EPA-600/2-75-031
September 1975
Environmental Protection Technology Series
ACTINOMYCETES OF
SEWAGE-TREATMENT PLANTS
Municipal Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
-------
RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development,
U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories were established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
:3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL PROTECTION
TECHNOLOGY STUDIES series. This series describes research
performed to develop and demonstrate instrumentation, equipment
and methodology to repair or prevent environmental degradation from
point and non-point sources of pollution, This work provides the
new or improved technology required for the control and treatment
of pollution sources to meet environmental quality standards.
This document is available to the public through the National
Technical Information Service, Springfield, Virginia 22151,
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EPA-600/2-75-031
September 1975
ACTINOMYCETES OF SEWAGE-TREATMENT PLANTS
by
Hubert A. Lechevalier
Waksman Institute of Microbiology
Rutgers, the State University of New Jersey
New Brunswick, New Jersey 08903
Grant No. R802003 (17050 GUJ)
Program Element No. 1BB043
Project Officer
Ronald F. Lewis
Municipal Environmental Research Laboratory
Wastewater Research Division
Cincinnati, Ohio 45268
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for
publication. Approval does not signify that the contents necessarily
reflect the views and policies of the U.S. Environmental Protection
Agency, nor does mention of trade names or commercial products consti-
tute endorsement or recommendation for use.
11
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FOREWORD
Man and his environment must be protected from the adverse effects of
pesticides, radiation, noise, and other forms of pollution, and the
unwise management of solid waste. Efforts to protect the environment
require a focus that recognizes the interplay between the components of
our physical environment--air, water, and land. The Municipal Environ-
mental Research Laboratory contributes to this multidisciplinary focus
through programs engaged in
• studies on the effects of environmental contaminants on the
biosphere, and
0 a search for ways to prevent contamination and to recycle
valuable resources.
As part of these activities, the study described herein presented
the isolation of numerous strains of actinomycetes from nuisance foams
at activated sludge plants, demonstrated in the laboratory that Nocardia
amarae may cause the kind of foam observed in the plants, studied factors
affecting the growth of N. amarae, and proposed a method of control of the
foam by addition of digester supernatant to the activated sludge tanks.
A. W. Breidenbach, Ph.D.
Director
Municipal Environmental
Research Laboratory
111
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ABSTRACT
In some sewage-treatment plants of the activated sludge type, a
thick foam may be formed at the surface of the secondary aeration
and settling tanks. Such foams have often been found to be rich in
actinomycetes. This report covers the work done on this problem
between April 1971 and May 1974.
Over 250 strains of actinomycetes have been isolated from foams
or activated sludge from 19 different sewage-treatment plants
located in 8 states. The actinomycete most commonly associated
with foams is a previously undescribed Nocardia which has been
given the name N. Amarae. It has been demonstrated experimentally
in the laboratory that N. amarae may cause the kind of foam observed
in the plants.
Factors affecting the growth of N. amarae have been studied and a
method was proposed for control of the foam by addition of digester
supernatant to the activated sludge.
This report was submitted in fulfillment of project number EPA
802003 by the Waksman Institute of Microbiology under the partial
sponsorship of the Environmental Protection Agency. The reported
work was completed as of May 1974.
IV
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CONTENTS
Page
Abstract ii
Tables vi
Acknowledgments viii
Sections
I Conclusions 1
II Recommendations 2
III Introduction 3
IV Microbiological Survey of Foams 5
V Production of Actinomycetic Foams in the 19
Laboratory
VI Search for Biological Inhibitors for 25
Nocardia amarae
VII Comparison of the Operating Conditions of 36
Foaming and Non-foaming Plants
VIII Antinocardial Activity of Anaerobic Digester 49
Supernatant
DC Discussion 57
X References 59
XI List of Inventions 62
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TABLES
NUMBER PAGE
1 Actinomycetes Isolated From Foam and/or...Activated Sludge 8
2 Sources of Strains Used in This Study . 9
10
3 A Comparison of the Physiological Characteristics of N. Amarae 11
With Other Species of Nocardia 12
4 A Comparison of the Physiological Characteristics of N. Amarae 13
With Other Species of Nocardia 14
5 Pathogenicity of Some Strains of Nocardia From Sewage Foam 18
6 Growth and Foam Formation of N. Amarae Strain SE 214 in Baffled 21
and Llnbaffled Flask Containing Czapek Medium
7 Effect of Aeration Rate on the Biomass Production and Foam of 21
N. Amarae Strain SE 6 in Czapek Medium After 1 Week of Incubation
8. Foam Formation By N. Amarae SE 110 in YCZ and YCZ/5 in Unbaffled 23
Flasks
9 5 Day Old Broth Culture of N. Amarae SE 110 Grown in Unbaffled 23
Flasks of YCZ Medium (220 RPM)
10 Morphological and Biochemical Properties of #3 Isolate 27
28
11 LB4 Vs. N. Amarae SE 110 in YCZ Medium (Poor Growth of Bacterium: 31
Good Growth of N. Amarae)
12 LB4 Vs. N. Amarae SE 110 in Bennett's Medium (Good Growth of Both 32
Strains)
13 LB 4 Vs. N. Amarae SE 110 in VP Medium (Poor Growth of Both 33
Strains)
14 100-9 Vs. N. Amarae SE 110 in YCZ Medium 34
15 Treatment Facility Comparison 38
39
16 Sewage Characteristics 41
17 Comparison of Process, Loading, and Operating Parameters 43
44
18 Chemical Analysis of Potable Water Supplies 46
19 Toxicity of Anaerobic Digest (AD) to N. Amarae SE 110 in YCZ 50
Medium
vi
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TABLES (Continued)
NUMBER PAGE
20 Toxicity of Autoclaved and Millipore Filtered Anaerobic 51
Digest (AD) to N. Amarae SE 110 in YCZ Medium
21 Toxicity of Anaerobic Digest (AD) to Growth of N. Amarae 52
SE 110 in YCZ Medium Neutralized With Excess CaCO
J
22 Toxicity of Anaerobic Digest (AD) to Growth of N. Amarae 53
SE 110 in YCZ Medium Neutralized With Excess CaCOj
23 Toxicity of a Chloroform-Methanol Extract of Autoclaved 55
Anaerobic Digest Solids to N. Amarae SE 110
vii
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ACKNOWLEDGMENTS
We thank the operators of all the plants listed in Table 1 who have
been most cooperative in furnishing samples for microbiological
analyses.
Mr. Paul E. Wyszkowski, P. E., has carried but the comparison
of the operation data of the waste water treatment plants of Ocean
Township, Middletown Township and Bernardsville. In turn we are
indebted to the Sewerage authorities of these municipalities for
placing their data at his disposal.
The microbiological studies here reported were carried out by Mrs,
Mary P. Lechevalier with some assistance by Dr. C. E. Lee. We
are greatly indebted to Dr. Francois Mariat of the Institut Pasteur
of Paris for testing the pathogenicity of representative sewage
Nocardias.
Vlll
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SECTION I
CONCLUSIONS
Thick foams formed on the surface of secondary aeration and
settling tanks in sewage-treatment plants of the activated sludge
type are often, but not always, rich in actinomycetes.
The dominant actinomycetes in these foams are usually members
of the genus Nocardia. Some human pathogens, such as N.
asteroides and N. caviae, may occasionally be involved but more
often the dominant organism is N, rhodochrous or even more
frequently, a new species which we have described as N. amarae.
Pure cultures of N. amarae were added to non-foaming sludge under
laboratory conditions and thick foams were produced which were
similar to those found in the plants thus proving the etiologic role
of the Nocardia.
The operation data for two non-foaming plants and one with a foam-
ing problem were compared. The only obvious difference noted was
that in the non-foaming plants the anaerobic digester supernatant
was returned without treatment into the system. In the foaming
plant, the supernatant was oxidized with chlorine (Purifax treatment)
before being returned to the system.
The supernatant from the anaerobic digesters of two different plants
were found to contain a nocardiotoxic principle which completely
prevented the growth of N. amarae when diluted 1 to 10"^ and which
was still partially inhibitory at a dilution of 10'°.
It was thus concluded that the return of the nocardiotoxic anaerobic
digester supernatant into the sewage treatment system might be an
effective way of preventing the formation of nocardial foams.
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SECTION II
RECOMMENDATIONS
The production of actinomycetic foams should be prevented because
the growth of nocardias in aeration tanks is a health hazard due to
the formation of nocardia-containing aerosols and, in addition, the
production of thick foams in activated sludge type plants interferes
with good sewage treatment and is a source of extra labor costs.
We feel that we may have discovered a proper method of control and
we recommend that two further types of studies be carried put to
prove this point: 1) pilot studies in cooperating plants equipped with
anaerobic digesters, in which the value of returning anaerobic
digester supernatant will be explored; 2) a study of the nature of
the nocardiotoxic principle found in the supernatant of anaerobic
digesters.
In addition, in plants affected with nocardial foam, the foam should
be skimmed off the secondary settling tanks and sent to the
anaerobic digester. We understand, however, that many plants are
designed in such a way that this operation may not be possible.
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SECTION III
INTRODUCTION
GENERAL
Ideally, sewage-treatment plants should run smoothly, raw sewage
entering at one end and an effluent, not damaging to the environment,
coming out at the other end. In practice, plant operators have to
face and master numerous crises which may be brought about by
excessive rain or by the dumping of large amounts of specific
wastes in the sewage to be treated. The resulting disturbances may
take many forms. For example, in the case of activated sludge
plants, one troublesome "disease" is "bulking" usually associated
with a bloom of Sphaerotilus. When bulking occurs, the sludge does
not settle and it is impossible to obtain a clear effluent by simple
gravity sedimentation (Wells and Garrett,
Another "disease" of the activated sludge is the formation of foam on
the surface of the aeration tanks. This foam, which is less dense
than >water, floats on the surface of the settling tanks. It may be-
come very thick, 6 inches to one foot is not uncommon, but it may be
even thicker. The foam that we are discussing should not be confused
with detergent suds. The foam is quite rigid, and where it
accumulates, a bucket may be dropped from a height of several feet
onto the surface of the foam without passing through it. The form-
ation of such foams is a nuisance, preventing normal gas exchange
at the interface and requiring many hours of labor on the part of
cleaning crews. Often, no sooner is an aeration tank cleaned than
the cleaning must be repeated. The foam may escape from the
treatment tanks and be distributed as smelly pieces by the wind. It
has been found by Dr. R. F. Lewis that such foams may be full of
branching hyphae of actinomycetes. As a matter of fact, one could
say that in some cases the foam is an actinomycetic mycelial mat
with entrapped air bubbles.
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OBJECTIVE
The purpose of this study was to determine, 1) which actinomy-
cetes were found in these foams, 2) the role they played there, and
3) what could be done to prevent the formation of foams.
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SECTION IV
MICROBIOLOGICAL SURVEY OF FOAMS
Direct microscopic examination of sludge and foam.
The presence of actinomycetes in the foam and in the sludge from
foaming tanks is very easy to demonstrate. It is enough to place a
small sample in a drop of water located between slide and cover slip
to be able to visualize the numerous hyphae with a phase contrast
microscope. In the field, visualization can be made in bright field
by staining lightly with methylene blue or cotton blue. The actino-
mycetic hyphae bear no sporulating structures but are usually
banded and may be very wide (2 p.).
The actinomycetes may be almost the only visible organisms in the
foam. In the sludge from foaming tanks, actinomycetes are
abundantly mixed with other bacteria and protozoa. Occasionally
yeasts can also be seen. The presence of Sphaerotilus spp. can
also be easily detected by this method.
Direct microscopic examination is an excellent and rapid diagnostic
method for the detection of sewage actinomycetic foam. As a matter
of fact, if one can detect numerous actinomycetic hyphae in a
sample of activated sludge from a non-foaming aeration tank, one
may predict that a foaming episode may soon occur.
Geographic distribution of sewage actinomycetic foam.
Dr. R. F. Lewis drew our attention to a number of plants which
were suffering from foaming problems. These included the
Metropolitan Jones Island Plant at Milwaukee, a plant in San Jose,
California, and plants in Miami, Florida, San Antonio and Austin,
Texas, and Ocean Township, New Jersey. Actinomycetes were
found in the foam from all these plants. Dr. Lewis also put us in
contact with Mr. Paul E. Wyszkowski, an engineer familiar with the
Ocean Township plant.
-------
Mr. Wyszkowski made a list of 49 sewage-treatment plants of the
activated sludge type located in New Jersey. A letter was sent to
all the operators of these plants asking if they were affected by the
formation of foam. About a dozen operators responded affirm-
atively. On the basis of phone conversations, some plants were
considered especially promising and were visited. Examination of
the foam was made in situ and the microscopic demonstration of the
actinomycetes to plant operators was always a success. The
following plants were visited and were found to have actinomycetic
foam: Bedminster Inn, Bordentown Township, Chatham/Madison,
Delran, East Windsor, Eve sham, Matawan, Ocean Township and
Roxbury. In the plant in Middletown Township there was no foaming
but a few actinomycetes could be seen in the sludge particles. Foam
without actinomycetes but with Sphaerotilus was found at the
Morristown (Florence Avenue) plant and one incident of foaming with
a lot of Sphaerotilus and very few actinomycetes was observed at the
Ocean Township plant.
Samples of foam from out-of- state found to contain actinomycetes
were received from: San Jose, California; Hartford and Enfield,
Connecticut; the Andover and Riverdale plants in Miami, Florida;
Jamaica Bay, New York; Austin, Texas; Arlington, Virginia; and
Milwaukee, Wisconsin. Only Sphaerotilus and no actinomycetes were
observed in a sample from the Newton Creek Plant of New York City.
New Jersey plants without foaming problems which were visited in
1971 and 1972 included those at Bernardsville, the Somerset-Raritan
plant in Somerville and that of the American Cyanamid Co. in Bound
Brook. During most of this study, the Bernardsville plant was used
as a source of sludge visually and analytically free of actinomycetes.
The following general conclusions can be drawn from this survey:
1) Plants with actinomycetic foams can be found in widely different
areas.
2) Although thick foams are not invariably associated with the
presence of actinomycetes, these organisms are a very common
cause of this troublesome problem.
3) From information received from the plant operators, it seems
that actinomycetic foaming occurs mainly when the weather is warm,
the aeration rate is high and the mixed liquor thick.
6
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Isolation of actinomycetes from foams
Foams were added to different liquid media, both shaken and
stationary which were known to support, in some cases, selectively,
the growth of soil actinomycetes (El-Nakeeb and Lechevalier .
Except for potato-dextrose (PD) these showed overgrowth of the
foam actinomycete by other jnicrobes (fungi, bacteria and protozoa)
present. Direct plating of the diluted foam was more successful.
The actinomycetes grew very slowly only on certain isolation media
(1-3 weeks at 28" . These media included "synthetic" agar, chitin
agar (El-Nakeeb and Lechevalier 2), tap water agar, thin Pablum
agar (Lechevalier *^), nutrient agar, and peptone-yeast agar (PY)
(Kolstad and Bradley H). The best media for isolation (growth in
5-7 days), were found to be Czapek's agar amended with 0.2% yeast
extract (YCZ) (Higgins and Lechevalier 9) and glycerol agar (Gordon
and Smith 8).
Foams were also purified by differential centrifugation and
thoroughly washed to concentrate the actinomycetic hyphae prior to
plating and remove some of the associated biota.
Identity of the actinomycetes present in foams
Samples of sludge and/or foam were received or collected from the
plants listed in Table 1. For our taxonomic study, strains listed in
Table 2 were used. The methods were as described in Lechevalier,
M. P. and Lechevalier, H. A. 17.
When the physiology of certain of the sewage strains was compared
with that of strains of N. asteroides, N. braailiensis, and N. caviae,
it was found to differ as indicated in Table 3. These sewage
organisms were closest to N. ("Mycobacterium") rhodochrous,
strains of which are also isolated from some foams. A comparison
of these two taxa, along with the reactions of strains of
N. rhodochrous^ isolated from sewage, N. vaccinii 3500, and
N. carnea 3419, are presented in Table 4.
The principal characteristics differentiating the new organism from
N. rhodochrous are the following: nocardomycolic acid type,
hydrolysis of tyro sine, growth at 10 C, growth on adenine and on 5%
NaCl, acid from inositol, maltose, rhamnose, salicin, and sorbitol,
utilization of benzoate and citrate, and survival at 50 C for 8 h. The
principal differences between the new organism and Gordon's
(Gordon4) rhodochrous-related Nocardia sp. (which she placed in
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Table 1. ACTINOMYCETES ISOLATED FROM FOAM
AND/OR ACTIVATED SLUDGE
Location
California
Connecticut
Florida
New Jersey
New York
Texas
Virginia
Wisconsin
San Jose
Hartford
Enfield
Miami (Andover and
Riverdale plants)
Bedminster Inn
Bordentown Township
C hatham / Madi son
Delran
East Windsor
Eve s ham
Matawan
Middletown (No foam)
Ocean Township
Roxbury
Jamaica Bay
Austin
Arlington
Milwaukee
Anaerobic
Predominant actinomycetes digester
Nocardia rhodochrous
Nocardia rhodochrous
Nocardia amarae
Nocardia amarae, N. asteroides
Streptomyces, Micromonospora,
Nocardia asteroides,
Actinomadura sp.
Nocardia amarae, N. rhodochrous
Nocardia amarae + a
Nocardia amarae, N. asteroides,
N. rhodochrous
Nocardia caviae, N. asteroides
Nocardia amarae
Nocardia amarae
Nocardia rhodochrous +
Nocardia amarae + c
Nocardia rhodochrous
Nocardia amarae + a
Nocardia amarae, N. asteroides
Nocardia amarae + a
Nocardia rhodochrous
'No supernatant back in system.
Supernatant returned into the system.
'Supernatant returned after heavy chlorination.
8
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Table 2. SOURCES OF STRAINS USED IN THIS STUDY
Strain
designation9
SN 5101
SN 5 104
A 1Z974
3639
1240
NC 8139
1082S
624
369
462
515
570
625
N 10
N 31
N 60
N 239
NC 1621
A 7005
A 11890
I 384
1549
I 564
I 1256
Mil 14
Mil 15
SJ2
Se 113
Se 135
Se 141
Se 167
Se 187
Se 188
Se 189
Se 192
Se 194
Se 3
Se 6
Se45
SeSl
Se 53
Se6l
Se64
Identity
Nocardia rhodocKrous
Nocardia rhodochrous0
Nocardia rhodochrousc
Nocardia rhodochrous0
Nocardia rhodochrousc
Nocardia rhodochrousc
Nocardia rhodochrousc
Nocardia rhodochrousc
Nocardia rhodochrousc
Nocardia rhodochrous6
Nocardia rhodochrousc
Nocardia rhodochrousc
Nocardia rhodochrous^
Nocardia rhodochrous
Nocardia rhodochrous
Nocardia rhodochrous
Nocardia rhodochrous
Strain related to N. rhodochrous
Strain related to N. rhouochrous
Strain related to N. rhodochrousd
Strain related to N. rhodochrous^
Strain related to N. rhodochrous
Strain related to N. rhodoehrous^
Strain related to N. rhodochrousd
N. rhodochrous
N. rhodochrous
N. rhodochrous
N. rhodochrous
N. rhodochrous
N. rhodochrous
N. rhodochrous
N. rhodochrous
N. rhodochrous
N. rhodochrous
N. rhodochrous
N. rhodocjirous
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia- amarae
Nocardia amarae
Nocardia amarae
R. Bofticke
R. Bonicke
R. E. Gordon
R. E. Gordon
R. E. Gordon
R. E. Gordon
R. E. Gordon
R. E. Gordon
R. E. Gordon
R. E. Gordon
R. E. Gordon
R. E. Gordon
R. E. Gordon
M. Goodfellow
M. Goodfellow
M. Goodfellow
M. Goodfellow
R. E. Gordon
R. E. Gordon
R. E. Gordon
R. E. Gordon
R. E. Gordon
R. E. Gordon
R. E. Gordon
Abnormal foam.
Abnormal foam.
Abnormal foam,
Abnormal foam,
Abnormal foam,
Abnormal foam,
Abnormal foam.
Abnormal foam.
Abnormal foam,
Abnormal foam,
Abnormal foam.
Abnormal foam.
Abnormal foam,
Abnormal foam,
Abnormal foam,
Abnormal foam,
Abnormal foam,
Abnormal foam,
Abnormal foam,
Source
Milwaukee, Wis. , R. F. Lewis
Milwaukee, Wis. , R.F. Lewis
San Jose, Calif. , R. F. Lewis
Delran, N. J.
Middletown, N. J.
Ocean Township, N. J.
Hartford, Conn.
Hartford, Conn.
Hartford, Conn.
Bordentown, N. J.
Bordentown, N. J.
Bordentown, N. J.
Andover (Miami, Fla.
Andover (Miami), Fla,
Andover (Miami), Fla.
Riverdale (Miami), Fla.
River dale (Miami), Fla.
Ocean Township, N. J.
Ocean Township, N. J.
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Table Z {continued). SOURCES OF STRAINS USED IN THIS STUDY
Strain
designation*
Se65
Se 85
Se 87
Se 90
Se 91
Se 96
Se 97
Se 102
Se 106
Se 107
Se 110
Se 111
Se 117
Se IIS
Se 119
Se 120
Se 1ZZ
Se 139
Se 144
Se 149
Se 149B
Se 151
Se 154
Se 156
Se 157
Se 160
Se 16Z
Se 164
Se 18
Se43
Se 73
Se 75
Se 93
Se 114
SeZOS
Se 7Z
SeBl
Se83
Identity
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia amarae
Nocardia asteroides
Nocardia asteroides
Nocardia asteroides
Nocardia asteroides
Nocardia asteroides
Nocardia asteroides
Nocardia asteroides
Nocardia caviae
Nocardia caviae
Nocardia caviae
Source
Abnormal foam, Ocean Township, N. J.
Abnormal foam, Austin, Tex.
"Normal" froth, Austin, Tex.
Abnormal foam, Austin, Tex.
Abnormal foam, Austin, Tex.
Sludge return, Austin, Tex.
Abnormal foam, Bordentown Township, N. J.
Abnormal foam, Bordentown Township, N. J.
Abnormal foam. Ever sham, N. J.
Abnormal foam. Eve re ham, N. J.
Abnormal foam, Delran, N. J.
Abnormal foam, Delran, N. J.
Abnormal foam, Matawan, N. J.
Abnormal foam, Matawan, N. J.
Abnormal foam, Matawan, N. J.
Abnormal foam, Matawan, N. J.
Artificial foam lab isolate, Bordentown, N. J.
Abnormal foam. Ocean Township, N. J.
Abnormal foam. Ocean Township, N. J.
Abnormal foam, Jamaica Bay, N. Y.
Abnormal foam, Jamaica Bay, N. Y.
Abnormal foam, Jamaica Bay, N. Y.
Abnormal foam, Jamaica Bay, N. Y.
Sample No. 1, abnormal foam, Arlington, Va.
Sample No. 1, abnormal foam, Arlington, Va.
Sample No. 1, abnormal foam, Arlington, Va.
Sample No. 2, abnormal foam, Arlington, Va.
Sample No. 2, abnormal foam,Arlington, Va.
Abnormal foam, Andover Plant, Miami, Fla.
Abnormal foam, Andover Plant, Miami, Fla.
Abnormal foam, East Windsor, N. J.
Abnormal foam, East Windsor, N. J.
Abnormal foam, Austin, Texas
Abnormal foam, Delran, N. 3,
Abnormal foam, Bedminster Inn, Eedraineter.NJ
Abnormal foam, East Windsor, N. J.
Abnormal foam, East Windsor, N. J.
Abnormal foam, East Windsor, N. J.
* American Type Culture Collection, Rockville, Md.: I » Institute of Microbiology, Rutgers
University; NC « National Collection of Type Cultures, London, England; N « Collection of
the University of Newcastle Upon Tyne (England).
b Received as N. Pellegrini.
C Received as "Mycobacterium" rhodochrous.
d Received as related to "Mvcobacterium" rhodochrous.
10
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Table 3. A COMPARISON OF THE PHYSIOLOGICAL, CHARACTERISTICS
OF N. AMARAE WITH OTHER SPECIES OF NOCARDIA
Determination
Hydrolysis of:
Casein
Hypoxanthine
Tyrosine
Xanthine
Adenine
Starch
Gelatin
Esculin
Urea
Production of:
Nitrate reductase
Growth at/on/in:
10 C (YD)
45 C (YD)
Lysozyme broth
Acid from:
A do nit ol
Arabinose
Dulcitol
Erythritol
Galactose
Glucoee
Glycerol
Inositol
Lactose
Maltose
Mannitol
Mannose
Melibioae
a -Methyl- D-glucoside
Raffinose
Rhamnose
Salicin
Sorbitol
Xylose
N. amarae
(35 strains)
ob
0
0
0
0
86(w)
0
100
100
100
6
0
9
0
0
0
0
0
100
100
92
0
100
100
100
0
0
0
92
100
0
0
N. asteroides
(137 strains*)
0
4
1
0
0
67
34°
100
96
86
15
41
100
0
0
0
7
27
98
99d
3
0
6
1
17
0
0
32
2SC
0
0
N. brasiliensis
(62 strains*)
98
94
100
0
3
55
100
100
90
37
2
100
0
0
0
2
94
97
98*
100
0
4
94
68
0
0
0
0
0
N. caviae
(24 strains4)
0
100
0
100
4
54
100
92
100
13
50
100
0
4
0
0
0
100
100d
100
0
18
90
36
0
0
0
5
0
5
11
-------
Table 3 (continued). A COMPARISON OF THE PHYSIOLOGICAL CHARACTERISTICS
OF N. AMARAE WITH OTHER SPECIES OF NOCARDIA
Determination
Utilization of:
Acetate
Benzoate
Citrate
Lactate
Malate
Oxalate
Propionate
Pyruvate
Succinate
Survival:
50 C/8 h
N. amarae
(35 strains)
100
0
0
100
100
0
100
100
100
0
N. asteroides
(137 strains3-)
iooa
0*
38
31e
97
Oe
100d
99d
92
94
N. brasiliensis
(62 strains2)
100d
2d
98
100
100d
100<*
100
0
N. caviae
(24 strains3-)
100d
Od
29
100
100d
100d
100
88
a Gordon and Horan (1968).'
Percent positive; w, weak.
c Gordon and Mihm (1957); (79 strains of B. asteroides).
d Gordon and Mihm (1962); (142 strains of N. asteroides; 15 strains of N. caviae; 62 strains
of N. brasiliensis).
R. E. Gordon, personal communication.
12
-------
Table 4. A COMPARISON OF THE PHYSIOLOGICAL CHARACTERISTICS
OF N. AMARAE WITH OTHER SPECIES OF NOCARDIA
N. amarae
Determination
Hydrolysis of:
Casein
Hypoxanthine
Tyro sine
Xanthine
Adenine
Starch
Gelatin
Esculin
Urea
Production of:
Phosphatase
Nitrate reductase
Growth at/in/on:
10 C (YD)
45 C (YD)
Lysozyme broth
5% NaCl (YD)
Adenine agar
Acid from:
Adonitol
Arabinose
Cellobiose
Duleitol
Erythritol
Fructose
Galactoce
Glucose
Glycerol
Inoaitol
Lactose
Maltose
N. rhodochrous
(35 (97
strains) strains)*
Oc
0
0
0
0
86(w)d
0
'100
100
83d
100
6d
0
9d
6d
6d
0
0
0
0
0
100
0
100
100
92d
0
100
0
74
0
97
73e
82e
100
46
8
0
99
21
0
27«
07 b
strains)"
0
35
35
'o
76
100
100
53
59
100
100
0
6
12
100
6
94
35
Nocardia sp.
related to
N. rhodochrous
(36 (7
strains)* strains)b
0
0
5
0
71
97 43
0
86
100
100
100
97 100
11 14
28
100
100
0
0
0
0
0
43
0
100
57
0
0
14
N. rhodo-
chrous
(sewage)
(12 .
strains)
0
0
58
0
75
25
0
75
83
100
58
94
66
94
83
100
0
0
0
0
0
100
17
100
100
75
0
8
N. vac- N.
cinii car-
nea
IMRU IMRU
3500b 3419b
— „
_ _
-
• _
_
» „
„, —
+ +
+
+
+ +
+
_
+ 4
+
+ +
.. _
_ _
_ _
. _
.
+ +
+ 4.
+ +
+ +
+
_ _
,
13
-------
Table 4 (continued). A COMPARISON OF THE PHYSIOLOGICAL CHARACTERISTICS
OF N. AMARAE WITH OTHER SPECIES OF NOCARDIA
Nocardia sp.
Determination
Acid from:
Mannitol
Mannose
Melibiose
a. -Methyl- D-
glucoside
Raffinose
Rhamnose
Salicin
Sorbitol
Sucrose
Trehalose
Xylose
P -Methyl- D-
xyloside
Utilization of:
Acetate
Benzoate
Citrate
Lactate
Malate
Oxalate
Propionate
Pyruvate
Succinate
Tart rate
Survival:
50 C/8 h
N. amarae
(35
strains)
100
100
0
0
0
92d
100
0
100
100
0
0
100
0
0
100
100
0
100
100
100
0
0
N. rhodochrous
(97
strains)*
99
99
0
0
9
100
97
,
70
90
100
99
0
100
strains)0
100
0
29
100
100
41
0
100
100
100
12
82
related to
N. rhodochrous
(36
strains)*
0
61
0
0
0
0
24
19
8
100
94
0
97
0 .
strains)0
0
0
0
0
29
0
0
100
100
100
0
86
N. rhodo- N. vac-
chrous cinii
(sewage)
(12 IMRU
strains) 3500°
100 +
100 +
0
0
0
0 +
25
100
100
92 +
42 +
0
100 +
83
100 +
100 +
100 +
0
100 +
100 +
100 +
0
92 ND1
N.
car-
nea
IMRU
3419b
+
.
-
-
_
_
.
+
.
+
-
-
+
-
_
_
+
.
+
+
+
-
ND
* R. E. Gordon (1966).
k Run by authors.
c Percent positive; w, weak.
d Reactions of type strain (ATCC 27808); starch hydrolysis -; phosphatase production +; growth
at 10 C -; growth in/on: lysozyme broth-, 5% NaCl (YD) - adenine agar -; acid from:
inositol+, rhamnose +.
* Based on tests of 60 strains (Gordon and Mihm, 1957).
f ND • Not determined.
14
-------
M. rhodochrous) are: no car domy colic acid type, hydrolysis of
adenine, growth at 10 C, growth on adenine and 5% NaCl, acid from
fructose, maltose, mannitol, rhamnose, salicin, sucrose, and
trehalose, and survival at 50 C.
The new organism also differs from the Russian N. ucrainica
(Krasnikov et al. ^) by eleven characters: acid from galactose,
lactose, a-methyl-d-glycoside, raffinose, rhamnose, and xylose,
utilization of citrate, decomposition of tyrosine and casein, and
growth on 5% NaCl.
Dr. Ruth E. Gordon of our Institute informed us (personal
communication, 1973) that she had no named strains of Nocardia in
the IMRU collection having the same pattern of physiological
reactions as our organism.
On the basis of the overall evidence, we felt these organisms
represented a new species; thus we have proposed the name
Nocardia amarae, to accommodate this new group (Lechevalier,
M. P. and Lechevalier, H. A.17).
All of the strains of the new organism had a cell wall of type IV
(Lechevalier, H. A. and M. P. Lechevalier ^) and a whole-cell
sugar pattern of type A (Lechevalier, M. P. *5j typical of members
of the genus Nocardia. They contained nocardomycolic acids
(Lechevalier, M. P. et al. *&) of a novel type whose main a branch
is mono-unsaturated. Thus, the mycolates of these strains give
rise, on pyrolysis, to major amounts of mono-unsaturated fatty
esters having 16 and 18 carbons, accompanied for some strains by
minor amounts of analogous saturated fragments. This is in con-
trast to mo st Nocardia strains we have examined to date which
contain nocardomycolates whose pyrolysis fragments are saturated
fatty esters having 12 to 18 carbons. Nevertheless, minor amounts
of unsaturated fragments have been noted in the pyrolysates of the
mycolic acids of numerous nocardiae, particularly members of the
N. rhodochrous group (Lechevalier, M. P., unpublished results).
We have found this novel type of nocardomycolate in two other
nocardial strains: N. vaccinii IMRU 3500 and N. carnea 3419.
As indicated in Table 1, not all actinomycetes isolated from sewage
foams were strains of N. amarae. Some of the organisms (Table 2)
had the properties of N. caviae and N. asteroides (Table 3)^ Strains
of mycobacteria could also be isolated with great regularity from
foams. These organisms, because of their high lipid content, tend
15
-------
to float and are probably entrapped in the network of the nocardial
hyphae. No work has been carried out to determine the taxonomic
status of these mycobacterial strains. It is conceivable, however,
that they may be a potential public health hazard. In the case of the
small package plant serving Bedminster Inn, strains of Streptomyces,
Micromonospora and Actinomadura were isolated in addition to
strains of N. asteroides. One could probably safely assume that in
that plant the percentage of soil contamination is minimal and that of
human contributions are maximal.
In summary, we have noted that the well-branched actinomycete most
often found associated with sewage foam is a new species of
Nocardia which we have named N. amarae. The next most common
species is N. rhodochrous. Strains of the pathogenic N. asteroides
and N» caviae are next in importance and the least frequently
encountered actinomycetes are strains of Streptomyces,
Mic romono spora and Actinomadura.
Antibiotic activity of Nocardia amarae
The antibiotic activity of N. amarae may have a certain ecological
importance in sewage-treatment plants since N. amarae may become
a dominant organism as a consequence of its antagonistic activity.
Twenty-one strains of N. amarae, 2 of N. caviae and 3 of N.
rhodochrous all isolated from sewage foams, were tested by cross-
streak test against 4 fungi (Prototheca portoricensis, Candida
albicans, Geotrichum sp., -Penicillium nptatum) and 4 bacteria
(Escherichia coli, Pseudomonas aeruginosa, My c oba ct e r ium
smegmatis and Staphy loco ecus aureus) on two media (Yeast extract- .
Czapek's, Nutrient glucose agar). Plates were incubated at 28 C
and zones of inhibition were observed at 24 hr for bacteria, 72 hr
for the Mycobacterium and 96 hr for the fungi.
The strains of N. caviae and N. rhodochrous exhibited no antibiotic
activity against any of the test organisms. Twenty strains of
N. amarae were active against C. albicans, 1 against P. notatum,
14 against M. smegmatis and 5 against S. aureus.
Three strains of N. amarae (Se 110, Se 110-DHMS-R, Se 151) were
selected for a more detailed study of antibiotic activity by cross-
streak test. The 8 test organisms listed above were used, but four
assay media were investigated. Zones of inhibition, when present,
were measured after 3, 5 and 9 days of incubation at 28 C.
Streptomyces fradiae strain 3535 known to produce both neomycin.
16
-------
and fradicin was used as a positive control. The 4 assay media
used were Yeast extract-Czapek's (YCZ), Bennett's Nutrient and
Soil extract agars. On all media, S. fradiae inhibited all the test
organisms, and at times, the inhibition was total.
The 3 strains of N. amarae never showed antibiotic activity against
the 2 gram-negative bacteria, but some antibiotic activity could be
demonstrated against all the other organisms on yeast extract-
Czapek's agar. As a rule the zones of inhibition were very narrow
(about 2 mm). The best activity was against C. albicans (up to 20
mm) and M. smegmatis (up to 6 mm). There were no obvious
differences between the antibiotic activities of the three strains of
N. amarae.
Strain Se 110-DHSM-R was grown in liquid Yeast extract-Czapek's
medium at 28 C and tested for antibiotic activity after 3, 8 and 14
days of incubation. Antibiotic activity was tested by the streak
dilution assay method against C. albicans, M. smegmatis and
S. aureus. Both the filtrates of the culture and the homogenate of
the disrupted cells were tested for antibiotic activity. No trace of
activity was detected in any case.
On the basis of these experiments we could conclude that it is most
unlikely that N. amarae gains a predominant position in activated
sludge through the production of antibiotics.
Pathogenicity of sewage actinomycetes
Eleven strains of Nocardia isolated from foams were sent to Dr. F.
Mariat at the Pasteur Institute in Paris for pathogenicity testing in
animals. The identity of the strains was withheld from Dr. Mariat
until the end of the test. The strains were grown on Bennett's
medium, suspended in physiological saline and ground in a tissue
grinder. Inoculation of the suspensions was carried out intra-
peritoneally. Guinea pigs received twice the dose given to mice.
For each strain a total of eight guinea pigs and eleven mice were
inoculated. After one month, surviving animals were sacrificed,
examined for lesions, and retrocultures were attempted. The results
are summarized in Table 5 and indicate that strains isolated from
foam and identified as N. caviae and N. asteroides have a tendency
to be pathogenic to laboratory animals whereas N. amarae seems,
fortunately, to be innocuous. N. asteroides and N. caviae are
opportunistic pathogens and the results obtained bv Dr. Mariat are
typical of what may be expected with members of these species
under the experimental conditions used (Kurup et al.
17
-------
Table 5. PATHOGENICITY OF SOME STRAINS OF NOCARDIA
FROM SEWAGE FOAM
Strain
No.
Identity
Nocardia
Guinea pigs
Mice
Sacrificed after 1 month
Deatha
lesions cultures
Death
Sacrificed after 1 month
lesions cultures
CO
Se 72
Se 18
Se 93
Se 3
Se 64
Se 85
Se 91
Se 102
Se 107
Se 111
Se 117
caviae
asteroides
asteroides
argarae
it
ii
ti
it
M
3,17
15,17,18
+
Days of death. Dead animals had numerous lesions, cultures were positive.
-------
SECTION V
PRODUCTION OF ACTINOMYCETIC FOAMS IN THE LABORATORY
Introduction
After a number of unsuccessful experiments, it was found that the
production of actinomycetic foam in the laboratory was easy to
obtain. At no time, however, were we able to obtain foams as thick
and as viscous as those produced in affected plants.
In the laboratory, foam was produced by bubbling air through
actinomycete-containing activated sludge. Four liter bottles were
used containing a liter and a half to two liters of sludge. Moist air
was bubbled through a bottom sparger at the rate of 1 to 3 liters per
minute. Aeration at 3 liters per minute was better than at 1 liter
per minute but excessive foaming often forced a reduction in the rate
of aeration. Air was permitted to escape from the aeration bottles
after passing through a cotton filter. Good foaming was obtained if
the sludge had a dry solid content of 0. 5 to 1%. It was possible to
keep the foaming going for several days in a giv&n aeration bottle by
stopping the aeration daily for one hour, aspirating the clear super-
natant and replacing it with raw sewage or with a nutritive solution.
The Bernardsville Sewage-treatment Plant was free of foaming
problems until recently (see pg. 48 ). At the time no foaming was
occurring, microscopic examination of its activated sludge (pH
usually slightly above 7. 0) revealed that it contained no visible
actinomycetic hyphae. Bernardsville sewage taken after primary
treatment was placed in aeration jars and aerated for more than a
month. Every day during this period, the aeration was stopped and
after settling, half of the volume of liquid was removed and was
replaced by fresh sewage from Bernardsville. Thus a small amount
of sludge was eventually formed and this laboratory-made sludge was
microscopically free of actinomycetic growth.
19
-------
In a second experiment, Bernardsville sludge, concentrated to 0. 7-
0. 9% solids by decantation, was inoculated with actinomycetic foam
received from Miami. The actinomycete could be maintained in a
healthy state (continuing to form foam) by adding fresh raw sewage
daily (obtained from the Rutgers University pumping station) after
aspiration of part of the contents of the aeration flasks as described
above. The same experiment was repeated with foam coming from
local sources such as Ocean Township and Bordentown. As a rule
during the aeration process, the pH of the sludge dropped to 6. 5-5. 5.
In addition, aerated sludge from Bernardsville fed with the same
raw sewage as in the second experiment, was inoculated with six
different pure cultures of actinomycetes (Se 3, Se 61, Se 65, Se 67,
Se 74, and Se 75) which had been isolated by plating out foams from
various plants. In all cases, foaming was obtained in the laboratory.
We conclude that the combination of raw sewage from Rutgers and
sludge from Bernardsville was capable of supporting the growth of
actinomycetes with resultant foaming.
Effect of aeration rate on the biomass production and the formation
of foam by strains of N. amarae
The growth of N. amarae in Czapek's medium dispensed in baffled
and unbaffled 250 ml Ehrlenmeyer flasks was compared. Foam was
simply recorded as being present or absent. Foam was observed
only in baffled flasks although growth was faster in the unbaffled type
(Table 6).
Similar results were observed by growing N. amarae Se 6 in
sparger-aerated two liter bottles containing Czapek's medium.
After four days of operation, the foam was thicker where the
aeration had been the most vigorous although growth was maximal
at a lower aeration rate (Table 7). This was due to the fact that in
the bottles or flasks where the aeration was greatest many cells were
carried away from the growth medium by the foam and deposited on
the sides of the bottles where they no longer grew.
Foaming factor
A surface active agent is produced by Npcardia amarae which favors
the formation of foam. It was found that in unbaffled flasks of YCZ
or low sucrose YCZ (YCZ/5), that the greater the amount of
no car dial growth, the greater the amount of the surface activity
20
-------
Table 6. GROWTH AND FOAM FORMATION OF N. AMARAE
STRAIN SE 214 IN BAFFLED AND UNBAFFLED FLASK
CONTAINING CZAPEK MEDIUM
Day of
incubation
0
1
2
3
4
5
6
Unbaffled flask
Klett Foam
units formation
23
56
125
310
570
700
750
Baffled
flask
Klett Foam
units formation
23
45
65
155
450
630
750
-
-
-
+
+
•f
+
Table 7. EFFECT OF AERATION RATE ON THE BIOMASS
PRODUCTION AND FOAM OF N. AMARAE STRAIN SE 6
IN CZAPEK MEDIUM AFTER 1 WEEK OF INCUBATION
Aeration
rate
1/min
0
1.8
2.4
3.3
Biomass production
in 1 week
pcva (ml/liter)
1.4
3.8
2.9
2.5
Foam formation
in 4 days
cm
0
10
24
65
Packed cell volume expressed as milliliters per liter
broth culture.
Distance between surface of solution and zone of biomass
accumulation.
21
-------
produced (Table 8). Since the increase in amount of the agent
parallels the increase in the amount of cells of the nocardia, it is
possible that it is a cytoplasmic protein or even a "soap" derived
from cell lipids. Also, as illustrated in Table 9, the foam
formation is enhanced and stabilized by the presence of the cells,
since when these are removed by centrifugation, the foam collapses
more readily.
Use of jmtifoam compounds in the laboratory
Once a method was developed to obtain actinomycetic foam in the
laboratory, a few antifoam compounds were tried in order to
determine if they had any effect. The following compounds produced
by Hodag Chemical Corp. of Skokie, Illinois, were investigated:
S-9, S-49, S-118 and PPG-2000. PPG-2000 is a polyglycol; the
other compounds are of unknown composition. Foaming was reduced
in all cases even at the lowest concentration used (0. 1 ml per l|-
liter per day), but the actinomycetic growth was not eliminated. The
most promising of these compounds seem to be the poiyglycol.
However, foaming was not completely eliminated, only reduced, at
the highest concentration used (0.4 ml per 1^ liter per day).
In the use of antifoam compounds one should make a distinction
between using these compounds as a preventive measure and as a
curative measure. Possibly prevention might be the most effective
method, however cost and potential deleterious effect on the treat-
ment of the sewage would have to be estimated.
Effect of addition of calcium carbonate on foaming
It had been observed that foam samples collected in a sewage-
treatment plant always have a pH lower than that of corresponding
sewage. In addition, it was noted that aeration of sewage and
activated sludge in the laboratory produced a lowering of the pH.
Powdered calcium carbonate, at the level of 10 and 20 grains per
liter, was added to Bernardsville activated sludge inoculated with
Bordentown foam. Foaming was abundant in all flasks and all showed
good growth of the actinomycetes. The addition of 10 gm per liter of
calcium carbonate did not affect the pH. Doubling the dose permitted
a differential of one pH unit between treated flasks and the control
(5.8 vs. 6.8). When the aeration was stopped, sedimentation of
sludge was more rapid in calcium carbonate-containing flasks than
in the untreated control.
22
-------
.Table 8. FOAM FORMATION BY N. AMARAE SE 110 IN
YCZ AND YCZ/5 IN UNBAFFLED FLASKS
Day
1
2
3
4
5
Uninoculated
broth
YCZ Medium YCZ/5 Medium
pcv pcv
(ml/liter) mm Foama (ml/liter) mm Foama
0 min 60 min o min 60 min
20 5 4 20 76
120 1.3 13 40 99
80 14 12 36 10 10
84 14 12 30 10 10
80 12 12 30 10 10
70 - 70
a
Foam formation in mm produced on 5 cc of whole broth (cells +
medium) mixed on vibromix for 30 sees, in standard 16 x 150 mm
tubes, measured immediately and after 60 min. standing.
b
pcv = packed cell volume.
Table 9. 5 DAY OLD BROTH CULTURE OF N. AMARAE SE 110
GROWN IN UNBAFFLED FLASKS OF YCZ MEDIUM (220 RPM)
mm of Foama
Time after vibromixing
0 min 1 man 60 min
Control medium
(uninoculated)
Broth culture
(cells present)
Broth culture
(cells removed by
centrifugation at
12,800 G/10 min)
12
14
12
11
12
See Table 8.
23
-------
The practice of maintaining a near-neutral pH by addition of calcium
carbonate to sewage is undoubtedly beneficial to the growth of
N. amarae; since this organism, like most actinomycetes, is
inhibited by pH's lower than 5. 0. In this case, the changing of the
pH of the foam from 5. 8 to 6. 8 by the use of calcium carbonate did
not appear to have any effect on the actinomycete growth or the
foam's stability.
24
-------
SECTION VI
SEARCH FOR BIOLOGICAL INHIBITORS FOR
NOCARDIA AMARAE
It seems possible that the activated sludge of plants without actinomy-
cetic foaming problems might be rich in some biological entities
which would prevent the proliferation of N. amarae. Conceivably,
these biological factors could be nocardiophages, parasitic bacteria
of the Bdellovibrio type, antagonistic microorganisms, or micro-
organisms producing nocardiolytic .enzymes or lipases capable of
degrading nocardial lipids.
In order to be able to measure the disappearance of N. amarae in a
given sludge quantitatively, it is helpful to incorporate a selective
inhibitor in the plating out medium in order to prevent the growth of
most sewage organisms while still permitting the proliferation of
N. amarae.
Twelve strains of N. amarae were tested for sensitivity to 10 anti-
microbial agents on Yeast-Czapek's agar. The disk assay method
was used with chloramphenicol, polymyxin B, triple sulfa, neomycin,
dihydrostreptomycin, erythromycin, penicillin, tetracycline,
bacitracin and streptomycin. All strains were resistant to triple
sulfa but were sensitive to the other antimicrobial agents. Since the
antimicrobial range of activity of triple sulfa is not wide enough to be
of use in repressing the sewage flora during the plating out
procedure, a highly dehydrostreptomycin-resistant mutant
(resistant to at least 400 fig/ml) was developed from one of the
strains of N. amarae (Se 110). This strain (Se 110-DHSM-R) can be
added to activated sludge from a non-foaming plant and its fate can
be followed quantitatively by plating out on a dihydr o streptomycin-
containing medium.
25
-------
a) Study of an antagonist from the sludge
Washed cells of N. amarae Se 110-DHSM-R were placed in a salt
solution fortified with vitamins to which was added some
Bernardsville sludge which has never been known to foam. Under
such conditions, sewage organisms had only cells of N. amarae as
sources of carbon and nitrogen. Flasks were incubated on a rotary
shaker at 28 C. At the start of the experiment, and after 4 and 10
days, counts of j^J. amarae were made on a medium containing 400
fig/ml of dihydro streptomycin. Other bacteria were counted on
antibiotic-less medium. During the incubation period, a reduction
in the viable population of No amarae was observed and the
establishment of a dominant bacterium was noted.
This bacterium (No. 3 isolate) was isolated and tested for antibiotic
activity by crosa streak test against a number of nocardiae, myco-
bacteria, bacteria and fungi. It was especially very active against
strains of Pseudomonas, and inactive against fungi. The properties
of the bacterium were investigated (Table 10) and the organism was
identified as an atypical strain of Enterobacter aerogenes.
In liquid shake cultures, bacterium No. 3 failed to produce any anti-
biotic. An investigation .of the differences observed between the anti-
biotic activity as shown by cross-streak test and the lack of activity
in liquid media revealed that the activity of the bacterium on solid
media was due to the production of acid(s). When the growth medium
was neutralized with solid CaCOj, no activity was noted. The nature
of this acid was not determined since it was felt that in the sludge,
which is maintained close to neutrality in most plants, bacteria, such
as the No. 3 isolate, could not play a role in the control of the
nocardiae.
b) Attempt at isolating nocardiolytic organisms from sludge
Direct plating
Fresh sludge samples obtained from Bernardsville and Middletown,
N. J. plants (neither of which showed foaming) were diluted and
plated out on yeast extract-peptone or vitamin salts medium contain-
ing washed cells of N. amarae Se 110. After incubation at 28° for
2, 6 and 10 days, the plates were examined for clearing of the amarae
cells. No lytic microorganisms were recovered.
26
-------
Table 10. MORPHOLOGICAL AND BIOCHEMICAL
PROPERTIES OF #3 ISOLATE
Properties
Results
Gram staina and morphology
5% sheep blood agar
EMB agar
MacConkey agar
Blood Phenylethyl alcohol agar
Oxidaseb
Catalasec
Motilityd
Nitrate to nitrite
Triple Sugar Iron Agar
Lysine-Iron Agar
Phenylalanine deaminase
Indolef
M.R.g
V.P.S
Simmon's citrate
Lysine decarboxylase
Ornithine decarboxylase
Arginine dihydrolase
Urease1
Esculin
•
OF-glucoseJ
OF-xylose
OF-inositol
OF-glycerol
OF-dulcitol
Gram-negative rods with rounded
ends occur mostly single, some in
pairs; occasionally very long wavy
rods not clearly consisting of
single cells.
No hemolysis, diameter 2-3 mm
Metallic sheen, diameter 2 mm
Red mucoid colony, diameter 2 mm
No growth
A/Ag.e
K/Kg. e No H2S.
No H2S.
F
Ag
Ag
Ag
27
-------
Table 10 (continued). MORPHOLOGICAL AND BIOCHEMICAL
PROPERTIES OF #3 ISOLATE
Properties Results
OF-cellobiose Ag
OF-sorbitol Ag
CTA-glucosek Ag
CTA-maltose . Ag
Purple Broth rhamnose Ag
Purple Broth raffinose Ag
YCZ agar containing 400 |j.g/ml No growth.
DHSM
a 18 hrs. YCZ agar slant.
Kovacs method, reagent 1% tetramethyl-p-phenylenediamine
aqueous solution.
c 24 hrs. nutrient agar slope, 10% H^O,.
° Young broth culture, wet mount.
e A = Acid; K = alkaline; g = gas.
f SIM medium, 48 hrs. 37 C.
° Culture incubated at room temperature (25 C) up to 3 days V. P.
O'Meara's method.
Moeller method.
Christensen's urea medium. Weak reaction compared to
Proteus vulgaris.
J = OF = Oxidation/Reduction Medium.
k = Cystine-Trypic Medium.
28
-------
Soil was also examined for antagonists toward N. amarae. In this
experiment dilutions of garden soil were incorporated into
Woodruff's agar along with washed log phase cells of No amarae
Se 110 or Se 149B and incubated for 3 weeks at 24° C. Most colonies
which showed antagonistic properties toward the N. amarae strains
were Streptomyces; one was a bacterium. Since it is rare to
encounter Streptomyces in sewage, and since they probably do not
survive long in that environment, it was felt that further work with
the bacterium (100-9) might prove more fruitful.
Enrichment techniques
Flasks containing washed cells of N. amarae Se 110-DHSM-R
suspended in a vitamin-salts solution and to which Bernardsville
sludge had been added were incubated on a shaking machine at 28 C
for up to two months. Periodically, the contents of one flask was
centrifuged and the supernatant filtered through a Seitz filter. Drops
of the filtrate were deposited on lawns of N. amarae in order to
detect nocardiophages. Thus far none have been detected.
In other experiments washed log phase cells of No amarae Se 110
were added to flasks of liquid yeast-extract peptone or vitamin-salts
medium along with either untreated or Millipore-filtered sludge from
Bernardsville, Enfield or Middletown. After shaking 4-5 days at
220 RPM, and after every subsequent 5 day period, further fresh
N. amarae cells were added and shaking continued 3-4 weeks.
Flasks were monitored microscopically for the disappearance of
amarae cells. Flasks which showed rapid disappearance of the cells
were plated out (with and without Millipore filtration) versus a lawn
of N. amarae Se 110 or Se 149 in a two-layer solid-semi-solid yeast
extract peptone medium and observed for lysed areas after a suitable
period of incubation. In this way two different antagonistic bacteria
were isolated from Enfield, one from Bernardsville and none from
Middletown. A protozoan, Colpoda sp., which caused the
disappearance (2-3 days) of amarae in certain flasks was also
isolated from both Enfield and Bernardsville sludge. Again no
Bdellovibrio-like strains or nocardiophage were found.
Antagonistic activity of bacterial isolates
Sludge isolates
The most rapidly growing and actively lytic bacterial strain (LB4)
from sludge (Enfield) was investigated. It is a yellow, gram-negative,
29
-------
motile, oxidative, catalase-positive, cytochrome oxidase-positive
rod. When LB4 was grown on an agar medium such as yeast-
peptone (YP) containing cells of N. amarae, microscopic
examination-after 3 days showed a dissolution of the amarae cells
next to the colonies of the bacterium. In shaken liquid culture it
was found that:
1. In a medium where good growth of N. amarae (Se 110) and very
poor growth of the bacterium takes place, control of the amarae
growth occurs only if the bacterium and the actinomycete are
inoculated simultaneously {YCZ Medium, Table 11).
2. In a medium where both organisms grow-well (Bennett's Table
12) or very poorly (YP, Table 13), the bacterium clumps, then lyses
the amarae cells even when added after the growth of amarae has
reached stationary phase. The bacterium appears to grow at the
expense of the Nocardia.
Inocula for both organisms (LB4 and Se 110) were 24 hr. old yeast-
dextrose (YD)-grown cells which were added at 7. 5 x 10° cells/flask
for N. amarae and 57 x 10° cells/flask for the bacterium. Packed
cell volumes were determined by centrifuging 5 cc. of whole broth
3 min. at 1, 200 x g.
We conclude that under proper nutritional conditions L.B4 stops
amarae growth and lyses its cells even when a substantial growing
biomass of amarae is already present.
Soil isolate
The bacterium 100-9 antagonistic to N. amarae which was isolated
from soil is a yellow, gram-negative non-motile, oxidative cyto-
chrome-oxidase positive rod which produces large amounts of slime
on sugar-containing media. The bacterium was tested in YCZ liquid
medium vs. N. amarae Se 110. The inoculum of the 24 hr. old, YD-
grown bacterial cells was added at 19.6 x 109 cells/flask. N. amarae
inocula were like those previously described for the sludge isolate
LB4.
In this medium, the bacterium inhibits the growth of N. amarae and
causes lysis of log phase but not stationary phase cells (Table 14).
We conclude that addition of these nocardiolytic bacteria to tanks with
amarae foams might help control this problem. We were unsuccessful
30
-------
Table 11. LB4 VS. N. AMARAE SE 110 in YCZ MEDIUM
u>
(POOR GROWTH OF BACTERIUM: GOOD GROWTH OF N. AMARAE)
pcva (ml/liter)-
Se 110
LB4
Day
1
2
3
4
6
a pcv =
N. amarae
Se 110
only
40.0
100.0
100.0
90.0
80.0
LB4
(bacterium)
only
6
4
4
4
2
both
Se 110 added day 0 Se 110 added day 0
added + +
day 0
8
8b
4b
4b
2b
LB4 added day 1 LB4 added day 2
_ _
100. Oc
100.0 100. Oc
90.0 100.0
100. 0° 120. 0C
packed cell volume.
Microscopic picture :
b No
amarae cells.
c amarae cells are
clumped but
healthy
looking, and viable.
pH values remained >6. 6 in all flasks.
-------
Table 12. LB4 VS. N. AMARAE SE 110 IN BENNETT'S MEDIUM
to
(GOOD GROWTH OF BOTH STRAINS)
Day
1
2
4
5
9
N. amarae
Se 110
only
44.0
42.0
36.0
32.0
40.0
LB4
(bacterium)
only
36.0
28.0
32.0
36.0
28.0
pcva (ml/liter)
Se 110 added day 0 Se 110 added day 0 Se 110 added day 0
+ f +
LB4 added day 0 LB4 added day 1 LB4 added day 2
36. Ob
28. Oc 38. Od
44. Oc 44. Ob 40. Od
32. Oc 40. Ob 40. Od
36. Oc 44.0° 24. Oc
a pcv = packed cell volume.
Microscopic picture:
b
c
Lysing amarae cells covered
No amarae cells.
with bacteria.
Clumped but healthy-looking amarae ceils.
pH's were >6. 7 in all flasks.
-------
Table 13. LB4 VS. N. AMARAE SE 110 IN VP MEDIUM
(POOR GROWTH OF BOTH STRAINS)
(ml/liter)
OJ
Day
N. amarae
Se 110
only
LB4
(bacterium)
only
Se 110 added day 0
+
LB4 added day 0
Se 110 added day 0 Se 110 added day 0
+ +
LB4 added day 1 LB4 added day 2
1
2
3
12.0
14.0
12.0
12.0
14.0
4
12.0
14. Ob
6.0d
,
14. Oc
10. Od
--
-
10. Od
pcv = packed cell volume.
Microscopic picture:
Lysing amarae.
c Clumping but healthy amarae.
"• Ghosts (empty cells) of amarae.
pH's were >7. 0 in all flasks.
-------
U)
Table 14. 100-9 VS. N. AMARAE SE 110 IN YCZ MEDIUM
pcva (ml/liter)
N. amarae Se 110 added day 0 Se 110 added day 0 Se 110 added day 0
Se 110 100-9 + -f f
Day only only 100-9 added day 0 100-9 added day 1 100-9 added day 2
1
2
3
5
50
120
84
78
4
2
2
N
8b
2C
2C
Nc
-
b
20
4C
Nc
-
-
102d
80d
st
pcv = packed cell volume.
Microscopic picture:
" Clumped amarae cells.
No to slight traces amarae cells.
Healthy amarae.
pH's in flasks were >6.0 throughout experiment.
N = Negligible.
-------
in detecting actively lytic nocardiophage and Bdellovibrio-type
bacteria active against nocardias. In particular, our repeated
failure to isolate nocardial antagonists from the non-foaming
Middletown plant makes it seem unlikely that the presence of lytic
microorganisms is responsible for the absence of nocardial foams
in some sewage treatment plants.
35
-------
SECTION VII
COMPARISON OF THE OPERATING CONDITIONS
OF FOAMING AND NON-FOAMING PLANTS
The purpose of this study was to compare the operating records of
similar wastewater treatment facilities: some having a significant
development of actinomycetic growth and the others free of this
problem. The aim of this comparison is to find parameters which
may have an effect on the actinomycetic growth pattern in waste-
water plants.
Wastewater treatment facilities selected for comparison
The wastewater treatment facilities that were selected for
comparison in this study are the Ocean Township Sewerage
Authority's Wastewater Treatment plant in Ocean Township,
Monmouth County, N. J. ; the Township of Middletown Sewerage
Authority's Wastewater Treatment Plant, in Middletown Township,
Monmouth County, N. J. and the Bernardsville Wastewater Treat-
ment Plant in Bernardsville, Somerset County, N. J. In addition,
some generalized comparisons were also made utilizing the waste-
water treatment plants located at Bordentown Township, N. J. , the
Madison-Chatham joint meeting plant at Chatham, N. J., and the
municipal wastewater treatment plant at Roxbury Township, N. J.
The wastewater treatment facilities utilized in this comparison were
all of the activated sludge process type. The treatment facilities at
Ocean Township consist of preliminary treatment, primary sedi-
mentation, activated sludge, effluent disinfection, and separate
anaerobic sludge digestion. The activated sludge process utilized is
of the sludge reaeration type (contact stabilization). Digested sludge
is dewatered via vacuum filtration and supernatant from the digesters
is oxidized with chlorine in a Purifax unit prior to being returned to
the preliminary treatment system. The Ocean Township Treatment
facility began operation in October, 1968.
36
-------
The Middletown Waste Treatment Facility began operation in July,
1971. This facility provides for preliminary treatment, primary
sedimentation, activated sludge, effluent disinfection, and separate
anaerobic digestion. The activated sludge system has been designed
for stepped aeration, however, conventional and sludge reaeration
can also be utilized. At the present time the conventional activated
sludge system is being employed at this facility. Digested sludge is
removed from the facilities via barging with ocean disposal. Super-
natant from the digestion system is returned to the preliminary
treatment facilities.
The Bernardsville Wastewater Treatment Facility was constructed in
1933. The facilities at the plant consist of preliminary treatment,
primary sedimentation, activated sludge, effluent disinfection and
anaerobic sludge digestion. The conventional activated sludge
process is utilized at this waste treatment facility. Digested sludge
is disposed via a dewatering centrifuge and supernatant is returned
to the primary treatment system.
Table 15 presents a comparison of the major aspects of these three
wastewater treatment facilities.
Actinomycetic problems
The treatment facility at Ocean Township has had an annual actinomy-
cetic foaming problem in the aeration tanks and final settling tanks
since 1970. The foam appears in late spring when the sewage
temperature rises above 55° F and continues through to the fall, when
the sewage temperature drops below 55 °F. Various means of
controlling the foam with water sprays, defoaming agents, and
chlorination have been unsuccessful. In more recent plant control
methods, the foam has been reduced to a manageable level by
reducing the solids and the dissolved oxygen in the aeration system.
It is assumed however, that some reduction of treatment efficiency
is also likely with this control method.
The treatment facilities at the Township of Middletown and at
Bernardsville, however, have not experienced the actinomycetic
foaming problems. Examinations of the activated sludge and scum
formed at the Bernardsville plant had not revealed the presence of
any actinomycete. Examinations of the Middletown sludge revealed
some actinomycete but these were of a very low concentration.
37
-------
Table 15. TREATMENT FACILITY COMPARISON
Item
Ocean Township
Middletown Township
Bernardsville
CO
Initial operation
Preliminary treat-
ment
Primary treatment
Primary sludge and
scum disposal
Secondary treatment
Aeration means
Waste sludge
disposal
Secondary sedi-
mentation
Activated sludge
return
October, 1968
Comminutor and grit
remover
Rectangular clarifier
To digester
Activated sludge
sludge-reaeration
(contact stabilization)
Mechanical turbine
To primaries
Circular clarifier with
scum removal
Variable speed pumps
July, 1971
Mechanical bar screen
and grit remover
Rectangular clarifier
To digester
Activated sludge
conventional
Mechanical turbine
To digester
Circular clarifier with
scum removal
Variable speed pumps
1933
Comminutor
Rectangular clarifier
To digester
Activated sludge
conventional
Diffused air
To digester
Square clarifier
Air ejector
-------
Table 15 (continued). TREATMENT FACILITY COMPARISON
Item
Qcean Towns hip
Middletown Township
Bernards ville
Secondary scum
disposal
Other treatment
Sludge treatment
Supernatant disposal
Digested sludge
disposal
To aeration tanks
Effluent chlorination
Anaerobic digesters
high rate
To wet well after
purifax treatment
Vacuum filter and land
fill
To digester
Effluent chlorination
Anaerobic digesters
high rate
To wet well
Ocean barging
None
Effluent chlorination
Anaerobic digesters
conventional
To primaries
Centrifuge and land
fill
-------
Technical review and comparison of operating records
The operating records covering a two year period for each of the
three wastewater treatment plants selected were obtained from the
operating agencies and reviewed. Based upon the completeness of the
available data and since experience in the New Jersey facilities has
indicated that the formation of actinomycetic foam occurred when the
temperature of the sewage is above 55 * F, the period of April through
November, 1972 was selected for detailed review and study.
In evaluating the records of this period, the following observations
were made regarding the wastewater characteristics at each treat-
ment facility as detailed in Table 16.
1. The average sewage temperature in the Middletown and Ocean
Township Plants were approximately the same and in the range of
63° to 66 *F.
2. The pH of the raw sewage for Ocean Township was somewhat
lower than the Middletown Township and Bernardsville Sewage
Treatment Plants. This was in the realm of a pH difference of 0. 1
to 0.2.
3. The BOD of the raw sewage and the BOD of the settled sewage
treated by the activated sludge system appeared to be reasonably
similar in the Ocean and Middletown Treatment Facilities. These
values for the raw BOD ranged between 187 and 217 mg/1 and for the
settled waste between 123 and 128 mg/1. The Bernardsville values
were estimated as 200 and 130 mg/1 respectfully.
4. The suspended solids content of the raw sewage and the settled
sewage in both the Middletown and Ocean Treatment Plants were
again reasonably similar. The raw sewage suspended solids for the
plants ranged between 161 and 247 and the settled sewage suspended
solids ranged between 107 and 141 mg/1. It is to be noted that the
higher values were found in the Middletown Township Plant and are
believed due to the influence of the returned supernatant from the
anaerobic digestion system..
In all three plants the wastewater received and treated at these
facilities was essentially domestic waste with very minor amounts of
industrial and/or commercial wastes.
40
-------
Table 16. SEWAGE CHARACTERISTICS
April to November, 1972
Item
General classification
Industrial and commercial
Average daily flow
Biochemical oxygen demand-
ing (BOD) raw
To aeration
Suspend solids raw
To aeration
Temperature
PH
Ocean Township
Domestic
Minor
2. 89 mgd
187 mg/1
123 mg/1
161 mg/1
107 mg/1
66'F
6.9
Middletown Township
Domestic
Minor
3. 94 mgd
21 7 mg/1
128 mg/1
247 mg/lb
141 mg/1
63°F
7.1
Bernardsville
Domestic
Minor
0.44 mgd
200 mg/1 Ea
130 mg/1 E
-
-
6.9+
a E = Estimated.
Possible excessive supernatant load.
-------
Further comparison of the three treatment facilities in regard to
process, loadings, and operating parameters are presented in Table
17 and can be summarized as follows:
1. The mixed liquor suspended solids maintained in the aeration
tanks ranged between approximately 2000 to approximately 3800
mg/1. In the reaeration portion of the Ocean Township facility the
suspended solids carried in the mixed liquor was approximately
6500 mg/1. A direct comparison with the Ocean Plant, in this
aspect, is difficult since the plant is separated into a mixing tank and
a stabilization tank with two separate concentrations of mixed liquor
suspended solids.
2. The dissolved oxygen maintained in the mixed liquor within the
aeration tanks for the Ocean and Middletown Treatment Plants ranged
between 2. 3 and 2. 6 mg/1. Although no information is available as to
the dissolved oxygen in the Bernardsville mixed liquor it is believed
that this figure would be very low.
3. The rate of return activated sludge utilized in the Ocean Township
and Middletown Treatment facilities is approximately the same in
ranges between 41 and 47%. The rate of return at the Bernardsville
facility is estimated to be 25%.
4. The organic loadings applied to the aeration tanks in the Ocean
and Middletown Plants were 14 and 10 Ibs. BOD per 100 Ibs. mixed
liquor suspended solids, respectively, and 30 Ibs. BOD per 100 Ibs.
MLSS in the Bernardsville Plant.
5. The detention times in the final settling tanks at the respective
plants were 2.2 hours for Ocean, 3. 7 hours for Middletown, and 2.6
for Bernardsville.
6. The sludge age in each of the treatment plants aeration tank were
7. 3 days for Ocean, 9.4 days for Middletown, and 3.1 days estimated
for Bernardsville.
In regard to a comparison of the physical plants (Table 15), each of
the three treatment plants has preliminary treatment and primary
sedimentation prior to the activated sludge system. The
Middletown and Bernardsville Treatment Plants operate a
conventional activated sludge system, whereas the Ocean Township
Treatment Plant operates under a sludge reaeration or contact
stabilization activated sludge system. The waste sludge from the
42
-------
Table 17. COMPARISON OF PROCESS, LOADING, AND OPERATING PARAMETERS
April to November 1972
Item
Primary tank-detention time
Aeration tank detention time
at Q
Activated sludge return rate
Mixed liquor suspended solids
Mixed liquor volatile suspended
solids
Mixed liquor dissolved oxygen
Loading #BOD/#MLSS
#BOD/#MLVSS
Ocean Township
1. 6 hrs
3. 9 hrs
47%
Mix. 2160 mg/1
Stab. 6535 mg/1
Mix. 3. 5 mg/1
Stab. 2.3 mg/i
0.14
Middletown Township Bernards ville
2.4 hrs
8.4 hrs
41%
3770 mg/1
2620, mg/1
2. 6 mg/1
0.10
0.14
2. 4 hrs
5. 1 hrs
25% Ea
1900 mg/1 E
Very low E
0.30E
-------
Table 17 (continued). COMPARISON OF PROCESS, LOADING, AND
OPERATING PARAMETERS
April to November 1972
Item
Ocean Township
Middletown Township
Bernardsville
Sludge age
7. 3 days
Secondary sedimentation tank 2. 2 hrs
detention time with return
9.4 days
(6.6 days VSS)
12.3 daysb
(8. 6 days VSS)
3.7 hrs
3. 1 days E
2.6 hrs E
E = Estimated.
Based on April to August - no excessive supernatant load.
-------
activated sludge system in the case of Ocean Township is returned
to the primaries. In the case of Middletown Township, the waste
activated is conveyed to the anaerobic digesters. The plants at
Ocean and Middletown are both provided with skimmers and scum
baffles on the final settling tanks. The scum collected from these
units is returned to the aeration tanks in the Ocean Plant; whereas,
the scum is directed to the anaerobic digesters in the Middletown
Plant. In all three plants, sludge removed from the system is
anaerobically digested with supernatant returned to the primary
treatment units. In the case of Ocean Township, the supernatant is
oxidized with chlorine prior to discharge into the primary units.
Comparison of domestic water supplies serving each area
A comparison of the water supplies that serve each of the treatment
plant service areas was considered, in order to determine whether
there were any unusual characteristic differences in the supplies
which might contribute to the development of the actinomycetic
growths. Analysis of the water supplies were obtained from the
purveyor serving each of the respective wastewater treatment plant
service areas and were evaluated for chemical composition. Table
18 lists the chemical analyses for the three areas.
In general, the evaluation indicates that the water supplies of the
Ocean Township, Middletown Township, and Bernardsville areas are
similar in chemical composition with the only notable exceptions
being a slightly higher nitrate nitrogen content in the Middletown and
Bernardsville waters over that of the water supplied to the Ocean
Township area. The analysis indicated the Ocean waters to contain
a nitrate nitrogen content of approximately 0. 6, the Middletown
waters 1.29, and the Bernardsville waters 15.5 mg/1. In a similar
manner, some differences were noted in the sodium content of the
waters; with Ocean Township having an average of 18, Middletown
having an average of 10 and Bernardsville having an average of 12
parts per million. The pH of each of the water supplies was 7. 7 for
Ocean, 7. 7 for Middletown and 7.8 for Bernardsville.
Summary and conclusions
The overall evaluation of the operating records and analysis at the
treatment plants in Middletown Township, Ocean Township and
Bernardsville does not disclose any significant difference in either
the raw sewage characteristics, the characteristics of the drinking
water supplied to the general area nor the operational
45
-------
Table 18. CHEMICAL ANALYSIS OF POTABLE WATER SUPPLIES
Substance
NH3 - N
NO3 - N
T - P
Hg
As
Cu
Pb
Se
Na
Zn
F"
Cd
pH
Ocean Township
<0.05
0.60
<0.05
<0. 00052
<0.0025
0.006
<0.0065
<0.0023
18
0. 0063
1.90
0.0011
7.7
Concentration (mg/1)
Middletown Township
<0.05
1.29
<0. 05
0.00063
<0.0025
0.029
0.008
<0.0023
9
0.0037
1.94
<0.001
7.7
Bernardsville
15.
0
0.
0
0.
0
12
0.
0.
0.
7.
5
009
01
05
11
002
8
(Based on analyses in period 1968 to 1973.)
-------
characteristics of the activated sludge processes used at each
treatment facility. There are some minor variations between the
parameters of loadings and the specific activated sludge process
used; however, these do not appear to be significant to the problem.
There is also some minor differentiation between the pH of the raw
sewage at each of the treatment facilities and a general indication
that perhaps the raw sewage at Ocean Township, which is experienc-
ing the actinomycetic growth problem, may be somewhat lower than
the Middletown and Bernardsville raw sewage. Again this minor
difference is relatively small and is not believed to be significant.
However, one item of difference was noted between the operation at
the Ocean Township facility and that at the Middletown and
Bernardsville facilities. This operational difference was in the
manner of disposal of the anaerobic supernatant for the digester
facilities. In the Bernardsville and Middletown facilities, the
supernatant is returned to the primary treatment units without any
pretreatment. This in turn creates a loading on the units and can be
seen in the,Middletown plant as a definite darkening of the mixed
liquor contents of the aeration tank. In the Ocean Township Plant,
the supernatant is either diverted from the treatment facility or is
treated with super-chlorination by a "purifax" unit prior to discharg-
ing into the primary unit. As such, the material exerts no loading or
other effects on the treatment process.
This treatment of anaerobic supernatant at the Ocean plant, in effect,
creates a similarity with several other plants observed to have an
actinomycetic foaming problem in the New Jersey area as noted in
the following:
a) The treatment facilities at Bordentown Township, are of the aerobic
type with aerobic digestion rather than anaerobic digestion. This
plant is experiencing problems with actinomycetic foaming.
b) Actinomycetic foaming was also observed at the Madison-
Chatham Joint Meeting Treatment Plant where supernatant from
anaerobic digesters was being conveyed directly to sand drying beds
and not being returned to the system.
c) In similar instances, the plants at Roxbury Twp., Matawan, and
East Windsor have had actinomycetic foaming and also have no
anaerobic digestion units.
47
-------
In addition, the latest developments at the Bernardsville plant
appear to also indicate a possible relationship between the absence
of anaerobic digestion and actinomycetic foaming. Recently this
plant process was modified and the anaerobic digestion facilities were
taken out of service. After a short operating period, some foaming
was observed in the aeration tanks. Examination of this foam
revealed the presence of actinomycetic growths.
In light of these findings, it appears conceivable that the presence
or absence of an anaerobic supernatant may have a bearing on
actinomycetic growth being experienced in some activated sludge
treatment plants. As can be seen by an examination of Table 1, all
the plants with foam problems either have no anaerobic digester or,
if they have one, do not return the untreated supernatant into the
system.
48
-------
SECTION VIII
ANTINGCARDIAL ACTIVITY OF ANAEROBIC
DIGESTER SUPERNATANT
We attempted to confirm a possible toxic effect of untreated
anaerobic digest on the growth of N. amarae.
In the first experiment, fresh, unchlorinated digest (from
Middletown, N. J.) was diluted in sterile water and distributed into
flasks of YCZ medium. These flasks were inoculated with 24 hr old
log phase N. amarae Se 110 cells and harvested periodically to
determine their cell content by packed cell volume. As can be seen
in Tables 19 and 20:
1. The anaerobic digest was toxic to amarae even at a final dilution
of 10-5 to 10-6, depending on the level of solids per ml in the
anaerobic digesj: used.
2. Removal of the solids of the anaerobic digest by filtering through
a Millipore Filter (0. 45 p. pore) removes the toxic effect.
3. Autoclaving the anaerobic digest partly destroys the toxic
principle.
As the pH's of the flasks in the first experiment on amarae inhibition
by anaerobic digest were quite low (/VpH 5. 7), a second experiment
was run using YCZ medium neutralized with excess CaCO- (Tables
21 and 22). Here again the Middletown digest showed toxicity when
diluted to 103'6 thus:
4. The inhibition of N. amarae by anaerobic digest is not caused by
lowered pH alone.
A sample of unchlorinated anaerobic digest supernatant from a second
plant (Ocean Township, N. J.) was assayed for toxicity to N. amarae
49
-------
en
o
Table 19. TOXICITY OF ANAEROBIC DIGEST (AD)a TO
N. AMARAE SE 110 IN YCZ MEDIUM
Total pcvb (ml/liter)
YCZ
control
/f* 1 1 /\
(Se 110
Day only)
1 44
2 116
3 70
4 68
5 76
6 70
25 mg solids (Iry
t> pcv = packed cell
YCZ
10"2 10~3 IO"4
10
50C
62C
90C
60C
44c
weight) per
volume.
10
40d
66d
80d
110d
60d
ml.
18
18d
90d
120d
100d
100d
+ AD diluted to
io-5 io-6
20 28
18e 20e
14e I6e
8e 30e
I6e I6e
8e I6e
io-7 io'8
44 46
64f 120f
62f 70f
80f 70f
68f 64f
76f ND
Microscopic picture:
c No N. amarae (yeasts + bacteria).
d 40% N. amarae
e 90% N. amarae
(estimated)
(estimated)
; 50% yeasts
; no yeasts,
, 10%
bacteria.
10% bacteria.
f 100% N. amarae.
-------
en
Table 20. TOXICITY OF AUTOCLAVED AND MILLIPORE FILTERED ANAEROBIC
DIGEST* (AD) TO N. AMARAE SE 110 IN YCZ MEDIUM
Total pcv (ml/liter)
Filtered AD
Day
1
2
3
4
YCZ
control
(Se 110
only)
60
150
90
84
YCZ + 10'1
dilution
millipore
filtered AD
60
130
70
60
YCZ
control
(Se 110
only)
56
104
ND
ND
Autoclaved AD
YCZ + 10-2
dilution
autoclaved
AD
10
86
ND
ND
YCZ + 10-3
dilution
autoclaved
AD
46
104
ND
ND
25 mg solids (dry weight) per ml.
pcv = packed cell volume.
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Table 21. TOXICITY OF ANAEROBIC DIGEST (AD) TO GROWTH OF
N» AMARAE SE 110 IN YCZ MEDIUM NEUTRALIZED WITH EXCESS CaCO,
Total pcv (ml/liter)
en
to
Day
1
2
3
5
7
Estimated
amarae
cellsc
YCZ + CaCO3
control
(Se 110 only)
16
40
44
60
52
100%
io-2
12
2
4
4
Neg.
5%
YCZ
io-3
16
4
16
20
16
10%
•f CaCO
io-4
16
20
20
18
4
30%
3+ AD
io-5
16
20
30
34
20
30%
diluted
io-6
8
16
16
20
20
30%
to
10- 7
16
20
12
8
20
70%
10~8
16
40
34
44
30
98%
25 mg solids (dry weight) per ml.
pcv = packed cell volume.
Other organisms were mostly bacteria.
-------
Table 22. TOXICITY OF ANAEROBIC DIGEST (AD)a TO GROWTH
OF 3M. AMARAE SE 110 IN YCZ MEDIUM NEUTRALIZED
WITH EXCESS CaCO
Corrected N. amarae pcv (ml/liter)c
Day
1
2
3
4
5
YCZ + CaGO3
(Se 110 only)
2
80
80
70
54
YCZ +
10-5
0
2
0
20
30
CaCO3
io-6
0
40
26
68
76
+ AD diluted to
10-7
0
90
80
64
60
10-8
0
90
70
72
70
rt
Contained 18 mg solids/ml (dry weight).
pcv = packed cell volume.
c Corrected by subtracting pcv figures obtained in YCZ + CaCO-
flasks inoculated with AD alone (measuring the natural
microbial population of the AD) from the total pcv (amarae +
other microorganisms). Corrected figures represent just the
growth of amarae.
53
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grown in YCZ + CaCO, as above. The digest showed toxicity even
when diluted to 10"', thus:
5. The toxicity of anaerobic digest supernatant to N. amarae is not
solely confined to one plant.
Attempt at isolation of the toxic principle
Solids from the anaerobic digest were extracted with methanol,
chloroform-methanol (2:1), isopropyl acetate or petroleum ether
(B. p. 30-40" C). Th&pH-ofthe mixture was 7.0. The extracts
were taken to dryness with nitrogen, dissolved in dimethyl sulfoxide
and Seitz-filtered. Added to YCZ liquid medium at a digest dilution
equivalent of 10~^ (see Table 23 for definition) no extract had
toxicity to growth of N. amarae Se 110. However, a chloroform-
methanol (2:1) extract of autoclaved anaerobic digest solids, was
dried und&r nitrogen, taken up in chloroform-methanol (2:1). Seitz-
filtered and assayed in YCZ versus N. amarae Se 110 at digest
dilution equivalents of 2 x 10" * and 10-1, it showed toxicity (Table
23). Thus:
6. Organic solvent extracts of anaerobic digest solids are not toxic
to N. amarae when assayed at a level equivalent to that normally
inhibitory when the solids themselves are used; however, an assay of
a chloroform-methanol (2:1) extract at higher concentrations shows
that the toxic factor can-be partially extracted, by organic solvents
under certain conditions.
Characterization of the toxic principle of anaerobic digest
The anaerobic digest was added to flasks of liquid YP and dilute YP
(YP/25) which contained hyphae of washed log phase N, amarae
Se 110 cells and these were shaken at 28" for 5 days. Although the
amarae hyphae appeared clumped they did not lyse. Attempts to
isolate organisms antagonistic to amarae from these flasks was
unsuccessful. Thus,
7. The toxic principle of anaerobic digest does not cause lysis of
amarae cells.
8. There do not appear to be organisms antagonistic to N. amarae
in anaerobic digest when tested under these conditions.
54
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Table 23. TOXICITY OF A CHLOROFORM-METHANOL
EXTRACT OF AUTOCLAVED ANAEROBIC DIGEST
SOLIDS TO N. AMARAE SE 110
pcva (ml/liter)
Day
1
2
3
8
a
pcv =
Solvent control
for
2x 10"1 10"1
16
82
86
82
packed cell
20
100
82
80
volume.
Digest dilution equivalent:
Solvent extract
(Digest dilution equivalent)
2X10-1 10'1
4
4
4
96
The extract coming
8
16
38
98
from the
amount of anaerobic digest solids contained in a given dilution
of the whole anaerobic digest supernatant.
55
-------
In general, it may be concluded that a thermolabile principle toxic
to Nocardia amarae is associated with the solids from anaerobic
digestion and that this principle is: 1) poorly soluble in water, 2)
soluble in organic solvents under certain conditions, and 3) capable
of being diluted to 10~^ or 10-? and still show toxic effects (dilution
depends on the amount of solids present in the original anaerobic
digest).
56
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SECTION IX
DISCUSSION
As we conclude this study, our final hypothesis is that nocardial
foams might be prevented by returning untreated anaerobic digester
supernatant into the system. This hypothesis is supported by the
following evidence: 1) as far as we know nocardial foaming occurs
only in plants which either do not have an anaerobic digester or in
plants having such facilities in which the supernatant is either not
returned or is returned only after some form of treatment such as
Purifax chlorination. 2) Samples from the supernatants of
anaerobic digesters from two different plants strongly inhibited the
in vitro growth of Nocardia amarae, the most common sewage
nocardia.
In general, this hypothesis makes sense from an historical point of
view. It is only recently that nocardial foams hav* been noticed by
plant operators j.n spite of the fact that the activated sludge process
has been known since 1914 (Anonymous, 1967). However, the
classical method of activated sludge treatment has included the return
of the supernatant from the anaerobic digesters into the system and
recently the trend has been to build more and more plants without
anaerobic digesters, to eliminate the digesters from those that have
them or to treat the supernatant before returning it into the system
in order to reduce the loading factor.
According to our hypothesis, it is thus not surprising that more and
more plants report nocardial foaming.
If one reviews the literature on the microbiology of activated sludge,
one is struck by the lack of attention paid to actinomycetes (Farquhar
and Boyle^; van Veen1**). We can predict that if action is not taken
to control nocardial foams, the actinomycetes will become a matter
of great concern to sanitation microbiologists.
57
-------
Logically the next steps in this study are, 1) the testing of our
hypothesis in cooperating plants and 2) the elucidation of the nature
of the nocardiotoxic compound. This last part of the study will be
especially important to operators of plants without anaerobic
digesters. If the nature of the toxic principle is known and if the
cost does not turn out to be prohibitive, one could conceivably add it
to the sewage of plants without anaerobic digesters.
58
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SECTION X
REFERENCES
1. Anonymous, "Pioneers of Activated Sludge: Arden, Lockett,
and Fowler, " The Surveyor and Municipal Engineer (London),
pp 28-29, 33, 17 June 1967.
2. El-Nakeeb, M. A., and Lechevalier, H. A., "Selective
Isolation of Aerobic Actinomycetes, " Appl. Microbiol, 11(2);
75-77, March 1963.
3. Farquhar, G. J., and Boyle, W. C., "Identification of
Filamentous Microorganisms in Activated Sludge, " J._..Wat»_
Pollut. Control Feder. U.S.A. ^3(4); 604-622, 1971.
4. Gordon, R. E., "Some Criteria for the Recognition of
Nocardia madurae, " J. Gen. Microbiol* (London), 45(2);
355-364, November 1966.
5. Gordon, R. E., and Horan, A. C., "Nocardia dassonvillei,
A Macroscopic Replica of Streptomycea griseus, " J. Gen.
Microbiol. (London), 50; 235-240, February 1968.
6. Gordon, R. E., and Mihm, J. M., "A Comparative Study of
Some Strains Received as Nocardiae, " J. Bacteriol. 73; 15-27,
January 1957.
7. Gordon, R. E., and Mihm, J. M., "Identification of Nocardia
caviae (Erikson) Nov. comb.," Ann. N. Y. Acad. Sci. 98(3);
628-636, August 1962.
59
-------
8. Gordon, R. E., and Smith, M. M., "Rapidly Growing, Acid
Fast Bacteria. I. Species Descriptions of Mycobacterium
phlei Lehmann and Neumann and Mycobacterium smegmatis
(Trevisan) Lehmann and Neumann, " J. Bacteriol. 66(1): 41-48,
July 1953.
9. Higgins, M. L. , and Lechevalier, M. P. , "Poorly Lytic
Bacteriophage From Dactyl ospo rangiumthailandensis, "
J. Virol. 3; 210-216, February 1969.
10. loneda, T., Lederer, E., and Rozani, J., "Sur La Structure
Des Diesters de Trehalose ('Cord Factors') Produits Par
Nocardia asteroides et Nocardia rhodochrous, " (On the Structure
of Diesters of Trehalose (Cord Factors) Produced by Nocardia
asteroides and Nocardia rhodochrous.) Chem. Phy s. Lipids
(Amsterdam). _4(3): 375-392, 1970.
11. Kolstad, R. A., and Bradley, S. G., "Factors Affecting
Replication of an Actinophage for Streptomycgs Venezuelan, "
Develop. Indust. Microbiol. 8; 198-205, 1967.
12. Krasnikov, E. I., Nesterenko, A., Romanovskaya, V. A., and
Kasumova, S. A., "Microorganisms of the Genera Nocardia
Trevisan and Mycobacterium Lehmann and Neumann Which
Utilize Natural and Individual Gaseous Hydrocarbons, "
Microbiology U. S. S. R. .40(2): 240-246, March-April 1971.
13. Kurup, P. V., Randhawa, H. S., Sandhu, R. S., and
Abraham, S., "Pathogenicity of Nocardia caviae, N. asteroides
and N. brasiliensis, " Mycopathol, Mycol. Appl. (The Hague).
40(2): 113-130, 1970.
14. Lechevalier, H. A., and Lechevalier, M. P., "A Critical
Evaluation of the Genera of Aerobic Actinomycetes, pp 393-405
in H. Prauser (ed.). " The Actinomycetales. Fisher. Gena.
1970.
15. Lechevalier, M. P., "Identification of Aerobic Actinomycetes
of Clinical Importance," J. Lab. Clin. Med. 71; 934-944, 1968.
16. Lechevalier, M. P., Koran, A. C., and Lechevalier, H.
"Lipid Composition in the Classification of Nocardiae and
Mycobacteria," J. Bacteriol. 105; 313-318, January 1971.
60
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17. Lechevalier, M. P., and Lechevalier, H. A., "Nocardia
amarae sp. nov., An Actinomycete Common in Foaming
Activated Sludge, " Inter. J. System Bacteriol. 24(2): 278-288,
April 1974.
18. Van Veen, W. L., "Bacteriology of Activated Sludge, In
Particular the Filamentous Bacteria, " Antonie van
Leeuwenhoek (Amsterdam). 39(2): 189-205, 1973.
19. Wells, W. N., and Garrett, M. T., "Getting the Most From
an Activated Sludge Plant, " Public Works, pp 63-68, May 1971.
61
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SECTION XI
LIST OF INVENTIONS
Lechevalier, M. P., and Lechevalier, H. A. . Nocardia amarae
sp. nov., an actinomycete common in foaming activated sludge.
Intern. J. Systematic Bacteriol. 24: 278-288, 1974.
62
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/2-75-031
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
ACTINOMYCETES OF SEWAGE-TREATMENT PLANTS
5. REPORT DATE
September 1975 (Issuing .Date")
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Hubert A. Lechevalier
8. PERFORMING ORGANIZATION REPORT NO,
9, PERFORMING ORGANIZATION NAME AND ADDRESS
Waksman Institute of Microbiology
Rutgers, the State University of New Jersey
New Brunswick, New Jersey 08903
10. PROGRAM ELEMENT NO.
1BB043 (ROAP 21-ASR, Task 038)
11. CONTRACT/GRANT NO.
R802003 (17050 GUJ)
12. SPONSORING AGENCY NAME AND ADDRESS
Municipal Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final. 1971-1974
14. SPONSORING AGENCY CODE
PA-ORD
15. SUPPLEMENTARY NOTES
16, ABSTRACT
In some sewage-treatment plants of the activated sludge type, a thick foam may be
formed at the surface of the secondary aeration and settling tanks. Such foams have
often been found to be rich in actinomycetes. This re'port covers the work done on
this problem between April 1971 and May 1974 . Over 250 strains of actinomycetes
have been isolated from foams or activated sludge from 19 different sewage-treatment
plants located in 8 states. The actinomycete most commonly associated with foams is
a previously undescribed Nocardia which has been given the name N. amarae. It has
been demonstrated experimentally in the laboratory that N. amarae may cause the kind
of foam observed in the plants. Factors affecting the growth of N. amarae have been
studied and a method of control of the foam by addition of digester supernatant to
the activated sludge is proposed.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
*Actinomycetales, *Nocardia, Activated
sludge process, Foam—cellular materials,
Microorganism control (sewage), *Aeration
tanks
Digester supernatant
13B
8. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
71
20. SECURITY CLASS (TMspage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
63
*USQPO: 1975 - 657-695/3306 Region 5-11
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