Recently, I ran a simulation study to evaluate increase aeration for a treatment system with high ammonia waste. In this case, ammonia was the criteria pollutant for surcharges at a downstream POTW. The existing system had very low DO even with all blowers functioning. Back-of-envelope calculations revealed that there should be more than enough D.O. for the ammonia loading to the system. What was wrong?

This is a case where TKN is evolving through chemical and biological processes into ammonia. In many wastewater systems proteins, amines and other nitrogenous compounds will eventually evolve into ammonia nitrogen - either as free ammonia (NH3-N) or ammonium (NH4-N). This process accelerates with warm water temperatures and with good microbial activity. So the oxygen depletion and lack of ammonia removal was actually caused by the TKN being higher than the influent ammonia. The biological processes were working, but ammonia formed from TKN conversion was outpacing the ability to oxidize ammonia into nitrite/nitrate.

​So, when presented with ammonia removal questions in wastewater, it is necessary to also evaluate TKN (total nitrogen), Total Organic Nitrogen, and Ammonia when evaluating the system.

After dissolved oxygen, farmers most closely monitor ammonia when raising either fin fish or shrimp when growing in intensive, highly stocked ponds. Ammonia in unionized forms (NH3-N) is the most toxic common pollutant in aquaculture. Even in its less toxic ammonium form (NH4-N), it increases stress on the animals. With increased stress, the shrimp or fish are much more susceptible to disease and have lower feed conversion rates.

When I interact with aquaculture researchers and farmers, I often hear that they need to add ammonia oxidizing bacteria (AOB) to combat high ammonia in the ponds. Multiple companies appear to offer blends of microbes containing AOB – usually NItrosomonas and Nitrobacter sp. For farmers the questions are:

• Are the currently marketed products effective?
• And, if not – what should I do to control ammonia & nitrite in the ponds?
  
  

For a quick review, let’s look at the pond nitrogen cycle. Ammonia is the byproduct of animal metabolism – it also comes from excess high protein feed decomposition but most is from animal waste. Ammonia converts into the more toxic unionized form (NH3-N) at higher pH. At this point, the ammonia can be detoxified in three ways:

1. Uptake by algae – preferably by eukaryotic (green algae). The algae uses nitrogen to build cells during photosynthesis.
2. Use by heterotrophic (carbon waste degrading microbes). Bacteria digest waste materials (even sludge) and use nitrogen (preferably ammonia) in building new cells. This can remove a substantial amount of ammonia.
3. Finally, we have ammonia oxidizing bacteria – they require sufficient D.O., pH, and are relatively slow growing. However, they are very efficient at converting ammonia into first nitrite and then nitrate in a two-step process.

Now to answer the question of existing AOB product effectiveness. AOB organisms are very delicate and in concentrated form MUST be refrigerated to get a 3 – 6 month self-life. Any exposure to high temperatures or freezing will kill a majority of the AOB in the product. Therefore, any blend with AOB stored at room temperature will likely have only a trace of the AOB.  Additionally, AOB in a healthy pond are only part of the ammonia removal process. Unlike a wastewater plant with 30+ mg/L ammonia, the pond ammonia does not support extensive AOB growth.

So what alternatives do farmers have for ammonia issues? In my experience ammonia can be combatted best through monitoring and good operations. With low water exchange and high stocking densities, I have found that using a heterotrophic microbial addition can help lower ammonia within  2 – 4 days. In addition to improving waste removal and restoring natural balances, the “cleaner” water now supports better eukaryotic algae growth that efficiently remove nitrogen from the water. Unlike cyanobacteria, the eukaryotic algae cause fewer problems with pH swings and off-flavors.
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So unless there are special factors and critical ammonia problems, the addition of true AOB microbes is rarely need in aquaculture. I prefer to see farmers add a balanced blend of probiotic and waste degrading microbes that along with beneficial algae form the majority of waste removal biomass in the pond. For best impact of adding heterotrophic microbes, they should be added early on before the pond becomes unbalanced. With small regular doses throughout the season (with higher does as feeding and animal weights increase), the pond will remain in better ecological balance and animals will experience less stress.

When early wastewater systems were built, the engineers believed that Zooglea were the primary organism and formed the best floc. As microbiologists started examining the microbial mix that we call MLSS - they determined that the MLSS consisted of many different organisms functioning in a unique ecosystem. Differences in influent makeup, temperature, pH, dissolved oxygen, and cell residence time all had a major impact on the organisms found in the MLSS. 

Today, all mentions of Zooglea tend to be in a negative light. Under the microscope mature Zoogleal type colonies appear as fingers. The key visual clue is the gelatinous matrix that contains the cells. The extracellular materials move from promoting floc formation to entrapping water and creating viscous bulking.

Now what conditions favor Zooglea or other microbes responsible for viscous bulking:

• High levels of soluble organics - (you can address this through step-feed of influent)
• Macronutrient (C:N:P) imbalance that favors organisms that an uptake scarce nutrients (often I see viscous bulking with low soluble phosphorus but nitrogen is also critical)
• Changes in influent makeup, temperature, pH, or any stress that causes microbes to encapsulate in a EPS matrix.

I have a great interest in microbial biosurfactants. If you are not familiar with biosurfactants, they are a number of compounds produced by microbes that lower the surface tension of water. In nature, they allow microbes access to non-polar, insoluble compounds. So in terms of wastewater treatment or bioremediation, it is the biosurfactant that initiates rapid decomposition of FOG, long chain fatty acids, paraffins, waxes, and other more complex hydrocarbons.



Most of the research indicated that biosurfactants were only produced using either oxygen or nitrate as terminal electron acceptors. However, when testing various microbial enhanced oil recovery (MEOR) formulations, I noticed signs of continued biosurfactant production after dissolved oxygen and added nitrate were depleted. We also suspected similar biosurfactant activity in collection systems and grease traps where conditions were anaerobic.

In the August issue of the Journal of Biotechnology, there is an article on the non-oxygen or nitrate production of biosurfactants. The study looked at oil field microbiotia and found microbes that could produce biosurfactants by fermentative respiration. The microbes identified by 16s and cpn60 testing were Pseudomonas sp (similar to a P. fluorescens) and Bacillus mojavensis. At Aster Bio we have been working with both strains for biosurfactant production. We have used both strains in high FOG wastewater, bioremediation, and microbial enhanced oil recovery (MEOR). It is great to see peer reviewed papers confirming our lab and field testing of microbes and their metabolic products.

Here is a link to the abstract: Yurly Kryachko et al. "Enrichment and identification of biosurfactant-producing oil field microbiota utilizing electron acceptors other than oxygen and nitrate," Journal of Biotechnology Vol. 231, August 2016. pp 9 - 15.