Trying to understand and make a sulfur denitrator work

CamGG

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The full story is below. The short story is I’ve become obsessed with trying to get a sulfur denitrator to work. I’ve started injecting vinegar into the reactor. I’m posting this because I would like to hear from others what they think the chemistry/ biochemistry is occurring here and the things I could try.



The tank is a 90gallon with a 15-gallon sump. Three fish, two clowns, and a tang. A handful of softie corals. Nitrates are always around 20 or higher. My corals hate water changes even though I match the salinity, alkalinity, calcium, pH, and temperature perfectly. This is the reason I’m trying to use a denitrator.



The chemical reaction for thiobacillus can be found here

https://www.sciencedirect.com/topic...-molecular-biology/thiobacillus-denitrificans

Here's an article discussing how Thiobacillus Denitrificans uses CO2 as its carbon source.
https://pubmed.ncbi.nlm.nih.gov/161...cies of,carbon source under anoxic conditions.


There are hundreds of articles on this. These bacteria can get carbon in different pathways. They can oxidize many different sulfur compounds as well as various metals including iron. I’ve found articles claiming that thiobacillus is dependent on another bacterium. I don’t think anyone fully understands how these denitrators work.

The Fully Story


I converted a Korallin C1502 calcium reactor into a sulfur denitrator a little over a year ago. I filled the reactor with korallin sulfur and limestone. I started off with a drop every two seconds and had no success after months. I went down to a drop every 4 seconds and began to see nitrites in the effluent.

This never changed though. This went on for months. I eventually took out some limestone and added some Fluval bio-media to try to make more surface area for bacteria. This had no effect on the reactor.

The only way 0 nitrate/nitrite water would come out of the reactor is if it were as slow as a drop every 10-20 seconds. The next thing I attempted was to peal the korallin sticker and glue off and submerge the reactor. The water level in the sump covered about ¾ of the reactor. I don’t believe these are meant to be submerged but the bolts were not in the water so it did not concern me. I submerged it to try to bring the temperature up for the biological processes. This had no effect.

In the course of this whole time, I looked at dozens of scientific papers regarding thiobacillus denitirificans as there has been extensive research for water treatment plants. I also looked for other people who could never get their reactors working. There must be some reason why they work for some and not at all for others. In my case, it was obvious that the bacteria existed in the reactor but were either not growing to a large enough population to treat the water. Something was limiting their growth.

I eventually stumbled on to a scientific paper discussing the carbon sources used by Thiobacillus and their primary is through the reduction of carbon dioxide. Maybe it’s possible that some tanks do not have a high enough CO2 concentration to support these reactors. I have a protein skimmer rated for 210g on a 90-gallon tank. I then attempted injecting 0.8ml of vodka into the feed pump. A few hours later the effluent was nitrate/nitrite-free.

Going this route, I changed out some plumbing parts to be able to inject straight into the reactor. Unfortunately, air entered the reactor and I had to start over again. After a few days of dosing vodka I got no results this time.

With the drip rate at one per 5 seconds, the effluent was saturated with nitrites. I read another report claiming that acetate can un-suppress the gene required for sulfur oxidation. I then attempted 5% vinegar always diluted with 10ml of clean saltwater. Five hours after the injection I got nitrate/nitrite-free water. This roughly coincides with the 4.4 hours replication rate I found for thiobacillus denitrificans(if under optimal conditions which no reef tank is). At this point, I put the drip rate to one drop per 4 seconds and waited 24 hours. The nitrites were present again so I repeated the injection of 0.5ml of vinegar diluted in saltwater. Five to six hours later the effluent was clean so I went down to two seconds. I repeated the injection but went to 0.6ml of vinegar.

The effluent became clear but a few hours later it was full of nitrites. I injected a 1-10ml vinegar-saltwater ratio into the reactor. An hour later the effluent was clear. Full of nitrites again the next morning. I think there could be a few things occurring here. Either the vinegar is going towards the production of new bacteria, to the production of thiobacillus, or this carbon source is entirely necessary for denitrification. If it were solely the replication of thiobacillus then when the vinegar dosing is stopped, the reactor should continue to work. While doing this, dozens of bubbles popped up in the reactor which I assume are nitrogen gas. To this point, the Fluval, sulfur, and limestone media look brand new still. The entire internals of the reactor looks spotless.

There is also no way for me to know what bacteria is actually in the reactor. There are dozens of denitrifying bacteria that exist.
 

Biglew11

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I was never able to get mine to produce 0 nitrates. In hind site i think my phosphates were bottomed out at this time. All organisms require some amount of nitrates phosphates and an organic carbon source. How much I don't know. It was my understanding at the time that the sulfer became the organic source of carbon. And the lack of o2 forced the bacteria to strip the oxygen molecules off the nitrate. This requires a negative orp to accomplish.

Organic carbon and co2 are 2 different compounds. Vodka, vinegar are organic carbon. co2 is the gas that we expel when we breath.

I'm going by memory from when I was trying mine out so I could be wrong on this next bit.
I thought in general the bacteria that fed on sulfur was different than the bacteria that fed on organic carbon. If this is the case then feeding the reactor vinegar was just plain carbon dosing. The reason they said you shouldn't carbon does and use sulfur or biopellets at the same time.
 
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CamGG

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I was never able to get mine to produce 0 nitrates. In hind site i think my phosphates were bottomed out at this time. All organisms require some amount of nitrates phosphates and an organic carbon source. How much I don't know. It was my understanding at the time that the sulfer became the organic source of carbon. And the lack of o2 forced the bacteria to strip the oxygen molecules off the nitrate. This requires a negative orp to accomplish.

Organic carbon and co2 are 2 different compounds. Vodka, vinegar are organic carbon. co2 is the gas that we expel when we breath.

I'm going by memory from when I was trying mine out so I could be wrong on this next bit.
I thought in general the bacteria that fed on sulfur was different than the bacteria that fed on organic carbon. If this is the case then feeding the reactor vinegar was just plain carbon dosing. The reason they said you shouldn't carbon does and use sulfur or biopellets at the same time.
Here is a quote from the second link "Thiobacillus denitrificans, a species of autotrophic facultative anaerobic bacterium, was found to be capable of oxidizing sulfide into elemental sulfur when nitrate was adopted as its electron acceptor and carbon dioxide as its sole carbon source under anoxic conditions"

this is only from the abstract so I'll have to give the whole article a read later but these researchers' goals were to convert sulfide back to elemental sulfur S 0. However, from reading studies of wastewater treatment plants comparing sulfur denitrification to heterotrophic denitrification, which requires carbon dosing, I found no experiments that provided any carbon to sulfur denitrification. I also stumbled upon this article which says anoxic (carbon dosed) heterotrophic denitrification produces nitrous oxide so I'm definitely done carbon dosing the reactor at this point.

here is this article if anyone is interested
 

Randy Holmes-Farley

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Here is a quote from the second link "Thiobacillus denitrificans, a species of autotrophic facultative anaerobic bacterium, was found to be capable of oxidizing sulfide into elemental sulfur when nitrate was adopted as its electron acceptor and carbon dioxide as its sole carbon source under anoxic conditions"

this is only from the abstract so I'll have to give the whole article a read later but these researchers' goals were to convert sulfide back to elemental sulfur S 0. However, from reading studies of wastewater treatment plants comparing sulfur denitrification to heterotrophic denitrification, which requires carbon dosing, I found no experiments that provided any carbon to sulfur denitrification. I also stumbled upon this article which says anoxic (carbon dosed) heterotrophic denitrification produces nitrous oxide so I'm definitely done carbon dosing the reactor at this point.

here is this article if anyone is interested

Not sure what you are getting at.

There should be almost no sulfide present in a sulfur denitrator or reef tank water as it is very toxic to many marine organisms. That is not a process you need to drive.

There's no need to reinvent the wheel. The sulfur denitrator process is well studied. This is the sulfur denitrator process:


8. Sulfur Denitrators

In these systems, bacteria use elemental sulfur and produce N2 from the sulfur and nitrate according the following equation (or something similar):

2 H2O + 5 S + 6 NO3– → 3 N2 + 5 SO42- + 4 H+

The production of acid (H+) in this reactor can tend to reduce the aquarium alkalinity. It has also been suggested to pass the effluent of such a reactor through a bed of aragonite to use the acid (H+) produced to dissolve the calcium carbonate, and thereby provide calcium and alkalinity to the aquarium. While that is a fine idea, it doesn’t add much calcium and alkalinity to most aquaria.

To estimate the magnitude of the effect, we start with a liberal estimate of how much nitrate might be removed. Say 10 ppm of nitrate per week.

10 ppm nitrate = 0.16 mmole/L of nitrate

Since 4 moles of H+ are produced for every 6 moles of nitrate consumed, this will produce

0.107 mmoles/L of H+ per week

How much calcium this could produce?

Assume that it takes one proton to dissolve one calcium carbonate:

CaCO3 + H+ → Ca2+ + HCO3–

Clearly, this is a substantial overestimate because much of the acid will be used up driving the pH down to the point where CaCO3 can even begin to dissolve. Consequently, we have an upside limit of 0.107 mmoles of Ca2+ per week. Since calcium weighs 40 mg/mmol, that’s 4.3 ppm Ca2+ per week.

For comparison, an aquarist adding 2% of the tank volume in saturated limewater daily is adding on the order of 16 ppm of calcium per day. Consequently, this method may not be especially useful for maintaining calcium. Additionally, the acid produced will have a long term lowering effect on the alkalinity. In fact, it is double dipping on the alkalinity depletion since alkalinity is consumed when the nitrate is produced, and again when it is removed in the denitrator. So if you use a sulfur denitrator, be sure to monitor the alkalinity in the aquarium.
 

Randy Holmes-Farley

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The above article also discusses the process of using a carbon denitrator, and organic carbon dosing (which are different processes):

6. Carbon Denitrators

There are a variety of different commercial systems available, none of which are especially popular in the United States at this time. However, they can do a good job of removing nitrate and some aquarists like them.

In one of these types of systems, a carbon source is added to a portion of tank water in a low oxygen environment. In many cases, the carbon source is methanol. The methanol is mixed with aquarium water in a controlled situation (such as fluid pumped through a coil) and the methanol is consumed by bacteria that use nitrate as an electron acceptor instead of oxygen:

12 NO3– + 10 CH3OH + 12 H+ → 10 CO2 + 6 N2 + 26 H2O

The end result is that nitrate is removed from the aquarium. The typical drawback to such a system is the need for careful control over the conditions, and the consequent complexity that often accompanies such a reactor. Note again that this process returns the alkalinity (by consuming H+) that was lost in the production of the nitrate originally.

This method is similar to organic carbon dosing, but is localized inside of a reactor. Such localization can have advantages (less chance for cyanobacteria to be driven to grow more by consuming the organic), but it is a bit harder to accomplish technically than simple dosing of organics to the aquarium (described below).
 
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CamGG

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Not sure what you are getting at.

There should be almost no sulfide present in a sulfur denitrator or reef tank water as it is very toxic to many marine organisms. That is not a process you need to drive.

There's no need to reinvent the wheel. The sulfur denitrator process is well studied. This is the sulfur denitrator process:


8. Sulfur Denitrators

In these systems, bacteria use elemental sulfur and produce N2 from the sulfur and nitrate according the following equation (or something similar):

2 H2O + 5 S + 6 NO3– → 3 N2 + 5 SO42- + 4 H+

The production of acid (H+) in this reactor can tend to reduce the aquarium alkalinity. It has also been suggested to pass the effluent of such a reactor through a bed of aragonite to use the acid (H+) produced to dissolve the calcium carbonate, and thereby provide calcium and alkalinity to the aquarium. While that is a fine idea, it doesn’t add much calcium and alkalinity to most aquaria.

To estimate the magnitude of the effect, we start with a liberal estimate of how much nitrate might be removed. Say 10 ppm of nitrate per week.

10 ppm nitrate = 0.16 mmole/L of nitrate

Since 4 moles of H+ are produced for every 6 moles of nitrate consumed, this will produce

0.107 mmoles/L of H+ per week

How much calcium this could produce?

Assume that it takes one proton to dissolve one calcium carbonate:

CaCO3 + H+ → Ca2+ + HCO3–

Clearly, this is a substantial overestimate because much of the acid will be used up driving the pH down to the point where CaCO3 can even begin to dissolve. Consequently, we have an upside limit of 0.107 mmoles of Ca2+ per week. Since calcium weighs 40 mg/mmol, that’s 4.3 ppm Ca2+ per week.

For comparison, an aquarist adding 2% of the tank volume in saturated limewater daily is adding on the order of 16 ppm of calcium per day. Consequently, this method may not be especially useful for maintaining calcium. Additionally, the acid produced will have a long term lowering effect on the alkalinity. In fact, it is double dipping on the alkalinity depletion since alkalinity is consumed when the nitrate is produced, and again when it is removed in the denitrator. So if you use a sulfur denitrator, be sure to monitor the alkalinity in the aquarium.
Apologies for the confusion. I wasn't trying to claim that in a reefs sulfur reactor they used sulfide. That research's goal was to convert sulfide back to elementaI sulfur with the same bacteria used in a sulfur denitrator. I posted that because those researcher's claimed that these bacteria do need a source of carbon,though a inorganic one. I've stopped carbon dosing the reactor for a day and the effluent is clear of nitrates/nitrites at a drop every 3 seconds. I know I should be doing an actual ml/min count but the flow changes anytime I move the tube. Prior to carbon dosing the reactor would only work at a drop every 12+ seconds. I'll be giving it a week to see if it can maintain this nitrate/nitrite free output. If it does, is that not indictive that the sulfur oxidizing bacteria in the reactor used some of the carbon source?
 

Randy Holmes-Farley

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Certainly, every organism needs a source of carbon to make organic molecules.

For many, including most photoautotrophs such as algae and chemoautotrophs such as those that oxidize elemental sulfur in a sulfur denitrator, that source is CO2, bicarbonate, or carbonate, as opposed to organic matter.

I do not think there is concern in a sulfur denitrator that carbon is in short supply, but I’ve never seen a study they looked at that question.

The measurement of the output from reactors is fraught with difficulty. Most notably, a tiny amount of nitrite will typically lead to a lot of falsely high nitrate.

I don’t know what happened in your reactor, and haven’t seen actual numbers, but it is certainly possible that organic carbon dosing caused the sulfur to be coated with bacteria that are unable to metabolize sulfur and thus the process was inhibited.
 
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