Bio-Pellets and Bacteria

[HighlandReefer]

"•Supplied in a state of suspended animation for maximum longevity."

The only method I have seen in research and what is used in practice by scientists & other commercial uses for storing bacteria in a so called state of suspended animation is to freeze dry bacteria in the spore form (extremely dry form with very little moisture). Moisture activates bacteria from the spore form. Other than that, when bacteria are stored in liquid formulations, they are rather short lived. If the manufacturer has a method of acutally accomplishing this, it would be worth a lot of money for other purposes. I doubt their statement is true for any extened periods of time, especially with both anaerobic bacteria and aerobic bacteria within the same enclosed (anaerobic) bottle. Keep in mind every time you open the bottle, you allow airbourne bacteria to enter it.

Keep in mind that a reef tank is inocculated with bacteria from air bourne spores and live bacteria constantly. I doubt bacterial diversity is a problem in a reef tank. The amount of airbourne bacteria constantly added to a reef tank would IMO far succeed what is added in any bottle, especially regarding diversity.

Scientific research has shown when used for other fields like waste water management, that introduced bacteria into an existing system, the introduced bacteria do not become the dominate bacteria. Perhaps dosing bacteria in a new tank is benefitial, but in existing tanks, the available research clearly indicates that it does not help, even when continually added on a regular basis.

Cyanobacteria produce some potent toxins to maintain their space in a reef aquarium. Cyanobacterial can utilitze photosynthesis which most bacteria can not. This makes cyano quite a competitor. Scrubbing rock on a conitunal basis will help eliminate the toxins produced by cyano which can allow bacteria to settle better into the areas the cyano currently occupy.

Cyano can utilize your dissolved organics that contain P as well as inorganic phosphate. So, limiting phosphate alone may not be enough. You will want to reduce dissolved organics as well, which can be done using GAC more vigorously, in addition to running GFO to help reduce phosphate.

 

[cromedogg33]

Other than that, when bacteria are stored in liquid formulations, they are rather short lived.

Are we talking minutes/hours/days/weeks/months? This is the major concern I have with any product like this. How can the bacteria survive coming from point A where it's 95F shipping over to Point B where it's 32F and still survive? I'm not sure keeping it refridgerated after it's been sitting in a warehouse and on a shelf at a LFS would do any of us any good, am I correct? I also thought bacteria needed to be stored in a glass vial or sorts.

I can't deny my ongoing success with bacteria in a bottle. I'm seeing it first hand....but this could be a matter just like anything else. How do I prove that it is doing what I THINK it's doing? I'm watching cyano in my tank start to turn grayish as though it's dying and I'm currently not seeing any new growth.

Granted, my tank is fairly new (almost to 6 months) and I seeded 100% with MB7 and never saw my tank "cycle" in a common hobby way. I never could record my ammonia spike, and I'm beginning to think back to other tanks that I've setup in the past without bacteria and everyone went from a diatom bloom to a cyano bloom, and then settled in. This was well before 6 months though, so I'm beginning to think while my tank may have been able to break down the ammonia, it was not cycled and I was just adding enough bacteria to keep my test results at an acceptable level.

Maybe I'm just seeing a delayed full cycle in terms of the algae blooms.

 

[bertoni]

There are many kinds of bacteria, so it's hard to generalize, but concentrated active heterotrophic bacterial cultures likely would deplete the oxygen or food in a container in a day or so. To last longer, they'd need to be kept cooled enough for the types of bacteria in the culture to be inactive, or the solution would have to contain dormant bacteria.

 

[Randy Holmes-Farley]

Bacteria can form many different types of stages for protection, such as spores.

This link has some discussion:

http://en.wikipedia.org/wiki/Endospore

 

[2thdeekay]

As far as testing you confirmed what I was thinking in general of testing but how could we test this as it relates to my algae problems other than observation? Or even test how it relates to our hobby.

Scientific testing as it relates to your current algae problems would be pretty complicated, but that's already been said.

For seeding a system containing only dead rock/sand, one could test effectiveness in establishing a bio-filter, but as Cliff said, it doesn't account for bacteria from the air, hands, fish, etc. You'd need lab conditions and controls to make it scientific. We've been over this before, elsewhere.

When you cycled your tank with MB7, and reported to observe no ammonia spike, and at the end no detectable NO3, you wanted to know the MB7 had quickly established denitrification, per the product description, perhaps aerobic denitrification.

You could've tested by dosing nitrite to observe if indeed the N arrives @ NO3, or just disappears. You asked for my help, but were put off by Randy's review of Brightwell products and declined the NaNO2 stock sol'n I offered to give you. A self described 'believer' very recently.

Ironic you'd like Randy and others to help come up with a way to scientifically prove that the Brightwell product works! LOL Not a bad idea, just interesting considering extensive discussions we've had so recently, & the 180 in a few of your newly expressed views.

Good you took my advice to post here. The place to ask good questions. Your politeness on the RC Chem forum IS wonderful. Following along...

 

[cromedogg33]

Bacteria can form many different types of stages for protection, such as spores.

This link has some discussion:

http://en.wikipedia.org/wiki/Endospore

Randy,

Thanks for the read. A bit out of my realm of really thinking about what the bacteria could be in my certain bottle right now.

I don't think this "endospore" would be correct in terms of the bottle I do currently have...otherwise why would any of the bottles have a reccomended usage date if according to the link they can live hundreds if not thousands of years?

Interesting for sure. I will try and snap some photos tonight and over the next few days to try and document my theory!

 

[HighlandReefer]

Raising bacteria in industrial setting is easier said than done. Bacteria mutate easily. Constant monitoring is necessary to keep cultures pure. The aquarium companies who produce these bacteria are not controlled or inspected by government entities. The effects of adding bacteria into aquariums have not been studied. I have yet to find any research which describes the effects of adding bacteria in an established tank nor any research which shows the dominant bacteria present after dosing for extended periods of time. This information is necessary to come to any kind of conclusions to determine the effectiveness of these bacterial products in a reef aquarium. We are just suppose to take their word for it. The research on adding the bacteria to new tanks left a lot to be desired as they did no bacterial ID in the tanks.

This article goes into details regarding the use of probiotics and the unknowns and complications that can occur.

Probiotic Bacteria as Biological Control Agents in Aquaculture

Laurent Verschuere,1 Geert Rombaut,1 Patrick Sorgeloos,2 and Willy Verstraete
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC99008/



From it:

"The scarcity of data about their effective implementation in practice makes the practical relevance of probiotics in aquaculture hazy as yet. Furthermore, there is still a lack of knowledge about the exact modes of action involved in probiotic effects. Typically, a correlation is made between, for instance, the in vitro observations that a pathogen is inhibited by the production of an antimicrobial or bacteriostatic agent or by competition for nutrients (e.g., production of a siderophore) and the in vivo probiotic effect of the same bacterial strain. It can be argued that it is unlikely from an evolutionary point of view that bacteria spend a great deal of energy and resources if this effort is not beneficial to the organism in one way or another, but the evidence is still circumstantial and not conclusive. More in-depth studies of the competitive processes between bacteria must be carried out, and the ecological relevance of the different processes in situ remains to be elucidated. Also, the interaction between the cultured aquatic species and its associated microbiota deserves further research into possible immunostimulative effects.

Most of the literature references on the use of probiotics report on probiotics consisting of single bacterial strains (Table ​(Table1).1). It stands to reason that probiotics based on a single strain are less effective than mixed-culture probiotics when microbial control is desired. The approach should be systemic, i.e., based on a mixture of versatile strains capable of acting and interacting under a variety of conditions and able to maintain themselves in a dynamic way. It has been argued above that in aquaculture the microbial habitat undergoes continuous alterations, allowing constant changes in the structural composition and the functions of the microbial community. It is unlikely that a single bacterial species will be able to remain dominant in a continuously changing environment. The probability that a beneficial bacterium will dominate the associated microbiota is higher when several bacteria are administered then when only one probiotic strain is involved.

At present, for a sample or situation, it is not feasible to examine the extent to which all niches are filled in or, alternatively, how many opportunities are left for putative pathogens to grow and become a threat. Hence, one useful approach is to carefully monitor shifts in the overall microbial communities. Methods which rapidly provide insights into how a community of microbial species evolves are of utmost value. They may signal instability and potential evolution of unwanted microbial associations. Examples of such approaches are the monitoring of the Biolog pattern and the denaturing gradient gel electrophores (DGGE) pattern (41, 60, 77, 78, 137). These approaches, coupled with appropiate information technology, may permit the microbial ecology of aquaculture systems to be monitored in the near future.

Techniques to indicate probiotic strains, such as the use of green fluorescent protein or immunoassay procedure or 16S rDNA probes (70, 137), may also be useful in distinguishing an exogenous probiotc strain in a mixed microbial community, in order to better locate and quantify the probiotic strain. From a practical point of view, however, comparison of the spectrum of activity and the duration of the effect with those of antimicrobials, vaccines, and immunostimulants should also be made.

In order to become of practical significance, probiotics applicable in large-scale aquaculture will have to be produced and formulated under industrial conditions that conform to quality control guidelines. Much effort obviously still has to be invested in terms of the production of such multispecies inocula and the methods needed to preserve them and to validate their quality."

 

[HighlandReefer]

Are we talking minutes/hours/days/weeks/months? This is the major concern I have with any product like this. How can the bacteria survive coming from point A where it's 95F shipping over to Point B where it's 32F and still survive? I'm not sure keeping it refridgerated after it's been sitting in a warehouse and on a shelf at a LFS would do any of us any good, am I correct? I also thought bacteria needed to be stored in a glass vial or sorts.

You are asking the big million dollar questions here.

Interactions between bacteria in liquid formulations is very complicated. There are lots of factors, such as pH that will cause problems. For the endospores, according to what I have read, they need to be stored in very pure form, 99.8% with various chemicals present, which they are currently experimenting with. For the studies where they did use very pure forms of endospores, they stored them at very low temperatures to save them for later use or freeze dryed them. Also from what I can tell, only gram postive bacteria produce endospores.

Another factor that needs to be taken into consideration with bacterial formulations is the pathogen bacteria, which can dominate mixes if not carefully monitored.

 

[cromedogg33]

Cliff,

You gave me an article and post to try and wrap my head around. I will try and give it it's due credit and read through a bit more thorough.

After the few posts by yourself/RHF/bertoni, it becomes a bit more clear as to the convoluted mess this companies are making.

I also have been re-reading how you stated that these companies are not regulated in any way, but you mentioned waste water management companies potentially using the same principal. I may be taking a shot in the dark on your vast knowledge but are they mandated in anyway?

You also say if added to an existing system they may not become the dominant bacteria. I have stated I used this to seed my system....As Randy has stated we are always trying to export from our system. Could this be a reason that when I stopped dosing that cyano showed up and as soon as I started dosing that within a week I started to see a noticeble difference? This possibly being that the bacteria I seeded was no longer becoming the dominant form?

 

[cromedogg33]

Also, to bring back the pellets back into the conversation so as I try to figure this whole thing out as well.....

Lets just assume the bacteria colonized on the pellets that I was dosing. As the pellets get used up and as my skimmer is taking out the "used up pellets" is the bacteria theoretically reproducing as fast as it's using up the pellets?

 

[Boomer]

Josh

Some of those bacteria will end up in the water column which increases water column bacterial destiny. Almost all reef tanks are excessively low in WC bacterial count. Bacterioplankon is a major food source for many animals and especially corals. Controlled lab tests have shown that freshly made seawater has 10x more bacteria in the WC than reef tanks and natural reefs have 10 x that of freshly made seawater. However, the issue still remains do we need them in the WC ?

 

[HighlandReefer]

Cliff,

I also have been re-reading how you stated that these companies are not regulated in any way, but you mentioned waste water management companies potentially using the same principal. I may be taking a shot in the dark on your vast knowledge but are they mandated in anyway?

You also say if added to an existing system they may not become the dominant bacteria. I have stated I used this to seed my system....As Randy has stated we are always trying to export from our system. Could this be a reason that when I stopped dosing that cyano showed up and as soon as I started dosing that within a week I started to see a noticeble difference? This possibly being that the bacteria I seeded was no longer becoming the dominant form?

The only time bacterial supplements are regulated is if they are used for humans (FDA) and if they are used as pesticides (EPA). Currently the only reef oriented product available to my knowledge that is regulated as a pesticide is AlgaeFix which has to undergo extensive testing to prove it is safe in a reef aquarium and to prove it is effective. The bacterial supplements sold are not regulated or monitored by any government entity. This means you are at the mercy of the manufacturing company as to how effective and safe these products are. We have some research provided by one company I am aware of but none from any of the others.

There is some research available from universities that are currently studying these bacterial products either in regards to using them to prevent disease in fish or other organisms grown or for ammonia, nitrate and nitrite control.

Keep in mind that the bacterial products used for scientific research are developed into very pure cultures of bacteria and are preserved by either extreme cold or freeze dried. Before the cultures are added to the experimental tanks the bacteria are carefully checked for species and viability. The doses used are extremely high and are repeated at least weekly at very high doses. The results vary substantially from one research project to another. Many state there may be some effect, but trying to eliminate all the organisms in a tank that can interfere is extremely difficult. The best results are appearing in Chinese research which are not explained as far as how they conducted the experiments at all & there response that I read was simply there was some improvement. In other research there seems to be little difference from the controls used, with the exception of clearer water. Mono-cultures do not seem to work at all. The theory is that multi-cultures may provide some good, but the bacteria used have to be able to displace existing bacteria present in the tank, which does not happen easily, especially where bacteria have started bio-films prior to bacterial supplement.

It is possible that the products you use may be able to displace cyano, but the theory is they would have to be pathogens of the cyano to do this, which there is no mention on the product labels. The idea of adding pathogenic bacteria to control different algae and cyano is being studied now.

One research project I read is working on a micro-algae species which according to preliminary results seems to be more effective at nitrate reduction than bacteria and actually has shown evidence of out-competing and displacing cyanobacteria. Who knows perhaps these micro-algae will soon be available to dose into tanks.

 

[HighlandReefer]

Also, to bring back the pellets back into the conversation so as I try to figure this whole thing out as well.....

Lets just assume the bacteria colonized on the pellets that I was dosing. As the pellets get used up and as my skimmer is taking out the "used up pellets" is the bacteria theoretically reproducing as fast as it's using up the pellets?

If you add the bacteria to a reactor with the pellets, first the bacteria need to adhere to the pellet surface. If there are no bacteria already adhering to the pellet surface and you allow the bacteria to stay in clean seawater for a while without circulation, this would provide the added bacteria a chance to adhere to the surface and establish themselves before other bacteria are introduced once tank water is circulated.

As the bacterial biofilms grow on the pellet surface and the bioflims increase in size, then parts of the biofilms will sluff off and be collected by your skimmer or filter bags. The bacterial will multiply very quickly on the pellets and continue growing on them until gone. The sluffed bacteria from the films are what is collected.

The species of bacteria that are best adapted to utilize these biopellets are the ones that will cover the surface & it is likely that species will change within weeks. The bacterial biofilms are not made up of just one type of bacteria. Instead the biofilms are composed of many types of bacteria. As the bio film grows in size, inner areas will become anaerobic and anaerobic bacterial will move into these areas. The outer layers will be aerobic bacteria. Also you will have cyano, dinos and other micro-organisms incorporated into these bioflims. The best way to describe biofilms is that they are like a miniature city with lots of activity going on inside them. Species of organisms change like populations in cities, except at a very fast pace since bacteria reproduce so quickly. Also, bacteria will mutate quickly, so it is very unlikely that any one strain will remain dominate in a bacterial biofilm for a long period of time.

Bacteria populations in the water column behave similarly, with frequent changes in species that may become dominate for periods of time. Every time water parameter conditions in the reef water change, it is very likely that the bacterial populations change as well. For example if nitrate increases or ammonia levels increase or if phosphate levels increase, this will cause changes in the populations of bacteria as well as many other micro-organisms.



Edit: I might add that waste products are likely removed through a series of different types of bacteria in a biofilm. For example, the outer layer of bacteria on a bioflim will utiize say ammona. The biproducts of these bacteria may be nitrite with other unwanted chemicals produced as well (bacterial bi-products). The bi-products of the first group of bacteria are then passed on and broken down by another species deeper in the biofilm. The passing on of bi-products of the different species can form quite a long chain of chemical reductions until the bacteria can no longer act on the final poduct like H2 which then rises as bubbles out of the tank.

 

[HighlandReefer]

Another aspect regarding bacteria that has not been discussed is the toxins that bacteria produce to protect their space. Let's say a species of bacteria colonizes a complete biopellet or rock (which is very unlikely to happen). In order for another species of bacteria to take over, it needs to be able to deal with (resistant to or able to break down) the current bacteria's toxins to over take it. If the introduced bacteria can do this it can become dominate or perhaps the new bacteria simply reduces the current bacteria's toxins to a point where they are gone which can allow other species to then take over.

 

[cromedogg33]

Awesome info Highland and in some terms I'm starting to understand a bit better.

How this relates to my situation and potentially more reefers and if I'm also thinking about this correctly. Theoretically speaking "fresh seaswater" really never is fresh especially in our hobby. As soon as the water hits our trash can/oxygen/hands it is already contaminated with bacteria.

While "seeding" I opened up my reactor during the seeding phase and dosed my bacteria directly into it.

So if I'm understanding this.... potentially, it wouldn't matter if I dosed bacteria at all. Since the bacteria from the air in my house had time from the moment I opened the container housing the pellets could be the dominate species.

I am confident there is absolutely bacteria on the pellets due to the fact of the sluffing off of the surface and the decrease or overall amount.

Another thought with how you are saying there are "layers" that may provide different situations at each time. Would this mean in the case of N and P pellets that whatever is more prominent in your tank that the bacteria on the pellets will essentially morph into whichever needs to be decreased? I think this goes back to exactly to, do we know which bacteria specifically is in the bottle?

 

[2thdeekay]

I think this goes back to exactly to, do we know which bacteria specifically is in the bottle?

That is the question--if you still assume that the bottle indeed includes bacteria. All the great info posted here can make one wonder if the product actually contain bacteria or, "is it possible to contain something entirely different while achieving a similar result, enough to make a product profitable?"

Any input on enzymes in regards to packaging and utility, that is advertised to be in Josh's bottle? I would, but really think it's better if someone else does.

Complex system of non-pathogenic aerobic and anaerobic microbes, as well as natural
enzymes, specifically formulated to establish biological filtration in new aquarium set-ups,
and to enhance the rate of nitrification, denitrification, and organic waste degradation in
marine and freshwater aquaria through complete nutrient remineralization.

 

[2thdeekay]

Another thought with how you are saying there are "layers" that may provide different situations at each time. Would this mean in the case of N and P pellets that whatever is more prominent in your tank that the bacteria on the pellets will essentially morph into whichever needs to be decreased?

Josh, must say this is also a good question you're asking. Nice.

 

[HighlandReefer]

So if I'm understanding this.... potentially, it wouldn't matter if I dosed bacteria at all. Since the bacteria from the air in my house had time from the moment I opened the container housing the pellets could be the dominate species.

In short, from tests for waste water management using bioreactors, if you add bacteria to the reactor initially, the added bacteria will be dominate, but after a month or less other bacteria will become dominate, even when continuing to dose the bacteria.

I don't see the need to dose these types of bacteria except possibly to get a new tank going quicker.

Boomer has brought up a good point regarding reef aquariums. The bacterial numbers in the water column are quite a bit less than the ocean in open water. Whether this is good or bad, I don't know. Adding vinegar or vodka will increase the bacterial populations in the water column. Simply adding new bacteria to a tank, may not increase these numbers much, since nutrients are the limiting factor. Instead of adding bacteria it may be more beneficial to increase the existing bacterial numbers using a carbon source, especially to try to out-compete other organisms present like cyano.

Perhaps putting skimmers on timers may aid in controlling cyano by allowing increased populations of bacteria in the water column?