Everyone knows that AOB & NOB cultures are among the slowest growing cultures in WW treatment plants.  Upsets of any kind may result in effluent ammonia breakthrough.  If your system has an MCRT lower than the washout or wasting rate, the. % nitrifiers in the biomass will decrease and eventually you will see increased effluent ammonia.  The key is to understand AOB & NOB growth rates and how you can try to keep them at their maximum rates while also managing all other biological unit processes.

I often get calls about a system where everything is fine - great COD removal and settling - except the nitrifiers are either dead or not working.  Here is the process for walking through the nitrifier problem without wasting operation budgets.  


First, if you lost nitrification don’t just blindly throw expensive nitrified concentrates at the problem!  
(1) Identify what happened in order of likelihood - you will often see multiple factors

• Higher influent TKN or ammonia - yes, both are needed
• Look at influent organic loadings (COD or BOD)
• Changes in MCRT, F/M, pH, Temperature, D.O., alkalinity
• Toxicity - phenol, sulfides, cyanides (Not as common as everyone thinks)
  
  

(2) Determine if you have really lost your AOB/NOB population by running a qPCR nitrifier panel.  Fast, low cost and gives quantitative numbers for both AOB & NOB. Make sure the qPCR primer library is based on actual wastewater AOB/NOB testing.

(3) Depending upon qPCR results, you select options

• Provide ideal conditions for growing indigenous AOB/NOB populations. If you have inhibitory compounds or higher than normal COD/BOD, we need to optimize the heterotrophic population and improve conditions to allow for full AOB/NOB activity.
• To speed the natural recovery process, adding nitrifiers may be recommended.  AOB/NOB concentrates are still slow growing chemotrophs just like indigenous nitrifiers.  Dose is based on how much ammonia needs to be oxidized, how long recovery time is available, and expected growth rate for the cultures.  AOB max growth rate is usually 12 hours vs 1 - 2 hours for heterotrophs.
  
  

(4) The key is don’t lose your AOB/NOB populations.  Don’t wait for effluent ammonia to increase!.  For systems with a history of nitrification issues, run qPCR checks on a routine basis to discover normal populations.  If you see a decrease, take actions to improve the environment for your nitrifiers.

We all know that microscopic exam tells you a lot about wastewater system microbiological health.  Even older microscopes, without phase contrast, allow for observation of protozoa and metazoa.  Both protozoa and metazoa are known as indicator organisms as different morphological types appear based on D.O., soluble BOD, and freedom from inhibitory compounds.  

In performing a daily microscopic exam, I like to see operators noting the following:

• Note floc size and density.  While phase contrast helps to see filaments, you can also see filaments with regular light microscopy.  You do not need to do full filament ID with staining here.  Just note floc and filaments that you see and compare your observations with SV30/SVI, turbidity, and other system data.
• Note protozoa present - you do not have to identify down to genus level! Just note amoeba, flagellates, free-swimming ciliates, crawling ciliates, and stalk ciliates. These are morphological types which only require quick observation.  Also note multi-cellular organisms if present.  

Now that you have the observations, you can note where the system is on the growth curve.  Remember, most system are designed to function best in the early stages of decline phase growth. This region of the growth curve gives low soluble BOD5, efficient nitrification, and good floc formation - all good things. 

While the bug chart says you will see a biomass dominated by stalk ciliates, crawling ciliates, and some metazoa, you may have system specific differences.  This is why you want to perform frequent microscopic exams on your system to know how a good, normal indicator organism population looks.  If you see changes in the population, you can look for movement along the growth curve towards "young sludge" or "old sludge" and make operational changes as needed.

Sometimes a normal vital MLSS organism can exhibit behaviors that we don't want.  From forming "Zoogleal" EPS to building sheathed filaments, these organisms respond to environmental factors and do things that we don't want in a treatment system.  From a science standpoint, this is a discussion of phenotype (what you see) vs genotype (what DNA is present).  However, we are increasingly able to predict what causes this bad behavior and can take corrective actions.

Example 1 - Zoogleal (Non-filamentous) bulking
In typical activated sludge and decline phase growth systems, often Zooglea type organisms are the most common genera found when looking at DNA.  In addition to degrading many organic components of BOD5, these organisms are also great for denitrification and making EPS needed for floc/biofilm formation.  Problems come when high Zooglea (or Thauera) populations meet conditions that cause excess EPS production.  Example triggers include:

• high soluble BOD5 (they store soluble BOD as EPS)
• Low N or P which slows cell metabolism - resulting in organics being stored in EPS

Example 2 - Sphaerotilus natans
A common sheathed filament, S. natans can often be seen in filamentous bulking sludges. Under normal conditions with sufficient D.O., S. natans grows as a typical floc forming organisms. In response to stress of low D.O., we see the formation of the sheath and filamentous growth.  

While the mathematics behind calculus can be complex, in nature we see organisms "taking the easy path" that yields the most energy.  This process of dynamic optimization can be described using mathematics. Fortunately for wastewater treatment, we just need to understand the concepts to predict what is going to happen.

Like children given broccoli or candy at Halloween, bacteria prefer to grow on the "tasty" compounds first.  In this case, heterotrophic organisms grow fastest on soluble compounds with glucose often being the "ideal" energy source.  The key concept here is - net energy yield!  Cells have to produce enzymes and undertake other biochemical processes all of which take energy when growing.  These enzymes and biochemicals transform organics into simple compounds that fuel cellular machinery and yield energy.

Here are a few general rules of bacteria calculus:

• Simple soluble organic compounds are degraded first.  Tells why insoluble FOG tends to accumulate.
• Aerobic processes yield more cellular energy than anaerobic.
• While complex organics such as lignin or Polynuclear Aromatics Hydrocarbons (PAH) are biodegradable, the bacteria using these compounds much be either slow growing niche organisms or have exhausted the "easy" energy sources
• AOB/NOB (nitrifiers) are among the chemotrophic organisms that occupy a niche - they may have some other pathways, but most of their energy comes from oxidation of ammonia and nitrite.  This has lower energy yield than most organics, so AOB & NOB have lower growth rates.

So how do you use bacterial calculus to predict behavior in wastewater?

• High energy reactions will happen first.  As energy sources are exhausted, the population will shift to other energy sources.
• Low F/M - means that you are forcing the bacteria to work on the lower energy reactions sooner than would be in a high F/M system.
• Low F/M - means that niche organisms have more of a chance to acquire macronutrients and other growth factors as competition with fast growing organisms is reduced.

If you see ammonia increasing in a normally nitrifying system, it is not automatically a sign that you have acute toxicity!  Here is my checklist that I use:

• Check influent TKN (Total Nitrogen)
  Most systems only run influent ammonia. However organic N is converted to ammonia in the wastewater treatment process and should be considered part of your total loading.  
• Look at the aeration basin environment 
  Alkalinity, pH, Temperature, D.O. - did anything change from when the system was working correctly?
• Acute toxicity
  Not as common as you would think -> Examples sulfides, phenols, organics with thiocarbonyl functional group (>C=S), cyanide
• Organic loading and combination of factors causing loss of nitrifier populations
  Remember that both AOB & NOB cultures grow at slower rates than heterotrophic cultures.  An old rule of thumb is that 80 of BOD/COD needs to be removed before nitrifiers can compete for nutrients and grow.  This means that you must have hydraulic residence time for the nitrifiers to grow and develop.  Also consider low temperatures or non-ideal pH factors that can slow nitrifier growth.  All of this can lead to a loss of nitrifiers as a % of biomass.

How can you check your nitrifier populations?
Most of us learned nitrifiers as Nitrosomonas (AOB) and Nitrobacter (NOB) - but in practice wastewater treatment systems we see a different mix of organisms than were isolated and grown in the lab.  Our use of next generation sequencing has identified that most systems rely on a mix of Nitromonas and Nitrospira (which can be NOB & COMAMMOX).  After running over 5 years of sequencing data, we now have developed primer sets to run qPCR on wastewater samples to get rapid quantitative data on AOB & NOB populations.  The qPCR nitrifier test gives information if you have a true toxicity event that killed the nitrifers or if you have conditions inhibiting their normal metabolism which eventually leads to washout.  This can save money and time in addressing nitrification problems.

Flow through membranes in MBR systems determines wastewater charge rates.  Key parameters for MBR operation include:

• Flux - (L/m2/hr or GFD)
• Transmembrane Pressure (Bar or PSI)
• Permeability (LMH/Bar or GFD/PSI)

During operation, membrane pores become plugged with inorganics, particulates, and even microbial EPS.  While backflushing usually restores the membrane, the loss of flow while backflushing reduces the system charge rate.  

For the past year, Aster Bio has been working with an MBR system having issues with biomass EPS reducing flux rates through the membrane.  Using both Aster Bio's Microbial Community Analysis (MCA) and customized qPCR based on MCA results, the facility was able to:

• Identify the organisms responsible for problem EPS generation
• Use qPCR testing to monitor the populations and discover impact of operational changes on both "good" and "bad" microbial populations

In this case, the problem organisms were from the Thauera genera.  While often the most common genera in stable wastewater treatment systems, under select conditions certain groups of Thauera and Zooglea can produce excess EPS which increases transmembrane pressure and lowers permeability.  The challenge is identifying the genetic sequences for the problematic organisms (which are often system specific) and then correlating the high EPS production with operational data.  For this facility, Aster Bio determined that one group of Thauera were responsible for most loss of permeability and other Thauera were benign.  In the graph above, you can see that high percentages of Group 2 Thauera were strongly correlated with reduced permeability.