water. Normally, it takes a few days to get K1 media to sink into clean tap water, but with this method it was taking a few hours. The same dip was performed with the Japanese matting we were using in the filter system. We did the same setup for a second test, but here we used live bacteria (Cleartech Filter Balance Pro) to help the start-up instead of using the potassium permanganate dip. The tanks used in the test were identical, holding 500 gallons of water with five koi 10in-long. The first chamber had Japanese matting and the last chamber had 50 litres of Kaldnes K1, aerated with an Airtech 40. The test results can be seen in diagrams 4 and 5, where you can see the ammonium peak in Tank 1 was not reaching higher then 0.4 mg/l.
However, in Tank 2 the peak was reaching almost 1.5 mg/l on day five.
The nitrite peak was starting in Tank 1 on day four – it was higher than 0.38 (maximum reading of the colorimeter) and was coming down on day 10. But in Tank 2 the peak started on day 6 and came down on day 18. This test made it clear that if the filter was started correctly, the nitrite problem was solved. Adding the live bacteria also helped to reduce the ammonium peak. So, now the important question was, 'why was the caustic soda not creating a good primer layer?' The reason for this was
quite simple, if you use it, even after cleaning, it leaves a thin film on the surface of the bio media. This thin film has a base soap effect – if you clean a microscopic slide with caustic soda, when it dries you will see a white film, which means the surface is not clean. This caustic soda residue of gives off a chemical effect that dissolves the EPS layer that the bacteria need to survive, and it also means the macro molecule film (primer) can not be developed. But potassium permanganate doesn’t have this problem as you can see in diagram 4 and 5.
Bio Fouling
When starting a new filter we want tocreate a bio film that is very thin in the beginning – most of the time this would be an aerobic process. However, when the bio film becomes thicker, the oxygen diffusion ability is going to be lower, therefore an anaerobic zone is generated in the bio film. Diffusion of oxygen is a very slow process and the bacteria will consume a lot of it. So, in the aerobic zone we will have nitrification and in the anaerobic zone we will get denitrifcation. But bio fouling is a biological process in the bio film because, in nature, other animals would be eaten by other living creatures and in this case, diatomeen and zoo plankton
would eat bacteria, and rotifers would feed themselves again to these living
creatures. Diagram 6 shows this. To prove this to you, Jan took a very special picture of the Kaldnes K1 media in situ where you can see the macro molecular film (primer) and the white spots in the water are diatomeen and plankton. This picture 11 is quite unique and demonstrates that there are a lot of living creatures in the media. In picture 12 you can see the plankton that was found in this K1 media.
EPS
If we feed our koi lots of food, we generate more waste which is the food source for the bacteria that generate EPS (Extracellulair Polymeric Substances) (i.e. bio film). If we generate more EPS we create a thicker bio film. In picture 12 you can see the intense red part that is the bacteria colonies with the transparent EPS, which is the food source for the bacteria. In picture 13 you can see a Scanning Electron Microscope image of these bacteria. If we analyse the EPS layer with an analytic REM/RMA (advanced methods for analyzing living tissue or materials) methods we can find the following elements in these substances. The most important ones are phosphorus and Kalium, which are needed to keep the EPS alive and are the food source for the bacteria.
PP treatments
So, after the big test with PP treatment to get a clean surface and a stable bio carrier material, we could now generate the macro molecular film (primer), needed as a sticky base for the bacteria.
However, most of us would say we use PP to kill our bacteria, not help it thrive. To prove it works we have carried out some tests on a live system where we used Japanese matting and Kaldnes K1 media. In this test, we counted the amount of bacteria in each hour of the test and the results are quite shocking! We found the Kaldnes K1 media recovered quickly after a PP treatment but it was much slower in Japanese matting. So again, we had more questions then answers...
Firstly, we looked at the chemical reaction after a PP treatment and we found the end residues are actually a nutrient for the bacteria and are the same as we have found in the REM/RMA. This was the reason the filter had a boost after a PP treatment. For the Japanese matting this result was less obvious and it was taking more time to recover from the treatment.
We think now that this is because of the protective surface area inside the Kaldness K1 media.
Conclusion
It is now clear that when you start a new filter that uses Kaldnes K1 moving bed, you should first treat the media with Potassium Permanganate as this will speed up the maturation process. If this is not fast enough, you could always add some live bacteria, such as the Cleartech Filter Start products, with some extra filter start tablets. We believe what is written in many books about nitrogen conversion is not correct and that in the bio film things are happening that we did not know about a few years ago. The methods used to analyse bio films are becoming more advanced and
we know now that 22 different groups of bacteria are involved in the protected bio film created in the K1. We believe protected bio films are the key for successful koi keeping as they mean less health problems for your koi.
Activated sludge is a home for SRB bacteria that could generate health problems and does not create a stable protective bio film. After three and half years of research it was now clear to us that Kaldnes K1 moving bed had much more to offer than we first thought..."
Ok - so given the above can you see how quick your
pond will mature and grow bio-film on every available surface if you PP the entire
pond....
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sorry 
om ook iets te se om my dag verder te versuur. Laat ek eers aangaan my personeel se reeds, nou hoekom het Mnr kom werk.
But you need a hellova lot (pee) to influence 30 000lt of water!
??