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from the Unox system does contain many actinomycete colonies.
2. The Marox system mixed liquor contains two types of filamen-
tous microorganisms. One. type appears as a series of rod-
shaped cells about 1.5 um in diameter. It was present in
the Marox mixed liquor during May and June 1976 and is still
present. The other type is more slender and appears as a
continuous filament.
3. Both types are also present in the air system mixed liquor.
4. Both types are also present in the influent, Actinomycete
colonies were also seen in the influent, but they were
judged to be derived from the filtrate from the. Unox sludge.
5. The activated sludges in both the Marox and air systems appear
to be poorly flocculated.
I prepared streak plates on corn meal agar and glucose-peptone-yeast
extract agar of the secondary influent, air mixed liquor., and Marox mixed
liquor. I also brought back samples of these materials for further examin-
ation. My laboratory results, so far, are as follows:
1. Filamentous bacteria did not develop on the streak plates.
However, I have the filamentous bacteria growing in a liquid
enrichment culture. I may be able to Isolate them in the
next few weeks.
2. The streak plates did develop unusually large numbers of
fungi. The dominant types are those which produce arthrospores
(Geotrichun, Oidiodendron, and Trtchosporon). I do have
these organisms in pure culture.
3. The arthrosporic fungi could give the appearance of chains of
rod-shaped cells in liquid culture; thus, these fungi could
account for the filamentous organisms which have been present
in the mixed luqour since last May.
4. The arthrosporic fungi are also present in the secondary
influent.
I have encountered arthrosporic fungi as nuisance organisms in activated
sludge twice before. They are extremely difficult to get rid of, but the
activated sludge system can be operated to give satisfactory performance
even when they are present. The other filamentous organism may be even more
of a problem, but I don't want to speculate further until I can find out
more about it.
I recommend that we undertake a program to accomplish the following
objectives:
1. Operate the system to give satisfactory performance in spite
of the high SVI and in spite of the poor flocculation.
97
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2. Determine If the same problem exists in the air system as in
the Marox system.
3. Determine if the secondary influent is the primary source of
filamentous organisms.
4. Find a technique for improving the flocculation of the activated
sludge.
5. Find a method of reducing the SVI.
The first thing we need is a method of measuring SVI which gives repro-
ducible results and which gives an SVI value independent of the mixed liquor
suspended solids concentration. I recommend using a one liter cylinder with
a stirrer operated at one rpm or less. 1 also recommend that you acquire
equipment so that four settled volume tests can be run simultaneously and
that SVI versus suspended solids concentration curves be developed for both
the Marox and air sludges. I discussed this test procedure with Dr. Rise
and Mr. Parker vhen T. was out there last Monday. If it can be shown that
the SVI versus suspended solids concentration curves are the same for both
th& Marox and air systems, this would go a long way toward demonstrating
that the problems are the same. In any case we need to get consistant SVI
values to deal with (-he problem in the Marox system.
We need to have a measure of how well flocculated the sludges are. The
only reasonable measure of flocculation that anyone has come up with so far
is the supernatant suspended solids from the settled volume test. However,
mixed liquor suspended solids concentration has an influent on the super-
natent suspended solids. After the SVI versus suspended solids curve has
been determined, pick a solids concentration (about 3000 mg/1) which gives
2 consistant settled volume and use it as a standard solids concentration
for determining both settled volume and supernatent suspended solids. After
several days of supernatent suspended solids data have been collected plot
these values versus the respective effluent suspended solids. There should
be a linear proportionality between the two values if the secondary settling
tank is being operated properly.
In order to stabilize operation of the system I recommend that the
secondary influent be introduced at a constant rate (the large daily fluctu-
ations in the aeration tank suspended solids may be part of the problem).
If it is impossible to obtain a constant influent rate, adjust the return
sludge flow rate in proportion to the influent flow rate. The mixed liquor
suspended solids should be
MLSS = Q ^ R (RAS)
Have your operator make this calculation each day and check it against
the measured MLSS. It will help him understand your operational objectives.
I recommend that the return sludge flow rate be made equal to the
influent flow rate (R=Q). Have the operator calculate a solids balance on
the secondary settling tank each day (pounds of solids in •* pounds of solids
out) and measure the height of the sludge blanket each day. Once the sludge
98
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blanket is gone and the solids balance works (within 10%) reduce the return
sludge rate slowly. I recommend operating at R = 0.9 Q for a few days to
see how the system responds, then going to R «= 0.8 Q for a few days, etc.
When the sludge blanket starts to build up again do not reduce R aj^V further.
Try to hold the sludge blanket at least two feet below the overflow'weirs.
I recommend that you allow the solids to build up in the system and try
to operate between 5000 and 6000 mg/1 in the aeration tank. This will re-
duce the F/M ratio which should improve flocculation. It will also increase
the SRT which could encourage the growth of actinoitycetes in your sludge.
Continue the periodic microscopic examination of your sludge. If the
actinomycetes show up reduce the SRT even if it requires increasing the
F/M ratio.
I suggest that you send me weekly data summaries for my review. If
an operating problem arises suddenly, you can call me and I will respond as
best I can on the telephone.
I believe that we need to isolate and identify the other filamentous
organism from your sludge in order to determine if the air system has the
same problem and if the source of the organism is the'secondary influent.
I will be working with my enrichment cultures for the next couple of weeks
to see what 1 can isolate. If I am able to isolate the organism, I will
need to get additional samples to demonstrate that it really is the
organism in your system. If I don't isolate the organism, I will need
additional samples for trying different isolation techniques.
The arthrosporic fungi need to be investigated further. There are
techniques tor obtaining reasonably precise counts of these organisms in
wastewater and iii sludge. However, this would require a significant amount
of laboratory time. I could get a student here and train him for that type
of work, but, if'the laboratory work were done here, the expense of trans-
porting the samples would be rather large. It might be more reasonable for
you to get a laboratory technician whom I could train to work out there.
These recommendations and suggestions should give you a good deal to
think about and I expect that you will have several questions. I will be
expecting a response from you after you have had time to consider all these
points. Looking ahead, it occurs to me that Monday, Jan. 31, might be a
suitable date for me to make another visit to Denver so that we could
review the progress and I could obtain additional samples.
Sincerely,
V. 0. Pipes
Beta Professor of Ecology
Department of Biological Sciences
WOP/sls
99
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WEBUEY O. PIPES. PH.D.-
»7 SHADY HILL. ROAD
MEDIA, PENNSYLVANIA IOOO3
(»IB) l_O •-**«•
February 1, 1977
Mr. Richard B. Weber
3999 South Kariposa Street
Englewood, Colorado 80110
Dear Mr. Weber:
Enclosed is ray report on examination of the samples which were sent
to me during January. The fungus counts in the system are high but
apparently not high enough to indicate that fungi are a major part of
the problem. The filamentous bacteria grow very slowly or not at all.
in the enrichment cultures T have tried so far. It'appears that they
will be difficult to isolate and identify them but 1 believe that we
should continue to try in order to show that the same problems exist
in both the air and Marox systems and to devise a ready-made solution
in case this probleia ever occurs in another Marox system.
In thie type of situation it is important to be able to correlate
the laboratory observations with the operating data and with experiments
with sludge samples. I would like to have the opportunity to analyze
the operating data for January for both the air and Marox systems.
Once your laboratory personnel arc set up to run settling tests at dif-
ferent solids concentrations, as I recommended Jn my letter of Jan." 17, 1977,
and we can get some data to determine the similarity of settling character-
istics of the air and Marox sludges, I will be able to recommend some
laboratory experiments which can be used to test some possible solutions
to the bulking problem. I have always found it expedient to try out ideas
in laboratory tests before trying them in the plant because a variety of
ideas can be tried out at the same time without running the risk of
upsetting plant performance.
There are no obvious simple solutions to this problem and it may take
Borne time and effort to find and demonstrate a solution. It is possible
that we may stumble across a simple solution such as just reducing the
tine the sludge is out of th? aeration tank; however, at this point we
can'/count on that. We really need operating data foi eoir.e extended period
when the operating conditions .ire not changed frequently and then a care-
ful trial of possible solutions suggested by knowledge of the nature of
the filamentous organisms .".r.d/or by laboratory tests with sludga samples.
Sincerely,
M o. /y'«*
W. 0. Vipes, Ph.D.
Bets Vxoffssor of Ecology
100
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W. 0. Pipes
Jan. 31, 1977
FMC-DENVER PROJECT
Examination of Samples for Filamentous Microorganisms
Introduction
This report covers the examination of samples collected from the
Metro Denver North plant on Jan. 10, Jan. 19, Jan. 25, and Jan. 27, 1977.
There is a demonstration Marox oxygen activated sludge system operating
at this plant. Both the air and the Marox systems have had operational
difficulties over the past six months apparently due to poorly floccu-
lated activated sludge and a growth of excessive numbers of filamentous
microorganisms which causes high SVI values. Ths objectives of these
examinations were to determine the indentities of the filamentous micro-
organisms and obtain some clues to the nature of the problem so that po-
tential solutions can be developed.
Samples
Jan. 10, 1977 Microscopic examination of the air and Marox mixed
liquor samples showed that the floe particles in both systems were loose
and open textured. Two types of filamentous microorganisms were seen in
both mixed liquor samples. Some filamentous microorganisms were seen in
the secondary influent sample.
Both mixed liquor samples and the secondary influent sample were
streaked on glucose-peptone-yeast extract agar (GPY) and on corn meal agar.
Filamentous bacteria did not grow on these plates; however, several fungus
colonies developed. Many of the fungi produced arthrospores or blasto-
spores or' both. On the four secondary influent streak plates a total of
15 fungus colonies developed of which 8 produced arthrospores, 3 produced
blastospores, and 2 developed both arthrospores and blastospores. On the
101
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2.
four air mixed liquor streak plates a total of 25 fungus colonies developed
of which 6 produced arthrospores, 12 pTeoduced blastospores and 2 produced
both. On the four Marox mixed liquor streak plates a total of—19 fungus
colonies developed of which 4 produced arthrospores, A produced blastospores,
and 2 produced both. Although these fungus counts can not be related to
the volume of liquid which contained them, they are unusually high numbers
of fungus colonies for streak plates. Arthrosporic fungi can sometimes
look very much like filamentous bacteria when growing in a liquid. Thus,
it was decided that future samples should be examined for fungus colony
counts.
The two types of filamentous bacteria from the mixed liquor samples were
observed to be growing on GPY plates which were flooded with 4 ml of mixed
liquor each. Many other microorganisms were also growing in these cultures.
Jan. 19, 1977 Samples of air mixed liquor and Marox mixed liquor col-
lected on Jan. 19 were received that evening and examined the following day.
The floe particles were still loose and open textured. Both types of
filamentous microorganisms were still present. A few short chains of what
could be arthrospores were seen in the Marox mixed liquor but not in the
air system mixed liquor. Clumps of cells which could be blastospores were
seen in both mixed liquor samples.
Pour plates of serial dilutions; 0.1, 0.01, 0.001, and 0.0001 ml per
plate; were made of the two mixed liquor samples on CPY agar. Microscopic
counts of floe particles in the agar and of floe particles developing
fungus mycelia were made on random fields after four days of incubation
et 20 C. The purpose of these counts was to determine the faction of the
floe particles which developed mycelia and thus to test the hypothesis
that most of the floe particles were developing around fungus mycelia.
In the Marox mixed liquor 1.3% of the floe particles developed mycelia and
5j> the air system mixed liquor 0.4% of the floe particles developed mycelia.
From this,, the hypothesis was rejected.
Total counts of fungus colonies developing on the pour plates were made.
102
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3.
In the air system mixed liquor, the fungus colony count was 1500 per ml
of which 500 had^arthrospores and 900 had blastospores. In the Marox
nixed liquor, the funcus colony count wivs 900 per ml of which 300 had
arthrospores and 600 had blastospores. Four plates using GPY agar is
not the best method of obtaining fungus colony counts, so any conclusions
about the numbers and role of the fungi in the systems were deferred
until after examination of additional samples.
Liquid enrichment cultures were made up in GPY broth and lactate-
glycerol broth and intioculatcd with mixed liquor from the Marox and air
systems. These enrichment cultures were aerated and incubated at 20 C.
Microscopic examination after four days of incubation showed that the
filamentous bacteria were not growing in these cultures.
Hicroscopic examination of the mixed liquor samples on Jan. 24, after
they had been sitting at room temperature for five days without aeration
showed that the two types of filamentous microorganisms were growing more
than the other sludge, organisms. The larger organism had formed filaments
up to 300 uro long. This organism is greater than 1 urn in diameter and
forms a continuous tube, not divided into cells, having occasional dark
inclusions. The other filamentous organism is less than 1 \im in diameter,
has many dark inclusions, and forms short filaments (about 40 pro long)
sometimes in a rosette formation. From the observation that these orga-
nisms are growing in the anaerated mixed liquor samples, it was hypothesized
that they are microaerophylic. The dark inclusions could be sulfur deposits.
Jan. 25, 1977 Samples of air mixed liquor,.Marox mixed liquor, and
raw sewage collected around noon were received at 7:00 p.m. and examined
that evening. Three samples of raw sewage were received; labeled Delganey
structure, Gibville Main, and Broadway Relief Main. The sample from the
Delganey structure is a mixture of sewage from four Interceptors two of
which are the Gibville Main and the Broadway Relief Main.
Microscopic examination of the mixed liquor showed that both types of
filamentous organisms were still present in both samples. The Marox mixed
103
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4.
liqu-ir appeared to be more poorly flocculated and the air mixed liquor
better flocculated than previous samples.
Spread plates were made of serial dilutions; 0.1, 0.01, 0.001, and
0.0001 ml per plate; 'on Cooke's Rose Bengal Agar with chlorotetrocycline.
This medium is the best known for Isolation of fungi from water and waste-
water. The fungus colony counts at four days of incubation at 20 C are
presented in Table I.
TABLE I
Fungus Colony Counts (per ml) on .Jan. 25 Samples
Sample Counts
Total Colonies Colonies Other
with with Fungus
Arttvrospores Blastospores Colonies
Air ML 2100 1300 800
Marox ML 1600 400 600 600
Delganey 1000 800 200
Gibville 300 100 200
Broadway 700 100 400 200
These counts are somewhat higher than are usually found in raw sewage
and mixed liquor samples. The question of their significance in the
system is deferred for the discussion below.
In an attempt to find a suitable enrichment techniques for the filamen-
tous orgeniGir.p seen in the mixed liquor samples, liquid cultures were made
up using 1) sodium acetate, 2) sodium acetate plus sodium sulfide, 3) sodium
acetate plus peptone, and 4) glucose, peptone, and sodium sulfide. The
cultures were incubated at 20°C and not aerated. The two types of filamen-
tous micro.organisms grew in all four enrichment media but only in the
cultures with glucose, peptone, and sulfide did they grow more than in the
unenriched, unaerated mixed liquor.
104
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5.
Heavy streaks of mixed liquor were placed on acetate-sulfide agar and
these plates were Incubated in air at 20°C. The filamentous microorganisms
grew for two days on these plates but other microorganisms prew wore
vigorously and after four days the filaments were breaking up.
Two hundred ml of sewage was placed in the bottom of a desslcator and
1 gm of dextrose, 1 gm of peptone, and 500 mg of M SO, were added to
stimulate the production of H~ S. Heavy streaks of mixed liquor were
placed on GPY agar and on acetate sulfide agar and these plates were incu-
bated in the dessicator for four days. The filamentous microorganisms
grew more vigorously on these plates than on the acetate-sulfide plates
Incubated in air. However, several protozoans developed on these plates
and spread the other bacteria to the extent that the isolation of the
filamentous microorganisms is impossible.
Jan. 27. 1977 Samples collected in the morning were received at
5:30 p.m. and examined that evening. The samples were air mixed liquor,
Marox mixed liquor, secondary influent and raw sewage.
The microscopic examination showed the mixed liquor samples to be
quite similar to those of Jan. 25. Both types of filamentous microorganisms
were still present.
Spread plates were made of serial dilutions; 0.1, 0.01, 0.001, and
0.0001 ml per plate; on Cooke's Rose Bengal Agar with chlorotetracycline.
The fungus colony counts at four days of incubation are presented in
Table II.
105
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TABLE II
Fungus Colony Counts (per ml) on Jan. 27 Samples
Sample Counts
Total Colonies Colonies Other
with with Fungus
Arthrospores Blastospores Colonies
Air ML 4300 400 2700 1200
Marox ML 5200 300 3100 1800
Secondary
Influent 1200 100 400 700
Raw
Sewage 300 100 200
Discussion
'Fungi The loose, open texture of the floe particles suggested that
the floe might be fromed by bacterial cells adhering to the outside of
fungus mycelia. The counts made on the pour plates of the Jan. 19 samples
shows that this is not the case. The vast majority of the floe particles
do not contain fungi. Some other explanation must be sought for the poor
flocculation.
The usual range for fungus colony counts both on raw sewage and on
mixed liquor samples is 10 to 100 colonies per ml. I have previously seen
mixed liquor samples which had obvious fungus myeella1 mats and in which
the fungus colony counts were in the range of 10,000 to 100,000 per ml.
In those cases the fungi clearly were associated with poor flocculation
and a high SVI.
In this case the fungus colony counts in the raw wastewater are in
the range of 100 to 1,000 per ml and in the mixed liquor samples are in
the range of 1,000 to 10,000 per ml. The only count made on the secondary
influent showed more fungus colonies than the influent. Except for the
samples of Jan. 27 the majority of the fungi are those which produce
blastospores. The blastosporic fungi are commonly called yeast and,
106
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7.
although they are not uncommon In sewage, It is a bit unusual to find them
making up over half of the fungi present.
Whether the fungi are growing in the aeration tank or not is .not clear
from these data. The fungus colony counts in the mixed liquor are almost
10 times higher than those of the raw sewage but the solids in the mixed
liquor should be about 10 times higher than in the raw sewage. Although
only one fungus colony count was made on the secondary influent the results
are suggestive of some source of fungus growth within the plant. There
is no other apparent reason tor a higher fungus count in the secondary
influent than in the raw wastewater.
At this point, it is apparent that the filamentous microorganisms in
the mixed liquor are not fungi. Further investigation of fungi in the
activated sludge system does not seem warranted except for investigation
of a possible source of fungi somewhere between the raw sewage and the
secondary influent.
Bacteria The two types of filamentous microorganisms in the mixed
liquor are bacteria. They do not grow on an agar medium with a simple
'substrate and the usual array of growth factors. They are not
Sphaerotilus natans. Bacillus cereus, Vitreoscilla, or Cytophaga. They
do not form true branches; thus, they are not any of the actinomycetes.
They seem to be stimulated by sulfide ion and thus could belong in the
Begglatoa - Thiothrix group. However, this awaits further confirmation.
The filamentous organisms appear to be microaerophylic because they
grow more vigorously in unaerated cultures. Why microaerophylic organisms
should be growing in an oxygen activated sludge system is rather puzzellng.
The only apparent reason is that there must be periods of anaerobic
conditions in the secondary settling tank and the return sludge lines
where they have an opportunity to grow.
Recommendations
1. In seeking a solution to a case of filamentous bulking when
-------
the causative organisms can not be isolated immediately, it is
very important to analyze the operating data to determine how
the activated sludge reacts under different conditions. I
recommend that the operating data for the month of January
for both the Marox and air systems be supplied to me for my
analysis.
2. It appears that the sludges in both the air system and the
Marox system are similar. The tests to demonstrate that they
do have stellar settling and flocculation characteristics should
be carried out as soon as possible. Please refer to my letter
of Jan. 17.
3. The entire flow diagram for the Metro Denver North Plant
should be analyzed to determine if there is a potential source
of fungi between the raw sewage and secondary influent.
A. From the information gleaned about the filamentous micro-
organisms in the nixed liquor, it appears that one possible
solution to the high sludge volume index could be to reduce
the amount of time that the sludge is held in the secondary
settling tank and the return sludge lines.
5. The efforts to isolate and identify the filamentous
microorganisms in the sludge should continue. Identification
of these organisms is essential for demonstrating thut the
problem in the Marox system is the same as in the air system
and for development of a ready-made solution for the problem if
it ever is encountered In other Marox systems.
108
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v-.l
torch 16, 1977
Mr. Richard B. Weber
Operations Manager
Havox Diffusion Systems
FMC Corporation
3999 South Marlposa Street
Enplewood, Colorado 80110
Dear Mr. Weber:
Enclosed Is a report on try exanlnatlon of the samples of March 10, 1977.
It Is claar that the filamentous organisms present now are different from
those present In January. The air systems are growing an organism tentatively
identified as Sph&erotiluE natans which does not appear to be growing In the
Mevox system. The fact that Sphaerotilus Is growing in the air eyatta but
not in the parallel Karox system could be quite significant if It continues.
The Marox mixed liquor contains large numbers of short, Irregularly
bent filaments. I have had no success In trying to prow this organism, It
could bo the sane organism which was present in the 1 farox nixed liquor during
June last year if I remember the stained slides from that period properly.
The filaments are ehort enough that they nay not interfer very much with
settling and compaction.
Sincerely,
W. 0. Pipes, Ph.D.
Bet* Professor of Ecology
WOP/fllB
enclosure
tic! Dr. Ratph His*
109
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FMC - DKNVER PROJECT
EXAMINATION OF MARCH 10 SAMPLES
Four samples were received on the evening of March 10, 1977; namely, Marox
mixed liquor, air mixed liquor from area 2, air mixed liquor from area 4, and
secondary influent.
Microscopic Exarnination
The Marox mixed liquor was poorly flocculated with many free bacterial cells
and the floe particles were loose, network-like structures. Many free swimming
and stalked ciliates were present. These protozoc are usually associated with
a well flocculated sludge and probably indicate that the flocculation will
improve. One filamentous organisra was present in large numbers. It is greater
than 1 urn in diameter and forms short, irregularly bent filaraents with dark
inclusions. It is an organism which was not present in any of the samples
examined during January. No other filamentous organisms.were observed.
The Marox nixed liquor was reexamined after sitting underated for four days.
The. short irregularly bent filaments were still present but gave no appearance
of having grown. A much longer, more slender filamentous organism had appeared.
It appeared to be a sheathed bacterium.
The air mixed liquor from area 2 was somewhat'better flocculated than the
Marox mixed liquor but not well flocculated. A variety of protozoa were seen
but noue in large numbers. Two filamentous organisms were seen. The short,
irregularly bent filaments seen in the llarox mixed liquor were present in small
numbers. The dominant filamentous organism was less than 1 vim in diameter and
appeared to be a sheathed bacterium. Reexamination of the sample after four
days without aeration showed that the longer, more slender, filamentous organism
had grown and many more filaments were present.
The air mixed liquor from area 4 was better flocculated than the other two
samples. However, it had many free swimming flagellated organisms which are
usually indicative of poor flocculation and low dissolved oxygen concentration.
Both types of filamentous organisms were present. After the sample had been
left four days without aeration the longer, more slender filamentous organism
had grown and many more filaments were present. Shorter filaments of the raore
slender organism showed motility.
The secondary influent sample showed both types of organisms present in
good numbers. This is probably due to some recirculation of material from the
activated sludge.
Sludge Test
The lysozyrae - detergent test was performed on all three sludge samples on
March 11, 1977. The longer, more slender organism is definately a sheathed
bacterium, probably Sphacrotilus natans. The irregularly bent filaments were
dissolved completely.
110
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2.
Cultures
Spread plates were made of serial dilutions (0.1, 0.01, vnd 0.001 ml per
plate) on Cooke's rose bengal agar, GPY agar, AcS agar, and tryptacase soy
agar (TSA). The fungus colony counts were in the same range ac the January
counts. It was not possible to obtain counts of filamentous bacterial colonies
due to overgrowth of the plates by fungi and protozoa.
Streak pJates were made on GPY, AcS, and TSA media. The short, irregularly
bent fil?ments started to grow on the AcS plates but were overgrown by other
bacteria in four days. Colonies of the sheathed bacterium were isolated from
TSA plates.
Summary
In terms of operational effect, the most disturbing feature of .the Marox
mixed liquor is the poor floccvlation. This tray be a feature of the start-up
process and the flocculation could improve greatly. However, if the floccula-
tion does not improve the F:M ratio should be reduced.
The sheathed filamentous bacterium in the air mixed liquor is tentatively
identified as Sphaerotilus natans. It definately was not present in the samples
examined during January. The fact that it is growing in the air systems but
not in the Marox system could be extremely significant. This needs more
.documentation.
The irregularly bent, filamentous organism which dominates the Marox mixed
liquor appears to be different from the two organisms which were present in
January. It could correspond to one of the filamentous organisms seen on the
stained slides from last summer. I have no information as to why it would be
showing up again at this time.
ni
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Department of Biological Sciences
(215) 895-2624
drexel university • Philadelphia 19104 • 215-895-2000
March 24, 1977
Mr. Richard B. Weber
Operations Manager
Marox Diffusion Systems
FMC Corporation
3999 South Mariposa Street
Englewood, Colorado 80110
Dear Mr. Weber:
This will complete my report on the examination of the samples received
on February 23, 1977. You may recall that in my report of March 2, 1977
I mentioned that I had made pour plates with nutrient agar to obtain actin-
omycete counts but that it would take four weeks for the colonies to develop.
The Englevood sewage had an actinomycete count of 10 per ml. Of the
two samples from the Metro Denver North Plant, the air mixed liquor had a
count of 300 per ml and the Marox Pilot Plant had a count of 3000 per ml.
These counts are- not high enough to indicate an operational problem, .how-
ever, they do indicate the potential for developing an actinomycete problem.
Keeping the sludge residence time in the activated sludge systems to less
than 12 days should prevent the problem from developing.
Sincerely,
W. 0. Pipes, Ph.D.
Betz Professor of Ecology
WOP/sls
cc: Dr. Ralph E. Rise
112
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Department of Biological Sciences
(215) 895-2624
drexel university • Philadelphia 19104 • 215-395-2000
July 19, 1977
Mr. Richard B. Weber
Operations Manager
Marox Diffusion Systems
KSC Corporation
3999 South Mariposa Street
Englewood, Colorado 80110
Dear Mr. Weber:
Enclosed is a report on my examination of the samples which were sent
to me on June 16, 1977. The Marox mixed liquor at that time was in better
condition than the air mixed liquor but 1 did not find anything unusual
about the microbiology of either sample.
I am also enclosing a summary of my findings on all the samples for
the January-June period. The summary describes some culture work which we
did during March and April and which had not been previously reported to
you. I believe that the filamentous bacterium which was causing the
problem last winter is an acid-producing, sheathed organism which has not
previously been described. However, there is another filamentous bacterium
which is still present in both systems and we never have been able to do
any culture work with it. Thus, I am not able to reach any firm conclusions.
Sincerely,
Wesley 0. Pipes, Ph.D.
Betz Professor of Ecology
WOP/sls
enclosure
cc: Dr. Ralph E. Hise
113
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FMC - DENVER PROJECT
Samples of .June 16, 1977
On June 16, samples from the Metro Denver North plant were received.
The samples were from the Marox system basin 11, the air system basin 10,
and the secondary influent flowing into basin 11. Spread plates of serial
dilutions (0.1, 0.01, 0.001, and 0.0001 ml per plate) were made on Cooke's
Rose Bengal agar, on glucose-peptone-yeast extract (GPY) agar, on acetate-
sulfide (AcS), and on trypticase soy (TS) agar. Heavy streaks of mixed
liquor samples were made on GPY agar and AcS agar. All plates were incu-
bated at 20°C.
An extra sample consisting of algae from the secondary settling tank
was also received. This sample was examined microscopically and then
streaked on GPY and AcS agar plates.
Microscopic Fxamination
The Marox mixed liquor sample showed a well flocculated sludge but the
particles were sir^all and stringy .(tassel-like). Many free swimming and
stalked ciliates were seen indicating a "well-oxidized" sludge. Only one
type of filamentous organise was seen. It was apparent as the short,
irregularly bent filament with dark inclusions which is more than one
micrometer in diaoieter and which has been seen in all the mixed liquor
samples from this plant. It apparently does not interfere with the settling
characteristics of the sludge.
The mixed liquor from the air system showed a loose textured sludge with
very small, stringy particles. The sludge was highly filamentous and the
filaments appear to be the same type as seen in the Marox system. Only a
few ciliated protozoa were seen.
The secondary influent samples had only a small amount of suspended
material in it. A few filair-uitous bacteria of the same type seen in the
mixed liquor were present. .he filaments in the secondary infJuent are
much, much longer than those in the njixed liquor.
The algap. from the secondary settling tank consisted of very long
filaments of Ulothrix, many green flogellated algae of the genus Euglena,
and many desmids of the genus Closterium. These are common types of
algae often found around sewage outfalls. Ulothrix grows attached to
surfaces and forms a microhabitat which provides conditions suitable for
the other algae. Many protozoa were also associated with the algne as
were filamentous bacteria of the type seen in the mixed liquor. A few large
filaments showed numerous dark inclusions and a definate gliding motility.
They are definately Eeggiatoa but they were not found in the mixed liquor
camples. Streak plates were made on GPY agar and AcS agar in an attempt
to isolate the Beggiatoa.
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2.
Streak Plates
The filamentous bacterium seen in the two mixed liquor samples and in
the secondary influent did not grow on any of the streak pistes. This is
consistant with previous results. One filamentous bacterium was isolated
from the Marcx mixed liquor. It was found to be a gram negative spore-
former and its morphology is consistant with that of Bacillus cereus. It
is clearly not the filamentous organism growing in the sludge.
The Beggiatoa. from -the sample of algae from the surface of the secondary
settling tank grew slowly on AcS agar. However, it was not isolated.
The fungus colony counts on Cooke's Rose Bengal Agar made after four
days of incubation are givaa in Table I. These counts are quite comparable
with the fungus found on samples obtained during the winter. They are
somewhat higher than normal for sewaee and mixed liquor samples but apparently
the fungi in this case are not associated with deterioration of the settling
properties of the sludge.
TABLE I
Fungus Colony Counts (per ml) on June 16 Samples
Sample Total Colonies Colonies Other
with with Fungus
Arthrospores Blastospores Colonies
Air ML 3,700 700 200 2,800
Marox ML 2,400 1,200 200 1,000
Secondary
Influent 300 100 200
Actinomycete Counts
Actinomycete counts were made on the TS plates after 28 days of incu-
bation. The actinomycete count on the secondary influent was 70 per ml,
on the air mixed liquor 580 per ml and on the Marox mixed liquor 1,100 per
ml. These counts are not high enough to indicate an operating problem.
Summary
The June 16, 1977 samples showed a well flocculated sludge in the Marox
system but a poorly flocculated, filamentous sludge in the air system. The
filamentous bacterium in both systems is the short, irregularly branched
organism which has been seen in all samples examined from this plant.
Fungi and actinomycetes are also present but in relatively small numbers.
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FMC - DENVER PROJECT
Summary of Samples Examined
January - June 1977
Introduction
This report summarizes the information obtained about the microbiology
of the air activated sludge and Marox systems at the Metro Denver North
plant for the period January through June 1977. Both systems have had
operation difficulties from time to time due to poor flocculation- and a
growth of excessive numbers of filamentous organisms which cause exces-
sively high SVI values. The Marox system is a demonstration project
operating in one basin of the activated sludge plant in parallel with the
air system. A pilot plant Marox system was also operated to provide
additional information for comparison purposes.
Samples
Samples of mixed liquor from the air activated sludge system and the
Marox system were obtained on January 10, 19, 25, and 27, February 23,
March 10, aiid June 16. The Marox sample on February 23 was from the
pilot plant. Secondary influent samples were examined on January 27 and
June 16. Raw sewage samples were examined on January 25 and January 27.
Kaw Englracod sewage samples were examined on February 23.
Filamentous Bacteria
One type of filamentous bacterium was seen In all mixed liquor samples.
It is a short, irregularly bent organism with dark inclusions. It is
between one and two micrometers in diameter and is not divided into cells.
We had no success attempting to grow this organism in the laboratory dispite
trying several different wet' a and a variety of cultural conditions. When
the mixed liquor samples art left unaerated for several days it grows form-
ing longer filaments. A lysozyme-detcrgent test failed to reveal the pres-
ence of a sheath and it reacted negatively to a sulfur deposition test.
Thus, it is neither Sphjaerocilus nor Bepgiatoa. It is present in the raw
sewage and secondary influent. Since it was present in samples with good
settling characteristics as well as in samples with poor settling character-
istics it apparently does not form filaments long enough to interfere with
the settling of the sludge.
A second filamentous bacterium was observed in both the air and Marox
mixed liquor samples during January and in the air mixed liquor but not
the Marox mixed liquor samples in February and March. It was not seen in
the June samples. It is less than one micrometer in diameter and forms
very long, straight filaments. It gave a negative sulfur deposition test
but a lypozyme-detergent test showed some sheath material. It would grow
some on GPY agar but died out after about one week. Good growth was ob-
tained on initial isolation plates by adding calcium carbonate to the
medium. In a GPY broth it lowered the pH to 3.0 in less than one day and
116
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2.
then died off. It appears to be an acid producing relative of Sphaerotilus.
It also appears to be the filamentous organism responsible for the high SVI
values found in the mixed liquor samples last winter.
The fungus colony counts on the raw sewage, secondary influent, and
mixed liquor samples were higher than is usual for sewage and activated
sludge. Also, an unusua!3y high proportion of fungi which produce either
arthrospores or blastospores were found. Howevei, the fungus counts were
approximately one tenth of the fungus counts I h.ive found in mixed liquor
when it was clear that the' fungi were growing in the system and interfering
with the settling characteristics of the sludge.
Actinomycetes
Actinoinycete colony counts were in the normal range for sewage and
mixed liquor samples except for the Marox pilot plant sample of February
23. That sample showed a mixed liquor consisting largely of actinouiycetes.
Otherwise there was no indication that the actinoiaycetes were causing a
continuing problem in either system.
Conclusion
A variety of different types of filamentous organisns are present in
both activated sludge systems. Most of the filamentous organisms are
present in the raw sewage and in the secondary influent as we.ll as in the
mixed liquoi". The funpi may be partially responsible for the poor flcc-
culation observed but are not present in high enough numbers to interfere
with the settling characteristics of the sludge. Actinomycetes could
cause settling problems if a long sludge residence time were used but
this is not usually the case. The filamentous bacteria present appear to
be microaerophylic and appear to grow more vigorously in the air system
than in the oxygen system. The filamentous bacterium which appears to
be the most likely cause of high SVI values 'is an acin-producing, sheathed
organism which has not been previously described.
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/2-79-012
2.
3. RECIPIENT'S ACCESSIOI*NO.
4. TITLE AND SUBTITLE
5. REPORT DATE
FULL-SCALE DEMONSTRATION OF OPEN TANK OXYGEN
ACTIVATED SLUDGE TREATMENT
May 1979 (Issing Date)
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
Stephen R. Pearlman
Donald G. Fullerton
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Metropolitan Denver Sewage Disposal District No. 1
6450 York Street
Denver, Colorado 80229
10. PROGRAM ELEMENT NO.
1BC822, SQS#3. Task D-l/27
11. CONTRACT/GRANT NO.
Grant No. S803910
12. SPONSORING AGENCY NAME AND ADDRESS
Municipal Environmental Research Laboratory--Cin.,OH
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
Final. June 1976-Sept. 1977
14. SPONSORING AGENCY CODE
EPA/600/14
15. SUPPLEMENTARY NOTES
Project Officer: Richard C. Brenner (513)-684-7657
16. ABSTRACT
This report presents an operating summary of a full-scale demonstration of the FMC
open tank pure oxygen (FMC 02) activated sludge system, conducted at the facilities of
the Metropolitan Denver Sewage Disposal District No. 1 (Metro) in Denver, Colorado.
The system was operated over a period of 15 mo at both steady state and diurnal flow
rates ranging from 22,700 m3/day (6 mgd) to 53,000 m3/day (14 mgd), with average F/M
loadings varying between 0.49 and 1.14 kg BOD/day/kg MLVSS and clarifier overflow rates
ranging from 16.5 to 32.2 mj/day/m2 (406 to 791 gpd/ft2).
The overall performance of the system was dictated in large part by the quantity of
filamentous organisms in the activated sludge, and the investigation of these organ-
isms, with attempts to control or eliminate them, was an ongoing activity during much
of the demonstration.
The system was evaluated as to oxygen transfer efficiency and oxygen consumption.
Further, it was compared to the Metro diffused air system running in parallel with the
demonstration system on the basis of sludge production and power requirements. Com-
parison of sludge settleabilities was made between these two systems and a closed tank
pure oxygen system (UNOX) also in operation at Metro.
In addition to allowing an evaluation of the FMC 02 system as a whole, the demon-
stration provided an opportunity to evaluate various component equipment items as to
their reliability and performance.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Sewage treatment, *Activated sludge pro-
cess, *0xygenation, Aeration tanks, Sedi-
mentation tanks, *Liquid oxygen, Upgrading
*0xygen activated sludge
system, *Rotating active
diffuser, *0pen tank,
Dissolution
13B
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
128
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
118
v LI S GOVERNMENT PRINTING OFFICE 1979-657-060/1640
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