Much larger than bacteria, protozoa (single cell eukaryotes) and metazoan (multiple cell organisms) are easily seen with light microscopy at 10x and 40x objectives. Both are usually very active and easily spotted as they feed and move about the sludge. Meanwhile, individual bacteria cells look like tiny specs that bounce with water flow on the slide.

Floc, often referred to as "bugs" is composed of:

• Living and dead bacteria
• Extracellular biopolymers (EPS) functioning as glue creating a biofilm that serves to trap stored insoluble organics being acted on by extracellular enzymes and protecting cells from surrounding environment.
• Inorganics - EPS also contains grit and other inorganics trapped in the matrix

Both protozoa and metazoan prey on either free bacteria or graze on the floc. This grazing and filter feeding helps improve wastewater operation.

• Filter feeders remove free bacteria cells from solution and encourage floc formation by consuming cells not in the biofilm (floc). You can watch stalk ciliates actively filtering free floating cells while attached to floc. In the case of "young sludge" - high F/M - you will see flagellates and amoeba moving along consuming free cells.
• Grazing feeders move along developing floc and consume outer layers of the biofilm. Grazing feeders include free crawling ciliates, rotifers, water bears, and other metazoan. By grazing on the outer layers, these organisms reduce excess EPS and help keep the biomass active and aerobic. All are good for improving floc density and settling.
  
  

Can protozoa and metazoan "eat" key organisms in the system?
While it is possible, a healthy ecosystem contains both primary producers and predators. This relationship between predator and prey ensures no overgrowth of any single organism and a more diverse biomass. So, just note the indicator organisms present and relate activity to where the system is on the growth curve. Don't worry about protozoa or metazoan eating all the biomass.

Poster with indicator organisms and how they relate to the growth curve is available for download here
https://asterbio.com/bug-poster/

Non-filamentous bulking in wastewater treatment plants (WWTPs) can be a challenging issue, affecting the quality of the final effluent. In the photo above, note the rounded colonies that appear to have more "gel" between the cells - these are high EPS zones. Also, the above photo is using India Ink. The carbon particles in the ink do no penetrate high EPS floc which proves that you have excess EPS and non-filamentous bulking. Let’s explore strategies for controlling this phenomenon:

1. Understanding Non-Filamentous Bulking:
   • Filamentous bulking occurs due to excessive growth of filamentous bacteria in WWTPs.
   • Ideally, there should be a balance between filamentous bacteria and floc-forming bacteria.
   • In moderate amounts, filamentous bacteria contribute to AS settle-ability by providing a structural base for robust floc formation.
2. The Role of EPS (Extracellular Polymeric Substances):
   • EPS is crucial for floc formation in suspended growth and biofilms.
   • It consists of polysaccharides, proteins, nucleic acids, and metabolic byproducts.
   • EPS protects cells and accumulates nutrients (N, P, metals).
   • Proper EPS levels are essential for solids removal and phosphate removal.
3. The Transition to Excess EPS:
   • Excess EPS leads to problems:
     • Waste solids retain more bound water.
     • Dewatering becomes difficult.
     • Secondary clarifiers may have floating scum and poor compaction.
   • Major contributors to non-filamentous bulking:
     • Thauera: Common in industrial activated sludge.
     • Zooglea: More common in domestic wastewater.
     • Azoarcus: Less common but efficient denitrifiers.
4. Control Strategies:
   • Nutrient Adjustment:
     • Turn up nutrients (N or P) if not already optimal. If C:N:P ratio is in the target zone, adding additional nutrients will not help with bulking.
   • Waste Solids Management:
     • Remove excess EPS through waste activated sludge (WAS).
     • Be cautious as it increases the F/M ratio.
   • Bioaugmentation:
     • Increases viable microorganisms in the mixed liquor suspended solids (MLSS) breaking bottlenecks created by overgrowth of high EPS producing organisms.
     • Aster Bio has identified and produced cultures having the ability to degrade EPS via extra-cellular enzymes and form beneficial capsular polymers that function much like chemical cationic coagulants.
5. Monitoring and Early Detection:
   • Use molecular tests including qPCR to detect changes in Zooglea populations.
   • Look for signs of increased soluble BOD loadings at the influent.

Remember that effective control strategies require a combination of traditional and innovative approaches.

Protozoa are eukaryotic residents of wastewater treatment plants that serve as primary predators of bacteria. (Some protozoa are free-living and not predatory). Since protozoa are much larger than bacteria and easily recognized by shape and motility, we use their diversity and populations as indicators of system health.
 
Since the protozoa “eat” bacteria and bacteria are the organisms that remove pollutants, are the protozoa detrimental to treatment efficiency?
 
Protozoa populations and diversity increase as you move along the growth curve where bacterial populations are higher and water quality improves. The protozoa feed on both free bacteria in solution and by grazing on the biofilm/floc. We know that by removing free bacteria in solution, the protozoa decrease turbidity and TSS. When grazing on floc, the protozoa contribute to biofilm turnover. We are concerned that protozoa predation will damage slower growing microbial populations such as Ammonia Oxidizing Bacteria (AOB) and Nitrite Oxidizing Bacteria (NOB) – Nitrifiers.
 
Looking at research, we find the following:

• Bacterial populations have evolved into syntrophic communities and predatory protozoa are an important part of this ecosystem.
  
  
• Protozoa predation increases diversity in sludge biomass with a greater mix of both r-strategists (fast growing organisms) and k-strategists (slow growing specialists).
  
  
• The “ideal” spot for predation and bacterial diversity is also the decline phase growth zone where waste treatment is optimized.
  
  
• Studies on AOB, NOB, and ANAMMOX biomass found that even with predation, ammonia and nitrite removal rates remained stable.

 
Good articles on protozoa impact on wastewater microbial populations:
 
Burian, A., Pinn, D., Peralta-Maraver, I. et al. Predation increases multiple components of microbial diversity in activated sludge communities. ISME J 16, 1086–1094 (2022). https://doi.org/10.1038/s41396-021-01145-z
 
Wu L, et al. Global diversity and biogeography of bacterial communities in wastewater treatment plants. Nat Microbiol. 2019 Jul;4(7):1183-1195. doi: 10.1038/s41564-019-0426-5. Epub 2019 May 13. Erratum in: Nat Microbiol. 2019 Dec;4(12):2579. PMID: 31086312.
​
Pogue AJ, Gilbride KA. Impact of protozoan grazing on nitrification and the ammonia- and nitrite-oxidizing bacterial communities in activated sludge. Can J Microbiol. 2007 May;53(5):559-71. doi: 10.1139/W07-027. PMID: 17668014.

This topic was brought to my attention last week when a facility that I am working with noticed that Thiothrix filaments appeared to be reducing oxygen transfer efficiency in the aeration basin. While low D.O. promoting filamentous growth is well known, I was not familiar with how much filamentous bulking can reduce OTE.
 
Oxygen Transfer Efficiency (OTE) refers to how effectively oxygen is delivered and dissolved in water under specific conditions. Systems are designed based on predicted OTE as conditions vary in working wastewater treatment systems.
 
When you compare floc forming sludge (FFS) versus filamentous bulking sludge (FBS), OTE decreases by up to 50%. Filament length also impacts the results with longer filaments, such as Thiothrix, having more impact on OTE than short filament that do not extend from the floc. The studies proposed that the loss in transfer efficiency related to:

• Viscosity – filamentous typically being more viscous.
• Filaments attaching to bubble surface reducing air/water interface.
• EPS on filaments acting in a manner similar to surfactants which reduce air/water interface.

Good reference articles on the topic:

Ken Campbell et al. Filamentous organisms degrade oxygen transfer efficiency by increasing mixed liquor apparent viscosity: Mechanistic understanding and experimental verification
https://www.sciencedirect.com/science/article/abs/pii/S0043135420301068
 
Guoqiang Liu et al. Formation of filamentous microorganisms impedes oxygen transfer and decreases aeration efficiency for wastewater treatment
https://www.sciencedirect.com/science/article/abs/pii/S0959652618311521
 
Xianwei Wu et al. Thiothrix eikelboomii interferes oxygen transfer in activated sludge
https://www.sciencedirect.com/science/article/abs/pii/S0043135418310364
 

Archaea are not bacteria! While still unicellular, they are as different from bacteria as a they are from you (a Eukaryote). Some cool facts about Archaea:

• First isolated from extreme environments where they compose a larger % of the total biomass. Extreme temperatures, salinity, pressures, and pH – think deep sea vents, highly anaerobic sediments, and acid mine waters.
  
  
• In wastewater, methanogens are archaea. They are found in aerobic digesters, digestive tracts, and deep within wetland soils – oxygen is toxic to these organisms.
  
  
• Ammonia Oxidizing Archaea are responsible for much ammonia oxidation in the ocean. They have a different metabolism than more prevalent wastewater AOB.
  
  

While interesting and common in certain environments, do archaea play a significant role in aerobic wastewater treatment systems? Early attempts to answer this with conventional microbiology resulted in – “we don’t know” as most organisms did not grow on petri dish plates. But using new molecular (DNA) techniques, we can evaluate the percentage of biomass related to archaea vs bacteria.
 
Last week, Paul Campbell used qPCR to test a random sample of four municipal treatment systems (some with upstream anaerobic digesters) and four industrial aerobic treatment systems. Here are the results (% of total biomass)

Note the relatively low levels of archaea in these wastewater treatment systems. Those with higher levels of archaea tended to be those with upstream anaerobic digesters where methanogens are very important. Or systems with longer sludge ages where we also find more niche bacteria in the MLSS. While low in frequency, these archaea may perform some vital functions, but this is not known. We are continuing to use molecular tools to learn more about the wastewater microbial community and will report more of our results as we discover new pathways and interesting organisms.

Biofilters consist of a bed of organic material, such as compost, wood chips, or synthetic materials, that provides a substrate for the growth of microorganisms. The media's surface area is crucial for microbial colonization and the adsorption/removal of odorous compounds.
 
Microbial Colonization:

• Microorganisms, including bacteria and fungi, naturally colonize the media in the biofilter.
• Specific microbial strains are selected for their ability to metabolize and break down hydrogen sulfide and volatile organic acids.
• Colonization by microorganisms takes time at startup or following a loss of viable biomass in an operating system. Aster Bio has successfully used bioaugmentation to reduce time required for startup using sulfur oxidizing and volatile organic acid degrading organisms.

 Air Distribution:

•   Odorous air containing hydrogen sulfide and volatile organic acids is introduced into the biofilter.
• The air is evenly distributed through the biofilter bed to ensure contact with the microorganisms.

Biological Degradation:

• Microorganisms on the biofilter media surface metabolize the odorous compounds through enzymatic reactions.
• For hydrogen sulfide, bacteria like Thiobacillus spp. oxidize H2S to sulfate, reducing its concentration.
• Volatile organic acids are typically broken down through aerobic microbial activity, producing carbon dioxide and water.

Moisture and Nutrient Management:

• Maintaining optimal moisture levels and providing essential nutrients (nitrogen, phosphorus, etc.) is crucial for microbial activity.
• Moisture is often controlled to prevent over-wetting or drying out of the biofilter bed.

 pH Control:

•  pH levels in the biofilter are monitored and controlled to ensure an environment conducive to microbial activity.
•  Some microorganisms may require specific pH conditions for efficient degradation.
• If you are treating both organic acid and H2S, greater care in maintaining pH in target ranges as SOX cultures tend to lower pH to ranges below ideal VOC culture growth.

Monitoring and Adjustments:

• Regular monitoring of the biofilter's performance includes measuring inlet and outlet concentrations of odorous compounds.
• Adjustments to operating parameters, such as airflow rate, temperature, and nutrient supplementation, may be made based on monitoring results.

 Periodic Maintenance:

•  Periodic maintenance involves replacing or replenishing the biofilter media and optimizing microbial populations.
• This ensures the long-term effectiveness of the biofilter.

 Final Treatment and Discharge:

•  Treated air exiting the biofilter should meet regulatory standards for odor control.
• The treated air can be discharged into the atmosphere, and in some cases, it may undergo additional polishing treatments if needed.

 
Biofilter systems are often chosen for their sustainability, cost-effectiveness, and ability to handle a wide range of odorous compounds. However, the efficiency of biofilter treatment can be influenced by factors such as temperature, pH, nutrient availability, and the specific characteristics of the odorous compounds being treated. Regular monitoring and proper maintenance are essential for optimal performance.