The microscope can give you great information on a wastewater system's biomass  health. While expensive phase-contrast, high magnification/resolution microscopes are great, a simple low-cost microscope can provide much of the same information. Let's review what can be done with a microscope similar to the one pictured above.

• Floc size & density - simply look at floc shape, size, and density. You are looking for changes and relating what you see under the microscope to effluent quality. To see changes, you need to look at the biomass often!
  
  
• Indicator organisms - bacteria are usually too small to see under a low cost microscope, and aside from general shapes and motility, you don't see much. So, we look at indicator organisms which are much larger and more active than bacteria. As with the floc, you are looking for changes in indicator protozoa and metazoa (if present). Don't worry too much about exact ID for the protozoa. Look for general location methods, size, and relate what is there to your own system's operations. I have attached a "bug poster" for easy use in classifying common wastewater protozoa. Insteand of photos, I use drawings which makes you focus on the locomotion and main characteristics rather than exactly matching photos. By looking at the MLSS frequently, you learn what should be in your system when it is in good shape.

The big step up in microscopes comes when you add phase contrast capabilities to your microscope. This requires phase contrast objectives and diaphragm condenser. Phase contrast allows you to see more detail - it is very valuable in performing filament ID, evaluation of EPS, and looking at individual bacterial cells. These tests also require a bit more experience for the microscope operation and take more time. While phase contrast will make daily exam slightly better - it is overkill for most coursory exams. 

So just dust off the old microscope and start using it daily. A quick 5 minute look under the microscope using 10x and 40x objectives will give you good information and help improve your monitoring program.

If you don't have a bug poster near your microscope, I have included one that we made for Aster Bio here. 
​

Your aerobic treatment system has high D.O., good floc formation, and is discharging great quality water. All of sudden, you start to notice small flying insects above the water which are soon followed by small red worms in the MLSS. The flying insects are midge, a native insect that lays eggs on decaying organic matter. It just happens that wastewater MLSS with high D.O. is a perfect environment for laying eggs and incubating larvae. Soon after seeing the midge flies, the worms "blossom".

Problems happen when the larvae consum the MLSS - which can cause loss of slower growing organisms such as Ammonia Oxidizing Bacteria (AOB). They can also create effluent TSS if they wash over the clarifier weir.

To control midge larvae, the EPA approves the use of Bacillus thuringensis (Bt) spore solutions (kill larvae by damanging their digestive tract) or Strike which is an insect growth regulator (similar to that used in pet flea/tick control).

Here is a bit more that I wrote on the midge problem in a previous post:
​ https://www.biologicalwasteexpert.com/blog/whats-eating-my-biomass-red-worms-blood-worms-midge-fly-larvae

Another post on ammonia and nitrite removal by microbes! Often grouped as nitrifiers, AOB & NOB cultures must be present if you require significant biological ammonia and nitrite oxidation. Both groups are obligate aerobes and are slower growing than most other wastewater bacteria. This makes AOB & NOB the most common bottleneck in wastewater treatment. 

As discussed in my last post, the organisms responsible for ammonia and nitrite removal are a diverse group. Instead of just Nitrosomonas and Nitrobacter, we have a variety of organisms that thrive under slightly different conditions. As they are all key microbes and slow growing, Paul Campbell - Aster Bio's molecular diagnostics guru - has developed a qPCR battery to monitor nitrifiers. qPCR works by fast quantificagtion of specific segments of genetic material. It can be customized to fit the actual poplations in the system. However, you need to make sure the qPCR is a good fit for you biomass which means off-the-shelf type kits are not always the right test.

What we now call a qPCR  nitrifier pannel can quantify levels of the following:

• Nitrosomonas amoA
• Nitrospira 16s (total Nitrospira)
• Nitrospira nxrB (measure of NOB)
• Multiple Nitrospira COMAMMOX - most common groups in WW as found by testing
• Nitrobacter nxrB
• Nitrosospira 16s
• AOA amoA - ammonia oxidizing archaea

We are working on development of qPCR for key ANAMMOX cultures. If anyone operates a system with ANAMMOX, we would like to obtain samples to calibrate the tests. Just contact me by email.

When talking about ammonia and nitrite treatment in wastewater, we often mention Nitrosomonas (ammonia oxidizing bacteria) and Nitrobacter (nitrite oxidizing bacteria). With the arrival of more advanced testing such as Microbial Community Analysis (MCA or a 16s DNA census) and customized qPCR for specific 16s segments or metabolic pathways, we have the ability evaluate the real organisms removing ammonia and nitrite in working wastewater treatment systems.

After testing industrial, municipal, and even Aster Bio's own "nitrifier" culture reactor, we have noticed that removing ammonia and nitrite from wastewater is a complex process that can involve many different microbes working as part of the microbial community. The most common "types" of nitrifiers are summarized below:

• Ammonia Oxidizing Bacteria (AOB) - the type organism here is Nitrosomonas. This group convert ammonia into nitrite. This has often been called the rate limiting step in nitrification. In pratice, the AOB cultures were lower than expected in testing of working systems. Meanwhile, a commercial nitrifier producition tank had 80 - 90% Nitrosomonas as given by 16s sequencing.
  
  
• Nitrite Oxidizing Bacteria (NOB) - the most common referenced organism is Nitrobacter. However outside the nitrifier reactor, the primary NOB is Nitrospira. Why the difference between a reactor and wastewater treatment units? It appears to be related to the mixed cultures and how nitrite concentrations are concurrently produced and oxidized which leads to lower nitrite concentrations than is seen in a lab reactor.
  
  
• Complete Ammonia Oxidation (COMAMMOX) - during MCA tests, Aster Bio has found Nitrospira reads at much higher levels than Nitrosomonas (often 5 - 10x higher concentrations). This fact led us to evaluate Nitrospira's metabolism. In addition to nitrite oxidation, a subset of Nitrospira can also oxidize ammonia into nitrite. These Nitrospira are the COMAMMOX bacteria, and appear to be very common in wastewater treatment.
  
  
• Ammonia Oxidizing Archaea (AOA) - archaea are a different kingdom than bacteria and are most often seen as methanogens in anaerobic digesters. In systems with lower pH resulting in more ammonium (NH4+), AOA which utilize NH4+ rather than NH3 become more important for ammonia oxidation. We are still exploring the role of AOA in wastewater and how vital they are for ammonia oxidation.
  
  
• Anaerobic Ammonia Oxidation (ANAMMOX) - relies on a novel biochemical pathway where organisms shunt nitrite and ammonia directly to nitrogen gas. This avoids the energy use oxidizing nitrite to nitrate while also accomplishing denitrification. ANAMMOX cultures have a slow rate of growth and much research is being done on how to optimize wastewater treatment systems to take advantage of this metabolic pathway. So far, it appears many of the ANAMMOX cultures are in the Planctomycete phylum.
  
  
• Heterotrophic nitrification - bacteria including some Pseudomonas, Paracoccus, and Alcaligines have the ability to obtain energy from ammonia oxidiation. However, the rate of ammonia oxidation is lower than that seen in obligate chemotrophic organisms.

​

After touring the Pearland, TX new wastewater treatment facility this week, I saw many newer treatment technologies in practice. The most amazing thing was the facility is very close to neighbors with no odors. Even standing near the headworks, there was no apprecialble odor. Best yet, they did not have any perfume or odor adsorbing misting. What was being used were two biological filters. One treated the lift station and a second served the headworks. 

Biofilters are an efficient way to treat H2S and volatile odorous organic compounds. Unlike chemical scrubbing or odor neutralizing, a biofilter maintains a biofilm on a fixed film media. The biofilm contains a mix of sulfur oxidizing cultures - often Thiobacillus, Paracoccus, Thiosphaera, along with organic acid cultures including Pseudomonas, Paracoccus, Thiosphaera (Note how Paracoccus and Thiosphaera are in both SOX & Organic Oxidizers).  In an active biofilter, the biofilm is maintained by a spray system that adds nutrients and pH adjustment to keep the biofilm at maximum activity. The biofilters are sized based on expected loadings and air volumes to be treated. If sized correctly, they are very efficient and almost appear as a magic box. 

Since Aster Bio has been performing Microbial Community Analysis (MCA) genetic testing on MLSS from a wide range of industrial and municipal samples, I have learned much more about the organisms causing non-filamentous bulking and why past control methods were not very effective. I don't know how many times I advocated adding nutrients, wasting high EPS sludge, and trucking in MLSS & using bioaugmentation cultures; only to have bulking return later.  Below is a summary of what causes non-filamentous bulking and why these bacteria can go from beneficial to a problem.

Non-filamentous bulking or excess extracellular polymeric substances (EPS). How do you know if you have excess EPS?

• Use India Ink with microscopic exam. Excess EPS appears as clear zones.
  
• Even without India Ink, high EPS  colonies can be identified by rounded, fingers with cells suspended in gelatinous matrix.
  
• Waste solids have increased bound water, higher polymer demand, difficulty in dewatering
• Secondary clarifiers may have increased floating scum, beds that tend to billow and not compact was well as normal.

Going Back to EPS – the good versus the bad
EPS is very important for floc formation in suspended growth and for biofilms in attached growth wastewater treatment plants. Wastewater plants were designed to operate in decline phase growth where microbial division slows and cells begin to form aggregates. Key to aggregate formation is the production of EPS. Consisting of polysaccharides, proteins, nucleic acids, and a variety of metabolic byproducts; EPS functions to keep cells protected and viable under the stress of low soluble organics (food). Another function of EPS is the accumulation/storage of nutrients including N, P, and metals. Under normal operations, the proper amousnt of EPS is needed for solids removal and helps remove phosphates in biological nutrient removal systems. The problems happen when the biomass generates excess EPS.

Main organisms responsible for non-filamentous bulking
Using Microbial Community Analysis (total MLSS microbial census) technology, we have identified the major contributors to non-filamentous bulking as:

• Thauera – usually the most common organisms in industrial activated sludge and lagoons in decline phase growth
• Zooglea – more common in domestic wastewater
  
• Azoarucs – a third genera in the Zooglea family. Less common than Thauera or Zooglea
  
  

While these organisms can be responsible for non-filamentous bulking, they are also the main contributors to the EPS needed for floc formation, removal of organics (BOD/COD), and are even efficient denitrifiers. So, what causes the transition from good EPS to excess EPS?

• High influent soluble organics such as organic acids. Excess soluble BOD is stored by cells in EPS. The EPS stores the energy for future use by the cells. If you don’t have sufficiently low F/M or time for metabolism of stored organics, the EPS accumulates and eventually will produce bulking.
  
• If you have excess soluble organics with insufficient N, P, or some other vital micronutrient. In this case, the cells store carbon in the EPS as they do not have enough of some key nutrient(s) to divide, produce enzymes, and support full cell metabolic activity.
  
  

What can be done to prevent & control non-filamentous bulking?
Past Methods

• Turn up nutrients - if nutrients (N or P) were not a problem, adding in excess will not help
• Wasting - removes EPS and stored carbon with WAS. But also increases the F/M ratio  which is not needed!
  
• Usually add bacteria via bioaugmentation or bringing in non-bulking sludge -> this helps reduce F/M. Both increase numbers of viable microorganisms in the MLSS.

 
New monitoring & control strategies

• Monitor for system specific Zooglea Forms (qPCR)
  • Populations often increase into critical zone before bulking develops
  • qPCR detects changes much earlier than microscopic exam & India Ink tests
• Look for signs of increased soluble BOD loadings at influent. Evaluate methods to keep F/M in target range and increase cellular metabolism (sufficient D.O., C:N:P ratios, utilize EQ capacity, improve pre-treatment if possible).