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.

With tighter effluent ammonia and total nitrogen limits on the horizon, many treatment plants are evaluating ways to improve biological nitrogen removal while keeping a cap on capital or operational costs.  Among the options is Anaerobic Ammonia Oxidation (ANAMMOX) and using partial nitrification followed by denitrification. 

ANAMMOX inovolves a unique biochemistry that existing in several slow growing genera that when in anaerobic conditions can utilize nitrite and ammonium as an energy source. 

• Step 1 (AOB)                            2NH4+ + 3O2 → 2NO2− + 4H+ + 2H2O
• Step 2 (ANAMMOX)                NH4+ + NO2− → N2 + 2 H2O
  
  

Partial Nitrification with denitrification relies on a more conventional process to yield nitrogen gas. Of course, there are intermediate steps not detailed below - but the final reaction products are what is important.

• Step 1 (AOB)                            2NH4+ + 3O2 → 2NO2− + 4H+ + 2H2O
• Step 2 (denitrification)           NO2− + CHO (organic) → N2 + CO2 + H2O

For ANAMMOX to work a system must have sufficient residence time and growth environment to favor building an ANAMMOX population. Systems capable of ANAMMOX include:

• Side stream ANAMMOX treatment reactor separate from the main biological treatment unit.
• Use of MBBR media in the existing system 
• Use of Aerobic Granular Sludge (AGS) technology
• MBR system with higher MLSS, longer MCRT

​The complexity of ANAMMOX including potential consruction costs, has led to interest in partial nitrification with immediate denitrification. Here you need to encourage AOB activity followed by suitable soluble BOD under anoxic conditions for cultures to convert NO2 into N2 gas. This can be done by closely monitoring redox potentials and using step-feed or other high soluble BOD source just prior to the anoxic zone. 

When a wastewater plant is making plans on receiving a new influent stream or removing an existing stream, it is good test for potential impact upon biological treatment units. If the change is minor, I have used rapid acute toxicity tests including a rapid heterotrophic organism respiration inhbition and Ammonia Oxidizing Bacteria (Nitrifer) screen - both of these tests take less than 4 hours to complete. But if the changes are greater or new compounds are of high concern, we can use a bench scale activated sluge unit to test specific waste streams, dilutions, treatment protocols, and best evaluate best operating practices. All the testing reduces likelihood of major problems in the actual wastewater treatment plant.

Key points for a bench scale reactor test

• Keep it simple - lab reactors are not your actual system but a close approximation. Don't ask a bench test to determine to many variables at one time
• Be ready to test various pre-treatment or dilutions
• Adding 16s MCA - total microbial census - helps to understand the changes inside the biomass that would take longer to manifest as visible problems and is more sensitive than turbidity and COD tests that are normally run on the effluent
• There is a learning curve to operating a lab scale activated sludge unit. It is good to do a few practice runs before doing even a simple evaluation.

The lab scale activated sludge can be as complex as you want. The most common type is often called an Eckenfelder Reactor which dates back to the 1960s. It uses gravity separation in a clarifier section with recycle via a port at the bottom of the clarifier. More complex systems include a separate clarifier with pump recycle to control RAS and MCRT with greater accuracy. In our lab, we select the reactor type to match closely the system being studied. We also adjust the test to meet the study objectives and information required.

Below is an example study done at Aster Bio for determining the impact of a various waste streams on AOB (Nitrosomonas) and NOB (Nitrospira) populations. The testing found that one of the streams was responsible for problems with nitrification and required adjustments to pre-treatment. So why run the more complex, multiple day test? Earlier screening with acute toxicity nitrifier concentrate (Tox-N) did not detect the suspected chronic toxicity - which is best detected using qPCR molecular testing.

When you have MLSS above 5,000 mg/L, SV30 tests often have solids with a very narrow band of supernatant. This can make it difficult to diagnose problems with pin floc, settling velocity, and compaction. If you see the water phase of less than 20% of the settleometer, the diluted SV30 test may prove useful. Here is how to run a diluted SV30:

• Fill your settleometer with 25 - 50% MLSS taken from the aeration basin. Make sure you mix the sample first to ensure you have solids all suspended.
• Use effluent or tap water to fill the settleometer. Again mix the MLSS/Water blend to suspend the solids.
• Start 30 minute timer. Note sludge level at time 5, 10, 15, 20, 25, & 30. Adjust dilution to have SV30 settled solids reading between 150 - 300 ml on the settleometer. After settling, note floating solids and pin-floc in the supernatant. You should also let the solids continue to compact for 2 - 4 hours. Evaluating the compacting solids gives information about how concentrated the RAS solids will become in the secondary clarifier.
• For reporting purposes and SVI calculations, remember to multiply by your dilution factor.

Most of the work on Aerobic Granular Sludge (AGS) has been either with synthetic wastewater (lab studies) or in domestic wastewater installations. In these cases, the AGS forms into irregular but tight granules that settle rapidly. AGS has great potential because:

• Allows for higher biomass concentrations than activated sludge - smaller footprint
• Removal of BOD5, Ammonia, Phosphate, and Nitrate/Nitrite all in one single basin
• Less energy use than conventional activated sludge

This brings me to think about the application of AGS to indsutrial wastewaters where you have lower concentrations of soluble organic compounds and more realcitrant/resistant chemicals that are traditionally difficult to treat. Fortuantely, I found a study that discusses AGS appearance and formation on simulated industrial influents. 

The authors created an excellent graphical abstract for evaluation of AGS appearance with different influents. 

Pronk, M., Abbas, B., Al-zuhairy, S.H.K. et al. Effect and behaviour of different substrates in relation to the formation of aerobic granular sludge. Appl Microbiol Biotechnol 99, 5257–5268 (2015). https://doi.org/10.1007/s00253-014-6358-3

Bacteria form biofilms (in wastewater we call this floc) for many reasons. Among the most important is to protect the individual cells from the surrounding environment and capture/concentrate nutrients necessary for growth. Montanta State University actually has a group focusing on biofilms - http://www.biofilm.montana.edu/

The researchers at Montana State have created a great infographic on why biofilms are important that I want to share. It is a very good read.