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Old 12/02/2011, 06:51 PM   #1
HighlandReefer
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Interesting article regarding current knowlege of anammox bacteria & the biochemistry

Critical Reviews in Biochemistry and Molecular Biology, 2009; 44(2–3): 65–84

Biochemistry and molecular biology of anammox bacteria

http://informahealthcare.com/doi/pdf...09230902722783

Mike S. M. Jetten1,2, Laura van Niftrik1, Marc Strous1, Boran Kartal1, Jan T. Keltjens1, and
Huub J. M. Op den Camp1
1Department of Microbiology, IWWR, Faculty of Science, Radboud University of Nijmegen, Toernooiveld 1, NL-6525
ED Nijmegen, The Netherlands, and 2Department of Biotechnology, Delft University of Technology, Julianalaan 67,
NL-2628 BC Delft, The Netherlands

Abstract (Enitre article is available to read)

Anaerobic ammonium-oxidizing (anammox) bacteria are one of the latest additions to the biogeochemical
nitrogen cycle. These bacteria derive their energy for growth from the conversion of ammonium and nitrite
into dinitrogen gas in the complete absence of oxygen. These slowly growing microorganisms belong to
the order Brocadiales and are affiliated to the Planctomycetes. Anammox bacteria are characterized by a
compartmentalized cell architecture featuring a central cell compartment, the “anammoxosome”. Thus far
unique “ladderane” lipid molecules have been identified as part of their membrane systems surrounding
the different cellular compartments. Nitrogen formation seems to involve the intermediary formation of
hydrazine, a very reactive and toxic compound. The genome of the anammox bacterium Kuenenia stuttgartiensis
was assembled from a complex microbial community grown in a sequencing batch reactor (74%
enriched in this bacterium) using a metagenomics approach. The assembled genome allowed the in silico
reconstruction of the anammox metabolism and identification of genes most likely involved in the process.
The present anammox pathway is the only one consistent with the available experimental data, thermodynamically
and biochemically feasible, and consistent with Ockham’s razor: it invokes minimum biochemical
novelty and requires the fewest number of biochemical reactions. The worldwide presence of anammox
bacteria has now been established in many oxygen-limited marine and freshwater systems, including
oceans, seas, estuaries, marshes, rivers and large lakes. In the marine environment over 50% of the N2 gas
released may be produced by anammox bacteria. Application of the anammox process offers an attractive
alternative to current wastewater treatment systems for the removal of ammonia-nitrogen. Currently, at
least five full scale reactor systems are operational.


------------------------------------------------------------------------

Perhaps the use of anaerobic reactors with acetate as a carbon source may be a more effective way for reducing nitrate in a reef aquarium.


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Old 12/02/2011, 07:43 PM   #2
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So am I correct to interpret this as bringing credence to plenums and DSB's employed in our hobby?


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Old 12/03/2011, 06:18 AM   #3
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This review discusses the benefits of anaerobic reactors which can be used to either replace or act in series with aerobic reactors for reducing nutrients in waste water management, aquaculture & other applications. They are saying that anaerobic reactors are cheaper & reduce more CO2 out of the water than aerobic reactors, which would be beneficial to reef aquariums. They state the carbon sources used are important when using anaerobic reactors since alcohols (including ethanol) inhibit ANAMMOX bacteria. Acetate (vinegar) is one carbon source that works well with ANAMMOX bacteria.

I have not seen anaerobic bioreactors used in the hobby much with the exception of sulfur reactors which have their concerns.

From threads I have read here at RC, there are questions regarding how effective deep sand beds are in a reef aquarium. ANAMMOX bacteria grow relatively slowly compared to aerobic bacteria without a carbon source, but when the scientists add acetate (vinegar) this greatly increases their effectiveness for reducing excess nutrients in the water column. Perhaps dosing vinegar in a reef aquarium with a deep sand bed will increase nitrate reduction to where it is a much more useful tool.


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Old 12/03/2011, 03:11 PM   #4
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I think Mr. Wilson is already doing this. I think I had read somewhere that he has an airline tube threaded down through a deep sand bed (in his overflow) and doses. I may be remembering this wrong, but I'll see if I can find it.

Very interesting.


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Old 12/03/2011, 03:12 PM   #5
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Cool article Cliff.


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Old 12/03/2011, 03:37 PM   #6
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Originally Posted by HighlandReefer View Post
They are saying that anaerobic reactors are cheaper & reduce more CO2 out of the water than aerobic reactors, which would be beneficial to reef aquariums. They state the carbon sources used are important when using anaerobic reactors since alcohols (including ethanol) inhibit ANAMMOX bacteria. Acetate (vinegar) is one carbon source that works well with ANAMMOX bacteria.

I have not seen anaerobic bioreactors used in the hobby much with the exception of sulfur reactors which have their concerns.

From threads I have read here at RC, there are questions regarding how effective deep sand beds are in a reef aquarium. ANAMMOX bacteria grow relatively slowly compared to aerobic bacteria without a carbon source, but when the scientists add acetate (vinegar) this greatly increases their effectiveness for reducing excess nutrients in the water column. Perhaps dosing vinegar in a reef aquarium with a deep sand bed will increase nitrate reduction to where it is a much more useful tool.
Does this have any implications for using vinegar exclusively as opposed to a vodka/vinegar blend (such as tmz discusses in his TOTM)? Or would the average aquarist still see benefits in dosing both?


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Old 12/03/2011, 04:17 PM   #7
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Most data available regarding nutrient reduction comes from waste fresh water treatment studies. There have been a few studies completed involving seawater waste removal which seems to follow suit with the freshwater studies.

This article provides a good comparison of anaerobic vs aerobic bacterial nutrient break-down for fresh water:

ANAEROBIC VERSUS AEROBIC TREATMENT IN THE U.S.A.
http://www.google.com/url?sa=t&rct=j...8jzpBnoVaaDE3Q

From the graphs in this article, anaerobic break-down is the winner as far as cost and provides the best nutrient break-down (significantly lower than aerobic alone and the combo of the two). The chemical pathways for reduction seem to be the most efficient IMHO using anaerobic methods. From the graphs the anaerobic bacteria provide the lowest final nutrient levels.

The studies I have read indicate that ethanol will inhibit the anaerobic bacteria which is not a good thing IMHO. On the other hand acetate (vinegar) will increase growth rates of both anaerobic and aerobic bacteria which is a win, win IMHO. So to answer your question, IMHO vinegar alone may be the clear winner based on what info I have read.

Vinegar is the cheapest carbon source as well, which makes it a winner as far as long term costs. The biopellets are just too expensive at this point and I see no need for their use. Vodka is more expensive than vinegar.


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Old 12/03/2011, 04:41 PM   #8
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As far as reactors that hobbyists can use to take advantage of the anaerobic pathway, while using vinegar as a carbon source for nutrient reduction, perhaps Randy is currently using a very good method.

It is my understanding that Randy is currently using a canister filter with GAC in it and when he cleans it out he finds lots of bacterial growth in it, which he rinses as necessary to keep flow going. Randy does a lot of vinegar as well. A canister filter with reduced flow that produces lots of bacterial masses (biolfims) will produce a lot more anaerobic bacteria than a reactor with biopellets in it as hobbyists are currently using, unless they really slow down the flow and allow a lot of bacterial biomass to develop. Most hobbyists don't do this with the pellet reactors from the pictures I have seen. Perhaps this is a reason why many hobbyists seem to have problems with the pellet reactors? For anaerobic bacterial production it is all about the production of large bacterial biomasses which contain the anaerobic areas needed for the bacteria to grow.


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Old 12/03/2011, 04:59 PM   #9
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Another advantage for using vinegar is that it will increase anaerobic break-down of nutrients within sand beds and the surface of rocks where cyano tends to grow if space is available which is a survival of the most fit situation. If enough anaerobic bacterial growth (biofilms) are produced by dosing vinegar in these sites than the anaerobic bacteria prevail and out-compete the cyano. On the other hand vodka will inhibit the anaerobic bacteria at these sights which may allow the cyano to growth instead which results in cyano out-competing the aerobic bacteria. Cyanobacteria utilize photosynthesis which aerobic bacteria can't in most cases, which gives the cyano a big edge to outcompete the aerobic bacteria. IMO, anaerobic bacteria are your best bet to out-compete the cyano. Therefore, acetate (vinegar) dosing Win-Win-Win-Win.


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Old 12/03/2011, 05:11 PM   #10
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Another advantage for using vinegar is that it will increase anaerobic break-down of nutrients within sand beds and the surface of rocks where cyano tends to grow if space is available which is a survival of the most fit situation. If enough anaerobic bacterial growth (biofilms) are produced by dosing vinegar in these sites than the anaerobic bacteria prevail and out-compete the cyano. On the other hand vodka will inhibit the anaerobic bacteria at these sights which may allow the cyano to growth instead which results in cyano out-competing the aerobic bacteria.
How is this theory when it comes to dinos? Do your theory still apply as it does concerning cyano?

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Old 12/03/2011, 05:26 PM   #11
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Most dinos are phototrophic like cyano and algae. Many dinos can consume algae, bacteria & other dinos as a food source depending on the specie involved, so limiting phosphate like for cyano does not work well for many dino species. So, again, IMO using anaerobic bacteria may be your best bet to try and out-compete dinos while using vinegar to increase the normal slow anaerobic bacterial growth.

Simply dosing vinegar will not be the total answer when trying to control dinos, you will still need to use the other means to reduce nutrients in a reef tank.

If you have areas where rock and sand beds are covered with cyano, algae or dinos it will not be easy for the anaerobic bacteria to establish themselves in place of the pests since these pests all produce toxins to inhibit other bio growth. By removing the pests, you than allow the bacteria bioflims to develop and perhaps out-compete the pests.


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Old 12/03/2011, 05:43 PM   #12
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Perhaps yet another advantage for using acetate (vinegar).

From this article two species of cyanobacterial (blue green algae) were tested using acetate and it did not increase the growth of these species.


The Metabolism of Acetate by the Blue-green Algae, Anabaena
variabilis and Anacystis nidulans
http://www.google.com/url?sa=t&rct=j...DaujP-P9TbjPnA

From it:

RESULTS

Growth arid respiratory studies

Short-term experiments. When the minimal salt (autotrophic) medium was supplemented
with sodium acetate (20 mM) neither Anabaena variabilis nor Anasystis
nidulans exhibited a significantly higher rate of growth. Variation of the concentration
of sodium acetate (10-50 mM) was without effect and attempts to grow A . variabilis
in the presence of sodium acetate and absence of carbon dioxide were unsuccessful.


The failure of acetate and other substrates to stimulate respiration in blue-green
algae has been known for some time (Kratz & Myers, 1955b) and in our experiments
A. variabilis was no exception.
Over a range of pH values (4-3-8.0) the endogenous
rate of respiration of this organism was not significantly affected by the addition of
sodium acetate to a concentration of 30 ]TIM. The oxygen uptake by A . nidulans was
slightly stimulated by the addition of glucose. Comparison of respiratory rates in the
mineral salt medium and 0.1 M-potassiutn phosphate buffer (pH 7.0) showed that the
growth medium was a superior environment for respiration by suspensions of washed
organisms. When the organisms were starved of carbon dioxide for 4 hr immediately
before harvesting, the rate of endogenous respiration was decreased ; the rate of
respiration was largely restored by the addition of sodium acetate or glucose; other
organic compounds had less effect (Table I ) .


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Old 12/03/2011, 06:05 PM   #13
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In this study ethanol stimulates the growth rate of cyanobacteria and acetate has no effect on cyano growth rate.

Characterization of an Alcohol Dehydrogenase from the Cyanobacterium Synechocystis sp. Strain PCC 6803 That Responds to Environmental Stress Conditions via the Hik34-Rre1 Two-Component System▿†

http://jb.asm.org/content/191/13/4383.full

From it:

"As shown in Table 2, the enzyme was active toward a wide variety of primary alcohols and their corresponding aldehydes, but neither ketones nor secondary alcohols were effective substrates."

Table 2:

http://jb.asm.org/content/191/13/4383/T2.expansion.html


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Old 12/03/2011, 06:18 PM   #14
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In conclusion:

Unless someone can provide scientific evidence I have not read, I see no reason to dose vodka (ethanol). Rather I have found plenty of advantages for utilizing vinegar (acetate) as the carbon source in the articles listed above.

FWIW, the above articles are just a few of the articles I have gone through to come up with this concludsion.


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Old 12/03/2011, 06:51 PM   #15
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I would point out that both bacteria and cyanobacteria produce PHA as an energy storage for use when times are lean, so it is only logical to conclude that the Biopellets can be utilized by cyanobacteria. Anaerobic bacteria do produce PHA as well, so I would assume that the anaerobic bacteria can utilize biopellets. The question I have is since the biopellets are a larger molecule than acetate, how well does PHA penetrate into larger bacterial biomasses where anaerobic bacteria exist?


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Old 12/03/2011, 10:06 PM   #16
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I think Mr. Wilson is already doing this. I think I had read somewhere that he has an airline tube threaded down through a deep sand bed (in his overflow) and doses. I may be remembering this wrong, but I'll see if I can find it.

Very interesting.
I asked Mr. Wilson if he was using this technique; here is his response:

Quote:
No, I'm not doing this now. What you may have read is my idea of using sand in an overflow with perforated feeding tubes to add a carbon or sulphur source. You could place some NP biopellets at the bottom of the tube. Just make sure it doesn't become too anaerobic and release hydrogen sulphide into the water column.
HighLandReefer, what do you think about a layer of biopellets at the bottom of a 6" sand bed?


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Old 12/03/2011, 10:26 PM   #17
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Highland,
You feed my brain.


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Old 12/04/2011, 06:15 AM   #18
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Quote:
Originally Posted by Telaverus View Post
HighLandReefer, what do you think about a layer of biopellets at the bottom of a 6" sand bed?
This was done in the patent listed below using PCL pellets:

Denitrification of aquarium water
http://www.google.com/patents?hl=en&...page&q&f=false

The author's findings were that it does not work well with fine sand like commonly used in reef tanks. Instead this method works better with crushed coral. Personally I would not use crushed coral in a reef tank.

According to the author's findings adding crushed coral to a pellet reactor reduces the amount of biopellets you need to use to accomplish the same amount of reduction. This sounds better to me.


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Old 12/04/2011, 06:18 AM   #19
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Highland,
You feed my brain.
I'll take this as a compliment.

You're welcome.


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Old 12/04/2011, 06:35 AM   #20
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Thanks Cliff


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Old 12/04/2011, 06:41 AM   #21
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You're welcome.


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Old 12/04/2011, 07:49 AM   #22
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Great finds, Cliff.


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Old 12/04/2011, 08:18 AM   #23
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Thanks Randy.

It's always nice to get a compliment from my mentor.


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Old 12/04/2011, 11:30 AM   #24
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Does this have any implications for using vinegar exclusively as opposed to a vodka/vinegar blend (such as tmz discusses in his TOTM)? Or would the average aquarist still see benefits in dosing both?
After reading the whole study once I,m not sure it's very relevant to aquarium applications. Specialized industrial reactors involving nitrite injections and many other manipulations are used in waste water studies and aplications noted and measured for cost efficiency vs methanol reactors.
Naturally occuring amanonox activity happens predominantly at the edge of sulfur reducing anoxic regions which is where the ammonium for the process originates, as I read it. So would you need SO4 reduction and concomitant sulfide and hydrogen sulfide production for anamonox bacteria to play a significant role in a closed system with or without a deep sand bed or plenum? It was first discovered in the large anoxic basin known as the Black Sea and subsequently in some large upwelling systems in the ocean where it is hypothesized that ammonium wells up from the sulfur reducing regions where it is produced along the way to mineralization. The tank water would normally be low in ammonium in a healthy aquarium. since oxidation via the usual aerobic nitrifying process would likely occur first, so it seems a source of ammonium to very hypoxic areas would be needed to encourage anamonox activity . I don't think that is worth the effort or the risks at least at the stage of knowledge now available . The anamonox bacteria involved are very slow growers(11 to 12 days for division) as far as bacteria go so any efect on nitrogen reduction would likely be very slow.

It's difficult to cross over waste water applications to biotypes like aquariums but the studies can be thought provoking .Personally, I don't use them as baseline material for aquariums.


Now for winners and losers, vodka and vinegar vs vinegar only. IMO, it's a tie.



Ethanol oxidizes to acetic acid(vinegar without the dilution) ,so it sources acetate after an extra step or two as well . These steps don't result in discernible harm and may or may not be of benefit. I'd guess they cause no significant benefit or harm based on my 3 year experience.


I'll have to read the study again because I didn't find a clear indication that ethanol suppresses anamonox bacteria but even if it does I'm not sure it matters in an aquarium since there may not be any significant number of them to start with.

Ethanol is also a neutral ph additive as opposed to vinegar with a low ph and CO2 additions.

I prefer bolus dosing as opposed to incremental dosing via dosing pump and timers needed with larger amounts of vinegar to mitigate precipitous ph effects. Bouls dosing is the routine I've followed for 3 years with satisfying results, a complete change over would change bacterial populations .
While I'm always looking for opportunities for improvement as per the shift to 25% vinegar from all vodka several years ago, a total change of carbon source and resuting changes in the bacterial cultures in my aquarium are not a hand I want to play right now..
I also dose some vodka at night without worry about introducing more CO2.
Bolus dosing may encourage more anaerobic denitrifiction activity than incremental dosing according to one study.
I haven't seen anything that would ease my reluctance to switch to only vinegar in my system in the amnamonox writings but I think vinegar only makes good sense for some systems and would be inclined to try it if I was just starting if for no other reason than to skip the extra bacterial steps ethanol needs to go to acetate andto see how the animals in t4h tank reacted vs the curent vodka/ vinegar method.

Vodka does cost more but not much when you don't count the extra water.
The vodka I use is $14 for 1.75 liters(about 59 ounces) ,40% of that is ethanol roughly 23.6 ounces ,for a per ounce cost of 59 cents.
I use the store brand vinegar in the economy size,It;s the cheapest around here @ $4 for 154 ounces, 95% of which is water leaving 7.7 ounces of acetic acid for $4 or 52 cents per ounce.


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Old 12/04/2011, 11:32 AM   #25
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Great stuff Cliff. It sure seems like dosing acetate is the way to go. Using a dosing pump or like you said just adding it to your top off makes it every bit as convenient as bio-pellets.


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