In wastewater treatment systems the referenced organisms responsible for ammonia and nitrite oxidation are Nitrosomonas sp.(AOB) and Nitrospira sp. (NOB). Both genera are chemolithotrophic organisms – they obtain energy from oxidation of ammonia or nitrite while using CO2 as carbon source. These and are other genera with similar chemolithotrophic nitrogen pathways responsible for ANAMMOX, COMAMMOX, and Ammonia Oxidizing Archaea (AOA). There are additional pathways for ammonia and nitrite oxidation via metabolic activities by heterotrophic organisms (heterotrophic organisms grown on organic compounds in wastewater and use this carbon to build cellular materials).
 
Heterotrophic Organisms with Ammonia Oxidation pathways
 
Methane Oxidizing organisms (Methanotrophs) are close cousins of Nitrosomonas and the enzymes produced for C1 oxidation can also oxidize ammonia. The bacteria do not gain any energy from this reaction and high ammonia concentrations can actually block methane degradation.
 
Paracoccus denitrificans is an interesting common wastewater organism that is capable of both sulfide and ammonia oxidation while also using nitrate/nitrite as alternative electron acceptors. P. denitrificans metabolism on ammonia was first investigated in the late 1980s and the ability to oxidize ammonia to nitrite coupled with simultaneous denitrification was noted in lab cultures. In real wastewater systems, this organism does aggressively denitrify, but often pursues higher energy yields using conventional heterotrophic pathways.
 
Similar to P. denitrificans, several Pseudomonas sp. have similar ammonia oxidation and denitrification pathways. To what extent these are responsible for ammonia oxidation in working wastewater systems is unknown. Other genera with potentially similar pathways include Alcaligenes and Bacillus.
 
What does this mean for wastewater operations
 

• Most ammonia and nitrite oxidation is accomplished via chemolithotrophic pathways by AOB & NOB cultures.

 

• When it comes to heterotrophic nitrification, just because an organism has the genes for ammonia oxidation pathways does not mean that the genes are turned on and the organism is oxidizing ammonia.
  
  
• Paracoccus denitrificans was one of the first heterotrophic organisms noted to have ammonia oxidation and denitrification pathways in a single organism. Paracoccus bacteria are commonly found in wastewater treatment systems – and are especially useful in improving nitrate/nitrite removal rates.

• Pseudomonas tend to also be aggressive denitrifiers and have been found to possess ammonia and nitrite oxidation genes. What we don’t know is how much Pseudomonas and other similar genera are responsible for primary ammonia oxidation in wastewater systems.

I always think of a wastewater unit in terms of biological population dynamics and how our actions can impact biomass composition. Using molecular testing (Microbial Community Analysis), we are now able to conduct a total bacterial census of MLSS - both quickly and for low cost. The resulting data has allowed for a deeper discussion on the differences among common functional groups with respect to their r/K strategy.

r-strategists are your fast-growing organisms thriving in high F/M and in changing environments due to influent and other rapidly changing environmental conditions. In wastewater, we previously thought of r-strategists as the young sludge heterotrophic organisms. K-strategists are typically slower growing organisms that have higher substrate affinity - a complex way to say they can continue to grow at lower concentrations of food.  

However, even among slower growing niche organisms you can have genera classified as r or K strategists. Such as nitrifiers (AOB & NOB), NOB genera Nitrobacter are r-strategists versus Nitrospira, a K strategist. If you have high nitrite, you favor Nitrobacter, but in most wastewater with low levels of NO2, you favor Nitrospira. Some of the growth rate and substrate affinity differences are slight in these niche organisms, but small changes in operations can shift which organism grows in the system.

For a particularly good discussion on r/K Strategists in wastewater check out the following article:
Qidong Yin , et al. "The r/K selection theory and its application in biological wastewater treatment processes," Science of the Total Environment, Issue 824 February 2022. 
https://www.sciencedirect.com/science/article/pii/S0048969722009287?via%3Dihub​

Operators often call the biological treatment units the "bug farm", where they monitor and care for a huge population of living microbes. The organisms growing in the system are present based on:

• Influent composition & concentration
• Operating environment - pH, Temperature, D.O., MCRT, F/M, system design

While there is a great degree of variation in the species of microbes, we can classify many organisms based on their ecological niche in the biological treatment unit. Here are the most common functional groups.

• Heterotrophic Floc Formers – want microbes that uptake soluble organics. Best represented by the sponge analogy. Rapid uptake of organics with actual metabolism as the organisms pass through the system. Ideal organisms form biofilm/floc. This is composed of polysaccharides, proteins, DNA, adsorbed particulate organics, and other biological polymers. Want the “perfect” slime – that gives stable floc without holding too much water (viscous bulking). Manage populations with F/M or MCRT
  • Keeping design F/M favors bacteria with rapid uptake of soluble BOD (organics)
  • Stored organics in EPS used as “food” outside the abundant zone
  • Too little EPS = pin floc or old sludge, too much EPS = non-filamentous or zoogleal bulking
• Niche Bacteria that require operator attention
  
  • AOB/NOB – managed by adjusting MCRT & F/M – Alkalinity, D.O. Key concept is that both AOB & NOB are slow growing cultures so need lower F/M and longer MCRT.
  • PAO – function in similar niche to Thauera/Zooglea. Rapid uptake of soluble organic acids for use as “food” later. To favor PAO over Thauera, have true anaerobic zone (no nitrate) with high organic acids.
  • Denitrifying – very common pathway – nitrate/nitrite removal optimized by low D.O. (anoxic conditions) with soluble organics. Relatively high energy yield so these are common and can even thrive inside “aerobic” reactors.
  • Sulfur Oxidizers/Sulfur Reducers - sulfur oxidizers have convert sulfides into sulfur and eventually sulfate. If you have sulfides in the influent, the SOX are important for AOB/NOB growth. The Sulfur Reducers (SRB) are found in anaerobic zones and generate sulfides - often found in collection systems & anaerobic digesters
    
  • Filaments - Microbes that can grow into high surface area filamentous morphology. Filamentous bacteria in small quantities help floc formation by acting as a biological rebar to strengthen the floc. Once the filaments are bridging floc or extending free into the solution, you start to see filamentous bulking that can be a problem in secondary clarification. 
    
  • Foaming bacteria - this includes Nocardia and M. parvicella. Problem foaming is caused when influent contains insoluble organics (fatty acids or grease). These organisms use hydrophobic EPS to accumulate the fatty acids to support their growth.  These are slower growing organisms than most heterotrophic wastewater bacteria, so they compensate for slow growth by exploiting their ability to grow on insoluble organics.

Biological phosphorus removal requires an operating environment that promotes organisms capable of storing soluble phosphorus inside their cells. Once in the cells, the phosphorus is removed by wasting the biological solids.
 
PAO – organisms with the ability to “store” polyphosphates as energy reserve inside the cell. When faced with abundant organics, they use bound energy (phosphate) to adsorb/absorb organics for later use.  Other organisms can store soluble organics, so the key to phosphorus removal is having the correct mix of organic acids and an anerobic/nitrate free zone before moving to an aerobic zone.  Once the environmental conditions are correct in A2O and other biological phosphorus removal systems PAO specific organisms become favored – most common in WWTP is Candidatus accumulibacter.
 
C. accumulibacter releases phosphorus to fuel the uptake of short chain VFA which are produced by anaerobic metabolism of soluble BOD by bacteria in either the collection system to a lesser extent in the anaerobic digester (methane production reduces VFA concentrations). Long residence time collection systems will yield needed VFA. However, odor control programs using nitrate can prevent VFA as well as H2S formation. Also, if nitrate makes it to the anaerobic zone, PAO organisms will have competition from NRB. 

PAO organism populations can be monitored using molecular tools like other biological nutrient removal cultures. We have noticed that PAO populations drift with changes in residence times, VFA concentrations, and nitrate/nitrite levels. Using a Microbial Community Analysis test for periodic monitoring helps operations improve system control.
 
Interesting note – aerobic/NRB bacteria in aerobic WW systems also use a similar organics uptake/storge mechanism. In conventional activated sludge systems, we see Thauera & Zooglea genera which uptake soluble organics near the influent, store the organics in EPS, and use the EPS to fuel growth in lower F/M conditions that exist outside the influent zone. It is when you don’t have enough time with low F/M that the EPS continues to build creating non-filamentous or zoogleal bulking.
 
Key Points to Promote PAO Growth

• PAO are favored when you combine VFA & Anaerobic (nitrate free) conditions before an aerobic zone.
• Phosphorus is released by the cells in the anaerobic zone and uptake soluble phosphorus in the aerobic zone.
• Need to ensure sufficiently low nitrate/nitrite in the return MLSS to the anaerobic zone.
• Check influent VFA concentrations – you must have enough to promote the unique PAO metabolism.

Do you recall seeing the microbial growth curve in wastewater training documents? More than teaching the basics of wastewater class, it is also useful for evaluating biological health.  

Let's start with a refresher on the growth curve:

• Lag - bacteria and their enzymes needed to degrade the influent are not common in the biomass. It takes time for populations to adjust and enzyme pathways to activate.  This is seen with massive changes in influent makeup or in totally new WWTP.
• Log - high F/M situation where bacteria grow rapidly. It is interesting to know that the bacteria thriving at this stage are often different from those seen further along the growth curve.  OUR is high and effluent often is turbid.
• Stationary - F/M has dropped and cell division slows. Microbial populations shift to organisms capable of exploiting ecological niches such as nitrifiers, phosphate accumulators, and even filaments. This is the target zone for operating most activated sludge units.
• Endogenous (death) - often seen in aerobic digesters or in the sludge layer in polishing ponds, the Endogenous phase is where low soluble BOD results in a loss in viable bacterial cells and most importantly, lysis of dead cells. This phase releases bound water and reduces sludge volume for disposal.

Idea of Steady-State
You may have seen the term Steady-State being used in wastewater systems. It simply means that the microbial population is at the target growth curve point for good effluent quality.  Steady-state conditions are not a singular point on the curve, but a range.  Adjusting wasting, recycle rates, aeration, and even influent flow are tools to adjust your position on the growth curve.

When not in SS, the system exhibits signs of biological issues

• Foaming
• Bulking
• Odors
• Increased polymer demand
• Effluent quality issues

The New Challenge of Multiple Steady-States
Moving along the old growth curve was not as complex when effluent targets were BOD and TSS.  We simply relied on natural population dynamics to reach a point where biomass removed soluble BOD and flocculated. Now with permits including BOD, Total Phosphate, and Total Inorganic Nitrogen (TIN) – we have systems with multiple steady-states as you need to have microbial populations balanced to achieve all the treatment goals.  The key to efficient operations is to keep the system in the “ideal” range using all monitoring and control tools available.

Often the control tools are determined by your system's design and setup. All operational controls work by encouraging the growth of ideal microbial populations while limiting the undesirable organisms. None work instantly to correct problems. So, good monitoring helps you keep the system operating efficiently.  Conventional monitoring with OUR, SV30, SVI, F/M, MCRT, and ATP is good for BOD removal information, but is not as effective at detecting changes in more niche organisms. For tracking ecological niche organisms, I suggest looking into molecular testing. With improved knowledge of niche organisms and lower costs for molecular testing, it can be a useful part of any monitoring program.

I have been a part of a team at Aster Bio has been using qPCR and metagenomic testing in wastewater treatment systems for over 7 years and have found that molecular tests are highly effective at monitoring slow growing niche organisms such as:

• PAO - phosphate accumulating organisms
• GAO - glycogen accumulating organisms 
• AOB & NOB - ammonia oxidizing bacteria & nitrite oxidizing bacteria
• NRB - nitrate & nitrite reducing bacteria
• Filaments - (can even be customized to a specific system's most problematic filaments)
• Non-filamentous bulking
• Foaming (Nocardia forms)

More information on molecular testing at www.environmentalgenomics.com

Many industrial wastewater systems have an influent with a C:N:P ratio outside the ideal ratio of 100:5:1.  For example, pulp & paper wastewater tends to be deficient in both N & P relative to their BOD5. Whereas petrochemical wastewater has sufficient N (often in excess) and lacks P. In order to have a healthy biomass which forms floc and removes pollutants, industrial facilities often benefit from adding macronutrients.  

How much N & P are needed
Textbook C:N:P ratios are given at anywhere from 100:10:1 to 100:5:1 for aerobic biological treatment. Personally, I prefer using nutrient residuals when working with a system adding nutrients. By using residuals, you are accounting for variation in ratios created by influent makeup and biomass composition. If you have biological unit effluent ammonia nitrogen at 0.25 - 0.5 and orthophosphate at 0.1 - 0.2, you have enough to maintain a stable biomass.  

Problem with running ammonia nitrogen residuals for macronutrient dosage
Running residuals on ammonia nitrogen seems like best practices, but with molecular testing we have seen issues with this method of adjusting nitrogen feeds.  In a healthy aerobic system, you tend to grow both AOB and NOB cultures if there is ammonia present. The AOB & NOB populations can reduce ammonia below operational targets, resulting in increased nitrogen feed. The problems is you are paying for both the added nutrient and the oxygen required to remove this ammonia - a vicious cycle.

What can be done
If you add urea or other form of nitrogen nutrient in a biological treatment unit, it is beneficial to run periodic Microbial Community Analysis (MCA) checks to see if you have AOB & NOB populations using DNA sequencing technology. If there is an increase in AOB or NOB and effluent ammonia residuals are below residual targets, the solution is not to add more nitrogen. We can monitor biomass health using molecular testing and microscopic exam while adjusting nutrient feeds downward to promote stable biomass.

The next question is does the same thing happen with orthoPhosphate?  The answer is having PAO cultures is less likely, but it does happen. Fortunately, the MCA picks up phosphate accumulating organisms too. 

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