Lesson 8:

Filamentous Bacteria

 

Objective

In this lesson we will learn the following:

  • What it means for filamentous bacteria to be in the wastewater treatment system.
  • What is bulking and foaming and how to take care of this problem.

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Reading Assignment

In addition to the online lecture, read chapter 7 in Wastewater Microbiology.




Lecture

Introduction

Filamentous bacteria serve as the backbone of floc formation. Sludge settles most efficiently when it contains a moderate number of filaments which provide structure for the floc and aid in the stripping of the water column. The floc cannot form properly if there are too few filaments, and the floc cannot settle properly if there are too many. The filamentous bacteria are analyzed in two ways: their effect on floc structure and their abundance. In small amounts, they are quite good to a biomass, but in large amounts they can cause many problems.

There are a number of types of filamentous bacteria which proliferate in the activated sludge process. Filamentous organisms perform several different roles in the process, some of which are beneficial and some are detrimental. When filamentous organisms are in low concentrations they serve to strengthen the floc particles. This effect reduces the amount of shearing in the mechanical action of the aeration tank and allows the floc particles to increase in size. Larger floc particles are more readily settled in a clarifier. Larger flow particles settling in the clarifier also tend to accumulate smaller particulates (surface adsorption) as they settle, producing an even higher quality effluent. Conversely, if the filamentous organisms reach too high a concentration, they can extend dramatically from the floc particles and tie one floc particle to another (interfloc bridging) or even form a filamentous mat of extra large size. Due to the increased surface area without a corresponding increase in mass, the activated sludge will not settle well. This results in less solids separation and may cause a washout of solid material from the system. In addition, air bubbles can become trapped in the mat and cause it to float, resulting in a floating scum mat. Due to the high surface area of the filamentous bacteria, once they reach an excess concentration, they can absorb a higher percentage of the organic material and inhibit the growth of more desirable organisms.

Filamentous bacteria have many positive aspects, such as:

  • They are very good BOD removers.
  • They add a backbone or rigid support network to the floc structure.
  • Helps the floc structure to filter ut fine particulate matter that will improve clarifier efficiency.
  • They help the floc to settle if in small amounts.
  • They reduce the amount of "pin" floc.

 

They also have severyal negative aspects, such as:

  • They can interfere with separation and compaction of activated sludge and cause bulking when predominant.
  • They can affect the sludge volume index (SVI) (Sludge Volume Index).
  • They can cause poor settling if dominant.
  • They can fill up a clarifier and make it hard to settle, causing TSS (Total Suspended Solids) carryover.
  • They can increase polymer consumption.
  • They can increase solids production and cause solids handling costs to increase significantly.

 

 

Filamentous Bulking

Since the introduction of continuous-flow reactors, sludge bulking has been one of the major problems affecting biological waste treatment. There are several problems regarding solid separation in activated sludge. Often in industrial and municipal activated sludge processes a nutrient deficiency may occur. The nutrients that are usually deficient in these processes are either nitrogen or phosphorus. This deficiency results in the production of nutrient deficient floc particles, loss of settleability, and possibly a billowy white or greasy gray foam on the surface of the aeration tank.

During a nutrient deficiency, the bacteria within th floc particles remove soluble BOD from the wastewater. However, when nitrogen or phosphorous is deficient, the soluble BOD is not degraded but it is stored within the floc particles as an exocellular polymer-like material. This slimy material interferes with settling and may cause foam upon aeration.

Bulking is a problem consisting of slow settling and poor compaction of solids in the clarifier of the activated sludge system. Filamentous bulking is usually caused by the excessive growth of filamentous microorganisms. Bulking is caused by the overgrowth of filamentous bacteria in activated sludge. These bacteria are normal components of activated sludge flocs but may outcompete the floc-forming bacteria under specific conditions.

 

Name of Problem Cause of Problem Effect of Problem
Dispersed growth Microorganisms do not form flocs but are dispersed, forming only small clumps or single cells Turbid effluent. No zone settling of sludge
Sime (jelly) Viscous bulking Microorganisms are present in large amounts of extracellulr slime Reduced settling and compaction rates. Virtually no solids separation, in severe cases in overflow of sludge blanket from secondary clarifier.
Pin floc (or pinpoint floc) Small, compact, weak, roughly spherical flocs are formed, the larger of which settle rapidly. Smaller aggregates settle slowly. Low sludge volume index (SVI) and a cloudy, turbid effluent.
Bulking Filamentous organisms extend from flocs into the bulk solution and interfere with compaction and settling of activated sludge. High SVI; very clear supernatant.
Rising sludge Denitrification in secondary clarifer releases poorly soluble N2 gas, which attaches to activated sludge flocs and floats them to the secondary clarifier surface. A scum of activated sludge forms on the surface of the secondary clarifier.
Foaming/scum formation Caused by (1) nondegradable surfactants and (2) the presence of Nocardia and sometimes (3) the presence of Microthrix parvicella Foams float large amounts activated sludge solids to the surface of treatment units. Foam accumulates and putrefies. Solids can overflow into secondary effluent or overflow onto walkways.

 

Sludge settleability is determined by measuring the sludge volume index (SVI), which is given by:

 

where SV = volume of settled sludge after 30 min (mL/L); and MLSS = mixed liquor suspended solids (mg/L).

The sludge volume index is expressed in mL per gram and is thus the volume occupied by one gram of sludge. A high SVI (>150 mL/g) indicates bulking conditions, whereas an SVI below 70 mL/g indicates the predominance of pin (small) floc. Based on the relationship between floc-forming and filamentous bacteria, three types of flocs are observed in activated sludge: normal flocs, pin-point flocs, and filamentous bulking.

Normal flocs: A balance between floc-forming and filamentous bacteria results in strong flocs that keep their integrity in the aeration basin and settle well in the sedimentation tank.

Pin-point flocs: In these flocs, filamentous bacteria are absent or occur in low numbers. This results in small flocs that do not settle well. The secondary effluent is turbid despite the low SVI.

Filamentous bulking: Filamentous bulking is caused by the predominance of filamentous organisms. The filaments interfere with sludge settling and compaction.

Filamentous bacteria have a higher surface-to-volume ratio than that of their floc-forming counterparts, which helps them survive under low oxygen concentration and low nutrient conditions. Filamentous bacteria are able to predominate under low dissolved oxygen, low F/M, low nutrient conditions or high sulfide levels. However, it appears that low F/M is the predominant cause of bulking in wastewater treatment plants. These differences between filamentous and floc-forming bacteria can be exploited to control filamentous bulking in activated sludge.

 

 

Types of Filamentous Microorganisms

Some 20 to 30 types of filamentous microorganisms are known to be involved in activated sludge bulking. A survey of bulking activated sludge plants in the U.S. has revealed that approximately 15 major types of filamentous microorganisms are responsible for bulking, one of the most predominant being Nocardia, which is responsible for foaming.

The application of conventional techniques for the identification of filamentous microorganisms is both difficult and time-consuming. Other problems are their slow growth and difficulties in obtaining pure cultures from activated sludge samples. Thus, filamentous microorganisms were first characterized by microscopic examination, mostly with a phase contrast microscope. For such identification, the following characteristics should be obtained. Filament shape, size and shape of the cells, branching, motility, presence of a sheath, presence of epiphytic bacteria on sufaces, size and diameter and presence of granules. Other tests can help detect the presence of these microorganisms, such as gram stain and Neisser stain techniques.

The dichotomous key is used to identify filamentous microorganisms.

 

 

 

Factors Causing Filamentous Bulking

Filamentous microorganisms are normal components of the activated sludge flocs. Their overgrowth may be due to one or a combination of the various factors. High carbohydrate wastes appear to be conducive to sludge bulking. Carbohydrates composed of glucose, maltose, and lactose support the growth of filamentous bacteria. Some filaments appear to be favored by redily biodegradable organic substrates, such as alcohols, volatile fatty acids and amino acids, while others are able to use slowly biodegradable substrates.

Low substrate concentration (low F/M ratio) appears to be the most prevalent cause of filamentous bulking. At low substrate concentration filamentous microorganisms have a higher substrate removal rate than that of floc-formers, which prevail at high substrate concentrations.

 

 

Sludge Loading and Sludge Age

The relationship between sludge loading and sludge age depends on whether the reactor is a completely mixed or plug flow system. In completely mixed systems, increasing sludge loading leads to a decrease of SVI and thus to a decrease of filamentous microorganisms. Some filamentous microorganisms occur over a wide range of sludge age (MCRT: mean cell retention time) values while others occur only at low or high values. The optimum pH in the aeration tank is 7.0-7.5. pH values below 6.0 may favor the growth of fungi and cause filamentous bulking. In laboratory activated sludge units, bulking caused by the excessive growth of fungi occurred after 30 days at pH 4.0 and 5.0.

The growth of certain filamentous bacteria is favored by relatively low dissolved oxygen levels in the aeration tank. A substrate overload in the tank may induce oxygen deficiency. Aeration tanks should be operated with a minimum of 2 mg O2/L to avoid a predominance of specific filamentous microorganisms. Deficiences in nitrogen, phosphorus, iron or trace elements may cause bulking. Some filamentous microorganisms display a high affinity for nutrients. Increased temperature supports the growth of filamentous bacteria associated with low dissolved oxygen concentrations. Moreover, there is a tendency of Microthrix parvicella to e the dominant filamentous microorganism during the winter season.

According to a sludge bulking hypothesis, activated sludge consists of three categories of "model" microorganisms: (1) fast-growing zoogleal type microorganisms; (2) slow-growing filamentous organisms with high substrate affinity; and (3) fast-growing filamentous organisms with a high affinity for dissolved oxygen. Intermittent feeding pattern creates favorable conditions for the development of nonfilamentous microorganisms that have high substrate uptake rates during periods of high substrate concentration and a capacity to store reserve materials during periods of starvation (endogenous metabolism).

Another hypothesis on filamentous bulking is based on the ability of filamentous bacteria to denitrify nitrate to only nitrite with no accumulation of toxic nitric oxide by the cells. This gives a competitive advantage over floc-forming bacteria.

 

 

Control of Sludge Bulking

Filamentous bacteria can be controlled by treating the return sludge with chlorine or hydrogen peroxide to selectively kill filamentous microorganisms. This approach is based on the fact that filamentous microorganisms protruding from the flocs are more exposed to oxidants, whereas most of the floc-forming microorganisms embedded inside the flocs are protected from the lethal action of the oxidants. Bulking control by chlorination was proposed over 50 years ago and this practice is probably the most widely used cost-effective and short-term method for controlling filamentous bacteria. Chlorine may be added to the aeration tank or to the return activated sludge (RAS). The method of choice is the addition of chlorine to the RAS line as chlorine gas or sodium hypchlorite about three times per day. Chlorine concentration should be 10-20 mg/L (concentrations greater than 20 mg/L may cause deflocculation and formation of pin-point flocs). However, chlorination is sometimes unsuccessful in bulking control.

Hydrogen peroxide is generally added tot he RAS at concentrations of 100-200 mg/L. However, as shown for chlorine, excessive levels of hydrogen peroxide can be deleterious to floc-forming bacteria. In addition to its role as an oxidizing agent, hydrogen peroxide may also act as a source of oxygen in the aeration tank. Ozone was also proposed for curing filamentous bulking.

Synthetic organic polymers, lime, and iron salts may be added to the mixed liquor to improve bridging between the flocs and thus promote sludge settling. However, the addition of lime and iron salts increases the solids load, and the use of polymers is costly. Although treatment with polymers and coagulants leads to an immediate improvement in sedimentation, their effect is of short duration because they exert no adverse effect on filamentous microorganisms.

 

 

Foaming in Activated Sludge

Foaming in activated sludge is a common operational problem in may plants. The foam can occur in the aeration tank, secondary clarifier, as well as in the anaerobic digester. This foam is normally sticky, viscous and brown in color. It floats and accumulates on top of the tanks, and can take up a large fraction of solids inventory and reactor volume, which decreases the effluent quality and control of sludge retention time (SRT). The foam can also overflow onto walkways and surrounding areas, making it hazardous for the operator, and causing risk to the operation and environment.

Many reasons are associated with foaming, including the following:

  • the presence of slowly biodegradable surfactants (ie. household detergents) from industrial or municipal wastewater,
  • excess production of extracellular polymeric substance (EPS) by activated sludge microbes under nutrient-limited conditions,
  • proliferation of filamentous organisms,
  • gas provided in the aeration tank or produced in the anoxic zone of aeration tanks, secondary clarifiers and anaerobic digesters.

 

Stable foaming in treatment plants is the product from interaction among gas bubble (generated by aeration, mechanical mixing and biological processes like denitrification and anaerobic digestion), surfactants (which come from the wastewater streams that contain slowly biodegradable surfactants, which reduce the surface tension of a liquid when dissolved), and hydrophobic particles (usually referred to as filamentous bacteria with long-chain structures and a hydrophobic surface). The hydrophobic particles congregate at the air-water surface and strengthen the water film between air bubbles. Meanwhile, the particles also serve as a collector for surfactants, which stabilizes the foam.

 

 

Physical and Chemical Environment

We know that for foaming to occur, gas bubbles are essential to foam generation. Gas bubbles are involved in many steps during the process. In the aeration tank, aeration and mechanical mixing is employed to ensure enough dissolved oxygen is available for the microbes to break down the pollutants, or go through nitrification. This creates an abundance of gas bubbles. Besides being introduced through external sources, gas bubbles can also be produced from the biological processes themselves. Both denitrification and anaerobic digestion produce gases such as nitrogen, methane and carbon dioxide. These gases all favor the generation of foam.

Most surfactants in treatment plants originate from the detergents, oil and grease that are used in households and industry. The extracellular polymeric substance (EPS) produced by bacteria is believed to contribute to part of the surfactants in the wastewater. Surfactants could stabilize the foam and allow it to accumulate.

Filamentous bacteria are important in forming stable foam. The growth rate of these bacteria was not significantly affected by a pH range of 6.7-8.0, and only slightly decreased in population at pH 8.4. The optimum temperature of Microthrix parvicella, associated with foaming, was reported to be around 25°C. These bacterium also prefer low oxygen concentrations. You can eliminate them from your system by increasing the DO to 2-3 mg/L as an effective control method for filamentous bacteria.

 

A brief review of activated sludge foams and their causes is given in the table below. Use of microscopic examination can readily diagnose most of these, particularly when filaments are involved.

Foam Description Cause(s)
thin, white to grey foam low cell residence time or "young" sludge (startup foam)
white, frothy, billowing foam once common due to nonbiodegradable detergents (now uncommon)
pumice-like, grey foam (ashing) excessive fines recycle from other processes (e.g. anaerobic digesters)
thick sludge blanket on the final clarifier(s) denitrification
thick, pasty or slimy, greyish foam (industrial systems only) nutrient-deficient foam; foam consists of polysaccharide material released from the floc
thick, brown, stable foam enriched in filaments filament-induced foaming, caused by Nocardia, Microthrix or type 1863

 

Three filamentous organisms can cause activated sludge foaming: Nocardia and Microthrix parvicella (commonly) and type 1863 (rarely). Nocardial foaming appears to be the most common and occurs at approximately 40% of activated sludge plants in the U.S.

Nocardial foam occurs as a thick, stable, brown foam or "scum" inches to many feet thick on aeration basin and final clarifier surfaces. Normal scum traps (too small) and water sprays (too weak) may be useless to control this type of foam. This foam consists of activated sludge solids (flocs) containing large amounts of Nocardia filaments growing from their surface and is quite stable, compared to most other foams, due to the physical "interlocking" of the Nocardia filaments. These foams are easy to diagnose microscopically - they are dominated by branched, gram positive filaments and a simple gram stain of the foam is all that is needed. The analysis should include comparison to the underlying MLSS.

Nocardial foams occur in all types of plants, with no particular association with specific modes of operation or aeration. These foams may be more severe in plants with fine bubble or jet aeration and in oxygen activated sludge plants. These foams also occur equally in plants treating domestic, industrial and mixed wastes. Industrial wastes promoting Nocardia growth (and foaming) include dairy, meat and slaghterhouse, food processing, pharmaceutical, and any others that contain a significant amount of grease, oil or fat. Nocardial foaming is also associated with high-density restaurant operation in recreational areas. Nocardial foaming has been observed to be caused by treatment of locomotive and truck washing wastes.

Severe Nocardial foams cause a number of operational problems. These include aesthetics, odors, and safety hazards if they overflow basins to cover walkways and handrails. In cold weather these foams can freeze, necessitating "pick and shovel" removal. Foam may escape to the effluent, increasing effluent suspended solids and compromising disinfection. In covered aeration basins, foam can accumulate to exceed the available hydraulic head for gravity flow of wastewater through the basin. Process control can be compromised if a significant fraction of plant's solids inventory is present in the non-circulating foam.

 

 

Identifying Filaments

There are only three filaments that are responsible for the majority of the foaming in activated sludge systems. This makes identification a lot easier than bulking filaments. A simple gram stain is all that is needed to identify these filaments.

  • Type 1863
  • Microthrix parvicella
  • Nocardia

 

Remember, gram negative bacteria stain pink and gram positive bacteria stain purple.

Type 1863 is the only foaming filament that is gram negative. It is very easy to identify because it looks like a pink dashed line. So, if the treatment system is foaming, grab a sample, make a smear and do a gram stain test. If you see pink dashed lines, your problem is type 1863.

 

Microthrix parvicella and Nocardia are both gram positive bacteria, but are very different from one another, structurally. This makes it very easy to distinguish one from the other. Microthrix is very thin and smoothly curved. When stained, it looks like purple spaghetti. Yum! Nocardia, on the other hand, is a short-branched filament and resembles a purple patch of branches.

 

All foaming filaments are "hydrophobic", which means they have water resistant cell walls. This gives them the ability to float upon aeration. They have a few other things in common. Each thrives in wastewater high in greases, oils and fats under low F/M conditions. The fact that they thrive in these conditions and have longer sludge ages give these filaments the advantage over floc-forming bacteria. Although they have a lot in common, there are conditions that will give one the advantage over the other.

Filament type 1863 is favored when there is a decline in the aeration basin pH. Microthrix is favored in colder temperatures and generally prefers animal and vegetable greases, oils and fats. Nocardia on the other hand is favored in warmer temperatures and longer sludge ages.

Identifying filaments that cause foaming is fairly simple. There are only three to choose from and they are all easily identifiable structural characteristics: a pink dashed line, purple shaghetti, or purple branches.

 

Operational Considerations

All foaming filaments generally thrive in wastewater that contains excess amounts of oils and grease. Microthrix is favored in colder temperatures and low F/M, whereas Nocardia is favored in warmer temperatures and longer sludge ages. It's important to know which filament you are dealing with to be able to control it. Influent oils and grease must be controlled before entering the plant. This may require restaurants to install grease traps and have their grease removed instead of sending it to the plant. Type 1863 likes a lower than normal pH environment, so the pH must be adjusted to eliminate foaming from this filament.

You can control Microthrix by raising the F/M ratio. The F/M ratio is a measure of how much "food" is available compared to the amount of microbes eating. If there is a little bit of food and a lot of microbes, the F/M ratio will be low. You can increase this ratio by increasing the rate of wasting.

Nocardia, on the other hand, is more difficult to eliminate since the foam it produces is a very stable foam. This filament can continue to live and reproduce within the foam. Since it's a soil bacterium, it is not affected by UV light from the sun, nor is it affected by dry conditions. While chlorination can help control foaming from type 1863 and Microthrix, it is not useful in conrolling Nocardia foaming. In fact, it makes it worse! May times an operator will spray chlorine on the foam to break it up, but doing this on Nocardia foam will cause it to multiply. The exact opposite of what you want to occur! Nocardia is more difficult to eliminate because of how it grows in the system.

Nocardia Growth Cycle

In the early stages of growth, it's harmless and doesn't cause foaming. As the wastewater temperature increases, and the Nocardia begin to grow, branches begin to form, elongating at a rapid pace once the temperature is higher than 16°C. Once Nocardia is fully branched the foaming problems may begin. A unique characteristic of Nocardia is that as the branches grow, they break up into cylindrical, short cells and the foam will begin to dissipate. The short cells will begin to grow and branch themselves and the growth cycle starts all over again. The increased number of branches will create a greater amount of foam than the generation before.

The addition of chlorine works well for most filamentous bacteria because they have a lot more surface area than floc-forming bacteria or other protozoa. This is not the case with Nocardia. Applying chlorine sprays to the foam will cause the Nocardia branches to prematurely break up into tiny cells, decreasing the surface area. So, in essence, you will be forcing them to reproduce at a faster rate, since each cell goes through it's own branching growth cycle, which will cause more foam. So what should you do? Remember that floc-forming bacteria grow rapidly in the activated sludge system, peaking in number early in the process. I, however, require a much longer time to grow from its early growth stage to fully branched. As long as the sludge age remains short enough, I will continue to exist as harmless, small cells.

What about all the foam on the tanks, in the wet wells and on the clarifiers? As long as the foam remains on the surface of these areas, Nocardia will continue to grow. The foam, once accumulated, also provides a source of seed for Nocardia growth. This means the foam must be removed: sucked or skimmed off the surfaces to prevent them from multiplying. DO NOT put the skimmed off foam back into the head of the treatment system. It is better to land apply it or digest it. Eliminating foaming problems caused by Nocardia will require time, patience and work. By decreasing the amount of time this microbe remains in the aeration basin, you prevent it from maturing into branching filaments, causing foam. By removing the foam from other surfaces, you prevent it from multiplying as well.

Foaming can give the appearance that the treatment plant is doing a poor job of treating the wastewater. Although it looks bad, aesthetically, foaming doesn't interfere with good treatment unless it's very severe.

 

 

Review

Filamentous bacteria serve as the backbone of floc formation. Sludge settles most efficiently when it contains a moderate number of filaments which provide structure for the floc and aid in the stripping of the water column. The floc cannot form properly if there are too few filaments, and the floc cannot settle properly if there are too many. Just like with Goldielocks, it has to be "just right"!

During a nutrient deficiency, the bacteria within the floc particles remove soluble BOD from the wastewater. However, when nitrogen or phosphorous is deficient, the soluble BOD is not degraded but it is stored within the floc particles as an exocellular polymer-like material. This slimy material interferes with settling and may cause foam upon aeration. Bulking is a problem consisting of slow settling and poor compaction of solids in the clarifier of the activated sludge system. Filamentous bulking is usually caused by the excessive growth of filamentous microorganisms. Sludge settleability is determined by measuring the sludge volume index (SVI), which is given by:

 

A high SVI (>150 mL/g) indicates bulking conditions, whereas an SVI below 70 mL/g indicates the predominance of pin (small) floc.

Filamentous bacteria can be controlled by treating the return sludge with chlorine or hydrogen peroxide to selectively kill filamentous microorganisms.

Chlorine concentration should be 10-20 mg/L (concentrations greater than 20 mg/L may cause deflocculation and formation of pin-point flocs). However, chlorination is sometimes unsuccessful in bulking control. Hydrogen peroxide is generally added tot he RAS at concentrations of 100-200 mg/L. However, as shown for chlorine, excessive levels of hydrogen peroxide can be deleterious to floc-forming bacteria.

Numerous measures for controlling foams in activated sludge have been proposed. Several of these cures are not always successful and have not been rigorously tested under field conditions. The control measures proposed include chlorination of foams, increase in sludge wasting, use of biological selectors, reducing air flow in the aeration tank, reducing pH and oil and grease levels, addition of anaerobic digester supernatant, water sprays, antifoam agents, physical removal and use of antagonistic microflora.

 

 

Assignment

Complete Assignment 8 on Filamentous Bacteria. You must be logged into Canvas to submit this assignment. Make sure you choose the appropriate semester.

 

 

Lab

Review the lab for wet mount and smear slide preparation for microbial identification.

 

 

Quiz

Answer the questions in the Lesson 8 quiz You will need to log into Canvas to take the quiz. You may take the quiz 3 times, and an average will be taken from your attempts for final grade calculation. Make sure you choose the appropriate semester.