Office of Research a n d Center for Environmental Research

CW3
' /—- LB _____
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g) a4 mime
D l Information
Cincinnati OH 4 5 2 6 8
'm—.—.—.—______—.—

TéchnSIOQy Transfer ___ EPA/625/8-87/O12

FEPA Summary Report

The Causes and

Control of Activated
Sludge Bulking and
Foaming
 ' Summary Report
The Causes and Control of
Activated Sludge Bulking
‘ ' andFoaming ‘ 1

July 1987 .. $3,233

Prepared for
U . S . Environmental Protection Agency
Center for Environmental Research Information
Cincinnati, Ohio 4 5 2 6 8
EPA Contract 6 8 - 0 3 - 3 2 5 2

Prepared by
Dynamac Corporation
The Dynamac Building
1 1 1 4 0 Rockville Pike
Rockville, Maryland 2 0 8 5 2
 Foreword

The United States Environmental Protection Agency (EPA) has a responsibility to

collect and transmit technical information that can be of use to members of the
public involved in operations that contribute to increasing or maintaining the
quality of the environment. In order to be most effective, this effort must be
documented in a manner that facilitates the transfer of the technical information
to the public for consideration and use. This report is an effort t o provide a
reference material on the causes and controls of sludge bulking and foaming in
activated sludge treatment that can be readily understood, but also includes suf—
ficient detail (in the appendices) to help plant Operators control their systems.
 Contents

Chapter ' Page

1 . 0 Introduction . . . . . . . . '. . . . . . I. . . . . . . . . . . . . . . . .. . . . . . . . . . . . . 1

1 . 1 Definition o f Problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
, 1 . 2 Overview of Activated Sludge Process . . . . . . . . . . . . . . . . . . . . . . . 1

2.0 ActivatedSludgeFloc . . . . . . . . . . . . . . . . . . . . . . '. ... . . . . . . . . . . . . 5

2 . 1 The Floc—-Contents and Components ....................... 5 '
2 . 2 Filamentous Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2 . 3 Definitions of Terms Used for Quantitative
Description o f Floc . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2 . 4 Effect of Number of Filamentous Microorganisms on
Quantitative Floc Parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

3 . 0 Activated Sludge Bulking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9

3 . 1 Symptoms of Sludge Bulking . . . . . . . . . . I. . . . . . . . . . . . . . . . . . . 9

3 . 2 Role of Filamentous Microorgahisms in Sludge Bulking . . . . . . . . . . . . 9
3 . 3 Factors Affecting the Presence of Filamentous .
Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9
3 . 4 Control of ActiVated Sludge Bulking . . . . . . . . . . . . . . . . . . . . ; . . . 13

4 . 0 Activated Sludge Foaming . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

4 . 1 Symptoms of Sludge Foaming . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4. 2 Role of Filamentous Microorganisms In Sludge Foaming . . . . . . . . . . . 29
4 . 3 Factors Affecting t h e Presence of Nocardia . . . . . . . . . . . . . . . . . . . 29
4 . 4 Control of Activated Sludge Foaming . . . . . . . . . . . . . . . . . . . . . . . 29

5 . 0 Appendix 1 —IVIeth0ds for Microscopic Examination of Filamentous

Microorganisms in Activated Sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5 . 1 Sampiing Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
5 . 2 Staining Procedures . . . . . . . . . . . . . . . . . _. . . . . . . . . . . . . . . . . . 34
5 . 3 Examination Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
5 . 4 Counting Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41

6 . 0 Appendix ZmMicroscopic Identification of Filamentous

Microorganisms . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

6 . 1 Observation of Microorganism Characteristics . . . . . . . . . . . . . . . . . 45

6 . 2 Identification of Microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . 61
6 . 3 Descriptions of Types of Filamentous Microorganisms . . . . . . . . . . . . 61

7 . 0 Appendix 3 - C a s e Histories of Bulking Control

Using Chlorination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

References . . . . . . . . . . . . , .................................... 85

iii
 Figures

Number Page

Microscopic appearance of activated sludge flocs .............. 6

Effect of filamentous microorganisms o n activated
floc structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7
Effect of filamentous microorganisms on floc
structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
Correlation of filament count and SVI . . . . . . . . . . . . . . . . . . . . . . 11
Activated sludge process schematic . . . . . '' . . . . .. . . . . . . . . . . . . . 15
Zone settling of activated sludge solids . . . . . . . . . . . . . . . . . . . . . 16
Clarification tank design and operation diagram . . . . . . . . . . . . . . . 18
Clarification tank thickening analysis example . . . . . . . . . . . . . . . . 21
Step feed Operation example schematic . . . . . . . . . . . . . . . . . . . . 23
Step feed Operation example— plot of parameters . . . . . . . . . . . . . . 24
Progressive effects of chlorination ........................ 27
Nocardia foaming In activated sludge . . . . . . . . . . . . . . . . . . . . . . 32
Floc “texture” in activated s!udge . . . . . . . . . . . . . . . . . . . . . . . . 39
Effect of filamentous microorganisms o n floc . .' . . . . . . . . . . . . . . . 4O
Filament abundance categories . . . . . . . . . . . . . . . . . . . . . . . . . . 42
Trichome branching . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48
Examples of filament shapes . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
“Color" of filamentous microorganisms . . . . . . . . . . . ._ . . . . . . . . 50
Attached growth of epiphytic bacteria . . . . . . . . . . . . . . . I, . . . . . . 51
Appearance of sheaths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52
Cell shapes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
Deposition of intracellular sulfur granules . . . . . . . . . . . . . . . . . . . . 55
Gram staining reaction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
Neisser staining reaction . . . . . . . ‘. . . . . . . . . . . . . . . . . . . . . . . . 57
Thiothrix I! . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
Type 0 2 1 N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 59
Thiothrix I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
Dichotomous key for filamentous microorganism
identification ....................................... 62
Sphaerotilus natans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
Type 0041 and 0 6 7 5 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
Type 0914- . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
Beggiatoa spp... type 1 8 5 1, 0 8 0 3 , and 0 0 9 2 . . . . . . . ; . . . . . . . . 69
Type 0 9 6 1 and Microthrix parvicella . . . . . . . . . . . . . . . . . . . . . . . 70
Nocardia spp., and Nostocoida Iimicola . . . . . . . . . . . . . . . . . . . . . 71
Haliscomenobacter hydrossis type 0 5 8 1 , 1 8 6 3 , and
041 1 ............................................ 73
Type 1 7 0 2 , 1 8 5 2 , 0 2 1 1 , Flexibacter spp., and
Bacillus spp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Fungus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75
Control of bulking by RAS chlorination . . . . . . . . . . . ; ......... 79
Use of target SVI to coritro'l RAS chlorination
dosage . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
Comparison of RAS chlorination effectiveness . . . . . . . . . . . . . . . . 81
SVI and chlorine dose to RAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82
SVI and chlorine dose to RAS . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83
SVI response to peroxide treatment . . . . . . . . . . . . . . . . . . . . . . . 84
 Tables

Number Page

1 Causes and effects of activatedsludge separation -

problems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
2 Dominant filament types as indicators of conditions
causingbulking............................- ........... 12
3 Bulking filamentous microoganisms that have been .
controlled by chlorination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 25
4 Use of hydrogen peroxide for bulking control . . . . . . . . . . . . . . . . . . . 26
5 Subjective scoring of filament abundance ..... -. . . . . . . . . . . . . . . . 41
6 Suggested format for filamentous microorganism _
identification worksheet. ... . ; . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
7 Summary of typical morphologicaland staining
characteristics ..... ............... 63

‘‘‘‘‘‘ mm“ ‘
 Acknowledgement

Major portions of this document are taken in whole or in part from the recent EPA
report, “Manual on the Causes and Control of Activated Sludge Bulking and
Foaming,“ authored by David Jenkins, Michael G . Richard, and Glen T . Daigger
under EPA Cooperative Agreement CR 8 1 1 8 1 0 .

vi
 = Chapter; 1 .
1 . 0 Introduction:
The reaction of wastewater with biologically active = and the hydraulic
sludge to remove degradable pollutants from the!
wastewater is a widely applied treatment technique.
The microscopic organisms in the sludge metabolize
the degradable constituents of the waste, utilizing the
products for growth and releasing less noxious by-
products into the treated water.
The activated sludge treatment process consists of
two basic unit ’operatiens: (1) aeration, and (2)
clarification. Ihe’se operations may be physically
separated or, as in a Sequencing Batch Reactor (SBR)
process, m a y take place in the same unit. I n aeration,
microorganisms are brought into contact with biode-
gradable wastes in t h e presence of a continuous
oxygen supply. I n clarification, the solids (consisting
mostly of the active microorganisms and metabolic
products) are allowed to separate from the liquid.
Some of this activated sludge is then returned to the
aeration tank, while the rest is removed from th e
system as waste activated sludge for further treat-
ment and final disposal. The clarified liquid (effluent)
flows from the clarifying tank to final processing
(such as filtration or disinfection) before discharge.
The efficiency of this process depends upon the
satisfactory functioning of both the biOIOQicaI oxida-
tion and t he solids separation processes. This report
concentrates o n t w o problems that inhibit satisfac-
tory separation of sludge solids: (1) bulking, and (2)
foaming. The report also discusses their causes and
presents means by which they may be controlled.
1 . 1 Definition of Problems
Several common problems may arise in the separation
of activated sludge from wastewater (see Table 1). Only
two specific problems are considered in this report.
1. 1. 1 Sludge bulking;
This is a condition in which the sludge becOmes very
light, increases in voiume, and will not settle. The
specific cause of sludge bulking considered here is the
overabundance of filamentous microorganisms in the
sludge that extend from the floc and interfere with the
compaction and settling of the sludge. The effects i o f
- the-bulking are a High sludge Volume Index, low
solids concentration in the return and waste sludge,
overloading of the solids handling
systems. Under these conditions, floating sludge
flows out of the tank with the effluent, producing a
poor quality product.
1. 1.2 Sludge foaming.
This is a condition in which various kinds of foams
appear on the surface of aeration and clarification
tanks. One type of foam occurs during startup of acti-
vated sludge plants and usually disappears once the
process is established. A more troublesome foam,
heavy and brown, can accumulate on the surface of the
aeration tank during normal Operation, migrate to the
clarification tank, and is then discharged with the efflu—
ent. It may become so thiqk that it overflows the clarifi-
cation tank. A s with bulking, the principal cause of this
type of foaming is the overabundance of filamentous
microorganisms. This report considers those foams
caused by the presence of specific filamentous micro-
organisms in the floc, i.e., Nocardia ssp. and Microthrix
parvicella.
The deleterious effects of sludge bulking and foaming
occur mainly in the clarification tank, and result in
degradation of effluent quality. The following is an
overview 9f t h e component steps of the activated
sludge process in which these two problems occur.
1 . 2 Overview of Typical Activated Sludge
Process
1.2. 1 Unit processes.
After preliminary treatment (e.g., screening and grit
removal) and primary treatment (settling), the influent
wastewater in a typical activated sludge treatment
plant flows into the aeration tank, along with acti-
vated sludge returned from the secondary clarification
tank. In the aeration tank, the mixture is aerated, pro-—
viding mixing to bring the wastewater and sludge into
close contact and supplying oxygen to the microorga—
nisms in the sludge. The bacteria digest t h e biode- '
gradable components of the wastewater. The mixture
of wastewater and sludge (mixed liquor) then fléws to
the clarification tank, where settling of the sludge oc—
curs. The solids settl’e to the bottom of the tank, while
the clear effluent is drawn off. Some of the settled
‘‘‘‘‘ “WNW
Table 1. Causes and Effects of Activated Sludge Separation Prpblems
Problem Cause
Dispersed growth Mibroorganisms do not form flocs but
are dispersed, forming only small
'clumps or single cells.
Slime (jelly) Microorganisms are present in large
Viscous bulking: (also pos’sibly amounts of eXocellular slime. I n
has been referred to as Non- severe cases the slime imparts a jelly-
filamentous bulking) ' like consistency td the activated
,sludge.
Pin floc or Pinpoint floc Small, compact, weak, roughly
spherical flocs are formed, the larger
of which settle rapidly: Smaller
aggregates settle slowly.
Buiking Filamentous organisms extend from
flocs into the bulk solution and
interfere with compaction and settling
of activated sludge.
Blanket rising Denitrification in secondary clarifier
releases poorly soluble N2 gas which
attaches to activated sludge flocs and
floats them to the secondary clarifier
sUrface. . “
Foaming/Scum formation Caused by (i) non-degradable
surfactants and by (ii) by the ,
presence of Nocardia spp. and
sometimes by the presence of
Microthrix parvicella.
Effect
Turbid effluent. No zone settiing of
sludge. . - .' .-
Reduced settling and compaction "
rates. Virtually n o solids separation in
severe cases resulting in overflow of
sludge blanket from secondary
clarifier. I n less severe cases a .
viscous foam often is present;
.Low sludge volume ihdéx (SV'I) and-a
cloudy, turbid effluent. ll
High SVI —very"clear s‘upernatan’fi..
Low RAS and WAS solids concentra-
tion. I n severe cases overflow of'
sludge blahket occiurs. Solids handling
processes become hydraulically .
ovefloaded. ' . I;
A scum of activatedisludge forms on
surface of secondary clarifier.
Foams§float large amounts‘of .
activated sludge solids to the surface
of treatment units. Nocardia and
Microthrix foams are pefisistent and
, difficUlt to break mechanically} Fo'ams
accumulate and can ‘putrify. Solids'
can overflow into secondary efflué'ntr
or overflow tank freeboard onto
walkwa ys.
' sludge is returned t o t h e aeration tank, while the ex— chlorine, some = a m m o n i a in the effluent increases t he
cess sludge is removed for further treatment and final amount of chlorine needed t o bring the effluent within
disposal. Sometimes the return sludge is aerated in a regulatory limits. Control of ammonia conversion is
separate tank before it is reintroduced to the main maintained by careful control of the level of oxygen in
- aeration tank, to reactivate th e microorganisms by the aeration tank, and by aging of the sludge to en-
allowing them ”co-complete consumption of waste re— sure the presence of nitrifying bacteria. '
maining in t he sludge.
Phosphorus is incorporated into therprotoplasm of t h e
1. 2. 2 Microbiological processes. new cells that are gerierated when the bacteria
The character of municipal wastewater is usually ex— multiply as they oxidize the organic waste. Fifty to 9 0
pressed in terms of two parameters: Five--day Bio- percent of phosphorus. in influent wastewater can be
chemical Oxygen Demand (BODS) and Suspended removed by the activated sludge process:
Solids. (88). BOD5 indicates the amount of oxygen
that would b e required for biological oxidation of the ‘ 1. 2. 3 Physical plant.
waste." -It is determined by a standard test wherein a Wastewater enters the typical plant and receives
known a m o u n t of wastewater is mixed with a known ‘ preliminary treatment. This may include use of
ambunt of oxygen, and the mixture is incubated for 5 screens (when 'there are many large floating solids),
days at 20°C. During that time bacteria in the comminutors (grinders to achieve size reduction), and
wastewater use the oxygen to oxidize organic matter grit chambers (when there are inorganic suspended
in the waste A t the end of that time t h e oxygen rev solids). This is usually, but not always, followed by
maining in the mixture is measured and frdm the primary treatment in sedimentation tanks (for removal
amount of oxygen consumed, the BOD5 in milligrams _ of settleable solids).
per liter is calculated. SS is expressed in milligrams“ '
The wastewatér then flows. to t he aeration tank. I n ‘
per liter, and is determined by filtering a sample of the
wastewater and weighing the dried, solid—containing some cases, the waste is mixed with return activated
sludge at a point outside t h e aeration tank, and the
filter. .= (Standard Methods 2 0 8 D ) " .
- mixture then enters the tank. I n other systems, th e
The objective of the activated sludge treatment proc- waste ‘and return" sludge are mixed in the aeration
333 is the reduCtion of both the BOD and 3 3 of the ‘tank. In the aeration tank, the mixture is aerated by
wastewater Bacteria in the sludge convert organic' either a diffused air system (in which compressed air
material in t he waste to more stable material and in- enters t he tank at t he bottom of the tank, causing th e
organic byproducts ; ( C O Z , H20) and cell protoplasm tank's cohtents t o circulate) or a mechanical aeration
in t h e presence of o x y g e n . I n o r d e r for t h i s process to system‘ (in w h i c h a b l a d e or pump i s u s e d to agitate
proceed at Optimum biological activity, th e plant the"surfac:e or pump the mixture and toss it in the air,
operator needs t o determine the appropriate ratio" to 'and"1:'o-.introduce oxygen and mix the sludge and
be maintained between t h e food supply,(BOD) in the wastewater). T h e _‘size of the aeration t a n k i s a f u n c -
incoming wastewater and the mass of bacteria in the tion of the length of time that the waste will remain in
aeration tank. the tank.

Other biologiCal conversions that can take place in the
aeration tank are phosphorus removal and nitrogen
rempval. Bothiof these materials stimulate natural
bionical activity i n _ t h e effluent-receiving waters,
causing a decrease 'in dissolved oxygen and thus
resulting in a decreased ability of receiving streams to
support fi_sh._ ' . vent it from accumulating and flowing out with the ef-
Nitrogen removal begins with the Conversion of am—
monia t o nitrites and nitrates in the presence of oxy—
gen (nitrification, conversion of Nitrogenous Oxygen
Demand [NOD]). Then the liquid containing the
nitrites and nitrates is placed under anaerobic condi—
tions with an organic carbon source for the denitrify-
ing bacteria (9.9., methanol or raw sewage) where the
nitlrites and nitrates can be converted to the N2 gas
that is lost from the liquid (denitrification). This two-
step process is not accomplished at 1 0 0 percent effi-
ciency—some ammonia remains in the effluent. Fur-
thermore, _in plants that disinfect effluent with
After a suitable contact period (9.g . 4 to 2 4 hours)
thé mixture of activated sludge and treated waste—
water; called: mixed liquor flows into t h e secondary
clarification t a n k , w h e r e t h e s l u d g e settles o u t . This
tank must be carefully monitored so that the settled
sludge is removed at frequent enough intervals to pre—
fluent, and to prevent anaerobic conditions.
The settled slxudge removed from the clarification tank
is us'ually split into two streams. Some is returned to
the aeration tank, and the rest is wasted and dis—
posed, usually after thickening by sedimentation,
centrifuging, or filtration. Sometimes the return ac-
tivated sludge is reaerated t o maintain aerobic condi—
tions before its return t o t h e aeration tank. The clear
effluent flows from the clarification tank t o be disin-
fected and discharged from the plant.
 1.2.4 Batch and continuous flow processes.
In a batch operation, material enters and leaves the
tank only at the beginning and end of a full treatment
cycle (including aeration and settling). All processes
are performed in the same tank, but sequentially
rather than simultaneously. The tank is filled, aerated
for a set time, aeration is shut off, the solids are
allowed to settle, and the clear supernatant is drawn" " :
off to allow room f o r ‘ t h e next ”batch" of raw
wastewater. A portion of the sludge is retained to
seed the next cycle. The excess sludge is wasted.

Most large activated sludge plants use the continuous

flow process. In this process, the wastewater flows
continuously from one tank to another through the
system with a discrete process occurring.in each unit.
Each process is therefore occurring continuously and
simultaneously with the other major treatment proc—
esses. I n such a system, a continuous flow of food
(wastewater) and return activated sludge enters the
aeration tank, and a continuous flow of effluent and
settled activated sludge leaves the clarification tank,
 Chaptér 2

2.0 Activated Sludge Floc Thus, filamentous (macrostructure) microorganisms .

are extremely important to a good floc and efficient
The activated sludge process produces a mass of
production of a clear effluent (see Figure 1).
active microorganisms, which agglomerates and floc-
culates in the system's aeration tank and secondary
> 2. 2. 1 Effect of filamentous microorganism count on
clarification tank, and which is capable of oxidizing
. floc.
organic matter in wastewater. Many of the operating
W h e n filamentous microorganisms are present in acti-
problems that occur in activated sludge systems can
vated sludge, they form a network to which the
be attributed to various characteristics of the acti-
smaller flee—forming bacteria can cling. This promotes
vated sludge floc. This section presents a description
the formation of larger flocs, which d o not readily
of the composition and contents of the floc.
break apart ih'the aeration tank, and which settle more
efficiently, mechanically removing smaller particles as
2 . 1 The Floc—Contents and Cbmponents
they settle. These large flocs are irregular in shape, in-
Activated sludge floc is normally composed of a stead of small and spherical.
widely varied assortment of microorganisms and par-
ticle sizes. Particles range from single bacteria, Activated sludge floc can have a good balance of fila-
measuring 0 . 5 to 5 m, to large flocs measuring 1 mm mentous and flee—forming bacteria, giving good set—
( 1 , 0 0 0 p m ) or more. tling characteristics and a clear effluent; have too few
filamentous microorganisms, resulting in pin floc and
The primary active population of the floc is hetero: a poor—quality effluent; or have too many filamentous
trophic bacteria (those that feed on organic matter), microorganisms, resulting in bulking (see Figure 2).
including Pseudomonas, Achromobac‘ter, Flavobacte-
rium, Alcaligenes, Arthrobacter, Citromonas, and 2.2.2 Examination of filamentous microorganisms in
2009193. Also present in smaller quantities are fungi, floc.
protozoa, and metazoa. Flocs also contain organic and I n order to effectiveiy manage Operational problems
inorganic particles from the influent wastewater, caused by filamentous microorganisms, it is important
”extracellular polymers” that seem to promote bio- to know not only how many are present, but also what
flocculation, and volatile matter. types (see Section 3 ) . Microscopic examination of floc
will yield data o n microorganism number and type,
Researchers have suggested that there are two levels and on physical fioc characteristics that influence set-
of structure in floc. The “microstructure,” which is tling. Appendices 1 and 2, present specific information
the basis for floc formation, consists of bacteria that about methods for performing such examination, in-
are able to “stick to” one another by microbial aggre- cluding procedures for sampling, staining, counting,
gation and bioflocculation. The “macrostructure,” and identifying organisms.
which consists of filamentous microorganisms, forms
a large network to which the flee-forming bacteria of 2 . 3 Definitions of Terms Used for
the microstructure can cling. '
Quantitative Description of Floc
2 . 2 Filamentous Microorganisms Several quantitative parameters exist for the descrip-
tion of activated sludge floc. These parameters are
I f a particular sludge contains only microstructure defined below.
bacteria, and n o macrostructure (filamentous) micro-
organisms, flocs are small (averaging approximately 2. 3. 1 Mixed Liquor Suspended Solids (MLSS).
7 5 p m ) , round, and easily broken up in the aeration
The contents of the aeration tank is referred to as
tank. This results in an operational problem known as
mixed liquor. It contains suspended microorganisms
”pin f l o c . ” Such floc will settle rapidly, but will leave and other sludge components. MLSS is an expression,
large amounts of smaller particles still suspended in in m g / l , of the amount of microorganisms suspended
the effluent.
Figura 1. Microscopic appearance of activated sludge flocs: a. small. weak flocs (pin-{loo} (100x phase contrast);b. small, weak flops. __
(1OOXphasa contrast): c. flocs containing microorganisms (100 X phase contrast); d. floc containing filamentous microorganisms ‘
"network" or “backbone" (1000 x phase contrast) (a and 0 bar: 100nm; b and d bar: 10pm).

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Figure 2 . Effect of filamentous microorganisms o n activated sludge floc structure.

A . Ideal. Non-bulking Activated Sludge Floc.

'
1. FLANENTOlB ORGANISMS Am FLOO
FWIIG ORGANISMS IN BALANCE

2. STROKE, LARGE FLOO

3. FILAMENTS DO NOT INTERFERE

4. CLEAR SIPERNATANT

5. LOW SVI

F ILAMENT BACKBONE

B. Pin-pofnt Floc.

_
. _0 0 0 ' . C? , 1.NOFI.AMEN1'OUSORGANISMS
.0 v
2. WEAK. SMALL FLOC
. .

3.mm swam-rm
033 0 & o g3 I
_ ' 4.Low svu
‘ O 1
0
(5 .,
C ‘ a: ° \
C}
DISPERSED PARTICLE
Q 0 R0

C. Filamentqus Bulking Activated Sludge.

“‘1
1. FILANENTOUS ORGANISJS PFEDOMINAN'I'
a V . I 2. smoua . LARGE FLOO
‘ ‘ 3. FILAIvENTs IN'I'ERFERE WITH 85mins,
I - count-xenon .
c , ‘ EXTENDED FILAMENT
4. CLEAR WTANT
lb
. Q I» o
'
FILAMENT BACKBOE

Reference: Jenkins, et 3 L , 1 984.

 in the mixed liquor, in mg/I, and is a measure of the
amount of microoyganisms available to degrade the
waste, or of the strength and density of the mixture as
it flows from the aeration tank to the clarification tank.
This parameter is determined by filtration of a known
volume of mixed liquor, and drying and weighing of
the filter and solids.(Standard Method 209C).
2.3.2 Sludge Volume Index (SVI).
This value measures the settling characteristics of the
floc (sludge), and aids in determining if any adjust-
ments should be made in operations.
SVI is the volume (in ml) of one gram of sludge after
30 minutes of settling. It is determined by taking one
liter of mixed liquor from a point near the outlet of the
aeration tank, allowing the sample to settle for 3 0
minutes in a graduated cylinder, and performing
following computation:
2.3.4 Dissolved Oxygen (DO).
This parameter is a measure of the amount of dis-
solved oxygen present in the process tanks. A mini-
mum of 0 . 5 m g f l of D O should be maintained in the
aeration tank. D 0 must be carefully monitored and
controlled to ensure efficient operation. This monitor-
ing may be accomplished with a portable oxygen
meter or by lab analysis of samples (the former is pre-
ferred because there is no delay in obtaining results).
Ideally monitoring takes place continuously at several
points in the aeration tank.
2.3.5 Food/Microorganism Ratio (F/M). _
This parameter indicates the ratio of food entering the
system to the mass of microorganisms being aerated.
I t 'IS computed as follows:
F/M pounds per day of BOD added to aeration tank
the
MLSS in aeration t a n k (lbs)

SVI = volume (sludge) after 30 min. (ml/l) x 1000 = fl
SVI is a measure of the settling characteristics of the
sludge—a sludge with a low SVI will settle and com;-
pact well. However, an SVI that is too l o w is indicative
of pin floc. A high SVI is indicative of bulking.
2.3.3 Sludge Age.
This parameter measures the average number of days
that solids remain in the system.
Sludge Age is computed using known parameters:
_da
VoI. aer. tank (gal.) x M L S S (mg/l) __
Recycle flow rate (gal.Iday) x recycle MLSS (mg/I)
A similar parameter, Sludge Retention Time, is com—
puted by determining the mass of microorganisms
under aeration or reaeration in the system, and divid-
ing that value by the mass of microorganisms released
in the waste activated sludge and lost in the effluent.
Sludge Age is important because it aids the operator in
maintaining a viable population of microorganisms.
The common range for Sludge A g e in a conventional
activated sludge plant is 3 to 1 5 days. Optimum
Sludge A ge can vary seasonally, as biological activity
increases in summer and decreases in winter. Thus
Sludge Age should generally be adjusted at least twice
a year, raised in the winter and lowered in the summer.
The normal range for F/IVI for a conventional plant is
0.15 to 0.5.
2 . 4 Effect of Number of Filamentous =
Microorganisms on Quantitative Floc
Parameters
SVI is the parameter most closely related to the number
of filamentous microorganisms in the floc. Pin floc (floc
with very few filamentous microorganisms) will. have
an SVI below 7 0 mI/g, while bulking sludge (with high
numbers of filamentous microorganisms) will have an
SVI above 1 50 ml/g.
3
Similarly, filamentous microorganisms affect the
amount of suspended solids in the effluent; Sludge
having few such microorganisms and thus resulting in
pin floc will yield a turbid effluerit, containingmany
suspended solids. A bulking sludge with too_ many fila-
mentous microorganisms will result in a very clear ef—
fluent b u t as t h e effluent flows from the clarification
tank it will carry the large light flocs increasing the
88 In the effluent.

A Sludge Age that is too low can cause floc to be light

and fluffy, settling slowly or escaping with the
effluent (a bulking condition). A high Sludge Age (too
many solids in the system) can result in pin floc and a
turbid effluent.
 Chapter 3
3.0 Activated Sludge Bulking
Sludge bulkingis one of the most common bperational.
problems experienced by activated sludge treatment
plants.
3.1 5 Symptoms of Sludge Bulking
Sludge bulkihg has visible symptoms as well as ef—
fects on the parameters used to assess the functions
of the activated sludge system.
3 . 1 . 1 Qualitative manifestations.
Sludge, bulking occurs w h e n sludge in the clarification
tank gains in volume and becomes light and fluffy,
flowing out over the weirs with the effluent. The ef— tions are summarized in Table 2 . The research that re—
fluent itself is otherwise very ciear.
3 1..2 Quantitative manifestations.
The most conspicuous quantitative symptom of
sludge bulking is an elevated Sludge Volume Index
(SVI). Bulking sludge generally has an SVI of over
1 5 0 m l / g . The density of bulking sludge is much
lower than that of ” i d e a l " sludge, and therefore fewer
Microorganisms are returned to the aeration tank in
the return: activated sludge (HAS). This interferes
drastically with treatment efficiency, because fewer
microorganisms are present in‘ the aeration tank to
digest organic matter.
3. 2 Role of Filamentous Microorganisms“ In
Sludge Bulking
As discussed in Section 2.2, filamentous microorgan-
isms play an important role in the flocculation of acti-
vated sludge, forming a “macrostructure” to which
smaller bacteria can cling (see Figure 3). Without fila-
mentous microorganisms, pin floc can develop.
However, if there are too many filamentous micro-
organisms, floc will be diffuse and will settle poorly,
resulting in bulking.
3.2.1 Relationship of filamentous microorganism
count to bulking.
The several methods of expressing counts of fila—
mentous microorganisms are discussed in Appendix
1 . In general, a filamentous microorganism count that
exceeds a known standard, generated through experi-
mentation and observation; will=result in a high SVI
and a bulking condition in the sludge (see Figure 4 ) .
Different filamentous microorganisms (and mixes of
such organisms as occur in activated sludge) do differ
in the number of such organisms necessary to impart
a bulking condition to the sludge.
3.2.2Relationship of specific filamentous
microorganisms to bulking. ,
Researchers have developed correlations between
bulking and various specific filamentous microorgan—
isms. Further, they- have shown that different micro—
‘ organisms that can cause bulking flourish when differ—
ent operating problems are present. 'These correla—
sulted in this table gathered data on plant operating
conditions only incidentally;-it was focused o n deter-
mining the presence of various microorganisms in
bulking sludge. However, t he table can be used to de-
termine probable operational causes of bulking, if the
dominant microorganisms in the bulking sludge are
identified. (See Appendix 2 for a guide to microscopic
identification of filamentous ‘microorganisms.’)
3.3 Factors Affecting the Presence of
Filamentous Microorganisms
A s Table 2- shows, changes in plant operating-condi—
tions, and resultant changes in the quantitative
parameters defined in Section 2.3, can pi'omote the
growth of different filamentous microorganisms.
A low pH in the aeration tank canpromote ”the growth
o f ‘ f u n g i . Based on Iaboratory eXperiments, the best
activated sludge performance can be obtained at pH
values from 7 . 0 to 7 . 5 although removals of oxygen
consumed, suspended solids and bacteria are good
at pH values ranging from 6 . 0 to 9 . 0 . The presence of
fungi indicates that t h e wastewater most likely con—
tains strong acid discharges and an aeration tank pH
value below 6.0. .
Low- D 0 in the aeration tank can promote the'growth of
S. natans and type 1 7 0 1 . Low D O concentrationin low
F/IVI systems can promote the growth of H. hydrossis.
Experiments have shown that the D 0 concentration re—
quired to prevent the growth of low D 0 filamentous
Figum3 . Effect of filamentous microorganisms on fine structure: a. aggregates observed foi a pure culture of an activated sludge floc
former: b. aggregates observed for a dual culture of a single flOc former and a‘single filamentous microorganism (100x phase
contrast: b a r : 100 pm) (Lau et aL. 1984).
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10
Figure 4. Correlation of filament count and SVI for secondary (first stage) and tertiary (second!stage) activated sludge systems at the San
Jose! Santa Clara Water Pollution Control Plant, CA (Beebe at al., 1982). '

140

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11
 Table 2. Dominant Filament Types as Indicators of Conditions Causing Activated Sludge Bulking

SuggestedCausative Conditions Indicative Filament Types

Low
DO type 1 7 0 1 , S. natans, H. hydrossis

Low
Fl M M. parvicella, H. hydrossis, Nocardia- sp
types 021N.r 0 0 4 1 , 0 6 7 5 , 0092, 0 5 8 1 , 0 9 6 1 ,
0803

Septic
WastewaterlSulfide Thiothrix sp., Beggiatoa and type 0 2 1 N

Nutrient
Deficiency Thiothrix sp., 8. natans, type 0 2 1 N, and possibly
H. hydrossis and types 0 0 4 1 and 0 6 7 5

fungi

Richard e t al. 1 9 8 2 3 ; Strom and Jenkins 1984.

microorganisms was a function of the FIM load. The wastewater at the point where the RAS and influent
greater the FIM load, the greater the D O concentration enter the tank. Emperical measures producing a car—
required to prevent the low D O bulking. b o n a c e o u s substrate concentration gradient i n the
aeration tank, or a high substrate concentration at the
Experiments have also shown that low D O bulking point where RAS and influent enter the aeration tank
can be caused and cured by manipulation of F/M load have Consistently controlled l ow F/ M induced bulking
and D O concentrations. However, in dealing with low in laboratory studies, but less effectively- in practice.
DO bulking problems, a basic economic question The correlation of cause and effect between low F/M
must lge addressed. To cure the problem, one must load and the presence of filamentous microorganisms
lower the FIM or increase the aeration tank D O con- is not completely understood and is being studied
centration. Each of these actions may have undesir- further. ' ‘
able consequences. Lowering the PM may cause the‘
onset of nitrification and increase MLSS levels to Treatment of septic waste can lead to growth of
values that exceed the solids capacity of the secon- Thiothrix spp. and type 0 2 1 N . This may result from
dary clarifiers. Increasing the aeration tank DO con- the ability of these microorganisms to grow on in-.
centration will require greater power input and may organic reduced sulfur compounds and organic acids,
8150 cause the onset of nitrification. Because of these which are produced by fermentation in septic
factors, it is necessary to consider the alternative of sewage. Under such conditions these microorgan—
operating at a low aeration tank D O concentration and isms produce intracellular sulfur granules that are
killing off the filamentous microorganisms that convenient for identification purposes.
develop using RAS chlorination. RAS chlorination is
discussed in Section 3 . 4 . 4 . 1 . Treatment of nutrient-deficient wastes (low in
nitrogen o r phosphorus) can lead to growth of
Low FIM load may result in M. parvicella and types Thiothrix spp. and type 0 2 1 N , and possibly types
0041, 0675, 0581, 0961, 0803, and 0092. The 0 0 4 1 and 0 6 7 5 . Under these conditions, Thiothrix
specific causes for the growth of filamentous spp. and type 0 2 1 N d o not contain'intracellular sulfur
microorganisms that appear in continuously fed, granules, but may contain other intracellular granules
completely—mixed, l-ow FIM load activated sludge such as polyhydroxybutyrate (PHB). Also under
systems are poorly understood. These systems pro- nutrient-deficient conditions, types 00,41/and 0675
duce poorer settling activated sludge than systems may show a Neisser—pos-itive eXtracellular élime'
that are fed intermittently or have aeration "tanks covering, although they normally _s"iain Neisser‘
when: there is a relatively high local concentration of negative (see Appendix 1 for stain procedures).

12
When microscopic examination suggests nutrient
deficiency, the BOD, COD, or TOC to nitrogen and
phosphorus ratio of the influent should be checked.
Generally, sufficient nutrients are present when the'
influent to be treated by activated sludge contains
BODs/N/P in the ratio of 100/ 5/ 1. If necesSary; the re-
quired nutrients should be added. Long sludge age
systems can treat wastes with’ less nitrogen and
phosphorus than indicated by this ratio because of
the recycling of nutrients by the endogenous decay of
activated sludge in these systems.
While the BODSIN/P ratio is a useful guideline for
detecting nutrient deficient wastes, the following fac-
tors also should be taken into account:
0 The availability of'nitrogen and phosphorus in th e
influent should be high enough for use during the
metabolism of the carbon source.
0 The nutrient supply should be paced so that
nutrients never run out in any part of the aeration
tank as a result of shock from influent carbon
loads.
0 Because each wastewater/activated sludge com-
bination has its own unique nutrient demand, an
influent BOD5/N/P ratio should be checked by
measurements- of effluent concentrations
dissolved ( 0 . 4 5 m p . filtered) orthophosphate,
-monia, and nitrate.
0 Ammonia and: nitrate are available as nitrogen
”sources f q r , activated sludge . g r o w t h . Ammonia.
added in nitrifying systems for nutrient supplema-
tion will be converted to nitrate and still remain
=available as a nitrogen source.
A Sludge Age that is too low can ' r e s u l t in “straggler
floc ” light buoyant particles that d o not settle in thé
clarification tank and flowover the weirs_in an other—
wise clear effluent.
3 . 4 Control of ActiiiatedSludge Bulking
Maintenance of Optimum levels ‘of filamentous
microorganisms in activated sludge is necessary to
ensure efficient settling. Although proper treatment
plant design is the best means of preventing the ex~
cessive growth of filamentous microorganisms that
cause bUIkihg, Some de'sign innovations are very re:
cent, and many existing activated sludge plants. d o
ndt have them. In such plants bulking sludge must be,
corrected by adjustments in operations .or by addition
of chemicals.
3 . 4 . 1 General approach to controlling filamentous
micrbbrganisms.
A good general approach to controlling bulking prob-
lems is as follows: First identify th e filamentous
microorganisms causing the bulking (for identification
procedures, see Appendices 1 and 2). Second, using
the information in Table 2 and familiarity with condi-
tions at the plant in question, determine the probable
cause of bulking. Third, determine whether the under—
lying problem can be immediately rectified by opera-
tional changes (such as prechlorination of septic
waste, addition of missing nutrients, or other adjust—
ment of parameters). Fourth, determine whether the
plant requires major design or operational changes
that might take a long time to implement (such as
changes in aeration capacity or unit configuration). I f
the plant does require such changes, sludge settling
will not immediately become normal when those
changes are made, but will improve only slowly
because sludge remains in the system for an extended
period of time (it could take as long as three times the
Sludge Retention Time for settling to become normal).
Because of the delays associated with major design
and Operational changes, methods are needed for
rapid correction of the problem while such changes
take effect. These methods include process manipuia-
tion, addition of chemicals to enhance settling, and
addition of toxicants to selectively kill filamentous
microorganisms.
3.4.2 Process manipulation to control bulking.
of: In many cases, the adverse effects of bulking sludge
am- can be minimized by proper management of the ac—
tivated sludge system, particularly if it is underloaded
or if certain process options were built into the
original- design. To use these process management
tools to deal with bulking sludge,- it is necessary t o :
°_ Sfudy in detail the teéhnical operating principles of
the activated sludge clarification tank, particularly
as they relate to the thickening function;
°_ Examine specific techniques for determinirig the
solids handling capacity of the clarification tank and
howthis is influenced by sludge settling charac—
teristics; and
0 Review techniques of clarification and aeration tank
operation, in order to maximize clarification tank
capacity in systems with bulking sludge.
3, 4. 2. 1 Design Calculations for Process
Modifications
Bulk sludge problems can often be corrected by
changes in design parameters.' This section reviews
system operations from a quantitative perspective,
and demonstrates caldulatiohs forchanges in design.
Clarifieri Operation Principles. Activated sludge
clarification tanks have two 'basic functions—
clarificati‘on and thickening._ Clarification is the
removal of activated sludge 'floqs-to produce . a clear
overflow that either meets diScharge standards or.
13.
does not overload downstream processes. Thickening 2. Calculation of Required HAS Flowrate -. ‘_
of the settled activated sludge solids (Le... RAS) is
required so that they can be returned to the aeration 0—, OX m
basin. I f the activated sludge solids are not thickened arm
and removed fro-m the clarification tank at a rate faster
than they are added, they will accumulate until the 'This relationship can be used to calculate the RAS
clarification tank is full and the excess solids will be flowrate required for a Specified influent flowrate, the
discharged into the effluent. Since bulking affects the current aeration tank JVILSS concentration, and an
ability of the activated sludge solids to thicken, the RAS SS concentratiOn that it is believed (perhaps bas-
thickening function and thickening capacity of the clari— ed on past history) can be achieved in the clarification
fication tank are affected by sludge bulking. . tank.

3. Clarifier Capacity Calculation-

Process Schematic and Definitions. Figure 5 is a proc-
ess schematic of the activated sludge system. The
aeration tank has a volume V, and the clarification
tank has a surface area A . Wastewater enters the
aeration tank at flowrate 0, together with RAS, at
.con-
flowrate 0‘. The aeration tank suspended solids
centration (MLSS) is X, and the RAS, SS is X“. Ac-
tivated sludge soilds are applied to the clarification
tank at a flowrate of Q + 0,.
I t is assumed here that sludge is wasted from the RAS
at a flowrate Ow, at the same 88 concentration as the
RAS, X“. Effluent is discharged from the clarification
tank at a flowrate of 0—Ow, and it contains an 88
concentration of X...
Process Operating Relationships. In developing rela-
tionships to describe the clarification tank thickening
function. certain simplifying assumptions can be
made. First, the WAS flowrate (Ow) is usually small
compared to the influent flowrate (Q), and its effect
on effluent flow can be neglected. Second, the quan—
tities of activated sludge solids discharged in WAS
and in the effluent are small compared to the quantity
of sludge solids applied to and withdrawn from the
clarification tank.
Using these assumptions and assuming that solids are
not accumulating in the clarification tank, a simplified
solids mass balance over the clarification tank is:
M+QM=0A (n
the top of the settling sludge and thé clear superna-
Equation 1 states that, at steady-state, the rate at ‘7 tant. When the height of the interface is plotted
.time,
which solids are applied to the clarification tank must against
be equal to the rate at which they are removed. Equa-
tion 1 can b e used to develop the following relation-
ships that are useful for assessing clarification tank
operation.
1. Degree o f Thickening that can be Achieved b y a
Clarification Tank
&=M+Q) (2)
X O.
This relationship illustrates that the degree of thicken-
ing is a function of the influent and RAS flowrates.
Q = OJXu -—‘X) (4)
X
This relationship can be used to calculate the capacity
‘of a clarification tank for a specified RAS flowrate and
_an aeration tank MLSS concentratiént and for an
achieveable HAS/SS concentration. Equations 1 - 4
assume that sufficient clarification tank area is
available to thicken the HAS to a concentration of X“.
Techniques for determining the clarification tank area
required t o achieve this are discussed in the following
section. ' ' ‘
Sludge Thickening Theory. Thickening of activated
sludge solids in a clarification tank occurs by gravity
Settling. A s the solids settle, their concentration in-
creases from t h e IVILSS concentration (X) t o the RAS
concentration (XU). Because of their flocculent nature
and concentration, activated sludge particles do not
settle individually, bu t as an entire mass. The solids
settle at a constant rate until they begin to “ p i l e u p ”
at the bottom of the container in which they are settl-
ing. The initial settling velocity of t he activated sludge
solids (Vi) decreases as the initial 8 8 concentration in-
creases. This type of behavior is called zone settling, '
or type III settling.
Zone settling is familiar to anyone who has run an SVI
test. The entire mass of activated sludge settles
together, producing a well-defined interface between
the line initially' is straight but later
begins to level off (Figure 6). The slbpe of the initial
straight line is the initial settling" velocity, V i . Sludges
with higher initial 58 concentrations settle slower
than those with lower initiél 5 8 concentrations
(Figure 6). ‘ ' ‘ ‘
Clarification tank thickening capacity is related to t he
tank’s ability to accept the applied solids load and to
convey all the solids to the underflow. This is ac—
complished in two ways: settling of the activated
sludge solids and withdrawal of RAS by pumping.
RAS withdrawal causes a general flow of fluid 10 the: ,_
bottom of the tank, and this general flow aiéo carries '

14
 SECONDARY
' AERATION BASIN ‘ CLARIFIER

a - , 0+ 0,,x Q-me
INFLUENT ? x, V MIXEDLIQUOR EFFLUENT
_§L

of! xu
t 0W, x“

RETURN ACTIVATED swoea WASTE ACTIVATED SLUDGE

(RAS) . (WAS) '-

' DEFINITIONS:
. A = CLARIFIER SURFACE AREA Qw = WASFwaATE .
V = AERATION BASIN VOLUME x = AERATION BASIN MLss CONCENTRATION
0 = INFLUENT FLOWRATE xu= RAS SUSPENDED SOLIDS CONCENTRATION
Q, = RAS FLOWRATE Xe = EFFLUENT SUSPENDED SOLIDS CONCENTRATION
Figure 6 . Zone Settling of Activated Sludge Solids.

t-
a:
g H I G H E R INITIAL C O N C E N T R A T I O N
m . A
2
ll.
a:
Ill
I-
_z_

LOWER INITIAL CONCENTRATIO N .

O
0 TIME I»
Refaronca: Jenkins, ataL. 1984.

16
 lppib .,

the settling activated sludge solids to the bottom corresponds to the. point at which thickening failure is
where they can be removed. just beginning to occur. The applied solids loading
rate under these conditions is calculated. This is the
The .vafio’Us feb'hfiiques ’that haVe been’ developed to “allowable solids Ioading rate“ for the selected RAS rate
determine the thickening capacity of clarification and the observed sludge settling characteristics. By
tanks generally compare the rate at which activated repeating this procedure at different:RAS rates and
sludge solids are applied t o the rate at which they are with sludges of different settling characteristics, the
conveyed to the bottom of th e tank by settling and clarification tank thickening capacity can be estab-
bulkwithdrawal. A s long as th e application rate is no Iished for a variety of conditions. ‘
greafger than the settling and withdrawal rate, the ap—
plied solids will be conveyed to the bottom of the tank While this technique has been used in practice, it is
and removed (i.e., thickening failure will not occur). I f rather cumbersome and time-consuming, and the ef-
the L s o l i d s application rate is greater, then excess fluent quality may be degraded during testing.
solids will accumulate in the tank until it is full and Moreover, bulking sludge must be available to allow
solids will overflow into the effluent. clarification tank capacity to be measured for bulking
conditions. The major advantage of this method is
Techniques for clarification tank thickening analysis ' that the determination of allowable thickening capaci-
are relatively complex. They account for SS concen— ty is direct and no assumptions are required to trans—
tratibn increases in the‘ clarification tank during settl- late the results to full-scale plant performance.
ing and t he effects of this on the rate at which solids
are Conveyed to the bottom of the tank by both settl— Calculai‘ion techniques fof determihing: allowable
ing and bulk withdrawal. These techniques are ciarification tank thickening capacity require the
discussed thoroughly in the literature (Keinath et al., measurement of sludge settling characteristics. The
..-relationship
1 9 7 7 ) ;Ethe‘y will _no,_t b e discussed in detail here. between 8 8 concentration and the initial
settling velocity must be established either by direct
The thickening capacity of a clarification tank can be measurement or b y using correlations between an in-
stated in terms of an allowableapplied Solids loading dex of sludge settleability and the 8 8 concentration/
rate '(G’a); which is the 'mass of solids applied per unit initial settling velocity relationship developed by
of clarification tank surface area. others. Correlations have been developed using in—
dices such as the standard SVI, the stirred SVI con-
Using the definitions presented earlier, the actual ducted at MLSS = 3.0 g/l, and DSVI.
solids loading rate is:
Ga Direct measurement of the SS concenfratipn/initial
=‘X(O + OVA (5)' settling velocity relationship can be quite cumber—
some and time-consuming. Initial settling velocities
Comparison of the actual solids loading rate with the
allowable rate will indicate whether the clarification must be measured for sludges with various initial 88
concentrations obtained by mixing various propor-
tank‘ is overloaded and whether it will experience
tions of mixed liquor, RAS, and effluent. Measure-
thickening failure.
ments must be made in settling columns: that are at
The' allowable' solidé 'loadin'g‘ fate is a} fUriction of léast 0 . 1 5 m ( 6 in.) diameter, 1 . 8 m ( 6 ft.) tall, and
seVeral factors including the RAS flowrate per unit of equipped with a stirring device. Bulking sludge must
clarification tank surface area (0 IA) and the settling be available before its thickening characteristics can
characteristics of the sludge. By varying these fac- be measured.
tors, it may be possible t o increase the capacity of th e
Figure 7 presents the reSults of a correflation tech-
clarification tank and/or of the activated sludge
nique and analysis developed by Daigger’. and Roper
system.
(1984) in which the allowable solids loading rate (Ga)
Clarification Tank Thickening Capacity. T h i s is plotted as a function of the RAS 88 concentration
parameter can be determined by direct‘measur-ement _ for sludges with SVI in the range 50—— 3 5 0 mI/g.
or by calculation using the techniques discussed Dashed lines that correspond to variOus underflow
above. ' ” r a t e s (0 fA) also are plotted. This correlation is based
on a broad data base and should be readily useable by
Direct measurement of thickening capacity is ac- plant Operators because it uses the standard SVI as
complished by manipulating the 'clarification tank the index of sludge settleability. To use this diagram,
solids loading until thickening failure occurs. For ex- the point that. corresponds to clarification tank
ample, the clarification tank influent flowrate can be operating conditions is located in the diagram. Two of
increased, while the RAS flowrate is maintained cons- the following pieces of information are needed to
tant. The clarifiéation tank sludge blanket depth is locate this point:
monitored and the point at which it begins to increase

17

. | ".1." I151-
 RA
l I I l | I I | | I l l l | l I '
50 mI/g~ 6'00 glpmd/fl-2 _
I .—

ALLOWABLE SOLIDS LOADING LINES / X 500 glp-d/ft2 __

70 FOR VARIOUS SVI VALUES . /
OPERATING LINES FOR VARIOUS / / -
UNDERFLOW RATES 400 gp-d/fl2 —‘

60
100 ml/g ~ g 300 gp-d/flz _

50.

/" 200rgpd/fl2 —
40
8L

l
30'

m.-
\ __ - 10-0 gpd/fl2 __

20 /

all-.-
10

nAs SUSPENDED soups CONCENTRATION, "cu—(g/n' _ _

—_—_J
' The actual solids-loading rate: Assflméithatj the clarification tank in the example
0 T h e u n d e r f l o w rate; a n d ' discussed a b o v e i s 4 5 feet ( 1 3 . 7 m ) i n d i a m e t e r a n d
0 T h e RAS 8 8 concentration. has a surface area of 1 , 5 9 0 ft2 ( 1 4 8 m2). From Equa—
tion 5 , the applied solids loading rate for the intitial
The third piece of’information can be used as a check. operating condition is:
When the point plots below the allowable solids
loading line of interest, the clarification tank is G :X(O+Q)/A
operating below its allowable thickening capacity and =;,(3000 mg/I)(1 0 + . O 33)IVIGD(8. 3 4 ) / 1 5 9 0 ft2
should not be subject t o thickening failure. When the = 20. 9 Ib/ftz——day ( 1 0 2 kg/m2 day)
point falls directly o n the allowable solids loading line
of interest, the clarification t a n k fi s operating at the This conditioh plots as point number 1 in Figure 41
failure point. W h e n the point plots above the and indicates that the clarificatioh tank could operate
allowable solids loading, thickening failure will occur. successfully with a sludge having an SVI of up to
A n example of the use of this *diagram is presented 1_ 5O ml/g.
later in this section;
For the second operating condition (RAS 8 : 8 8 000
Clarifier; Analysis and Operation. The first steps in", . mg/l RAS flowrate = 0 . 6 MGD) the applied solids
clarification tank analysis often are to use the relation- loading rate is:
ships in Equations‘1—4. For example, sludge bulking
generally will cause a decrease in the RAS SS concefi— (3,000 mgfl)(1.0 + O.6)MGD(8.34)/1,590 ft2
tration. Equation 1 predicts that a decrease iri the -' :_— 2 5 . 2 Ib/ftZ—day ( 1 2 3 kg/m2 day)
RAS 88 concentration (Xu) requires an increase in This condition plots as point number 2 in Figure 4 1
RAS flowrate. This increaSe is necessary to enSure and indicates that the clarification tank now can
that sludge applied t o the clarification tank IS removed operate successfully with a sludge having an SVI just
in t h e RAS. over 200 m m .
For example, consider an activated sludge system that NOW-assume that the‘ maximum RA'S pumping capac—
operates at an influent flow of 1 . 0 MGD ( 0 . 0 4 4 ma/sec) ity is . 0 . 9 5 IVIGD ( 0 . 0 4 2 m3/sec). A t the maXimum
and an RAS flow of 0 . 3 3 M G D ( 0 . 0 1 4 m3/sec). The . RAS flowrate, the bulk withdrawal rate is 0 . 9 5
MLSS concentration is 3000 mg/I, and the BAS 8 8 MGD/1; 590 ft2 or 597 gpd/f’c2 (24.3 m/day) and the
concentration is 1 2 000 mg/l. A s a check calculate the applied solids loading rate is:
required RAS flowrate using Equation 1 .
(3 000 mg/|)(1..0+O 95(MGD{8.34)/1 590 ft2
= 3 0 . 7 lb/ftZ-day (1 50 kgfmz—day)
0_ =OX/(X— X) = (1.0 MGD) (3 000)/(12 000- 3 000)
= 0 33 MGD (0.0143/3901 This cohdition plOts as point number 3 on Figure 4 1
and indicates that the -' clarification tank could be
This agrees with , t h e actual RAS flowrate. Now
operated successfully with a sludge having an SVI of
assume that ”the sludge settleability deteriorates and about 2 5 0 m l / g and that the RAS 8 8 concentration
that the clarifier ‘sludge blanket begins to inCrease.
would be about 6 300 mg/I. ' .
The RAS 88 concentration is found to be 8,000
m g / I . Equation 1' indicates that the RAS flowrate Now assume that th e RAS flowrate is maintained at
must be: . - ' . 0.95 MGD (‘0.042 'm3/sec) {equivalent to a bulk with—
draWal rate of 597 gpd/ftz) ( 2 4 . 3 m/day) but that the
0r 2 OX/(XU-X) ='=.,(1.0'MGD)(3,000)/(8,000-3,000)
SVI will be controlled to a valqe of n o more than 1 5 0
= 0.60 MGD.(O.026m3/seb) , . ‘ ‘ , m l l g by RAS chlorination.
Moving along the operating
line (for 5 9 7 gpd/ftz) to point number 4 indicates that
Thus, for these conditions increasing the RAS * the clarificatio‘n tank could be Operated at a .solids
flowrate to O.6 0 MGD should prevent clarification loading rate of up to 4 2 . 8 Ib/ftZ—day (209 kg/mZ-day)
tank failure. , ~ ‘énd with an RAS 88 concentration of up to 8,700
mg/_I at this loading.
The use of Equations 1 —4 to develop alternative
clarification tank Operating strategies must be accom-1
panied b y an analysis of clarification tank thickening
capacity. I n this manual the correlation method (with
SVI) of Daigger and Roper ( 1 9 8 4 ) will be used; as in—
dicated previously, ‘other somewhat less convenient
techniques are available. ’

19
 ‘sblidé
Equation 5 can be arranged to calculate for these The use of step fee'd‘to lower the loading épJ
conditions: plied to the clarification tank is illustrated using the
previous example. In addition to an influent flowrate}
a. The maximum allowable influent flow rate at (Q) of 1 . 0 MGD ( 0 . 0 4 4 m3/sec) an RAS flowrate of;
M L S S : 3000 mg/l ( Q ) of 0 . 3 3 M G D ( 0 . 0 1 4 m3/sec) an IVILSS concen—n
, t r a t i o n (X) of 3,000 mg/l an RAS concentration (X 1:
= (GaA/X) -- Q 2' of 1 2 , 0 0 0 m g / l , and a clarification tank solidus,
= (43.5 lblftz-day(1,590 : loading rate of 2 0 . 9 lb/ft2/day ( 1 0 2 kg/mZ/day), it is;
ft2)/(3.000 mg/l)(8.34))
assumed that the aeration t a n k h a s a total v o l u m e o f “
-- 0.95 MGD
" 0 . 2 5 MG ( 3 7 8 0 m3) and that it can be operated-in...
.1:1.8 MGD (0.79 m3/sec) either the plug flow (conventional) or the two-pass-
step feed mode (Figure 9). .
The maximum allowable MLSS at an influeht flow Point number 1 'in
Figure 1 0 indicates that these
of 1.0 MGD (0.079 m3/sec.) operating conditions are acceptable when the SVI is_
less than 1 5 0 m l / g .
= (GQAMQ + C1,)
= (43.5 lblftZ-day)” , 5 9 0 ft2) When the operating mode is changed to two--pas:-:.f
+ (1.0 + 0.95)MGD(8.34) step feed at the same influent and RAS flow rates all
of the RAS but only one——half of. the influent f l o w '18 ad»
£4,250 mgll dad to the first pass. The remainder of the influent_
f l o w is added to the second pass. A redistribution of-
These examples illustrate how the general principles mixed liquor solids inventory; Occurs so that it is-
of clarification tank analysis can be used to optimize higher in the first pass than .in the second pass, This
the existing activated sludge clarification tank opera- arises because less dilution of the RAS by influehf oc—
tion. curs in the first pass than in the second pass: I f t h e :
total mixed liquor inventory is maintained the sa'r'ne as
System Analysis and Operation. In addition to
in the plug flow mode, then the MLSS concentration
manipulation of RAS flowrates, the effects of bulking
in t h e second pass will be less than When the system
sludge can be ameliorated by reducing lVlLSS' concen—U.
. was Operated in the plug flow mode.
tration in the clarification tank feed. This reduces the
applied solids loading rate to the clarification tank .' ,The IVILSS concentrations in each of the two pasSeS'
(Equation 5). Reducing applied solids loading rate can be calculated for step feed by writing solids mass
while maintaining the same RAS bulk withdrawal rate balances for each pass. These equations then are sub-fl
(Le... move downward and to the left along an RAS ‘stituted into a n equation for the total solids inventory,
bulk withdrawal operating line in Figure 8) increases w h i c h i s solved for the s'olids” conce'htrations ih‘ each
the allowable SVI. . pass and in the RAS.‘ For our example, the aeration'
tank NILSS inventory in t he plug f l o w mode is:
MLSS concentration in the.clarification tank feéd can
5"

be reduced by reducing the mixed liquor solids inven- ,:‘ Aeration Tank .Inventory (3 000 mg/l)‘
tory and by changing the activated sludge operating- (.0 2 5 MG)(8.34);1.
mode. Mixed liquor solids inventory can b e reduced v
, A
.

by increasing the sludge wasting ra‘te. This may nét 6 2 5 5 lb ( 2 8 4 0 kg)“-

be feasible if sludge handling capacity is not available’ W?

or if reducing the mixed liquor solids inventory would _: T h i s inventory will be maintained in the two- pas's step I
lead to an unfavorable FM (3.g. a low P M may be re-- feed mode.
quired to achieve nitrification)
The solids mass balance for the point ’at which the in- .
The objective of changingoperatiohal méde to cOm- fluent flow to the first pass (0 5 MGD) IS mI-xed Wlth
:concentration
bat sludge bulking is to reduce MLSS in the RAS flow (0. 3 3 MGD) is:
the ciarification tank feed without redUcing the mixed
liquor solids inventory. The step feed (step aeration) 3 (0. 3 3 MGD) (Xu) (0.5 + 0.33MGDRXT)’
and contact stabilization configurations‘are particular- and ‘= " "
ly useful in this regard. Needless to say, they can only x.1 (0.33/o..833)xu
be employed to .‘combat bulking sludge if the plant is 0.4 xu (6)”.
designed with the flexibility to operate in these con-
figurations. Both Operating configurations allow the
operation of part of the aeration tank at a higher
MLSS concentration (for a given F/M) than would be
acheived were the aeration tank completely miXed.

20K .
 80
ll-ll__|l|| l.llll I l I
. Somllg-f 600 sap-fill"2

.-
‘ ALLOWABLE SOLIDS LOADING LINES
FOR VARIOUS sw VALUES X 500 gpdm2
70 OPERATING LINES FOR VARIOUS

60

50

150 mI/g

40

200 ml/g "

. 30.7 ), ' 3
30-

.
' 25.2
250 ml/g ,4-
a '- ‘ ‘ s \ ; 10.0 gpd/fl2
- 300‘mI/g a C
20.9, A _. g 4"“.

20. .2—
J

350ml/g ': E,

10 ///Iz// / __;_..=——-e---—
»/ i " "'- \ so gpdm2
/ @ m/ ' \
’-
/ _. "i I arr-12 000 m" / |
I-fifl'fl I | ’I | 9 l I I I I | I l l 1 | I
5 10 15 20 25

HAS SUSPENDED SOLIDS CONCENTRATION, Cu(g/l)

,LZ
 figure 9 . S t e p Feed Operation Example Schematic
(After Dafgger and Raper ( 1 9 8 4 ) ) .

1.0 M 6 0
I I X = 3000 mg/l D

SLR = 20.9 Ib/ttz-day

1.33 MGD, sooo mg/I

0.33 MGD,xu = 12,000 mg/l

PLUé FLOW (CONVENTIONAL) MODE

mSMGDv .
D
;_ _
LOMGD _
x1= 3690 mg/l , : ' -i
9-5
”GD x2 = 2310 mg/l D
. SLR = 16.1lb/ft2~day
1.33 M G D , 2310 m g / l

0.33 MGD, xu = 9230 mg/l

S T E P FEED M O D E

7 22
 80
II lllllgllll III Il|:_:l ll
-' 50 m I / g ~. 600 g p d m 2 ;
ALLOWABLE SOLIDS LOAfiiNGLINES ’ K
FOR VARIOUS sw VALUES / 500 gpdm2
70
OPERATING LINES FOR VARIOUS . / I
_ UNDERFLOW RATES / /
400 gpd/I‘t2

60
100 ml/g ~
g? 300 gpd/ft2
4mmmmflu

/
CD
01
-

O
A
sz '9

, / h. J ~ / . /'QL zoo g p d m z - w
zooII/g / __ -- ‘_ 203g/pdmz/
(mg = 0.95 _ GD) ‘ g _ ‘
00
O

250 mI/g 2 ..
.

O
N

10

RAS SUSPENDED SOLIDS CONCENTRATION, Cu(g/l)

T he solids mass balance for the point at which the an RAS flowrate of 0 . 3 3 MGD ( 0 . 0 1 m3/sec), as cal—
flow from the first pass (0.83 MGD) mixes with the in- culated above. These operating points show that a
fluent flow to t h e second pass ( 0 . 5 MGD) is: sludge with an SVI of approximately 2 2 0 — 2 3 0 m l / g
requires an RAS flowrate of 0.95 IVIGD when the aera—
( 0 . 8 3 MGDHXJ = ( 0 . 8 3 + 0.5 IVIGDHXZ) tion tank is plug f l o w and 0 . 3 3 MGD (0.01 m3/sec)
when the aeration tank is two—pass step feed. This re-
and sults both in lower RAS pumping costs and higher
RAS, 8 8 concentration (9230 m g / l vs. 6300 mg/|)——
X2:(O.83/1.33)X1= 0.625X1 (7) which may reduce waste sludge processing and dis-
Combining Equations 6 and 7 gives: posal costs.

X2= 0.625X1= O.625(0.4Xu) = 0.25Xu (8) This example illustrates the advantage of two-pass
step feed over plug flow operation for dealing with
The total aeration tank mixed liquor solids inventory bulking sludge. Operation in other step feed modes
can be expressed as a function of Xu as follows, re- .(such as three— or four-pass), contact stabilization, 0r
membering that the volume of each aeration tank pass step feed with RAS’ reaeration (a combination of stép
is 0.25 MGIZ or 0.125 MG (1890 m3): ‘ feed and contact stabilization) offer similar advan—
tages. Indeed, any change which results in less dilu—
Aeration Tank Solids Inventory 3 tion of RAS by influent wastewater will allow storage
of activated sludge solids in the aeration tank and a
(xmon25 MG)(8.34) + (xznm 25 Mensa) decrease in the MLSS concentration in t h e clarifica-
=-.1.0425 (x1+ x2) (9) tion tank feed. For example, if t h e system depicted in
Figure 4 2 had been placed in t he contact stabilization
Substituting for X1 and X2 from Equations 6 and 8 mode, using the first pasg for sludge stabilization and
gives: the secohd pass for contact (While maintaining the
same mixed liquor solids and inventory), the MLSS
Aeration Tank Solids Inventory 2
concentration in the clarification tank feed would have
1.0425(0.4Xu + 0.25Xu)
been reduced to 1,200_mg/| and t h e Clarification tank
= 0.6776xu solids l o a d i n g r a t e w o u l d be 3 . 4 l b / f t ‘ U d a y
(41 kg/mZ/day).
Setting this equal to the total solids inventory of 6 2 5 5
lb gives: These examples have illustrated calculation pro-
cedures for determining the Operating conditions
6255 lb = 0.6776Xu when the mode of operation is changed. I n some
cases a simplified calculation procedure can be
and developed for a particular system operating uhder
defined operating conditions, or operating diagrams
Xu = 9,230 mg/I can be developed for a particular system. Alternative-
ly, these concepts can be used in a plant by changing
X , can be calculated using Equation 6 :
plant operation a n d observing the results. The'general
X 1 : 0.4xu z (0.25)(9,230 mg/l) = 3,690 mg/l principle that changes resulting in less dilution of RAS
b y influent: wastewater will decrease the IVILSS con-
and X; can be calculated using Equation 8: I centration o f the Clarification tank feed can be: used to
guide changes in operating mode. After making
X; = 0.25Xu = (0.25)(9,230 mg/l) = 2,210 mg/I c h a n g e s , t h e i r i m p a c t s o n solids d i s t r i b u t i o n i n t h e
system c a n b e m o n i t o r e d . I n m o s t cases r e d i s t r i b u t i o n
The clarification tank solids loading rate for this oper— of the solids takes less than 1 day, so that the results
ating condition would be: can be determined in a timely manner. The resulting
clarification tank solids loadings can then be deter—
GIi= (2,310 mg/l)(1.33 MGD)(8.34/1,590 ft2 mined and the need for further changes assessed.
= 1 6 . 1 lb/ftz-day (78.6 kg/mz-day) (see Figure 1 0 )

The benefits of two-pass step feed operation in alloW-
ing an activated sludge system to operate success-
fully with a poor settling sludge are illustrated in Figure
1 0 using the data from the previously calculated ex-
ampies. Points numbers 1 through 3 are for plug flow
operation with RAS flowrates of 0 . 3 3 , 0.6, and 0 . 9 5
MGD ( 0 . 0 1 , 0.026, and 0 . 0 4 m3/sec), respectively.
Point number 4 is for two-pass step feed Operation 'at
3 . 4 . 3 Addition of chemicals to enhance settling.
T h e intent of t h i s t e c h n i q u e i s t o i m p r o v e s e t t l i n g of
sludge without destroying the filamentous microorga—
nisms. Synthetic polymers, added to the mixed liquor
as it leaves t h e aeration tank or t o the clarification tank
entry point, aid in settling of. diffuse filamentous floc.
The polymer used is usually a high molecular weight,
cationic charge polymer, and may be applied alone or

24
Table 3. Bulking Filamentous Microorganisms That Have Been in combination with an anionic polymer. In some
Successfully Controlled by Chlorination
cases, lime, ferric‘chloride, or other inorganic precipi-
Location Major Filamentous Orgahismts) ianté have been added to systems, producing a heavy
Causing Bulking precipitate that forces the sludge flee to settle. This
method greatly increases the solids load the system
City of Albany, GA type 0041 must bear.
type 0092
H. hydrossis Because overdosing with polymers can degrade
type 1 7 0 1
system performance, careful testing must be done to
Malibu Mesa, CA type 1701 estimate appropriate doses. -
type 0 2 1 N
The use of polymers is ' a n expensive method of bulk-
City 'of Los Angeles type 0 2 1 N
Terminal Island, CA type 0 5 8 1 ing control. Chemical costs of up to $ 4 5 0 / M G have
been reported.
Carrousel Pilot Plant type 0041
Brewery Waste type 0092 3.4.4 Addition of chemicals to selectively kill
City of Pacifica, CA S. natans filamentous microorganisms,
type 1 7 0 1 T w o main chemicals—chlorine and hydrogen
type 1 8 6 3 peroxide—have been successfully used in the selec—
City of San-Jose, CA H. hydrossis tive destruction of filamentous microorganisms.
type 1 7 0 1
type 021 N 3 . 4 . 4 . 1 Chlorine. .
type 0 8 1 2 The use of chlorine is pepular in part because it is fre-
type 0041 quently used in effluent disinfection, and is thus
type 0 6 7 5 available at most activated sludge plants. Chlorine
may be added at three points in the system: directly
Derry.Township Municipal Thiothrix sp
Authority, Hershey, PA M. parvicella into the aeration tank; into-a “loop” in which mixed
liquor is withdrawn from the aeration tank, chlorin—
Ormo Lorna Sanitary H. hydrbssis ated, and returned to t h e tank; or into the return ac-
District, CA type 1 7 0 1 tivated sludge (RAS) stream. The last is the most
Weyerhaeuser type 0675 common and preferred choice, except in plants where
Longview, WA (NaOCI) type 1 8 5 1 aeration time is very long or where t h e RAS line is in-
Miller-Brewing C0. type 0041
accessible. -
Fulton, N Y type 0675
The following guidelines must be 'followed for suc—
Stroh Brewing Co. ' ' - type 1851 cessful use of chlorination:
Longview, TX type 0 2 1 N
(NaOCl to Aeration Basin) Nostocoida Iimicola II 1) A target value of SVI or some other floc parameter
Champion Ihtern'ational Thiothrix sp must be set.
(Pulp and Paper) _ type 1 7 0 1
Coufiland, AL 2) Chlorination must be us‘ed only when the target
value is regularly exceeded; a careful, frequent plot of
Mohterey Regional Water Nostocoida Iimicola I! the parameter should b e made to determine whether
Pollution Control Plant . type 1 701
it consistently exceeds the target value.
Monterey, CA Thiothrix sp
City of Redding, CA type 1 7 0 1 3) Chlorine doses must be k n o w n and carefully con-
S. natans trolled, and must be added to the sludge at a point
WestcheSter County type 1 7 0 1
where it will m i x efficiently. I f it‘ does not mix well,
Yonkers, NY , Nostocoida Iimicola parts of the sludge will overdose while parts will not
be chlorinated at all. Poor mixing will cause turbid ef—
Tharries Water Authority _ type 0 2 1 N fluents, reduction in treatment efficiency due to killing
(UK) (NaOCl)
of floC—forming organisms, and consumption of large
Central Contra Costa type 021N amounts of chlorine without control of the bulking
Sanitary District " ' M. parvicefla problem.
Concord, CA Thiothn‘x sp
City of Pacifica, CA 8. natans. 4) Ch‘lo'rine should be added where chlorine demand is
type 1 7 0 1 at minimum—that is, where the a m o u n t ' b f organic
matter is lowest. Chlorine reacts quickly with am—
monia and organic matt‘er,_ a n d b e c o m e s ‘ u n a v a i l a b l e
for killing filamentpus microorganisms. It should not

25
be added to influent wastewater, or to RAS after it dose. However, the dose concentration must be low
has begun to mix with influent waste just outside the enough so that a small part of the sludge inventory is
aeration tank. not exposed t o excessively high chlorine doses. I n
such cases, the exposed sludge floc can be complete-
‘5)
Appropriate parameters must be considered in ly destroyed while the bulk of t h e solids inventory is
selecting a dosing point. These are: unaffected. Also, the frequency of exposure of the
k9 C ' z sludge inventory t o the chlorine dose must be high
Overall Mass Dose enough t o control t h e filamentous microorganisms
Expressed in (generally greater than once per day). If proper control
of filamentous microorganisms cannot be achieved
Local Mass Dose kg Cl2 because of insufficient frequency of exposure or
Expressed in because the local mass dose cannot be achieved at a
103 kg 8 8 per day
reasonable dose concentration, than a change in the
Frequency of Exposure day/“(times per day dosing point or the addition of a dosing point(s) may
sludge passes point be necessary.
of dose)
6) Reliable control tests must be performed t o assess
Overall Mass Dose is based on the plant’s total sludge the effectiveness of chlorination. These incIude a
measure of sludge settling rate (such as SVI); a
inventory and tells how much chlorine must be added
per day. However, it does not indicate how often and measure of final effluent turbidity (such as a tur-
in what concentration the chlorine should be applied bidimeter or a Secchi disc); and microscopic examina—
tion of the sludge, to observe progressive deforma-
for a given application point in the system. The other
such information. tion of filamentous microorganisms or to detect signs
three parameters provide
of overdosing (such as total elimination of filamen-
The local mass dose indicates the quantity of chlorine tous microorganisms and presence of broken flocs).
which must be added at a dosing point in order t o The progressive effects of chlorination on type 1 7 0 1
control the filamentous microorganisms. The dose and Thiothrix spp. are illustrated in Figure 1 1 . Types
concentration is t h e concentration of c h l o r i n e to b e ' of filamentous microrganism successfully controlled
added at the dosing point t o achieve the local mass b y chlorination appear in Table 3 .

Tabb 4. Use of Hydrogen Pea-oxide for Bulking Control

Location Reference Successful

concentration, m g / l

PETALUMA ANON, FMC Corp. (1973) 9—68

fulla-scale plant Caropreso et al. (1974) average 3 1 . 5

ST. AUGUSTINE ANON, FMC Corp. (1976) 1 2 (basis unclear)

full-scale plant

S A N JOSE
lab scale, Strunk and Shapiro (1976) 40—200
fill and draw

PRINCETON Caropreso et al. (1974) 100b

small fun-scale

WILMINGTON Cole e t al. (1973) 40—200”

labtscale

WASHINGTON Cole e t al. (1973) ' 2-0—40"

pilot plant 200-—400a

TEXTILE MILL Keller and Cole (1973) 60 (basis unclear)

a“Based
on Wastew ater inflbw.
"Based on volume of aeration basin and clarifier (single dose).
Figure 1 1 . Progressive effects of chlorination on type 1 7 0 1 (a, b, and c) and Thiothrix sp. (d, e, and f): a. and d. no chlorination; b. and e. moderate
chlorination effects; anti 0. and f. severe chlorination effects (all 1000)( phase contrast).

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27

‘ JIIIIMMII'J
3.4.4.2 Hydrogen peroxide.
The use of hydrogen peroxide (H202, 50 percent by
volume) has been effective in both continuous and
batch dosing when added towthe aeration tank, to the
RAS line, and to the mixed liquor as it passes between
the aeration tank and the clarification tank. It
presumably attacks the sheath of the filamentous
microorganism, destroying its shape, and resulting in
progressive deformation like that seen in chlorination;

Mixing is important with H5102 as it is with chlorine.

Somewhat higher doses and longer contact times. may
be needed.
I t is possible that as H202 kills filamentous
microorganisms, it also releases oxygen. I f bulking was '
a result of low D C , this could provide additional im—
provement in operations. However, if sludge oxidizes
[-1202 before it has a chance to kill filamentous
microorganisms, this treatment will not be effective.

Examples of the use of hydrogen peroxide in bulking

control are shown in Table 4.

28
 Chapter 4
4.0 Activated Sludge Foaming
Several different types of foam have been observed in
activated sludge treatment systems. Some are nofmal
and harmless, while others are signs of operating
problems and need to be controlled.
4 . 1 Symptoms of Sludge Foaming
A t the startup of an activated sludge plant, while
solids concentrations and Sludge A g e are l o w , white
froth usually occurs. A s the process stabilizes and
solids build up, this type of foam usually disappears. I f
excessive, sudsy white foam persists, the Sludge Age
may be too l o w , or there ma y be nonbiodegradable
substances in t h e waste (industrial wastes or
cleaners). A more troublesome foam, heavy, viscous,
and brown, can appear on t h e surface of both aera-
tion and clarification tanks, degrading the effluent and
spilling over onto walkways. While. this type of foam
has been attributed t o a high Sludge Age, filamentous
microorganisms may also be involved in its produc—
tion.
4 . 2 Role of Filamentous Microorganisms in
Sludge Foaming
Analysis o f samples of sludge from systems contain-
ing heavy brown foam has revealed large numbers of
the filamentous microorganism Nocardia (see Figure
12). While the mechanisms of Nocardia foaming are
not fully understood, it is believed that Nocardia cell
walls are hydrophobic, and sufficient quantities of
Nocardia can make sludge flocs hydrophobic. Thus the
floc would not be wettable and would cling to air bub-
bles, resulting in a scum.
4 . 3 Factors Affecting the Presence of
Nocardia
The causes of Nocardia growth in activated sludge are
not well understood. However, researchers have found
correlations between high Nocardia counts and vari—
ous sludge parameters. These include high Sludge Age
and warm temperature oil and grease in th e waste-
water, low F/lVl ratio, and high 8 8 levels.
4.4 Control of Activated Sludge Foam'ing
Because the mechanisms of this phenomenon are as
yet so little understood, there is no single proven
29
method of controlling it. However, some standard
methods have had some success.
4 . 4 . 1 Physical methods.
The most common physical method of foam control is
the use of low—volume water spray to rupture t h e
bubbles in the foam. This method is not effective with
very stable foams, which usually must be collapsed
and diluted by high—volume sprays. Use of this method
is often complicated by the fact that the scum traps in
most secondary clarifiers are too small to receive the
amount of foam Nocardia can cause, and t h e scum
trap drainage pipes are usually too narrow for such
foam to flow out.
Grease and oil related problems are best solved by a
strong industrial waste enforcement program and
control of commercial grease traps'. High amounts of
grease and oil coupled with high temperatures and
high suspended solids contribute to the presence of
Nocardia and may act as a causitive agent to keep the
population alive.
4.4.2 Process manipulations.
The most common approach to Nocardia foaming
control is to reduce the Sludge A ge by increasing the
sludge wasting rate, thus washing out the Nocardia,
and concurrentiy inpreasing the H M loading. This
process is affected by temperature—the higher the
waste temperature, the further the Operator must
lower the Sludge 'Age. This approach works only
when the plant is capable of handling the increase in
waste activated sludge; and when nitrification will not
be performed (the Sludge A g e required for nitrification
makes Nocardia washout impossible b y this means).
Another process manipulation involves the addition of
sludge from an anaerobic digester. Such sludge may
contain material that is toxic to Nocardia. This method
has had only mixed success in laboratory studies.
4.4.3 Chemical methods.
The use of chemical antifoam agents in combatting
Nocardia fo‘am does not have a good record of
success.
However, a combination of physical removal,
c h l o r i n a t i o n , a n d addition of ferric chloride has been
used to reduce Nocardia populations to a manageable
level.

The most important consideration in control of Nocar-

dia, regardless of the method chosen, is the preven-
tion of foam recycling. I f such foam is recycled
through the system, it will reseed the sludge. W h e n
the contents of scum traps are fed back to gravity
thickeners or the RAS, while wasting continues to
remove the mixed liquor and sludge from which the
scum separated, Nocardia becomes concentrated in
the system.

30
 '
W
Figure 1 2 . Nocardia foaming in activated sludge: a. and b. foam on the aeration basin; 0. and d. microscopic appearance of Nocardia foam
(0. 400 x phase contrast; bar: 25 pm: d. 1000 X phase contrast; b a r : 10 pun).

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31

“madam 1mm
 Chapter 5

5 . 0 Appendix 1— Methods for Microscopic tank and the clarification tank. Mixed liquor samples
Examination of Filamentous should be taken below the surface, excluding any
Microorganisms in Activated Sludge foam or other floating material. I f the activated sludge
is foaming, a separate foam sample should be taken
Microscopic examination of activated sludge is an im- from the surface of the effluent end of the aeration
portant tool' for the identification of sludge settling tank, from the surface of the mixed liquor channel, or
problems. Determining t h e number and type of fila- from the surface of the clarification tank. Subsurface
mentous microorganisms is the first step in controlling liquid should be excluded from foam samples. Al—
bulking and foaming. though foams can be thick and viscous and difficult to
transfer to sample bottles, they should not be diluted
Such examination requires a research-grade phase to ease sampling, because this will prevent compari-
contrast microscope with 1 0 0 x and 9 0 0 — 1 0 0 0 x son of the relative abundance of filamentous micro—
phase contrast objectives. A mechanical stage is es— organisms in the foam and the mixed liquor.
sential for controlled scanning. A binocular head (or
trinocular head if photography is desired) and built—in Some activated sludge process modifications contain
light source are highly recommended. A n ocular more than one tank, e.g., step-feed, contact stabiliza-
micrometer should be inserted into one of the eye- tion, or “plug-flow” systems. I n such systems, char-
,pieces and calibrated at each magnification employed acteristics of the sludge in all tanks are usually similar.
using a stage micrometer, Thus it is usually not necessary to sample all tanks,
only the effluent end of the aeration tank. Where an
A photographic record of sample appearance at 1 0 0 X activated sludge plant consists of separate and paral-
is recommended for reference purposes. A 3 5 m m lel systems, there may be differences in floc and fila-
camera mounted o n the trinocular head is most con— mentous microorganism characteristics in each sys-
venient. The camera should have a built—in light meter tem, especially if there is not complete admixture of
and a replaceable, fine ground-glass focusing screen. the return activated sludge (RAS) streams. I n this situ-
Polaroid instant film cameras can be used. It is con— ation, a sample is required from each system. Simi—
venient and highly efficient to take all photographs larly, for two-stage activated sludge systems (9.9.,
using color slide film (e.g., Kodak tungsten-balanced “carbonaceous” first-stage followed by “nitrifica—
type II professional film designated KPA), and produce tion” second—stage), a sample from the aeration tank
color or black-and—white prints from the slides using a of each stage is necessary for proper characterization
slide printer employing Polaroid instant film. of floc structure and filamentous microorganism pop-
ulations. Some activated sludge systems treat a
Because many of the observations to be made in these
waste that has been pretreated in another type of bio-
procedures are close to the limit of resolution of the
logical treatment system (Iagoon or trickling filter).
light microscope, and many of the features to b e
studied are difficult to detect, the microscope must be These units may seed the activated sludge with fila—
in good - condition. Regular adjustment of the phase mentous microorganisms. To determine the extent of
rings is required, the microscope objectives must be this seeding, a sample should be taken of the waste
kept clean, and a dust-free envircnment is necessary. entering the activated sludge system.
Professional servicing of the microscope by the manu—
Sampling and examination frequency will be dictated
facturer or recommended agent should be performed by plant circumstances and by the location of exami-
at least once per year. nation of samples. During critical periods (when bulk-
ing is occurring or is anticipated, during the use of
5 . 1 Sampling Methods RAS chlorination for bulking control and during
Samples of activated sludge mixed liquor should be periods of experimental operation), daily onsite exami—
taken from the effluent end of the aeration tank, or nation can be justified. Routine onsite examination
from the mixed liquor channel between the aeration may be performed once or twice per week. Offsite

33
examination may be performed weekly, monthly, or overnight delivery services, transit time should pose
seasonally, depending o n the severity of problems no problem in offsite analysis.
being encountered o r the desire to establish an operat-
ing history (and on the budget available for this Filamentous microorganism staining reactions can be
activity). quite sensitive to prolonged sample storage. I f much
time is expected to pass between sampling and exam-
Samples should be examined as soon as possible after ination, two air-dried smears on microscope slides
they are taken. When examination is performed on— should be prepared at the time of sampling (using
site, samples not examined within several hours .techniques
outlined below) and sent together with the
should b e stored at 4 ° C in a refrigerator. When sample. In this way, the original characteristics of the
samples must be transported offsite for analysis, they activated sludge will be preserved for conducting
should be sent in sample containers in which there is Gram and Neisser stains. The slides should be marked
an air space at least equal to the volume of the sample, with sample identification, date, and G or N (Gram or
to avoid septicity. (Five to 1 0 ml are needed for typical Neisser), on the side of the slide containing the smear.
microscopic examination, s o a 20—25 ml vial will be The same procedure should be followed if samples
adequate.) The sample should be neither chemically cannot be analyzed immediately upon receipt.
preserved nor frozen, since these procedures can aiter
the characteristics of the flocs and filamentous micro—
5 . 2 Staining Procedures
organisms. The longer the time that elapses between
sampling and examination, the more difficult and un- Two primary staining procedures are used in the
certain sample examination and data interpretation examination of activated sludge samples—the Gram
become. Samples from plants with low organic load- stain and the Neisser stain. Other procedures with
ings (long Sludge Ages) maintain their characteristics more specialized uses are the ” 8 ” test, for sulfur oxi-
longer than samples from plants with high organic dation to detect intracellular sulfur granules; India Ink
loadings (short Sludge Ages). For long Sludge Age reverse staining, to detect the presence of large
samples, a satisfactory examination can be obtained if amounts of extracellular polymers; PHB staining, to
the sample is looked at within 7 to 1 0 days of sam- detect the presence of intracellular storage products
pling; for sludges from highly loaded plants, it is wise such as polyhydroxybutyrate (PHB); and staining with
to examine the activated sludge within 3 to 4 days of crystal violet, to allow examination of sheaths. The
its sampling. With the availability of express mail and procedures are discussed in the following subsections.

34
5.2. 1 Gram stain, modified Hacker method.
Preparation: ;

Solution 1 : Prepare the following separately, then combine:

A ._B

Crystal Violet 2 g Ammonium oxalate 0.8 g

Ethanol, 9 5 % 2 0 ml , Distilled water 8 0 ml

Solution 2 :

Iodine 1 9
Potassium iodide 2 g
Distilled water 3000 ml

Solution 3 :

Safranin O (2.5 percent in 9 5 percent ethanol) 1 0 ml

Distilled water _ 1 0 0 ml

Procedure: _

1 . Prepare thin smears on microscope slides and thoroughly air dry (do not heat fix).

Stain 1 min. with Solution 1 ; rinse 1 sec. with water.

Stain 1 min. with Solution 2; rinse well with water.

Hold slide at an angle and décolorize with 9 5 percent ethanol added drop by drop to the smear for 2 5 sec. Do
n o t over decolo'rize. Blot dry.
.0"

Stain with Solution 3 for 1 min.; rinse well with water and blot dry.

Examine under oil immersion at 1 0 0 0 >< magnification with direct illumination (not phase contrast): blue-violet
is positive; red is negative.

35
5.2.2 Neisser stain.
Preparation:

Solution 1:

Separately prepare and store the following:

A B

Methlylene Blue 0.1 9 Crystal Violet ( 1 0 percent w/v in 9 5 %

Ethanol. 9 5 % 5 ml ethanol) - 3 . 3 ml
Acetic acid, glacial 5 ml Ethanol, 9 5 % 6 . 7 ml
Distilled water 1 0 0 ml Distilled water 1 0 0 ml

Mix 2 parts by volume of A with 1 part by volume of B ; prepare fresh monthly.

Solution 2:

Bismark Brown (1 percent w/v aqueous) 3 3 . 3 ml

Distilled water 6 6 . 7 ml

Procedure:

1 . Prepare thin smears on microscope slides and thoroughly air dry. Do not heat fix.

2. Stain 3 0 sec. with Solution 1; rinse 1 sec. with water.

3. Stain 1 min. with Solution 2; rinse well with water; blot dry.

4. Examine under oil immersion at 1 0 0 0 x magnification with direct illumination (not phase contrast): blue—violet
is positive (either entire cell or intracellular granules); yellow-brown is negative.

36
 5.2.3 ”8” test (sulfur oxidation)
Test A (modified from Eikelboom 1975)

Solution: Sodium sulfide solution (NaZS-QHZO) 1 . 0 g / I (prepare weekly)

Prbcedure:

1 . On a microscope slide mix. 1 drop of activated sludge sample and 1 drop sodium sulfide soiution.

2 . Allow to stand open to the air 10—.- 2 0 min. ’

3 . Place a coverslip on the preparation and gently press to exclude excess solution: remove expelled solution
with a tissue. .

4. Observe at 1000>< using phase contrast. A positive 8 test is the observation of highly refractive, yellow—
colored intracellular granules (sulfur granules) (Figure 22b). -—

This test at times gives variable results. This is due to methodological problems involving the relative concen—
trations of sulfide and oxygen present (sulfur oxidation is an aerobic process). A n alternative sulfur oxidation
test developed by Nielsen ( 1 9 8 4 ) may be used:

Test B (modified from Neilsen 1 9 8 4 )

Solution: Sodium thiosulfate (N323203-5H20) 1 9 / 1 0 0 ml

Procedure: - I - I = .

1 . Allow activated sludge sample to settle, and transfer 20; ml of clear supérnatant to a 1 0 0 ml Erlenmeyer flask.
plat-sap

>-
Add 1—2 ml of activated sludge to the flask. > _ ,

Add-1 ml of thiosulfafe solution to the flask (final fhiosulfafe concentration is 2 m M ) .

Shake the flask overnight at room temperature.

Observe at 1 0 0 0 ' x phase contrast, as above.

37

- a n III-WW...“
5.2.4 India Ink reverse stain. 5 . 3 Examination Procedures
Solution: India ink (aqueous suspension of carbon Upon sample receipt, or at least within several hours
black particles) prior to sample examination, spread 1 drop of the
sample evenly over approximately 5 0 percent of the
Procedure: area of each of two 2 5 x 7 7 m m microscope slides.
Allow these slides to air dry at room temperature (do
1 . Mix one drop of India ink and one drop of activated not heat fix). The slides can be stored and stained
sludge sample on a microscope slide. Depending later. Mark t he slides with a sample identification code
on the ink used, the sample volume may need to b e and a G or N (Gram or Neisser) on the side on which
reduced. the smear has been made (frosted end slides are rec-
ommended). Perform the Gram and Neisser staining
Place on the cover slip and observe at 1 0 0 0 x using
procedures (5.2.1 and 5.2.2).
phase contrast.
Withdraw one drop (approximately 0 . 0 5 ml) of sample
In “normal" activated sludge, the India ink par-
with a l00p or a clean, disposable Pasteur pipette and
ticles penetrate the flocs almost completely, at
place o n a 2 5 X 7 5 m m microscope slide. Place a
most leaving a clear center.
2 2 mm No. 1 cover slip on the drop, and press down
In activated sludge containing large amounts of gently on the cover slip with a blunt object. Remove
exocellular polymeric material, there will be large, the liquid that is expelled from the sides of the cover
clear areas containing a low density of cells. slip with a tissue. This procedure ensures a thin prepa-
ration, which is necessary because of the limited
5.2.5 PHB (polyhydroxybutyrate) stain. depth of focus of the microscope optical system.

Solution 1 : Sudan Black B (IV), 0 . 3 percent w/v in 60 Examine the wet mount under phase contrast at 1 0 0 x
percent ethanol magnification for the following characteristics:

Solution 2: Safranin 0 0 . 5 percent w/v aqueous 1 . The general size and shape of flocs present;
measure approximately 10—20 flocs and place
Procedure: them in the following categories:
1 . Prepare thin smears on a microscope slide and a. Size range: small < 1 5 0 m diameter
thoroughly air dry. - . ‘ ' medium 1 5 0 — 5 0 0 p m diameter
large 2 5 0 0 mm diameter
2. Stain 1 0 min. with Solution 1 ; add mqre stain if the
slide starts to dry out. b . Shape: rounded and compact, or irregular and
diffuse; note whether texture is firm or weak
3. Rinse 1 sec. with water. (Figure 13a and 13b).
4. Stain 1 0 see. with Solution 2; rinse well with . The presence of protozoa, organic or inorganic
water; blot dry. particles, and fingered or amorphous zoogleal
Examine under oil immersion at 1000 x magnifica-
organisms (Figure ‘130 and 13d).
tion with transmitted light: PHB granules will ap— The presence of free (dispersed) cells in the bulk
pear as intracellular, blue-black granules while solution (Figure 13b). Samples containing signifi-
cytoplasm will be pink or clear. cant amounts of dispersed cells will appear turbid.
5.2.6 Crystal Violet sheath stain. The presence and effect of filamentous micro—
Solution: Crystal Violet, 0 . 1 percent w/_v acqueous organisms on floc structure, as follows:
solution
8. None
Procedure:
b . Bridging—filaments extend from the floc sur-
1. Mix 1 drop activated sludge sample and 1 drop face into the bulk solution, and bridge between
Crystal Violet solution on a micrOSCOpe slide, the flocs (Figures 1 4 a and 14b).
cover and examine at 1 0 0 0 x magnification phase
Open floc structure—the floc population at—
contrast. Cells stain deep violet while the sheaths
are clear to pink. taches to and grows around filamentous micro-
organisms, leading to a large, irregularly
shaped floc with substantial internal voids
(Figures 1 4 0 and 14d).

38
Figure 1 3 . Floc "texture” in activated sludge ( a and b):— a. rounded, firm compact: b. irregular and diffuse with substantial free cells.
Appearance of fingered (c) and amorphous (d) zoogleal organisms in activated sludge (all 100'>< phase contrast: b a r : 1 0 0 p m ) .

5 - .IF-
_ .41 ..-

gas-3.34;“
—
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hr;
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54 .. . 19mm:.L 2.... " z.»fl u. 3;, : i. . . ‘'

._ _‘ . _
w——-"-_
_ _ “H. _.- r aA ;f" ‘ ='“. :23;.".

Reference: Jenkins. et.a!.

39
Flgme 14. Effect of filamentous microorganisms in activated sludge on floc morphology 5nd settleability: a. and b. inter-floc bridging; c.
and d. diffuse fine structure (al! 100 x phase contrast: bar: 100 um).

um

[I

”fig 1 "L m
-%
HIE
I g l ' .
W ...... u ‘ .
Iqa‘mq- . .1’ 51.: .ul
. _ 43*
r ...2'" ’
5.... i -V
.- 1r 3" 1 ul '5 ‘
“Nah ‘

3%
4L
" ImmimL- IHII.I:_.:..I.maI
A
in.

5"
llllll .-:‘w
F

1 3‘ L’ ....
A.
.«-
u. ”..' t
"I""

!.|

16%.
Iv

Reference: Jenkins. anal.

40
Table 5 . Subjective Scoring of Filament Abundancea

' Numerical Abundance Explanation

Value

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—5 filaments per
floc)
4 very common Filaments observed in all flocs at medium density (9.9., 5—20 per floc)

5 abundant Filaments observed in all flocs at high density (9.9., 2 0 per floc)

6 excessive Filaments present in all flees—appears more filaments than floc and/or
filaments growing in high abundance in bulk solution

3This
scale from O to 6 represents a 1 0 0 to 1 ,OOO-fold range of total extended filament length.

5 . The abundance of filamentous microorganisms. 5. 4. 1 Filament measurement technique of Sezgin

This can b e quantified by several methods. For th e
purpose of th e type of analysis required here, a
subjective scoring! system is used. Filamentous
microorganisms are observed at 1 0 0 x and subjec—
tively rated for overall abundance o n a scale from 0
(none) to 6 (excessive) ( T a b l e 5 ) . A n a b u n d a n c e
rating is determined for the sample as a whole (all
filament types together) and for each fiiamentous
microorganism ‘ observed.- Individual filamentous
microorganisms are considered dominant (and
probably most responsible for bulking problems) if
they are scored “very co mmo n ” or higher. Organ-
isms are considered secondary (present, but not in
sufficient abundance to account for a bulking
problem) if they are scored ”common” or lower.
This method is rapid and is suitable for establishing
whether a filamentous organism is dominant or
secondary. Abundance categories generally are re-
producible to within i one abundance category be-
tween observers. Examples of filament abundance
categories are shown in Figure 1 5 .
Other methods of microorganism counting
scribed below.
et al. (1978).
1 . Transfer 2 ml of a well—mixed activated sludge
sample of known suspended solids concentration
using a wide-mouth pipette ( 0 . 8 mm diameter tip)
t o 1 liter of distilled water in a 1.5-liter beaker a n d
stir at 9 5 rpm o n a jar test apparatus (G = 8 5 sec-1)
for 1 min.
Using the same pipette, transfer 1 . 0 m l diluted
sample to a microscopic counting chamber cali—
brated to contain 1 . 0 m| and cover with a glass
cover slip.
.1 Using a binocular microscope at 1 0 0 x magnifica—
tion with an ocular micrometer scale, count the
number of filaments present in the whole chamber
or a known portion of it and place these in the
following size classifications: 0 to 1 0 ,um, 1 0 to 2 5
pm, 2 5 to 5 0 um, 5 0 to 1 0 0 pm, 1 0 0 to 200 p m ,
200 to 400 gm, 400 to 8 0 0 u m , and greater than
800 ,um. Measure filaments of length greater than
800 um individually.
are de— Express results as the total g m of filament length
per g or m l of MLSS:

5 . 4 Counting Procedures iota] extended filament length (TEFL) pm/gMLSS

I n addition to the subjective scoring method for count— = t o t a l filament length, mm in the 1 . 0 m l diluted
ing filamentous microorganisms, other more detailed s a m p l e X t h e dilution factor ( x 500 in the above
procedures exist for measuring the total extended fila— example) + MLSS concentration, 9/!
ment length (TEFL) in activated sludge.

.41'

.. . . . : . n m . n m w . . l l l l . .
Flgma 15. Ffiament abundance categories using subjective scoring system: a. few; b. some: a. common: dcvery common; a. abundant:
and f. excessive (all 100 x phase contrast; b a r : 100 pm).

i." uu
"val:
b II hl:l::fl“|iln i‘ufllHI'." "MIMI"Il1m.nl-:nI’ :I':l :lil‘ III; I I Hu u II I I .: ” I I I I||||.||| I" "
I 'uwnllrflwlqlnmpllwflfl‘lrllhll '. ' ' ,
I . - I .
.' I ' .IHIIIIIIIJInu'HIlFlI‘llmlh’l'| :
. - y: I - J. |.: |||||||_umnl|ll|1llll|l|l|ll|ll|||1|||||| .
:n:l:im III|

. .. . '5‘. ... . | . I," ”I" _ ..

I'
*
~-
- E' " "h M "W""'MH'IILL.1‘r '

w?
rm “Mm".dilfimk" 1."..H 59.1“ .
¥ H. _
mimf‘l'“. 1
II I ITfiMfih

”"'ilfl,l
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wfi“
. WI HII WIW WMJ
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.... n; ' a
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. ,. as“

|"||Ilmlllnflflmflllflllfllmflflfllll IWHMMIFIEIRMI I II
mmlllllllll II:
|:|”||:|H|!m| :FIJ‘TIF:'"Illlmnlllllll" l | | lI||||'|
Illrniinlul !|Ii|||||:| IIIIIIIIEII

. n

| | | | | I| | | I| | | | | | | | |I|I I I I I I JL.7H .!!.!!.!!.!|. . . . . .LI."l. L!..l. !. !. !!m.m. . !. !.l.lm L'!!H!!!!:"”!!!!!H .. _. _: - .a .p- ,- '
\"WHII HHH WW "..nm|.. ..uw. m ""'.-U E m m y ” Hulllinnnmm “ will I 1 .
WM|""' mm “gumM

- Wkrvwnu' Jma'rarum -!--""-I|

... u. .||.'_ 'L"_

'Hl-i li-:-”'-""u$

:'
W I in!::II!!.|||"...m..I|u" =
In "a???“ WWW .. M
'"-' W I

3‘ WI-|\ | i fl|i HHWHHHIH‘

w

T‘s.
"innhi'"1.

_.. q . :.h_ . —‘
liil _ml ”I". l ~ Mu .. _.'

I “"”'MII””|”W””"'H1"
.HI pP | mg *3mi"
WW3“ if »
WWW ' "

d mulnhfimlill 1W

afi l ‘fillm
|H'| m.-'| ml uM 'Illlhll'lln
Iliuh'fllunmu
mmmflfll
' —
. d|!|y|h|”rlflmhll% j - . .. .. £"murflm " fi'ngm
WWMWW
A":..T..llfl?«".... W W
5- || ' ' |||||||||||||||||||||||
I“ Illllulllllm II I II
_.
. 9+ ’ y ‘ m m n r Junhmlnll" W W W " |||'
“1]" "III” hm“
uluuluwmww
lw‘flwi” 4F
.. Jr i- Immi-

Iml IIIIIIIILII||I||I||||IIII.I||II||..“IlllllllllllllllIIIIIIIMIIIIIIIIIIII Ill IIIIIIIIIIIIIII IHIILIILIIIII..... "IIIIIIIIIIIIII

uuur ... uuuummlumfimuununullufiflflluu
.Imlmmummml‘fimmInmmi‘ imrm-il‘mmnllnl

Rafemnce: Jenkins. anal.

42
 total extended filament length (TEFL) u m / m l 4. The eyepiece is fitted with a single hairline. Count
M L S S : total filament length m, in the 1 . 0 ml the number of times that any filamentous micro—
diluted s a m p l e x t h e dilution factor ( X 500 in the organism intersects the hairline.
above example)
5 . Total the number of intersections for all fieids
examined. This is the Filament Count. I f a ”unit
count” is desired, the Filament Count must be
5.4.2 Diluted SVI procedure of Stobbe (1984}. multiplied b y - t h e number of fields in the 2 2 mm
Several researchers have found close correlations be-
width of the slide. At San Jose this is 1 2 fields.
tween the number of filamentous microorganisms in
Thus
sludge and a parameter known as Diluted Sludge
Volume Index (DSVI). The DSVI can be used to predict (filament intersections in) °
sludge settling properties and is well correlated to ( field counted ) x 12
Filament Count/pl =
TEFL. 50 u l

1 . Set up several 1-liter graduated cinders (the SJ ISCWPCP has correlated filament count with SVI.
number will depend on prior knowledge of the set— However, filament count cannot provide an early
tleability of the sludge). warning for problems caused by increased SVI, be-
cause the two are temporally correlated.
Using well-clarified secondary effluent, prepare a
series of two—fold dilutions of the activated sludge
(i.e., n o dilution, 1 : 1 dilution; 1 : 3 dilution).

. Stir the graduate cylinders individually for 30—60

sec. using a plunger to resuspend and uniformly
distribute t he sludge solids.

Allow the activated sludge to settle for 30 min.

under quiescent conditions.

. Observe the 'settled sludge volume (SVao) in the

graduated cylinder where the settled volume is
‘ less than and closest to 200 ml (SV30 < 2 0 0 ml).

Calculate the diluted SVI using:

8 8 (g/l)
where n is the number of two—fold dilutions re-
quired to obtain a settled sludge volume (SVso) less
than 200 m l and SS is the suspended solids con-
centration of the undiluted activated sludge.

5.4.3 Simplified filament counting technique used by

the San Jose/Santa Clara (California) Water
Pollution Control Plant (SJ/SCWPCP).
This method does not directly measure TEFL. Rather,
it counts the number of times filaments intersect a
single line on the microscope eyepiece, for several
fields of View across a wet mount preparation of the
activated sludge sample.

1 . Transfer 5 0 p l mixed liquor sample to a glass slide.

2 . Cover completely with a 2 2 x 30 mm cover slip.

3. Using 1 0 0 X total magnification and starting at the

edge of the cover slip, observe consecutive fields
across the entire 3 0 mm length of t h e cover slip.
At SJ lSCWPCP this is 1 7 fields.

43

M-m‘lm
|||||||
 Chapter
6 . 0 Appendix 2 — Microscopic Identification
of Filamentous Microorganisms
The other important part of microscopic study of acti-
vated sludge samples is identification of the type(s) of
filamentous microorganisms present in the sludge.
6 . 1 Observation of Microorganism
Characteristics
Scan the sample at 1 0 0 x using phase contrast to
ascertain the number of different types of filamentous
microorganisms present. Carefully characterize each
filamentous microorganism present by looking at
severe! filaments of each type and expressing the re-
sults as an “ a v e r a g e . " Use count sheets such as the
ones presented in Table 6 t o record and summarize
observations.
This task can be simplified by accepting only a limited
number of descriptions, and learning to recognize spe-
cial features that provide clues to the filamentous
microorganism type. These features are:
1. Branching—present or absent; if present,
whether true or false branching.
True branching is cell branching where there is a
contiguous cytoplasm between branched trik—
chomes (Figure 1 6 a , 16b, and 160). I n activated
sludge t h e o n l y branched trichome-forming
microorganisms are fungi and Nocardia spp., and
Nostocoida Iimicola (observed incidentally).
False branching occurs when there is n o contin—
uous cytoplasm between trichomes; two tri-
chomes have merely stuck together and grown
outward (Figure 16d). In activated sludge! false
branching usually is only ‘observed for Sphaero—
tilus natans, but also has been reported inciden-
=tally for type 1 7 0 1 .
2 . Motility— none, or, if present, describe.
Only a few filamentous microorganisms in acti-
vated sludge are motile. Beggiatoa spp., Flexi-
bacter s p p . , and some blue-green bacteria
(Cyanophyceae) are motile b y gliding; Thiothrix
spp. and type 0 2 1 N m a y display limited “twitch-
ing” or swaying motions.
45
6
3 . Filament s h a p e — s t r a i g h t , s m o o t h l y - c u r v e d ,
bent, irregularly—shaped “ c h a i n of cells,” coiled;
or mycelial (Figure 17).
. Color—transparent, m e d i u m , d a r k (Figure 1 8 3 ,
18b, and 180). .
. Lobation4extending from floC surface, found
mostly within the floc, or free in the liquid be-
tween flocs (Figure 18d, 1 8 9 and 1 8 f ) .
. Attached growth o f epiphytic unicellular bacteria
—present or absent; if present, whether substan—
tial growth or incidental (Figure 19)._
. Sheath— present or absent.
The presence of a sheath is one of the most diffi-
cult characteristics to establish. A true sheath is
a clear structure (hence, hard to observe) exterior
to the cell wall. Sheaths can be seen best in un—
stained preparations when they are empty of the
cells, or when some of the cells are missing. I n
the latter case the outline of the sheath can be
seen continuing along either side of the empty
space (Figure 20). Several features of fila-
mentous microorganisms can be confused with
sheaths. A yelloWish ” h a l o ” observed around
filaments under phase contrast observation is not
a sheath, but an artifact of phase contrast illumi-
nation. Short, empty spaces in a‘trichome or at
the trichome apex‘ should be used to indicate t h e
presence of a sheath. The cell wall of some fila-
mentous microorganisms may remain after cell
lysis; however, this can be distinguished from a
sheath because some evidence of pre-existing
cross-walls usually remains. This is commonly
observed for type 0 2 1 N.
Presence of substantial attached growth gener—
ally indicates the presence of a sheath. Staining
wet mounts with Crystal Violet (Section 5.2.6)
may aid in sheath detection (Figure 2 0 f ) .
. Cross-walls (cell septa)——~present, absent.
This feature can be variable for some filamentous
microorganisms, and detection is dependent o n
the quality and adjustment of the microscope. It
Tabla 6 . Suggested Format for Filamentous Microorganism Identification Worksheet

No. Sample

SAMPLE DATE I / OBSERVATION DATE? / /

FWENTABUNDANCE E 0
[:1 D1 2
:1
' 3 4
E! E 5 6
E.
None Few Some Common Very Abundant Excessive
Common

FILAMENT EFFECT ON FLOC STRUCTURE: Little or None Bridging Open Floc Structure

MORPHOLOGY OF FLOC: |:| Firm |:| Round, Compact

l:| Weak _ |:| Irregular, Diffuse

FLOC DIAMETER ( m ) FEATURES:

150 150—500 500 Free cells in suspension

I I I I Zoogloea’s
Inorganic/ Organic Particles

FILAMENTOUS MICROORGANISM SUMMARY:

Rank Abundance Rank Abundance
Nocardia sp. M. parvicella
type 1701 type 0 5 8 1
S. natans ‘ Y type 0092
type 021N type 0803
Thiothrix sp. type 1851
type 0041 type 0 9 6 1
H. hydrossis other
N. limicola other

x = Dominant O 2 Secondary

REMARKS:

Reference: Jankins. a t 3!... 1 9 8 4 .

46
Table 6 (continued).

No. Sample

COMMENTS:

OBSERVATION 0F:
Protozoa
Metazoa:

WET MOUNT OBSERVATION,7000X,PHASE CONTRAST:

FILAMENT # A B

BRANCHING
MOTILITY

FILAMENT SHAPE
COLOR
LOCATION

ATTACHED UNICELLS
SHEATH
CROSSWALLS

FILAMENT DIAMETER
LENGTH

CELL SHAPE
SIZE

SULFUR DEPOSITS
OTHER GRANULES

COMMONNESS
RANK

STAINS, TOOOX
GRAM
NEISSER

|.D.

Reference: Jenkins. e t al., 1 984.

47

... Wu» I-.

Figuw16. Trichome branching observed foi' filamentous microorganisms in activated sludge: a . , b. and 0. true branching (fungus,
Nocardfa sp., and Nostocoida limicola II respectively]; d. false-branching (Sphaerotilus natans) (all 1000>< phase contrast;
batm10pm). -
ӣ51 m . Ir'
‘ ”3' ..::'.
u 15”.-I' " ' -*'
--i:w.i:';r¢;
.||I! '11all“

'-'I"|Hi|'
' hi." '

III. Ilul'

Reference: Jenldns. 92.31.

Figure 1 7 . Examples of filament shapes: 3. straight: b. smoothly-curved; c. bent; d. irregularly-shaped "chain of 'cells”: 9. coiled: and
f. mycelial (all 1 0 0 0 x phase contrast).

-. . . , ‘. , ' '. ' ‘93: L

. Qi/glwkfigg‘w M3, . ';. ,1" , ; ._ ._ - ‘fi:_rj4.,.«t.§rgtl-IW‘; W.
INu .1” ‘
‘9'. . ' - ' ' II b J - b h». v
1:.

d g;
W
7 ”w.
1‘».
.
2a.,
a “44..- aflvnfi'fifii‘mm , f»
wfiuau 915$:e '
.- v 5 "-9.1

_ .P
r.

M
Iv‘l
+

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I dew
. 4*? M «.1
_...Is:-1. . L;-mm&
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_.,____,._ .3
1.; . '.
;:.§-4fi1v‘_r|nr. r" :
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J

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, y a.

P.
w. .3: - .
- . ) '9

é.

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4

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g‘
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15mm. fi x

1.. . , .7“:t ’1 I n u..~

n. ..
. c-
‘r 'Kwfi.m‘§'§-}§y}5‘ 3;“iz'i' I
{(5

4
”3. . _ __ ’ .'
mmmmmwwflf'

J
lk—Il*u‘
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49
III
fig

Reference: Jenkins. etal.

49
Figum 1 8 . “Colo-r" of fflamentous microos'ganisms (a, b and c): a. transparent; b. medium; and c. dark: Location of filaments in activated
sludge (d, e and f): d. extending from floc surface: 9. most within the flue: and f. free (all 1 0 0 0 >< phase contrast).

0
"

I .IIIII.::IIIIIIIHII. "I
—-— ... . flflfin'l m

................ ”m

I.||||W't.
. . . \"l'MHI HI| |HH|| | IM
I l'i"Ill!” law
. . , "II M“ ‘lilllulmuw fig:
1.. ! I" llillll‘mll
NIHMM‘H‘W! ”1k twig .' :
I. I I gill-III“ ""I
.

|"'
nunnmnll

Reference: Jenkins, a t a!.. 1 984.

50
Figure 1 9 . Attached growth of epiphytic bacteria on filamentous microorganisms: 3., b. and c. substantial (types, 0041 , 0 6 7 5 and 1 7 0 1
respectively): d. incidental {type 1851) (all 1000X contrast).

'Jfl'll
ll'|"-..
'u lu-
31' I'. I F u: 2-,

[+
in 11151

IE4
u
¥

“a
4'):
F"

H
r
It

E,
,i
'43:. '5'

I
II
t
«I'm-v _ m—

I
|
; _..- _
QC ‘_.."' ._ _ _ = - _ — ‘3; ,_,

a.

.Ifi‘hj : IF‘ __ Li '1

H" :u _‘ i
I a“ :
I - _
.?‘II
J

h-n-:.m.._,w
2. a” __ -=—__—‘|_,? _ 1
fl. .0. a ‘ _= '—_ -
Q3—: 1 if 'W‘r — — : "_§
—"”' W a! m- , — —_... —_ 1—“

IdF
:1 ,. a _ 1"; 27‘ ’:‘= —— —- - '
_._
_.. . $ 1 . w: ‘ ; —-—-"
m “an

Q“
‘.

“Hi
ik'l
“i

F1.

*1

I
J
‘
EH|

Reference: Jenkins, e t a]... 1 9 8 4 .

51
Flgmc 20. Appearance of sheaths: a. Sphaerotilus natans; b. type 1701; c. Thiothrix II; d. Thiothrixl; 9. type 0041; and 1’. type 1701
stained with cwstal violet (all 1000 x phase contrast).

.hhhh
.........

”mild ,iiHP'.‘
llllll
ilmqrfllmmnim ..: ..:i='

Reference: Jenkins, a t 3].. 1984.

52
Figure 21. Cell shapes observed for filamentous microorganisms in activated sludge: a. square: b. rectangular: 0. oval; d. barrel:
e. discoid: and f. round-ended rods (all1000>< phase contrast). _

“E.
Hr . :-

‘3' '

as
it, , «
.. .‘v w. ' } ~
~ ' ' n
r ' .r . , .-
‘ t

t I . ' : ., ‘ i ‘H - I 1
.
., ,

35‘.
..
J'

.‘s _ *"
2
.'=,- fix...

Z. ‘

i .

r)
.

.
- I . “5 ¢ - , -
,. .. .. . .

.. .{ -- _ . _ a. ‘v.4
-_ .v -
- v .._.. -
9..
105:,
.41-, .If,

Reference: Jenkins, et al., 1 984.

53
 is important to determine whether a true tri-
chome is present (Figure 17b) or whether the fila-
ment is made up of a chain of cells (Figure 17d).
Filament diameter—Both the average diameter
and its range in mm should be measured; it is im-
portant to note whether the diameter is greater
than 1 nm or less than 1 pm.
10. Filament lengthwrange in pm.
11. Ce” shape—wsquare, rectangular, oval, barrel,
discoid, round-ended rods (Figure 2 1 ) .
It is important to note whether there are indenta-
tions at cell septa (Figure 2 1 c, 2 1 d, 2 1 e, and
2 1 f ) or whether trichome walls are straight at the
cell junctions (Figure 2 1 a and 21b).
12. Size-uaverage length and width of cells in pm.
13. Suh‘ur deposits—present or absent in situ and
present or absent after the 8 test (Section 5 . 2 . 3 )
(Figures 228 and 22b).
Under phase contrast observation, sulfur gran-
ules appear as bright yellow-colored cell inclu-
sions, either in the shape of spheres observed for
Thfothrix spp., Beggiatoa spp., and type 021N
(Figure 220, 22d, and 22a); or in the shape of
squares for type 0 9 1 4 (Figure 22f). Type 0 9 1 4
does not respond to the 8 test.
14. Other granules-«present or absent. Commonly
observed granules are polyphosphate (Neisser
positive granules), and PHB (confirmed by PHB
staining) {Section 5.2.5).
15. Staining reactions—Each filamentous micro-
os'ganism present ié separately evaluated for
Gram staining and Neisser staihing reaction by
observing the stained smears at 9 0 0 — 1 0 0 0 x
using transmitted light (not phase contrast). The
position and length of filamentous microorgan-
isms in the wet mount and the presence or
absence of attached growth should be carefully
noted so that the same filament types can be
examined in the stained smears. Care is required
in this observation because some filamentous
microorganisms change size Upon drying and
staining (6.9., type 0 0 9 2 appears much wider
when Neisser stained than in wet mounts).
The Gram stain requires much practice. Reagents
should b e reasonably fresh (3—6 months) and, if
possible, should be tested on fresh cultures of
known Gram reaction. The decolorization step
should b e controlled precisely to avoid over-
decolori’zation. Also, large flocs do not decolorize
fully, so the Gram reactions inside large flocs
should be ignored.
54
Score the Gram reaction as positive, negative, or
variable. Most filamentous microorganisms ob-
served in activated sludge are Gram negative
(Figure 2 3 a and 23b). Nostocoida Iimicola and
types 0 0 4 1 and 0 6 7 5 most often are Gram posi-
tive but can be Gram variable or Gram negative
(Figure 230). Type 1 8 5 1 ,stains weakly Gram
positive, and generally is observed as a chain of
.Gram positive “beads" (Figure 23d). Thiothrix I ,
Beggiatoa spp., type 0 2 1 N , and type 0 9 1 4
generally stain Gram negative, but may stain
Gram positive when they contain substantial
intracellular sulfur deposits. Microthrix parvicella
and. Nocardia spp. are generally strongly Gram
positive (Figure 23a and 23f).
Neisser staining is a straightforward technique.
Score as negative, positive (entire trichome is
stained), o r negative w i t h Neisser-positive
granules (Figure 2 4 ) .
Type 0 0 9 2 (light purple—Figure 240) and N.
Iimicola (dark purple—Figure 24d) stain entirely
Neisser—positive. M. parvicella and Nocardia spp.
stain Neisser-negative but generally contain
Neisser-positive intracellular granules (Figure
249 and 24f). Beggiatoa spp., Thiothrix spp., and
types 0 0 4 1 , 0 6 7 5 , 0 2 1 N , 0 9 1 4 and 1 8 6 3 may
contain Neisser-positive granules (infrequently).
In addition, H. hydrossis and types O 6 7 5 a n d
0041 may have a Neisser-positive trichome
“covering” (Figure 24b) when present in acti-
vated sludge that is nutrient—deficient.
1 6 . Additional o b s e r v a t i o n s — T w o f i l a m e n t o u s
microorganisms, Thiothrix spp. and type 0 2 1 N
(uncommonly) may display rosettes and gonidia.
A rosette deveIOps w h e n trichomes radiate out—
ward from a common origin (Figure 25). Gonidia
are ovalor rod-shaped cells present at the tri-
chome apex that are distinctively different in ap-
pearance from vegetative cells (Figures 2 5 , 2 6 ,
and 2 7 ) .
The f o l l o w i n g observations may indicate a
nutrient deficiency (9.9., N and/or P) in activated
sludge systems:
0 Large amounts of intracellular PHB granules;
0 Unusual Neisser—staining reactions of some
filamentous microorganisms (see above); and
0 Large amounts of extracellular material in the
flocs. This is detected by conducting the
India ink-negative staining procedure (Section
5.2.4). When observed at 1 0 0 x phase con—
trast, the Indian ink particles normally can be
seen to penetrate deeply into the flocs, leav—
ing only a clear center; when there are large,
 :m
Figure 2 2 . Deposition of intracellular sulfur grafiulés during the 8 test ( a afid b): béfore (a) and after (b) adding sodium sulfide. Appearance
of intracellular sulfur granules In filamentous miCroorganisms (c, d e and f): c. Thiothrix I; d. Thiothrix ll; 9. type 0 2 1 N ; and f.
type 0 9 1 4 (all 1 0 0 0 x phase contrast). "

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‘55 ’
Figure 23. Gram stain reaction of filamentous microorganisms: a. and b. Gram negative (types 021m and 0092 . r e s p e c t i v e l y ) : c._Gram
vmiabla (type 0041); d. weakly Gram positive (type 1851-): and e. and f. Gram positive (Microthrix parwcella and Nocardta spp.
respectiveiy} (all 1000): transmitted light).
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Reference: Jenkins. at a!" 1 9 8 4 .

56’
Figure 24. Neisser staining reaction of filamentous microorganisms: a. negative: b. Neisser positive trichome covering observed atypically
. for type 0 0 4 1 ; c. and d. Neisser positive (type 0092 and Nostocoida limicola II respectively); and e. and f. Neisser positive
granules (Micrathrix parvicella and Nocardia spp. respectively) {all 1000_x transmitted light).
m— w... . u" -- .. .. . - «. .............

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Reference: Jenkins. et al., 1 984.

57
Figure 25. Thr’orhrix I! (a and b 1 0 0 x phase contrast; b a r : 100 pm; 0—1” 1000 X phase contrast: b a r : 10 um).

Ili'

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58
 Figure 2 6 . Type 021M (a 1 0 0 x phase contrast: bai= 100 um: b—HOOO x phase contrast; bar= 1 0 pm).
up— - — h-w ., wpm-«w Ni ’0- -v-p‘— «~—
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Reference: J e n k i n s , e t 31., 1 9 8 4 .

59
Hgma 27. mmrhrbr I (a 100 x phase contrast; bar:100pm;b-f 1000 x phase contrast: bar: 16‘um).

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60”
 clear areas with low cell density, the pres- curved and most often has substantial attached
_ 'ence of large amounts of extracellular mate— grth.
. rial (probably polysaccharide) is indicated; f
Occasionally, a filamentous microorganism is ob-
Additional clués pointing to thetpi'esénce of'large served that IS not represented by a type or genus des-
amounts of this highly water-retentive extracellu- ignation, Such a filamentous microorganism should be
lar material are: the activated sludge settles and‘ reported as “not identified”; do not try to ‘_‘force-fit”
compaCts podrly even though filamentous. m'icr'o- the microorganism into existing filament types.
organism abundance is low; the 'actiVated sludge
is slippery or slimy to the teach and appears
ViSCOUS when poured. . 6.3“.iDescriptions of Types of Filamentous
Microorganisms
These short descriptions of each filamentous micro-
6.2 Identificatiqh of Microorganisms . - organism Commonly observed in activated sludge are
Observed and recorded filament charaCteristi’cs are based on information given by Eikelboom and van
used to Characterize the filamentous micrOOrganisms Buijsen (1981) as modified by experience with fila-
by genus or by a numbered type using the dichoto- mentous ‘ m i c r o o r g a n i s m characteristics in activated
mous key shown in Figure 2 8 . This key lists the 2 2 sludges from the USA and South Africa. The following
filamentous bacteria most commonlybbserved in, acti— filament typ‘es . u s u a l l y are observed in domestic
vated sludge. To simplify this key, several filamentous WasteWater'treatment plants at conventional organic
microorganisms having readily identifiable specific loading rateé:
characteristics are not listed, but are described later.
These include: fungi, CyanOphyceae, Flexibacter S.néiabé‘ , = _'‘Beggiatoa spp. H. hydrossis
spp. and Bacillus spp In addition, some filamentous type
1701
Nocardia spp. type 1863
microorganisms observed only occasionally. in acti— type
021N N. limicola ll, . -'
vated sludge are not included in thé key. These include Thiothnix- I and II:
filament types 1702 1852 and “0211.
Filar’fiént tyfies I observed in industrial or domestic
This dichotomous key is a modification of the fila-a wastewéter treatment plants Operated at low organic
mentous microorganism identification key given by , loading rates are:
Eikelboom and van Buijsen (1981), with changes to:
type 0041 I Beggiatoa spp. N]. parvicella '
0 Deemphasiz'e the need _fOI‘_' .observat‘idnj of gel] type 0 6 7 5 s type 1851 Nocardia spp.
septa (crosswalls), which can depend o n the qual- type 02-1N type 0803 N. IimiCoIa I ; II, III
ity and adjustment of the microscOpe used;.and‘ ' Thiothrixl and II; typ'e 0092 H. hydrossis
.lnclude type 0 9 1 4 " type 0961 type 058‘]
some filamentous microorganisms in the'
key twice where an important characteristic is Filament types that are observed infrequently include:
- variable, e. 9 . , Gram stain reaction for N. limicola II
and the observation of intracellular sulfur granules fungi A " 'Flexibacter spp. type 0 2 1 1
.—
for types 0 9 1 4 and 0 2 1 N . ' Cyanophybege ‘ type 1702 type 041 1
Bacillussp‘p. = type 1 8 5 2
The_ use of this key is not without risk because some
filamentous microorganism characteristics vary, and 1 . Sphaerotilus natans. (Figure 29a, b , and 0; see
the key cannot always address all of these variables. also a F i g p r e s 1 6 d and 20a.) Relatively long
The filament type arrived at using the key should be (100-11000 m ) straight or smoothly—curved fila-
carefully checked.>against 'the typical ;microorgénis'ms ments composed of round—ended,' rod—shaped
characteristics listed in Table 7 and pfesented in the cells (].O——1.8><1.5—3.0 pm) contained in a
short descriptions and the photographs of each organ- clear, tightly-fitting sheath. Cell septa are clear
ism that follow. If characteristics given in Table .7 or --with indefitations at septa. Filaments radiate out-
in the photographs do not correSpond to the filament ".Ward' from the floc surface into the bulk solution.
type arrived at using the key, careful re-examinatiqn of False brainéhing frequently is observed, giving a
characteristiCs used'In the key IS in order. For example, '“tree—brahch"—Iike appearance. Gram negative,
type 0041- generally gives a weak, Gram. positiVe ‘or a Neisser negatix’xe, n o sulfur granules: PHB fre-
Gram variable reaction, and as such, is keyed'cqrrectly quehtjy qbserved. Cell shape can be rectangular
from Figure 2 8 . However, if strongly Gram positive, it when the cells are tightiy packed within the
would be keyed as N. limicola II. Reference to Table 7 sheath. 'An exocellular slime coating may occur
shows N. Iimicola II to be coiled and to possessno'at- ‘at nutrient limitation. Attached growth uncom—
tached growth—type. 0041 is straight or smoothly- mon; . b u t m a y occur when not growing.

61
 "Pal! Ciao -’ 1 . 5 pl Thlothrix l

...._.;..."ullur mules '5 an" if”

“*l’h 1 ”m-
3 9r an G? ‘6 M m *Ccll a n . s 1 . 2 p-
PI! of: can i n sulfur
granular. I n 5 u or «mar
plan; an 111-0 “Ms-P
1| --—a--Suuur granules “spherical"
*flo’t mil I e iypc OZIH
win-flat
sheathed—“‘1
*Hofllo 8399! M o n spp

r-D-True branching Nocardiat 59p.

C e l l 61a. 0 . 8 - 1 . 2 pm N . limlcola I I
all???”
Gram— +05! I "septa present —[:
pas ye -—II-Ho branching—.— C e l l a l a . 1.5 - 2 . 2 pm N . llmlcola I I I

* N e i s s e r ‘ posHive frlchome

--iI-No ca! 1 sepfa

*Naisser negative frlchome
(Neisser p o s i t i v e granules) M. parvicella

1y " Gram variable or weak!

. Ce!l d i a . 1.4 - 1.6 gm ? e 0041
Filamenfs dolggf confaln Gram posi+ive Y usuaggy heavy affached yp
sulfur granu 9 row
L ., 1-0
Heavy attached grou+h
cells square-
“type 0675 '
Cell dia. s um
Liffle or no affached
. ; fype 1851
r.::2¢e°§.553.2§°*3"9"'ar
«7.9

Trichome c o i l e d N. limicola II
Gram negative C e l l d i a . l . 0 - 2 . 2 um
c e l l sepfa_presenf -- ShéaThed, f a l s e branching
Trichome sfraighf or ‘
smoofhly curved No? sheafhed
I

Cell dia. < 1 . 5 pm C e l l s b a r r e l , recfaqgular

Ceils rec+angular Cells square
"fransparen?" C e ] ! d i a . 1 , 0 - 1.2 pm or discoid; cel daa.
no Indenfaflons at sepfa 1 . 2 - 2 . 0 pm: i n d e n f a f i o n s
a + sepfa

type 0961 Type 0914 Type 021M

Néisser posi+ive Neisser negafive
Type 0092 .

Trichome s f r a i g h f , smoofhly Trichome irregularly ben+ Trichome coiled

c u r v e d or benf "chain of c e l l s "
fype 0581
No cel'u se 'l'a C e l l sep'I'al presen'l' Ceiis oval Cells eicxéafed rods
p . 0.8 Klee-1.531 0.8x ‘41]!!!

type 0411
H . hydrossns Sheafhea; usually No? sueafhed +ype ‘ 8 6 3
heav¥ affached n o a++ached ”
grow h

r'l'ype 1701 I Wpe 08037

 a.

Table 7 . Summary of Typical Morphological and Staining Characteristics of Filamentous Microorganisms Commonly Observed in
Activated Sludge

BRIGHT FIELD OBSERVATION P H A S E CONTRAST OBSERVATION 1000)(

an
z E

CELLSHAPEAND
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1g;
TYPE 5 s m NOTES
H a:

s. natans — — — — — PHB 1'.o - 1.4 500 , St E + + + — round-ended rods False branching

1.4 x 2.0

type 1 7 0 1 ~— — — — — - PHB 0 . 6 - 0.8 2 0 - 80 St,B LE + + + + + round-ended rods cell septa hard

0 . 8 x 1 2. to discern

type 0041 +,V -—~ —.+ — — — 1.4-1.6 100-500 St LE + — + ++,—— squares Neisser positive
1.4><1.5-2.0 reaction occurs

typeOB75 +,V — —,+ —- -— — 0.8—1.0 50-150 _St I + — + ++,- squares Neisser positive
1,0 x 1.0 reaction occurs

type 021N - — —, + —, + + PHB 1.0 - 2.0 50 - 500 St.SC E + + — — barrels,‘ rectan— rosettes, gonidia
gles, discoid
1—2 x 1.5 - 2.0

Thiothrixl -— . + — - .+ +,— + PHB 1.4 - 2.5 100 - 500 -St,SC E + — + - rectangles rosettes, gonidia
2.0 x 3—5

Thiothrix I! — — —, + + ,- + PHB 0.8 - 1 . 4 5 0 - 200 . St,SC E + — + — rectangles rosettes, gonidia

0x 1 5

type 0 9 1 4 -- , + — —, + — _. + — PHB 1.0 5 0 - 200 St 5E,F + -- -—- —- squares sulfur granules

1.0 x 1.0 ”square”

Beggiatoa - .+ - - .+ + ,— + PHB 1.2 - 3.0 100 — 500 St F —, + - — — rectangles motile: flexing and
spp. 2.0 x 6.0 gliding
_.
type 1851 + — — — 0.3 100-300 St,B ‘- E +,— — + -,+ rectangles trichome bundles
weak 8x 1 5

type 0803 ' ... — — — -— - 0.3 50- 150 st 5,1: 4. _ _ _ rectang1es

0.8 x 1.5

type 0092 — + — — — + 0.8 - 1 . 0 20 - 6 0 8’03 I + .- - - - rectangles

0.8 x 1.5

.—
type 0961 _. — — — - 0.8—1.2 40- 80 St E + ‘- — -- rectangles ‘_'transparent"
1.0 x 2 0

M. parvicella + — + - — PHB 0.8 1 0 0 - 400 C i —- — -—- — large "patches”

Nocardia spp. + - + — - PHB 1.30 1 0 - 20 ,I 1 + . -- - - - variable true branching

1.0 x 1 - 2

N . Limicola I + + — — — — . 0.8 100 C LE — — — —

N . Limicola II -- , + + . — — — — PHB 1.2 «1.4 100 - 200 C LE + + —- — discs, o v a l s Incidental branching

2 X 1 0 G r a m a n d Neisser
variable
N . Limicola 11! + + — — .. PHB 2.0 200 - 300 C LE + + -— — disc, o v a l s
2.0 x 1.5

H . hydrossis —- — — - — — 0.5 20 - 100 St,B ’ E,F — — + —— . 4- “rigidly straigh "

type 0581 — — — — — —- 0.5 - 0.8 100 — 200 C | ‘ — — — -—

type 1863 — — —.+ —- - — 0.8 20-50 3.1' E,Fl + + — — oval rods “ c h a i n of cells"
0.8 x1-1.5

type 0411 — — — — — -— 0.8 50 - 150 3,1 F. + + — — elongated rods ” c h a i n of cells"

0.8 x 234

notation: + 2 positive; — = negative: V = variable; single symbo! invariant; + , -— or -— , + , variable, the first being most observed.
Trichome shape: St = straight; B = bent; S C = smoothly curved; C = coiled; I z lrregularly—shaped.
Trichome location: E = extends from flat: surface; I : found mostly within the flue; F = Free in liquid between t h e fi n e s .

Reference: Jenkins, e t 3]., 1 984.

63.
2. Type 1 7 0 1 . (Figure 29c, d, and e; see also
Figures 190 and 20f.) Relatively short (10—100
mm), curved or bent filaments composed of
round-ended, rod-shaped cells (0.7—1.0><
1.0—2.0 um) contained in a clear, tightly fitting
sheath. Cell septa are clear with indentations at
septa. Filaments found predominantly inter-
twined within the floc interior with only short fila-
ments extending into the bulk solution. No
bmnching. Gram negative, Neisser negative, n o
sulfur granules; PHB frequently observed. At-
tached growth of epiphytic bacteria is almost
always observed, making observation of indi-
vidual cells difficult.
Type 0041. (Figure 30a, b, and 0; see also
Figures 1 9 a . 199, and 230.) Straight, smoothly—
curved or bent filaments, 1 0 0 — 5 0 0 p m in length,
composed of square-shaped cells ( 1 . 2 — 1 . 6 x
1.5-2.5 um) contained in a clear, tightly fitting
sheath. In domestic waste systems, it is most
often observed inside the floc and covered with
heavy. attached growth. In industrial
systems, it may occur extending from the flee
surface or free in the bulk solution, and may not
have any attached growth. Gram positive or
Gram variable, tending to Gram positive when
found within the floc and Gram negative when
e x t e n d i n g into t h e b u l k s o l u t i o n . Neisser
negative; Neisser-positive granules are observed
infrequently; and a Neisser—positive (light purple
color) slime coating may be observed in some in—
dustrial waste systems (Figure 24). Intracellular
granules are rarely observed. No sulfur granules.
The sheath is difficult to detect and is observed
when cells are missing, particularly at the tri— -
chome apex (Figure 3 0 ) .
Type 0675. (Figure 30d, 9, and 1‘.) Very similar to
type 0041, only smaller in trichome length
(50—150 mm) and cell dimension (0.7—1.0 um).
Covered with heavy, attached growth in do‘mes-
tic waste activated sludge; may lack attached
growth in some industrial waste activated
sludge. A sheath is present. Gram positive to
Gram variable, Neisser negative. Neisser—positive
granules occur; no sulfur granules.
Type 0 2 1 M (Figure 26a—f; see also Figures 2 1 d
and e, 3 1 9 , and 2 3 a . ) Filaments typically are
1.0—2.0 mm in width and 1 0 0 — 5 0 0 p m in length
and taper from a thicker basal region, often ex-
hibiting an inconspicuous hold-fast, to a thinner
apical region, often terminating in loosely-
attached gonidia. Trichomes a r e straight, -.
smoothly-curved or sometimes slightly coiled
and are found extending from the floc surface.
Cell shape ranges from- ovoid to rectangular or
barrel-shaped with clear cell septa and indenta-
tions at septa. Gram negative and Neisser nega-
tive; may contain Neisser—positive granules. Cells
may stain slightly Gram positive when they con-
tain sulfur granules. Spherical intracellular sulfur
granules are observed in situ infrequently; re—
sponse to the 8 test generally is' positiveQ
Rosettes are observed infrequently. No attached
growth. N o sheath is present; however, a heavy
cell wall (still showing cross septa) often remains
after cell lysis.
. Thiothn'x I. (Figure 27a—f; see also Figure 22c.)
Straight or smoothly curved trichomes, 1.4—2.5
porn in width and 100—500 p m in length, found
extending from the floc surface. Cells are rec—
tangular (1 .4—2.5 x 3—5 pm) with clear cell septa
without indentations at septa. N o attached
growth. A sheath is present. Gram negative and
Neisser‘ negative; however, a Gram—positive reac-
tion may occur when sulfur granules are present.
Neisser—positivg granules may occur. Cells fre-
' q u e n t l y cOntain sulfur granules in situ and this
waste organism responds strongly to the 8 test. Apical
gonidia commonly are observed, and an incon-
spicuous hold—fast is present.
. Thiothrix II. (Figure 25a—f; see also Figures 20::
and 22d.) Straight or smoothly-curved filaments,
0.8—1.4 p m in width and-50—200 pm in length,
found extending from the floc surface. No at—
tached growth. Cell septa without indentations
are present. Cells are rectangular ( 0 . 8 — 1 . 4 x 1—2
= p m ) . Gram negative and Neisser negative, with
-, Neisser-positive granules sometimes. present.‘
Cells frequently contain spherical sulfur granules
in situ and this organism responds to the 8 test.
Apical gonidia and rosettes are commonly ob—
served. Trichomes may taper somewhat from
base to tip. A sheath is present, :but difficult to
detect.- '
Type 0 9 1 4 . (Figure 3 1 a , b, and 0.) Straight or
smoothly-curved filaments, 0.7—1.0 p m in width
and 5 0 - 2 0 0 pm in length, found extending from
the floc surface or more commonly free t i n the
bulk solution. May have incidental attached
growth. Cells are- square—shaped ( 1 . 0 x 1 . 0 pom)
without constrictions at septa. No sheath ' i s
present. G r a m negative a n d Neisser negative, but
may stain Gram positive when substantial‘ sulfur
granules are present. Neisser positive granules
m a y occur. M a y contain intracellular sulfur
granules, w h i é h appear square rather than
-spherical as observed for other filamentous
microorganisms. Does nOt respond to the 8 test.
. Beggiatoa spp. (Figure 3 2 3 and b.') Large, straight
filaments, 1.0—3.0 u m in width and 100—500
p m in Length, found free in the bulk solution and
64
Figure 2 9 . Sphaemtilus natans (a, b and c) and type 1 7 0 1 (d, e and f} ( a and d 1 0 0 x phase constrast; b a r : 100 m; b, c, e and f 1 0 0 0 x
phase contrast, b a r : 1 0 pm}.

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Reference: Jenkins. e t al.. 1984.

65
Figure 30. Type 0041 (a, b a n d c) and type 0 6 7 5 ( d e and f) ( a and d 1 0 0 x phase contrast: b a r : 1 0 0 p m ; b c, e and f 1 0 0 0 x phase
conflast; b a r : 10 u m ) .

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66
 a

Figure 3 1. Type 0914 {a 100 X phase contrast; bar: 100 mm b and c 1000 x phase contrast: bar= 1 0 p m ) (note sulfur granules in b).

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Reference: Jenkins, et al., 1 9 8 4 .

67
 actively motile by gliding and flexing. Generally
contains substantial spherical sulfur granules;
cell septa are not visible when sulfur is present.
Without sulfur, cells are rectangular (1—3 X 4—8
pm). No attached growth. No sheath. Gram neg-
ative and Neisser negative; however, cells may
stain Gram positive when substantial sulfur is
present. Neisser-positive granules may occur.
10. Type 1 8 5 1 . (Figure 320 and d; see also Figures
1 9 d and 23d.) Straight or bent trichomes, 0.8
p m in width and 100-300 p m in length, ob-
served extending from the floc surface, or more
commonly in bundles of intertwined filam'ents'. A
sheath is present, but hard to observe. Cells are
rectangular (0.8 x 1.5—2.5 pm) without indenta-
tions at septa. or with indentations that are diffi-
cult to observe. Attached growth-occurs, which
is distinctly perpendicular to the trichome sur—
face. Weakly Gram positive (a Gram-positive
"beaded" effect) and Neisser negative. N o s u l f u r
granules. . - 'without constrictions are present. Gram positive
11. Type 0803. (Figure 32e.) Straight or smoothly-
curved filaments of uniform diameter ( 0 . 8 pm)
and 50-4 50 pm in length, found extending from
the flat: surface or at times free in the bulk solu—
tion (industrial waste activated - sludges). No
sheath and no attached growth. Cells are rectan-
gular ( 0 . 8 x 1 . 5 — 2 . 0 u m ) without constrictions
at septa. Gram negative and Neisser negative. N o
sulfur granules.
12. Type 0092. (Figure 321‘; see also Figures 2 3 b and
240.) Straight, irregularly-curved or bent fila—
ments, 0 . 8 - 1 . 0 p m in diameter and 10—60 pm
in length, found mostly within the floc. Cells are
rectangular (0.8x 1 . 5 p m ) without constrictions
at septa, or with constrictions that are hard to ob—
serve. Neither attached growth nor a Sheath is
present. CelIs stain Gram negative and thé entire
trichome stains Neisser positive (purple). No
sulfur granules. ‘ -
This filament often is overlooked, or its abun-
dance underestimated, until the Neisser'stained
slide is examined. Filaments appear wider
(1.0—1.2 um) in dried, stained smears. -
13. Type 0961. (Figure 33a and b.) Straight tri-
cho-mes, 0 . 8 - 1 . 4 pm in diameter and, 50—150
um in length, observed extending from the floc
surface. No attached growth. Cells are rectangu-
llr ( 0 . 8 - 1 . 4 x 1 . 5 - 4 pm). A true sheath is not
present; however, a slime coating may be
present, appearing as a n empty “cuff” at the tri—
chome apex. Gram negative and Neisser nega-
tive and no sulfur granules. Cells appear ”trans-
parent" without any intracellular contents.
68
14; Microthrix parvicella. (Figure 330, d, and e; size
also Figures 2 3 9 and 2 4 a . ) Irregularly-coiled fila-
ments, 0.6—0.8 pm in diameter and 100-400.
p m in length, found in tangles in the floc or as
loose ”patches" free in the bulk solution. Neither
attached growth nor a sheath is present. No
branching. Cell septa are not observed; however,
substantial intracellular, granules may occur, giv—
ing a ” b e a d e d ” effect. Gram—positive, Neisser-
negative, and Neisser-positive granules com-
monly occur. Short, clear spaces may occur in
‘the filament.
1'5.'Nocardia spp..(Figure 34a, b, and 0; see also
Figures ~16b, 23f, and 241‘.) Irregularly—bent,
short filaments, 1 . 0 pm in diameter and 10—20
um in length, found mostly within the floc, but
may occur free in the bulk solution. A branched
(true branching) mycelium often is observed. No
sheatH and no attached growth. Cell shape is
somewhat irregular ( 1 . 0 x 1.0—2.0 p m ) and septa
a n d Neisser negative, and Neisser—positive
..
granules commonly are observed. No sulfur
granules. PHB commonly is observed. g A b u n -
dance of this organism is best assessed frOm the
Gram stained preparation. '
1 6 . Nostocoida limicola l . Bent and irregularly—coiled
f i l a m e n t s , 0 . 6 — 0 . 8 pm i n d i a m e t e r a n d
100—200 p m in length, found within the flocs
and free in the bulk solution. Cell septa are hard
to observe; when - observed, , cells are. oval
(0.6—0.8 u m diameter). N o sheath and no at-
tached g r o w t h . Gram positive a n d ‘ Neisser—
positive trichome. N o sulfur granules. Resembles
M. parviceII-a, e x c e p t i n N e i s s e r - s t a i n i n g
properties.
1 7 . Nostocoida Iimicola II. (Figure 3 4 d , e,‘ and f ; see
also Figures 1 6 0 and 24d.) Bent and irregularly-
coiled filaments, 1.2—1.4 p m in' diameter and
100—200 pm in length, found mostly within the
floc. Cell septa are clear, with oval cells (1 .2—1 . 4
um in diameter) and indentations at septa. No
sheath and no sulfur granules. PHB granules
commonly are observed. Gram and Neisser stain-
ing reactions are variable. Most observed is Gram
negative, but a Gram-positive reaction occurs.
Most often the entire trichome stains Neisser
positive (purple), b ut can be Neisser negative at
times. Incidental branching is observed. Note:
Eikelboom and van Buijsen (1981) state =that‘N.
Iimicola II is both Gram positive and Neisser nega—
tive, and that a bacterium closely resembling N.
limicola l l occurs in some industrial waste acti—
vated sludge systems, differing from N. Iimicola II
by being Gram negative and Neisser negative.
Both forms are considered N. Iimicola l l here.
Figure 3 2 . Beggiatoa spp. ( a and b), type 1 8 5 1 ( c and d). type 0 8 0 3 (e) and type 0 0 9 2 (f) ( a and c 1 0 0 x phase contrast: b a r : 1 0 0 um; b, d,
’ e and f 1000 x- phase contrast:= b a r : 10 y,m)_.
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69
Figure 3 3 . Type 0 9 6 1 (a and b} and Micrathrb: pamicella (c, dand e) (a and c 100 x phase contrast;bar: 100 pm: b, dand e 1000 x phase
. =
contrast; barn 10 pm).

Reference: Jenkins. ems!u 1984.

70
 ....
'Figure 34. Nacardia spp. (a, b and c) and Nostoco Eda (mice la ll (d, e and f) (a and d 1 0 0 x phase Contrastln bar: 100pm: b 400 x phase
contrast: II b a r : 25 pm; 0, e and f 1 0 0 0 X phase contrast II bar: 10pm).

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71
18. Nostocofda limicola Ill. Bent and irregularly-coiled Gram negative and Neisser negative. No sulfur
filaments, 1.6-—~2.0 pm i n diameter and granules.
200—300 mm in length, found mostly within the
floc. No attached growth and n o sheath. Cell 24.‘ Type 1852. (Figure 36b.) Straight or'slightly bent
septa are clear, with oval cells (1.6—2.0 p m filaments, 0.6—0.8 pm in diameter and 2 0 - 8 0
diameter) and indentations at cell septa. PHB , p m in l e n g t h , f o u n d e x t e n d i n g f r o m t h e flat: sur—
granules are commonly observed. Gram positive face. Cells are rectangular (O.6——0.8><1.0—-2.0
and Neisser positive. No sulfur granules. um) without constrictions at septa. No sheath,
n o attached growth. Gram negative and Neisser
19. Haliscomenobacter hydrosis. (Figure 35a, b, and negative. No sulfur granules. This filament ap-
0.) Straight or bent, thin filaments, 0.5 pm in pears “transparent,” as does type 0 9 6 1 .
diameter and 2 0 - 1 00 p m in length, found radiat—
ing outward from t h e floc surface or free in the 2 5 . Type 0 2 1 1 . (Figure 3 6 0 . ) Bent and twisted fila-
bulk solution. A sheath is present. No cell septa ments, 0 . 3 — 0 . 5 am in diameter and 20—100 pm
are observed; however, empty spaces in t he tri- in length, found extending from the floc surface.
chome are commonly observed. Gram negative Cells are rod—shaped with clear constrictions at
and Neisser negative. N o sulfur granules. Fila- cell septa. No sheath and n o attached growth.
ments ma y occur in bundles, and attached Gram negative and Neisser negative. No sulfur
growth is variabIe, ranging from rare to abun— granules. 1
dant. This filament can be easily overlooked at
1 0 0 x observation. 2 6 . Flexibacter spp. (Figure 36d.) Short, straight or
smoothly—curved filaments, 1 . 0 um in diameter .
20. Type 0581. (Figure 35d.) Smoothly—coiled fila— and 2 0 — 4 0 p m in length, found free in the bulk
ments, 0.4—0.7 [1m in diameter and 100—200 s o l u t i o n . T h i s o r g a n i s m i s m o t i l e by s l o w gliding
gm in length, found mostly within the floc but and flexing. N o sheath and n o attached growth.
may occur in "patches” free in solution. No Cell septa may be lacking. Gram negative and ’
sheath and no cell septa. No sulfur granules; no Neisser n e g a t i v e . PHB granules c o m m o n l y
attached growth. Gram negative and Neisser observed. '
negative. This filamentous microorganism ap—
pears similar to M. parvicella, but differs ' i n its
2 7 . Bacillus spp. (Figure 3 6 9 . ) Rounded-end rods in”
Gram and Neisser staining reactions.
irregularly—shaped chains of cells, 0.8—1.0 p m in
diameter and 2 0 — 5 0 u m in length, found mostly
21. Type 7863. (Figure 358.) Short, irregularly-bent
at the edges of the floc. Gram positive and.
filaments, 0.8 pm in diameter and 5 0 p m in
Neisser negative. N o sheath and n o sulfur
length, found extending from the floc surface
granules.
and free in the bulk solution. N o sheath and n o at-
t a c h e d g r o w t h . Cells are oval-shaped rods 2 8 . Cyanophyceae. (Figure 361‘.) Straight, large fila-
(0.8 X 1 . 5 nm), and appear as a “ c h a i n of cells,” m e n t s , 2.0—5.0 pm in d i a m e t e r a n d 1 0 0 — 5 0 0
with indentations at the septa. Gram negative
p m in l e n g t h , f o u n d free in t h e b u l k s o l u t i o n . C e l l s
and Neisser negative; Neisser-positive granules
are square to rectangular (2—5>< 2—8 porn) with
may occur. No sulfur granules. clear septa. N o sheath and n o attached growth.
22. Type 0 4 1 7 . (Figure 3 5 f . ) lrregularly—bent tri- Often motile by slow gliding. Distinct green color
chomes, 0 . 8 p m in diameter and 50—150 p m in under transmitted light observation. Gram nega-
from the floc surface. tive but sometimes with a slight Gram—positive
length, found extending
Filaments composed of elongated, rod-shaped reaction; Neisser negative. N o sulfur granules.
cells ( 0 . 8 - 2 . 4 p m ) ; constrictions at septa give
the appearance of a ” c h a i n of cells.” No sheath 29. Fungi. (Figure .37a, 3 7 b , and 370; see also Figure
and no attached growth. Gram negative and 168.) Very large trichomes, 3—8 pm in diameter
Nelsser negative. N o sulfur granules. and 300—1000 ,um inylength, found mostly in the
floc. Cells are rectangular ( 3 — 8 X 5—15 porn). and
23. Type 1702. (Figure 368.)Short, straight or bent contain intracellular granules and organelles;
filaments, 0.6—0.7 gm in diameter and 20—80 cytoplasmic streaming may be observed. True
pm in length, found within the floc and extending branching. Gram negative and Neisser negative.
from the floc surface. Cell septa are absent and a N o sulfur granules and n o sheath, although a
sheath is present. Incidental attached growth. heavy cell wall is present.

72
Figure 3 5 . Haliscomenobacter hydrossr's (a, b and 0) type 0 5 8 1 (d)_ type 1 8 6 3 (e) and type 0 4 1 1 (1‘) (all 1 0 0 0 x phase contrast:
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73
‘at‘ii
Figure 3 8 . Type 1702 (a), type 1 8 5 2 {b}, type 0 2 1 1 (c), Flexibacter sp. (d), Bacillus sp. (9) and a blue green bacterium (cyanophyceae) ( 0
(all 1000 x phase contrast; b a r : 1 0 pm).

Rafomnce: Jenkins. a t a!” 1 9 8 4 .

74
Figt'lré' 37. Fungus (a 100 X ”phase contrast: bar: 100 pun: b 400 X phase contrast: bar: 25 pm; 0 1000 x phase contrast; b a r : 10 um).

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75

"w;"WWW"
 Chapter 7
7 . 0 Appendix 3—Case Histories of Bulking
Control Using Chlorination.
City o f Albany, G A. The City of Albany, G A , is a
20-MGD (0.88 m3/sec) (design) activated sludge
plant currently receiving 1 3 t o 1 6 MGD ( 0 . 5 7 to 0 . 7
m3/sec) of a waste in which approximately 5 0 percent
of t he BOD5 and SS loads are from industry (paper
processing and convenience foods manufacturing).
The plant experienced bulking problems to such a
degree that, with only approximately 1 2 M G D (0.5
m3/sec) of influent flow, attainment of secondary ef~
fluent criteria ( 3 0 mg/I monthly average for BOD5 and
8 8 ) was not possible.
A1: Albany, t he permanent RAS chlorination system
injects chlorine solution into a series of well—mixed
RAS wet wells. The installation consists of a Wallace
and Tiernan Chlorinator 2,200 lb/day ( 1 , 0 0 0 kg/day)
capacity, and a 2—inch (50mm) injector with 2-inch
(50mm) PVC injector water and chlorine solution
lines. The chlorine injection system is enclosed in a
sheet metal housing for protection. The chlorine solu-
tion line is a 2—inch (50mm) PVC line leading to a
16—foot (5m) long, 2—inch (50mm) PVC header with
1/4-t0 3/8-inch (6 to 10mm) diameter holes at 4—inch
(100mm) spacing. The system was constructed in
November 1 9 7 7 for $4,953.
Figure 3 8 presents the history of RAS chlorination at
Albany. When chlorination for bulking control was in-
itiated, the RAS chlorination system did not exist.
Because of the urgency of controlling SVI, ’ c h l o r i n e
wés added to a wet well where the entire HAS stream
mixed with primary effluent. This mode of chlorina-
tion was commenced in Week 1 5 and continued until
Week 2 6 , w h e n the change to RAS chlorination was
made. Chlorine ‘was being added t o the mixture of
RAS and primary effluent, and doseé of 5 to 1 5 lbs
C|2/103|bs (5 to 15 kg CIZ/1O3kg) SS/day were
needed to control bulking. Immediately after the
change from chlorinating t h e mixture of RAS and
primary effluent t o chlorinating RAS alone, chlorine
doses that satisfactorily controlled bulking for the
mixture of RAS and primary effluent were “over-
doses” for RAS alone. Figure 3 8 s h o w s that even
though the chlorine dose declined during the change-
over of the chlorine dose point [from 4 . 7 to 2 . 9 lbs
77
C l2/103Ibs (4.7 to 2 . 9 kg C I2/1O3kg) SS/day] there
was an increase in secondary effluent SS (and to a
lesser extent in secondary effluent BODS) that
resulted from floc breakup caused b y the chlorine
overdose.
RAS chlorination has been practiced with generally
excellent results at Albany for a period o f over
7 years. A t various times, the target SVI has been
changed, and in some instances (Figure 3 8 , Weeks
1 8 0 to 200), RAS chlorination was not initiated soon
enough to prevent secondary effluent deterioration by
a loss of sludge blanket solids in the effluent during
peak f l o w periods. To illustrate the chlorine dosing
technique for bulking control, as employed by the City
of A l b a n y , 3 b u l k i n g i n c i d e n t i s e x a m i n e d i n d e t a i l in
Figure 3 9 . A t this time, the target SVI w a s 2 3 0 ml/g.
The chlorine dose was started at low levels and grad—
ually increased until the SVI responded by stabilizing
and then falling. I n the middle of this period ( 3 0 M a y
through 6 J u n e ) , the SVI fell t o below the target
value. The RAS chlorine dose was reduced in
response to this. On 18—20June, the SVI dropped to
approximately 1 0 0 to 1 3 0 ml/g. The chlorine dose to
the RAS was reduced and then turned off.
City o f San Jose/Santa Clara Water Pollution Control
Plant (SJ/ SC WPCP), CA. The SJ/SC WPCP provides
t e r t i a r y t r e a t m e n t t o 1 0 0 M G D ( 4 . 4 m3/sec) o f
domestic sewage; during Ju|y~September (the peak
load season), flows increased approximately 2 0 % and
BOD5 loading approximately doubled due to cannery
waste discharges. Effluent discharge criteria include
30—day average BOD5 and SS of 1 0 mg/I each and
receiving water undissociated ammonia standards
that dictate virtually complete nitrification. The plant
has two stages of activated sludge with complete
nitrification and tertiary effluent filtration. I t had plant
upsets due to bulking in the secondary activated
sludge system during the peak load seasons of 1 9 7 9
and 1 9 8 0 . Interim measures included RAS chlorina-
tion in both t h e secondary and tertiary activated
sIudge systems, and provision of supplemental oxy-
gen and ammonia to prevent bulking in the 1 9 8 1 and
1 9 8 2 peak load seasons (Beebe et al., 1 9 8 2 ) .
The importance of chlorine solution mixing into the
RAS for providing effective filamentous microorgan-
ism control was illustrated at the SJ/SC WPCP. I n the
firstwstage activated sludge system, the initial chlorine
injection point to the RAS was through a PVC pipe
with a 4~foot (1.2m) freefall into a -20 x 1 6 x 1 3 foot
(6m x 5 m x 4m) wet well. It soon became evident that
this arrangement was ineffective. Extension of the
2-inch ( 7 5 mm) PVC pipe to a point 5 feet (1.5m)
below the RAS surface in this wet well virtually
eliminated loss of gaseous chlorine, but SVI control
was still inefficient. Doses of 8 to 1 0 lbs Cl2/103Ibs (8
”to 1 0 kg C l z l 103kg) SS/day had little effect o n SVI
but created a turbid secondary effluent.
Following this failure, two alternate RAS chlorination
devices were installed:
(i) Diffusers about mid—depth across the full width of
the aerated mixed, liquor channels leading to the
secondary clarification tanks;
(ii) Single outlet injectors through the clean-out ports
about 6 inches ( 1 5 cm) upstream of closed-
Impeller, low-speed centrifugal RAS pumps.
The data presented in, Figure 40 show that the pump
clean-out po’rt injection point, with its superior mix—
ing, provided more rapid, predictable, and efficient
SVI control. Operating data indicated that approx-
imately 20 times more chlorine was added when
chlorine was dosed to the RAS well rather than to the
RAS pump clean-out port. A s a result of this success,
a similar chlorine injection system was installed in the
second-stage activated sludge system. It too has
been used successfully for bulking control without
compromising the nitrifying ability of the second—
stage activated sludge system. Injection of chlorine
solution into the first- and second-stage activated
sludge system RAS pumps for over 3 years has had
n o deleterious effects o n these pumps, even though
the seco‘nd-stage RAS pumps have brass bearings.
Impellers in a " HAS pumps are cast iron, and great
care is taken to make certain that chlorine solution is
never fed to an out-of-service pump.
RAS chlorination for control of filamentous bulking
has been developed to a high degree at the SJ/SC
WPCP (Figure 4 1 ) . Chlorine doses are regulated
routinely on the basis of a target SVI ’value with input
from the plant microbiologist w h o observes activated
sludge samples o n a daily basis during periods of RAS
chlorination. During past peak load seasons, extreme—-
Iv low target SVI values ( 6 0 to 8 0 mI/g) have been
used in the first stage activated sludge system so that
high solids loading rates ( 3 0 — 5 0 lbs SS/ft.2/day)
( 1 5 0 - 4 5 0 kg SS/mzlday) could be applied to the
secondary clarifier. To maintain SVI values this low,
chlorine doses to the first-stage activated sludge
system often were high—in the range of 8 to 1 6 lbs
Ola/1O3Ibs (8 to 1 6 kg C|21103kg) VSS/day. These
73
high chlorine doses produced significant floc destruc—
tion, resulted in turbid secondary effluents, 'and
caused increases in secondary effluent dissolved total
organic carbon (TOC) concentrations. The elevated
turbidity and TOC increase from approximately 1 5 to
3 5 m g / l could be tolerated at the SJ/SC WPCP.,
because the downstream second-stage activated
sludge system readily polishes the effluent. Applying
such high chlorine doses and obtaining a high—quality
secondary effluent would be difficult if the first—stage
activated sludge plant were operated without the
second—stage activated sludge system to capture the
fine solids.
RAS chlorination also is effective for bulking control in
the second—stage (nitrifying) activated sludge system
(Figure 42). At t he chlorine doses used [24 lbs
Clz /1O3Ibs ( 2 - 4 kg CI2/103kg)VSS/day]; 1 . 5 to 3 . 0
m g Clz /l dose in the RAS stream), nitrification effi-
ciency was unaffected. This observation is consistent
with that of Strom and Finstein ( 1 9 7 7 ) . I n the nitrify-
ing system, the decrease in SVI with commencement
of RAS chlorination was m u c h faster than it was in
the first-stage activated sludge system. Whether this
was due t o t he presence of the more germicidal free
chlorine in the nitrifying system, to differences in the
types of filamentous microorganisms causing bulking
(type 0 0 4 1 in the second stage, type 1 7 0 1 in the first
stage), or t o differences in system sludge growth
rates is not k n o w n .
Case history o f bulking control using hydrogen perox—
ide. City o f Petaluma, CA (Caropresso e t 31., 1974).
During 1 9 7 4 , t h e City of Petaluma, C A , Water Pollu-
tion Control Plant, which treated mixed domestic/
industrial‘ wastes, experienced severe bulking prob-
lems with SVI values in the range of 400 1:0 7 0 0 ml/g.
Hydrogen peroxide (as a 5 0 % V N - H202 solution) was
dosed to the final quadrant of a two—pass aeration
tank that w as being fed primary effluent at the 1 / 4
points and RAS at the head end. Figure 43 shows the
doses a n d cOncentrations of hydrogen peroxide u s e d
over a 4-day period and the effect o n SVI. The
hydrogen peroxide ( 1 0 0 % as H202) doses ranged be-
tween 9—68 m g / l and were variously applied for 8 - 2 4
hr/day. During the dosing of hydrogen peroxide,
2 3 0 0 lb (1050 kg) H202 (100% basis) was used to
bring the SVI under control (from 5 5 0 ml/g to 300
ml/g). The SVI continued t o decrease following the
termination of hydrogen peroxide dosing.
Figure 38. Cdntrol of bulking by RAS chlorination at the City of Albany, GA. Wastewater Treatment Plant (Jenkins et a!" 1983).

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79
Flguw 3 9 . Use of tatget SVI to control RAS chlorination dosage for bulking control at the Albany, GA, Wastewater Treatment Plant
(Jenkins. 1980].

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Figure 42. SVI and chlorine dose to RAS in the two second-stage activated sludge systems at the San Jose/Santa Clara Watér Pollution
Control Plant, C A — 1 9 8 1 peak load season (Beebe et al., 1 9 8 2 ) .

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 References
This section, besides containing references cited in the
text, also contains a more complete coverage of the
literature on activated sludge bulking, filamentous-
organism growth and Nocardia growth and foaming.
AI—Diwamy, L.J; and Cross, T . (1978), “Ecological
Studies of Nocardia Foams and Other Actinomycetes
in Aquatic Habitats.” In. Nocardia and Streptomyces;
M . Modarski W . Kurytowicz and J Jeljaszewicz
(Eds.) Gustav Fischer Verlag, Stuttgart Germany,
1 53.
Anon. (1969) ”Milwaukee M y s t e r y ” Unusual Operat-
ing Problem Develops.” Water and S e w . ' Works,
716, 2 1 3 .
Anon. FMC Corporation (1973) “Bulking Control w i t h
Hydrogen Peroxide: Case History, Water Pollution
Control Plant, City of Petaluma, Sonoma, C A . ”
Technical Data Polln. Control Release No. 4 1 .
Anon. ( 1 9 7 4 ) Bergey’s Manual o f Determinative Bac-
teriology 8th Ed.‘, R.E. Buchanan and N E Gibbons,
Eds. The Williams and Wilkins Co., Baltimore.
Anon. FMC Corporation (1976) ”Sludge Bulking Cure:
Hydrogen P e r o x i d e . ” Polln. Control Release No. 9 5 .
Municipal South Nov. /Dec.
A n o n . (1979) f ‘ H y d r o g e n Peroxide Solves Bulking.
P r o b l e m at Coors W a s t e T r e a t m e n t P l a n t . ” Food
Engineering, November 1 9 7 9 and FMC Corporation
Technical Data Polln. Control Release No. ‘1’] 7.
A n o h . ( 1 9 8 1 ) Manual o f Methods for General Bacteriol-
ogy. American Society for Microbiology, Washing-
ton, D.C.
Anon. (1983) ”Thames Water Uses Chlorine t o Control
Bulking Sludge.” Water Research News, 10, 6,
W a t e } Research Centre, Medmenham, Bucks,
England.
Adamse, A.P. ( 1 9 6 8 ) “Bulking of Dairy Waste Acti~
vated Sludge.” Water Research, 2, 7 1 5 .
Albagnac, G . , and Morfaux, J . N . (1980) “Traitabilitie
C o m p a r e e en A e r a t i o n P r o l o n g e e et e n Contact—
Stabilisation des Eau Residuaires de Brasierie.” Trib.
Cebedeau, 33, 6 3 .
Allen, L A . (1944) ” T h e Bacteriology of Activated
Sludge.” J. Hyg., 43, 4 2 4 .
Ardern, E. and Lockett, W . T . (1914a) ”Experiments o n
the Oxidation of Sewage Without the A i d of Filters.”
J. Soc. Chem. Ind., 33, 5 2 3 .
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 Matschei N.F. (1982) “Control of Bulking Sludge—— Nurdogan, Y.'and Jenkins, D . (1983)‘Unpublished data
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van Leeuwenhoek, 29, 1-21. Pittman, A.R. ( 1 9 8 4 ) “Settling of Nutrient Removal
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Chemical and 'Mibrobiological Aspects o f Filamentous ference lntl. Assoc. for Water Polln. Res. Amster-
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#U.S.GOVERNMEN'!'PRIN11NGOFFICE:1991.5#8.187,I§0550