Denitrification, carbon, oxygen etc..

Well.. first, I relaxed skimmer air intake by fully open cabinet doors for 24 hrs. No noticable difference..

I am further experimenting with siporax, and want to share some results.

While siporaxes were sitting in a small container, I decided to follow ph and orp status in it.

After flooding the container, ph & orp were equalized with tank, then, ph decreased first for a couple of hours and then started to increase, steadily (From around 7.8).
I let it climb up to 8.2 while tank water were 7.9 max.
I flooded the container and followed ph again. Same repeated in 12 hours. Then I remember what I read in articles, denitrification results with increase of alkalinity, so ph. This time I measured Alk. of tank and container, tank was 8.3, siporax was 10.2 (siporax ph was slightly over 8)
This repeated few times and finally I connected ph controller and a small pump to flush the container when it reaches to 8.0 ph :)
This was 5 days ago. Since then, pump works twice about every 24 hours and supplies the tank with alkalinity. Dosage pump is also working and its providing Ca and Alk. also (placed after siporaxes).

Any idea how this happens? Will it continue to provide alkalinity forever? :)

If it is from denitrification, yes it will continue as long as the tank has nitrate.

But it is adding back only what the nitrifiers took away when they converted ammonia into nitrate.

So it cannot be used as a supplement system. :D
 
Thats correct it not adding extra Alk., but Cant we see it this way; its adding the stolen alkalinity by nitrifiers, back in to service of corals.
But, (there is, as always) a problem.:)
While I am adding 8 ph water into tank this way, tank pH seems not Improving over time. It even seems very slightly lowering:) If co2 from denitrification is the cause(how can it be, if the container ph is over 8), I guess it may be the right time to start using kalkwasser, in rodi reservoir, which is a part of the sump. Currently 4-6 liters of water evaporating daily and I allowed DIY top off to energize every 6 hours.
 
Thats correct it not adding extra Alk., but Cant we see it this way; its adding the stolen alkalinity by nitrifiers, back in to service of corals.

Yes, certainly. But other nitrate reduction methods also do that, including organic carbon dosing and even macroalgae growth.

I think in terms of pH, you are just not adding enough to notice much effect.

Limewater is a fine way to go. It is all I use for calcium and alkalinity.
 
Yes, certainly. But other nitrate reduction methods also do that, including organic carbon dosing and even macroalgae growth.

I think in terms of pH, you are just not adding enough to notice much effect.

Limewater is a fine way to go. It is all I use for calcium and alkalinity.

Thanks Randy, mind clarified. I will follow limewater route.
There are different suggestions for limewater usage, like, dissolving it in vinegar first, etc. What makes me worry on use of it in ro/di topoff container is, I will initially add some percent vinegar of topoff water, and then calcium hyroxide, again recommended percent of total volume. Then, rodi water will be consumed and filled back via pipeline, which will ruin all the rates, percentages etc since I dont normally measure how much ro/di added, to topoff the ro/di container.
Is this means I should everytime measure amount of water added to the container and add relevant amount of vinegar and Ca_hydroxide? Or is there any easy,bulletproof and secret method for all this? :) (my rodi container is actually a section of my sump, at back, approx 40 liters).
 
I wouldn't add vinegar to it in most cases.

The easy method, if your tank can handle full strength limewater, is to mix in enough to saturate it (2 teaspoons per gallon or more, although I do it by conductivity, not measurement of the solids) stir for a while (5 min to overnight) while closed, let it settle, and then use the clear liquid.
 
I wouldn't add vinegar to it in most cases.

The easy method, if your tank can handle full strength limewater, is to mix in enough to saturate it (2 teaspoons per gallon or more, although I do it by conductivity, not measurement of the solids) stir for a while (5 min to overnight) while closed, let it settle, and then use the clear liquid.

Very well then, so you are not using it by adding to topoff, dosing pump?

I have access to lab grade conductivity meter, what numbers should I aim for?
 
I have used limewater for 20+ years. I've only dosed vinegar for the past few years, and I did that by manual dosing (once or twice a day) and now by dosing pump. :)

Saturated limewater is about 10.3 mS/cm. There's no magic number, since it depends on what your tank actually needs, but I aim for at least 8 mS/cm before I stop stirring and let it settle before using it. :)
 
An advantage of a batch denitrator versus adding a carbon source directly to the aquarium is that the carbon is consumed in a controlled environment before it makes its way to the main tank. I have seen many tanks develop STN and RTN in a couple of days after adding a carbon source, no matter which one, bio pellets, ethanol, methanol, acetic acid and no matter how many ml are dosed. One thing that people does not pay attention to is that we introduce in our systems a myriad of bacteria strains from every corner of the world every time we buy a coral or live rock. Carbon sources fuel "bad"and "good" bacteria, it doesn't discriminate. Large counts of opportunistic bacteria "fueled" by a carbon sourced disturbed the coral holobiont causing RTN. If the denitrification process is done in a separate chamber the likelihood of developing RTN is largely reduced.
 
The current way I am following for safe denitrification is using a siporax container isolated from aquarium water. I am keeping the glass container within sump but tank water not mixing to it, until ph controller says ph increased to a certain level, as a result of rise in kH level, which is an indication of denitrification. Then a small pump fills the siporax container to overflow into sump, until ph drops under setpoint.

A bit speculation then. For some reason, this process may also contributing corals' health and happiness, too. Experienced a good Ca and kH drop lately (340 & ~5.3) for a short period, and observed no complaints from corals.
 
I will leave this here for all of you to consider.
The Coral Holobiont
get_file.php
Corals are host to a wide diversity of organisms, including endosymbiotic algae, protists, fungi, Bacteria, Archaea, and viruses. Together, these organisms make up the coral holobiont. In our lab, we are interested in understanding the physiological roles of these players in their interaction with the coral animal, and how this relates to coral reef health. Incidences of coral death and disease are highly correlated with human impact, and we propose that anthropogenic stresses induce microbes normally associated with the coral to become opportunistic pathogens. Alternatively, opportunistic or specific pathogens from the water column might attack the weakened coral. To differentiate between these possibilities, my lab has had to determine if healthy corals have characteristic microbiotas. To do this, we have employed a variety of techniques ranging from electron microscopy (e.g., Johnston and Rohwer 2007) to metagenomics (e.g., Wegley et al. 2007).

In order to look at the diversity and specificity of coral microbes, our lab used high-throughput sequencing of bacterial 16S rDNAs associated with three coral species. This culture-independent study of coral-associated Bacteria found 430 (mostly novel) bacterial species in 14 samples from 3 coral species. The coral-associated microbial communities were ecologically structured: different coral species had different bacterial communities, even when physically adjacent, while bacterial communities from the same coral species separated by time (~1 year) or space (3000 km) were similar. We also found that some bacterial species were present only in a subset of spatial niches within individual coral colonies (Rohwer, et al., 2002).

get_file.php
In order to look at the function of microbes on corals, we use metagenomic sequencing (454 Life Sciences) to identify the microbes and their functional genes. Our work found that bacteria associated with corals are primarily heterotrophic. Our metagenomic data showed an abundance of sugar and protein utilization and uptake pathways in the microbial community. These microbes are likely utilizing the complex polysaccharides and peptides from the coral mucus. Several types of cyanobacteria were also found associated with the coral, and may be providing fixed carbon and nitrogen to the coral. In addition, an abundance of fungi were associated with corals, including those involved in nitrogen cycling, indicating that fungi may be fixing nitrogen and making it available to members of the coral holobiont (Wegley et al. 2007).

We have also looked at the viruses associated with healthy and bleaching corals, and find viruses with a wide variety of hosts including many of the various members of the coral holobiont. These viruses include plant and algal viruses, herpes-like viruses, and cyano- and vibriophage, to name a few (Wegley et al. 2007, Marhaver et al. 2008). Due to the abundance of viruses and the wide variety of host ranges they possess, we expect that they play an important role in coral health and structuring of the coral holobiont.

In summary, the associations of the coral animal, prokaryotes, zooxanthellae, viruses, fungi, and other undefined components will define the niche that any coral colony occupies on a reef. This system is almost certainly exemplary of many other interactions between microbes and their higher eukaryotic hosts, and our studies will make predictions that can/will be tested in other complex host-microbial flora systems.

Stressors Alter Microbial Dynamics on Corals
get_file.php
An important implication of the coral holobiont model is that disrupting any one of these components may cause the whole community to collapse and lead to coral death. In order to test this hypothesis, we have performed several experiments exposing corals to different stressors and then looked at the changes in microbial dynamics and diversity, as well as coral pathology. In collaboration with Dr. Nancy Knowlton and Davey Kline at the Scripps Institution of Oceanography, we applied stresses to different coral species in the presence and absence of antibiotics. Our data showed that of the many commonly cited stressors of corals, organic carbon (OC) loading is the most problematic. Coral death induced by OC can be delayed with antibiotics. Additionally, OC loading causes the coral-associated microbial communities to grow much faster then normal. This strongly suggests that changes in the bacterial community, and not the stresses themselves, are responsible for coral mortality. (Kline et al. 2006, Kuntz et al. 2005). Additionally, when corals are placed next to algae with a filter impervious to viruses and bacteria, corals mortality is high. This mortality is also inhibited by antibiotics (Smith et al. 2006).

In a separate experiment, corals were exposed to one of four types of stressors currently threatening coral reefs: elevated nutrients, temperature, and organic carbon, and lowered pH. We then isolated the microbial and viral communities and performed whole-genome sequencing (pyrosequencing, 454 Life Sciences) to look at how the diversity and function of these organisms changed following stress. Our data showed that stress led to a shift towards a more pathogenic microbial community in all cases, with pathogen-associated genes also increasing in abundance (e.g. motility, virulence, and secondary metabolite genes) (Vega Thurber et al. Env Micro 2009). The viral assemblages also changed on the coral, with viruses related to the Herpesviridae family greatly increasing in abundance (Vega Thurber et al. PNAS 2008). We found that one herpes-like virus was undetectable by quantitative PCR (qPCR) prior to stress, but then increased dramatically within 1 hr of stress exposure, indicating an increase in production of the virus under stress. "Forest Rohwer, PhD Linda Wegley Kelly, PhD http://coralandphage.org/research_coral.php"
 
I will leave this here for all of you to consider.
The Coral Holobiont
get_file.php
Corals are host to a wide diversity of organisms, including endosymbiotic algae, protists, fungi, Bacteria, Archaea, and viruses. Together, these organisms make up the coral holobiont. In our lab, we are interested in understanding the physiological roles of these players in their interaction with the coral animal, and how this relates to coral reef health. Incidences of coral death and disease are highly correlated with human impact, and we propose that anthropogenic stresses induce microbes normally associated with the coral to become opportunistic pathogens. Alternatively, opportunistic or specific pathogens from the water column might attack the weakened coral. To differentiate between these possibilities, my lab has had to determine if healthy corals have characteristic microbiotas. To do this, we have employed a variety of techniques ranging from electron microscopy (e.g., Johnston and Rohwer 2007) to metagenomics (e.g., Wegley et al. 2007).

In order to look at the diversity and specificity of coral microbes, our lab used high-throughput sequencing of bacterial 16S rDNAs associated with three coral species. This culture-independent study of coral-associated Bacteria found 430 (mostly novel) bacterial species in 14 samples from 3 coral species. The coral-associated microbial communities were ecologically structured: different coral species had different bacterial communities, even when physically adjacent, while bacterial communities from the same coral species separated by time (~1 year) or space (3000 km) were similar. We also found that some bacterial species were present only in a subset of spatial niches within individual coral colonies (Rohwer, et al., 2002).

get_file.php
In order to look at the function of microbes on corals, we use metagenomic sequencing (454 Life Sciences) to identify the microbes and their functional genes. Our work found that bacteria associated with corals are primarily heterotrophic. Our metagenomic data showed an abundance of sugar and protein utilization and uptake pathways in the microbial community. These microbes are likely utilizing the complex polysaccharides and peptides from the coral mucus. Several types of cyanobacteria were also found associated with the coral, and may be providing fixed carbon and nitrogen to the coral. In addition, an abundance of fungi were associated with corals, including those involved in nitrogen cycling, indicating that fungi may be fixing nitrogen and making it available to members of the coral holobiont (Wegley et al. 2007).

We have also looked at the viruses associated with healthy and bleaching corals, and find viruses with a wide variety of hosts including many of the various members of the coral holobiont. These viruses include plant and algal viruses, herpes-like viruses, and cyano- and vibriophage, to name a few (Wegley et al. 2007, Marhaver et al. 2008). Due to the abundance of viruses and the wide variety of host ranges they possess, we expect that they play an important role in coral health and structuring of the coral holobiont.

In summary, the associations of the coral animal, prokaryotes, zooxanthellae, viruses, fungi, and other undefined components will define the niche that any coral colony occupies on a reef. This system is almost certainly exemplary of many other interactions between microbes and their higher eukaryotic hosts, and our studies will make predictions that can/will be tested in other complex host-microbial flora systems.

Stressors Alter Microbial Dynamics on Corals
get_file.php
An important implication of the coral holobiont model is that disrupting any one of these components may cause the whole community to collapse and lead to coral death. In order to test this hypothesis, we have performed several experiments exposing corals to different stressors and then looked at the changes in microbial dynamics and diversity, as well as coral pathology. In collaboration with Dr. Nancy Knowlton and Davey Kline at the Scripps Institution of Oceanography, we applied stresses to different coral species in the presence and absence of antibiotics. Our data showed that of the many commonly cited stressors of corals, organic carbon (OC) loading is the most problematic. Coral death induced by OC can be delayed with antibiotics. Additionally, OC loading causes the coral-associated microbial communities to grow much faster then normal. This strongly suggests that changes in the bacterial community, and not the stresses themselves, are responsible for coral mortality. (Kline et al. 2006, Kuntz et al. 2005). Additionally, when corals are placed next to algae with a filter impervious to viruses and bacteria, corals mortality is high. This mortality is also inhibited by antibiotics (Smith et al. 2006).

In a separate experiment, corals were exposed to one of four types of stressors currently threatening coral reefs: elevated nutrients, temperature, and organic carbon, and lowered pH. We then isolated the microbial and viral communities and performed whole-genome sequencing (pyrosequencing, 454 Life Sciences) to look at how the diversity and function of these organisms changed following stress. Our data showed that stress led to a shift towards a more pathogenic microbial community in all cases, with pathogen-associated genes also increasing in abundance (e.g. motility, virulence, and secondary metabolite genes) (Vega Thurber et al. Env Micro 2009). The viral assemblages also changed on the coral, with viruses related to the Herpesviridae family greatly increasing in abundance (Vega Thurber et al. PNAS 2008). We found that one herpes-like virus was undetectable by quantitative PCR (qPCR) prior to stress, but then increased dramatically within 1 hr of stress exposure, indicating an increase in production of the virus under stress. "Forest Rohwer, PhD Linda Wegley Kelly, PhD http://coralandphage.org/research_coral.php"

Organic carbon dosing does carry a theoretical risk of such issues, but fortunately, it seems rather rare for actual bad effects to be noted by reefers (but there are some). FWIW, the organic carbon sources used in some of those studies ((D-glucose, lactose, galactose, starch, arabinose; 5 to 25 mg l–1)) are not the types or doses that many reefers use, but some of the effects may be similar. :)
 
Interesting setup! Keep us updated! :)

This setup is primarily planned to benefit from the support siporaxes provide to denitrificating bacteria, in the hope of lowering high nitrates. I can say it is a great success, and the best, works in a controlled way.
I don't know if its valid to link another forum here, but dont want to repeat everything, you can take a look to my detailed results here, see post 696.
 
The link is fine. :)

How is it doing at controlling the tank nitrate?

Hi Randy, well, same feeding regime for last two years, no changes in food types (dry + frozen cubes), It took about three months to break in, now nitrates are nearly undetectable (salifert test barely pink-ish from side), I am quite happy with the result, since it fall here from 15-20 ppm range.

Ph controller triggers two or three times in 24 hrs. Flushes about 5-6 liters of water out of 10, everytime. I am measuring ORP also, ORP readings were initially (right after three month period) low, like 260-300, now (2 more months) around 330 plus minus 15. Alkalinity is always increasing with the rising pH within the siporax box, then flushes to aquarium. There is no light source around sump, so siporax hoops sit in darkness.

Basically, my system's major difference is the waiting period to allow denitrification occur. And its upper range is limited by increased alkalinity (pH).
 
This setup is primarily planned to benefit from the support siporaxes provide to denitrificating bacteria, in the hope of lowering high nitrates. I can say it is a great success, and the best, works in a controlled way.
I don't know if its valid to link another forum here, but dont want to repeat everything, you can take a look to my detailed results here, see post 696.
Thank you! Out of curiosity, have you been following phosphate levels as well?
Thanks again!
 
How much does the pH change in the container during a cycle? Is it being aerated?
Ah, I think this comes to excess CO2 issue :). The container is not mixed in any manner to prevent aerobic team win over anaerobs. For the pH swing, my current set point is like 8.10 and it falls somewhere around 7.96 after flush, then rises again.

Since I dont have a pH logger, I am using another way to log whats going on, a sample 24 hour below, upper is pH and lower num. is ORP. Enjoy:)

 
Thank you! Out of curiosity, have you been following phosphate levels as well?
Thanks again!

As expected, there is not much help from denitrification for phosphates, rowaphos recently added since levels were around 20 (Hanna ultra low phosphorus checker (equals to 0.06 I guess)).
 
I will leave this here for all of you to consider.
The Coral Holobiont
....

Very nice documentation, showing us the level of complexity of natural equilibrium, and also indicating the elasticity of organisms and whole ecosystem in response to disturbances.
 

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