Methods 23

TABLE 2.15
Subjective Scoring of Filament Abundancea,b
Numerical Value Abundance Explanation
0 None No Þlaments observed
1 Few Filaments present, but only observed in an occasional ßoc
2 Some Filaments commonly observed, but not present in all ßocs
3 Common Filaments observed in all ßocs, but at low density (1 to 5 Þlaments per ßoc)
4 Very common Filaments observed in all ßocs at medium density (5 to 20 per ßoc)
5 Abundant Filaments observed in all ßocs at high density (>20 per ßoc)
6 Excessive Filaments observed in all ßocs (more Þlaments than ßoc and/or Þlaments
growing in high abundance in bulk solution)
a This 0 to 6 scale represents a 100- to 1000-fold range of total extended Þlament length.
b See Figure 2.8 for examples of Þlament abundance scores.

from a common origin (Figure 2.19a and Figure 2.19b).
Gonidia are oval- or rod-shaped cells present at the Þla-
ment tip that are distinctively different in appearance from
the rest of the vegetative cells further down the Þlament
(Figure 2.19c and Figure 2.19d). Rosettes and gonidia
indicate that the organisms are growing rapidly.
11. Filamentous Organism Identification
a. Using the Dichotomous Key
Characterize the Þlamentous organisms to genus or to a
numbered “type” using the dichotomous key shown in
Figure 2.20. This procedure is simpliÞed in two ways.
First, only a limited number of characteristics are used to
describe the organisms (Figure 2.20 and Table 2.18). Sec-
ond, only the 23 Þlamentous bacteria most commonly
observed in activated sludge are listed. The infrequently
observed Types 1702 and 1852 are omitted.
To further simplify the key, several Þlamentous organ-
isms having readily identiÞable speciÞc characteristics are
not included and are described separately. These include
fungi, Cyanophyceae, Flexibacter spp., Bacillus spp., and
actinomycetes.
This dichotomous key is a modiÞcation of the Þla-
mentous organism identiÞcation key of Eikelboom and
van Buijsen (1981). Changes have been made to de-empha-
size the need for the observation of cell septa (cross-walls),
which can depend on the quality and adjustment of the
microscope used, and to include some Þlamentous organ-
isms in the key twice where an important characteristic is
variable, e.g., the Gram stain reaction for N. limicola II
and the observation of intracellular sulfur granules for
Types 0914 and 021N.
Using this key is not without risk because some Þla-
mentous organism characteristics vary and the key cannot
always address all variables. Carefully check all the Þla-
ment types found by using the key (Figure 2.20) against
the typical organism characteristics listed in Table 2.18
and presented in the descriptions and photographs of each
organism. If the characteristics cited in Table 2.18 or
shown in the photographs do not correspond to the Þla-
ment type determined by using the key, carefully reexam-
ine the characteristics used in the key. For example, Type
0041 usually produces a weak Gram-positive or Gram-
variable reaction and may be correctly identiÞed from the
dichotomous key. However, if strongly Gram-positive, it
would be keyed as N. limicola II. Table 2.18 shows that
N. limicola II is coiled and free of attached growth, while
Type 0041 is straight or smoothly curved and most often
has substantial attached growth. The Gram stained slide
should be re-examined.
Occasionally, a Þlamentous organism observed cannot
be placed in a type or genus designated in the key.
Describe the organism characteristics and report it as “not
identiÞed.” Do not try to “force Þt” an organism into
existing Þlament types.
b. Building Your Skills
One of the hardest parts of Þlamentous organism identiÞ-
cation is building the conÞdence to make an identiÞcation.
To develop this conÞdence (1) characterize the Þlamen-
tous organisms in a sample and send the same sample to
someone skilled in the technique (some treatment plants
in a particular area established round-robin Þlament iden-
tiÞcation procedures to assist each other); and (2) take a
course on microscopic characterization of activated sludge
and Þlamentous organism identiÞcation.
The task is not as daunting as it might at Þrst appear
to someone assigned to a particular treatment plant. It is
doubtful that someone in such a situation would encounter
all the Þlamentous organism types listed in Table 2.18. It
is common to deal with about half a dozen that eventually
can be recognized easily.
12. Filamentous Organism Descriptions
The individual Þlamentous organism descriptions presented
here are based on our experience with characteristics of
Þlamentous organisms found in activated sludges all over

Copyright 2004 by CRC Press LLC.

 24 Manual on Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems

FIGURE 2.8 Phase contrast micrographs of Þlament abundance categories using the subjective scoring system: (a) few, (b) some,
(c) common, (d) very common, (e) abundant, and (f) excessive. (Original magniÞcation 100¥.)

the world. Data from Eikelboom and van Buijsen (1981)
and Eikelboom (2000) on samples from northern Europe
are also incorporated.
a. Sphaerotilus natans (Figures 2.9c, 2.13b,
2.14f, and 2.21a)
Filament Description — Straight or smoothly curved
Þlament that extends from the ßoc surface. Filaments are
generally 100 to ≥500 mm in length. Cells are sausage-
shaped with indentations at the cell septa and typically
1.6 ¥ 2.5 mm in dimension. Cell shape can be rectangular
when cells are packed tightly within the sheath. A sheath
is present; false branching is usually present. Attached
growth is generally absent, but may be present if the
Þlament is not growing. Not motile.
Staining Reactions — Gram-negative and Neisser-
negative.
SulÞde Oxidation — Negative.
Key Characteristics — Large size, false branching,
and sheath.
b. Type 1701 (Figure 2.21b)
Filament Description — Straight, smoothly curved, or
bent Þlament commonly found within the ßoc or can
extend from the ßoc surface. Filaments are generally 10 to
150 mm in length. Cells are sausage-shaped and typically

Copyright 2004 by CRC Press LLC.

 Methods 25

TABLE 2.16
Floc Characterization/Filamentous Organism Identification Worksheet

Sample number ___________________ Sample location ___________________

Sample date ______/______/______ Observation date ______/______/______

Comments:

Observations of:
Protozoa
Metazoa
Wet mount observation: 1000¥ phase contrast for Þlament characteristics
1000¥ direct illumination for stains

Filament number 1 2 3 4 5
Branching

Motility

Filament shape

Filament location

Attached growth

Sheath

Cross walls

Filament diameter, mm

Filament length, mm

Cell shape

Cell size, mm

Sulfur deposits

Other granules

Gram stain

Neisser stain

Commonness

Rank

IdentiÞcation

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 26 Manual on Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems

TABLE 2.17
Floc Characterization/Filamentous Organism Identification Worksheet (Page 2)

Sample number ___________________ Sample location ___________________

Sample date ______/______/______ Observation date ______/______/______

Filament Abundance:

0 1 2 3 4 5 6
None Few Some Common Very Common Abundant Excessive

Little or Bridging Open Floc

Filament Effect on Floc Structure: None Structure

Morphology of Floc:

Firm Weak Round Irregular Compact Diffuse

Free cells in suspension Neisser positive cell clumps

Zoogloeas India ink test
Spirochaetes Chlorine damage to Þlaments
Inorganic/organic particles

Filamentous Microorganism Summary:

Rank Abundance Rank Abundance

Nocardioforms M. parvicella

Type 1701 Type 0581

S. natans Type 0092

Type 021N Type 0803

Thiothrix spp. Type 1851

Type 0041 Type 0691

H. hydrossis Type 0675

N. limicola Other

Fungus Other

Remarks: ______________________________________________________________________________________________________________
_______________________________________________________________________________________________________________________
_______________________________________________________________________________________________________________________
_______________________________________________________________________________________________________________________
_______________________________________________________________________________________________________________________
_______________________________________________________________________________________________________________________

Copyright 2004 by CRC Press LLC.

 Methods 27

FIGURE 2.9 Phase contrast micrographs of branching of

Þlamentous organisms in activated sludge: (a) and (b) true
branching (fungus and nocardioform organism), (c) false
branching (Sphaerotilus natans). (Original magniÞcation
1000¥.)

0.8 mm to 1.0 ¥ 1.5 mm in dimension with indentations at
the cell septa. A sheath is present and attached growth gen-
erally is present. False branching occurs rarely. Not motile.
Staining Reactions — Gram-negative and Neisser-
negative.
SulÞde Oxidation — Negative.
Key Characteristics — Small size, usually sausage-
shaped cells; usually inside ßocs with ßocs formed around
Þlaments.
c. Haliscomenobacter hydrossis (Figure 2.21c)
Filament Description — A rigidly straight or bent Þla-
ment that extends from the ßoc surface; may be found
within the ßoc or free in suspension. Filaments are 10 to
100 mm in length and 0.5 mm in diameter. Cell septa are
not present; however, empty spaces in the Þlament may
appear as cells. A sheath is present and attached growth
can be present. False branching occurs rarely. Not motile.
Staining Reactions — Gram-negative and Neisser-
negative.
SulÞde Oxidation — Negative.
Key Characteristics — A very thin, small Þlament
that can be overlooked if not examined at magniÞcations
greater than 100¥; has “pins in a pincushion” appearance.
d. Type 021N (Figures 2.11b, 2.13d, 2.14d,
2.14e, 2.15c, 2.18a, 2.19b, and 2.22a)
Filament Description — A smoothly curved Þlament that
extends from the ßoc surface. Filaments are 50 to
≥500 mm long and 1.6 to 2.5 mm in diameter. Indentations
are present at the cell septa. Cell shape is quite variable
(rectangular, oval, barrel-shaped). Individual cell dimen-
sions are typically 2.0 ¥ 1.5–2.0 mm. Filaments often taper
from a thicker basal region with an inconspicuous holdfast
cell to a thinner apical region, often terminating in loosely
attached gonidia (sausage-shaped) cells. Rosettes (many
Þlaments radiating outward from a common ßoc) may
occur. A sheath is absent and attached growth does not
occur. No branching and not motile.
Staining Reactions — Gram-negative and Neisser-
negative.
SulÞde Oxidation — Positive but variable. Intracel-
lular spherically shaped sulfur granules may be present in
situ or after performing the S test.
Key Characteristics — One of the largest and longest
Þlaments. Indentations at cell septa. Cell shape varies from
rectangular to oval or barrel-shaped. Rosettes and gonidia
may occur. If growing on sulÞde, intracellular sulfur gran-
ules are usually present and the S test will be positive.
e. Thiothrix I (Figures 2.14b, 2.15a, 2.19c,
and 2.23a)
Filament Description — A straight or smoothly curved
Þlament that extends from the ßoc surface. Filaments are
100 to 500 mm in length and 1.6 to 2.5 mm in diameter.
The cell shape is rectangular and cells are 1.6 to 2.5 mm ¥
2.0 to 4.0 mm in dimension. There are no indentations at
the cell septa. Filaments often taper from a thicker basal
region with an inconspicuous holdfast cell to a thinner

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 28 Manual on Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems

FIGURE 2.10 Phase contrast micrographs of Þlament shapes: (a) straight, (b) smoothly curved, (c) bent, (d) irregularly shaped chain
of cells, (e) coiled, and (f) mycelial. (Original magniÞcation 1000¥.)

apical region, often terminating in loosely attached
gonidia (sausage-shaped) cells. Rosettes (many Þlaments
radiating outward from a common ßoc) may occur. A
relatively thick sheath is present generally without
attached growth. Attached growth only occurs if this Þla-
ment is not growing. No branching and not motile.
Staining Reactions — Generally Gram-negative and
Neisser-negative; may stain Gram-positive when growing
on sulÞde.
SulÞde Oxidation — Positive but variable. Intracel-
lular spherically shaped sulfur granules may be present in
situ or after performing the S test.
Key Characteristics — One of the thickest Þlaments,
typically 2.0 to 2.5 mm, but sometimes as large as 4 mm
in diameter. Intracellular sulfur granules are usually
present and the S test is positive if growing on sulÞde.
f. Thiothrix II (Figures 2.19d, 2.23c, and 2.23d)
Filament Description — A straight or smoothly curved
Þlament that extends from the ßoc surface. Filaments are
typically 50 to 200 mm in length and 0.8 to 1.4 mm in
diameter. The cell shape is rectangular and cells are 0.8 to
1.4 mm ¥ 1.5 to 3.0 mm in dimension. There are no inden-
tations at the cell septa. Filaments often taper from a thicker

Copyright 2004 by CRC Press LLC.

 Methods 29

FIGURE 2.11 Phase contrast micrographs showing loca-

tions of Þlamentous organisms: (a) free ßoating (dispersed),
(b) extending from ßoc surface, (c) within ßoc. (Original
magniÞcations 1000¥, (a); 1000¥, (b) and (c).)

FIGURE 2.12 Phase contrast micrographs showing attached growth of bacteria on Þlamentous organisms: (a) heavy, (b) incidental.
(Original magniÞcation 1000¥.)

basal region with an inconspicuous holdfast cell to a thin-
ner apical region, often terminating in loosely attached
gonidia (sausage-shaped) cells. Rosettes (many Þlaments
radiating outward from a common ßoc) may occur. A rel-
atively thin (hard to observe) sheath is present, generally
without attached growth. Attached growth may occur when
the Þlament is not growing. No branching and not motile.
Staining Reactions — Generally Gram-negative and
Neisser-negative; may stain Gram-positive when growing
on sulÞde.
SulÞde Oxidation — Positive but variable. Intracel-
lular spherically shaped sulfur granules may be present in
situ or after performing the S test.
Key Characteristics — Thiothrix II is generally about
1.0 mm in diameter and can be confused with several other
Þlaments of the same diameter. It differs from other sim-
ilarly sized Þlaments in that it is straight or smoothly
curved and extends away from the ßoc surface. Intracel-
lular sulfur granules are usually present and the S test is
positive if growing on sulÞde.
g. Type 0914 (Figures 2.15d, 2.24a, and 2.24b)
Filament Description — A straight or smoothly curved
Þlament that extends from the ßoc surface, is inside the
ßoc or is dispersed in suspension. Filaments are typically
50 to 200 mm in length and 1.0 to 1.2 mm in diameter.

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 30 Manual on Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems
FIGURE 2.13 Phase contrast micrographs showing appearances of sheaths: (a) and (b) Sphaerotilus natans, (c) Type 0041, and (d)
empty cells in Type 021N. (Original magniÞcation 1000¥.)
The cell shape is square without indentations at the septa
and cells are 0.8 to 1.2 mm ¥ 1.0 mm in dimension. A
relatively thin (hard to observe) sheath is present. Attached
growth is common but may be absent, particularly when
growing dispersed. No branching and not motile.
Staining Reactions — Generally Gram-negative and
Neisser-negative; may stain Gram-positive when growing
in the presence of sulÞde.
SulÞde Oxidation — Positive but variable. Square-
shaped intracellular sulfur granules may be present in situ.
This Þlament does not respond to the S test.
Key Characteristics — Square-shaped irregular sul-
fur granules. Type 0914 can be confused with several
Þlaments of similar size if attached growth is present and
sulfur granules are absent. Its sheath is difÞcult to observe
but is indicated by the presence of attached growth. Eikel-
boom (2000) noted that Types 0914 and 0803 are often
complementary, i.e., when Type 0914 disappears, type
0803 appears, and vice versa. Based on this observation,
he suggests that Types 0914 and 0803 may be two forms
of the same organism.
h. Beggiatoa sp. (Figures 2.15b, 2.22c, and 2.22d)
Filament Description — A straight or smoothly curved
Þlament that extends from the ßoc surface or is dispersed
Copyright 2004 by CRC Press LLC.
in suspension. Filaments are typically 100 to >500 mm in
length and 2.0 to 4.0 mm in diameter. The cell shape is
rectangular without indentations at the septa; however, the
cell shape is masked when intracellular sulfur granules
are present. The cells are 2.0 to 4.0 mm ¥ 6.0 to 8.0 mm
in dimension. Sheath, attached growth, and branching are
absent. Beggiatoa spp. are usually motile and exert ßexing
and gliding motions.
Staining Reactions — Generally Gram-negative and
Neisser-negative; may stain Gram-positive when contain-
ing many intracellular sulfur granules.
SulÞde Oxidation — Generally positive. Intracellular
spherically shaped sulfur granules may be present in situ
or after performing the S test.
Key Characteristics — Gliding, ßexing motility and
large size.
i. Nostocoida limicola I (Figure 2.25a)
Filament Description — An irregularly curved Þlament
intertwined within the ßocs. Filaments are typically 40 to
100 mm in length and 0.8 to 1.0 mm in diameter. The cell
shape is oval and cells are 0.8 to 1.0 mm ¥ 0.8 mm in
dimension. Indentations occur at the cell septa. No sheath
is present and no attached growth occurs. No branching
and not motile.
 Methods 31

FIGURE 2.14 Phase contrast micrographs of Þlamentous

organism cell shapes: (a) square, (b) rectangular, (c) oval,
(d) barrel, (e) discoid, (f) sausage, and (g) irregular. (Orig-
inal magniÞcation 1000¥.)

Copyright 2004 by CRC Press LLC.

 32 Manual on Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems
FIGURE 2.15 Phase contrast micrographs of intracellular sulfur granules: (a) Thiothrix sp., (b) Beggiatoa sp., (c) Type 021N, and
(d) Type 0914. (Original magniÞcation 1000¥.) (See color insert following page 70.)
FIGURE 2.16 Direct illumination micrograph of PHA staining.
PHA granules are dark blue/black. (Original magniÞcation
1000¥.) (See color insert following page 70.)
Staining Reactions — Gram-positive and Neisser-
positive; may be Gram-negative and Neisser-negative in
activated sludge treating some industrial wastewaters.
SulÞde Oxidation — Negative.
Key Characteristics — Typically intertwined within
the ßocs; readily identiÞed by its Neisser-positive staining
reaction and the presence of individual cells. This Þlament
can be confused with Type 0092 because both are Neisser-
positive and M. parvicella because both are Gram-positive,
Copyright 2004 by CRC Press LLC.
but N. limicola I has individual cells that are not observed
for these other Þlaments.
j. Nostocoida limicola II (Figures 2.10e, 2.11c,
2.17b, 2.18e, and 2.25b)
Filament Description — An irregularly curved Þlament
that extends from the ßoc surface and is present within
the ßoc. Filaments are typically 50 to 200 mm in length
and 1.4 mm in diameter. The cell shape is oval and cells
are 1.4 ¥ 1.0 mm to 1.5 mm in dimension. Indentations
occur at the cell septa. No sheath is present and no attached
growth occurs. No branching and not motile.
Staining Reactions — Gram-positive or -negative and
Neisser-positive; generally Gram-positive in municipal
wastewater activated sludge but often Gram-negative in
industrial wastewater activated sludge (especially in pulp
and paper wastewater activated sludge); is occasionally
Neisser-negative in industrial wastewater activated sludge.
SulÞde Oxidation — Negative.
Key Characteristics — Neisser-positive staining
reaction and the presence of individual cells. If Neisser-
negative, its cell structure is usually sufÞcient for identi-
Þcation. In stained preparations, the structures appear like
“runs” in nylon stockings. Branching does not occur. An
actinomycete may be confused with this Þlament, but the
branching of the actinomycete separates these Þlaments.
 Methods 33

FIGURE 2.17 Direct illumination micrographs of Gram staining of Þlamentous organisms: (a) improperly decolorized ßoc retaining
Crystal Violet, (b) Gram-negative Nostocoida limicola II, (c) Gram-variable Type 0041, (d) Gram-variable Type 1851, (e) Gram-
positive nocardioform organism, and (f) Gram-positive Microthrix parvicella. (Original magniÞcation 1000¥.) (See color insert
following page 70.)

k. Nostocoida limicola III (Figure 2.25c)
Filament Description — An irregularly curved Þlament
that extends from the ßoc surface. Filaments are typically
100 to 300 mm in length and 2.0 mm in diameter. Cell
shape is oval to discoid and cells are 2.0 mm ¥ 1.5 mm in
dimension. Indentations occur at the cell septa. No sheath
is present and attached growth does not occur. No branch-
ing and not motile.
Staining Reactions — Gram-positive or -negative and
Neisser-positive; is generally Gram-positive in municipal
wastewater activated sludge and generally Gram-negative
in industrial wastewater activated sludge (especially pulp
and paper wastewater activated sludge); is occasionally
Neisser-negative in industrial wastewater activated sludge.
SulÞde Oxidation — Negative.
Key Characteristics — Neisser-positive staining
reaction, large size and individual discoid-shaped cells. If
Neisser-negative, its cell structure is usually sufÞcient for
identiÞcation. Eikelboom (2000) no longer recognizes
N. limicola II as separate from N. limicola III.

Copyright 2004 by CRC Press LLC.

 34 Manual on Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems

FIGURE 2.18 Direct illumination micrographs of Neisser staining of Þlamentous organisms. (a) Neisser-negative Type 021N, (b)
and (c) Neisser-negative with positive granules (nocardioform organisms and Microthrix parvicella, respectively), (d) and (e) Neisser-
positive (Type 0092 and Nostocoida limicola II, respectively), (f) false Neisser-positive stain covering Þlament, Type 0041. (Original
magniÞcation 1000¥.) (See color insert following page 70.)

l. Type 0411 (Figure 2.26a) SulÞde Oxidation — Negative.

Filament Description — An irregularly curved Þlament
that extends from the ßoc surface. Filaments are typically
50 to 150 mm in length and 1.0 mm in diameter. The cells
are shaped like elongated rods and are 0.8 to 1.2 mm ¥
2.0 to 5.0 mm in dimension. Indentations occur at the cell
septa. No sheath is present and attached growth does not
occur. No branching and not motile.
Staining Reactions — Gram-negative and Neisser-
negative.
Key Characteristics — Long sausage-shaped cells;
irregular Þlament shapes.
m. Type 0961 (Figure 2.26b)
Filament Description — A straight or smoothly-curved
Þlament that extends from the ßoc surface. Filaments are
typically 40 to 150 mm in length and 1.0 to 1.4 mm in
diameter. The cell shape is rectangular and cells are 1.0 to
1.4 mm ¥ 2.0 to 4.0 mm in dimension. No indentations

Copyright 2004 by CRC Press LLC.

 Methods
FIGURE 2.19 Phase contrast micrographs of rosettes and gonidia: (a) rosette, (b) Type 021N rosette, (c) Thiothrix I gonidia, (d)
Thiothrix II gonidia. (Original magniÞcations 100¥, (a); 1000¥, (b), (c), and (d).)
appear at the cell septa. No sheath is present but a slime
covering on the Þlament that looks like an empty “cuff”
may be present at the Þlament tip. No attached growth;
no branching; not motile.
Staining Reactions — Gram-negative and Neisser-
negative.
SulÞde Oxidation — Negative.
Key Characteristics — Long rectangular cells;
“transparent” appearance; no cell inclusions.
n. Type 0092 (Figures 2.18d and 2.26c)
Filament Description — A straight or bent Þlament
always inside the ßoc. Filaments are typically 10 to 80 mm
in length and 0.8 to 1.0 mm in diameter. Individual cells
usually cannot be seen within the Þlament. No sheath is
present and no attached growth occurs. No branching and
not motile.
Staining Reactions — Gram-negative and Neisser-
positive.
SulÞde Oxidation — Negative.
Key Characteristics — Location inside the ßoc and
Neisser-positive staining reaction. Filament is often over-
looked in a microscopic examination of a wet mount but
becomes recognizable when a Neisser-stained preparation
is examined. Filaments appear wider (1.0 to 1.2 mm) in
Copyright 2004 by CRC Press LLC.
35
dried Gram- and Neisser-stained preparations than in wet
mounts.
o. Type 0581 (Figure 2.26d)
Filament Description — Curved or coiled Þlament inside
the ßoc. Filaments are typically 100 to 200 mm in length
and 0.5 to 0.8 mm in width. Individual cells cannot be seen
within the Þlament. No sheath is present and attached
growth does not occur. No branching and not motile.
Staining Reactions — Gram-negative and Neisser-
negative.
SulÞde Oxidation — Negative.
Key Characteristics — Location within the ßoc and
Gram-negative staining reaction. This Þlament can be con-
fused with M. parvicella but has a Gram-negative staining
reaction; M. parvicella is Gram-positive.
p. Type 0041 (Figures 2.10a, 2.12a, 2.13c, 2.14a,
2.17c, 2.18f, and 2.27a)
Filament Description — A straight Þlament usually
inside the ßoc. Filaments are typically 100 to 500 mm in
length and 1.8 to 2.0 mm in width. Individual cells are
square-shaped and 1.8 to 2.0 mm ¥ 2.0 to 3.0 mm in
dimension. A heavy sheath is present; branching is absent.
Large amounts of attached growth are present. The
 36
Cell dia. 1.6-2.5 µm Thiothrix I
Filaments contain sulfur Sulfur granules "square" Type 0914 Sheath
granules in situ or after Cell dia. 0.8-1.4 µm Thiothrix II

Manual on Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems
applying the S test Sulfur granules "spherical"
Not motile Type 021N
True branching Nocardioforms No sheath
Strongly Gram Motile Beggiatoa spp.
positive
Cell dia. 0.8-1.0 µm N. limicola I
Cell septa present
No branching Cell dia. 1.4 µm N. limicola II
Filaments do not contain
sulfur granules
Cell dia. 1.8-2.0 µm; Cell dia. 2.0 µm N. limicola III
Gram variable or
weakly Gram positive
usually heavy Type 0041
attached growth No cell septa M. parvicella
Attached growth;
cells square
Type 0675
Cell dia. ≤ 1.0 µm
Attached growth;
cells rectangular; Type 1851
trichome bundles
Cell dia. 1.4 µm N. limicola II
Trichome coiled
Cell dia. >1.0 µm;
cell septa present Cell dia. 2.0 µm N. limicola III
Gram negative

Cell dia. ≤ 1.0 µm Cell dia. 1.6 µm;

usually false branching
S. natans
Sheath
Trichome straight or
smoothly curved
No sheath Cell dia. 0.8-1.2 µm;
usually attached growth
Type 0914
Neisser positive;
Neisser negative Cells barrel, rectangular or
inside floc Cells rectangular;
"transparent" discoid; cell dia. 2.0-3.0 µm;
Type 0092 indentations at septa
Type 0961
Type 021N

Trichome straight, Trichome irregularly bent No cell septa;

smoothly curved or bent "chain of cells" trichome coiled
Type 0581

Cell dia. 0.5 µm; Cell septa present

no cell septa Cells oval; Cells elongated sausages; Cells oval;
H. hydrossis 0.8-1.0 x 1.0-1.5 µm 0.8-1.2 x 2.0-5.0 µm cell dia. 0.4-0.6 µm;
free in suspension
Sheath; usually No sheath; no Type 1863 Type 0411
attached growth attached growth Type 0211
Type 1701 Type 0803

FIGURE 2.20 Dichotomous key for the identiÞcation of Þlamentous organisms in activated sludge.

Copyright 2004 by CRC Press LLC.

 Methods
absence of attached growth suggests a rapid growth rate.
No branching and not motile.
Staining Reactions — Gram-variable (usually
weakly Gram-positive) and Neisser-negative, sometimes
with rows of round Neisser-positive granules. Occasion-
ally stains lightly Neisser-positive in industrial wastewater
activated sludge — a false reaction caused by precipitation
of Neisser stain on the surface of the Þlament.
SulÞde Oxidation — Negative.
Key Characteristics — Large size, Gram-variable
(weakly Gram-positive) staining reaction, attached
growth, and location within the ßoc.
q. Type 0675 (Figure 2.27b)
Filament Description — A straight Þlament usually
inside the ßoc. Filaments are typically 50 to 150 mm in
length and 1.0 mm in width. Individual cells have a square
shape and are 1.0 mm ¥ 1.0 mm in dimension. A thin sheath
is present. Branching is absent; heavy amounts of attached
growth are usually present. No branching and not motile.
Staining Reactions — Gram-variable (usually
weakly Gram-positive) and Neisser-negative.
SulÞde Oxidation — Negative.
Key Characteristics — Gram-variable (usually weakly
Gram-positive) staining reaction, attached growth and loca-
tion inside ßocs. The Þlament is similar to but smaller than
Type 0041. Eikelboom (2000) no longer differentiates Types
0041 and 0675 and combines them as Type 0041/0675.
r. Type 1851 (Figures 2.17d and 2.27c)
Filament Description — A straight or smoothly curved
Þlament usually inside the ßoc. Filaments are typically
50 to 200 mm in length and 0.8 mm in width. They may
intertwine to form twisted “ropes” or “bundles”. Individ-
ual cells are rectangular and 0.8 mm ¥ 1.5 to 2.0 mm in
dimension. A thin sheath is present and extensive attached
growth is usually present. The attached growth is typically
orientated perpendicularly to the cell surfaces of the Þla-
ment. No branching and not motile.
Staining Reactions — Gram-variable (usually
weakly Gram-positive) and Neisser-negative.
SulÞde Oxidation — Negative.
Key Characteristics — Formation of twisted Þlament
“ropes” or “bundles” by intertwined Þlaments; perpendic-
ular attached growth and Gram-variable staining reaction.
s. Type 0803 (Figure 2.27d)
Filament Description — A straight Þlament that may
extend from the ßoc surface and be dispersed in the bulk
solution. Filaments are typically 50 to 150 mm in length
and 0.8 mm in width. Individual cells are square with no
indentations at the septa and 0.8 ¥ 1.0 mm in dimension.
No sheath; no attached growth; no branching; not motile.
Staining Reactions — Gram-negative and Neisser-
negative.
Copyright 2004 by CRC Press LLC.
37
SulÞde Oxidation — Negative.
Key Characteristics — Dispersed growth. This Þla-
ment resembles Type 0675 but has neither sheath nor
attached growth. It stains Gram-negative, while 0675
stains weakly Gram-positive. Eikelboom (2000) suggests
that Types 0914 and 0803 may be the same organism.
t. Microthrix parvicella (Figures 2.17e, 2.18c,
2.28a, 5.1e, and 5.1f)
Filament Description — An irregularly coiled Þlament
found inside the ßoc, surrounding the ßoc, and dispersed.
Filaments are typically 50 to 200 mm in length and 0.8 mm
in width. Individual cells cannot be seen. Sheath and
attached growth are absent. No branching and not motile.
Staining Reactions — Strongly Gram-positive and
Neisser-negative. Neisser-positive intracellular granules
are commonly observed.
SulÞde Oxidation — Negative.
Key Characteristics — Strongly Gram-positive
staining reaction; Neisser-positive granules; coiled
growth; inability to see individual cells; “beaded” effect
caused by intracellular granules. No branching and not
motile.
u. Nocardioforms (Figures 2.9b, 2.10f, 2.14g,
2.17f, 2.18b, 2.28b, 5.1a, 5.1b, 5.1c and 5.1d)
Filament Description — Irregularly shaped true-branch-
ing Þlaments occurring inside the ßoc and dispersed in
the bulk solution. Filaments are typically 5 to 30 mm in
length and 1.0 mm in width. Individual cells are present
and shape is irregular. Sheath and attached growth are
absent. Not motile.
Staining Reactions — Strongly Gram-positive and
Neisser-negative. Neisser-positive intracellular granules
are commonly observed.
SulÞde Oxidation — Negative.
Key Characteristics — True branching and strongly
Gram-positive staining reaction. The true abundance in
activated sludge can only be assessed after examining a
Gram-stained preparation. Because nocardioforms com-
prise many genera (e.g., Nocardia, Gordona and Skerma-
nia), characteristics described can vary considerably. One
type, Skermania pinensis (the pine-tree-like-organism or
PTLO) is distinguishable by its characteristic branching
(see Figure 5.1c and Figure 5.1d). Many nocardioforms
can grow in a fragmented or single cell form.
v. Type 1863 (Figures 2.10c, 2.14c, and 2.28c)
Filament Description — An irregularly shaped Þlament
dispersed in the bulk solution. Filaments are typically
10 to 50 mm in length and 0.8 to 1.0 mm in width. Indi-
vidual cells are present, and the cell shape is oval, 0.8 to
1.0 mm ¥ 1.0 to 1.5 mm in dimension. No sheath; no
attached growth. No branching and not motile.
 38
Manual on Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems
TABLE 2.18
Summary of Typical Morphological and Staining Characteristics of Filamentous Organisms Commonly Observed in Activated Sludge
DIRECT ILLUMINATION OBSERVATION
¥
AT 1000¥ ¥
PHASE CONTRAST OBSERVATION AT 1000¥
Neisser Stain Sulfur Granules Filament Filament Cell Septa
Gram Other Cell Diameter, Length, Filament Filament Clearly Indentations Attached Cell Shape
Filament Type Stain Filament Granules In situ S test Inclusions mm mm Shape Location Observed at Cell Septa Sheath Growth and Size, mm Notes

S. natans – – – – – PHA 1.4–1.6 100–>500 St E + + + – Sausage-shaped False branching

1.6 ¥ 2.5
Type 1701 – – – – – PHA 0.8–1.0 20–80 St, B I,E + + + ++ Sausage-shaped Cell septa sometimes
1.0 ¥ 1.5 hard to see
H. hydrossis – – – – – – 0.5 10–100 St, B E,F – – + –,+ – Rigidly straight
Type 021N – – –,+ –,+ + PHA 1.6–2.5 50–1000 St, SC E + + – – Barrels, Rosettes, gonidia
rectangles,
discoid
1.6–2.5 ¥ 2.0
Thiothrix I –,+ – –,+ +,– + PHA 1.6–2.5 100–>500 St, SC E + – + – Rectangles Rosettes, gonidia
0.8–1.4 ¥ 1.5–3.0
Thiothrix II –,+ – –,+ +,– + PHA 0.8–1.4 50–200 St, SC E + – + – Rectangles Rosettes, gonidia
0.8–1.2 ¥ 1.0
Type 0914 –,+ – –,+ –,+ – PHA 1.0–1.2 50–200 St E,F + – + –,+ Squares Sulfur granules, square
1.0 ¥ 1.0
Beggiatoa spp. –,+ – –,+ +,– + PHA 3.0–4.0 100–>500 St F –,+ – – – Rectangles Motile: ßexing and
2.0–4.0 ¥ 6.0–8.0 gliding
N. limicola I + + – – – PHA 0.8–1.0 40–100 C I,E + + – – Ovals _
0.8–1.0 ¥ 0.8
N. limicola II –,+ +,– – – – PHA 1.4 50–200 C I,E + + – – Discs, ovals Incidental branching;
1.4 ¥ 1.0–1.5 Gram- and Neisser-
variable

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 Methods
DIRECT ILLUMINATION OBSERVATION
¥
AT 1000¥ ¥
PHASE CONTRAST OBSERVATION AT 1000¥
Neisser Stain Sulfur Granules Filament Filament Cell Septa
Gram Other Cell Diameter, Length, Filament Filament Clearly Indentations Attached Cell Shape
Filament Type Stain Filament Granules In situ S test Inclusions mm mm Shape Location Observed at Cell Septa Sheath Growth and Size, mm Notes

N. limicola III –,+ + – – – PHA 2.0 100–300 C I,E + + – – Discs, ovals _

2.0 ¥ 1.5
Type 0411 – – – – – – 0.8–1.2 50–150 B,I E + + – – Elongated rods Chain of cells
0.8–1.2 ¥ 2.0–5.0
Type 0961 – – – – – – 1.0–1.4 40–150 St E + – – – Rectangles Transparent
1.0–1.4 ¥ 2.0–4.0
Type 0092 – + – – – + 0.8–1.0 10–80 St, B I –,+ – – – Rectangles Wider when Neisser
0.8–1.0 ¥ 1.0 stained
Type 0581 – – – – – – 0.5–0.8 100–200 C I – – – – – Coiled in ßoc
Type 0041 +,V – –,+ – – – 1.8–2.0 100–500 St, SC I,E + – + ++,– Squares Neisser-positive
1.8–2.0 ¥ 2.0–3.0 reaction occurs
Type 0675 +,V – –,+ – – – 1.0 50–150 St, SC I + – + ++,– Squares Neisser-positive
1.0 ¥ 1.0 reaction occurs
Type 1851 + weak – – – – – 0.8 50–200 St, SC E +,– – + –,+ Rectangles Filament bundles
0.8 ¥ 1.5–2.0
Type 0803 – – – – – – 0.8–1.0 50–150 St E,F + – – – Squares –
0.8 ¥ 1.0
M. parvicella + – + – – PHA 0.8 50–200 C I – – – – – Large patches
Nocardioforms + – + – – PHA 1.0 5–30 I I + – – – Variable
1.0 ¥ 1.0–2.0 True branching
Type 1863 – – –,+ – – PHA 0.8–1.0 10–50 B,I E,F + + – – Oval rods Chain of cells
0.8–1.0 ¥ 1.0–1.5 Usually free in bulk

LEGEND: + Positive Filament Shape Filament Location

– Negative St Straight C Coiled E Extends from ßoc surface
V Variable B Bent I Irregularly shaped I Mostly within the ßoc
+,- or -,+ Variable; Þrst is most observed SC Smoothly curved F Free in liquid between the ßocs
Single symbol Invariant

39
Copyright 2004 by CRC Press LLC.
 40 Manual on Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems

FIGURE 2.21 Phase contrast micrographs of: (a) Sphaero-

tilus natans, (b) Type 1701, (c) Haliscomenobacter hydros-
sis. (Original magniÞcation 1000¥.) (See color insert
following page 70.)

FIGURE 2.22 Phase contrast micrographs of: (a) Type 021N, (b) Type 021N with sulfur granules, (c) Beggiatoa sp., (d) Beggiatoa
sp. with sulfur granules. (Original magniÞcation 1000¥.) (See color insert following page 70.)

Copyright 2004 by CRC Press LLC.

 Methods 41

FIGURE 2.23 Phase contrast micrographs of: (a) Thiothrix I, (b) Thiothrix I with sulfur granules, (c) Thiothrix II, and (d) Thiothrix II
with sulfur granules. (Original magniÞcation 1000¥.) (See color insert following page 70.)

FIGURE 2.24 Phase contrast micrographs of: (a) Type 0914, (b) Type 0914 with sulfur granules. (Original magniÞcation 1000¥.)
(See color insert following page 70.)

Staining Reactions — Gram-negative and Neisser-
negative with Neisser-positive intracellular granules.
SulÞde Oxidation — Negative.
Key Characteristics — A ßexible chain of irregular-
shaped cells often containing Neisser-positive granules.
w. Type 0211 (Figure 2.28d)
Filament Description — An irregularly shaped Þla-
ment that occurs dispersed. Filaments are typically 10 to
50 mm in length and 0.4 mm in width. Individual cells are
present, and the cell shape is oval, 0.4 mm ¥ 0.6 mm in
dimension. Sheath and attached growth are absent. No
branching and not motile.
Staining Reactions — Gram-negative and Neisser-
negative.
SulÞde Oxidation — Negative.
Key Characteristics — A very thin ßexible chain of
cells; the thinnest Þlament found in activated sludge.

Copyright 2004 by CRC Press LLC.

 42 Manual on Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems

FIGURE 2.25 Phase contrast micrographs of: (a) Nosto-

coida limicola I, (b) Nostocoida limicola II, (c) Nostocoida
limicola III. (Original magniÞcation 1000¥.)

FIGURE 2.26 Phase contrast micrographs of: (a) Type 0411, (b) Type 0961, (c) Type 0092, and (d) Type 0581. (Original magniÞcation
1000¥.)

Copyright 2004 by CRC Press LLC.

 Methods
FIGURE 2.27 Phase contrast micrographs of: (a) Type 0041, (b) Type 0675, (c) Type 1851, and (d) Type 0803. (Original magniÞcation
1000¥.)
x. Flexibacter sp. (Figure 2.29a)
Filament Description — An elongated, rod-shaped bac-
terium rather than a true Þlament. Straight or smoothly
curved and 1.0 mm ¥ 20 to 40 mm in dimension. It occurs
dispersed. No sheath; no attached growth; no branching.
Motile with a ßexing and gliding motion.
Staining Reactions — Gram-negative and Neisser-
negative.
SulÞde Oxidation — Negative.
Key Characteristics — Flexing and gliding motility.
y. Bacillus sp. (Figure 2.29b)
Filament Description — A long chain of rod-shaped cells
rather than a true Þlament. Straight or irregularly curved
and 1.0 mm ¥ 20 to 50 mm in dimension. Occurs dispersed
or attached to the ßoc. A sheath may be present but
attached growth is absent. No branching and not motile.
Staining Reactions — Gram-positive and Neisser-
negative.
SulÞde Oxidation — Negative.
Key Characteristics — Gram-positive staining reac-
tion; “chain of cells” appearance.
Copyright 2004 by CRC Press LLC.
43
z. Cyanophyceae (Figure 2.29c)
Filament Description — Large, straight Þlamentous
blue-green photosynthetic bacteria (often called blue-
green algae), 2.0 to 5.0 mm wide and 100 to 500 mm long.
They occur dispersed. No sheath; no attached growth; no
branching. May have slow gliding motility.
Staining Reactions — Gram-positive or -negative
and Neisser-negative.
SulÞde Oxidation — Negative.
Key Characteristics — Large size and a distinct
green color when viewed under direct illumination.
aa. Fungi (Figures 2.9a and 2.29d)
Filament Description — Large, truly branched Þlaments,
5.0 to 10 mm wide and 100 to 1000 mm long, usually
inside the ßocs. A sheath is absent; however, a heavy
cellulose cell wall is usually present. Attached growth is
absent. True branching occurs. Intracellular vacuoles,
organelles and granules are present and internal cytoplas-
mic streaming occurs. Not motile.
Staining Reactions — Gram-positive or -negative
and Neisser-negative.
SulÞde Oxidation — Negative.
 44 Manual on Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems
FIGURE 2.28 Phase contrast micrographs of: (a) Microthrix parvicella, (b) Nocardioforms, (c) Type 1863, and (d) Type 0211.
(Original magniÞcation 1000¥.) (See color insert following page 70.)
Key Characteristics — Very large, true branching
Þlaments containing vacuoles, organelles, and granules
and exhibiting cytoplasmic streaming.
C. PROGRESS IN IDENTIFYING
FILAMENTOUS ORGANISMS
Table 2.19 is a summary of the status of identifying and
classifying the Þlamentous organisms found in activated
sludge. More information about the identities of Þlaments
and types of organisms in activated sludge continuously
becomes available through the application of genetic
methods. Indeed, it is not beyond the realm of possibility
that by the time the next edition of this manual is written,
rapid test kits for identifying Þlamentous organisms in
activated sludge will replace the microscopic methods
described this chapter.
An especially promising genetic method for detecting
Þlamentous (and other) microorganisms in activated
sludge is ßuorescent in situ hybridization (FISH). A gene
probe (a fragment of genetic material approximately 15 to
30 bases in length) complementary to (binds speciÞcally
with) a unique portion of one of the RNA molecules in a
cell's ribosomes (usually the 16SrRNA molecule) is
prepared. The probe is then reacted with a ßuorescent mol-
ecule that, when illuminated with ultraviolet (UV) light,
Copyright 2004 by CRC Press LLC.
can be visualized. When the probe is added to a sample,
it will bind only to the 16SrRNA to which it is comple-
mentary. The extent and location of the probe binding can
be detected by observing the sample through a microscope
with UV illumination. FISH probes have been used exten-
sively in the characterization and identiÞcation of activated
sludge Þlamentous organisms. Other examples of their use
in activated sludge are described below.
Bouchez et al. (2000) showed that bacteria used to
“bioaugment” a laboratory sequencing batch reactor (SBR)
activated sludge system (with the objective of increasing
its denitrifying capacity) was very rapidly eaten by stalked
ciliated protozoa.
For many years, Nitrosomonas (ammonia oxidized to
nitrite) and Nitrobacter (nitrite oxidized to nitrate) micro-
organisms were credited with nitriÞcation in activated
sludge. Several workers recently called these assumptions
into question. For example, Wagner et al. (1998) found
that in an activated sludge treating an industrial waste-
water with very high ammonia content, the predominant
ammonia oxidizer was an organism resembling Nitroso-
coccus mobilis. They were unable to Þnd signiÞcant num-
bers of Nitrobacter spp., leading them to believe that
nitrite oxidation was carried out by another organism.
FISH studies by Juretschko et al. (1998) and Mobarry
et al. (1996) demonstrated that the nitriÞers did not grow
 Methods
FIGURE 2.29 Phase contrast micrographs of: (a) Flexibacter sp., (b) Bacillus sp., (c) Oscillatoria sp. (cyanophyceae), and (d)
fungus. (Original magniÞcation 1000¥.) (See color insert following page 70.)
in dispersed form and were not distributed evenly throughout
the activated sludge ßoc. Rather they grew as dense col-
onies (Figure 2.30a) and the ammonia oxidizer colonies
were surrounded by the nitrite oxidizer colonies
(Figure 2.30b). These Þndings have signiÞcant implica-
tions, both for bioaugmentation and for the modeling of
nitriÞcation in activated sludge.
The discrepancy between these Þndings and those from
previous work in which Nitrosomonas and Nitrobacter spp.
were identiÞed as the principal nitrifying organisms in
activated sludge results from the bias introduced by cul-
turing microorganisms from environmental samples.
Because many environmental microorganisms will not
grow on laboratory culture media, the only way to be
certain whether an organism is important in an environment
such as activated sludge is to demonstrate its presence by
in situ methods that do not involve culturing and isolation.
An excellent example of mistaken identity caused by
the bias introduced by culture and isolation methods was
the conclusion that organisms of the Acinetobacter genus
were largely responsible for the phenomenon of enhanced
biological phosphorus removal (EBPR) in activated sludge
systems with initial anaerobic zones. Recent work has
clearly shown that this is not so. Microorganisms that are
not closely related to Acinetobacter spp. are important in
this role (Hesselmann et al., 1999; Crocetti et al., 2000).
Copyright 2004 by CRC Press LLC.
45
D. PROTOZOA AND METAZOA
1. General
Microscopic observation of protozoa and other higher life
forms in activated sludge is a common and widespread
practice. In a very general way, the types of these organisms
present can be related to plant performance and efßuent
quality. They are useful for toxicity assessment but they
are of little or no value for determining the properties of
activated sludge that inßuence its behavior in solids sep-
aration processes.
From a morphological view, activated sludge is a rel-
atively simple microbial community consisting of free and
ßocculated bacteria (and at times Þlamentous bacteria),
protozoa, rotifers, nematodes, and a few other inverte-
brates. Protozoa and other higher life forms are usually
aerobic and bacteriovorous (they eat bacteria). A few anaer-
obic ßagellates and a number of saprophytic ßagellates
occur in activated sludge. The saprophytic ßagellates use
soluble organic matter for growth. Carnivorous free ciliates
and attached ciliates (suctorians) feed on other protozoa.
Chlorophyll-bearing ßagellates are incidentally observed
and are usually derived from aeration basin walls.
Protozoa and other higher life forms may constitute
approximately 5% by weight of the activated sludge biomass
and are represented by about 200 species (Curds, 1973;
 46 Manual on Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems

TABLE 2.19
Status of Activated Sludge Filamentous Organism Taxonomy
Name in Key FISH Current Name and
(Table 2.18) Pure Culture Probe Taxonomic Position References

S. natans Yes Yes Sphaerotilus natans Richard et al. (1982); Williams and Unz (1985a, 1985b); Ziegler et al.
(1990); Corstjens and Muyzer (1993); Wagner et al. (1994)
Type 1701 Yes No Sphaerotilus spp. Richard et al. (1982); Williams and Unz (1985a); Kämpfer et al. (1995);
Howarth et al. (1998)
H. hydrossis Yes Yes Haliscomenobacter van Veen (1973); van Veen et al. (1982); Ziegler et al. (1990); Wagner
hydrossis et al. (1994)
Type 021N Yes Yes Several Thiothrix spp. Howarth et al. (1999); Wagner et al. (1994); Ziegler et al. (1990);
Kanagawa et al. (2000); Aruga et al. (2002)
Thiothrix I and II Yes Yes Thiothrix spp. Howarth et al. (1999); Richard et al. (1982); Williams and Unz (1985a,
1985b, 1989); Tandoi et al. (1994); Kanagawa et al. (2000); Aruga et al.
(2002)
Type 0914 No No Unknown —
Beggiatoa Yes ? Beggiatoa spp. Williams and Unz (1985a, 1985b)
N. limicola I Yes Yes? Tricococcus spp. Liu et al. (2000, 2002); Scheff et al. (1984)
N. limicola II Yes Yes New genus, high GC van Veen (1973); Eikelboom (1975); Nowak and Brown (1990); Blackall
bacterium et al. (2000)
N. limicola II Yes Yes New genus, Snaidr et al. (2002)
Proteobacteria,
Alisphaeira europa
N. limicola II Yes Yes New genus, green sulfur Schade et al. (2002)
bacteria
N. limicola III Yes Yes? Isosphaera spp. Seviour et al. (2002)
Type 0961 No No Unknown Richard et al. (1982)
Type 0092 No No Unknown Bradford et al. (1996)
Type 0581 No No Unknown —
Type 0041 No Yes Member of TM7 group; Thomsen et al. (2002); Hugenholtz et al. (2001); Richard et al. (1982);
Bacterial domain Williams and Unz (1985a, 1985b)
Type 0675 No No New genus Hugenholtz et al. (2001)
Type 1851 Yes ? New genus; Beer et al. (2002); Kohno et al. (2002); Bjornsson et al. (2002)
Chloroßexus group
Type 0803 Yes Yes New genus, b Blackall et al. (1996); Bradford et al. (1996); Williams and Unz (1985a)
proteobacteria
M. parvicella Yes Yes New genus, Microthrix Slijkhuis (1983a, 1983b); Slijkhuis and Deinema (1982, 1988); Slijkhuis
parvicella et al. (1984); Blackall et al. (1994b, 1996); Rossetti et al. (1997a)
Nocardioforms Yes Yes for Many genera Goodfellow et al. (1998); Soddell et al. (1998)
some
Type 1863 Yes Yes Acinetobacter spp., Rosetti et al. (1997); Seviour et al. (1997)
Moraxella spp.

Curds, 1975). Total numbers range from 100 to 2. Microscopic Evaluation

>100,000/mL. Protozoa are generally dominant, with 500 to
To observe these organisms, place one drop (0.05 mL) of
several thousand/mL observed commonly. These organisms
activated sludge on a microscope slide, add a cover slip,
perform several important functions in activated sludge, the
and examine it at 100¥ using phase contrast illumination.
most important of which is the removal of nonßocculated
Count all protozoa and other higher life forms present by
and loosely ßocculated bacteria from wastewater to produce
scanning the entire cover slip area using the mechanical
a clariÞed efßuent (Curds et al., 1968; Curds and Fey, 1969).
stage of the microscope. Average the results of 4 to 5
Additionally, these organisms may contribute to biomass
separate preparations.
ßocculation through production of fecal pellets and mucus
(Curds, 1975) and may function to break up large ßoc Total number of organisms present
masses and encourage a more active biomass through their per mL activated sludge culture =
motility (Javornicky and Prokesova, 1963). average count per cover glass area ¥ 20

Copyright 2004 by CRC Press LLC.

 Methods
FIGURE 2.30 In situ identiÞcation of nitrifying bacteria in activated sludge: (a) simultaneous in situ hybridization with Cy3-labelled
probe NmV and FLUOS-labelled probe NEU. Nitrosococcus mobilis cells appear yellow; (b) simultaneous in situ identiÞcation of
Nitrosococcus mobilis and Nitrospora-like bacteria after in situ hybridization with FLUOS-labelled probe NmV (green) and Cy3-
labelled probe S-*-Ntspa-1026-a-A18 (red). (From Juretschko, S. et al. (1998), Appl. Environ. Microbiol., 64, 3042. With permission.)
(See color insert following page 70.)
If a large number of organisms are present (at times
observed for ßagellates), count the number present in each
Þeld of view (at 100¥) for 10 to 20 Þelds of view, calculate
an average number per Þeld of view, and multiply this
number by the number of Þelds of view for the cover slip
area (this is typically about 300 at 100¥ magniÞcation for
a 22 mm ¥ 22 mm cover slip). Follow this procedure
frequently (several times a week or even daily) because
protozoan populations in activated sludge can change rap-
idly in certain circumstances (e.g., an upset caused by
toxicity).
3. Taxonomic Classification
Taxonomic classiÞcation of these organisms is based pri-
marily on motility. The six basic groups observed in acti-
vated sludge are ßagellates, amoebae, free-swimming cil-
iates, attached (stalked) ciliates, rotifers, and a few other
invertebrates. IdentiÞcation to species is not necessary, but
recognition of the major groups of protozoa and higher life
forms can be useful in activated sludge operation. The most
common protozoan and higher life forms observed in acti-
vated sludge are shown in Figure 2.31 through Figure 2.33.
These photos can be used as simpliÞed identiÞcation keys.
More detailed taxonomic identiÞcation keys can be
found as follows:
Protozoa Curds (1969 and 1975); Jahn et al. (1980);
Mudrack and Kunst (1986)
Rotifers Calaway (1968); Doohan (1975); Gerardi (1987a)
Nematodes Calaway (1963); Schiemer (1975); Tarjan (1977);
Gerardi (1987b)
Annelids De L. G. Solbe (1975)
Copyright 2004 by CRC Press LLC.
47
The major groups of protozoa and higher life forms
found in activated sludge are described below.
a. Flagellates
These are small (5 to 20 mm), oval or elongated forms
actively motile via one or more long, whip-like ßagellae.
Many species found in activated sludge feed on soluble
organic matter and their presence can indicate signiÞcant
soluble biochemical oxygen demand (BOD) levels. Many
of these occur at low dissolved oxygen (DO) and high
organic load.
b. Amoebae
These vary in shape and size (10 to 200 mm) and are motile
via pseudopodia (“false feet”). Some species have a hard,
ornate shell called a test (and the organisms are known as
testate amoebae, e.g., Arcella). Amoebae grow well on
particulate organic matter and are able to tolerate low DO
environments. A bloom of amoebae can indicate a high
amount of particulate organic matter such as starch (pulp
and paper wastewater), yeast (brewery wastewater), and
septage (municipal wastewater).
c. Free-Swimming Ciliates
These are round to oval in shape (20 to 400 mm) and are
actively motile via rows of short, hair-like cilia. Some
species (“crawlers”) have cilia fused into spikes that aid
them in crawling on the surfaces of activated sludge ßocs.
Ciliates are usually found under conditions of good ßoc
formation and generally indicate satisfactory activated
sludge operation. Ciliates are sensitive and their presence
or absence can indicate toxicity.
 48 Manual on Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems
FIGURE 2.31 Phase contrast micrographs of common ßagellates and amoebae found in activated sludge: (a) Monas sp., (b) Bodo
sp., (c) Polychaos sp., and (d) Arcella sp. (Original magniÞcations 1000¥, (a) and (b); 400¥, (c), and (d).) (See color insert following
page 70.)
d. Attached Ciliates
These appear under conditions similar to those producing
free-swimming ciliates but are found attached to ßocs by
stalks that may be either rigid or contractile. Some species
have one organism per stalk (e.g., Vorticella spp.) while
others are colonial (e.g., Epistylis spp. and Opercularia
spp.). Stalked ciliates generally occur at low organic load
(high MCRT). Individual species can be used as indicators
of approximate MCRT. The colonial forms occur at higher
MCRTs with more “heads” per colony as the MCRT
increases.
e. Rotifers
These have a variety of shapes and are much larger (50 to
500 mm) and have more complex structures than protozoa
(Figure 2.34). Most are motile and attach to activated
sludge ßocs with contractile “feet.” These organisms occur
over a wide range of MCRT; some species are indicative
of high MCRT.
f. Higher Invertebrates
These include nematodes (Figure 2.35d), tardigrades such
as Macrobiotus sp. (Figure 2.35b), gasterotrichs
(Figure 2.35c), and annelids such as Nais sp. and Aeleo-
soma sp. (Figure 2.35a) which can impart a reddish color
Copyright 2004 by CRC Press LLC.
to activated sludge due to their red- or orange-colored
“eyespots.” Because of their low growth rates, nematodes
are generally observed only at higher MCRTs. Tardi-
grades, gasterotrichs and annelids appear to occur only in
nitrifying activated sludge systems, probably due to their
susceptibility to ammonia toxicity.
4. Use of Protozoa and Metazoa
as Indicator Organisms
Various protozoan and invertebrate groups develop in acti-
vated sludge according to growth conditions. Protozoa
have maximum growth rates of ≥1 d-1 at 20∞C (Curds,
1975) and rotifers have growth rates of 1 to 2 d-1 at 20∞C
(Doohan, 1975). Thus, the activated sludge growth rate
(or MCRT) rarely limits the development of these organ-
isms in most activated sludge systems. Nematodes have a
lower maximum growth rate and generally develop only
in high MCRT systems (Water Pollution Control Federa-
tion, 1990). Food availability principally through freely
dispersed bacteria or turbidity is the primary determinant
of which group predominates.
Flagellates, amoebae, and small, free-swimming cili-
ates require high prey densities (>106 to 107 bacteria/L)
because their chase and capture feeding mechanisms are
 Methods
FIGURE 2.32 Phase contrast micrographs of free and stalked ciliates: (a) Aspidisca sp., (b) Paramecium sp., (c) Tokophyra sp., and
(d) Podophyra sp. (Original magniÞcations 400¥, (a); 200¥, (b), (c), and (d).) (See color insert following page 70.)
inefÞcient. These groups appear during plant startup and
at low MCRT (high organic load) conditions. Attached
ciliates, rotifers, and other invertebrates develop at lower
prey densities because of their attachment to the activated
sludge ßoc and their ability to feed by ciliary action (Þlter
feeding). These organism groups are selected for at high
MCRT (low organic load). These factors lead to marked
differences in the populations of various protozoa and
other higher life forms as activated sludge process oper-
ating parameters change (Table 2.20).
Satisfactory activated sludge performance occurs
when there is a balance among free-swimming and
attached ciliates and rotifers. An overabundance of ßagel-
lates, amoebae, or free-swimming ciliates is an indication
of high F /M (low MCRT) while an overabundance of
attached ciliates, rotifers, and other higher life forms,
especially nematodes, is an indication of low F/M (high
MCRT). See Table 2.20. Because sludge settling often
deteriorates at organic loading extremes, many plants
attempt to adjust process parameters based on the types
of protozoa and other higher life forms observed in the
activated sludge. This is not a very sophisticated approach
to activated sludge settleability control, because many
other factors besides organic loading contribute to the
growth of the Þlamentous organisms that cause deteriora-
tion of activated sludge settling.
Copyright 2004 by CRC Press LLC.
49
One of the most valuable uses of microscopic observa-
tion of these organisms is toxicity assessment. These organ-
isms, particularly the ciliates and rotifers, are generally the
Þrst to be impacted by toxic materials and can serve as in
situ biomonitoring indicators for toxicants and other adverse
stresses on the activated sludge process. The Þrst noticeable
sign of toxicity or stress is usually the slowing or cessation
of cilia movement in the ciliates. Next, the predominant
protozoan groups shift toward ßagellates and small, free-
swimming ciliates that often “bloom” to high numbers
(>10,000/mL). This is an indication of activated sludge ßoc
breakup and the production of large numbers of dispersed
bacteria (turbidity) utilized as a food source by the ßagellates
and free-swimming ciliates. Finally, in severe cases, these
protozoa die, lyse, and release their cell contents, sometimes
producing a white foam that contains dead protozoans and
protozoan fragments. Stresses other than toxicity that induce
these responses include low DO, pH outside the range of
6.5 to 8.5, and high temperatures. Protozoa and other higher
life forms are generally absent from activated sludge systems
operated at temperatures above 37 to 40∞C.
E. PHYSICAL AND CHEMICAL METHODS
In this section, we present specialized methods for mea-
suring the physical properties of activated sludge related
to solids separation.
 50 Manual on Causes and Control of Activated Sludge Bulking, Foaming, and Other Solids Separation Problems

FIGURE 2.33 Phase contrast micrographs of stalked ciliates: (a) Opercularia sp., (b) Vaginicola sp., (c) Vorticella sp., and (d)
Epistylis sp. (Original magniÞcations 100¥, (a); 200¥, (b), (c), and (d).) (See color insert following page 70.)

FIGURE 2.34 Phase contrast micrographs of rotifers. (Original magniÞcation 100¥.) (See color insert following page 70.)

1. Settling Tests 2. Foaming Tests

Three activated sludge settling tests are presented: (1) the
standard unstirred sludge volume index (SVI), (2) the
stirred SVI at a standard initial SS concentration (SSVI
or SVI3.5) (in this case, 3.5 g SS/L), and (3) the diluted
SVI (DSVI). The relative merits of these methods are
discussed in Chapter 3. The SVI method is detailed in
Table 2.21, the SSVI method is found in Table 2.22, and
the DSVI technique is shown in Table 2.23.
Two foaming tests are presented. The Þrst test (Table 2.24)
is suitable for activated sludge mixed liquor samples and
aerobic and anaerobic digester contents. The Alka-Seltzer®
(Bayer Corporation, Morristown, NJ) test (Table 2.25) is
designed to determine the foaming potential of inßuent
and efßuent streams. It is not suitable for use with samples
containing high SS levels (such as mixed liquor and
digester contents).

Copyright 2004 by CRC Press LLC.

 Methods 51

FIGURE 2.35 Phase contrast micrographs of invertebrates: (a) bristle worm (Aeleosoma sp.), (b) tardigrade (water bear, Macrobiotus
sp.), (c) gasterotrich (Chaetonotus sp.), and (d) hydrachnid nematode. (Original magniÞcations 100¥, (a); 200¥, (b) and (d); 400¥,
(c).) (See color insert following page 70.)

TABLE 2.20
Predominant Higher Life Forms Observed at Various Activated Sludge Organic
Loading Levels
Condition Predominant Groups
Organic Loading MCRT

High Low Flagellates, amoebae and small free-swimming ciliates

Moderate Moderate High diversity of organisms, dominated by free-swimming ciliates
Low High Stalked ciliates, rotifers, and higher invertebrates, especially nematodes

Sources: From Reynoldson, T.B. (1942), Nature, 149, 608; Baines, S. et al. (1953), Sewage Industr. Wastes,
25, 1923; Curds, C.R. and Cockburn, A. (1970a), Water Res., 4, 225; Curds, C.R. and Cockburn, A. (1970b),
Water Res., 4, 237; Curds, C.R. (1975), in Ecological Aspects of Used Water Treatment: The Organisms and
Their Ecology, Academic Press, New York, Chap. 5; and Mudrack, K. and Kunst, S. (1986), Biology of
Sewage Treatment and Water Pollution Control, John Wiley & Sons, New York. With permission.

3. Methods for Differentiating Microbiological
and Process-Related Solids Separation Problems
The methods described in the following section were
developed by Wahlberg et al. (2001).
a. Dispersed SS (DSS)
DSS are the SS remaining in the supernatant of an acti-
vated sludge sample that has been settled quiescently for
30 min in the container used to take the sample. This
avoids any changes in ßoc structure that could be caused
by sample transfers. This test indicates the state of ßoc-
culation existing at the location in the treatment train from
which the sample is taken (Table 2.26).
b. Flocculated SS (FSS)
FSS are the SS remaining in the supernatant of an activated
sludge sample that has been stirred with rotating paddles

Copyright 2004 by CRC Press LLC.