'
Office of Research and
Development
Washington DC 2O46O
Center for Environmental Research
Information
Cincinnati OH 45268
Technology Transfer
Summary Report
The Causes and
Control of Activated
Sludge Bulking and
Foaming
EPA/625/8-87/012
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Summary Report
The Causes and Control of
Activated Sludge Bulking
and Foaming
July 1987
Prepared for
U.S. Environmental Protection Agency
Center for Environmental Research Information
Cincinnati, Ohio 45268
EPA Contract 68-03-3252
Prepared by
Dynamac Corporation
The Dynamac Building
11140RockvillePike
Rockville, Maryland 20852
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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 to 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.
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Contents
Chapter Page
1.0 Introduction 1
1.1 Definition of Problems 1
1.2 Overview of Activated Sludge Process 1
2.0 Activated Sludge Floe 5
2.1 The Floe—Contents and Components 5
2.2 Filamentous Microorganisms 5
2.3 Definitions of Terms Used for Quantitative
Description of Floe 5
2.4 Effect of Number of Filamentous Microorganisms on
Quantitative Floe Parameters 8
3.0 Activated Sludge Bulking 9
3.1 Symptoms of Sludge Bulking 9
3.2 Role of Filamentous Microorganisms 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 the Presence of Nocardia 29
4.4 Control of Activated Sludge Foaming 29
5.0 Appendix 1—Methods for Microscopic Examination of Filamentous
Microorganisms in Activated Sludge 33
5.1 Sampling Methods 33
5.2 Staining Procedures 34
5.3 Examination Procedures 38
5.4 Counting Procedures 41
6.0 Appendix 2—Microscopic 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—Case Histories of Bulking Control
Using Chlorination 77
References 85
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Figures
Number
Page
1 Microscopic appearance of activated sludge floes 6
2 Effect of filamentous microorganisms on activated
floe structure 7
3 Effect of filamentous microorganisms on floe
structure 10
4 Correlation of filament count and SVI 11
5 Activated sludge process schematic . . 15
6 Zone settling of activated sludge solids 16
7 Clarification tank design and operation diagram 18
8 Clarification tank thickening analysis example 21
9 Step feed operation example schematic 23
10 Step feed operation example—plot of parameters 24
11 Progressive effects of chlorination 27
12 Nocardia foaming in activated sludge 32
13 Floe "texture" in activated sludge 39
14 Effect of filamentous microorganisms on floe . 40
15 Filament abundance categories 42
16 Trichome branching 48
17 Examples of filament shapes 49
18 "Color" of filamentous microorganisms 50
19 Attached growth of epiphytic bacteria 51
20 Appearance of sheaths 52
21 Cell shapes 53
22 Deposition of intracellular sulfur granules 55
23 Gram staining reaction 56
24 Neisser staining reaction 57
25 Thiothrix II 58
26 Type 021N 59
27 Thiothrix I 60
28 Dichotomous key for filamentous microorganism
identification 62
29 Sphaerotilus natans 65
30 Type 0041 and 0675 66
31 Type 0914 67
32 Begg/atoaspp.,type 1851, 0803, and 0092 69
33 Type 0961 and Microthrix parvicella 70
34 A/ocard/aspp., and Nostocoida limicola 71
35 Haliscomenobacter hydrossis type 0581, 1863, and
0411 73
36 Type 1702, 1852, 0211, Flexibacterspp., and
Bacillus spp 74
37 Fungus 75
38 Control of bulking by RAS chlorination 79
39 Use of target SVI to control RAS chlorination
dosage 80
40 Comparison of RAS chlorination effectiveness 81
41 SVI and chlorine dose to RAS 82
42 SVI and chlorine dose to RAS 83
43 SVI response to peroxide treatment 84
IV
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Tables
Number
1
Page
Causes and effects of activated sludge separation
problems 2
2 Dominant filament types as indicators of conditions
causing bulking • 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 morphological and staining
characteristics , 63
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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 811810.
vi
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Chapter1
1.0 Introduction
The reaction of wastewater with biologically active
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 operations: (1) aeration, and (2)
clarification. These operations may be physically
separated or, as in a Sequencing Batch Reactor (SBR)
process, may take place in the same unit. In aeration,
microorganisms are brought into contact with biode-
gradable wastes in the presence of a continuous
oxygen supply. In 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 the
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 biological oxida-
tion and the solids separation processes. This report
concentrates on two 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.
7.7.7 Sludge bulking.
This is a condition in which the sludge becomes very
light, increases in volume, 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 floe and interfere with the
compaction and settling of the sludge. The effects of
the bulking are a high Sludge Volume Index, low
solids concentration in the return and waste sludge,
and the hydraulic overloading of the solids handling
systems. Under these conditions, floating sludge
flows out of the tank with the effluent, producing a
poor quality product.
7.7.2 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 thick that it overflows the clarifi-
cation tank. As 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 floe, 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 of the component steps of the activated
sludge process in which these two problems occur.
1.2 Overview of Typical Activated Sludge
Process
7.2.7 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 the biode-
gradable components of the wastewater. The mixture
of wastewater and sludge (mixed liquor) then flows to
the clarification tank, where settling of the sludge oc-
curs. The solids settle to the bottom of the tank, while
the clear effluent is drawn off. Some of the settled
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Table 1. Causes and Effects of Activated Sludge Separation Problems
Problem
Cause
Effect
Dispersed growth
Microorganisms do not form floes but
are dispersed, forming only small
clumps or single cells.
Turbid effluent. No zone settling of
sludge. ,
Slime (jelly)
Viscous bulking; (also possibly
has been referred to as Non-
filamentous bulking)
Pin floe or Pinpoint floe
Microorganisms are present in large
amounts of exocellular slirne. In
severe cases the slime imparts a jelly-
like consistency to the activated
sludge. ,
Small, compact, weak, roughly
spherical floes are formed, the larger
of which settle rapidly. Smaller
aggregates settle slowly.
Reduced settling arid compaction
rates. Virtually no solids separation in
severe cases resulting in overflow of
sludge blanket from secondary
clarifier. In less severe cases a .
viscous foam often is present.
Low sludge volume index (SVI) and a
cloudy, turbid effluent.
Bulking
Blanket rising
Filamentous organisms extend from
floes into the bulk solution and
interfere with compaction and settling
of activated sludge.
Denitrification in secondary clarifier
releases poorly soluble N2 gas which
attaches to activated sludge floes and
floats them to the secondary clarifier
surface.
High SVI —very clear supernatant.
Low RAS and WAS solids concentra-
tion. In severe cases overflow of
sludge blanket occurs. Solids handling
processes become hydraulically'
overloaded. '
A scum of activated sludge forms on
surface of secondary clarifier.
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.
Foams float large amounts of > .
activated sludge solids to the surface
of treatment units. Nocardia and
Microthrix foams are persistent and
difficult to break mechanically. Foams
accumulate and can putrify. Solids
can overflow into secondary effluent1
or overflow tank freeboard onto
walkways.
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sludge is returned to the aeration tank, while the ex-
cess sludge is removed for further treatment and final
disposal. Sometimes the return sludge is aerated in a
separate tank before it is reintroduced to the main
aeration tank, to reactivate the microorganisms by
allowing them to complete consumption of waste re-
maining in the sludge.
1.2 2 Microbiological processes.
The character of municipal wastewater is usually ex-
pressed in terms of two parameters: Five-day Bio-
chemical Oxygen Demand (BOD5) and Suspended
Solids (SS). BOD 5 indicates the amount of oxygen
that would be required for biological oxidation of the
waste. It is determined by a standard test wherein a
known amount of wastewater is mixed with a known
amount of oxygen, and the mixture is incubated for 5
days at 20°C. During that time, bacteria in the
wastewater use the oxygen to oxidize organic matter
in the waste. At the end of that time, the oxygen re-
maining in the mixture is measured, and from the
amount of oxygen consumed, the BOD5 in milligrams
per liter is calculated. SS is expressed in milligrams
per liter, and is determined by filtering a sample of the
wastewater and weighing the dried, solid-containing
filter. (Standard Methods 208D).
The objective of the activated sludge treatment proc-
ess is the reduction of both the BOD and SS of the
wagtewater. Bacteria in the sludge convert organic
material in the waste to more stable material and in-
organic byproducts (CO2 , H2O) and cell protoplasm
in the presence of oxygen. In order for this process to
proceed at optimum biological activity, the plant
operator needs to determine the appropriate ratio to
be maintained between the food supply. (BOD) in the
incoming wastewater and the mass of bacteria in the
aeration tank.
Other biological conversions that can take place in the
aeration tank are phosphorus removal and nitrogen
removal. Both, of these materials stimulate natural
biological activity in the effluent-receiving waters,
causing a decrease in dissolved oxygen and thus
resulting in a decreased ability of receiving streams to
support fish.
Nitrogen removal begins with the conversion of am-
monia to 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 (e.g., methanol or raw sewage) where the
nitrites and nitrates can be converted to the N2 gas
that is lost from the liquid (denitrification). This two-
step process is not accomplished at 100 percent effi-
ciency—some ammonia remains in the effluent. Fur-
thermore, in plants that disinfect effluent with
chlorine, some ammonia in the effluent increases the
amount of chlorine needed to bring the effluent within
regulatory limits. Control of ammonia conversion is
maintained by careful control of the level of oxygen in
the aeration tank, and by aging of the sludge to en-
sure the presence of nitrifying bacteria.
Phosphorus is incorporated into the protoplasm of the
new cells that are generated when the bacteria
multiply as they oxidize the organic waste. Fifty to 90
percent of phosphorus in influent wastewater can be
removed by the activated sludge process.
1.2.3 Physical plant.
Wastewater enters the typical plant and receives
preliminary treatment. This may include use of
screens (when there are many large floating solids),
comminutors (grinders to achieve size reduction), and
grit chambers (when there are inorganic suspended
solids). This is usually, but not always, followed by
primary treatment in sedimentation tanks (for removal
of settleable solids).
The wastewater then flows to the aeration tank. In
some cases, the waste is mixed with return activated
sludge at a point outside the aeration tank, and the
mixture then enters the tank. In other systems, the
waste and return sludge are mixed in the aeration
tank. In the aeration tank, the mixture is aerated by
either a diffused air system (in which compressed air
enters the tank at the bottom of the tank, causing the
tank's contents to circulate) or a mechanical aeration
system (in which a blade or pump is used to agitate
the surface or pump the mixture and toss it in the air,
and to .introduce oxygen and mix the sludge and
wastewater). The size of the aeration tank is a func-
tion of the length of time that the waste will remain in
the tank.
After a suitable contact period (e.g., 4 to 24 hours)
the mixture of activated sludge and treated waste-
water,' called mixed liquor, flows into the secondary
clarification tank, where the sludge settles out. This
tank must be carefully monitored so that the settled
sludge is removed at frequent enough intervals to pre-
vent it from accumulating and flowing out with the ef-
fluent, and to prevent anaerobic conditions.
The settled sludge removed from the clarification tank
is usually 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 to maintain aerobic condi-
tions before its return to the aeration tank. The clear
effluent flows from the clarification tank to be disin-
fected and discharged from the plant.
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7.2.4 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 for the 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. In 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.
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Chapter 2
2.0 Activated Sludge Floe
The activated sludge process produces a mass of
active microorganisms, which agglomerates and floc-
culates in the system's aeration tank and secondary
clarification tank, and which is capable of oxidizing
organic matter in wastewater. Many of the operating
problems that occur in activated sludge systems can
be attributed to various characteristics of the acti-
vated sludge floe. This section presents a description
of the composition and contents of the floe.
2.1 The Floe — Contents and Components
Activated sludge floe is normally composed of a
widely varied assortment of microorganisms and par-
ticle sizes. Particles range from single bacteria,
measuring 0.5 to 5 m, to large floes measuring 1 mm
(1,000 ^m) or more.
The primary active population of the floe is hetero-
trophic bacteria (those that feed on organic matter),
including Pseudomonas, Achromobacter, Flavobacte-
rium, Alcaligenes, Arthrobacter, Citromonas, and
Zooglea. Also present in smaller quantities are fungi,
protozoa, and metazoa. Floes also contain organic and
inorganic particles from the influent wastewater,
"extracellular polymers" that seem to promote bio-
flocculation, and volatile matter.
Researchers have suggested that there are two levels
of structure in floe. The "microstructure," which is
the basis for floe formation, consists of bacteria that
are able to "stick to" one another by microbial aggre-
gation and bioflocculation. The "macrostructure,"
which consists of filamentous microorganisms, forms
a large network to which the floe-forming bacteria of
the microstructure can cling.
2.2 Filamentous Microorganisms
If a particular sludge contains only microstructure
bacteria, and no macrostructure (filamentous) micro-
organisms, floes are small (averaging approximately
75 ^m), round, and easily broken up in the aeration
tank. This results in an operational problem known as
"pin floe." Such floe will settle rapidly, but will leave
large amounts of smaller particles still suspended in
the effluent.
Thus, filamentous (macrostructure) microorganisms
are extremely important to a good floe and efficient
production of a clear effluent (see Figure 1).
2.2.1 Effect of filamentous microorganism count on
floe.
When filamentous microorganisms are present in acti-
vated sludge, they form a network to which the
smaller floe-forming bacteria can cling. This promotes
the formation of larger floes, which do not readily
break apart in the aeration tank, and which settle more
efficiently, mechanically removing smaller particles as
they settle. These large floes are irregular in shape, in-
stead of small and spherical.
Activated sludge floe can have a good balance of fila-
mentous and floe-forming bacteria, giving good set-
tling characteristics and a clear effluent; have too few
filamentous microorganisms, resulting in pin floe and
a poor-quality effluent; or have too many filamentous
microorganisms, resulting in bulking (see Figure 2).
2.2.2 Examination of filamentous microorganisms in
floe.
In order to effectively manage operational problems
caused by filamentous microorganisms, it is important
to know not only how many are present, but also what
types (see Section 3). Microscopic examination of floe
will yield data on microorganism number and type,
and on physical floe characteristics that influence set-
tling. Appendices 1 and 2 present specific information
about methods for performing such examination, in-
cluding procedures for sampling, staining, counting,
and identifying organisms.
2.3 Definitions of Terms Used for
Quantitative Description of Floe
Several quantitative parameters exist for the descrip-
tion of activated sludge floe. These parameters are
defined below.
2.3.1 Mixed Liquor Suspended Solids (MLSS).
The contents of the aeration tank is referred to as
mixed liquor. It contains suspended microorganisms
and other sludge components. MLSS is an expression,
in mg/l, of the amount of microorganisms suspended
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1. Microscopic appearance of activated sludge floes: a. small, weak floes (pin-floe) (100 x phase contrast); fa. small, weak floes
(100 X phase contrast); c. floes containing microorganisms (100 x phase contrast); d. floe containing filamentous microorganisms
"network" or "backbone" (1000 x phase contrast) (a and cbar=100ftm; fa and dbar=10^un).
§Jn, ~ii-, : tj-r;, ijifiii;^wfil
, i 2 , iigf* •
fit i;! ifiBSSEffiSjjS
'-= ' : » :ils si • ! a* Pl
' • l! • !
(V f •'»»-•!=". ' "' ' '"'""r "'|l:':l, --' "'""•''""-"ri!* ^Pfiipiwfi1;*
'Sfjt^,:1 ^jpVy;; ;,;;, ^^ti.i;: :^^SS3S ^Sjii+ffif
i!1 r '& * ;""i""! '" ; " : ' !:i11"""" ?T^!rtr!st!WK
-;;-;- ?"ftSSHim'S
Reference: Jenkins, era/., 1984.
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Figure 2. Effect of filamentous microorganisms on activated sludge floe structure.
A. Ideal, Non-bulking Activated Sludge Floe.
1. FILAMENTOUS ORGANISMS AND FLOG
FORMMG ORGANISMS IN BALANCE
2. STRONG, LARGE FLOC
3. FILAMENTS DO NOT INTERFERE
4. CLEAR SUPERNATANT
5. LOW SVI
FILAMENT BACKBONE
B. Pin-point Floe.
• 'C1
* (r * f'x
U Cx
£'<2
^sX
p . ;
/~\ -
Q tf ^
. P-O 1. NO FILAMENTOUS ORGANISMS
; ^^^
* ^ 2. WEAK, SMALL FLOC
A ' 3. TURBID SUPERNATANT
°\ }
fc ( J 4. LOW SVI
<
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in the mixed liquor, in mg/l, and is a measure of the
amount of microorganisms 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
floe (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 30
minutes in a graduated cylinder, and performing the
following computation:
gyj _ volume (sludge) after 30 min. (ml/1) x 1000_ rn]
MLSS (mg/l) ~~jf
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 low is indicative
of pin floe. 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:
Vol. aer. tank (gal.) x MLSS (mg/l)
- = days
Recycle flow rate (gal./day) x recycle MLSS {mg/l)
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 Age in a conventional
activated sludge plant is 3 to 15 days. Optimum
Sludge Age 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.
A Sludge Age that is too low can cause floe 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 floe and a
turbid effluent.
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 mg/l of DO should be maintained in the
aeration tank. DO 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.
It is computed as follows:
_pounds per day of BOD added to aeration tank
MLSS in aeration tank (Ibs)
The normal range for F/M for a conventional plant is
0.15 to 0.5.
2.4 Effect of Number of Filamentous
Microorganisms on Quantitative Floe
Parameters
SVI is the parameter most closely related to the number
of filamentous microorganisms in the floe. Pin floe (floe
with very few filamentous microorganisms) will have
an SVI below 70 ml/g, while bulking sludge (with high
numbers of filamentous microorganisms) will have an
SVI above 150 ml/g.
Similarly, filamentous microorganisms affect the
amount of suspended solids in the effluent. Sludge
having few such microorganisms and thus resulting in
pin floe will yield a turbid effluent, containing many
suspended solids. A bulking sludge with too_ many fila-
mentous microorganisms will result in a very clear ef-
fluent, but as the effluent flows from the clarification
tank it will carry the large, light floes, increasing the
SS in the effluent.
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Chapter 3
3.0 Activated Sludge Bulking
Sludge bulking is one of the most common operational
problems experienced by activated sludge treatment
plants.
3.1 Symptoms of Sludge Bulking
Sludge bulking 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, when sludge in the clarification
tank gains in volume and becomes light and fluffy,
flowing out over the weirs with the effluent. The ef-
fluent itself is otherwise very clear.
3.7.2 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
150 ml/g. The density of bulking sludge is much
lower than that of "ideal" sludge, and therefore fewer
microorganisms are returned to the aeration tank in
the return activated sludge (RAS). 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 floe can develop.
However, if there are too many filamentous micro-
organisms, floe 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.
3.2.2 Relationship 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-
tions are summarized in Table 2. The research that re-
sulted in this table gathered data on plant operating
conditions only incidentally;-it was focused on deter-
mining the presence of various microorganisms in
bulking sludge. However, the 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
As Table 2 shows, changes in plant operating condi-
tions, and resultant changes in the quantitative
parameters defined in Section 2.3, can promote the
growth of different filamentous microorganisms.
A low pH in the aeration tank can promote the growth
of. fungi. Based on laboratory 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 the wastewater most likely con-
tains strong acid discharges and an aeration tank pH
value below 6.0.
Low DO in the aeration tank can promote the growth of
S. natans and type 1701. Low DO concentration in low
F/M systems can promote the growth of H. hydrossis.
Experiments have shown that the DO concentration re-
quired to prevent the growth of low DO filamentous
-------
FifluroS. Effect of filamentous microorganisms on floe structure: a. aggregates observed for a pure culture of an activated sludge floe
former; b. aggregates observed for a dual culture of a single floe former and a single filamentous microorganism (100x phase
contrast; bar= 100 pm) (Lau at a/., 1984).
* If''" ' Jt * •**
•?>* • .j'?.?,x. i '*'" M-*^ v
swas^w>5ss
- , ~. -- ••
:./;
'
'
BB**1'«*<(I7*****T*'"1T*" "T**^**1** * * "
Roforcnco: Jenkins of al.
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 et al, 1982).
140
120
100
E
>
100
FILAMENT COUNT
11
-------
Table 2. Dominant Filament Types as Indicators of Conditions Causing Activated Sludge Bulking
Suggested Causative Conditions
Indicative Filament Types
Low
DO
Low
F/M
Septic
Wastewater/Sulfide
Nutrient
Deficiency
Low
PH
type 1701, S. natans, H. hydrossis
M. parvicella, H. hydrossis, Nocardia sp
types 021N, 0041, 0675, 0092, 0581, 0961,
0803
Thiothrix sp., Beggiatoa and type 021 l\l
Thiothrix sp., S. natans, type 021N, and possibly
H. hydrossis and types 0041 and 0675
fungi
Richard eta/. 1982a; Strom and Jenkins 1984.
microorganisms was a function of the F/M load. The
greater the F/M load, the greater the DO concentration
required to prevent the low DO bulking.
Experiments have also shown that low DO bulking
can be caused and cured by manipulation of F/M load
and DO concentrations. However, in dealing with low
DO bulking problems, a basic economic question
must be addressed. To cure the problem, one must
lower the F/M or increase the aeration tank DO con-
centration. Each of these actions may have undesir-
able consequences. Lowering the F/M may cause the
onset of nitrification and increase MLSS levels to
values that exceed the solids capacity of the secon-
dary clarifiers. Increasing the aeration tank DO con-
centration will require greater power input and may
also cause the onset of nitrification. Because of these
factors, "rt is necessary to consider the alternative of
operating at a low aeration tank DO concentration and
killing off the filamentous microorganisms that
develop using RAS chlorination. RAS chlorination is
discussed in Section 3.4.4.1.
Low F/M load may result in M. parvicella and types
0041, 0675, 0581, 0961, 0803, and 0092. The
specific causes for the growth of filamentous
microorganisms that appear in continuously fed,
completely-mixed, low F/M load activated sludge
systems are poorly understood. These systems pro-
duce poorer settling activated sludge than systems
that are fed intermittently or have aeration tanks
where there is a relatively high local concentration of
wastewater at the point where the RAS and influent
enter the tank. Emperical measures producing a car-
bonaceous substrate concentration gradient in the
aeration tank, or a high substrate concentration at the
point where RAS and influent enter the aeration tank
have consistently controlled low F/M induced bulking
in laboratory studies, but less effectively in practice.
The correlation of cause and effect between low F/M
load and the presence of filamentous microorganisms
is not completely understood and is being studied
further.
Treatment of septic waste can lead to growth of
Thiothrix spp. and type 021N. This may result from
the ability of these microorganisms to grow on in-
organic reduced sulfur compounds and organic acids,
which are produced by fermentation in septic
sewage. Under such conditions these microorgan-
isms produce intracellular sulfur granules that are
convenient for identification purposes.
Treatment of nutrient-deficient wastes (low in
nitrogen or phosphorus) can lead to growth of
Thiothrix spp. and type 021N, and possibly types
0041 and 0675. Under these conditions, Thiothrix
spp. and type 021N do not contain intracellular sulfur
granules, but may contain other intracellular granules
such as polyhydroxybutyrate (PHB). Also under
nutrient-deficient conditions, types 00,41 /and 0675
may show a IMeisser-positive extracellular slime
covering, although they normally slain Neisser
negative (see Appendix 1 for stain procedures).
12
-------
When microscopic examination suggests nutrient
deficiency, the BOD, COD, or TOG 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
BOD5/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 BOD5/N/P ratio is a useful guideline for
detecting nutrient deficient wastes, the following fac-
tors also should be taken into account:
• The availability of nitrogen and phosphorus in the
influent should be high enough for use during the
metabolism of the carbon source.
• 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.
• Because each wastewater/activated sludge com-
bination has its own unique nutrient demand, an
influent BODS/N/P ratio should -be checked by
measurements of effluent concentrations of
dissolved (0.45 m/t filtered) orthophosphate, am-
monia, and nitrate.
• Ammonia and nitrate are available as nitrogen
sources for activated sludge growth.. 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 result in "straggler
floe," light buoyant particles that do not settle in the
clarification tank and flow over the weirs in an other-
wise clear effluent.
3.4 Control of Activated Sludge 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 bulking, some design innovations are very re-
cent, and many existing activated sludge plants do
not have them. In such plants, bulking sludge must be.
corrected by adjustments in operations or by addition
of chemicals.
3.4.7 General approach to controlling filamentous
microorganisms.
A .good general approach to controlling bulking prob-
lems is as follows: First, identify the 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). If
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 manipula-
tion, addition of chemicals to enhance settling, and
addition of toxicants to selectively kill filamentous
microorganisms.
3.4.2 Process manipulation to control bulking.
In many cases, the adverse effects of bulking sludge
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 to:
• Study in detail the technical operating principles of
the activated sludge clarification tank, particularly
as they relate to the thickening function;
• Examine specific techniques for determining the
solids handling capacity of the clarification tank and
how this is influenced by sludge settling charac-
teristics; and
• 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 calculations for changes in design.
Clarifier Operation Principles. Activated sludge
clarification tanks have two basic functions—
clarification and thickening. Clarification is the
removal of activated sludge floes to produce a clear
overflow that either meets discharge standards or
-------
does not overload downstream processes. Thickening
of the settled activated sludge solids (i.e., RAS) is
required so that they can be returned to the aeration
basin. If the activated sludge solids are not thickened
and removed from the clarification tank at a rate faster
than they are added, they will accumulate until the
clarification tank is full and the excess solids will be
discharged into the effluent. Since bulking affects the
ability of the activated sludge solids to thicken, the
thickening function and thickening capacity of the clari-
fication tank are affected by sludge bulking.
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 Q, together with RAS, at
flowrate Q,. The aeration tank suspended solids con-
centration (MLSS) is X, and the RAS, SS is Xu. Ac-
tivated sludge soilds are applied to the clarification
tank at a flowrate of Q + Qr.
It is assumed here that sludge is wasted from the RAS
at a flowrate Qw, at the same SS concentration as the
RAS, Xu. Effluent is discharged from the clarification
tank at a flowrate of Q—Qw/ and it contains an SS
concentration of X0.
Process Operating Relationships. In developing rela-
tionships to describe the clarification tank thickening
function, certain simplifying assumptions can be
made. First, the WAS flowrate (QJ 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:
{Q + Q,)X = QrXu
(1)
Equation 1 states that, at steady-state, the rate at
which solids are applied to the clarification tank must
be equal to the rate at which they are removed. Equa-
tion 1 can be used to develop the following relation-
ships that are useful for assessing clarification tank
operation.
1. Degree of Thickening that can be Achieved by a
Clarification Tank
= (Q + Q,)
(2)
x
2. Calculation of Required RAS Flowrate
Qr = Qx
(X..-X)
(3)
This relationship can be used to calculate the RAS
flowrate required for a specified influent flowrate, the
current aeration tank MLSS concentration, and an
RAS SS concentration that it is believed (perhaps bas-
ed on past history) can be achieved in the clarification
tank.
3. Clarifier Capacity Calculation
Q = Qr(Xu-X)
X
(4)
This relationship illustrates that the degree of thicken-
ing is a function of the influent and RAS flowrates.
This relationship can be used to calculate the capacity
of a clarification tank for a specified RAS flowrate and
an aeration tank MLSS concentration, and for an
achieveable RAS/SS concentration. Equations 1-4
assume that sufficient clarification tank area is
available to thicken the RAS to a concentration of Xu.
Techniques for determining the clarification tank area
required to achieve this are discussed in the following
section.
Sludge Thickening Theory. Thickening of activated
sludge solids in a clarification tank occurs by gravity
settling. As the solids settle, their concentration in-
creases from the MLSS concentration (X) to the RAS
concentration (Xu). Because of their flocculent nature
and concentration, activated sludge particles do not
settle individually, but as an entire mass. The solids
settle at a constant rate until they begin to "pile up"
at the bottom of the container in which they are settl-
ing. The initial settling velocity of the activated sludge
solids (V.) decreases as the initial SS 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 top of the settling sludge and the clear superna-
tant. When the height of the interface is plotted
against time, the line initially is straight but later
begins to level off (Figure 6). The slope of the initial
straight line is the initial settling velocity, V, . Sludges
with higher initial SS concentrations settle slower
than those with lower initial SS concentrations
(Figure 6).
Clarification tank thickening capacity is related to the
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 to the
bottom of the tank, and this general flow also carries
14
-------
Figure 5. Activated Sludge Process Schematic.
o>
X
Q u.
ZE
O <
o 3
Ul O
UJ
3
u.
LL
111
i!
F OC
<
cc
UJ
o
ui
O
Z
o
o
uj O
o co
CO CO
9 a
-I Ul
OQ
5 co z
•9 O
CO
X
o
6
CD
O
•S
OC
Ul
OC
o
O
Q
111
>
x"
Reference: Jenkins, eta/., 1984.
X
< CO O S2
OC < Z ;«
503 Ul"
SgSiZ
—i O 3 ui
< < co nl
^SoUfa
11 •* II II
* " 3 01
o x x x
Ul
o
Q
o o
15
-------
Figure 6. Zone Settling of Activated Sludge Solids.
HIGHER INITIAL CONCENTRATION
LOWER INITIAL CONCENTRATION
TIME
Reference: Jenkins, etal., 1984.
16
-------
the settling activated sludge solids to the bottom
where they can be removed.
The various techniques that have been developed to
determine the thickening capacity of clarification
tanks generally compare the rate at which activated
sludge solids are applied to the rate at which they are
conveyed to the bottom of the tank by settling and
bulk withdrawal. As long as the application rate is no
greater than the settling and withdrawal rate, the ap-
plied solids will be conveyed to the bottom of the tank
and removed (i.e., thickening failure will not occur). If
the solids application rate is greater, then excess
solids will accumulate in the tank until it is full and
solids will overflow into the effluent.
Techniques for clarification tank thickening analysis
are relatively complex. They account for SS concen-
tration increases in the clarification tank during settl-
ing and the effects of this on the rate at which solids
are conveyed to the bottom of the tank by both settl-
ing and bulk withdrawal. These techniques are
discussed thoroughly in the literature (Keinath et al.,
1977);, they will not be discussed in .detail here.
The thickening capacity of a clarification tank, can be
stated in terms of an allowable applied solids loading
rate (Ga), which is the mass of solids applied per unit
of clarification tank surface area.
Using the definitions presented earlier, the actual
solids loading rate is:
Ga = X(Q + Qr)/A
(5)
Comparison of the actual solids loading rate with the
allowable rate will indicate whether the clarification
tank is overloaded and whether it will experience
thickening failure.
The allowable solids loading rate is a.- function of
several factors including the RAS flowrate per unit of
clarification tank surface area (Qr/A) and the settling
characteristics of the sludge. By varying these fac-
tors, it may be possible to increase the capacity of the
clarification tank and/or of the activated sludge
system.
Clarification Tank Thickening Capacity. This
parameter can be determined by direct measurement
or by calculation using the techniques discussed
above.
Direct measurement of thickening capacity is ac-
complished by manipulating the clarification tank
solids loading until thickening failure occurs. For ex-
ample, the clarification tank influent flowrate can be
increased while the RAS flowrate is maintained cons-
tant. The clarification tank sludge blanket depth is
monitored and the point at which it begins to increase
corresponds to the point at which thickening failure is
just beginning to occur. The applied solids loading
rate under these conditions is calculated. This is the
allowable solids loading rate for the selected RAS rate
and the observed sludge settling characteristics. By
repeating this procedure at different RAS rates and
with sludges of different settling characteristics, the
clarification tank thickening capacity can be estab-
lished for a variety of conditions.
While this technique has been used in practice, it is
rather cumbersome and time-consuming, and the ef-
fluent quality may be degraded during testing.
Moreover, bulking sludge must be available to allow
clarification tank capacity to be measured for bulking
conditions. The major advantage of this method is
that the determination of allowable thickening capaci-
ty is direct and no assumptions are required to trans-
late the results to full-scale plant performance.
Calculation techniques for determining allowable
clarification tank thickening capacity require the
measurement of sludge settling characteristics. The
relationship between SS concentration and the initial
settling velocity must be established either by direct
measurement or by using correlations between an in-
dex of sludge settleability and the SS concentration/
initial settling velocity relationship developed by
others. Correlations have been developed using in-
dices such as the standard SVI, the stirred SVI con-
ducted at MLSS = 3.0 g/l, and DSVI.
Direct measurement of the SS concentration/initial
settling velocity relationship can be quite cumber-
some and time-consuming. Initial settling velocities
must be measured for sludges with various initial SS
concentrations obtained by mixing various propor-
tions of mixed liquor, RAS, and effluent. Measure-
ments must be made in settling columns, that are at
least 0.15 m (6 in.) diameter, 1.8 m (6 ft.) tall, and
equipped with a stirring device. Bulking sludge must
be available before its thickening characteristics can
be measured.
Figure 7 presents the results of a correlation tech-
nique and analysis developed by Daigger! and Roper
(1984) in which the allowable solids loading rate (Ga)
is plotted as a function of the RAS SS concentration
for sludges with SVI in the range 50 — 350 ml/g.
Dashed lines that correspond 'to various underflow
•'rates (Qr/A) also are plotted. This correlation is based
on a broad data base and should be readily useable by
plant operators because it uses the standard SVI as
the index of sludge settleability. To use this diagram,
the point that corresponds to clarification tank
operating conditions is located in the diagram. Two of
the following pieces of information are needed to
locate this point:
77
-------
Figure 7. Clarification Tank Design and Operation Diagram (after Daigger and Roper (1984)).
I I I I I I I I I I I I I I I I I I I I I I I I .I I I I I I I I I I I I I I
U? «» CO
-------
• The actual solids loading rate;
• The underflow rate; and
• The RAS SS concentration.
The third piece of information can be used as a check.
When the point plots below the allowable solids
loading line of interest, the clarification tank is
operating below its allowable thickening capacity and
should not be subject to thickening failure. When the
point falls directly on the allowable solids loading line
of interest, the clarification tank'is operating at the
failure point. When the point plots above the
allowable solids loading, thickening failure will occur.
An example of the use of this diagram is presented
later in this section.
Clarifier Analysis and Operation. The first steps in
clarification tank analysis often are to use the relation-
ships in Equations 1-4. For example, sludge bulking
generally will cause a decrease in the RAS SS concen-
tration. Equation 1 predicts that a decrease in the
RAS SS concentration (Xu) requires an increase in
RAS flowrate. This increase is necessary to ensure
that sludge applied to the clarification tank is removed
in the RAS.
For example, consider an activated sludge system that
operates at an influent flow of 1.0 MGD (0.044 m3/sec)
and an RAS flow of 0.33 MGD (0.014 rrWsec). The
MLSS concentration is 3000 mg/l, and the RAS SS
concentration is 12,000 mg/l. As a check, calculate the
required RAS flowrate using Equation 1.
Qr = QX/(X-X) = (1.0 MGD) (3,000)/( 12,000-3,000)
= 0.33 MGD (0.0143/sec)
This agrees with the actual RAS flowrate. Now
assume that the sludge settleability deteriorates and
that the clarifier sludge blanket begins to increase.
The RAS SS concentration is found to be 8,000
mg/1. Equation 1 indicates that the RAS flowrate
must be:
Qr = QX/(XU-X) = (1.0 MGD) (3,000)/(8,000-3,000)
= 0.60 MGD (0.026m3/sec) ,
Thus, for these conditions , increasing the RAS •
flowrate to 0.60 MGD should prevent clarification
tank failure. , •
The use of Equations 1-4 to develop alternative
clarification tank operating strategies must be accom-
panied by an analysis of clarification tank thickening
capacity. In this manual, the correlation method (with
SVI) of Daigger and Roper (1984) will be used; as in-
dicated previously, other somewhat less convenient
techniques are available.
Assume that the clarification tank in the example
discussed above is 45 feet (13.7m) in diameter and
has a surface area of 1,590 ft2 (148 m2). From Equa-
tion 5, the applied solids loading rate for the intitial
operating condition is:
Ga = X(Q + Qr)/A
= (3,000 mg/l)(1.0 + 0.33)MGD{8.34)/1,590 ft2
, ' = 20.9 Ib/ft2-day (102 kg/m2 day)
This condition plots as point number 1 in Figure 41
and indicates that the clarification tank could operate
successfully with a sludge having an SVI of up to
150 ml/g.
For the second operating condition (RAS, SS = 8,000
mg/l, RAS flowrate = 0.6 MGD) the applied solids
loading rate is:
(3,000 mg/l)(1.0 + 0.6)MGD(8.34)/1,590 ft2
= 25.2 Ib/ft2-day (123 kg/m2 .day)
This condition plots as point number 2 in Figure 41
and indicates that the clarification tank now can
operate successfully with a sludge having an SVI just
over 200 ml/g.
Now-assume that the maximum RAS pumping capac-
ity is ,0.95 MGD (0.042 m3/sec). At the maximum
RAS flowrate, the bulk withdrawal rate is 0.95
MGD/1,590 ft2 or 597 gpd/ft2 (24.3 m/day) and the
applied solids loading rate is:
, (3,000 mg/l)(1.0 + 0.95(MGD(8.34)/1,590 ft2
= 30.7 Ib/ft2-day (150 kg/m2-day)
This condition plots as point number 3 on Figure 41
and indicates that the clarification tank could be
operated successfully with a sludge having an SVI of
about 250 ml/g and that the RAS SS concentration
would be about 6,300 mg/l.
Now assume that the RAS flowrate is maintained at
0.95 MGD (0.042 m3/sec) (equivalent to a bulk with-
drawal rate of 597 gpd/ft2) (24.3 m/day) but that the
SVI will be controlled to a value of no more than 150
,ml/g by RAS chlorination. Moving along the operating
line (for 597 gpd/ft2) to point number 4 indicates that
the clarification tank could be operated at a .solids
loading rate of up to 42.8 Ib/ft2-day (209 kg/m2-day)
'and with an RAS SS concentration of up to 8,700
mg/l at this loading.
19
-------
Equation 5 can be arranged to calculate for these
conditions:
a. The maximum allowable influent flow rate at
MLSS = 3000mg/l
Q = (G«A/X)-ar
= (43.5 Ib/ft2-day(1,590 ft2)/(3,000 mg/l)(8.34))
-0.95 MGD
= 1.8MGD(0.79m3/sec)
b. The maximum allowable MLSS at an influent flow
of 1.0 MGD (0.079 m3/sec.)
X = (G8A)/(Q + Qr)
= (43.5 Ib/ft2-day)(1,590 ft2)
-M1.0 + 0.95)MGD(8.34)
= 4,250 mg/l
These examples illustrate how the general principles
of clarification tank analysis can be used to optimize
the existing activated sludge clarification tank opera-
tion.
System Analysis and Operation. In addition to
manipulation of RAS flowrates, the effects of bulking
sludge can be ameliorated by reducing MLSS concen-,
tration in the clarification tank feed. This reduces the
applied solids loading rate to the clarification tank
(Equation 5). Reducing applied solids loading rate
while maintaining the same RAS bulk withdrawal rate
(i.e., move downward and to the left along an RAS
bulk withdrawal operating line in Figure 8) increases
the allowable SVI.
MLSS concentration in the.clarification tank feed can
be reduced by reducing the mixed liquor solids inven-
tory and by changing the activated sludge operating
mode. Mixed liquor solids inventory can be reduced
by increasing the sludge wasting rate. This may not
be feasible if sludge handling capacity is not available
or if reducing the mixed liquor solids inventory would
lead to an unfavorable F/M (e.g., a low F/M may be re-
quired to achieve nitrification).
The objective of changing operational mode to com-
bat sludge bulking is to reduce MLSS concentration in
the clarification tank feed without reducing the rhixed '
liquor solids inventory. The step feed (step aeration)
and contact stabilization configurations are particular-
ly useful in this regard. Needless to say, they can only
be employed to (combat bulking sludge if the plant is
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.
The use of step feed to lower the solids loading ap-
plied to the clarification tank is illustrated using the
previous example. In addition to an influent flowrate';
(Q) of 1.0 MGD (0.044 m3/sec), an RAS flowrate of
(Qr) of 0.33 MGD (0.014 m3/sec), an MLSS concen-
tration (X) of 3,000 mg/l, an RAS concentration (Xu);
of 12,000 mg/l, and a clarification tank solids
loading rate of 20.9 Ib/ft2/day (102 kg/m2/day), it is;
assumed that the aeration tank has a total volume of •
0.25 MG (3780 m3) and that it can be operated in
either the plug flow (conventional) or the two-pass
step feed mode (Figure 9).
Point number 1 in Figure 10 indicates that these
operating conditions are acceptable when the SVI is
less than 1 50 ml/g.
When the operating mode is changed to two-pass
step feed at the same influent and RAS flow rates, all
of the RAS but only one-half of the,influent flbw is ad-
ded to the first pass. The remainder of the influent
flow is added to the second pass. A redistribution, of
mixed liquor solids inventory occurs so that it is.
higher in the first pass than in the second pass. This
arises because less dilution of the RAS by influent oc-
curs in the first pass than in the second pass. If ,the'
total mixed liquor inventory is maintained the same as
in the plug flow mode, then the MLSS concentration
in the second pass will be less than when the system
was operated in the plug flow mode. -''-''''['
The MLSS concentrations in each of the two passes
can be calculated for step feed by writing solids mass
balances for each pass. These equations then are sub-
stituted into an equation for the total solids inventory,
which is solved for the solids concentrations in each
pass and in the RAS. For our example, the aeration
tank MLSS inventory in the plug flow mode is:
Aeration Tank Inventory = (3,000 mg/l),
(0.25 MG)(8.34): ./
= 6255 Ib (2840 kg)-
This inventory will be maintained in the two-pass step
feed mode. ' ... ..
The solids mass balance for the point at which; the in-
fluent flow to the first pass (0.5 MGD) is mixed with
the RAS flow (0.33 MGD) is: • -; ' ;
(0.33 MGD) (Xu) = (0.5 + 0.33 MGDHX,)
and
X, = (0.33/0.833)XU =
0.4 X,, (6)
20
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Figure 8. Clarification Tank Thickening Analysis Example
(after Daigger and Roper (1984)).
3
O
cc
H
UJ
O
z
o
o
a
LU
O
z
Ul
Q.
V)
(O
<
cc
o '
ONiavon sanos
-------
Figure 9. Step Feed Operation Example Schematic
(After Daigger and Roper (1984)).
1.0 MGD
X = 3000 mg/l
SLR = 20.9 Ib/ft2-day
1.33 MGD, 3000 mg/l
0.33 MGD,XU = 12,000 mg/l
PLUG FLOW (CONVENTIONAL) MODE
1.0 MGD
0.5 MGD
0.5 MGD
X1 = 3690 mg/l .
X2 = 2310 mg/l
SLR = 16.1 Ib/ftz-day
1.33 MGD, 2310 mg/l
0.33 MGD, Xu = 9230 mg/l
STEP FEED MODE
22
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Figure 10. Step Feed Operation Example Plot of Parameters (after Daigger and Roper (1984)).
3
3
o
DC
z
111
o
z
o
o
CO
a
o
CO
Q
111
O
z
UJ
Q.
CO
CO
CO
(Aep-zu/qi) O '3JLVU ONIOVOH SQIIOS
23
-------
The solids mass balance for the point at which the
flow from the first pass (0.83 MGD) mixes with the in-
fluent flow to the second pass (0.5 MGD) is:
(0.83 MGDHX,} = (0.83 + 0.5 MGD)(X2
and
(7)
X2 = (0.83/1 .33)X, = 0.625X,
Combining Equations 6 and 7 gives:
X2 = 0.625X, = 0.625(0.4XU) = 0.25XU (8)
The total aeration tank mixed liquor solids inventory
can be expressed as a function of Xu as follows, re-
membering that the volume of each aeration tank pass
is 0.25 MG/2 or 0.1 25 MG (1890m3):
Aeration Tank Solids Inventory =
(X,)(0.1 25 MGK8.34) + (X2)(0.1 25 MGH8.34)
= 1.0425(X, + X2) (9)
Substituting for X, and X2 from Equations 6 and 8
gives:
Aeration Tank Solids Inventory =
1.0425(0.4XU + 0.25XU)
= 0.6776XU
Setting this equal to the total solids inventory of 6255
Ib gives:
6255lb = 0.6776X,,
and
Xu = 9,230 mg/l
X] can be calculated using Equation 6:
X, = 0.4XU = (0.25)(9,230 mg/l) = 3,690 mg/l
and X2 can be calculated using Equation 8:
X2 = 0.25XU= (0.25)(9,230 mg/l) = 2,210 mg/l
The clarification tank solids loading rate for this oper-
ating condition would be:
G, = (2,310mg/l)(1.33MGD)(8.34/1,590ft2
= 16.1 Ib/ft2-day (78.6 kg/m2-day) (see Figure 10)
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
10 using the data from the previously calculated ex-
amples. Points numbers 1 through 3 are for plug flow
operation with HAS flowrates of 0.33, 0.6, and 0.95
MGD (0.01, 0.026, and 0.04 m3/sec), respectively.
Point number 4 is for two-pass step feed operation at
an RAS flowrate of 0.33 MGD (0.01 m3/sec), as cal-
culated above. These operating points show that a
sludge with an SVI of approximately 220-230 ml/g
requires an RAS flowrate of 0.95 MGD when the aera-
tion tank is plug flow and 0.33 MGD (0.01 nWsec)
when the aeration tank is two-pass step feed. This re-
sults both in lower RAS pumping costs and higher
RAS, SS concentration (9230 mg/l vs. 6300 mg/l) —
which may reduce waste sludge processing and dis-
posal costs.
This example illustrates the advantage of two-pass
step feed over plug flow operation for dealing with
bulking sludge. Operation in other step feed modes
.(such as three- or four-pass), contact stabilization, or
step feed with RAS reaeration (a combination of step
feed and contact stabilization) offer similar advan-
tages. Indeed, any change which results in less dilu-
tion of RAS by influent wastewater will allow storage
of activated sludge solids in the aeration tank and a
decrease in the MLSS concentration in the clarifica-
tion tank feed. For example, if the system depicted in
Figure 42 had been placed in the contact stabilization
mode, using the first pass for sludge stabilization and
the second pass for contact (while maintaining the
same mixed liquor solids and inventory), the MLSS
concentration in the clarification tank feed would have
been reduced to 1,200 mg/l and the clarification tank
solids loading rate would be 3.4 Ib/ft2/day
(41 kg/m2/day).
These examples have illustrated calculation pro-
cedures for determining the operating conditions
when the mode of operation is changed. In some
cases a simplified calculation procedure can be
developed for a particular system operating under
defined operating conditions, or operating diagrams
can be developed for a particular system. Alternative-
ly, these concepts can be used in a plant by changing
plant operation and observing the results. The general
principle that changes resulting in less dilution of RAS
by influent wastewater will decrease the MLSS con-
centration of the clarification tank feed can be used to
guide changes in operating mode. After making
changes, their impacts on solids distribution in the
system can be monitored. In most cases redistribution
of the solids takes less than 1 day, so that the results
can be determined in a timely manner. The resulting
clarification tank solids loadings can then be deter-
mined and the need for further changes assessed.
3.4.3 Addition of chemicals to enhance settling.
The intent of this technique is to improve settling of
sludge without destroying the filamentous microorga-
nisms. Synthetic polymers, added to the mixed liquor
as it leaves the aeration tank or to the clarification tank
entry point, aid in settling of diffuse filamentous floe.
The polymer used is usually a high molecular weight,
cationic charge polymer, and may be applied alone or
24
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Table 3. Bulking Filamentous Microorganisms That Have Been
Successfully Controlled by Chlorination
Location
City of Albany, GA
Malibu Mesa, CA
City of Los Angeles
Terminal Island, CA
Carrousel Pilot Plant
Brewery Waste
City of Pacifica, CA
-City of San Jose, CA
Derry Township Municipal
Authority, Hershey, PA
Ormo Loma Sanitary
District, CA
Weyerhaeuser
Longview, WA (NaOCI)
Miller Brewing Co.
Fulton, NY
Major Filamentous Organism (s)
Causing Bulking
type 0041
type 0092
H. hydrossis
type 1701
type 1 701
type 02 1N
type 02 1N
type 0581
type 0041
type 0092
S. natans
type 1701
type 1863
H. hydrossis
type 1 701
type 021 N
type 08 12
type 0041
type 0675
Thiothrix sp
M. parvicella
H. hydrossis
type 1 701
type 0675
type 1851
type 0041
type 0675
Stroh Brewing Co.
Longview, TX
(NaOCI to Aeration Basin)
Champion International
(Pulp and Paper)
Courtland, AL
Monterey Regional Water
Pollution Control Plant
Monterey, CA
City of Redding, CA
Westchester County
Yonkers, NY
Thames Water Authority
(UK) (NaOCI)
type 1851
type 021N
Nostocoida limicola II
Thiothrix sp
type 1701
Nostocoida limicola II
type 1701
Thiothrix sp
type 1701
S. natans
type 1701
Nostocoida limicola
type 021N
Central Contra Costa
Sanitary District
Concord, CA
City of Pacifica, CA
type 02 1N
M. parvicella
Thiothrix sp
S. natans
type 1701
in combination with an anionic polymer. In some
cases, lime, ferric chloride, or other inorganic precipi-
tants have been added to systems, producing a heavy
precipitate that forces the sludge floe to settle. This
method greatly increases the solids load the system
must bear.
Because overdosing with polymers can degrade
system performance, careful testing must be done to
estimate appropriate doses.
The use of polymers is an expensive method of bulk-
ing control. Chemical costs of up to $450/MG have
been reported.
3.4.4 Addition of chemicals to selectively kill
filamentous microorganisms.
Two main chemicals —chlorine and hydrogen
peroxide —have been successfully used in the selec-
tive destruction of filamentous microorganisms.
3.4.4.1 Chlorine.
The use of chlorine is popular in part because it is fre-
quently used in effluent disinfection, and is thus
available at most activated sludge plants. Chlorine
may be added at three points in the system: directly
into the aeration tank; into a "loop" in which mixed
liquor is withdrawn from the aeration tank, chlorin-
ated, and returned to the tank; or into the return ac-
tivated sludge (RAS) stream. The last is the most
common and preferred choice, except in plants where
aeration time is very long or where the RAS line is in-
accessible.
The following guidelines must be followed for suc-
cessful use of chlorination:
1) A target value of SVI or some other floe parameter
must be set.
2) Chlorination must be used only when the target
value is regularly exceeded; a careful, frequent plot of
the parameter should be made to determine whether
it consistently exceeds the target value.
3) Chlorine doses must be known and carefully con-
trolled, and must be added to the sludge at a point
where it will mix efficiently. If it' does not mix well,
parts of the sludge will overdose while parts will not
be chlorinated at all. Poor mixing will cause turbid ef-
fluents, reduction in treatment efficiency due to killing
of floe-forming organisms, and consumption of large
amounts of chlorine without control of the bulking
problem.
4) Chlorine should be added where chlorine demand is
at minimum—that is, where the amount of organic
matter is lowest. Chlorine reacts quickly with am-
monia and organic matter, and becomes unavailable
for killing filamentous microorganisms. It should not
25
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be added to influent wastewater, or to RAS after it
has begun to mix with influent waste just outside the
aeration tank.
5) Appropriate parameters must be considered in
selecting a dosing point. These are:
Overall Mass Dose
Expressed in
Local Mass Dose
Expressed in
Frequency of Exposure
kgCI,
per day
103kgSS
kgCI2
103 kg SS per day
day ~1 (times per day
sludge passes point
of dose)
Overall Mass Dose is based on the plant's total sludge
inventory and tells how much chlorine must be added
per day. However, it does not indicate how often and
in what concentration the chlorine should be applied
for a given application point in the system. The other
three parameters provide such information.
The local mass dose indicates the quantity of chlorine
which must be added at a dosing point in order to
control the filamentous microorganisms. The dose
concentration is the concentration of chlorine to be'
added at the dosing point to achieve the local mass
Table 4. Usa of Hydrogen Peroxide for Bulking Control
dose. However, the dose concentration must be low
enough so that a small part of the sludge inventory is
not exposed to excessively high chlorine doses. In
such cases, the exposed sludge floe can be complete-
ly destroyed while the bulk of the solids inventory is
unaffected. Also, the frequency of exposure of the
sludge inventory to the chlorine dose must be high
enough to control the filamentous microorganisms
(generally greater than once per day). If proper control
of filamentous microorganisms cannot be achieved
because of insufficient frequency of exposure or
because the local mass dose cannot be achieved at a
reasonable dose concentration, then a change in the
dosing point or the addition of a dosing point(s) may
be necessary.
6) Reliable control tests must be performed to assess
the effectiveness of chlorination. These include a
measure of sludge settling rate (such as SVI); a
measure of final effluent turbidity (such as a tur-
bidimeter or a Secchi disc); and microscopic examina-
tion of the sludge, to observe progressive deforma-
tion of filamentous microorganisms or to detect signs
of overdosing (such as total elimination of filamen-
tous microorganisms and presence of broken floes).
The progressive effects of chlorination on type 1701
and Thiothrix spp. are illustrated in Figure 11. Types
of filamentous microrganism successfully controlled
by chlorination appear in Table 3.
Location
PETALUMA
full-scale plant
Reference
ANON, FMC Corp. (1973)
Caropreso eta/. (1974)
Successful
concentration, mg/l
9-68
average 31.5
ST. AUGUSTINE
full-scale plant
SAN JOSE
lab scale,
fill and draw
PRINCETON
small full-scale
WILMINGTON
lab-scale
WASHINGTON
pilot plant
TEXTILE MILL
ANON, FMC Corp. (1976)
Strunk and Shapiro (1976)
Caropreso eta/. (1974)
Coleefa/. (1973)
Cole eta/. (1973)
Keller and Cole (1973)
12 (basis unclear)
40-200
100b
40-200b
20-40"
200-4003
60 (basis unclear)
•Based on Wastewater inflow.
"Based on volume of aeration basin and clarifier (single dose).
26
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Figure 11. Progressive effects of chlorination on type 1701 (a, b, and c) and Thiothrix sp. (d, e, and fl: a. and d. no chlorination; b. and e. moderate
chlorination effects; and c. and f. severe chlorination effects (all 1000X phase contrast).
^«**".
Reference: Jenkins ef at.
27
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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 to "the 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 H2O2 as it is with chlorine.
Somewhat higher doses and longer contact times, may
be needed.
It is possible that as H2O2 kills filamentous
microorganisms, it also releases oxygen. If bulking was
a result of low DO, this could provide additional im-
provement in operations. However, if sludge oxidizes
H2O2 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
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Chapter 4
4.0 Activated Sludge Foaming
Several different types of foam have been observed in
activated sludge treatment systems. Some are normal
and harmless, while others are signs of operating
problems and need to be controlled.
4.1 Symptoms of Sludge Foaming
At the startup of an activated sludge plant, while
solids concentrations and Sludge Age are low, white
froth usually occurs. As the process stabilizes and
solids build up, this type of foam usually disappears. If
excessive, sudsy white foam persists, the Sludge Age
may be too low, or there may be nonbiodegradable
substances in the waste (industrial wastes or
cleaners). A more troublesome foam, heavy, viscous,
and brown, can appear on the surface of both aera-
tion and clarification tanks, degrading the effluent and
spilling over onto walkways. While, this type of foam
has been attributed to a high Sludge Age, filamentous
microorganisms may also be involved in its produc-
tion.
4.2 Role of Filamentous Microorganisms in
Sludge Foaming
Analysis of 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 floes hydrophobic. Thus the
floe 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 the waste-
water, low F/M ratio, and high SS levels.
4.4 Control of Activated Sludge Foaming
Because the mechanisms of this phenomenon are as
yet so little understood, there is no single proven
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 the
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 the 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 Age by increasing the
sludge wasting rate, thus washing out the Nocardia,
and concurrently increasing the F/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 Age required for nitrification
makes Nocardia washout impossible by 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 foam does not have a good record of
success.
However, a combination of physical removal,
chlorination, and addition of ferric chloride has been
29
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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. If such foam is recycled
through the system, it will reseed the sludge. When
the contents of scurn traps are fed back to gravity
thickeners or the HAS, while wasting continues to
remove the mixed liquor and sludge from which the
scum separated, Nocardia becomes concentrated in
the system.
30
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Figure 12. Nocardia foaming in activated sludge: a. and b. foam on the aeration basin; c. and d. microscopic appearance of Nocardia foam
(c. 400x phase contrast; bar = 25 jun; d. 1000x phase contrast; bar= lOyttm).
31
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Chapter 5
5.0 Appendix 1 —Methods for Microscopic
Examination of Filamentous
Microorganisms in Activated Sludge
Microscopic examination of activated sludge is an im-
portant tool for the identification of sludge settling
problems. Determining the number and type of fila-
mentous microorganisms is the first step in controlling
bulking and foaming.
Such examination requires a research-grade phase
contrast microscope with 100x and 900-1000 X
phase contrast objectives. A mechanical stage is es-
sential for controlled scanning. A binocular head (or
trinocular head if photography is desired) and built-in
light source are highly recommended. An ocular
micrometer should be inserted into one of the eye-
.pieces and calibrated at each magnification employed
using a stage micrometer.
A photographic record of sample appearance at 100 X
is recommended for reference purposes. A 35mm
camera mounted on the trinocular head is most con-
venient. The camera should have a built-in light meter
and a replaceable, fine ground-glass focusing screen.
Polaroid instant film cameras can be used. It is con-
venient and highly efficient to take all photographs
using color slide film (e.g., Kodak tungsten-balanced
type II professional film designated KPA), and produce
color or black-and-white prints from the slides using a
slide printer employing Polaroid instant film.
Because many of the observations to be made in these
procedures are close to the limit of resolution of the
light microscope, and many of the features to be
studied are difficult to detect, the microscope must be
in good condition. Regular adjustment of the phase
rings is required, the microscope objectives must be
kept clean, and a dust-free environment is necessary.
Professional servicing of the microscope by the manu-
facturer or recommended agent should be performed
at least once per year.
5.1 Sampling Methods
Samples of activated sludge mixed liquor should be
taken from the effluent end of the aeration tank, or
from the mixed liquor channel between the aeration
tank and the clarification tank. Mixed liquor samples
should be taken below the surface, excluding any
foam or other floating material. If the activated sludge
is foaming, a separate foam sample should be taken
from the surface of the effluent end of the aeration
tank, from the surface of the mixed liquor channel, or
from the surface of the clarification tank. Subsurface
liquid should be excluded from foam samples. Al-
though foams can be thick and viscous and difficult to
transfer to sample bottles, they should not be diluted
to ease sampling, because this will prevent compari-
son of the relative abundance of filamentous micro-
organisms in the foam and the mixed liquor.
Some activated sludge process modifications contain
more than one tank, e.g., step-feed, contact stabiliza-
tion, or "plug-flow" systems. In such systems, char-
acteristics of the sludge in all tanks are usually similar.
Thus it is usually not necessary to sample all tanks,
only the effluent end of the aeration tank. Where ah
activated sludge plant consists of separate and paral-
lel systems, there may be differences in floe and fila-
mentous microorganism characteristics in each sys-
tem, especially if there is not complete admixture of
the return activated sludge (RAS) streams. In this situ-
ation, a sample is required from each system. Simi-
larly, for two-stage activated sludge systems (e.g.,
"carbonaceous" first-stage followed by "nitrifica-
tion" second-stage), a sample from the aeration tank
of each stage is necessary for proper characterization
of floe structure and filamentous microorganism pop-
ulations. Some activated sludge systems treat a
waste that has been pretreated in another type of bio-
logical treatment system (lagoon or trickling filter).
These units may seed the activated sludge with fila-
mentous microorganisms. To determine the extent of
this seeding, a sample should be taken of the waste
entering the activated sludge system.
Sampling and examination frequency will be dictated
by plant circumstances and by the location of exami-
nation of samples. During critical periods (when bulk-
ing is occurring or is anticipated, during the use of
RAS chlorination for bulking control and during
periods of experimental operation), daily onsite exami-
nation can be justified. Routine onsite examination
may be performed once or twice per week. Offsite
33
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examination may be performed weekly, monthly, or
seasonally, depending on the severity of problems
being encountered or the desire to establish an operat-
ing history (and on the budget available for this
activity).
Samples should be examined as soon as possible after
they are taken. When examination is performed on-
site, samples not examined within several hours
should be stored at 4°C in a refrigerator. When
samples must be transported offsite for analysis, they
should be sent in sample containers in which there is
an air space at least equal to the volume of the sample,
to avoid septicity. (Five to 10 ml are needed for typical
microscopic examination, so a 20-25 ml vial will be
adequate.) The sample should be neither chemically
preserved nor frozen, since these procedures can alter
the characteristics of the floes and filamentous micro-
organisms. The longer the time that elapses between
sampling and examination, the more difficult and un-
certain sample examination and data interpretation
become. Samples from plants with low organic load-
ings (long Sludge Ages) maintain their characteristics
longer than samples from plants with high organic
loadings (short Sludge Ages). For long Sludge Age
samples, a satisfactory examination can be obtained if
the sample is looked at within 7 to 10 days of sam-
pling; for sludges from highly loaded plants, it is wise
to examine the activated sludge within 3 to 4 days of
its sampling. With the availability of express mail and
overnight delivery services, transit time should pose
no problem in offsite analysis.
Filamentous microorganism staining reactions can be
quite sensitive to prolonged sample storage. If much
time is expected to pass between sampling and exam-
ination, two air-dried smears on microscope slides
should be prepared at the time of sampling (using
techniques outlined below) and sent together with the
sample. In this way, the original characteristics of the
activated sludge will be preserved for conducting
Gram and Neisser stains. The slides should be marked
with sample identification, date, and G or N (Gram or
Neisser), on the side of the slide containing the smear.
The same procedure should be followed if samples
cannot be analyzed immediately upon receipt.
5.2 Staining Procedures
Two primary staining procedures are used in the
examination of activated sludge samples—the Gram
stain and the Neisser stain. Other procedures with
more specialized uses are the "S" test, for sulfur oxi-
dation to detect intracellular sulfur granules; India Ink
reverse staining, to detect the presence of large
amounts of extracellular polymers; PHB staining, to
detect the presence of intracellular storage products
such as polyhydroxybutyrate (PHB); and staining with
crystal violet, to allow examination of sheaths. The
procedures are discussed in the following subsections.
34
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5.2.1 Gram stain, modified Hucker method.
Preparation:
Solution 1: Prepare the following separately, then combine:
A B
Crystal Violet
Ethanol, 95%
2g
20ml
Ammonium oxalate 0.8 g
Distilled water 80ml
Solution 2:
Iodine
Potassium iodide
Distilled water
Solution 3:
1 g
2g
3000 ml
Safranin O (2.5 percent in 95 percent ethanol) 10 ml
Distilled water 100 ml
Procedure:
1. Prepare thin smears on microscope slides and thoroughly air dry (do not heat fix).
2. Stain 1 min. with Solution 1; rinse 1 sec. with water.
3. Stain 1 min. with Solution 2; rinse well with water.
4. Hold slide at an angle and decolorize with 95 percent ethanol added drop by drop to the smear for 25 sec. Do
not over decolorize. Blot dry.
5. Stain with Solution 3 for 1 min.; rinse well with water and blot dry.
6. Examine under oil immersion at 1000 X magnification with direct illumination (not phase contrast): blue-violet
is positive; red is negative.
35
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5.2.2 Neisser stain.
Preparation:
Solution 1:
Separately prepare and store the following:
A
B
Methlylene Blue
Ethanol, 95%
Acetic acid, glacial
Distilled water
0.1 g Crystal Violet (10 percent w/v in 95%
5 ml ethanol) 3.3 ml
5ml Ethanol, 95% 6.7ml
100 ml Distilled water 100 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) 33.3 ml
Distilled water 66.7 ml
Procedure:
1. Prepare thin smears on microscope slides and thoroughly air dry. Do not heat fix.
2. Stain 30 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 1000X magnification with direct illumination (not phase contrast): blue-violet
is positive (either entire cell or intracellular granules); yellow-brown is negative.
36
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5.2.3 "S" test (sulfur oxidation)
Test A (modified from Eikelboom 1975)
Solution: Sodium sulfide solution (Na2S-9H2O) 1.0 g/l (prepare weekly)
Procedure:
1. On a microscope slide mix 1 drop of activated sludge sample and 1 drop sodium sulfide solution.
2. Allow to stand open to the air 10-20 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 X using phase contrast. A positive S 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). An alternative sulfur oxidation
test, developed by Nielsen (1984), may be used:
Test B (modified from Neilsen 1984)
Solution: Sodium thiosulfate (Na2S2O3-5H2O) 1 g/100 ml
Procedure:
1. Allow activated sludge sample to settle, and transfer 20 ml of clear supernatant to a 100 ml Erlenmeyer flask.
2. Add 1 -2 ml of activated sludge to the flask.
3. Add 1 ml of thiosulfate solution to the flask (final thiosulfate concentration is 2mM).
4. Shake the flask overnight at room temperature.
5. Observe at 1000X phase contrast, as above.
37
-------
5.2.4 India Ink reverse stain.
Solution: India ink (aqueous suspension of carbon
black particles)
Procedure:
1. Mix one drop of India ink and one drop of activated
sludge sample on a microscope slide. Depending
on the ink used, the sample volume may need to be
reduced.
2. Place on the cover slip and observe at 1000 X using
phase contrast.
3. In "normal" activated sludge, the India ink par-
ticles penetrate the floes almost completely, at
most leaving a clear center.
4. In activated sludge containing large amounts of
exocellular polymeric material, there will be large,
clear areas containing a low density of cells.
5.2.5 PHB (polyhydroxybutyrate) stain.
Solution 1: Sudan Black B (IV), 0.3 percent w/v in 60
percent ethanol
Solution 2: Safranin 0 0.5 percent w/v aqueous
Procedure:
1. Prepare thin smears on a microscope slide and
thoroughly air dry-
2. Stain 10 min. with Solution 1; add more stain if the
slide starts to dry out.
3. Rinse 1 sec. with water.
4. Stain 10 sec. with Solution 2; rinse well with
water; blot dry.
5. Examine under oil immersion at 1000 x magnifica-
tion with transmitted light: PHB granules will ap-
pear as intracellular, blue-black granules while
cytoplasm will be pink or clear.
5.2.6 Crystal Violet sheath stain.
Solution: Crystal Violet, 0.1 percent w/v acqueous
solution
Procedure:
1. Mix 1 drop activated sludge sample and 1 drop
Crystal Violet solution on a microscope slide,
cover and examine at 1000 x magnification phase
contrast. Cells stain deep violet while the sheaths
are clear to pink.
5.3 Examination Procedures
Upon sample receipt, or at least within several hours
prior to sample examination, spread 1 drop of the
sample evenly over approximately 50 percent of the
area of each of two 25 X 77 mm microscope slides.
Allow these slides to air dry at room temperature (do
not heat fix). The slides can be stored and stained
later. Mark the slides with a sample identification code
and a G or N (Gram or Neisser) on the side on which
the smear has been made (frosted end slides are rec-
ommended). Perform the Gram and Neisser staining
procedures (5.2.1 and 5.2.2).
Withdraw one drop (approximately 0.05 ml) of sample
with a loop or a clean, disposable Pasteur pipette and
place on a 25 X 75 mm microscope slide. Place a
22 mm No. 1 cover slip on the drop, and press down
gently on the cover slip with a blunt object. Remove
the liquid that is expelled from the sides of the cover
slip with a tissue. This procedure ensures a thin prepa-
ration, which is necessary because of the limited
depth of focus of the microscope optical system.
Examine the wet mount under phase contrast at 1 00 X
magnification for the following characteristics:
1 . The general size and shape of floes present;
measure approximately 10-20 floes and place
them in the following categories:
a. Size range: small ^
medium
large ^.
1 50 m diameter
150-500 pm diameter
500 /xm diameter
b. Shape: rounded and compact, or irregular and
diffuse; note whether texture is firm or weak
(Figure 13a and 13b).
2. The presence of protozoa, organic or inorganic
particles, and fingered or amorphous zoogleal
organisms (Figure ~13c and 13d).
3. The presence of free (dispersed) cells in the bulk
solution (Figure 13b). Samples containing signifi-
cant amounts of dispersed cells will appear turbid.
4. The presence and effect of filamentous micro-
organisms on floe structure, as follows:
a. None
b. Bridging— filaments extend from the floe sur-
face into the bulk solution, and bridge between
the floes (Figures 14a and 14b).
c. Open floe structure— the floe population at-
taches to and grows around filamentous micro-
organisms, leading to a large, irregularly
shaped floe with substantial internal voids
(Figures 14c and 14d).
38
-------
Figure 13. Floe "texture" in activated sludge (a and fa): 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 lOOx phase contrast; bar= 100/xm).
«&fet*;j" "
rlFl*?1"*"-
-•-•- ,<*•:_j^- . \.^_:-:*iym^«-*sf-f¥~^
.. - __£ff-f-;.f*^jf,t^'A~-,-_^f$pf>!^~±-,.~: ':.'... -
y^-Sx'-S^^^" ~ "" "* mftfto^ %'J"i!L:'i,f Mb-ir
Reference: Jenkins, et.a/.
39
-------
Figure 14. Effect of filamentous microorganisms in activated sludge on floe morphology and settleability: a. and b. inter-floe bridging; c.
and d. diffuse floe structure (all lOOx phase contrast; bar= 100 /xm).
W&SK^&W
Reference; Jenkins, et.al.
40
-------
Table 5. Subjective Scoring of Filament Abundance8
Numerical
Value
Abundance
Explanation
0
1
2
3
4
5
6
none
few
some
common
very common
abundant
excessive
Filaments present but only observed in an occasional floe
Filaments commonly observed, but not present in all floes
Filaments observed in all floes, but at low density (e.g., 1-5 filaments per
floe)
Filaments observed in all floes at medium density (e.g., 5-20 per floe)
Filaments observed in all floes at high density (e.g., 20 per floe)
Filaments present in all floes — appears more filaments than floe and/or
filaments growing in high abundance in bulk solution
This scale from 0 to 6 represents a 100 to 1,000-fold range of total extended filament length.
5. The abundance of filamentous microorganisms.
This can be quantified by several methods. For the
purpose of the type of analysis required here, a
subjective scoring system is used. Filamentous
microorganisms are observed at 100 x and subjec-
tively rated for overall abundance on a scale from 0
(none) to 6 (excessive) (Table 5). An abundance
rating is determined for the sample as a whole (all
filament types together) and for each filamentous
microorganism observed. Individual filamentous
microorganisms are considered dominant (and
probably most responsible for bulking problems) if
they are scored "very common" 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 ± one abundance category be-
tween observers. Examples of filament abundance
categories are shown in Figure 15.
Other methods of microorganism counting are de-
scribed below.
5.4 Counting Procedures
In addition to the subjective scoring method for count-
ing filamentous microorganisms, other more detailed
procedures exist for measuring the total extended fila-
ment length (TEFL) in activated sludge.
5.4.1 Filament measurement technique of Sezgin
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)
to 1 liter of distilled water in a 1.5-liter beaker and
stir at 95 rpm on a jar test apparatus (G = 85sec~1)
for 1 min.
2. Using the same pipette, transfer 1.0 ml diluted
sample to a microscopic counting chamber cali-
brated to contain 1.0 ml and cover with a glass
cover slip.
3, Using a binocular microscope at 100 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 10 pm, 10 to 25
/j,m, 25 to 50 j«m, 50 to 100 ^m, 100 to 200 fj.m,
200 to 400 pm, 400 to 800 //.m, and greater than
800 fj,m. Measure filaments of length greater than
800 /*m individually.
4. Express results as the total /*m of filament length
per g or ml of MLSS:
total extended filament length (TEFL) /ttm/gMLSS
= total filament length, n
-------
Figure 15. Filament abundance categories using subjective scoring system: a. few; b. some; c. common; d. very common; e. abundant;
and f. excessive (all 100x phase contrast; bar= 100 ywi).
Reference: Jenkins, of.a/.
42
-------
total extended filament length (TEFL) ^m/ml
MLSS = total filament length, m, in the 1.0 ml
diluted sample X the dilution factor ( X 500 in the
above example).
5.4.2 Diluted SVI procedure of Stobbe (1984).
Several researchers have found close correlations be-
tween the number of filamentous microorganisms in
sludge and a parameter known as Diluted Sludge
Volume Index (DSVI). The DSVI can be used to predict
sludge settling properties and is well correlated to
TEFL.
1. Set up several 1 -liter graduated cylinders (the
number will depend on prior knowledge of the set-
tleability of the sludge).
2. Using well-clarified secondary effluent, prepare a
series of two-fold dilutions of the activated sludge
(i.e., no dilution, 1:1 dilution; 1:3 dilution).
3. Stir the graduate cylinders individually for 30-60
sec. using a plunger to resuspend and uniformly
distribute the sludge solids.
4. Allow the activated sludge to settle for 30 min.
under quiescent conditions.
5. Observe the settled sludge volume (SV30) in the
graduated cylinder where the settled volume is
less than and closest to 200 ml (SV30< 200 ml).
6. Calculate the diluted SVI using:
SS (g/l)
where n is the number of two-fold dilutions re-
quired to obtain a settled sludge volume (SV30) less
than 200 ml and SS is the suspended solids con-
centration of the undiluted activated sludge.
4. The eyepiece is fitted with a single hairline. Count
the number of times that any filamentous micro-
organism intersects the hairline.
5. Total the number of intersections for all fields
examined. This is the Filament Count. If a "unit
count" is desired, the Filament Count must be
multiplied by the number of fields in the 22 mm
width of the slide. At San Jose this is 12 fields.
Thus
Filament Count/^l = -
(filament intersections in)
field counted
50/tl
-X12
SJ/SCWPCP has correlated filament count with SVI.
However, filament count cannot provide an early
warning for problems caused by increased SVI, be-
cause the two are temporally correlated.
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 50 /xl mixed liquor sample to a glass slide.
2. Cover completely with a 22 X 30 mm cover slip.
3. Using 100 X total magnification and starting at the
edge of the cover slip, observe consecutive fields
across the entire 30 mm length of the cover slip.
At SJ/SCWPCP this is 17 fields.
43
-------
-------
Chapter 6
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 100x using phase contrast to
ascertain the number of different types of filamentous
microorganisms present. Carefully characterize each
filamentous microorganism present by looking at
several filaments of each type and expressing the re-
sults as an "average." Use count sheets such as the
ones presented in Table 6 to 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 tri-
chomes (Figure 16a, 16b, and 16c). In activated
sludge the only branched trichome-forming
microorganisms are fungi and Nocardia spp., and
Nostocoida limicola (observed incidentally).
False branching occurs when there is no 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 1701.
2. Motility— none, or, if present, describe.
Only a few filamentous microorganisms in acti-
vated sludge are motile. Beggiatoa spp., Flexi-
bacter spp., and some blue-green bacteria
(Cyanophyceae) are motile by gliding; Thiothrix
spp. and type 021N may display limited "twitch-
ing" or swaying motions.
3. Filament shape —straight, smoothly-curved,
bent, irregularly-shaped "chain of cells," coiled,
or mycelial (Figure 17).
4. Co/or—transparent, medium, dark (Figure 18a,
18b, and 18c).
5. Location—extending from flbc surface, found
mostly within the floe, or free in the liquid be-
tween floes (Figure 18d, 18e and 18f).
6. Attached growth of epiphytic unicellular bacteria
— present or absent; if present, whether substan-
tial growth or incidental (Figure 19).
7. 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. In
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 "halo" 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 the
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 021N.
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 20f).
8. Cross-walls (cell septa) — present, absent.
This feature can be variable for some filamentous
microorganisms, and detection is dependent on
the quality and adjustment of the microscope. It
45
-------
Table 6. Suggested Format for Filamentous Microorganism Identification Worksheet
No.
. Sample.
SAMPLE DATE.
FILAMENT ABUNDANCE
0
None
1
Few
FILAMENT EFFECT ON FLOC STRUCTURE:
MORPHOLOGY OF FLOC: | | Firm
| | Weak
FLOC DIAMETER ( m)
OBSERVATION DATE.
23456
Some Common Very Abundant Excessive
Common
Little or None
Bridging
Open Floe Structure
| | Round, Compact
Irregular, Diffuse
FEATURES:
150 150-500
500
Free cells in suspension.
Zoogloea's
Inorganic/Organic Particles
FILAMENTOUS MICROORGANISM SUMMARY:
Nocardia sp.
type 1701
S. natans
type 021 N
Thtothrixsp.
type 0041
H. hydrossis
N. limicola
Rank
Abundance
M. parvicella
type 0581
type 0092
type 0803
type 1851
type 0961
other
other
Rank
Abundance
REMARKS:
x = Dominant
0 = Secondary
Reference: Jenkins, eta/., 1984.
46
-------
Table 6 (continued).
No.
. Sample .
COMMENTS:
OBSERVATION OF:
Protozoa
Metazoa:
WET MOUNT OBSERVATION, 1000X, 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, 1000X
GRAM
NEISSER
I.D.
Reference: Jenkins, eta/., 1984.
47
-------
Figure 16. Trichome branching observed for filamentous microorganisms in activated sludge: a., b. and c. true branching (fungus,
Nocardta sp., and Nostocoida limicola II respectively); d. false-branching (Sphaerotilus natans) (all 1000X phase contrast;
Reference: Jenkins, of,a/.
48
-------
Figure 17. Examples of filament shapes: a. straight; b. smoothly-curved; c. bent; d. irregularly-shaped "chain of cells"; e. coiled; and
f. mycelial (all 1000 x phase contrast).
'Y !
i Y \v
;. •-» M
a " - * - %. i
A
\
;
h
Reference: Jenkins, et.al.
49
-------
Figure 18, "Color" of filamentous microorganisms (a, b and c): a. transparent; b. medium; and c. dark. Location of filaments in activated
sludge (d, eand fl: d. extending from floe surface; e. most within the floe; and f. free (all 1000 x phase contrast).
Roforonco: Jenkins, eta/., 1984.
50
-------
Figure 19. Attached growth of epiphytic bacteria on filamentous microorganisms: a., b. and c. substantial (types, 0041, 0675 and 1701
respectively); d. incidental (type 1851) (all 1000x contrast).
Reference: Jenkins, eta/., 1984.
-------
Ffgwo 20. Appearance of sheaths: a. Sphaerotilus natans; b. type 1701; c. Thiothrix II; d. Thiothrix I; e. type 0041; and f. type 1701,
stained with crystal violet (all 1000x phase contrast).
Roforenco: Jenkins, era/., 1984.
52
-------
Figure 21. Cell shapes observed for filamentous microorganisms in activated sludge: a. square; b. rectangular; c. oval; d. barrel;
e. discoid; and f. round-ended rods (all 1000x phase contrast).
Reference: Jenkins, eta/., 1984.
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).
9. Filament diameter—Both the average diameter
and its range in fim should be measured; it is im-
portant to note whether the diameter is greater
than 1 pm or less than 1 /mi.
10. Filament length—range in fim.
11. Cell shape—square, rectangular, oval, barrel,
discoid, round-ended rods (Figure 21).
It is important to note whether there are indenta-
tions at cell septa (Figure 21c, 21d, 21e, and
21 f) or whether trichome walls are straight at the
cell junctions (Figure 21 a and 21 b).
12. Size—average length and width of cells in /j.m.
13. Sulfur deposits—present or absent in situ and
present or absent after the S test (Section 5.2.3)
(Figures 22a 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 22c, 22d, and 22e); or in the shape of
squares for type 0914 (Figure 22f). Type 0914
does not respond to the S 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-
organism present is separately evaluated for
Gram staining and Neisser staining reaction by
observing the stained smears at 900-1000 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 (e.g., type 0092 appears much wider
when Neisser stained than in wet mounts).
The Gram stain requires much practice. Reagents
should be reasonably fresh (3-6 months) and, if
possible, should be tested on fresh cultures of
known Gram reaction. The decolorization step
should be controlled precisely to avoid over-
decolorization. Also, large floes do not decolorize
fully, so the Gram reactions inside large floes
should be ignored.
Score the Gram reaction as positive, negative, or
variable. Most filamentous microorganisms ob-
served in activated sludge are Gram negative
(Figure 23a and 23b). Nostocoida limicola and
types 0041 and 0675 most often are Gram posi-
tive but can be Gram variable or Gram negative
(Figure 23c). Type 1851 .stains weakly Gram
positive, and generally is observed as a chain of
Gram positive "beads" (Figure 23d). Thiothrix\,
Beggiatoa spp., type 021N, and type 0914
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 23e and 23f).
Neisser staining is a straightforward technique.
Score as negative, positive (entire trichome is
stained), or negative with Neisser-positive
granules (Figure 24).
Type 0092 (light purple-Figure 24c) and N.
limicola (dark purple —Figure 24d) stain entirely
Neisser-positive. M. parvicella and Nocardia spp.
stain Neisser-negative but generally contain
Neisser-positive intracellular granules (Figure
24e and 24f). Beggiatoa spp., Thiothrixspp., and
types 0041, 0675, 021N, 0914 and 1863 may
contain Neisser-positive granules (infrequently).
In addition, H. hydrossis and types 0675 and
0041 may have a Neisser-positive trichome
"covering" (Figure 24b) when present in acti-
vated sludge that is nutrient-deficient.
16. Additional observations —Two filamentous
microorganisms, Thiothrix spp. and type 021N
(uncommonly) may display rosettes and gonidia.
A rosette develops when 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 25, 26,
and 27).
The following observations may indicate a
nutrient deficiency (e.g., N and/or P) in activated
sludge systems:
• Large amounts of intracellular PHB granules;
• Unusual Neisser-staining reactions of some
filamentous microorganisms (see above); and
• Large amounts of extracellular material in the
floes. This is detected by conducting the
India ink negative staining procedure (Section
5.2.4). When observed at 100x phase con-
trast, the Indian ink particles normally can be
seen to penetrate deeply into the floes, leav-
ing only a clear center; when there are large,
54
-------
Figure 22. Deposition of intracellular sulfur granules during the S test (a and fa): before (a) and after (fa) adding sodium sulfide. Appearance
of intracellular sulfur granules in filamentous microorganisms (c, d, e and fl: c. Thiothrix I; d. Thiothrix II; e. type 021IM; and f.
type 0914 (all 1000 x phase contrast).
1 •'*«
•x-:
Nfei
^jLfflSf^^;.* f^!,^ '.^.c!t'i 'fri, •'
%
Reference: Jenkins, eta/., 1984.
55
-------
Figure 23. Gram stain reaction of filamentous microorganisms: a. and b. Gram negative (types 021N and 0092 respectively); c. Gram
variable (type 0041); d. weakly Gram positive (type 1851); and e. and f. Gram positive (Microthrix parvicella and Nocardia spp.
respectively) (all 1000X transmitted light).
Reference: Jenkins, eta/., 1984.
56
-------
Figure 24. Neisser staining reaction of filamentous microorganisms: a. negative; ft. Neisser positive trichome covering observed atypically
for type 0041; c. and d. Neisser positive (type 0092 and Nostocoida limicola II respectively); and e. and f. Neisser positive
granules (Microthrix parvicella and Nocardiaspp. respectively) (all 1000 x transmitted light).
-i!
Reference: Jenkins, eta/., 1984.
57
-------
Figure 25. Thtothrlx 11 (a and b 100x phase contrast; bar= 100pm; c-f 1000x phase contrast; bar=
Reference: Jenkins, eta/., 1984.
58
-------
Figure 26. Type 021N (a 100x phase contrast; bar= 100 ju,m; b-f lOOOx phase contrast; bar= 10/im).
- •• -I _ ' jt - .-^e'-™
"' . ; ,^'- ^ ^^
«*
Reference: Jenkins, eta/., 1984.
-------
FIguro27. Thtothrlx\ (a 100x phase contrast; bar= 100jtm; 6-MOOOx phase contrast; bar-
Reference: Jenkins, eta/., 1984.
60
-------
clear areas with low cell density, the pres-
ence of large amounts of extracellular mate-
rial (probably polysaccharide) is indicated.
Additional clues pointing to the presence of large
amounts of this highly water-retentive extracellu-
lar material are: the activated sludge settles and
compacts poorly even though filamentous micro-
organism abundance is low; the activated sludge
is slippery or slimy to the touch and appears
viscous when poured.
6.2 Identification of Microorganisms
Observed and recorded filament characteristics are
used to characterize the filamentous microorganisms
by genus or by a numbered type using the dichoto-
mous key shown in Figure 28. This key lists the 22
filamentous bacteria most commonly observed in acti-
vated sludge. To simplify this key, several filamentous
microorganisms having readily identifiable specific
characteristics are not listed, but are described later.
These include: fungi, Cyanophyceae, Flexibacter
spp., and Bacillus spp. In .addition, some filamentous
microorganisms observed only occasionally in acti-
vated sludge are not included in the key. These include
filament types 1702, 1852, and 0211.
This dichotomous key is a modification of the fila-
mentous microorganism identification key givep by
Eikelboom and van Buijsen (1981), with changes.to:
• Deemphasize the need for observation of cell
septa (crosswalls), which can depend on the qual-
ity and adjustment of the microscope used;.and,'
• Include some filamentous microorganisms in the
key twice where an important characteristic is
variable, e.g., Gram stain reaction for A/, llmicola II
and the observation of intracellular sulfur granules
for types 0914 and 021N. '
The use of this key is not without risk because some
filamentous microorganism characteristics vary, and
the key cannot always address all of these variables.
The filament type arrived at using the key should be
carefully checked against the typical microorganisms
characteristics listed in Table 7, and presented in,the
short descriptions and the photographs of each organ-
ism that follow. If characteristics given in Table 7, or
in the photographs, do not correspond to the f jlament
type arrived at using the key, careful re-examination of
characteristics used in the key is irv order. For example,
type 0041 generally gives a weak Gram, positive.or a
Gram variable reaction, and as such, is keyed correctly
from Figure 28. However, if strongly Gram positive, it
would be keyed as N. limicola II. Reference to Table 7
shows N. limicola II to be coiled and to possess no at-
tached growth—type 0041 is straight or smoothly-
curved and most often has substantial attached
growth.
Occasionally, a filamentous microorganism is ob-
served that is not represented by a type or genus des-
ignation. Such a filamentous microorganism should be
reported as "not identified"; do not try to "force-fit"
the microorganism into existing filament types.
6.3 Descriptions of Types of Filamentous
Microorganisms
These short descriptions of each filamentous micro-
organism commonly observed in activated sludge are
based on information given by Eikelboom and van
Buijsen (1981) as modified by experience with fila-
mentous microorganism characteristics in activated
sludges from the USA and South Africa. The following
filament types usually are observed in domestic
wastewater treatment plants at conventional organic
loading rates:
S. natans
type 1701
type 021N
ThiQthrix I and II
Beggiatoaspp.
Nocardia spp.
N, limicola II
H. hydrossis
type 1863
Filament types observed in industrial or domestic
wastewater treatment plants operated at low organic
loading rates are:
type 0041
type 0675
type021N
Thiothrix. I and II
type 0914
Beggiatoaspp.
type 1851
type 0803
type 0092
-type 0961
M, parvicella
Nocardia spp.
N. limicola I, II,
H. hydrossis
type 0581
Filament types that are observed infrequently include:
fungi
Cyanophyceae
Bacillus spp.
Flexibacter spp.
type 1702
type 1852
type 0211
type 0411
1. Sphaerotilus natans. (Figure 29a, b, and c; see
also Figures 16d and 20a.) Relatively long
(100-1000 /iifi) straight or smoothly-curved fila-
ments composed of round-ended, rod-shaped
cells (1.0-1.8x1.5-3.0 /mi) contained in a
clear, tightly-fitting sheath. Cell septa are clear
with indentations at septa. Filaments radiate out-
ward from the floe surface into the bulk solution.
False branching frequently is observed, giving a
"tree-branch"-like appearance. Gram negative,
Neisser negative, no sulfur granules: PHB fre-
quently pbserved. Cell shape can be rectangular
when the cells are tightly packed within the
sheath. An exocellular slime coating may occur
at ,1-iutrient limitation. Attached growth uncom-
mon^,but may occur when not growing.
61
-------
Ffguro28. Dtehotomous key for filamentous microorganism "identification" in activated sludge.
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Reference: Jenkins, eta/., 1984.
-------
Table?. Summary of Typical Morphological and Staining Characteristics of Filamentous Microorganisms Commonly Observed in
Activated Sludge
BRIGHT FIELD OBSERVATION
FILAMENT
TYPE
S. natans
type 1701
type 0041
type 0675
type 021 N
Thiothrix I
Thiothrix II
type 0914
Beggiatoa
spp.
type 1851
type 0803
type 0092
typo 0961
M. parvicella
Nocardia spp.
N. Limicola I
N. Limicola II
N. Limicola III
H. hydrossts
type 0581
type 1863
type 041 1
z
GRAM STO
+ .V
+ .V
_
_i +
_
+
-•-<-
+
weak
_
-
-
+
+
-1-
-.+
+
-
-
_
_
z
1
co
ijj
o
1
_
-
_
_
_
_
_
-
_
+
-
-
_
+
+ ,-
+
-
-
_
_
_
C
OJ
_
-•+
_I +
_J +
_1 +
_l+
_1 +
-•+
_
_
-
-
+
+
-
-
_
-
-
_J +
_
PHASE CONTRAST OBSERVATION 1000X
<
tr
o
CO
a
.s
_
-
_
_> +
+r_
+/_
_< +
+ •-
_
_
-
-
-
_
-
-
_
-
-
_
_
!
co
_
-
_
+
+
+
_
+
„
_
-
-
-
_
-
-
„
-
-
_
_
_JCO
OTHER CB
INCLUSIOI
PHB
PHB
-
_
PHB
PHB
PHB
PHB
PHB
_
-
+
-
PHB
PHB
-
PHB
PHB
-
-
_
_
QC
£
tij
TRICHOME DIA
1.0-1.4
0.6 - 0.8
1.4-1.6
0.8-1.0
1.0-2.0
1.4-2.5
0.8-1.4
1.0
1.2-3.0
0.8
0.8
0.8-1.0
0.8-1.2
0.8
1:0
0.8
1.2-1.4
2.0
0.5
0.5 - 0.8
0.8
0.8
1
TRICHOME LE
500
20-80
100-500
50 - 1 50
50-500
100-500
50 - 200
50 - 200
100 - 500
100-300
50-150
20-60
40-80
100-4OO
10-20
100
100 - 200
200-300
20-100
100-200
20-50
50-150
UJ
O-
s
TRICHOME S
. St
St.B
St
St
St.SC
St.SC
St.SC
St
St
St.B
St
St.B
St
C
.I
C
C
C
St.B '
C
B,l "
B.l
•z.
o
p
TRICHOME LOI
E
I.E
I.E
I
E
E
E
E.F
F
E
E.F
I
E
1
1
I.E
I,E
I.E
E.F
1
E.F
E
>
S
CELL SEPTA Cl
OBSERVED
+
+
+
+
+
+
+
+
-' +
+ |_
+
+ -
+
-
+ ,_
-
*
•f
-
'-
+
+
CO
INDENTATIO
CELL SEPTA
+
-
_
+
_
_
_
-
_
_
-
--
-
_
-
+
+
-
-
+
+
X
H-
CO,
+
+
+
+
_
+
+
_
-
+
_
-
-
-
_
-
-
_
+
-
_
_
f.
>
5
ATTACHED GR
+ +
+ +,-
+ +,-
_
_
_
_
-
_,+
_
-
-
-
-
-
-
-
-.+
-
_
_
Q
2
to
-jui
JTJN
uto
round-ended rods
1.4x2.0
round-ended rods
0.8x1.2
squares
1.4X1.5-2.0
squares
1,0X1.0
barrels,, rectan-
gles, discoid
1-2x1.5-2.0
rectangles
2.0x3-5
rectangles
1.0x1.5
squares
1.0X1.0
rectangles
2.0 X 6.0
rectangles
0.8X1.5
rectangles
0.8x1.5
rectangles
0.8X1.5
rectangles
1.0X2.0
-
variable
1.0X1 -2
-
discs, ovals
1.2X1.0
disc, ovals
2.0X1.5
-
-
oval rods
0.8x1-1.5
elongated rods
0.8 X 2:4
NOTES
False branching
cell septa hard
to discern
Neisser positive
reaction occurs
Neisser positive
reaction occurs
rosettes, gonidia
rosettes, gonidia
rosettes, gonidia
sulfur granules
"square"
motile: flexing and
gliding
trichome bundles
"transparent"
large "patches"
true branching
Incidental branching
Gram and Neisser
variable
"rigidly straight"
"chain of cells"
"chain of cells"
notation: + = positive; — = negative; V = variable; single symbol invariant; +, — or —, +, variable, the first being most observed.
Trichome shape: St = straight; B = bent; SC = smoothly curved; C = coiled; 1 = Irregularly-shaped.
Trichome location: E = extends from floe surface; I = found mostly within the floe; F = Free in liquid between the f Iocs.
Reference: Jenkins, eta/., 1984.
63
-------
2. Type 1701. (Figure 29c, d, and e; see also
Figures 19c and 20f.) Relatively short (10-100
Hm), curved or bent filaments composed of
round-ended, rod-shaped cells (0.7-1.Ox
1.0-2.0 urn] contained in a clear, tightly fitting
sheath. Cell septa are clear with indentations at
septa. Filaments found predominantly inter-
twined within the floe interior with only sfjort fila-
ments extending into the bulk solution. No
branching. Gram negative, Neisser negative, no
sulfur granules; PHB frequently observed. At-
tached growth of epiphytic bacteria is almost
always observed, making observation of indi-
vidual cells difficult.
3. Type 0041. (Figure 30a, b, and c; see also
Figures 19a, 19e, and 23c.) Straight, smoothly-
curved or bent filaments, 100-500 ftm in length,
composed of square-shaped cells (1.2-1.6X
1.5-2.5 nm) contained in a clear, tightly fitting
sheath. In domestic waste systems, it is most
often observed inside the floe and covered with
heavy, attached growth. In industrial waste
systems, it may occur extending from the flbc
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 floe and Gram negative when
extending into the bulk solution. 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 30).
4. Type 0675. (Figure 30d, e, and f.) Very similar to
type 0041, only smaller in trichome length
(50-150 /*m) and cell dimension (0.7-1.0 jim).
Covered with heavy, attached growth in domes-
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.
5. Type 021N. (Figure 26a-f; see also Figures 21 d
and e, 31 e, and 23a.) Filaments typically are
1.0-2.0 ;tm in width and 100-500 jim 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 are straight,
smoothly-curved or sometimes slightly coiled
and are found extending from the floe 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 S test generally is positive.
Rosettes are observed infrequently. No attached
growth. No sheath is present; however, a heavy
cell wall (still showing cross septa) often remains
after cell lysis.
6. Thiothrix I. (Figure 27a-f; see also Figure 22c.)
Straight or smoothly curved trichomes, 1.4-2.5
pirn in width and 100-500 pirn in length, found
extending from the floe surface. Cells are rec-
tangular (1.4-2.5 X 3-5 jim) with clear cell septa
without indentations at septa. No 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-positive granules may occur. Cells fre-
quently contain sulfur granules in situ and this
organism responds strongly to the S test. Apical
gonidia commonly are observed, and an incon-
spicuous hold-fast is present.
7. Thiothrix II. (Figure 25a-f; see also Figures 20c
.and 22d.) Straight or smoothly-curved filaments,
0.8-1.4 jim in width and 50-200 /^m in length,
found extending from the floe surface. No at-
tached growth. Cell septa without indentations
are present. Cells are rectangular (0.8-1.4 X 1-2
jim). 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 S 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.
8. Type 0914. (Figure 31 a, b, and c.) Straight or
smoothly-curved filaments, 0.7-1.0 jim in width
and 50-200 jim in length, found extending from
the floe surface or more commonly free in the
bulk solution. May have incidental attached
growth. Cells are square-shaped (1.0x1.0 jim)
without constrictions at septa. No sheath is
present. Gram negative and Neisser negative, but
may stain Gram positive when substantial sulfur
granules are present. Neisser positive granules
may occur. May contain intracellular sulfur
granules, which appear square rather than
spherical as observed for other filamentous
microorganisms. Does not respond to the S test.
9. Beggiatoa spp. (Figure 32a and b.) Large, straight
filaments, 1.0-3.0 (irrt in width and 100-500
jim in length, found free in the bulk solution and
64
-------
Figure 29. Sphaerotilusnatans (a, band c) and type 1701 (d, eand fl (aand d lOOx phase constrast; bar = 100
phase contrast, bar= 10 ftm).
*
Reference: Jenkins, eta/., 1984.
65
-------
Figure 30. Typo 0041 (a, b and c) and type 0675 (d, e and fl (a and d 100 x phase contrast; bar = 100 pm; b, c, e and f 1000 x phase
contrast; bar= 10fim).
Roferonco: Jenkins, etal., 1984.
66
-------
Figure 31. Type 0914 (a 100x phase contrast; bar= 100/im; b and c 1000x phase contrast; bar= 10/im) (note sulfur granules in b).
JTt« s'^i -••&-- -%-&^=' -,, / "^'.r -,j r^'^.
$ ! 1*9..if **- — • " -*^"_* itii. f
Reference: Jenkins, eta/., 1984.
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 (1-3x4-8
/tm). 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 1851. (Figure 32c and d; see also Figures
19d and 23d.) Straight or bent trichomes, 0.8
/tm in width and 100-300 /im in length, ob-
served extending from the floe surface, or more
commonly in bundles of intertwined filaments. A
sheath is present, but hard to observe. Cells are
rectangular (0.8 x 1.5-2.5 /im) 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. No sulfur
granules.
11. Type 0803. (Figure 32e.) Straight or smoothly-
curved filaments of uniform diameter (0.8 /urn)
and 50-150 /tin in length, found extending from
the floe surface or at times free in the bulk solu-
tion (industrial waste activated sludges). No
sheath and no attached growth. Cells are rectan-
gular (0.8x1.5-2.0 /tm) without constrictions
at septa. Gram negative and Neisser negative. No
sulfur granules.
12. Type 0092. (Figure 32f; see also Figures 23b and
24c.) Straight, irregularly-curved or bent fila-
ments, 0.8-1.0 /im in diameter and 10-60 /im
in length, found mostly within the floe. Cells are
rectangular (0.8 x 1.5 /urn) without constrictions
at septa, or with constrictions that are hard to ob-
serve. Neither attached growth nor a sheath is
present. Cells stain Gram negative and the entire
trichorne 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 ftm) in dried, stained smears.
13. Type 0961. (Figure 33a and b.) Straight tri-
chomes, 0.8-1.4 /im in diameter and 50-150
/tm in length, observed extending from the floe
surface. No attached growth. Cells are rectangu-
lar (0.8-1.4x1.5-4 /tm). A true sheath is not
present; however, a slime coating may be
present, appearing as an 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.
14. Microthrix parvicella. (Figure 33c, d, and e; see
also Figures 23e and 24e.) Irregularly-coiled fila-
ments, 0.6-0.8 /im in diameter and 100-400
/im in length, found in tangles in the floe 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 "beaded" effect. Gram-positive, Neisser-
negative, and Neisser-positive granules com-
monly occur. Short, clear spaces may occur in
the filament.
15. Nocardia spp. (Figure 34a, b, and c; see also
Figures 16b, 23f, and 24f.) Irregularly-bent,
short filaments, 1.0 /im in diameter and 10-20
Mm in length, found mostly within the floe, 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 (1.0x1.0-2.0 /tm) and septa
without constrictions are present. Gram positive
and Neisser negative, and Neisser-positive
granules commonly are observed. No sulfur
granules. PHB commonly is observed. Abun-
dance of this organism is best assessed from the
Gram stained preparation.
16. Nostocoida limicola I. Bent and irregularly-coiled
filaments, 0.6-0.8 /tm in diameter and
100-200 /tm in length, found within the floes
and free in the bulk solution. Cell septa are hard
to observe; when observed, cells are oval
(0.6-0.8 /tm diameter). No sheath and no at-
tached growth. Gram positive and Neisser-
positive trichome. No sulfur granules. Resembles
M. parvicella, except in Neisser-staining
properties.
17. Nostocoida limicola II. (Figure 34d, e, and f; see
also Figures 16c and 24d.) Bent and irregularly-
coiled filaments, 1.2-1.4 /im in diameter and
100-200 /im in length, found mostly within the
floe. Cell septa are clear, with oval cells (1.2-1.4
/tm 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), but can be Neisser negative at
times. Incidental branching is observed. Note:
Eikelboom and van Buijsen (1981) state that N.
limicola II is both Gram positive and Neisser nega-
tive, and that a bacterium closely resembling N.
limicola II occurs in some industrial waste acti-
vated sludge systems, differing from N. limicola II
by being Gram negative and Neisser negative.
Both forms are considered N. limicola II here.
68
-------
Figure 32. Beggiatoa spp. (a and fa), type 1851 (c and d], type 0803 (e) and type 0092 (fl (a and c 100 X phase contrast; bar = 100 jim; fa, d,
e and f 1000 x phase contrast; bar = 10 /K. m).
Reference: Jenkins, eta/., 1984.
69
-------
Figure 33. Typa0961 (aand b) and MterothrixparviceHa (c, dande) (aand c100x phase contrast; bar =100//.m; fa, dand e 1000x phase
contrast; bar= 10/tm).
Reference: Jenkins, era/., 1984.
70
-------
Figure 34. Nocardia spp. (a, b and c) and Nostocoida limicola II (d, e and fl (a and d 100 x phase contrast; bar = 100 /«m; b 400 x phase
contrast; bar=25/£m; c, eand MOOOx phase contrast; bar=10/im).
Reference: Jenkins, eta/., 1984.
-------
18. Nostocoida limicola III. Bent and irregularly-coiled
filaments, 1.6-2.0 p,m in diameter and
200-300 urn in length, found mostly within the
floe. No attached growth and no sheath. Cell
septa are clear, with oval cells (1.6-2.0 fim
diameter) and indentations at cell septa. PHB
granules are commonly observed. Gram positive
and Neisser positive. No sulfur granules.
19. Hallscomenobacter hydrosis. (Figure 35a, b, and
c.) Straight or bent, thin filaments, 0.5 /tm in
diameter and 20-100 pirn in length, found radiat-
ing outward from the floe surface or free in the
bulk solution. A sheath is present. No cell septa
are observed; however, empty spaces in the tri-
chome are commonly observed. Gram negative
and Neisser negative. No sulfur granules. Fila-
ments may occur in bundles, and attached
growth is variable, ranging from rare to abun-
dant. This filament can be easily overlooked at
100x observation.
20. Type 0581. (Figure 35d.) Smoothly-coiled fila-
ments, 0.4-0.7 jim in diameter and 100-200
/*m in length, found mostly within the floe but
may occur in "patches" free in solution. No
sheath and no cell septa. No sulfur granules; no
attached growth. Gram negative and Neisser
negative. This filamentous microorganism ap-
pears similar to M. parvicella, but differs in its
Gram and Neisser staining reactions.
21. Type 1863. (Figure 35e.) Short, irregularly-bent
filaments, 0.8 /tin in diameter and 50/im in
length, found extending from the floe surface
and free in the bulk solution. No sheath and no at-
tached growth. Cells are oval-shaped rods
(0.8 X 1.5 /tm), and appear as a "chain of cells,"
with indentations at the septa. Gram negative
and Neisser negative; Neisser-positive granules
may occur. No sulfur granules.
22. Type 0411. (Figure 35f.) Irregularly-bent tri-
chomes, 0.8 (im in diameter and 50-150 j«m in
length, found extending from the floe surface.
Filaments composed of elongated, rod-shaped
cells (0.8-2.4 fim); constrictions at septa give
the appearance of a "chain of cells." No sheath
and no attached growth. Gram negative and
Neisser negative. No sulfur granules.
23. Type 7702. (Figure 36a.) Short, straight or bent
filaments, 0.6-0.7 /tin in diameter and 20-80
ftm in length, found within the floe and extending
from the floe surface. Cell septa are absent and a
sheath is present. Incidental attached growth.
Gram negative and Neisser negative. No sulfur
granules.
24. Type 1852. (Figure 36b.) Straight or slightly bent
filaments, 0.6-0.8 /tm in diameter and 20-80
Mm in length, found extending from the floe sur-
face. Cells are rectangular (0.6-0.8x1.0-2.0
/^m) without constrictions at septa. No sheath,
no attached growth. Gram negative and Neisser
negative. No sulfur granules. This filament ap-
pears "transparent," as does type 0961.
25. Type 0211. (Figure 36c.) Bent and twisted fila-
ments, 0.3-0.5 /urn in diameter and 20-100 /tm
in length, found extending from the floe surface.
Cells are rod-shaped with clear constrictions at
cell septa. No sheath and no attached growth.
Gram negative and Neisser negative. No sulfur
granules.
26. Flexibacter spp. (Figure 36d.) Short, straight or
smoothly-curved filaments, 1.0 ^m in diameter
and 20-40 /urn in length, found free in the bulk
solution. This organism is motile by slow gliding
and flexing. No sheath and no attached growth.
Cell septa may be lacking. Gram negative and
Neisser negative. PHB granules commonly
observed.
27. Bacillus spp. (Figure 36e.) Rounded-end rods in
irregularly-shaped chains of cells, 0.8-1.0 /^m in
diameter and 20-50 /ttm in length, found mostly
at the edges of the floe. Gram positive and
Neisser negative. No sheath and no sulfur
granules.
28. Cyanophyceae. (Figure 36f.) Straight, large fila-
ments, 2.0-5.0 jum in diameter and 100-500
/tm in length, found free in the bulk solution. Cells
are square to rectangular (2-5x2-8 /tm) with
clear septa. No sheath and no attached growth.
Often motile by slow gliding. Distinct green color
under transmitted light observation. Gram nega-
tive but sometimes with a slight Gram-positive
reaction; Neisser negative. No sulfur granules.
29. Fungi. (Figure 37a, 37b, and 37c; see also Figure
16a.) Very large trichomes, 3-8 /tm in diameter
and 300-1000 /«m in length, found mostly in the
floe. Cells are rectangular (3-8 X 5-15 /im). and
contain intracellular granules and organelles;
cytoplasmic streaming may be observed. True
branching. Gram negative and Neisser negative.
No sulfur granules and no sheath, although a
heavy cell wall is present.
72
-------
Figure 35. Haliscomenobacter hydrossis (a, b and c), type 0581 (d), type 1863 (e) and type 0411 (fl (all 1000 x phase contrast;
bar=10/im).
Reference: Jenkins, eta/., 1984.
73
-------
Figure 36. Type 1702 (a), type 1852 (b), type 0211 (c), Flexibacter sp. (d). Bacillus sp. (e) and a blue-green bacterium (cyanophyceae) (fl
(alMOOOx phase contrast; bar=
Roforanco: Jenkins, eta/., 1984.
74
-------
Figure37. Fungus (a 100x phase contrast; bar=100jtm; 6400x phase contrast; bar = 25/zm; clOOOx phase contrast; bar=10/*m).
Reference: Jenkins, eta/., 1984.
75
-------
-------
Chapter 7
7.0 Appendix 3—Case Histories of Bulking
Control Using Chlorination.
City of Albany, GA. The City of Albany, GA, is a
20-MGD (0.88 m3/sec) (design) activated sludge
plant currently receiving 13 to 16 MGD (0.57 to 0.7
m3/sec) of a waste in which approximately 50 percent
of the 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 12 MGD (0.5
m3/sec) of influent flow, attainment of secondary ef-
fluent criteria (30 mg/l monthly average for BOD5 and
SS) was not possible.
At Albany, the permanent HAS 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 Ib/day (1,000 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-to 3/8-inch (6 to 10mm) diameter holes at 4-inch
(100mm) spacing. The system was constructed in
November 1977 for $4,953.
Figure 38 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, chlorine
was added to a wet well where the entire RAS stream
mixed with primary effluent. This mode of chlorina-
tion was commenced in Week 15 and continued until
Week 26, when the change to RAS chlorination was
made. Chlorine "was being added to the mixture of
RAS and primary effluent, and doses of 5 to 15 Ibs
CI2/103lbs (5 to 15 kg Cl2/103kg) SS/day were
needed to control bulking. Immediately after the
change from chlorinating the mixture of RAS and
primary effluent to chlorinating RAS alone, chlorine
doses that satisfactorily controlled bulking for the
mixture of RAS and primary effluent were "over-
doses" for RAS alone. Figure 38 shows that even
though the chlorine dose declined during the change-
over of the chlorine dose point [from 4.7 to 2.9 Ibs
C !2/103lbs (4.7 to 2.9 kg C !2/103kg) SS/day] there
was an increase in secondary effluent SS (and to a
lesser extent in secondary effluent BOD5) that
resulted from floe breakup caused by the chlorine
overdose.
RAS chlorination has been practiced with generally
excellent results at Albany for a period of over
7 years. At various times, the target SVI has been
changed, and in some instances (Figure 38, Weeks
180 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 flow periods. To illustrate the chlorine dosing
technique for bulking control, as employed by the City
of Albany, a bulking incident is examined in detail in
Figure 39. At this time, the target SVI was 230 ml/g.
The chlorine dose was started at low levels and grad-
ually increased until the SVI responded by stabilizing
and then falling. In the middle of this period (30 May
through 6 June), the SVI fell to below the target
value. The RAS chlorine dose was reduced in
response to this. On 18-20 June, the SVI dropped to
approximately 100 to 130 ml/g. The chlorine dose to
the RAS was reduced and then turned off.
City of San Jose/Santa Clara Water Pollution Control
Plant (SJ/SC WPCP), CA. The SJ/SC WPCP provides
tertiary treatment to 100 MGD (4.4 m3/sec) of
domestic sewage; during July-September (the peak
load season), flows increased approximately 20% and
BOD5 loading approximately doubled due to cannery
waste discharges. Effluent discharge criteria include
30-day average BOD5 and SS of 10 mg/l 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. It had plant
upsets due to bulking in the secondary activated
sludge system during the peak load seasons of 1979
and 1980. Interim measures included RAS chlorina-
tion in both the secondary and tertiary activated
sludge systems, and provision of supplemental oxy-
gen and ammonia to prevent bulking in the 1981 and
1982 peak load seasons (Beebe et a/., 1982).
The importance of chlorine solution mixing into the
RAS for providing effective filamentous microorgan-
77
-------
isrn control was illustrated at the SJ/SC WPCP. In the
first-stage 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 16 x 13 foot
(6m x 5m x 4m) wet well. It soon became evident that
this arrangement was ineffective. Extension of the
2-inch (75 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 10 Ibs CI2/103lbs (8
to 10 kg CI2/103kg) SS/day had little effect on 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 (15 cm) upstream of closed-
Impeller, low-speed centrifugal RAS pumps.
The data presented in Figure 40 show that the pump
clean-out port 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. As 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
no deleterious effects on these pumps, even though
the second-stage RAS pumps have brass bearings.
Impellers in all RAS 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 41). Chlorine doses are regulated
routinely on the basis of a target SVI value with input
from the plant microbiologist who observes activated
sludge samples on a daily basis during periods of RAS
chlorination. During past peak load seasons, extreme-
ly low target SVI values (60 to 80 ml/g) have been
used in the first stage activated sludge system so that
high solids loading rates (30-50 ibs SS/ft.2/day)
(150-250 kg SS/m2/day) 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 16 Ibs
CI2/103lbs (8 to 16 kg CI2/103kg) VSS/day. These
high chlorine doses produced significant floe 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 15 to
35 mg/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 the chlorine doses used [2-4 Ibs
CI2/103lbs (2-4 kg CI2/103kg) VSS/day]; 1.5 to 3.0
mg CI2 /I dose in the RAS stream), nitrification effi-
ciency was unaffected. This observation is consistent
with that of Strom and Finstein (1977). In the nitrify-
ing system, the decrease in SVI with commencement
of RAS chlorination was much faster than it was in
the first-stage activated sludge system. Whether this
was due to the presence of the more germicidal free
chlorine in the nitrifying system, to differences in the
types of filamentous microorganisms causing bulking
(type 0041 in the second stage, type 1701 in the first
stage), or to differences in system sludge growth
rates is not known.
Case history of bulking control using hydrogen perox-
ide. City of Petaluma, CA (Caropresso et al., 1974).
During 1974, the City of Petaluma, CA, Water Pollu-
tion Control Plant, which treated mixed domestic/
industrial' wastes, experienced severe bulking prob-
lems with SVI values in the range of 400 to 700 ml/g.
Hydrogen peroxide (as a 50% V/V H202 solution) was
dosed to the final quadrant of a two-pass aeration
tank that was being fed primary effluent at the 1/4
points and RAS at the head end. Figure 43 shows the
doses and concentrations of hydrogen peroxide used
over a 4-day period and the effect on SVI. The
hydrogen peroxide (100% as H202) doses ranged be-
tween 9-68 mg/1 and were variously applied for 8-24
hr/day. During the dosing of hydrogen peroxide,
2300 Ib (1050 kg) H202 (100% basis) was used to
bring the SVI under control (from 550 ml/g to 300
ml/g). The SVI continued to decrease following the
termination of hydrogen peroxide dosing.
78
-------
Figure 38. Control of bulking by RAS chlorination at the City of Albany, GA, Wastewater Treatment Plant (Jenkins et at., 1983).
6/ ILU 'X3QNI
|/6iu 'SS
3oarns
'31Vd 3SOQ
3NldOlHO SVd
79
-------
Fifluro 39. Use of target SMI to control RAS chlorination dosage for bulking control at the Albany, GA, Wastewater Treatment Plant
(Jenkins, 1980).
en o o
c\i
o
s
LU
a
a
•^
u
en
co
co
5.0
2.5
0
300
200
100
TARGETSVI 230 ml/g
_L
11
MAY 1979
21
31
10
I
20
JUNE 1979
30
80
-------
Figure 40. Comparison of HAS chlorination effectiveness when dosing chlorine to either the mixed liquor channel or the HAS pump (Beebe
eta/., 1982).
I 1 1 T
in
CM
CO
CNl
UJ
CO
y
CM
CO
U-l
X
CM
csi
CM CO
CM
'3SOO
-------
Figure 41. SVI and chlorine dose to RAS in the two first-stage activated sludge systems at the San Jose/Santa Clara Water Pollution Control
Plant, CA-1981 peak load season (Beebe et a/., 1982).
IL
i ,•
CO
CM
oo
LU
CD CD
UD UD
CsJ
GO
in
CO
UD
CsJ
CM
UD CSJ
UD
CD
CO
UD
CM
Nl
UD UD
-------
Figure 42. SVI and chlorine dose to RAS in the two second-stage activated sludge systems at the San Jose/Santa Clara Water Pollution
Control Plant, CA—1981 peak load season (Beebe et al., 1982).
1 1 1'
-
1 1 1
LO
0 SJ
m
CO
esj
Nl
00
C9
its
CO
m
CM
-------
Figure 43. SV1 response to peroxide treatment at the Petaluma, CA Water Pollution Control Plant (Caropreso et al., 1974).
TT
8OO
700 ~
600
5OO
4OO
300 —
200 —
100 —
I I I I i I I i in
START
TREATMENT
84
-------
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This section, besides containing references cited in the
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*U.S. GOVERNMENT PRWnNC OFFICE: 19 91 - 5 it e. 18 7/1. o 5 5 0
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