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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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.