Saturday 26 July 2014

Biofilm

Well “Biofilm” it’s there  all around us .. As well as inside us ...Any damp dark area it thrives ….. What causes Body order or your mouth to stink or teeth to decay.. What is the relation between Koi , and bad breath --well bacteria .. 
Someone truly said , Just keep the Bacteria ---the bacterial will keep your Koi ..
Now again let’s turn to our beloved Koi Guru Mike for what he has to say …

I also have to thank My other Sensais  Saiicu ,Yerrag ,Rick and Ray Jordan for the contribution to Mike's discussion ..

Biofilm

We often talk about beneficial bacteria and nitrifying bacteria when discussing koi pond filtration systems. Looking back in the popular literature, there is little or no mention of "biofilm". About a dozen or so years ago, there was quite a bit of discussion about biofilm on the old NI board and the term began being used with frequency in koi circles. I now find that a number of people use the term without having any real idea of anything more than 'biofilm is where the good bacteria live'. So, a bit of discussion of biofilm seems worthwhile.

I often talk about a 'mature pond' when discussing nitrification, greenwater and other biological aspects of the koi pond. I tend to think of a mature pond as one that is a year or two old. What I actually mean, however, is a pond with an established
 biofilm. Biofilm is an assemblage of microbes that are associated with one another in a matrix of polysaccharide material produced by bacteria in the assemblage, attached to a substrate. It is the 'slime' you can feel on a surface that has been submerged for a while. It is this polysaccharide matrix that is crucial to appreciation of biofilm, because it creates an hospitable environment for the resident microbes and allows intercellular communication. Just like people get under a roof (or cave, etc.) to avoid exposure to sun, rain and harsh temperatures, this 'slime' material allows survival, growth and reproduction in a hostile environment. 

In Nature,
 biofilm is all around. Virtually all bacteria live in multi-species communities in which there are mutual interdependencies. We are familiar with some bacteria converting ammonia to nitrite and others converting nitrite to nitrate. This is a simple example. The interdependencies are innumerable and complex. It is enough for us to understand that few bacteria can thrive if truly alone. The species need other species to supply one need or another. By associating with one another in the biofilm matrix, the resident bacteria are much more effectively able to take/share/get what they need from the others. All have a role to play, a contribution to make. And, in the process the biofiilm community creates its own micro-system, with an internal environment that differs from the external, and something of an internal eco-system. It is far more complex than simply a home for nitrifiers. There can be hundreds of species of bacteria thriving within a biofilm. The biofilm can be multiple layers thick, with the resident species varying between the outer layer and the deeper layers. Many of the species have evolved together over the eons to the point where there is a sort of communication among them. Chemical substances released by one will trigger responses in other species. In many ways, the matrix of creatures lives as if it was a single organism with the constituent species playing specialized roles.

Biofilm will form on just about any substrate. The plaque on your teeth is a type of biofilm. So, in our ponds every submerged surface is territory forbiofilm formation... the pond walls, the interior surfaces of pipes, the walls of the filter units and, of course, the filter media selected because it has great surface area in a small volume. When we set up a new pond, free floating bacteria are immediately present, arriving from the air, from any object that was submerged, from the fish placed in the pond. These bacteria have appendages called fimbraiae. The fimbraiae give the the little germ the ability to attach to a surface. They are very effective in attaching as long as the surface is not bactericidal itself. In short order, micro-colonies develop that are glued in place. These are not easily removed. Scrubbing will certainly reduce the size of the colony greatly, but it would have to be a super-human scrubbing to get rid of all the bacteria. As soon as the micro-colony is established, the bacteria begin producing EPS... extra-cellular polymeric substances. This material forms a barrier around the micro-colony. This EPS barrier also becomes a substrate, with microbes joining it. So, with an infinite number of micro-colonies being formed by numerous varieties of bacteria, it takes little time before the surface of a new pond will have a slimy feel to it. But, this biofilm is immature. Over a period of weeks and months, more species join the matrix. In time, the beneficial nitrifiers we talk about so much are present, but so are numerous other species, including ones we would consider harmful. But, all of them form one biofilm in a mutually beneficial community. 

This slime is not as simple as it at first seems. Over time, multiple layers form, and there can be different bacterial populations within the layers. Most of the reproductive activity, however, occurs in the outermost couple of layers. The lower layers within the
 biofilm can go into a reproductive stasis. If something comes along and scrapes off the outer layers, the bottom layer re-energizes and can recreate the lost layers. Bacteria also seem able to move between layers to some extent in response to changes in nutrient availability, environmental conditions and other factors. At the same time, there is death occurring within the biofilm. As bacteria die, their corpses supply nutrient to the community as a whole. With age, the polysaccharides will degrade/decompose producing a calcified deposit at the base of the biofilm. This deposit creates an extraordinarily strong attachment to the substrate. Curiously, this single biofilm develops structures as if it was itself a single organism. There are tiny 'canals' that form, which you can think of as being something like pores in human skin. These canal structures carry water (and its oxygen and minerals) to the lower layers. Some waste products can even be discharged from the biofilm through these structures, which form something of a labyrinth within the biofilm. Just as bacteria join the biofilm as it develops, bacteria can move out of it. Some scientists believe the biofilm is capable of expelling redundant bacteria that are not contributing to the community. 

Once a
 biofilm is fully established, it becomes very tough to eliminate. They can be setback by exposure to chemicals, by drying out and physical force, but it is really tough to eliminate the biofilm altogether. Even if dried out thoroughly for several weeks, put the dried out biofilm back in water and it will come back robustly in a comparatively short time. Some refer to biofilm as being nearly indestructible because studies have shown that complete removal requires the equivalent of sandblasting. But, pondkeepers should not put much weight on such ideas. Bacteria within the biofilm are also sensitive to environmental changes. The efficiency of nitrification can be affected by shifts in temperature or pH, by medical treatments and very mild chlorination. So, there is both fragility and strength in the structure. In a sense, the structure endures, but our concern is with it functioning efficiently to maintain the pond water. 

And, as a concluding comment, do not get the idea that all
 biofilm is the same. It is not. The constituent species will vary according to the environment in which it exists. In our ponds, there will even be some forms of algae taking up residence in the biofilm. 

So, when we talk about a mature pond, a large part of what is meant is that the
 biofilm is mature.... it has developed all of the interacting species suited to the environment of that pond, in the proportions that function well in that pond, and have the strong attachment and mutiple layers that come with time. Such a pond can withstand all the little stresses so much better, because its biofilm can withstand them and has even adapted to them in the range of species present.

But then does It mean that you need biofilm needs to be brownish always..  what if your media is not covered with slush
Yes, a dark color is not better than a light color. I do not know all the things that can affect the color of biofilm. I know that in a pond stained with tannins, the biofilm will be brownish. Mats I place in the top tray of my Bakki stay light colored except where algae glarf is captured. That portion of the mat captures fines and will be stained yellow-brown. In my Nexus filters the moving bed K1 media seems white until new K1 gets added. Then it is obvious that the older K1 is yellowish. I have seen K1 that was very dark where used with heavily loaded tanks in commercial facilities. I think a lot of what we see is often not so much the biofilm itself as debris that sticks to the surface of the biofilm. In your situation, the frequent removal of debris from the system is probably minimizing the attachment of fines, resulting in the mats being lighter. 

It is easy enough to see that used media is not the bright white of new media, but sometime take out a piece of K1 (or similar media) and visually examine it closely. There are so many different shades of color. And, you may even see a speck of green. ...How does a speck of what seems to be algae colonize a piece of K1 that is in an enclosed container, churning with aeration? It's an amazing world on every piece of media.

It does seem that I've been seeing a lot of threads around the 'net lately where pondkeepers have been dosing chemicals or blasting ponds clean. I guess it is 'Spring cleaning' in June? They end up with the various symptoms of 'new pond syndrome'... greenwater they've supposedly never had before, sick fish, cloudy water, etc., and they ask what they can use to make everything alright .... make it be like it was before chemicals and pressure washers were used to decimate the bio-community of the pond. Of course, they cleaned the pond because it was not properly designed (maintained) and there was a build-up of gunk on the bottom (rocks on the bottom/ no bottom drain), or some other frustration. They react with a burst of energy to make everything pretty, like when the pond was new, with no appreciation for the fact that problems will recur if you just treat symptoms and not causes.

It takes work to maintain a pond, but it is so much easier if you do not work against Nature and instead let Nature work with you. [And, I'm not talking about bog gardens! 
http://www.koi-bito.com/forum/images/smilies/smile.png ]
Now when did the bio film start ??
It began forming as soon as water was added to your pond with low enough chlorine levels that bacteria could survive. It is on all the surfaces of the pond, the filter mats, the pipes ...everything. What you may not have is a fully mature biofilm. That comes in time. You may recall discussion of how a forest forms? Think about Mt. Helens, wiped bare by the volcanic eruption back in the 1980s. First there were various small plants that colonized... mosses, lichens, a few clumps of grass. In time various shrubs and seedling trees took root. What was barren became green. Over the years ahead, the trees will grow and eventually there will be a forest. The accumulation of leaves over decades provides soil for more types of plants and trees to thrive, and with the expanding variety of plants comes an expanding variety of animals. A real forest comes into existence. The forest on the rocky mountainside is not the same as a forest in a river valley nearby, even though they are one continuous swath of forest. Each area within the forest differs according to the nutrient available, the type of soils, the exposure to drying conditions, etc. The main trees will differ as you go up to higher elevations. Biofilm is similar. There is a progression. It takes time for everything to come together. Some microbes that 'want' to join thebiofilm cannot do so until the biofilm has progressed to a state where its residents are producing the nutrients and environment needed. The more variable the environment (climate zone, sun-shade, chemical exposures), the longer it will take to reach a mature state with multiple layers. 

In a sense you can see the 
biofilm on pipes and such in the pond, but you will not really see it without using a microscope. We speak of biofilm'thickness', but it is very thin. Just that slimy smooth surface you feel with your finger..

Biofilm is a function of the totality of the pond environment. If the water is lacking oxygen, the biofilm will have anerobic bacteria in ascendance. The anaerobic bacteria are 'bad', but using PP to eliminate them does nothing to address the environmental condition - oxygen depletion. Disease causing bacteria are likely in residence to some degree. The proportion will be higher or lower in response to the environment. If there is a lot of organic waste to rot, the biofilm engaged in the decomposition will have a high population of organisms ready to attack a scratch and start an ulcer. Anyone who has torn out an old pond has seen how slimy the interior of the plumbing can be. And, a lot of gunk can be captured in the biofilm. The plumbing from bottom drain to filter system often collects debris that has to be flushed out regularly to minimize the rot occurring in the pond. The biofilm in those pipes will undoubtedly have a high proportion of the microbes we would prefer not be around. But, they are present because of the waste in the lines. If eliminated by a strong dose of PP, they will return as long as waste is going through the lines.

I have to disclose my bias against using chemicals in the pond. And, when the use of a chemical is necessary (such as treating for parasites), I strongly prefer whatever will do the job with the least collateral impact. So, I have an especially strong bias against PP, because it is so very thorough and effective in oxidizing everything. With that bias disclosed....

IMO, a situation would have to be extraordinarily extreme to 'bomb' the pond with PP to the point of destroying the 
biofilm. Other than sterilization to eradicate KHV, I cannot think of a circumstance where I think it would be justified.

I'll use the forest analogy again. Let's say we want a forest of mixed tropical hardwood trees, but we get a forest of eucalyptus. We can burn away the eucalyptus over and over, but we will not get a mixed tropical hardwood forest unless the conditions are right AND we stop burning everything down so the hardwoods can grow and reproduce. PP is like using fire to burn away the forest. If instead, rainfall is increased (and other environmental elements made conducive to tropical hardwoods), the eucalypts will decline in proportion and either disappear or have a minor presence. The tropical hardwoods will prevail because the conditions suit them.

Likewise, the more we can make the conditions in our ponds suited to our koi, the more the 
biofilm will be composed of the microbes suited to those conditions. Our main focus is on the nitrifiers, although biofilm is affecting many components of the water. So, we want the water to be highly oxygenated for the benefit of both our koi and the nitrifiers. We want to avoid extreme temperatures and fluctuations in pH. We want water parameters best suited to our koi. Those parameters will also result in a biofilm best suited to our pond and the needs of our koi. It is all connected.

But doesn’t biofilm buildup also live deposits like Calcium indie of water tubing..
"Deposits in the form of biofilm and biofilm with entrapped debris are generally easy to comprehend, but biofilms may often lead to the formation of mineral scales as well. Calcium ions are fixed into the biofilm by the attraction of carboxylate functional groups on the polysaccharides. In fact, divalent cations, such as calcium and magnesium, are integral in the formation of gels in some extracellular polysaccharides. If we can imagine these calcium ions being fixed in place by the biofilm at the heat transfer surface, then it would make them more readily available to react with carbonate or phosphate anions that are present. This would then provide nucleation or crystal growth sites that would not normally be present on a biofilm free surface. Additionally, biofilms may entrap precipitated calcium salts and corrosion by-products from the bulk water that will act as crystal growth sites.

A typical biofilm-induced mineral deposit that we are all familiar with is the calcium phosphate scale the dental hygienist removes from our teeth. When biofilms grow on tooth surfaces, they are referred to as plaques. If these plaques are not continually removed, they will accumulate calcium salts, mainly calcium phosphate, and form tartar (scale). One could make a comparison between rinsing your mouth twice daily with an antiseptic mouthwash to control plaque, with feeding microbicides and biodispersants to control 
biofilm related deposition in heat exchangers. If biofilms in heat exchangers are not controlled, then, like dental plaques, mineral scale may result."

So, it seems 
biofilm can increase the rate of scale formation.

And, in another study, the article abstract indicates that at high hardness levels the rate of calcium precipitation increases greatly:

Effect of Calcium on Moving-Bed 
Biofilm Reactor Biofilms
Authors: Goode, C; Allen, D. G
Source: Water Environment Research, Volume 83, Number 3, March 2011, pp. 220-232(13)
Abstract:
The effect of calcium concentration on the 
biofilm structure, microbiology, and treatment performance was evaluated in a moving-bed biofilm reactor. Three experiments were conducted in replicate laboratory-scale reactors to determine if wastewater calcium is an important variable for the design and optimization of these reactors. Biofilm structural properties, such as thickness, oxygen microprofiles, and the composition of extracellular polymeric substances (EPS) were affected by increasing calcium concentrations. Above a threshold concentration of calcium between 1 and 50 mg/L, biofilms became thicker and denser, with a shift toward increasingly proteinaceous EPS at higher calcium concentrations up to 200 mgCa2+/L. At 300 mgCa2+/L, biofilms were found to become primarily composed of inorganic calcium precipitates. Microbiology was assessed through microscopy, denaturing grade gel electrophoresis, and enumeration of higher organisms. Higher calcium concentrations were found to change the bacterial community and promote the abundant growth of filamentous organisms and various protazoa and metazoan populations. The chemical oxygen demand removal efficiency was improved for reactors at calcium concentrations of 50 mg/L and above. Reactor effluents for the lowest calcium concentration (1 mgCa2+/L) were found to be turbid (>50 NTU), as a result of the detachment of small and poorly settling planktonic biomass, whereas higher concentrations promoted settling of the suspended phase. In general, calcium was found to be an important variable causing significant changes in biofilm structure and reactor function. 

So, yes, those instances of scale build-up in plumbing lines restricting flow are directly related to 
biofilm interacting with the hardness of the water.

And What about the effect of sunlight on the media ….??

Direct sunlight does kill bacteria. The EPS of biofilm provides some protection. So, yes, filter media should not be exposed to direct sunlight, and darkness is better still. After all, we do not need algae growing on and clogging the media...


Sunday 20 July 2014

Koi Growth -Old and new Pond videos

We ll we surely can grow Koi fish in India
Well the water in India certainly has the capability of growing good koi What we need to add are the husbandry skills..

Please find  shot of the Old Gosanke Pond


And the New One

All the Koi's have grown Well , Some more than the others of course