- 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.
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.
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.
ANAMMOX & Partial Nitrification/denitrification - ways to remove wastewater nitrogen while lowering utility costs
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.
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:
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
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:
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:
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.
Erik Rumbaugh has been involved in biological waste treatment for over 20 years. He has worked with industrial and municipal wastewater facilities to ensure optimal performance of their treatment systems. He is a founder of Aster Bio (www.asterbio.com) specializing in biological waste treatment.
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