Nitrate Removal by Bacteria In Aquatic Systems

Its more complicated than you think!

Nitrate, its one of the big two bad guys in aquatics systems that people don’t want, and seem to struggle with removing.

The terminology out there to describe both its removal, and the modes of action by which it is removed, is so vastly incorrectly used and misunderstood, that no wonder it causes so much confusion, and therefore difficulty for people to achieve effectively.

First of all, removal of nitrate does not only occurs in anoxic zones (oxygen free) in the aquarium, it also occurs as a process of the metabolic production of cell mass (by algae and bacteria) in Oxic (oxygen rich) environments.

It is important to realize the differences between the forms of biological nitrate removal and how they work, in this way you can then choose a nutrient reduction system. And determine what processes will work with one another.

The processes by which the BULK of nitrate is removed in aquatic systems are:

Anaerobic denitrification (in anoxic conditions, a dissimilatory nitrate reduction process)

Aerobic denitrification (in oxic conditions, an assimilatory nitrate reduction process, that will also remove, and requires phosphate for the process to work effectively)

Anammox – Anaerobic Ammonia Oxidation (in anoxic conditions, a dissimilatory ammonia and nitrite reduction process in which nitrate is not produced by way of removing the ammonia and nitrite before it can be converted to nitrate)

Descriptions before we go further:

Oxic – Oxygen rich environmental conditions

Anoxic – Oxygen poor (or absent) environmental conditions

Aerobic – the process in which organisms metabolize their food in the presence of oxygen

Anaerobic – the process in which organisms metabolize their food in the absence of oxygen

I.e.
Oxic and anoxic describes the environmental conditions

Aerobic and anaerobic describes the process that occurs in these conditions

Carbon Source/fuel – either organic carbon (used by heterotrophic Bacteria) or inorganic carbon (used by autotrophic bacteria), is the primary ingredient in bacterial metabolism and needs to be present for bacterial metabolism to occur

Electron Donor – the ion that receives the electrons and is broken down.
Electron Acceptor – The ion that gives up electrons (Oxygen O2, Nitrate NO3, Sulphur SO4, Carbon Dioxide CO2, etc) and is reduced, in turn oxidizing the electron donor

Anaerobic De-nitrification

In this process, predominantly heterotrophic bacteria, take Organic carbon, nitrate and its preferred waste product to produce an end process of denitrification resulting in di-nitrogen gas.

In this way:

Fuel Source = Organic Carbon (OR inorganic carbon depending on if they are heterotrophs or Autotrophs, but this is the realm of mostly heterotrophic bacteria)

Electron Acceptor = Nitrate NO3

Electron Donor = whatever waste product it is digesting, solid waste, fats, lipids, cellulose, it is widely varied.

More accurately, the organic carbon is liberated from the waste product being assimilated by the bacteria, which they use for the reduction. The breakdown of the waste is a positive by-product of the reaction, and the breakdown of these by products are then processed by other bacteria and so on.

It is adding organic carbon artificially that is ENHANCING this process, and helping to break down excess nitrate, if not enough organic carbon is present.

Here, bacteria take NO3, dissimilating the nitrate, through several steps such as nitric oxide/nitrous oxide for example, to dinitrogen gas, which is then gased off to the atmosphere.

The Nitrate, along with the organic carbon is what they use to process their food source (waste of any description in the anoxic zone they are residing in). The Nitrogen Gas, now liberated from the oxygen in the NO3 molecule, now gases off, eventually, into the atmosphere after it has been dissimilated.

Some of these bacteria can operate as facultative anaerobes, meaning they can function both aerobically and anaerobically. They can be either Heterotrophic bacteria (and therefore benefit from the addition of organic carbon) or Autotrophic bacteria (and use carbonate, bi-carbonates and CO2 as their carbon source), autotrophic denitrifying bacteria however, are not as prolific or as common as heterotrophic bacteria.

Dosing organic carbon into the water WILL help this process if the predominate species of denitrifying bacteria are heterotrophic (weather or not they are obligate aerobes, obligate anaerobes of facultative anaerobes), but generally, there is enough organic carbon released from the breakdown of solid waste (that is their food source) to fuel this process, at least in an aerobic environment.

If they are Autotrophic (which is less common and less prolific) they will use inorganic carbon sources present in the water column, or inorganic carbon sources that are taken from the surrounding environment, either as CO2 or carbonaceous material from calcium carbonate based substrate, as their carbon source.

Because there is such a vast surface area in Aquariums in substrates such as live rock and deep sand beds, or substrate beds of significant depth in freshwater tanks that are commonly applied to promote Biological activity, the amount of waste caught up in these areas, when broken down, liberates enough organic carbon to be able to fuel anaerobic denitrification without adding organic carbon sources, which is usually only added to fuel aerobic assimilatory denitrification.

However, to augment these systems, and promote additional dissimilatory denitrification, additional organic carbon may need to be added into Aquatic systems with limited organic carbon reserves.

Anaerobic denitrifying bacteria will also use suplhur SO3, for their electron acceptor, as is the case with Sulphur based denitrators.

As this process is most often than not reliant on natural organic carbon sinks being liberated through the breakdown of waste, it is obviously a system that needs to mature these reserves of waste in order to fuel nitrate removal long term, and also provide an oxygen free zone to allow fermentation and removal of nitrate.

Aerobic De-nitrification

Aerobic De-nitrification occurs in oxygen rich environments. The bacteria involved are heterotrophic as with anaerobic denitrification, however they do not take in nitrate as their electron acceptor.

In this process, the heterotrophs take Nitrate and phosphate to build cell structure (as part of their metabolism, just like ALL bacteria do), along with organic carbon as a fuel source, oxygen as their electron acceptor and Varied Waste products floating around in the system as their electron donor.

Fuel Source = Organic Carbon

Electron Acceptor = Oxygen

Electron Donor = Varied Waste Products

In this way they build cell mass using the nitrate and phosphate, which then increases the size of the microbial herd in the system. The assimilation of nitrate and phosphate is carried out by all bacteria, it is in this application however, that we take advantage of the extreme growth rate of heterotrophs to remove bulk amounts of N and P.

This is then removed, mostly by a protein skimmer/foam fractionator. Organisms such as sponges and corals and other filter feeding organisms also feed on this Bacterioplankton and it constitutes a large percentage of their food source, both in nature and in tanks running organic carbon driven systems.

In phytoplankton, the ratio of uptake is 106ppm of organic carbon : 16ppm nitrate : 1ppm phosphate. 106:16:1.

In bacteria, it is theorized and has been shown in preliminary studies to be a ratio of around 50ppm organic carbon : 10ppm Nitrate : 1ppm Phosphate

50:10:1

This means that unlike anaerobic denitrification, which uses the pre-exisiting nutrient sinks in the anoxic zones of the tank, Aerobic denitirifcation requires the constant dosing of Organic carbon, and sometimes, in nitrate limited environments, nitrate sources as well. You can read about nitrate limitation here (insert link to blog post here)

This process, is therefore termed an Assimilatory denitrification process, as it assimilates nitrate (and phosphate) into its cell structure as nutrition, rather than dissimilating nitrate to scavenge the oxygen in dissimilatory Anaerobic denitrification.

This process of assimilatory denitrification Is beneficial if you are trying to combat both nitrate and phosphate issues. Sometimes you can become nitrate and/or phosphate limited (such as in the link above) and this can effect the overall systems performance.  

The first reason for this system not working (assimilatory nitrate reduction) is because they are nitrate limited. Being nitrate limited to bacteria is like when we do not have enough iron in our blood to allow the hemoglobin to carry oxygen in our red blood cells.

It is a limiting factor.

The second reason, is that people run GFO, or some other form of nutrient export for phosphate, which effects the redfield ratio and limits the uptake of nitrate.

Trying to run an enhanced nitrate (and phosphate) removal system where you actively dose organic carbon for Assimilatory nitrate reduction, and then use a system that interferes with that system, is bound to fail.

Anammox – Anaerobic Ammonia Oxidiation      

This is a process that is not very much considered.

It is the process in which ammonia (NH4+) is combined with Nitrite (NO2-) by AUTOTROPHIC bacteria (that use inorganic carbon, either carbonates or CO2).

Although several chemical conversions and reduction processes occur within the bacterial cell, the overall conversion representing Anammox is as follows:

NH4+  +  NO2-  =  N2  +  2H2O

Fuel Source = Inorganic Carbon
Electron acceptor = Nitrite

Electron donor = Ammonium

Although this is, in the way of describing it, NOT in fact a denitrification process as we in the Aquatic industry term it (denitrification being the removal of NITRATE) because there is no nitrate actually being removed, it is important as the net result is the same and the process is similar to that of other forms of nitrogen removal.

In this process Nitrous and nitric oxide, and sometimes ammonia (through reduction processes) can be produced during the conversion of ammonia and nitrite to dinitrogen gas and H2O.

Although this usually occurs in the bacterial cell itself, it has been proposed that in systems experiencing high levels of Anammox, that ammonia can in fact spike.

In an aquatic system with livestock, this could potentially be an issue.

HOWEVER, as discussed, the process of anaerobic bacterial metabolism (fermentation) is in fact much slower than ammonia uptake, one reason why you need a large deep sand bed, to provide an oxygen free zone, and also to reach a critical mass to ensure you have enough bacteria to perform anaerobic denitrification.

Uptake of ammonia by aerobic heterotrophs and Autotrophs is much faster. By design we not only want, but also achieve, much larger volumes of oxygen rich surface area in our systems. Any ammonia produced from this process would logically, and in my opinion, be quickly chewed up before a spike actually occurred. And would most likely only occur in localized zones, and not to any noticeable or even detectable levels.

If you were to get a crash of this bacterial population however, through disturbing and introducing oxygen into the anoxic zone, killing any anaerobic bacteria, or by means of toxicity from, for example, supplements or changes in water conditions, the ammonia bound up in the cells could, and more than likely will be released, resulting in mass transfer of ammonia into the system.

The killing of any bacteria creates a Glut of biomass in the system which then proceeds to break down, weather it is annamox bacteria, denitrifying anaerobes or denitrifying aerobes.

This can also happen when an overdose of organic carbon in Aerobic organic carbon driven systems occurs, as there Is not enough oxygen to support the bacterial respiration and bacteria starts to die, releasing the protein into the water column, which then breaks down into ammonia.

My point here is, avoid bacteria die offs, crashes, and just generally, don’t screw up.

Each of these systems has an effect on the other, and every other biological process then has an effect on these systems as nitrate is the final end product of nitrification.

Educating yourself and being aware of ALL denitrifying systems, and their associated bacteria, will allow you to understand how your system works and how what you are adding is impacting your mass removal of nitrate.

Further to this, firstly understanding how your biological system works in general, and how waste moves within, and in and out of your system, will allow you to fundamentally understand just how important you job of farming bacteria actually is.

As always, any questions or comments leave them in the comments section below.

Suffice to say its bloody great to be back, after life got crazy stupid (really really frikken stupid) I've now settled into a new job, got my mojo back, feeling PUMPED, and this is the first of, I hope, many posts that are going to occur regularly.

So thank you for anyone that has taken the time to read this after my extended absence. 

On Another Note......

W.O.W.

I have finally found a way you can cook with carp, finally an answer to the scourge of Australian waterways.

Step 1. Catch carp, and dispatch humanly.

Step 2. Soak carp in large tub with water from the river you caught it in, 5% rock salt and a selection of your favorite herbs, for 3 days

Step 3. After day 3, drain water, wrap in canvas or muslin, and take a rock, roughly the size of a grapefruit (preferably also form the same river system, but as long as it is clean it is fine), and weigh down the carp in the pot.

Step 4. Add a can of buttermilk for every liter of water you add to the pot to cover the carp

Step 5. Bring to a slow simmer and boil for 2 and half hours.

Step 6. Remove the rock from the pot, so you can then remove the carp easily.

Step 7. Take the carp, throw it in the bin, and eat the rock.

You’re welcome.