There are no general 1 size fits all solutions for troubleshooting biological wastewater treatment system
issues and while it is always preferred to address the root source of a problem, there are simply times in
which this may not be feasible, or logistically practical. A common instance of these scenarios includes
troubleshooting challenges with filamentous bacteria bulking.

damaged filaments cropped from 1000x phase contrast

Filamentous Bulking Defined

Textbook definition generally defines filamentous bulking as conditions in which the mixed liquor
possesses an SVI (sludge volume index) value of >150 mL/g. This definition often generally applies to
many systems, however the actual SVI values in which clarifier failure is reached (as solids entering the
clarifier at a higher rate than can be removed with corresponding suspended solids carry-over) has many
significant factors such as the size of the clarifier (most specifically the surface area) and the hydraulic
flow rate. For example, there may be treatment plants that are already near or beyond their design
hydraulic capacity and solids loss from the final clarifier (s) may be reached at significantly lesser SVI
values than 150 mL/g in these instances. Vice versa, in situations where the plant is well within range of
its hydraulic capacity and if the clarifier (s) are large enough, higher SVI values may be possible while still
maintaining a low sludge blanket and optimal effluent treatment parameters.

Risk Assessment

It is critical to be able to determine the differences within each system and determine on a scale of 1-10
how severe, or how much risk a potential scenario poses on clarifier performance. In systems such as
municipal systems with high I and I (inflow and infiltration) potential, these large increases of flowrate
must often be taken into consideration in context of how saturated the ground is, and how likely events
such as a heavy rain of a snow melt are to significantly impact the hydraulic flow rate.

RAS Chlorination

RAS chlorination is the process of specifically targeting undesirable filamentous bacteria with
disinfectant (generally sodium hypochlorite) to systematically kill filamentous bacteria to improve SVI
values, while reducing damage to bacteria within the floc as much as possible. In addition to risk
assessment, the choice of RAS chlorination should also be compared to other methods such as sludge
juggling (adding additional basins online etc.), chemical settling aids, and others. RAS chlorination is
generally most successful when flocs are strong and there are high amounts of filamentous extending
from or bridging the flocs together. Microscopic evaluation is important to determine the strength of
the floc structure, the location of the filaments, the overall health of the biomass, and ideally the rank
and abundance of the filamentous bacteria morphotypes that are present in order to assist with
changes in the wasting rate during the period of RAS chlorination.

General Dosing Guidelines

It is important to remember that each system is different, and the chlorine dose required for targeted
kill of filamentous bacteria varies depending upon the chlorine demand brought upon by factors such as
competing reactions. Generally, municipal systems are able to dose much closer to general guidelines,
while various industrial wastewater processes may require significantly higher amounts of chlorine.
Remember to start conservatively with chlorination as the dose can easily be increased, while if the
chlorine is applied to aggressively, this can cause many negative impacts to the health and settling
characteristics of the biomass. It is common for “light” RAS chlorination to involve addition of 2-3 lbs. of
active chlorine per 1000 lbs. MLVSS of the mixed liquor in the aeration basin. Moderate dosages
generally range between 5-6 lbs./1000lbs. MLVSS, and what would be considered generally higher
dosages are >8 lbs./1000lbs. MLVSS.

It is worth mentioning that in rare instances (such as lagoons that are converted to large extended
aeration activated sludge systems) that in these instances we prefer to chlorinate only the estimated lbs.
of MLVSS expected to pass through the clarifier within a 24-hour period. Note that RAS chlorination is
most effective when each “bug” comes into contact with chlorine 1-2x per day minimum. In instances in
which the 1-2x chlorine to “bug” exposure may not be possible options include multiple chlorination
addition points, a hypochlorite “bomb” (which due to high risk is only offered through consulting) or
understanding that a significantly longer period of time may be needed for any potential success (along
with the increased potential that chlorination may not be frequent enough to see beneficial results).
When viewing aerobic biological flocs under fluorescent microscopy, it is common for the majority of
the bacteria within the flocs to be dead or non-viable and the majority of the filaments (often 95% or
greater) to be healthy/viable. In most scenarios, a decrease in the wasting rate is warranted (i.e., 10% or
more) when RAS chlorination is applied due to the fact that filamentous bacteria are extremely efficient at oxidizing/treating carbonaceous organic material (BOD).

Application and Monitoring

It is essential that chlorine is added to the RAS in an area of high mixing. Most often these locations
include areas such as the RAS piping or the center well of the clarifier. The addition of chlorine to locations of poor mixing (such as RAS wet wells) does not allow for even distribution of the chlorine. When calculating the RAS chlorine dose, it is important to adjust the calculations to include the chlorine source (i.e., sodium hypochlorite) to 100% “purity”. The type (s) of filament that is being chlorinated (sheathed versus non-sheathed) have a significant impact in the time needed for improvement in the settleability as empty sheaths with dead filaments often still negatively impact SVI values. When chlorinating the RAS, daily microscopy with phase contrast oil immersion 1000x is desired to monitor the health of filaments. Once 50-60% of filaments show signs such as damaged cells or empty sheaths, the RAS chlorine is generally reduced slightly to prevent over-chlorination.

Additional Notes

Chlorination for filamentous bacteria control is only a “band-aid” option and if the conditions remain in
which filaments outcompete floc forming bacteria, it is common for filaments to grow back rapidly once
chlorination is ceased. Some systems use light RAS chlorine “maintenance doses” to help offset these
challenges, however, this is case specific and depends upon other options available, economic logistics,
availability of operational personnel etc.


Wastewater Microbiology book

There is an interesting and very important correlation with notable differences between the common terms “Low DO” (dissolved oxygen) and septicity. Due to this relatively complex relationship, there is often confusion regarding these terms. The goal of this blog is to briefly explain these differences and how each term should be thought of.

What is Septicity?

In my experience a good way to envision septicity is that “once wastewater has been exposed to septic conditions” it is then forever septic until biological oxidation (treatment). Without going deeply in depth, once available oxygen electron receptors are depleted anaerobic fermentation reactions occur in which carbonaceous “food” is “broken down” into smaller “pieces”, in which we call organic acids or volatile fatty acids. In other terms, once there is no available free dissolved oxygen, or forms of combined oxygen, food (BOD) is converted into simpler and more readily available forms. A good analogy is oatmeal versus Kool-Aid and the differences in how both of these are taken up in your body (Kool-Aid being the organic acids).

Septicity/ Organic Acid Impact on Microbe Competition/ Selection

While the overall loading rate (i.e., lbs./day of BOD) are a factor, the most significant factor appears to be the concentration of organic acids/volatile acids. Literature from Dr. Jenkins and Dr. Richard suggests that bulking from “organic acid filament types” is recognized to occur at volatile acid concentrations >100 mg/L. In addition to filaments, other microbe types that are undesirable at significant abundance (i.e., zoogloea bacteria types, single cell bacteria) appear to have a similar threshold. Based on my personal experience, the 100 mg/L volatile acids concentration appears to be a “ballpark” number due to conditions such as the individual various organic acids that encompass the total volatile acids test. Generally, if organic acid/volatile acid concentrations are diluted below the threshold in which the undesirable microbe gains a competitive advantage, significant changes in the microbes’ present may occur over a long-term period.  Potential control strategies for diluting organic acid concentrations include higher RAS rates, higher internal recycle flows (i.e., BNR plants), implementation of step feed (if possible), and in some instances recirculation of various % of effluent flow to dilute the strength of the incoming wastewater.

Low DO by Definition

Sphaerotilus Low DO Filament Type

The term “Low DO” is relative in that factors such as the oxygen uptake rate and the surplus available oxygen are generally most significant for selection for microbe types we recognize to grow at “Low DO”. A common way to envision this is that when we measure DO, we are measuring “residual DO” that is outside of the floc matrix. The DO setpoint needed to discourage “Low DO” microbe types is based upon the amount of dissolved oxygen needed to maintain aerobic conditions within the interior of the flocs. For example, in lightly loaded municipal systems it is common for DO concentrations as low as 0.5 mg/L to be adequate DO, while in highly loaded systems that are not designed to nitrify the oxygen uptake rate, bacterial growth rates at the front end of the aeration basin are so significant that DO concentrations of 6 mg/L or higher are often needed to discourage Low DO microbes.  For a Low DO microbe type, the applied DO is the most significant factor in controlling the growth of this organism.

Inter-Lap Between Low DO and Septicity

By definition, organic acid microbe types are selected by factors such as the concentration of various organic acids. Applying dissolved oxygen, once organic acids are already formed, is not an effective strategy to oxidize organic acids. Therefore, when troubleshooting a filament or other microbe type that is correlated with organic acids, increasing the DO setpoint is generally not successful if the organic acids are already present in the wastewater being treated prior to the aeration basin.  The only instance in which increasing the available DO is generally successful is if the front end of the aeration basin is septic and organic acids are being formed in the aeration basin itself.

In simple terms, the relationship between low DO and septicity is that once DO is low enough that forms of dissolved oxygen are depleted, fermentation reactions occur, in which organic acids/volatile acids are then produced. In these instances (such as eq basins, collection systems etc.) maintaining aerobic conditions may prevent the formation of fermentation reactions and corresponding formation of organic acids.


Wastewater Microbiology Paper book and Digital copy by Ryan Hennessy

It is highly common and often successful to operate activated sludge treatment systems based on maintaining a targeted MLSS (mixed liquor suspended solids) concentration, however as with just about everything in wastewater, there are notable exceptions to the rule. 

What makes up the MLSS?

MLSS Components chart displaying how we would see the existing MLSS components.

As can be seen from the chart above there are several main components that constitute the mixed liquor suspended solids (MLSS) 

It is important to recognize that MLSS concentration values consist of many variables that may change rapidly depending upon environmental conditions. In our experience the most useful way to envision this is that we are generally attempting to correlate the MLSS concentration to the number of alive/viable bacteria present. Based upon fluorescent microscopy it is common for approximately 40-80% of the mixed liquor to illuminate green (indicating “alive/viable”). 40-80% is a very broad range and depending upon the plant and circumstances that are present these percentages may vary. 

Figure 1: Fluorescent Viability 1000x

The Picture above Demonstrates Alive/Viable (Green) and Dead/Non-Viable (Red)

If we think about the bacterial growth curve ideally in most activated sludge systems, optimal treatment conditions are not optimal growth conditions for the bacteria (specifically that they are on the verge of starvation/endogeny). In our experience it is common for extended aeration systems to have as low as 35-40% bacterial viability, while in highly loaded industrial wastewater processes viability % of 75% or more may be common. Each system is different depending upon the treatment goals, sludge age, temperatures present, and many other conditions. 

Explanation of Other Variables

An influx of inert materials may significantly change the MLVSS (mixed liquor volatile suspended solids) to MLSS ratio. Dead or non-viable bacteria also may possess a biological oxygen demand and if they are now available substrate they may weigh as volatile solids. Higher life form organism weight proportion to the overall MLSS concentration is also recognized to vary between 5-20%. Among other notable variables is temperature and its metabolic impact on the bacteria. In general, at higher temperatures, less bacteria are needed for treatment while when temperatures deplete higher amounts of viable bacteria are commonly needed to achieve treatment goals. 

The Bugs Don’t Lie 

It is always useful to have process control data as part of the “puzzle” to help guide operational changes, however despite what the numbers say on paper, the bacteria present will be representative of actual conditions within the plant should microscopy be performed properly by a trained professional. In summary, when the plant is not behaving as suggested by the MLSS concentration values, it is often due to a change of one of the variables above. Microscopic evaluation, along with in house process control testing, and the input of an operator familiar with the treatment plant and how it generally behaves are most often the best things to get together to help make decisions that are practical and optimize or troubleshoot the system. 


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Higher life form organisms such as stalked ciliates and rotifers may be easily viewed under brightfield microscopes. They are fascinating, motile, and easy to recognize however process control changes should not be based strictly upon higher life form organisms and their abundance. The best analogy to use is that “you are what you eat” and if a higher life form organism type is predominant the root cause is that the environmental conditions and the substrate availability favor the selection of this organism. 


While some general correlations (such as predominance of flagellates often correlating with left over available substrate) often hold true, there are many common situations that may lead operators in the wrong direction. A common example is that rotifers often prey on small floc “fragments”. At higher SRT values when pin floc is present, rotifers are often common, however, if there is a stress that creates de-flocculation it is common for rotifers to proliferate. Nematodes (often associated with high sludge age values) may grow within treatment plants, but also commonly enter treatment processes through the influent, and have been viewed in systems with sludge ages as low as 2 days. There are many other examples that may be viewed at:

How Fast do Higher Life Form Organisms Grow? 

The mix of higher life-form organisms can change quickly depending on their available prey. The longest it takes protozoa to reproduce is 1 day at 20 degrees C (Curds, 1975) so sludge retention time rarely limits their competition. Certain species of rotifers can grow in sludge ages of less than even 4 days (Richard, 2018). In short, there is no proven correlation between higher life form organisms and sludge age, and because there are many other factors besides organic loading that can contribute to changes in sludge condition, basing process control decisions solely on higher life form organisms is not recommended. (Jenkins, 2004)

Impact of Environmental Stresses

From a stress perspective, free swimming and stalked ciliate species are typically the most sensitive to stresses and toxicity while testate amoebae and rotifers can compete well in tougher environments. Note that not all potential stresses are equal and depending on what type of stress is present these can have varying, and sometimes unusual impacts on the microbes present. In most instances, the higher life form organisms are the first to be impacted when there is stress or toxicity. 


In summary, there is value in looking at the higher life form organisms but is also important to put their emphasis in perspective as they only represent a “piece of the puzzle”. A shift in predominant higher life form organisms should generally suggest a “deeper look” at what is going on under the microscope with an evaluation that documents factors such as floc structure, dispersed growth, zoogloea bacteria type abundance, filament bacteria type rank, abundance, and suspected growth causes, health of filamentous bacteria, impact of filaments on the floc structure, and bacterial viability within the mixed liquor. 


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One of the most challenging problems commonly encountered in biological wastewater treatment processes is sludge bulking due to overabundance of Microthrix filament types.

What is Microthrix?

Microthrix filament types are recognized due to their Gram-positive staining characteristics and are generally 0.8 µm diameter with no visible septa (cell walls). Microthrix are recognized to grow on lipids and long chain fatty acids.  The MIDAS field guide ( recognizes 6 individual species of Microthrix including Microthrix parvicella, Microthrix calidas, and Microthrix subdominas. There are also several closely genetically genera that are strong candidates for Microthrix morphology (characteristics) and similar growth conditions. 

Microthrix Preferred Growth Conditions

Common Troubleshooting Strategies


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