How many living bacteria are in my MLSS? I get this question often and it is often difficult when people try to use F/M ratios during a system startup. (Often they wonder why a very high F/M (Low MLSS) - that the living biomass can remove 90% COD/BOD from influent). Understanding the technology and limits behind each test will give you an idea of the pros and cons of each test.
Operating a system using a single number is not feasible. Each test works great over a certain range of conditions. Outside that range, the test results do not provide an accurate picture of biomass activity or health. So, combine multiple tests and use good judgement in operating your system.
Handing and disposing of secondary biological solids can be difficult, expensive, and at times limited by equipment. Many of us are taught that by running a longer sludge age, the cell yield will decrease and make fewer solids requiring wasting. Therefore, I have seen a tendency to move toward long sludge ages which goes with very low F/M and high levels of MLSS. You may ask - "What can go wrong with very long sludge ages?" Let me list my top observations:
Often taught during coursework on ammonia oxidation, the biological conversion of nitrite to nitrate is a separate biological process. When I learned nitrite conversion to nitrate in wastewater, we were told that Nitrobacter sp. was the primary organism for nitrite removal. Since we have been using molecular testing (Environmental Genomics), I have seen many interesting datasets revealing that Nitrobacter sp are not the primary nitrite oxidizers in most wastewater systems.
To gain a better understanding, we began looking into the microbes growing in various bench and production scale nitrifier reactors (used to make AOB/NOB bioaugmentation products). Using substrates such as ammonium chloride and sodium nitrite, we found interesting microbial communities that were quite different from some of the wastewater treatment systems.
Major AOB in activated sludge systems include expected Nitrosomonas and Nitrospira (interesting organisms that can do both ammonia and nitrite oxidation). However obligate nitrite oxidizing organisms were much more rare than expected. In the pure sodium nitrite substrate reactor and mixed culture ammonium substrate reactors, we found large populations of Pseudomonas and Paracoccus which are also capable of nitrite oxidation. We are continuing research on these organisms in wastewater plants with emphasis on discovering new ways to enhance nitrite oxidation in activated sludge systems.
Everyone in wastewater treatment has seen the growth curve graphic during training classes. I find using the growth curve a great way to discuss wastewater system changes and dynamics. And, with our newer genetic/molecular wastewater testing, I am seeing how the system's microbial species change as we move along the growth curve.
For those in need of refresh, the Growth Curve describes microbial populations by dividing growth into (1) Lag, (2) Log, (3) Stationary, and (4) Decline (endogenous) phases. Species of microbes differ based on influent composition and environmental factors, but we also have a changes in species based on waste concentrations relative to microbial populations - this is also known as F/M ratio.
The growth curve is easiest to see in aerated lagoon systems. Near the influent, we find lag and log phase growth as abundant food favors fast growing k-rate strategist microbes. As you increase Dissolved Oxygen (DO) and BOD/COD (food) declines, you see a change to r-rate strategists such as ammonia oxidizing bacteria (AOB), Thauera/Zooglea (biopolymer producing low F/M microbes), and other niche organisms. What I find interesting is how much change we see in our Microbial Community Analysis (a total census of all microbes in the system) with slight variations in F/M or influent makeup at inflection points along the growth curve.
We call MLSS healthy when we meet the following:
But at a bacterial level, what is a healthy MLSS? Using Aster Bio's Environmental Genomics testing, we now have an understanding of what a healthy MLSS looks like with respect to microbial species. In this case we are looking at extended aeration activated sludge which is the most common system that we encounter.
Early in the system startup or following a major upset, we see a reversion to k-rate organisms such as Pseudomonas, Bacillus, and a number of less known fast growing organisms that thrive with high COD/BOD with associated low D.O. and perhaps quasi toxic components. As fast growers, these organisms have a high OUR (respiration rate) and grow rapidly to reduce COD/BOD. As the the MLSS OUR drops, the cells aggregate lowering free bacteria in solution. If an upset, they colonize on the existing floc mass which includes extracellular polymers, dead biomass, particulates, and a lower fraction than normal living biomass.
As the system matures, the mix of bacteria begins to favor r-rate strategist microbes that thrive in low F/M conditions seen in activated sludge or when water quality reaches effluent targets. The r-rate strategists tend to form EPS to store food and vital nutrients. This builds and maintains floc. Common r-rate wastewater strategists include Thauera, Zooglea, and AOB/NOB bacteria (in systems with nitrogen removal)
All bacteria in wastewater treatment have DNA. With molecular testing, such as Aster Bio's Environmental Genomics, we use specific DNA to ID populations of specific microorganisms (qPCR) or do a total microbial census (Microbial Community Analysis MCA). Identification gives us information on the genotype or the specific DNA sequences present.
Now comes the twist that can lead to some confusion. When we look under a microscope, we look at microbial appearance. The growth pattern or appearance of the microbe is called phenotype. For example, all human DNA sequences as species Homo sapiens. However we appear quite different which is due to phenotype.
Bacteria like humans can have the same genetic makeup but appear very different. Some examples here include an organisms going from floc forming to filamentous when D.O. drops. Others such as Thauera and Zooglea are important floc forming microbes in activated sludge but under certain conditions their growth can cause non-filamentous bulking. Other bacteria such as Nocardia are not always the foaming mess that stresses many operators.
Molecular testing needs to be coupled with other monitoring techniques such as SV30, F/M, MCRT, Respiration Rates, and microscopic exam to fully system microbial dynamics and make predictions on system performance.
Aquatic toxicity tests, often called bioassay or biomonitoring, were developed to estimate the effluent impact on receiving streams. Using living higher life-forms such as fat head minnow and daphnia (water flea), the test looks for both acute and chronic toxicity when subjected to various concentrations of wastewater effluent.
Often the chronic toxicity which is reproduction and weight gain are the harder part of the test to pass. But what causes failure?
Obvious causes of failure
Non-obvious causes of failure:
How to handle test failure:
I have received several questions on MLSS & MLVSS testing and how to calculate volatiles vs non-volatile fractions. Instead of regurgitating formulas, I will walk through the test calculations the way I remember the math.
Many of us in industrial wastewater have experienced a toxic shock event. In most cases, toxic shock is noted by loss of nitrification and deflocculation (turbidity/floating solids). Today, I will work thorough my process for identifying what caused or is causing the upset.
I like it when a facility looks under the microscope daily. Even if you do not have a high dollar phase contrast microscope, you can make valuable observations with they standard light microscope that is common to many high school classrooms. But what are you looking at when you observe samples at 100x, 400x, and even 1000x magnification? Today, I'll cover what you see when using a microscope.
First, the "bugs" we note using the microscope are mostly single celled protozoa and a few multicellular lifeforms. The bacteria - or the actual workers - can be seen as part of the floc or as very, very small particles in the water. Some bacteria can become filamentous in form or are large enough to be seen with microscope, but most even at 1000x appear as small rods or spheres.
We rely on the protozoa and higher life forms as indicator organisms for the underlying bacteria populations. For example, stalk ciliates are only present and active when there is sufficient dissolved oxygen and low levels of inhibitory or toxic compounds. This just happens to be where we are in decline phase growth or target F/M for most wastewater treatment plants. Multicellular forms such as rotifers, worms, or tardigrades appear even further along the F/M curve and can indicate old sludge or too low an F/M where you are carrying too much dead or inactive biomass.
I recommend looking under the microscope daily for the following:
While I use the microscopic exam daily, I also like to run newer molecular testing that looks directly into the floc's microbial community. With high throughput sequencing, we look at DNA in the system and find our which microbes are present and at what % of total biomass reads (identifying DNA segments). This is a total microbial census of the MLSS and is good to run when establishing a baseline population database, making operational changes or on a quarterly basis for tracking long term changes. Following Microbial Community Analysis (MCA), we identify key microbes that are most important for good treatment. We can then use qPCR technology to track these specific microbes. qPCR is faster, highly quantitative, and cheaper than the full microbial census (MCA).
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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