It is often taught that bacteria can double every 20 minutes. This 20 minute number comes from common food spoilage organisms such as E. coli. In wastewater with diverse microbial populations doubling time is different for each species but we can use trends in growth rates to see how doubling time relates to F/M, MCRT, and problems with washout. First, let's step back and look at some of the general principals of microbial ecology as it relates to wastewater.

Two General Strategists

• r-strategists - which are faster growing organisms
  • Log phase in WWTP
  • Seen in high F/M
  • Changing influent concentrations or makeup
  • Lag & Log phase growth biomass
• K-strategists -  are the slower growing organisms vital for treatment
  • Tend to be slower growing
  • Exploit a niche
    1. Substrates for growth
    2. pH extremes
    3. Temperatures extremes
       
       

The Microbial Growth Curve
If you have read this site before, you know that I always like to relate microbial populations and biological treatment unit performance to your location on the Microbial Growth Curve. This gives us a convenient visualization to help explain they why bacteria are behaving as they do.

• During startup or near a lagoon influent you have a “bloom” of favoring fast growing organisms that thrive in high F/M environments. This tends to be your r-strategist microbes.
• As the easily used soluble biodegradable organics (BOD5) decreases, you see overall growth rates slow and organisms that exploit niche substrates emerge. This is where we use the terms "mature biomass", "steady-state", and "decline phase growth". At this point log growth stops and you have low F/M.
  
  

Examples in WW

1. Fast growing in WW
   1. Many common genera – well known since the also grow fast in the lab!
   2. Doubling rate is usually in the hours for WWTP
2. Niche organisms
   1. AOB & NOB - nitrifiers
   2. Phosphate Accumulating Organisms (PAO)
   3. Sulfur Oxidizing
   4. Nocardia forms (foaming bacteria)
   5. Some filamentous bacteria (low F/M)
      
3. Environments promoting niche organisms
   1. Longer sludge age – must be long enough to avoid wash out
   2. Presence of substrate at levels promoting their growth
   3. Environmental factors that favor their growth - examples include organic acids, low D.O., high temperature, low N or P, etc.
   4. F/M conditions that favor development of these niche organisms. In the case of nitrifiers, the rule of thumb is 80% of COD must be removed before you start to see substantial growth in the MLSS.

Over the past year, I have seen an increasing number of systems having zero to low populations of known nitrifiers yet they are still removing total inorganic nitrogen. Driven by data generated from molecular testing, we are learning a lot about the various pathways for nitrogen transformations in wastewater. And now we are seeing the role the Phosphate Accumulating Organisms (PAO) can also play in the process.

Nitrification Pathways

• AOB/NOB - this process is the well-known chemoautotrophic bacteria. This is the familiar oxidation of ammonia to nitrite by Nitrosomonas. And the most common NOB is actually Nitrospira. 
  
  
• COMAMMOX - Complete Ammonia Oxidation - A subset of Nitrospira can oxidize ammonia to nitrate. This reduces the number of single step AOB seen in the system.
  
  
• ANAMMOX - Anaerobic Ammonia Oxidation - organisms from the phylum Planctomycetes utilize NO2 + NH4 --> N2 + H2O. This requires AOB for nitrite, but the short cut to N2 gas results in substantial energy savings. The challenge here is ANAMMOX cultures have an extremely slow growth rate and require anaerobic conditions to have a metabolic advantage.
  
  
• Heterotrophic Nitrification - some heterotrophic organisms can oxidize ammonia and nitrite. These organisms grow various organic substrates but also have the ability to oxidize ammonia and nitrite.
  
  
• Simultaneous Nitrification Denitrification - building on heterotrophic nitrification with organisms such as Paracoccus denitrificans, Pseudomonas sp, and Alcaligenes sp having pathways for ammonia oxidation and denitrification even under aerobic conditions.  Factors impacting SND pathways include carbon source, C/N ratio, pH, temperature, and Dissolved Oxygen.
  
  
• Simultaneous Removal of Nitrogen and Phosphorus - SNDPR - this is often seen in low COD systems and aquaculture, but some PAO also have the ability to take a role in removal of inorganic nitrogen in wastewater. 

Challenge of using the above pathways in wastewater treatment

1. Just because an organism has metabolic capabilities or genes, it does not mean that they will be turned on or used. For example, Paracoccus denitrificans can oxidize ammonia and grow on organic acids. If you have sufficient organics (COD), it will obtain more energy using standard heterotrophic pathways and you may see little ammonia oxidation. This is why C/N ratios can become very important for heterotrophic nitrification - in WW, this means lower F/M may trigger other energy yielding pathways.
   
   
2. Do you need separate zones with various D.O. levels, or can we have multiple zones inside biofilms or granules. This is the mechanism seen in newer granular sludge systems where the individual granule has aerobic/anoxic/anaerobic zones. This also happens to a lesser extent in conventional floc/biofilm.
   
   
3. Perhaps a mix of all of the above will be more robust and less susceptible to upset from changing conditions. We actually see this in working systems where organisms that can exploit a ecological niche will grow and populations do change in response to loading and environmental/operational conditions.

Good articles on heterotrophic nitrification, SND, and SNDPR

Pertti J. Martikainen, “Heterotrophic nitrification – An eternal mystery in the nitrogen cycle” Soil Biology and Biochemistry Volume 168, 2022. https://www.sciencedirect.com/science/article/pii/S0038071722000682?via%3Dihub
 
Shivani Shukla, et al. “Simultaneous Nitrification–Denitrification by Phosphate Accumulating Microorganisms” World Journal of Microbiology and Biotechnology (2020) 36:151
 
Zhao W, Bi X, Peng Y, Bai M. Research advances of the phosphorus-accumulating organisms of Candidatus Accumulibacter, Dechloromonas and Tetrasphaera: Metabolic mechanisms, applications and influencing factors. Chemosphere. 2022 Nov;307(Pt 1):135675. doi: 10.1016/j.chemosphere.2022.135675. Epub 2022 Jul 13. PMID: 35842039