Periodically systems with facultative or anaerobic holding ponds experience blooms of bacteria that transform the water to a pink of purple color. This color change comes from the growth of purple sulfur bacteria and/or purple non-sulfur bacteria. Both grow in the anoxic/anaerobic zone and are a product of a lagoon without high levels of mixing. 

Purple Sulfur Bacteria
The PSB is an interesting phototrophic organism that uses sunlight to power the conversion of sulfides (including hydrogen sulfide) into sulfur. The carbon source for their growth is usually carbon dioxide, but they can also assimilate carbon from organic acids or alcohols. They are obligate phototrophs, so they tend to be more common in summer months. As they remove H2S and some volatile organic acids, the PSB can be useful in lowering odor problems with lagoons.

Purple Non-Sulfur Bacteria
These are found in lagoons with lower sulfide (H2S) levels and can use organic compounds as an electron donor. They are still found in anoxic environments and use photosynthetic reactions to obtain energy.

On paper denitrification should be easy. All you need is a "food" source, common facultative heterotrophic bacteria, and anoxic conditions (no free D.O.). However, in practice you need to have a balance of conditions to ensure denitrification. If you are having problems maintaining denitrification, it helps to look at the process from an ecological perspective. 

What is required for bacteria to use nitrate/nitrite as electron acceptor (AKA alternative oxygen source)

• Many bacteria can use nitrate but not all can use nitrite. Your goal is to develop cultures that an convert all NOx into N2 gas. This process actually h as multiple steps that include 4 enzyme pathways.
  
  
• Denitrification happens with many substrates (electron donors) - this includes soluble organics (sugars, alcohols, starches) but can also include chemoautotrophic organisms such as sulfur and metal oxidizing bacteria. This last group is being researched as Autotrophic Denitrification (AuDen). However, most systems denitrify best with typical wastewater heterotrophic bacteria including Thauera, Zooglea, & Pseudomonas.
  
  
• Anoxic conditions are necessary to trigger denitrification. When D.O. is present, the bacteria obtain more energy using oxygen as an electron acceptor. Inside dense floc, biofilms, or aerobic granular sludge can have anoxic zones that denitrify inside aerobic basin. This is part of the appeal of MBBR and aerobic granular sludge systems. You do not have to manage the anoxic zone redox potentials (ORP) or residence time as closely as you would in conventional aerobic activated sludge.
  
  
• Always consider residence time, temperature, and availability of suitable electron donor (food) for the bacteria in the denitrification zone. Failure in any variable can be a problem.  Denitrification to N2 gas often takes action by several different organisms (although a few can take it to N2 gas inside a single cell).

Ammonia Oxidizing Bacteria (AOB) and Nitrite Oxidizing Bacteria (NOB) are often grouped into what we call nitrifiers.  These organisms grow by converting ammonia into nitrite and finally nitrate.  Organisms working in the nitrogen cycle are diverse and include the following:

• Heterotrophs that degrade organic nitrogen compounds (proteins, amines, etc) into ammonia. They also use nitrogen to build proteins used in cellular activities and reproduciton. Under anoxic conditions, many heterotrophs use nitrate and nitrite as alternative electron acceptors converting the NO3/NO2 into N2 gas.
• Chemoautotrophs - this includes AOB/NOB, COMAMMOX (funtions as both AOB & NOB in one organism), ANAMMOX (under anoxic conditions takes NO2 + NH4+ directly to N2 gas)
• Heterotrophic ammonia oxidation (slower than obligate AOB/NOB but does take place).

So given such diversity, toxicity screens become a complex undertaking.  So, here is what we are doing (while the COVID19 virus has travel and field work at minimum levels).

• Sampling diverse WW systems to see makeup of their nitrifying populations using Mirobial Community Analysis (MCA)
• Using qPCR tests created from MCA data for individual systems to develop baseline data for operating wastewater treatment plants - this involves desigining and refining oligos used in qPCR tests.
• Testing suspected compounds using concentrated AOB cultures for acute toxicity using Tox-N testing. In this we spike buffered water with suspect compounds and add AOB. We compare ammonia oxidation rates versus a control to get % inhibition. 
• More advanced simulation in pilot activated sludge units where we use a healthy MLSS and subject it to influents spiked with compounds of interest. We then monitor ammonia, nitrite, nitrate, and use molecular testing to see drifts in AOB/NOB populations.

Probably the best quick test for evaluataing your biological treatment system's health, microscopic exam does not have to be a long, complex test. Just note the following:

• floc appearance - size, density, filaments
• Protozoa present & relative numbers
• Any higher life forms 

The above exam takes 5 - 10 minutes while you are running SV30 tests.

Now there are times where you need more complex evaluation such as filament ID, EPS evaluation, and troubleshooting. Here is where I suggest sending samples out for evaluation while you also learn about the test and how the observations are interpreted. I have worked with Steve Leach of Leach Microbial Consulting for over 20 years and he combines microscopic exam experience with field operation of both municipal and industrial wastewater systems. He has finally set up a website with resources for performing micorscopic exam - this includes good documents and photos for doing everything from a cursory exam to filament ID.

His microscopic exam reports focus on your system's issues and helps you improve your operations. I find this much more useful than the standard outside microexam report.

Go check out his website and if you have a need for outside microscopic exam contact Steve.

www.leachmicrobial.com/ 

I will start with something that puzzeled me for years. In the lab, ammonia oxidizing bacteria (AOB or nitrifiers) have excellent activity at a D.O. of 2.0. Moving up from 2.0 mg/L DO to say 4.0 mg/L gives a slight boost in growth rates, but is not worth the energy cost required to increase the oxygen residual. Meanwhile, I have seen working activated sludge systems where a D.O. residual below 3.0 mg/L results in a loss of nitrification. So what  is going on?

The answer came after we started running molecular tests that look directly at the microbial makeup of MLSS. Running these tests, we found obligate anaerobic bacteria growing inside high D.O. basins. We then did extensive work on AOB/NOB inhbition with various influents and MLSS from different systems. Here is what we found:

• D.O. meter placement
  D.O. meters read oxygen residual in the water phase at the probe/water interface. D.O. residuals are subject to probe placement and mixing. Is your D.O. meter giving you a true average residual or D.O. you do manual checks at other locations to confirm readings?
  
  
• Impact of MLSS concentrations
  The aeration basin is not a homogenous mixture. We have water phase and solids. For oxygen to get inside the floc, it must pass through EPS and move from the water column into the biofilm. Larger floc or higher biosolids mean lower oxygen residuals inside the floc compred to the water on the exterior.
  
  
• Floc EPS levels
  Floc includes insolube organics, biopolymers, and inorganics. Increasing amounts of non-living materials result in lower oxygen transfer efficiency. Remember that high EPS levels can reduce how much oxygen moves into the floc. Check your EPS using India Ink for an easy on-site EPS test.
  ​

How do you get an idea on working D.O. (from a bacteria point of view)?

• Check D.O. residual manually to confirm in basin meter numbers
• Monitor D.O. meter numbers and see where you get needed activity from obligate aerobes (AOB/NOB). You can also look at denitrification or phosphate release/uptake that require anoxic and anaerobic zones.
• Run Microbial Community Analysis (MCA) which is a molecular test that identifies all bacteria present. The MCA tells you what is growing and functioning in the system.

 How do you estimate bacterial activity in a wastewater system? This is a question, we often ask and a number of methods have been used. I figured a list of common monitoring methods could be useful.

• Respiration Rate (Oxygen Uptake Rate)
  This can be complex using respirometer equipment or simple using a BOD bottle/DO probe on the benchtop. Here you use oxygen consumption as a measurement of microbial activity.
• Plate Counts
  Using various plate count media, labs can evaluate numbers of microbes in wastewater plants. Most often used for coliforms, it can also be used for heterotrophic and specific organisms using selective media. Problems with plate counts include the number of organisms that don't grow on plates, plate development time, and expense/labor required to do pate counts.
• ATP 
  Microbes store energy as ATP. Using a luminometer and various luciferase procedures, you can determine free ATP and cellular (bound) ATP. The more ATP present the more light produced in the reaction. A quick test, ATP can be used as a proxy for total plate count and a measure of non-specific microbial activity.
• Flow Cytometry
  Requring specialized equipment, flow cytometry uses fluorescent dyes and various techniques to quantify individual cells in the sample. Flow cytometry allows for evaluation of cell viability. 
• qPCR (molecular testing)
  qPCR or real-time PCR is a rapid test method for quantifying DNA present in samples. As dead cellular DNA is rapidly degraded in wastewater, PCR favors viable cells. While total bacteria qPCR tests are similar to rich media plates counts in looking at all bacteria present. qPCR can be used to evaluate key organisms. For example, qPCR can look at specific DNA sequences such as those for nitrifiers, filaments, nocardia, or any other group of interest. qPCR is a relatively quick test (hours versus days) and is highly accurate.