Filaments and Bacteria... Volume - 1...

When floc particles first develop in the activated sludge process, that is, at a relatively young sludge age, the particles are small and spherical. Because filamentous organism do not develop or elongate at relatively young sludge ages, the floc-forming bacteria can only "stick" or flocculate to each other in order to withstand shearing action. Bacterial flocculation and the absence of filamentous organisms result in spherical floc particles. As the sludge age increases and the short filamentous organism within the floc particles began to elongate, the floc forming bacteria now flocculate along the lengths of the filamentous organisms. These organism provide increased resistance to shearing action and permit a significant increase in the number of floc-forming bacteria in the floc particles. The presence of long filamentous organisms results in a change in the size and shape of floc particles. The floc particles increase in size to medium and large and change from spherical to irregular. Young sludge age (< 3 days) Toxicity (heavy metals etc.) Slug discharge Lack of active and abundant ciliated protozoan population Excessive shearing Excessive surfactant Dispersed growth is a population of bacteria that is suspended in the liquid portion of the mixed liquor. These bacteria are still growing rapidly and have not begin to flocculate. Most dispersed growth is bacterial. Only a little dispersed growth should be present in a properly operating activated sludge process. Ciliated protozoa play an important role in the removal of dispersed growth. Dispersed growth is also removed from the bulk medium by its adsorption to the surface of floc particles. A significant amount of dispersed growth is present at the start-up of an activated sludge process. A lot of food is available, and the bacteria are very active and are multiplying rapidly. The presence of significant or excessive dispersed growth within the mixed liquor can also be due to the interruption of proper floc formation. 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 the floc particles remove soluble BOD from the wastewater. However, when nitrogen or phosphorus 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. The solution usually involves addition of the limiting nutrient, such as ammonia to provide nitrogen, or phosphoric acid to provide phosphorus. There is usually enough nutrient if the ammonia plus nitrate in filtered (0.45 um) effluent is greater than 1 mg/L and the soluble orthophosphate is greater than 0.5 mg/L. However, in cases where easily degradable, soluble BOD is available, higher N and P concentrations may be necessary. Toxicity assessment is one of the most valuable applications of microscopic observation of microorganisms in activate sludge. The higher life forms, particularly the ciliates and the rotifers, are generally the first to be impacted by toxic materials and may serve in essence as an in-plant biomonitoring test for toxicants or other adverse stresses. The first noticeable sign of toxicity or stress is usually the slowing or stopping of cilia movement for these organisms and small flagellates and ciliates begin to predominate. This is an indication of the break up of the floc and an over abundance of free bacteria used by these organism as a food source. Indications of toxicity upset include : Loss of the higher life forms in the activated sludge (these are the most toxic sensi tive microbial components). A dispersed activated sludge biomass with poor floc formation and pin floc. Unusually low oxygen use, caused by poor biomass growth. Poor BOD removal. What can you expect to see under the microscope if toxic conditions exist ? There will be a sudden increase in flagellates. This is sometimes called a flagellate "bloom" The of protozoa and higher life forms will begin to die off Break-up of floc, sometimes accompanied by foaming Loss of BOD removal Filamentous bulking upon process recovery. Filamentous bacteria are very often the first to recover after a toxic upset. Toxic wastes generally do not favor filaments directly (except in the case of H 2S), rather upset conditions

Floc Particles : Sizes and Shapes...

Factors Interrupting Floc Formation...

Dispersed Growth...

Slime Bulking...

Operational Considerations...

Toxicity...

allow filaments to grow. Microscopic examination of activated sludge can diagnose toxicity, however, this is usually "after the fact". A better method of toxicity detection is the use of oxygen uptake rate testing to detect toxicity early and to find the source.

Filamentous Bulking and Foaming...

Filamentous Organism Haliscomenobacter hydrosis, Sphaerotilus natans, type 1701 Haliscomenobactor hydrosis, Microthrix parvicella, Nocardia spp., type 021N, type 0041, type 0092, type 0581, type 0675, type 0803 and type 0961 Sphaerotilus natans, Thiothrix spp. fungi, type 0675 and type 021N Nocardia spp, fungi Type 0041, type 0092, and Microthrix parvicella Beggiatoa spp, Thiothrix spp and type 021N Factors Promoting Rapid Growth Low D.O.

Low F/M

Low Nutrients (Nitrogen or Phosphorus) Low pH Low Organic Load Septic Wastewater / Sulfides

Foam Producing Filaments Microthrix parvicella Nocardia spp type 1863

Factors Promoting Rapid Growth Low F:M and high wastewater grease and fat,colder temperatures Longer MCRT, excess grease, oils and fats and warmer temperatures Low D.O., excess grease and fat, and low pH

To determine if aeration tank or clarifier foam is due to the growth of foam- producing filamentous organisms, a sample of fresh foam should be spread thinly and evenly over a clean microscope slide, and the slide stained by Gram staining. Microthrix parvicella and Nocardia spp each stain Gram positive (staining purple or dark blue). Microthrix is a long thin filament while Nocardia is a short branched filament. So, if you have foam and slide of mixed liquor stains Gram positive, you can easily determine which filament is responsible. Type 1863 stains Gram negative (staining pink). Type 1863 is a long filament which looks like a dashed line. If the foam is not due to foam-producing filamentous organisms, it may be due to the presence of a nutrient deficiency. To determine if a nutrient deficiency is the cause of foam production, a representative sample of mixed liquor should be treated with India ink and examined under phase contrast microscopy for the presence of nutrient deficient floc particles.

Volume - 2...
Make up about 95% of the activated sludge biomass. These single celled organisms grow in the wastewater by consuming (eating) bio-degradable materials such as proteins, carbohydrates, fats and many other compounds. Enzymes are compounds that are made by living organisms. Their purpose is to help biochemical reactions to occur. Almost all biochemical reactions require the presence of enzymes to cause the reaction to occur. Enzymes help bacteria in the process of breaking down nutrients, and in rebuilding broken down nutrients into the new compounds that they require for growth and reproduction. Enzymes only do what they are supposed to when environmental conditions are right. If the conditions are not right the enzymes will not function properly, thus, the bacteria will not function properly, and they will not survive. If conditions are right the bacteria will live and prosper. When there is plenty of food available, bacteria use the food mostly for growth and some for energy. A growing bacterium have flagella (hair-like structures on the outside of the cell) which makes it motile, able to move in search of food. A bacterium reproduces into two bacteria. The cell splits into two smaller cells and this process occurs over and over again. When there is very little food available, the bacteria use the limited food to produce energy and to maintain the cell. Very little is available for growth so less reproduction occurs. With little food available, and in an attempt to conserve energy, the bacterium lose s it flagella and thus, its motility. The waste products start to form a thick slime layer outside the cell wall, making the cells stick together. The growth characteristics of bacteria are better understood by studying the growth curve. Lag-phase : During this phase bacteria become acclimated to their new surroundings. They are digesting food, developing enzymes and other things required for growth. Accelerated Growth-phase : The bacteria are growing as fast as they can, since there is an excess of food. The cells are mostly dispersed, not sticking together. Declining Growth-phase : Reproduction slows down because there is not an excess of food. A lot of food has been eaten and there are now a large number of bacteria to compete for remaining food, so the bacteria do not have enough remaining food to keep the growth rate at a maximum. Stationary-phase : The number of bacteria is the highest possible, but not much food is left, so the bacteria cannot increase in number. There is some reproduction, but some cells are also dying, so the number of bacteria remain relatively constant. The bacteria have now lost their flagella and have a sticky substance covering the outside of the cell, allowing them to agglomerate into floc. In fact, the floc get big enough that if aeration and mixing were stopped, the floc could settle to the bottom. Death-phase : The death rate increases with very little if any growth occurring. Therefore, the total number of living bacteria keeps reducing. The bacteria are just trying to keep alive. We measure the amount of biodegradable matter the bacteria use for food by measuring the amount of BOD (biochemical oxygen demand) or COD (chemical oxygen demand) in the influent to the aeration basin. We estimate the weight of microorganisms in the mixed liquor by measuring the amount of volatile suspended solids (VSS) in the activated sludge. We use this information to form a relationship called food to microorganism ratio (F/M ratio). The F/M ratio tells us something about growth and cell condition. If the F/M ratio is high, the bugs normally grow quite rapidly (because this means there is a lot of "food" available in comparison to the amount of microorganism); if the F/M ratio is low, the bug normally grow very slowly (because little food is available for growth). Microorganisms need oxygen to live. Oxygen use and be used to determine the activity of the organisms. ( a ) Actively growing organisms are rapidly metabolizing the food, so they are use oxygen at a rapid rate. ( b ) We measure the rate at which oxygen is used by a test called the Oxygen Uptake Rate (OUR), or the Respiration Rate. It is measured in mg O2/hr.gm of MLSS. ( c ) Normally a higher uptake rate is associated with high F/M ratios and younger sludges and a lower uptake rate is associated with lower F/M and older sludges. So, if you want a higher uptake rate, more sludge should be wasted. Less should be wasted if you want a lower F/M ratio. As bacteria begin growing, they generally develop into small chains or clumps. They are very active and motile and it is difficult for them to settle. They have not yet developed the slime layer which aids in their sticking together. So, when mixing occurs, the small chains or clumps are broken up and the bugs are dispersed, and they will not flocculate or settle. As the sludge is allowed to age, the bugs lose their motility and accumulate more slime. Then the clumps and chains are better able to stick together. The clumps grow bigger and bigger until they form a floc. If the organisms are allowed to develop properly, under the right conditions, the floc get large and compact and begin to settle. The mixing in the aeration tank tends to keep the floc small since,

Bacteria...

The Role of Enzymes...

Growth Characteristics...

F:M (Food to Microorganism Ratio)...

The Use of Oxygen...

The Formation of Floc...

even though the bugs are sticky, the bond formed holding the organisms together is not very strong. This is good because it allows the cells, food, and oxygen to contact each other. Oxygen is required by these bugs to metabolize food for cell maintenance and growth. Although the bugs need oxygen, some bugs can get along with less oxygen than others. Each bug must have a dissolved oxygen of at least from 0.1-0.3 mg/L to function properly. So, it is important to maintain about 2 mg/L of D.O. in the activated sludge so that the bacteria that are contained in the floc can get oxygen. If the DO is less than 2 mg/L, the bugs on the outside of the floc use the DO before it can get to the center of the floc. If this happens, the bugs in the center may die causing the floc to break up. Mixing is required to bring organisms, oxygen, and nutrients together, and to remove metabolic waste products. If there is not enough mixing, proper treatment will not take place because of lack of contact between the bugs, their food and oxygen. If too much mixing is provided, it can cause break up of floc or formation of unstable floc particles. The enzymes which regulate many of the biochemical reaction in bacteria are very pH dependent. The optimum pH should be between 7.0 and 7.5 for the proper activated sludge microorganisms to dominate. Biochemical reactions are very temperature dependent. Lower temperatures cause such reactions to be much slower. Thus, more bugs are required to do the same job during the winter than in the summer. Microorganisms require certain nutrients for growth. The basic nutrients of abundance in normal raw sewage are carbon (C), nitrogen (N), phosphorus (P), with the ratio of C:N:P ratio approximately equal to 100:10:1. In addition to C, N, and P, trace amounts of sodium (Na), Potassium (K), magnesium (Mg), iron (Fe), and many others are required. In normal municipal sewage, most of these nutrients are provided. Most problems with nutrient deficiency occur when there is a lot of industrial wastes present. When proper nutrients are not available, the metabolism fails and a kind of bacterial fat (slime) will begin to accumulates around the cell. The cell slows down in activity because it cannot produce enough enzymes and because needed nutrients cannot penetrate the slime layer as they should. The sludge will not settle and BOD removal slows down. The presence of particular types of protozoans is related to effluent quality and plant performance. Protozoan play secondary but important role in purification of aerobic wastewater. The protozoans in the activated sludge treatment process fall into four major classes: amoebae, flagellates, and ciliates (free-swimming, crawling, and stalked). Amoebae : Amoebae are the most primitive, single-celled protozoans. They move by false feet. They are frequently present in raw influent, and their presence is short in the aeration basin. Amoebae can only multiply when there is an abundance of nutrients in the aeration tank. They move very slowly and it is difficult for them to compete for food the there is a limited amount available. They are only dominant in the aeration basin for a short time. They feed on small organic particulates. When amoeba are present in large numbers in the aeration basin this usually indicates that there has been some sort of shock loading to the plant (there must be a lot of food available). Their presence may also indicate that there is a low D.O. environment in the aeration basin, because they can tolerate very low amounts of D.O. Flagellates :Most flagellates absorb dissolved nutrients. Soon after amoebae begins to disappear and while there is still high concentrations of soluble food. Flagellates and bacteria both feed on organic nutrients in the sewage so as the nutrient level declines they have difficulty out competing the bacteria for soluble food so, their numbers begin to decrease. If large amounts of flagellates are present in the later stages of the activated sludge development this usually indicates that the wastewater still contains a large amount of soluble organic nutrients. Ciliates :Ciliates feed on bacteria not on dissolved organics. While bacteria and flagellates compete for dissolved nutrients, ciliates compete with other ciliates and rotifers for bacteria. The presence of ciliates indicate a good sludge, because they dominate after the floc has been formed and after most of the organic nutrients have been removed. Free-swimming ciliates :These ciliates appear as flagellates begin to disappear. As the bacterial population increases, a lot of dispersed bacteria is available for feeding and as a lightly dispersed floc appears, freeswimming ciliates begin to dominate and feed on the increased numbers of bacteria. Crawling ciliates :As floc particles enlarge and stabilize, crawling ciliates graze on floc particles. Crawling ciliates out compete free-swimming ciliates for food because they can find food within the floc. Stalked ciliates :Stalked ciliates appear in the mature sludge. Within the mature sludge the crawling and stalked ciliates compete for dominance. Temperature :Most protozoans can survive and reproduce in a temperature range at which activated sludge processes are carried out. They grow best in ambient temperatures (15-25 oC).

Dissolved Oxygen...

The Effects of Mixing...

The Effects of pH...

The Effects of Temperature... The Effects of Nutrients...

Protozoa & Rotifers...

Factors Influencing Protozoa...

pH :Protozoans are more sensitive to pH than floc-forming bacteria. They have an optimum pH range of 7.27.4 and a tolerance range of 6.0-8.0. Dissolved Oxygen :Like bacteria, protozoan must have oxygen to survive. Thus lack of DO will severely limit both the kind and number of protozoans. Nutrition :Most municipal wastewater treatment plants, however dilute, contains sufficient nutrients to support most of the protozoan associated with wastewater. Rotifers are rarely found in large numbers in wastewater treatment processes. The principal role of rotifers is the removal of bacteria and the development of floc. Rotifers contribute to the removal of effluent turbidity by removing non-flocculated bacteria. Mucous secreted by rotifers at either the mouth opening or the foot aids in floc formation. Rotifers require a longer time to become established in the treatment process. Rotifers indicate increasing stabilization of organic wastes.

Rotifers...

Filaments and Bacteria... Volume - 3...

Introduction to Filament Identification...
In order to identify many of the following filament characteristics, the mixed liquor must be examined under 100 X using immersion oil. It is difficult to see many of these characteristics under lower magnifications. Filaments may be long, short, smoothly curved, coiled, irregularly bent, straight, or bundled. Filamentous bacteria are made up of a chain of cells. The shape of the individual cells is a characteristic that can help us to identify the different filamentous bacterial types. Cell shape may be round, square, rectangular, oval, or discoid. The cell septa is the "line" which separates each individual cell which makes up the bacterial filament. The septa are clearly seen in some filaments an is very difficult to see in others. Some septa are "indented" and some are not. Indentations and the ability to clearly see the cell septa are other characteristics which can help us to identify the different filamentous bacteria. Motility is the ability of an organism to produce motion or to move. Beggiatoa spp is only one filamentous bacterium found in activated sludge that is motile. Some filaments store by-products as intercellular granules (mostly sulfur granules). Sulfur granules can be seen very clearly under phase contrast and are found usually in septic wastes. Sulfur granules are commonly found in Beggiatoa, Thiothrix and type 021N. Branching may be "true" or "false". If a filament has true branching the intercellular fluids will flow freely throughout all the branches of the filament. Intercellular fluids cannot flow through false branches. In false branching the filament are simply attached to each other simulating a branch. There are only two filaments which exhibit branching; one has true branching and the other false. Nocardia spp has true branching and Sphaerotilus natans exhibits false branching. The cells of some filamentous organisms are contained in a tight fitting sheath. The easiest way to detect a sheath is to look for "missing spaces" between the cells. Some filaments which have a sheath are Haliscomenobactor hydrosis, Sphaerotilus natans, type 1701, type 0041, and type 0675. Some filaments have bacterial cells attached along the side, perpendicular to the filament. There are three filaments on which this commonly occurs. Type 0041, type 0675, and type 1701.

Filament Identification...

Filament Shape and Length... Individual Cell Shape...

Cell Septa...

Motility...

Intercellular Granules...

Branching...

Sheath...

Attached Growth...

Filaments and Bacteria... Volume - 4...

Filamentous Bulking...
The start of any problem solving has to involve microscopic examination of the activated sludge. This reveals whether the problem is, or is not, caused by filaments. If caused by filaments, identification of the causative filament(s) yields a direction or approach to take for a remedy. As shown in Table 1, the causes for filament growth include low oxygen concentration, low F/M, septicity, nutrient deficiency, low pH and high grease and oil. Control methods, based on the specific type(s) of filaments causing the problem, follow.

Introduction...

Filament Types as Indicators of Conditions Causing Activated Sludge Bulking...

Causative Condition (1) Low Dissolved Oxygen (for the applied organic loading) Low Organic Loading Rate (low F/M) Septic Wastes / Sulfides (high organic acids) Nutrient Deficiency - N and/or P (industrial wastes only) Low pH (< pH 6.0) High Grease / Oil

Filament Types S. natans, type 1701 and H. hydrossis M.parvicella, Nocardia spp., and types 0041, 0675, 1851 and 0803 Thiothrix I and II, Beggiatoa spp., N. limicola II*, and types 021N, 0092*, 0914*, 0581*, 0961* and 0411 Thiothrix I and II and type 021N and N. limicola III Fungi Nocardia spp., M. parvicella and type 1863

(1) Note that some filaments occur at several conditions. * These filaments occur at lower F/M at septic conditions.

Low aeration basin dissolved oxygen (DO) concentration for the applied organic loading (F/M) leads to filamentous bulking by several filaments (see Table 1). The required aeration basin DO concentration to prevent "low DO bulking" is not a constant, rather, is a function of the F/M rate. Simply, higher bulk DO is required to prevent the growth of these filaments as the F/M increases, due to faster oxygen use within the floc at higher F/M, oxygen depletion inside the floc, and the need to maintain aerobic conditions in the interior of the floc. A higher bulk DO concentration increases the diffusion of oxygen into the floc interiors. In general, a bulk DO concentration of 2.0 mg/L is recommended for F/M values up to 0.5, typical of most domestic waste plants. This DO concentration should be maintained at the point of greatest oxygen demand in the system, for example, at the headend of a plug-flow system (not the backend). Some industrial waste systems and high rate domestic plants operated at higher F/M may need higher DO values than 2.0 mg/L due to oxygen diffusion limitations. Type 1701 bulking has occurred in an oxygen activated sludge plant operated at high F/M at a DO concentration of 12-14 mg/L, which was cured by raising the DO to 20 mg/L. As a rule, always trust the microscopic observation of low DO filaments to indicate oxygen limitation rather than the aeration basin DO values. Control of low DO bulking is by raising the aeration basin DO concentration and by raising the aeration basin MLSS concentration (decreasing the F/M). Note that this action is opposite to what intuition directs: to reduce the MLSS concentration, since less biomass needs less oxygen (wrong! -the F/M is increased at lower MLSS concentration and oxygen needs increase). Filamentous bulking is common in completely-mixed, lower F/M systems. Here, a number of filaments can cause bulking (see Table 1) because they grow better than most activated sludge floc-forming bacteria at low aeration basin BOD concentration. Intermittently-fed and plug-flow systems are more resistant to this type of bulking. Control of low F/M bulking is by reducing the aeration basin MLSS concentration and increasing the F/M (manipulating the "M" component). Lowering the MLSS concentration may not be suitable for many plants as this may cause the loss of nitrification and increase waste sludge production. Any change in operation that effectively increases the substrate concentration available to the activated sludge and introduces batch or plug-flow characteristics to the aeration basin, even on a short term basis, will help control low F/M bulking. These include: compartmentalization of aeration basins; fed-batch operation; intermittent feeding of wastes; and use of a selector. These latter methods do not reduce the MLSS concentration in the system. A selector is a mixing basin or channel where RAS and influent wastes mix prior to the aeration basin. Selector design is empirical at this time. Successful examples involve a 15-30 minute contact time of the RAS and influent waste; are aerated; and achieve at 70-80% removal of soluble BOD5 through the selector. Several newer designs are either operated anoxic (no free oxygen but nitrate present) or anaerobic, however, these are too new to state their general usefulness. A selector can be too large or too small in size to properly function. The goal is to provide a short term, high substrate condition which favors certain floc-formers but which discourages filaments. These floc-formers appear to rapidly store BOD as cellular storage products in the selector, which they use later for growth in the main aeration basin (they pack their own "lunch bags" in the selector). If the selector is too large, the substrate concentration achieved may not be high enough to encourage these special floc-formers and discourage filaments. If too small, insufficient time may be available for substrate uptake and storage. Also, a selector that is too small may cause the floc-formers to shunt carbonaceous substrate to exocellular polymer which can increase the SVI of the sludge ("slime bulking") and pose problems in waste sludge dewatering. The best approach is to try several selector sizes, using a larger basin or channel with movable baffles or exit gates. Use of a variable wastewater bypass around the selector can achieve the same objective and allow the operator some control over the selector. Selectors are specific tools to combat low F/M filaments and are not needed by all plants. Inappropriate selector use may made the problem worse (for example, where bulking is caused by low DO, nutrient deficiency or waste septicity). Influent wastewater septicity is usually indicated by odors (H2S or "rotten egg" smell) and a dark color to the wastewater, caused by precipitated ferric sulfide. Septic wastes contain elevated amounts of sulfides and low

Low Dissolved Oxygen Concentration...

Septicity...

molecular weight organic acids (such as acetic and butyric acids), both of which encourage the growth of certain filaments (see Table 1). Observation of these filaments with intracellular sulfur granules is a tip-off of a septicity problem. Septicity is more common in systems in warmer climates and in those with large wastewater collection systems that have lift stations and force mains. Waste septicity can be treated by preaeration (which releases odors), by chemical oxidation (chlorine, hydrogen peroxide or potassium permanganate), by chemical precipitation (ferric chloride), or use of sodium nitrate in the collection system as an "oxygen source". Septicity can originate within plant processes. Common sources of septicity include equalization basins, primary and secondary clarifiers, andreturn streams from sludge processing. These can be tested for sulfide or organic acid concentration to determine whether they are a significant source of septicity. A sulfide concentration >1-2 mg/L and an organic acid concentration >100 mg/L favors filament growth. Sulfide can be tested for using one of the simple sulfide test kits available commercially (such as by HACH). Organic acids can be tested for using distillation and titration as per Standard Methods (the same test as for digesters). Nitrogen and phosphorus can be growth limiting if not present in sufficient amounts in influent wastewater, a problem with industrial wastes and not domestic wastes. In general, a BOD5:N:P weight ratio in the wastewater of 100:5:1 is needed for complete BOD removal. Other nutrients such as iron or sulfur have been reported as limiting to activated sludge, but this is not common. Signs of nutrient deficiency include: filamentous bulking (see Table 1); a viscous activated sludge which exhibits significant exopolysaccharide ("slime") when "stained" with India ink; and foam on the aeration basin which contains exopolysaccharide (which has surface active properties). One check for nutrient deficiency is to be sure that at least 1.0 mg/L total inorganic nitrogen (TIN = ammonia + nitrite + nitrate) and 0.5-1.0 mg/L ortho-phosphorus remains in the effluent at all times. In systems treating mixed domestic and industrial wastes, only TIN and orthophosphorus should be used to calculate nutrient availability. Organically combined nitrogen and phosphorus (Kjeldahl nitrogen and total phosphorus) may not be hydrolyzed fast enough by the microorganisms in the activated sludge to keep pace with BOD use. The aeration basin pH should be maintained in the range 6.5 to 8.5. Low pH, <6.5, may cause the growth of fungi and fungal bulking. The aeration basin pH can be adjusted using caustic, lime or magnesium hydroxide. Control methods for filamentous bulking are based on, first, confirmation that the problem is indeed caused by filaments (some are not) and, second, identification of the causative filament(s). This information leads to specific remedies that can be used, appropriate for the filament(s) involved.

Low Nutrients...

Low pH...

Summary...

Filaments and Bacteria... Volume - 5...

There exist several methods of chemical addition to enhance activated sludge settling. Most used are synthetic, high molecular weight, cationic polymers alone or in combination with an anionic polymer that serve to overcome the physical effects of filaments on sludge settling. These are usually added to the MLSS as it leaves the aeration basin or to the secondary clarifier centerwell. Use of polymer does not significantly increase waste sludge production but can be quite expensive, up to $450. per million gallons treated. A polymer supply company should be consulted for selection of a polymer. Jar testing should be performed at your plant to determine the type of polymer needed and its dosage, which are quite plant specific. In some instances, inorganic coagulants/precipitants such as lime or ferric chloride can be beneficial. These produce a voluminous precipitate that sweeps down the activated sludge, improving settling. Sludge production may be significantly increased if these are used. The weighting action of inert biological solids has also been used to aid sludge settling in activated sludge modifications such as the Hatfield or Kraus processes that recirculate anaerobic digester contents through the aeration basin. Clay and fiber addition have been used by some industries (e.g. papermills) to help sludge settling on a short-term basis. Two toxicants, chlorine and hydrogen peroxide, have been used successfully to control filaments. Chlorine is the most widely used as it is relatively inexpensive and available on-site at most plants, and only this will be discussed here. Chlorination for bulking control is widespread, used by more than 50% of plants. The goal of chlorination is to expose the activated sludge to sufficient chlorine to damage filaments extending from the floc surface while leaving organisms within the floc largely untouched. Filamentous and floc-forming bacteria do not appear to significantly differ in their chlorine susceptibility. Chlorine dosage is adjusted such that its concentration is lethal at the floc surface but is sublethal within the floc, due to chlorine consumption as it penetrates into the floc. This is analogous to "peeling an orange" and removing the filaments attached to its surface. Chlorination is not a cure-all for all activated sludge microbiological problems, as this will actually make problems worse if the problem is non-filamentous, e.g. slime bulking, zoogloeal bulking or poor floc development. Chlorine can be applied from a chlorinator using chlorine gas feed or as a liquid hypochlorite. A separate chlorinator should be dedicated to bulking control and an independent rotameter and sampling point in this chlorine line is needed. The chlorine addition point is of most importance and should be at a point where the sludge is concentrated, raw wastes are at a minimum, and at a point of good mixing. Poor initial mixing results in the consumption of large amounts of chlorine without bulking control. Three common chlorine

Inert Material and Polymer Addition...

Chlorination...

addition points are: (1) into the RAS stream at a point of turbulence (elbows in pipes; into the volute or discharge of RAS pumps; and into and below the liquid level in a riser tube of an airlift RAS pump); (2) directly into the final clarifier center well or feed channel; and (3) in an installed sidestream where the MLSS is pumped from and returned to the aeration basin. Chlorine addition to the RAS line(s) is the method of choice and most generally successful. Chlorine addition to the aeration basin does not work and only causes floc dispersion and system damage. The two most important parameters are chlorine dosage and frequency of exposure of the activated sludge to chlorine. Chlorine dose is measured conveniently on the basis of sludge inventory in the plant (overall chlorine mass dose). Effective chlorine dosages usually are in the range 1-10 pounds chlorine/1,000 pounds MLVSS inventory/day (2-4 should work). Chlorine dosage should be started low and increased until effective. Most domestic waste plants can achieve a frequency of exposure of the activated sludge inventory to chlorine of three or greater times per day (the optimum) in the RAS line. The needed frequency is a function of the relative growth rates and efficiencies of kill of filamentous and floc-forming organisms. Success has been achieved at frequencies as low as one per day, however, not below this. In plants with long aeration basin hydraulic residence times (usually industrial waste plants), the daily passage of solids through the RAS line is generally too low (<1/day) for successful bulking control using chlorine at this point. Here, most success has been achieved using multiple chlorine addition points such as the RAS line(s) and the final clarifier(s). Chlorination controls filament extension from the floc surface and merely reduces the symptoms of bulking. Filaments will regrow rapidly, often with a vengeance, after termination of chlorination since the cause of the bulking has not been addressed. Signs of overchlorination are a turbid (milky) effluent, a significant increase in effluent TSS, a loss of the higher life forms (protozoa), and a reduction in BOD removal. It is normal to see a small increase in effluent suspended solids and BOD5 when using chlorine for bulking control. Microscopic examination of the activated sludge during chlorination is recommended to control chlorine application. Chlorine effects on filaments include, in order: a loss of intracellular sulfur granules (in those filaments that have these); cell deformity and cytoplasm shrinkage; and finally filament lysis. For filaments that don't have a sheath, the sludge SVI usually declines within a few days of chlorine use, if the dosage and frequency requirements are met. For sheathed filaments, the sheath is not destroyed by chlorine. Here, sludge settleability remains poor until the sheaths are washed out of the system by sludge wasting, which requires 1-2 sludge ages. Chlorine use for sheathed filaments should be stopped when mostly empty sheaths remain (60-80% of filaments) and not continued until the SVI falls, which can result in overchlorination. Activated sludge foaming is caused mostly by two filaments: Nocardia spp. and Microthrix parvicella (there are other non-filament causes of foaming). Both of these filaments have three causes in combination: (1) high grease and oil; (2) longer sludge age; and (3) low oxygen conditions or septicity. Nocardia appears to be favored at higher aeration basin temperature and M. parvicella at lower temperature. Antifoam chemicals are not effective to control this type of foam, due to the physical interlocking of the filaments in the foam. RAS chlorination is of limited use in Nocardia foam control, but is more useful for M. parvicella. This is because Nocardia is found mostly within the floc, and the higher chlorine dosages needed to get at Nocardia may destroy the activated sludge floc. For Nocardia foams, surface spraying of a 50 mg/L chlorine solution can be effective. Both these filaments grow on grease and oil. Systems that lack primary clarification (the main grease and oil removal mechanism) appear to suffer more foaming problems. Communities with enforced grease and oil ordinances appear to suffer less from foaming problems. Also, treatment of septage, which contains substantial grease and oil, has been associated with foaming problems. There is some relationship of foaming by these filaments and low oxygen concentration and septicity in the system. Septicity appears to cause the breakdown of grease and fat to organic acids, which specifically favor these filaments. Successful foam control may need control of septicity and low oxygen conditions. The most used control method for foaming is to reduce the system MCRT. M. parvicella can usually be controlled by a MCRT reduction to between 8 and 10 days. Nocardia can often be controlled by MCRT reduction to <8 days, but this is variable and somewhat temperature dependent. Plants in warmer climates have had to reduce the MCRT to <3 days for Nocardia control. Many foams reach problem levels because they are not removed and buildup on the surface of aeration basins and final clarifiers. Needed are enlarged surface scum traps and forceful water sprays to carry this material out of the aeration basin or the clarifier. Foam should be removed entirely from the system and not recycled back into the plant (for example, into the headworks). Foam disposal into aerobic or anaerobic digesters can result in foaming there, so this should be avoided. Filamentous bulking and foaming problems require the identification of the causative filament(s). This information leads to specific remedies, appropriate for the filament(s) involved. Short term control methods are often used to quickly stop a bulking problem. However, the best approach is to investigate the long term control methods suitable for your plant to arrive at long-term, trouble free operation.

Activated Sludge Foaming...

Summary...

Filaments and Bacteria... Volume - 6...

Glossary of Terms...

ACTIVATED SLUDGE : A flocculent microbial mass, produced when sewage is continuously aerated. BIOCHEMICAL OXYGEN DEMAND (BOD) :The amount of dissolved oxygen consumed in the oxidation of biodegradable organic matter by microbiological action when a sample is incubated, usually for 5 days at 200 o C. It is often referred to as BOD5. BULKING SLUDGE :A phenomenon, which occurs in, activated sludge plants when sludge/biomass occupies an excessive volume and does not settle readily so that in extreme cases the effluent from the final settlement tank contains an excessive amount of suspended solids. Usually associated with the presence and predominance of filamentous micro-organisms in the biomass. Appendix I explains bulking sludge in more detail. COD :The amount of oxygen consumed from a specific oxidizing agent in the oxidation of the polluting matter present in a sample. As normally determined i.e. as from the silver catalyzed dichromate, it approximates to the oxygen theoretically required for complete oxidation of the carbonaceous matter in the sample to carbon dioxide and water. DEFLOCCULATION :Flocs disperse as : Sludge ceases to react with waste. Protozoa cease activity and allow single cell bacteria etc upon which they feed to multiply. Bacteria in the floc die and floc breaks up. Excessive oxygenation. Shock load. Low pH. Low dissolved oxygen. DISSOLVED AIR FLOTATION (DAF) :A process that can be used for oil and grease removal, for concentration of activated sludge, or as a pre-treatment in physical chemical methods of treatment. In the concentration of activated sludge, flotation is achieved by the attachment of microscopic sized air bubbles to the activated sludge flocs thereby reducing their specific gravity and causing them to rise to the surface where the concentrated activated can be skimmed off. DENITRIFICATION :The reduction by microbial or other of Nitrate to Nitrogen gas, or in some cases to Nitrous oxide. DISSOLVED OXYGEN :Oxygen dissolved in a liquid, the solubility depending on the temperature, partial pressure and salinity, expressed in mg/l. FLOC FORMERS / FLOCCULATION :Micro-organisms in activated sludge usually agglomerate with each other and with suspended solids into clumps. This is called flocculation by biological action. Individual clumps are termed flocs. This phenomenon may be due to : Free cells becoming embedded in a gelatinous mass. Bacteria, which grow as free cells, flocculate under certain conditions. Bacteria adhering to extra cellular material produced by fungi. Bacteria being flocculated by protozoa. Possibly all of the above plus some other mechanism. Flocculation can also be achieved by the addition of a chemical or by stirring the waste -water by mechanical means. FOAMING :The formation of a foam or froth on the surface of : The mixed liquor in an aeration tank. The sludge in an anaerobic digestion tank. A body of water, caused by detergents, surfactants and/or polyglycols lowering the surface tension of the liquid. Foaming can also be caused microbiologically by certain filamentous bacteria particularly actinomycetes e.g. Nocardia. F/M RATIO :Ratio of Food to Mass ( Biomass ). In activated sludge process, it is the ratio of loading in kg of BOD/day to weigh in kg of either volatile or total suspended solids in the mixed liquor. At start up of an aeration basin, the concentration of the biomass is low while nutrient concentration is high. Thus the F/M is high. During the growth of biomass, cells multiply and compete for the available food/nutrients. As a result the food/nutrients supply decreases and the F/M ratio becomes much lower a low F/M. F/M = BOD of Sewage ( kg/m3 ) x Influent Flow ( m3 /d ) : Reactor volume ( m3 ) x Reactor Solids ( MLSS ) ( kg/m3 ) F/M VALUES FOR ACTIVATED SLUDGE SYSTEMS : High Rate : 0.6 1.8 kg BOD/kg MLSS.day Conventional : 0.1 - 0.6 kg BOD/kg MLSS.day Low Rate : 0.05 0.2 kg BOD/kg MLSS.day HIGH RATE :Any plant which is operated purposely at a hydraulic or organic loading significantly greater than that usually employed, without special regard to the quality effluent which the plant produces or to the type of waste being treated. - High Rate Filter : A biological filter containing a coarse medium or synthetic medium operating with a hydraulic load exceeding 3 m3/m3.day or an organic load exceeding 2 kg of BOD/m3.day. Also called a High Rate Tower. - High Rate Activated Sludge Process : A modification of the activated sludge process whereby a much shorter aeration period than usual and possibly a high mixed-liquor suspended solids concentration are used. HYDRAULIC LOADING :Also termed Volumetric Loading. When applied to biological filters e.g. percolating filter, it is the rate of application expressed in cubic meters of wastewater per cubic meter of media per day. Hydraulic Loading = Flow rate ( m3/day ) : Media volume ( m3 ) The normal loading rate for this application is 0.65 m3/m3.day. When applied to biological filters e.g. High

Rate Tower, it is the rate application expressed in cubic meters of wastewater per surface area ( m 2 ) of media per hour. Hydraulic Loading = Flow rate (m3/hr ) : Surface area of media ( m2 ) The normal loading rate for this application is 1.5 m 3/m2.hr. MIXED LIQUOR :A Mixture of wastewater and activated sludge undergoing aeration and circulation in an aeration basin or channel of an activated sludge plant. MIXED LIQUOR SUSPENDED SOLIDS ( MLSS ) :The weight of dry solids in mg/l of mixed liquor in the aeration tank or channels of an activated sludge plant. Determination of MLSS : Filter a know volume of mixed liquor through an Buchner Funnel onto predried and preweighed filter paper e.g. GFC. Dry at 103 oC to a constant weight ( usually one hour ) and reweigh : Result : MLSS = mg/l Typical MLSS values for the main activated sludge systems are as follows : MLSS ( mg/l ) High rate : 4,000 - 5,000 Conventional : 1,200 - 3,500 Nitrifying : 3,500 - 5,000 VITOX : 3,000 - 12,000 Oxidation ditch : 3,500 - 5,000 MIXED LIQUOR VOLATILE SUSPENDED SOLIDS ( MLVSS ) :This refers to mixed liquor solids, which volatilise in 20 minutes at 600 oC. The solids, which remain after ignition, are referred to as ash. MLVSS are usually expressed as a % of MLSS. NITRIFICATION :The oxidation of Ammonia to Nitrite and Nitrate . Usually carried out by autotrophs , which obtain their energy from the oxidation reaction. ORGANIC LOAD :The organic load is expressed as the kg of BOD per unit volume of aeration basin / filter medium. It is a measure of the amount of BOD, which is applied per unit volume of aeration tank capacity or per unit volume of filter medium. Organic Load = Influent Flow ( m3/day ) x BOD of Sewage ( kg/m3 ) : Reactor Volume ( m3 ) Typical organic load values for a Fixed Film ( Biological Filter ) are as follows : Organic Load High Rate : 0.32 1.0 kg BOD/m3 media.day Intermediate Rate : 0.04 0.48 kg BOD/m3 media.day Low Rate : 0.08 0.32 kg BOD/m3 media.day In general a BOD loading in excess of 0.5 kg BOD/m3.day is High Rate. The organic load will vary over a day with fluctuation in both influent flow rate and strength. Most accurate values ( BOD ) will be obtained from a composite sample, of samples of equal volume taken at 1 hour intervals over a 24 hour period. OXIDATION DITCH :A method of 6treating crude or settled wastewater in witch two parallel channels are joined at the ends to form a closed circuit. It can be equipped with one or two aeration rotors, used for aerating a mixture of the wastewater and activated sludge prior to separation in a settlement tank. ( First introduced in Holland in 1958 ). PIN POINT FLOCS :Small particles remaining suspended after sludge has settled. In the process, pin point flocs pass out in the effluent. Two types : Grey, fly ash type, no BOD, inert. Pink/Brown, doesnt rise/settle, has high BOD. PERCOLATING FILTER :A bed of material relatively inert to natural processes of degradation ( such as slag, molded plastics, clinker etc. ) usually contained within circular or rectangular walls and so constructed that air is continuously present throughout the bed. In use, settled sewage is distributed uniformly over the upper surface and trickles through the bed to underdrains, thus giving rise to the development on the packing material of a biological film containing aerobic bacteria and fungi which about oxidation and clarification of the sewage. Essentially, the process does not rely on filtration except at the microscopic level and the bed is incorrectly described as a filter. The first as above were installed at Salford in 1897. RETENTION TIME :The period during which wastewater is retained within a unit. This is based on : Maximum Flow Average Flow or Dry weather Flow. Retention Time = Reactor Volume ( m3 ) : Total Daily Flow ( m3/day ) This is the amount of time which sewage undergoes aeration in the reactor. Also knows as Hydraulic Retention Time. Usually 6-10 hours. RETURN ACTIVATED SLUDGE :That portion of the activated sludge separated from the mixed liquor in the secondary settlement tanks, which is returned to the aeration tanks. Normally expressed as RAS. RETURN SLUDGE RATE :25% of influent is the normal rate. Sometimes reasserting sludge before mixing with more wastewater improves settlement. ROTATING BIOLOGICAL CONTACTOR :This is a fixed film type of effluent treatment system. A unit consisting of a series of closely spaced, parallel discs are mounted on a shaft, which is supported just above the surface of the wastewater to be treated. The lower parts of the discs therefore extend to the wastewater, as the discs are slowly rotated. A biological film or slime layer develops on the wetted surfaces as the are alternatively submerged, exposed to nutrients and raised out of the liquid to oxidize the absorbed nutrients. SELECTOR : Selected systems are more resistant to low F/M bulking. A selector is a mixing basin/channel where the RAS and influent wastewater mix before entering the aeration basin.

 Selectors can be aerobic, anaerobic ( DO, Nitrate absent ), anoxic ( DO absent, Nitrate present). Selectors should be designed to remove virtually all the soluble BOD in the wastewater. To do this selectors can be divided up into compartments. Sludge developed in successive compartments exhibit several important characteristics. Soluble BOD5 uptake and where appropriate, oxygen uptake rate is more rapid than for sludges developed in a non selector completely mixed system. The aim of the selector is to provide a short term, high substrate condition that favours certain floc formers, but discourages filaments. Hydraulic retention time in a selector varies from 30 minutes to about 2.0 hours depending on whether it is an aerobic or anaerobic selector. SETTLED VOLUME ( SV ) :Volume occupied by sludge after settling. Normal the 30 minutes Settled Volume is determined. Place 1 liter of mixed liquor in a clean ( glass ) graduated cylinder, sit for 30 minutes and read volume of sludge settled from graduations on cylinder. Result : ml of sludge per liter of mixed liquor. SLUDGE AGE :Number of days over which total mass of sludge wasted mass of sludge undergoing aeration when an activated sludge plan is operating in equilibrium. Sludge age ( days ) is calculated as follows : Volume of liquid in aeration tank ( m3 ) x MLSS in aeration basin ( mg/l ) : {Sludge waste rate ( m3/day ) x MLSS of waste sludge ( mg/l )} + {Effluent discharge rate ( m 3/day ) x TSS in final effluent ( mg/l )} Note : Effluent discharge rate = Influent flow rate. Typical sludge age values for the main activated sludge systems are as follows : Sludge Age ( days ) High rate : 1 - 3 Conventional : 3 - 8 Nitrifying : 8 - 12 VITOX : 5 - 25 Oxidation ditch : 60 - 90 Reciprocal of Sludge Age = Specific growth rate of sludge. SLUDGE DENSITY INDEX ( SDI ) :Prepared by Donaldson in the USA. It is a measure of the settleability of activated sludge. It is the weight of dry solids contained in 100 ml of sludge settled for 30 minutes as for SVI. The results are expressed as g/100 ml. SDI = MLSS (%) x 100 : Settled volume of sludge (%) after 30 min Good Settling = 2.0 g/100 ml, poor Settling = 0.3 g/100 ml It is the reciprocal of SVI x 100. SLUDGE LOADING :Weight ( quantity ) of BOD applied to an activated sludge plant per day per unit weight of activated sludge undergoing aeration. Same as F/M. SLUDGE VOLUME INDEX ( SVI ) :Is the volume ( ml ) occupied 1 gram of activated sludge after settling aeration basin mixed liquor for 30 minutes in a 1 litre graduated cylinder. It is used for day to day monitoring of settlement in an activated sludge plant. SVI is calculated by dividing the 30 minutes Settle Volume ( SV ) by Mixed Liquor Suspended Solids ( MLSS ). Units are ml/g. It was described by Mohlman in 1934. It is sometimes to as the Mohlman Index. e.g. for a MLSS of 3,000 mg/l ( 3.0 g/l ). STIRRED SPECIFIC VOLUME INDEX ( SSVI ) :This is a more accurate measure of sludge settleability. The settleability of a sludge may be monitored using SSVI at an MLSS concentration of 3.5 g/l. SSVI = *Height of sludge ( cm ) x 100 : Initial height of sludge ( cm ) x MLSS ( g/l ) ( * : Height after 30 minutes ) Sludge from an average activated sludge plant operating well should have an SSVI of less than 100 ml/g. Sludge with very good settling characteristics will have an SSVI of 40-50 ml/g. Sludge with poor settling will have SSVI in excess of 120 ml/g and very poor settling will be observed with an SSVI greater than 250 ml/g. This test utilizes a 10 cm WRC settling apparatus. To follow are normal SSVI values for the main types of activated sludge systems : SSVI ( mg/l ) High rate : 120 - 140 Conventional : 40 - 100 Nitrifying : 40 - 80 VITOX : 40 - 70 Oxidation ditch : 100 - 200 UNOX :This is an activated sludge process recently develop in the USA, in which a surface aerator is used for oxygenation the mixed liquor in an oxygen enriched atmosphere within an enclosed tank. VITOX :The VITOX system utilizes pure oxygen to mix the mixed liquor in the aeration basin and to provide a source of oxygen for bio-oxidation. The VITOX system was originally intended as a method of uprating the oxygenation capacity of an overloaded sewage treatment plant. In addition it has proved very effective in the treatment of high strength industrial wastes. Many operators of this system report very low sludge yields. More recently the potential of VITOX units as the first stage activated sludge process has been realized.

Volume - 7...
Appendix - 1...
A phenomenon, which occurs in, activated sludge plants when sludge/biomass occupies an excessive volume and does not settle readily so that in extreme cases the effluent from the final settlement tank contains an

Bulking Sludge...

excessive amount of suspended solids. It is usually associated with the presence and predominance of filamentous micro-organisms in the biomass. To Be More Specific : - The SSVI ( Stirred Specific Volume Index ) is greater than 150 ml/gram. ( Less than 150 ml/gram = "Good Settlement" and greater than 250 ml/gram = "Poor Settlement" ) - Sludge settles slowly. - Sludge does not thicken readily. - Sludge contains large numbers of filamentous micro-organisms. - Water associated with sludge quality is high with respect to BOD. - Insufficient dissolved oxygen for long periods of time. Do less than 0.5 mg/l ( Should be 1.0 3.5 mg/l ). - Septic sewage e.g. presence of sulphides. - Low pH. pH should be 6.0 8.0 ( Low pH can lead to fungal selection ). - Low F:M ratio i.e. Food is diminishing, biomass is increasing, F/M should be greater than 0.05, normally. - Influent nutrient levels are low. Ideally C : N : P should be 100 : 5 : 1. ( C is normally BOD C ). - Other trace metals may also be missing e.g. Iron, Sulpur.

Reasons for Bulking Occuring...

Filament Types as Indicators of Conditions Causing Activated Sludge Bulking...

Suggested Causative Condition Low D.O. ( For the applied organic loading ) Low Organic Loading Rate ( F:M ) ( In completely mixed aeration basins ) Septic Wastes / Sulphides Nutrient Deficiency ( N and / or P ) Low pH ( Below pH 6.5 ) Indicator Filament Type S.natans, Type 1701 and H.hydrossis M.parvicella, Nocardia species, H.hydrossis, Type 021N, Type 0041, Type 0675, Type 0092, Type 0581, Type 0961 and Type 0803 Thiothrix species, Beggiatoa species, Type 021N Thiothrix species, Type 021N, Type 0041 and Type 0675 Fungi

Volume - 8...
Appendix - 2...
Microscopic observation, for analysis purposes, of protozoa and other higher life forms in activated sludge is common. Generally the types present are indicative of plant performance and effluent quality. These organism can also be used as indictors of toxicity. These organisms are a necessary component of activated sludge and perform a number of functions : Coliform and pathogen removal through feeding activated which yield a clarified effluent. Contribute to biomass flocculation by production of mucus. Contribute to the break up of large floc masses due to motility. The main higher life forms observed are as follows : Protozoa Rotifers Nematodes and Annelids The protozoa are the main group of higher life forms looked for upon microscopic examination of an activated sludge sample. They are unicellular organisms and comprise the following : Flagellates e.g. Euglena Rhizopoda e.g. Amoebae Ciliates. These can be further divided into : - Free swimming ciliates e.g. Paramecium sp. - Attached/stalked ciliates e.g. Vorticalla sp., Carchesium sp. - Creeping ciliates e.g., Aspdisca sp.

Higher Life Forms...

Protozoa...

Protozoa are useful indicator organisms is an activated sludge, since in general, a sludge is suggested to be in a poor condition when there are few ciliates and many flagellates present. The latter diminish as the sludge improves, until, in a good sludge, ciliates predominate. These are much larger and more complex in structure than the Protozoa. They are multicellular organisms. They tend to be able to expand and contract. They possess a tail and a head. Most are motile and attach to flocs by a contractile root. Long cylindrical bodies. Multicellular, the end of the body is thinner than the central part. Nematodes are large enough to be seen at low magnification. They will be very motile and difficult to maintain in view. Most have growth rates of one per day or higher. Food availability larger determines the predominance of a group. ( a ) High prey density : Flagellates, amoebae and some small free swimming ciliates require a high prey density greater than 106 or 107 per ml. They require a high prey density because of inefficient chase and capture feeding mechanism. ( b ) Lower prey densities : At long SRT, low F/M, and lower prey densities, attached ciliates and rotifers predominate as they attached to the flocs and they also feed by ciliary action ( filter feed). The table below summaries this : Higher Life Forms Observed Versus Organic Loading ( F :M) Conditions Low Organic Loading Optimum Organic Loading High Organic Loading Predomimant Groups Stalked Rotifers and Nematodes Good diversity of organisms, dominated by free-swimming and stalked ciliates Flagellates, Rhizopoda, Free swimming ciliates

Rotifers...

Nematodes...

What Determines the Presence of Higher Life Forms ?...

Toxicity assessment is on of the most valuable applications of microscopic observation of microorganisms in activated sludge. Ciliates and rotifers are amongst the first to be impacted by toxic materials. In severe cases they die, which can result in foaming. Heavy metals and cyanide in particular have a stressful effect on these organisms.

Volume - 9...
Appendix - 3...
- The influent pH should be in the range pH 6.0 8.5. - Too low : Add NaOH or Ca(OH)2. - Too high : Add HCl ( Never H2SO4 ), sometimes Phosphoric acid depending on P. The aeration tank temperature should be in the range 8 oC 35 oC. Too high : Introduce cooler water if possible. Influent nutrient levels should ideally be C : N : P = 100 : 5 : 1. Introduce N and/or P if there is a deficiency. D.O. should between 0.5 3.5 mg/l. Too low : Increase aeration. Too high : Reduce oxygen supply or reduce MLSS by wasting sludge. Hydrophobic substrates such as oils and fats promote filamentous growth. Improve pre-treatment e.g. use a D.A.F/ system or divert temporarily it feed streams are multi-sourced.

Some Optimum Operating Conditions at a Treatment Plant... pH...

Temperature...

Nutrient Levels...

Dissolved Oxygen...

Hydrophobic Substrates...

Volume - 10...
Appendix - 4...
Assessment of a Plant Problem...
Questions to be considered : ( 1 ) The natural of the influent to be treated.

( 2 ) They type of plant e.g. Fixed Film, Oxidation Ditch, Activated Sludge. ( 3 ) The natural the problem ( a ) Is the problem temporary e.g. seasonal ? ( b ) Is the problem associated with inadequate biological activity, solids/liquid separation, inadequate pre-treatment ? ( 4 ) The hydraulic Load. The peak hydraulic load and when or how often does it peak. The average hydraulic load. ( 5 ) The BOD loading to the plant ( or COD). ( 6 ) Effluent Quality: Actual: BOD, COD, SOLIDS, AMMONIA. Required: BOD, COD, SOLIDS, AMMONIA. ( 7 ) The volume of the aeration basin/filter. ( 8 ) Is there pH control ? ( 9 ) Are flocculants added to improve settleability ? ( 10 )Are nutrients added to ensure C : N : P is about 100 : 5 : 1 ? ( 11 )Is there an effective process to remove solids ? ( 12 )What is the flow rate ( a ) Through the plant ? ( b ) To the aeration basin ? ( 13 )If an activated sludge plant. ( a ) What is the MLSS mg/l ? ( b ) D.O. mg/l ? ( c ) Sludge age ? ( d ) Temperature ? ( 14 )What is settlement like e.g.. from the aeration basin/oxidation ditch ? ( 15 )The sludge age.

Ciliates...
For the past few months we have examined the staples of the home fish breeders: the culture of phytoplankton species and the use of rotifers are fish frys first foods. Also we discussed the fact that a number of fish fry (such as Gobisomas sp., Centropyge sp. angels, Scorpaenopsis sp. and Dascyllus sp. Chromis sp. damsels) are too small to utilize rotifers as a primary food item and therefore we needed to consider the culture of helper food items to assist in the development of the fish larvae. One such helper organism is ciliates (this species and its home culture is described in detail below). While the culture of ciliates is not traditional for the home breeders they do appear to have useful applications, in so far as ciliates seem to have potential as a larval food or a bridging food item for marine fish and also as a planktonic food for some invertebrates. However, this optimism must be tempered with a dose of reality: rotifers adequately serve the basic needs of most commercial aquaculture programs and therefore serious research into ciliate culture has been neglected. Additionally, a species of ciliate that is suitable as a first food for small marine larvae may or may not be found, but it seems to be worthwhile to look for one. So why would we even consider writing a column on raising a food item which may not be useful to the home breeder? The answer is that in nature, ciliates are a critical (helper) food item. Ciliates are important in the transfer of nutrient material through coastal food webs, as these organisms act as a link between small phytoplankton and larger zooplanktons (Reid et al. 1991). Ciliates graze between 30 - 50 % of primary production in many marine systems and may be the dominant group (up to 100 %) of micro zooplankton in temperate coastal waters (Pierce & Turner 1992). The quantitative importance of protozoa as a food source of zooplankton is well established, and studies indicate that qualitative aspects of a protozoan diet may enhance survival and fecundity of some zooplankton species. The perfect food for marine larval fish has the following requirements. 1. It is just the right size for capture and ingestion by the larval fish. 2. It displays the proper behavior to stimulate larval fish to feed upon it. 3. All larval marine fish will avidly feed on this organism. 4. It can be cultured with little effort in great numbers in small containers. 5. Its reproductive cycle is completed in only a few days so that immense numbers are quickly attained. 6. It contains, or can be enriched to contain, all the proper nutrients for strong and healthy larval development. 7. The variations in size of the food organism are great enough to adequately feed a wide size range during development of the larval fish. 8. It can be maintained with a simple media that does not require extensive algae culture. The perfect food organism for larval marine fish has not yet been found. At least I dont know about it if it has. The rotifer, Brachionus plicatilis, is the closest that fish breeders have come to this ideal. It fulfils, for the most part, requirements 1, 2, 4, 5, 6, and to some extent 8, but it isnt perfect. Many first feeding larval marine fish are too small to take rotifers, the larvae of some species of fish will not feed on rotifers (although they are large enough to take them), and most larval fish outgrow the size range of rotifers before they no longer require a planktonic food organism. Culture of a substantial algal base as well as the vast numbers of food organisms required to feed even modest numbers of marine fish larvae can also be problematic with rotifers, especially for small hobbyists hatcheries. Brine shrimp, Artemia, are historically the quintessential food organism for larval fish and invertebrates. For some species, especially freshwater fish, Artemia fulfils all the above requirements, but for many species of marine fish it falls woefully short. Artemia nauplii are notoriously too large for the early larvae of most species of marine fish and the nutritive content of the nauplii are often not compatible with the requirements for normal larval development. For many species, however, rotifers followed by brine shrimp is a feeding protocol that can be made to work with nutritional enrichment

The Culture of Ciliates...

The Perfect Food...

and this is currently the paradigm for feeding marine fish larvae. A photomicrograph of brine shrimp nauplii, about 250 microns long, rotifers, about 125 microns long, and oyster larvae (veliger, bivalve mollusk larvae), about 20 - 30 microns long. The oyster larvae are in the size range of many ciliates. This photo is useful to compare the size of various food organisms suitable for various larval fish. Obvious to anyone that has reared, or tried to rear, the larvae of a number of species of marine fish, it is highly unlikely that any perfect food organism exists. Probably the closest one can come is some species of copepod since copepods have many desirable characteristics, especially wide size range and excellent nutritional qualities. But the long reproductive cycle is a formidable barrier in copepod culture. Because of the slow reproductive cycle, about 25 days, a relatively small culture vessel cannot produce enough copepods to satisfy the demands of very many fish larvae. A single species of copepod may have a size range from about 50 to 70 microns from the early instar to about 700 microns or more in the adult. But even at 50 microns the smallest copepod nauplii may be just a bit too large for some species of marine fish. Although most marine fish larvae select first prey organisms in the 50 to 100 micron range, many marine fish species, pygmy angels, tangs, some wrasses, parrot fish, butterfly fish, some damsels, and others have eggs that are between about 500 and 800 microns, and these small eggs produce small first feeding larvae that seem to require a first food organism in the 20 to 30 micron range, a bit below a copepod instar, quite a bit smaller than a rotifer at 100 microns, and very much smaller than a brine shrimp at 250 microns wide and 400 microns long. There are other issues in rearing these small-egged species of marine fish, but providing an acceptable first food organism, of the proper size and nutrition, and available in acceptable numbers, is the biggest bear in the woods. A range of sieves suitable for sizing food organisms for larval fish and various invertebrates. The sieves range from 25 microns to about 1000 microns in mesh size. A functional sieve with ample water volume above the mesh for concentrating organisms of the desired size can be made from various plastic containers by cutting off the bottom, cutting out the center of the screw on lid, and then fastening the lid back on the container with the mesh cloth between the lid and the container. Now it is quite possible for any marine aquarist to easily rear a marine organism much smaller than rotifers in incredibly vast numbers. These would be ciliates. There are about 8,000 named species in the Phylum Ciliophora, Kingdom Protoctista, and many more still unknown. The name Cili ophora means bearing eyelashes and this is a good description of the tiny, short, whip shaped flagella that cover most species of ciliates. These short, threadlike cilia function in feeding and locomotion. Typically, ciliates feed on bacteria and small algal cells, as well as take up nutrients from the surrounding aquatic environment. Most are free living, a relative few are parasitic or commensal. Most ciliates reproduce by transverse binary fission dividing along the shorter width of the cell, although stalked ciliates that attach to a substrate usually reproduce by budding. Ciliates are among the most complex of the eukaryotic single celled microorganisms. Ciliates have even developed a method for exchange of genetic material called conjugation. Two cells attach together, sometimes for several hours, and exchange micronuclei, which results in two individuals with essentially the same genetic complement. A free living ciliate, rather than a stalked species, has the greater potential as a first food organism for the smallest of marine fish larvae. The hypotrichid ciliate, Euplotes sp. so often found in rotifer cultures measures about 20 by 40 microns, a size that seems to be in the range of many small fish larvae. Dinoflagellates are also a potential food organism. Ciliates are animals and Dinoflagellates are classed as algae, but their rRNA relates these diverse groups. Many dinoflagellates are in the same size range as ciliates and are photosynthetic, but dinoflagellates may be more difficult to culture and some species are quite toxic, which could be a problem. Both ciliates and dinoflagellates are part of the microbial loop in the marine food web. The microbial loop is relatively new concept developed to explain and explore the interactions of the smallest elements of life in the sea, essential minerals, viruses, bacteria, small phytoplankton, etc., that are too small to be consumed by copepods, but are actively consumed by ciliates and flagellates. This cyclic food web at the foundation of the food chain supports the copepods that fuel the classical food chain. The point is that the sea is full of organisms that are below the average size of copepod instars and that these organisms may form a food base for the early larvae of the smallest egged fish. And on an elemental basis, the understanding of this microbial loop in the food web of the sea may have some bearing on the basic functions of marine aquariums, but I digress. There are many species of ciliates capable of living in the marine environment, both planktonic and benthic, and some, particularly in the genera Tintinnopsis and Euplotes, have potential as food organisms for very small fish larvae and perhaps invertebrates as well. One of the key requirements of any good larval food organism is that it must be capable of rapid reproduction, and must be able to sustain dense cultures in order to supply the quantity of food required to feed large numbers of larvae. Ciliates reproduce by division and so in the proper culture environment, reproduction can be very rapid. Other requirements, however, such as nutritive value and acceptability by larval fish as food organisms, are not as encouraging. Whether ciliates would work as an initial food to rear the tiniest of marine fish larva, or various invertebrates, is entirely another story. I have not had success with ciliates as a first food for marine fish larvae and I dont know of anyone else that has had success with them, but this certainly does not mean that no one has been successful with ciliates or that it is not possible to utilize ciliates as a first food. There are many variables. It may not be possible under relaxed culture conditions to maintain a specific species of ciliate. Contamination from other species may reduce or eliminate the target species in the culture, a species of the proper size or nutritive value may not develop in the culture, and ciliate cultures, just as rotifer cultures, can crash for no apparent reason. And even if the ciliates cultured are of the right size and have adequate nutrition and are actually consumed by the larvae, bacterial and/or fungal contamination of the ciliates may destroy the ciliate culture and/or the larva within a day or two. But if a species of ciliate is found that can serve as a first food organism for small fish larvae, all these difficulties can be resolved. I dont think that a current culture of an acceptable ciliate species exists. As far as I know, aquaculture labs do not have a useful species of ciliate (or

All about Ciliates...

dinoflagellate) under culture that is shared or researched as is rotifers. To this point, most aquacultured species do not require a food organism smaller than a rotifer and thus not much effort has been expended in finding and developing a smaller version of the rotifer. Marine ornamental breeders may have to find a suitable species of small food organism and develop the culture techniques for this species with little help from the commercial food fish and scientific sectors if the small-egged ornamental fish are to be widely bred. A small organism is needed that will thrive within the nutritional, temperature, and salinity parameters of a captive marine breeding system. So it makes sense to use these parameters as the foundation for efforts to find and maintain such an organisms. For the most part, ciliate culture is very similar to rotifer culture. This reminds me of an incident that occurred at the Aqualife Research Corporation facility at Walkers Cay in the Bahamas some years ago. We were culturing macro algae in some of the 300 gallon fiberglass grow-out tanks and on one morning when I was scheduled to return to Ft. Lauderdale for the weekend, I observed something interesting in one tank that had been scheduled for cleaning and had remained several days with aeration, but no water exchange. It was swarming with a tiny creature, apparently a ciliate, about half the size of a rotifer. The plane was warming up on the runway so all I had time to do was to leave strict instructions that the tank was not to be touched and to leave a sign on the tank, Do Not Clean. All weekend I thought of those little creatures and wondered if they could be the Holy Grail of small fish larval culture. Of course you know what I found when I returned in a few days. The tank was clean, the sign was still on the tank, and no one knew who cleaned the tank. I never saw that organism again, even though I tried a few times to replicate the situation that had developed that culture.The vegetable juice formula for culturing ciliates and rotifers can be handled much like a rotifer culture based on a phytoplankton food source. Instead of feeding the phytoplankton, the vegetable juice based formula is added periodically. In the photo, the upper jars are phytoplankton cultures and the two lower jars are young, about 3 days old, vegetable juice rotifer culture; and an old, about 2 weeks, vegetable juice culture. These vegetable juice cultures are generally useful for about two weeks.This is a clue, however, as to how to go about finding a microorganism that might, just might, fill that gap before rotifers or copepods. I ran across another potential piece to this puzzle during the time that I was working with culture of the orchid dottyback (Moe, 1997). I cultured this species, Pseudochromis fridmani, as a hobbyist would, in a small, modified bathroom in a house far from the sea (OK, just 20 miles). I started with the typical phytoplankton cultures for rotifers but early on, as do many hobbyists with limited time and facilities, I had difficulty maintaining the quality and quantity of phytoplankton cultures required to produce the vast numbers of rotifers that hordes of hungry larval dottybacks required. So during that project I developed a formula based on a popular commercial vegetable juice that I used to feed and maintain rotifer populations without, or at least greatly reducing, dependency on phytoplankton cultures. The formula for this vegetable juice based rotifer food is reproduced below with permission from the publisher of my dottyback book (Barbara). 1. Take one 11.5 oz. (340 ml) can of XX juice (I suppose any brand of vegetable juice would be acceptable) and strain it through a 500 micron sieve. Typical window screening is 1000 microns and those little stainless steel strainers you can buy in the supermarket are about 500 microns. This straining removes the larger particles that do not help the culture. 2. Dilute the strained juice with about one quart (950 ml) of cold fresh water. It is easier to strain the juice if it is diluted first or during the straining process. 3. Add two teaspoons of bakers yeast. The yeast is optional, it is mainly a feeding supplement to the juice particles, but I find that the culture is more stable in that food remains in suspension longer and this helps the rotifers maintain high population levels, and reduces the need for more frequent feedings. The amount, or even the use of yeast is a subject for future experimentation. 4. I then add several drops of an omega-3 fatty acid supplement (Super Selco, another type of fish food supplement or even an Omega-3 or fish oil supplement from a health food store) to the juice solution and also add a pre dissolved B vitamin complex tablet and a vitamin C tablet. Put the top tightly on the container and shake very well. It may well be that different supplements or different amounts of these supplements will produce a better rotifer food. Much experimentation remains to be done. This mixture is then kept in the refrigerator and a portion is fed to the rotifer cultures each day in an amount fitting to the purpose of the culture. I feed about 30 to 50 ml per day to each gallon jar of rotifers to maintain rotifer populations at low levels during periods between breeding projects. High production would require at least two, perhaps three similar feedings each day. Stir the formula well before feeding. One of the good news/bad news developments in working with this rotifer formula was that it was a superb media for ciliates, several different species, and several different sizes. One was approximately 10 microns and one was about 30 microns with some in-between and they occasionally flourished in vast numbers. I had to develop methods for screening out the rotifers and beginning new cultures when the rotifers begin to diminish. Allowing the culture to settle, siphoning off the rotifer/ciliate mix above the sediment and then passing the culture through a mesh of 53 microns separated the rotifers and ciliates quite well. (An interesting aside is that some aquaculture interests in Japan use ciliates to enhance the health of rotifer cultures since the ciliates feed on the bacteria in the cultures.) This gives us a tool to use in the search for a ciliate that may be useful in culture of some marine fish larvae. Other organic preparations, potatoes, straw, fruit juice, algae, etc., could also be used and there may well be a

Preparing the Rotifer Feeding Formula...

better base, but I would start with the vegetable juice formula above just because it worked well before. After preparation of the vegetable juice formula, the next step would be to make up several gallon jars of the formula and add light aeration to keep the mixture suspended and oxygenated. Only 30 to 50 ml of the mix is needed in each jar of salt water. Now all we have to do is to find a source for a species of ciliate that may be useful. Some ciliate species may be available from commercial educational cultures, such as Didinium, Paramecium, and Euplotes, and these can be tried, but a better possibility for a marine species may be a natural source. These experimental cultures can be seeded with live sand, live rock, or even water from a natural marine source. A bit of live sand and/or rock from an old established reef tank could also be tried. Experimentation with different salinities, temperatures, and sources of potential ciliates will probably result in a wide variety of cultured ciliates, which can be selected for the larger species. A microscope will be a most useful tool for this work, but a 10x loop might be adequate. Once a possible candidate species is found, right size, large numbers, one should try to develop a pure culture of that species. Seeding a new culture with a pure sample of only that organism should be attempted. However, without good laboratory technique, this may not be possible. In fact, it may be that ciliate cultures do better when some rotifers are present in the culture. Under primitive conditions, sometimes the best one can do is to start a new culture with as massive an inoculation of the target organism as is possible and hope that the head start given to the desired species will be enough to out grow the competition, at least initially. Keep the culture rolling gently with an air stone and watch it for a week or so. I'm sure you will get a wild culture of ciliates (who knows what species). Whether they will work as a successful larval food is another story. It is not difficult these days to keep a breeding pair or harem of pigmy angelfish, damselfish, occasionally mandarinfish, maybe a wrasse species or two, and some of the small egged gobies. These species, and others, can provide plenty of larvae for experimental first feeding trials. Add the food organisms at about 3 per ml to the larval tank maybe a day or the night before first feeding is expected. This is about the time the yolk sac on the demersally spawned larva is absorbed and about three days after pelagically spawned larvae hatch. At the time first feeding begins, two things should happen. The larval fish should have a full gut at all times except first thing in the morning, and the larval fish should grow noticeably in two or three days after feeding begins. Again, a 10X loop or better still, a dissecting microscope is very important. If these two things happen then the fish larvae are able to take the food organism and the food organism is at least nutritionally adequate. It is then time to break out the Champagne.