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).
Microbes consume dissolved oxygen (DO) when growing on wastewater pollutants. The rate at which the microbes consumer oxygen is the respiration rate. During periods of high loadings the respiration rates increases as the microbes become more active and cellular division rates increase.
OUR rates increase in response to higher soluble organic loadings (BOD5), faster microbial division (low F/M), and following upset conditions when microbes are moving back towards stable population - this is log phase growth.
Another facet of OUR testing includes a potential for a drop in respiration rates due to acute toxicity where previously respiring microbes are inactivated (killed). I have seen this with phenol, cyanide, and tall oil releases in industrial wastewater.
So any large change in OUR rates should be investigated. If you are changing MLSS concentrations, you should standardize the OUR by using the SOUR calculation (divide OUR by MLSS or MLVSS in grams).
Here is a link to the OUR test protocol that I have used for the past 25 years.
Being both deadly in enclosed spaces and a nuisance at even low levels, hydrogen sulfide is among the most problematic of compounds in wastewater treatment. Instead of covering the whole geochemical sulfur cycle, I want to look into the wastewater specific cycle that converts benign sulfates and sulfur into the problematic reduced sulfide species. Instead of graphics, I want to detail each form of sulfur found in wastewater.
Activated sludge systems operate by moving a suspended biomass (MLSS) through an aerated basin and then separating solids for recycle back to the aeration basin. This process was invented in the early 20th century for treating wastewater and many variations on the process exist including contact stabilization, extended aeration, batch reactors, oxidation ditches, and pure oxygen systems. A key operating parameter has been the Food to Microorganisms (F/M) ratio. F/M is traditionally calculated using MLSS or MLVSS as the M and BOD5 as the F. Key is to be consistent and always know what you are using to calculate F/M and identify the best F/M ratio for operations.
Now we move to biofilm type systems versus suspended growth. Here we have tricking filters (old but still good), MBBR, and fixed film media. All relay on a biofilm which is actually just floc grown in attached form. Calculated F/M in this system is difficult since the biomass is attached to media. So we operate any biofilm based system on loading per unit of surface area - this can be BOD5/square meter or Ammonia/square meter. Again just be consistent in calculating.
The key benefit of a biofilm vs suspended growth system is the ability to hold more biomass than comparable activated sludge units which rely totally on solids separation to keep biomass in the aeration tank. Of course this advantage is not present for all influents. For example, systems with significant influent oil & grease can foul (coat) the media which can be a big problem.
Nitrosomonas species also known as Ammonia Oxidizing Bacteria (AOB) have always been discussed as obligate aerobic chemoautotrophs. Under aerobic conditions, the Nitrosomonas convert ammonia to nitrite while using carbon dioxide and carbonate as a carbon source.
I have recently read multiple peer reviewed papers that document the ability of Nitrosomonas to utilize various organic compounds as an energy source with nitrite as an alternative electron acceptor - producing N2 gas as a final product. This ability to use their waste product from aerobic reactions as an electron acceptor reveals how Nitrosomonas can survive in systems with substantial anoxic periods.
Here is a link to a 2009 paper with more details:
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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