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Activated Sludge Analysis...

Evaluation of Settling and Foaming Properties of Activated Sludges...

Several quantitative characteristics of activated sludge settling, agglomeration, and foaming properties have been developed and employed in practice.

Zone Settling Velocity...

The basic settling characteristic is the so-called zone settling velocity (ZSV). The zone settling velocity represents a maximum rate of sedimentation and can be expressed as a slope of the linear part of the sedimentation curve. The sedimentation curve can be obtained by measuring the velocity of movement of a sludge-supernatant interface motion in high calibrated cylinders. ZSV depends on sludge concentration. This is treated by the solids flux theory.

Volume of Settled Sludge - Sludge Volume Index...

The volume of settled sludge after 30 minute sedimentation ( V30 ) is a base for calculating a sludge volume index SVI :

SVI ( mL / g ) = V30 / X

where ; X : is biomass concentration ( MLSS g / L ). The procedures of measuring V30 values and calculating SVIs unfortunately differ from author to author and in different countries as well. Variations of measuring V30 include unstirred SVI, SSVI (stirred sludge volume index), SSVI3.5 (stirred specific volume index measured at 3.5 g / L or the DSVI (diluted sludge volume index). Values reported in the literature are thus not easily comparable. There are many attempts in literature to find a relationship between the SVI and ZSV values. For practical needs, rough estimates from table given below can be applied.

Type of sludge SVI ( mL / g ) ZSV at 3.5 g / L ( m / h )
Well settling < 100 > 3
Light 100 - 200 2 - 3
Bulking > 200 < 1.2

Using the Data Obtained from the Settleometer Test...

The values you read during the settleometer test are usually read as SSVt. SSV stands for settled sludge volume and the subscript "t" represents a specific time during the test. For exmple, the SSV30 means the settled sludge volume at 30 minutes of settling time. The SSV values are usually recorded on the data sheet. At time = 0 min, the sludge occupies the entire volume of the settleometer, the SSV = 1,000 mL.

Obtained results are given in table shown below ;

Time ( minute ) SSV ( mL / L )
0 1,000
5 500
10 400
15 325
20 290
25 260
30 250
40 220
50 200
60 200

"Graphical Representation of the Results Obtained from Settleometer Test"...

Calculation of the SSC...

The second column on the data sheet is the calculated value Settled Sludge Concentration or SSC. The SSC is an estimation of the concentration of the sludge at each point in time during the settleometer test. This value helps you understand the concentrating characteristics of the sludge. The SSC is calculated using the Aeration Tank Concentration (ATC) from the centrifuge test. The ATC is an estimated concentration of the sludge (in percent) in the aeration tank or at time = 0 of the settling test. The SSC is calculated using the following formula ;

For example, if the ATC = 3 % and the SSV at 5 minutes is 500 mL / L, the calculated SSC at 5 minutes of the test would be ;

If all the SSC values are calculated, the data sheet will look like ;

Time ( minute ) SSV ( mL / L ) SSC ( % )
0 1,000 3.0
5 500 6.0
10 400 7.5
15 325 9.2
20 290 10.3
25 260 11.5
30 250 12.0
40 220 13.6
50 200 15.0
60 200 15.0

"The Graph of SSV and SSC"...

Using the RSC Value...

The Return Sludge Concentration (RSC) is also a value generated from the centrifuge test. The RSC represents the estimated concentration of the sludge as it is pumped from the bottom of the clarifier. If the RSC is located on the settleometer curve you can estimate the time the sludge is held in the clarifier before it is removed. In this example, it was used an RSC value of 12 %. If it was located the 12 % RSC value on the SSC curve, it can be found an estimated sludge detention time of 30 minutes. This means that the sludge was held in the clarifier for 30 minutes before it was removed. In this example, it might be wanted to leave the sludge in the clarifier a little longer to get it to a higher concentration before it is removed.

"The Graph of RSC Value on the Graph"...

"More Settleometer Curve Examples"...

Length of Filaments...

The most exact way to quantify the occurrence of filamentous microorganisms is by directly counting the total length of filaments protruding from flocs to bulk liquid. The total length is expressed per unit of volume or biomass (e.g., 103 m / g MLSS). The counting technique was developed and is frequently used for this purpose. As the microscopic measurement of the total length of all filaments in the sample would be too laborious and time-consuming, the filaments are divided into seven size groups (in micro-meter) ; (1) 0 - 10, (2) 10 - 25, (3) 25 - 50, (4) 50 - 100, (5) 100 - 200, (6) 200 - 400, (7) 400 - 800.

Then the numbers of filaments within the above intervals are counted. The length of filaments greater than 800 micro-meter is measured individually. The total length of the filaments found in the microscopic sample is multiplied by the dilution factor if the sample was diluted before the measurement. The total length of filaments of 104 m / g is considered to be an appropriate boundary between non-bulking and bulking activated sludges.

The counting technique was further simplified, especially for the quantification of filaments with an irregular shape (e.g. branched Nocardia spp filaments). The (diluted) mixed liquor sample of a known volume is placed on a glass slide and covered completely with a cover slip. The covered area is divided into fields. The eyepiece of the microscope is equipped with a simple hairline. By moving the objective from one edge of the cover slip to the other we observe the consecutive fields and count how often any filamentous microorganism intersects with the hairline. Then we sum up the number of intersections of all fields examined, multiply by the number of fields on the slide, and divide by the volume / concentration of the sample. The final result can be expressed as a filament count per volume or biomass unit. This method is suitable for evaluating some remedial actions in a particular plant against filamentous microorganisms.


For a routine examination of the extent of filamentous growth, a rapid and simple method was elaborated. The "subjective scoring of filament abundance" is a counting technique based on microscopic observation of a wet mount under phase contrast at 100 x magnification. The abundance of filamentous microorganisms is classified according to the scale given in table shown below from "none" to "excessive". Naturally, this method is not as exact and reproducible as the above methods of direct length measuring or filament counting. Nevertheless, if the subjective scoring is performed by the same individual for a long time, it provides a true picture of the biocenosis of a given activated sludge. Using this method, an experienced observer can reveal the tendency to bulking much earlier than the SVIs start to deteriorate, so that appropriate remedial actions can be accepted in advance.

Numerical value Abundance Explanation
0 None -
1 Few Filaments present, but only observed in an occasional floc
2 Some Filaments commonly observed, but not present in all flocs
3 Common Filaments observed in all flocs, but at low density (e.g. 1 to 5 filaments per floc)
4 Very common Filaments observed in all flocs at medium density (e.g. 5 - 20 per floc)
5 Abundant Filaments observed in all flocs at high density (e.g. 20 per floc)
6 Excessive They present in all flocs, appears more filaments than flocs and / or filaments growing in high abundance in bulk solution

Scum Index...

The so-called scum index (SI) was proposed for the quantification of biological foaming :

SI = ( 100 ) ( Mass of biomass in foam / Total mass of biomass )

The portion of biomass in the foam is estimated by means of fractionary flotation performed with a standard aeration rate of 10 m3 / m3 . h. The flotation is repeated with the sediments after separating the scum from the settleable biomass several times until all foam-forming microorganisms are transferred into the scum. The scum index test can be used for the prediction of operational problems expected as a result of the presence of foam-forming microorganisms in activated sludge, as shown in table given below.

SI ( % ) The extent of problems
0 - 0.5 Negligible
0.5 - 6 Small
6 - 10 Medium
10 - 15 Serious
> 15 Catastrophic

Filamentous Microorganisms...

Some thirty distinctly different filamentous microorganisms are important in activated sludge systems.

Role of Filamentous Microorganisms...

The role of filamentous microorganisms in the biocenosis of activated sludge can be evaluated from different viewpoints, but the main aspects are as follows ; (1) The filamentous microorganisms are believed to form a "backbone" of activated sludge flocs on which floc - forming bacteria are fixed by means of extracellular polymers. (2) The deterioration of activated sludge settling properties is caused by an excessive occurrence of filamentous microorganisms in the biocenosis. (3) The increased occurrence of filamentous microorganisms in the biocenosis of activated sludge indicates that the activated sludge system is not designed or operated properly.

It was suggested that the presence of certain filaments can indicate the conditions causing activated sludge bulking, such as DO, low F : M, low pH, increased concentration of sulfides, and nutrient deficiency. Unfortunately, at the present state of knowledge, the indicative role of filamentous microorganisms is rather questionable. For instance, Type 1701, a "typical" low DO microorganism, was observed in an aeration tank operated at high DO levels. Thiothrix caused serious bulking problems in a lab-scale activated sludge system with anaerobic zone. After the reinoculation, however, the activated sludge did not display any excessive growth of Thiothrix filaments although all operational parameters remained equal as during the bulking period. Furthermore, as will be shown later, there are certain filaments that are connected with both bulking and foaming problems and it is difficult to predict in advance what kind of nuisance they will cause. On the other hand, if we were able to recognize more exactly all growth and nutrition requirements of filamentous microorganisms, then the correlation between their occurrence and bulking cause should be tighter. Clearly, better understanding of the causes of bulking is still needed.

Needs for Identification and Classification...

In the case of filamentous bulking, it is also useful to know the kind of filamentous microorganisms present and the extent of their growth. The identification of filamentous microorganisms and their role in a proper plant operation was discussed. The identification of filamentous microorganisms according to types by Eikelboom is now the most common method and Eikelboom's and Jenkins' manuals have become indispensable tools for wastewater treatment plant operators. The quantification of the occurrence of filamentous microorganisms in activated sludge biocenosis is necessary for the exact prediction of bulking problems intensity. It should be stressed, however, that the knowledge of the quantitative occurrence always has to be combined with the identification of the filaments. It is a well-recognized fact that different types of filamentous microorganisms cause different intensity of bulking problems (e.g., Type 021N vs Microthrix parvicella bulking).

A correct finding of the taxonomic position is necessary for the documentation of a microorganism and for its preservation in microbiological collections. Once the microorganism is positioned taxonomically, the identification of isolates from activated sludge becomes much faster and easier. The knowledge of the taxonomic position will also help in recognizing growth and nutrition requirements of a given microorganism by comparison to other strains within a genus or family. Such correct taxonomic identification is still very difficult and expensive. Fortunately, the real needs of engineers are more prosaic. A microbiological examination of the bulking and/or foaming activated sludge must result in a kind of information that enables the engineers ;

(1) To find out or predict the extent of filamentous bulking or foaming, i.e., not only to quantify the number or length of filaments but also to specify the type of filaments present in a particular sludge. It is a recognizable fact that all filamentous microorganisms do not deteriorate settling properties of activated sludge in the same manner. Much more severe bulking problems are to be expected when Sphaerotilus spp., Type 021N, and Thiothrix are the dominating filaments in comparison with, for example, Microthrix parvicella, Haliscomenobacter hydrossis, and Nostocoida limicola bulking. The foaming problems threaten only when particular filaments are present.

(2) To estimate the causes of the presence of filamentous microorganisms in the biocenosis. By making such an estimate other factors should also be considered, especially wastewater character and composition, aeration basin configuration, and all basic operational parameters. It is evident that neither a microbiologist nor an engineer, however experienced, can trace the causes himself. A close cooperation hetween the specialists is of extreme importance.

(3) The rectify the bulking and foaming problems. This is in fact the ultimate objective of our effort. The information resulting from the identification of filamentous microorganisms must help in selecting proper measures against excessive growth.

Possibilities of Identification...

The conventional method of identification of bacteria or other microorganisms is based on their isolation from mixed cultures and on subsequent exhaustive tests of morphological, biochemical, and physiological features. The results of these tests are then compared with standard references given in the manual such as "Bergey's Manual of Determinative Bacteriology". The main advantage of the conventional method seems to be in the unambiguous taxonomic location obtained in this way. But at present the taxonomic position of a given microorganism without genetic studies can hardly be considered as "definite". For the purpose of isolating the filamentous microorganisms from the diverse biocenosis of activated sludge, the conventional methods are too cumbersome, time-consuming, and mostly unnecessary or even inappropriate because of the following reasons ;

(1) There is a need of at least thirty stable features and as many as possible variable features (e.g., morphology of cells and cell agglomerates including trichomes) for a formal recognition of nomenclatoric taxon.

(2) There is still a severe lack of pure cultures of filamentous microorganisms originating from activated sludges that isolates can be compared with.

(3) There is a great probability that many morphological, physiological, and genetic changes may occur in the filamentous microorganisms within the pure cultures from bacteriological collections as a result of various undefinable influences on them in wastewater treatment plants.

(4) There are many difficulties in preparing a pure culture inoculum from the mixed culture of activated sludge, especially due to the removal of accompanying cells of other microorganisms.

(S) The low growth rates of most filamentous microorganisms cause the isolation and identification to take weeks or months. Thus, the results obtained do not correspond with the actual state of the biocenosis in the treatment plant the isolates originate from.

Identification of Types...

The identification and classification of types represented a great break in the identification of activated sludge filamentous bulking and foaming. Eikelboom's proposal was immediately accepted by most of the researchers and practitioners interested in the field throughout the world, though there were several modifications to the method.

The techniques are based on phase contrast microscopic observations of morphology, the relationship to other organisms present, and staining characteristics (Gram stain, Neisser stain and observation of sulfur and PHB granules). The filamentous microorganisms are classified (with several exceptions) into so-called types according to the following features ; cell shape dimensions, presence of sheath, morphology of filaments, staining characteristics, and presence or absence of polyphosphate, sulfur, and PHB granules. The taxonomic position of most of the types is uncertain. From twenty-nine categories of filamentous microorganisms only a few represent undoubted taxonomical species and most of them are not yet fitted into the accepted microbiological classification systems. However, this fact in no case contradicts the enormous advantages of this classification system. The identification of types according to Eikelboom and Jenkins is fast and can be performed even by trained non-microbiologists. Despite the fact that the types do not represent taxonomical species, their description meets all the practical needs for identification.

Classification According to Morphology and Metabolic Properties...

The nuisance filamentous microorganisms can be divided into four groups according to morphological, physiological, and metabolic similarity, their occurrence under the same operational conditions, and problems they cause ;

(1) Oxic zone growers S : Symbol S means sheathed, Sphaerotilus - like filamentous microorganisms. They are capable of utilizing organic substrate only under oxic conditions. Their occurrence in activated sludges is connected with saccharidic or other readily biodegradable substrates, higher sludge ages, and lower DO concentrations.

(2) Oxic zone growers C : Symbol C means Cyanophycae - like filamentous organisms, utilizing organic substrate under oxic conditions. Contrary to the first group, some of these filaments are connected with sulfur metabolism (Type 021N and especially Thiothrix spp.), so that the energy can be obtained from anaerobic desulfatation and oxic oxidation of reduced sulfur compounds. The causes of filamentous bulking in this group are mainly the presence of readily biodegradable compounds in wastewater, higher sludge ages, and nutrient deficiency.

(3) All zone growers A : Symbol A means filamentous microorganisms capable of utilizing substrate under all, i.e. anaerobic, anoxic, and oxic conditions. The metabolic and kinetic properties of these filaments from group A are very similar to those of floc - forming bacteria, which may explain the exceptionally observed failures of selectors in suppressing the growth of filamentous microorganisms.

(4) Foam - forming microorganisms F : These microorganisms exhibit the ability to produce biosurfactants and have hydrophobic cell membranes, which enable them to froth and create scum. The problems with biological foam are often increased by unsuitable design and operation of plants (entrapment of the foam in the system and its recirculation).


For all of those dedicated activated sludge microscopists out there, these images will remind you of the quick snap of a contractile myoneme that catches your eye at the edge of a field, the scurrying of out of view of a grazing ciliate and the smooth glide of a Paramecium in the otherwise immobilized world beneath a coverslip. For many of us, protozoa are usually a pleasant diversion during filament identification.

To commence this part of the ASP we, at the Biotechnology Research Centre, decided to present some images that were originally taken to show the clearest view of the organisms concerned, with little regard for the "artistic" or "esoteric" aspects that are possible. All of the images below are light micrographs, but we will happily accept electron micrographs as well.

We include these micrographs to encourage others send us images that we can include in this segment of the ASP, and we will provide prizes for the best/most artistic micrographs (what ever that actually is). We ask that the images are not computer enhanced, and the format we have decided upon is not bigger than approx. 300 pixels square at 72 pixels per inch (28 pixels per cm) resolution. JPEG compression should be used to produce a high (not maximum) quality image using, for example Adobe Photoshop or a similar program. The final image should have a file size of not more than 30k.

We will ask visitors to our site to cast votes by e-mail on the presented images in the ASP, failing that, the editorial group will make the choice. When you send us an image by post, or as an JPEG e-mail attachment, we will prepare it to suit our format. We cannot promise to present all images received, but we will do our best. Also send us any technical data, magnification etc. All of the images shown here were scanned at 150 pixels per inch from original micrographs prepared on Kodak 25 ASA colour print film or Ilford FP4 Plus. Images here were taken by Beth Seviour or Janelle Trewella as indicated.

Not being specialists in this area we are assuming that many people can do a lot better than us and look forward to reader contributions. We will offer a prize for the best contribution in each issue. Depending upon the response we may extend the gallery to include light and electron micrographs of other organisms.