WATER POLLUTION CONTROL RESEARCH SERIES • 17010 DDQ 11/71
MECHANISMS OF BIOLOGICAL
LUXURY PHOSPHATE UPTAKE
U.S. ENVIRONMENTAL PROTECTION AGENCY
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WATER POLLUTION CONTROL RESEARCH SERIES
The Water Pollution Control Research Series describes
the results and progress in the control and abatement
of pollution in our Nation's waters. They provide a
central source of information on the research, develop-
ment, and demonstration activities in the Environmental
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Inquiries pertaining to Water Pollution Control Research
Reports should be directed to the Chief, Publications
Branch, Research Information Division, Research and
Monitoring, Environmental Protection Agency, Washington,
D. C. 20460.
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MECHANISMS OF BIOLOGICAL LUXURY PHOSPHATE UPTAKE
by
The University of Arizona
Department of Microbiology and
Medical Technology
Tucson, Arizona 85721
for the
ENVIRONMENTAL PROTECTION AGENCY
Project #17010 DDQ
November -1971
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, B.C. 20402 - Price $1.00
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EPA Review Notice
This report has been reviewed by the Environmental
Protection Agency and approved for publication.
Approval does not signify that the contents neces-
sarily reflect the views and policies of the
Environmental Protection Agency, nor does mention
of trade names or commercial products constitute
endorsement or recommendation for use.
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ABSTRACT
Activated sludges obtained from the Rilling Road plant
located at San Antonio, Texas and from the Hyperion treatment
plant located at Los Angeles, California have the ability to
remove large amounts of phosphorus from Tucson sewage and
other liquors by means of biological mechanisms. Most of the
phosphorus seems to accumulate within the sludge cells as
orthophosphate. Tucson sludge seems to take up phosphorus
by biological mechanisms but removes considerably less from
its medium than does Rilling sludge. However, phosphorus up-
take by Tucson sludge is improved if the sludge is starved
prior to the addition of sewage.
The bacteria isolated from Rilling sludge do not individually
seem to account for a high phosphorus affinity when compared
to those from Tucson sludge. A culture of Sphaerotilus
natans was isolated from Rilling but not from Tucson sludge.
This organism had a higher affinity for phosphorus than
others tested but not sufficient to account for the superior
removal properties exhibited by the Texas sludge.
A known sludge bacterium, Zoogloea ramigera formed volutin
granules when excess orthophosphate was added to a phosphate
starved culture. However, the conditions necessary to pro-
duce these granules in this organism probably do not exist
in normal sewage.
This report was submitted in fulfillment of Project number
17010 DDQ under the partial sponsorship of the Environmental
Protection Agency.
111
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CONTENTS
Section Page
I CONCLUSIONS 1
II RECOMMENDATIONS 3
III INTRODUCTION 5
IV MATERIALS AND METHODS 7
WHOLE SLUDGE EXPERIMENTS 7
ISOLATION OF SLUDGE BACTERIA 12
VOLUTIN GRANULES IN ZOOGLOEA RAMIGERA 15
V RESULTS 17
WHOLE SLUDGE EXPERIMENTS 17
ISOLATION OF SLUDGE BACTERIA 43
VOLUTIN GRANULES IN ZOOGLOEA RAMIGERA 56
VI DISCUSSION 65
VII ACKNOWLEDGMENTS 69
VIII REFERENCES 71
IX LIST OF PUBLICATIONS 75
X GLOSSARY 77
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FIGURES
NO,
32 45
UPTAKE OF P, Ca, AND ORTHOPHOSPHATE
FROM SEWAGE BY TUCSON SLUDGE UNDER NORMAL
CONDITIONS.
32 45
UPTAKE OF P, Ca, AND ORTHOPHOSPHATE
FROM SEWAGE BY STARVED TUCSON SLUDGE.
32
DISTRIBUTION OF RADIOACTIVITY FROM P OR
45Ca IN FRACTIONS OBTAINED FROM STARVED OR
NORMAL SLUDGE BY EXTRACTIONS USING MODIFIED
WIAME TECHNIQUE.
UPTAKE OF 32P RADIOACTIVITY BY RILLING
THE EFFECT OF 2,4-DINITROPHENOL (2,4-DNP) 25
ON THE UPTAKE OF P, 45Ca, AND ORTHOPHOS-
PHATE FROM SEWAGE BY TUCSON SLUDGE.
32
DISTRIBUTION OF RADIOACTIVITY FROM P OR 26
45Ca IN FRACTIONS OBTAINED FROM 2,4-DINITRO-
PHENOL TREATED SLUDGE BY EXTRACTIONS USING
MODIFIED WIAME TECHNIQUE.
32 0_,
A COMPARISON OF UPTAKES OF P RADIOACTIVITY 27
FROM SEWAGE BY TUCSON AND RILLING SLUDGES.
PER CENT DISTRIBUTION OF 32P RADIOACTIVITY 28
AMONG FRACTIONS EXTRACTED FROM ACTIVATED
SLUDGES BY THE OGUR-ROSEN PROCEDURE.
8 EFFECT OF INCUBATION TEMPERATURE ON THE 34
UPTAKE OF 32p RAD:
ACTIVATED SLUDGE.
9 EFFECT OF VARYING TEMPERATURES AND TIMES 35
ON THE UPTAKE OF 32P RADIOACTIVITY BY
RILLING ACTIVATED SLUDGE.
10 EFFECT OF VARYING pH ON THE UPTAKE OF P 36
RADIOACTIVITY BY RILLING ACTIVATED SLUDGE.
11 EFFECT OF VARIOUS CONCENTRATIONS OF META- 38
BQLIC INHIBITORS ON THE PER CENT UPTAKE OF
P RADIOACTIVITY BY RILLING ACTIVATED
SLUDGE.
VI
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FIGURES
No. Page
12 GROWTH OF ZOOGLOEA RAMIGERA IN ARGININE 57
BROTH AND INOCULATING BROTH.
13 PHOTOMICROGRAPHS OF VOLUTIN GRANULES IN 58
ZOOGLOEA RAMIGERA.
14 GRANULATION IN ZOOGLOEA RAMIGERA AT 59
DIFFERENT GLUCOSE CONCENTRATIONS.
15 GRANULATION IN ZOOGLOEA RAMIGERA AT 61
DIFFERENT INITIAL PHOSPHATE CONCENTRATIONS.
16 GRANULATION IN ZOOGLOEA RAMIGERA AT 62
DIFFERENT MAGNESIUM CONCENTRATIONS.
17 GRANULATION IN ZOOGLOEA RAMIGERA AT 63
DIFFERENT CONCENTRATIONS OF GLUCOSE,
INITIAL PHOSPHATE, AND MAGNESIUM.
Vll
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TABLES
No. Page
1 EFFECT OF STORAGE TIME ON THE ABILITY OF 17
RILLING
SEWAGE.
RILLING SLUDGE TO REMOVE PO4~P FROM TUCSON
REMOVAL OF ORTHOPHOSPHATE FROM SEWAGE BY 19
TUCSON ACTIVATED SLUDGE.
3 2
RADIOACTIVITY (RA) RELEASED FROM P- 23
LABELED TUCSON SLUDGE DURING THE UPTAKE
OF ORTHOPHOSPHATE.
EFFECT OF SUSPENDING MEDIUM ON REMOVAL OF 30
ORTHOPHOSPHATE AND 32P RADIOACTIVITY (RA)
BY RILLING ACTIVATED SLUDGE.
EFFECT OF VARIOUS SALT CONCENTRATIONS ON 31
THE UPTAKE OF 32P RADIOAC1
RILLING ACTIVATED SLUDGE.
THE UPTAKE OF 32P RADIOACTIVITY (RA) BY
6 UPTAKE OF RADIOACTIVITY (RA) AND ORTHOPHOS- 32
PHATE RELEASED FROM 32P LABELED RILLING
SLUDGE.
7 EFFECT OF VARIOUS ANTIMETABOLITES ON THE 37
UPTAKE OF P RADIOACTIVITY (RA) BY RILLING
SLUDGE.
8 EFFECT OF 2,4-DINITROPHENOL (DNP) ON 40
RESPIRATION AND UPTAKE OF 32P RADIOACTIVITY
(RA) BY RILLING SLUDGE.
9 NUCLEIC ACID CONTENT OF ACTIVATED SLUDGES. 41
10 SPECIFIC ACTIVITIES (COUNTS/MIN./MG.) OF 42
32P LABELED RIBONUCLEIC ACID (RNA) ISOLATED
FROM ACTIVATED SLUDGES.
11 EFFECT OF VARIOUS TREATMENTS ON THE RECOVERY 44
OF BACTERIA FROM TUCSON ACTIVATED SLUDGE.
12 PHYSIOLOGICAL PROPERTIES OF BACTERIA ISOLATED 45
FROM TUCSON ACTIVATED SLUDGE.
Vlll
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TABLES
No. Pag
13 SOURCE AND DISTRIBUTION OF GRAM-NEGATIVE 46
ISOLATES FROM VIABLE PLATE COUNTS.
14 SOURCE AND DISTRIBUTION OF GRAM-NEGATIVE 47
ISOLATES FROM ENRICHMENT MEDIA.
15 REMOVAL OF RADIOACTIVE PHOSPHORUS (32P) 48
FROM SEWAGE BY ACTIVATED SLUDGE BACTERIA.
16 THE EFFECT OF GLUCOSE ON THE UPTAKE OF 32P 49
RADIOACTIVITY FROM SEWAGE BY SELECTED
SLUDGE BACTERIA.
17 UPTAKE OF 32P RADIOACTIVITY (RA) AND PO4~P 51
FROM TUCSON SEWAGE BY BACTERIA ISOLATED
FROM TUCSON SLUDGE AND KNOWN ORGANISMS.
18 A COMPARISON OF 32P AFFINITY RANGES FOR 52
BACTERIAL TYPES ISOLATED FROM VARIOUS
ACTIVATED SLUDGES.
32
19 UPTAKE OF P RADIOACTIVITY (RA) AND PO -P 54
FROM TUCSON SEWAGE BY SPHAEROTILUS NATANS
ISOLATED FROM RILLING SLUDGE.
20 AMOUNT OF PO4~P REMOVED FROM TUCSON SEWAGE 55
PER MG. (DRY WEIGHT) OF ORGANISM
IX
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SECTION I
CONCLUSIONS
1. Activated sludges from the Rilling Road plant at San
Antonio, Texas and the Hyperion plant at Los Angeles, Calif.
are capable of luxury phosphorus uptake from Tucson sewage
during the course of laboratory experiments. Phosphorus
removal was independent of externally supplied sources of
energy and ions since orthophosphate and P radioactivity
were readily removed from tap water, glass distilled water,
and deionized water. Phosphorus uptake by Rilling sludge
seems to be wholly biological since it has an optimum p_H
range, an optimum temperature range, and is inhibited by
various antimetabolites that affect enzymes involved in
the synthesis of adenosine triphosphate in bacteria.
2. Tucson sludge also removes phosphorus from sewage by
biological mechanisms. However, its net removal is con-
siderably less than that of the high uptake sludges because
of a high turnover of phosphorus occurring within the sludge
cells. When the sludge is starved of phosphorus before
adding sewage, turnover is almost eliminated and net uptake
is improved considerably.
3. Rilling and Tucson sludges were extracted and the various
cell fractions containing P radioactivity were analyzed.
Most of the radioactivity appeared to be in orthophosphate
within the cells. Little if any net synthesis of nucleic
acids occurred during a 6 hr. exposure to sewage. This con-
firms the luxury nature of the phosphorus uptake. A some-
what larger percentage of radioactive polyphosphate was
found in Rilling sludge than in Tucson but not enough to
account for the high removal capability of the former sludge.
4. Bacteria were isolated from high uptake sludges from
Rilling and a plant located in Houston, Texas and compared
to those isolated from Tucson sludge. A total of 229 pure
bacterial cultures were screened bv-a qualitative procedure
to determine their affinities for P radioactivity. No
significant distributions of high affinity bacteria were
found for Rilling sludge as compared to Tucson. However,
Sphaerotilus natans was isolated from Rilling sludge which
had a significantly greater phosphorus affinity than did the
others. This organism was not isolated from Tucson sludge
which also had filamentous bacteria. The amount of phos-
phorus removed by S. natans was about the same as that
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removed by Tucson sludge but not sufficient to account for
the superior abilities of Rilling sludge.
5. Zoogloea ramigera, a bacterium isolated from sludge, had
the ability to remove phosphorus and form volutin granules
under certain conditions. These conditions included more
glucose than is normally present in most waste waters. Low
phosphorus affinity bacteria isolated from sludge became high
affinity organisms when small amounts of glucose were added
to sewage.
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SECTION II
RECOMMENDATIONS
This program was limited to laboratory experimentation
although the conditions used were designed to approach those
that might exist in the field under optimum circumstances.
Further studies should take place first with a pilot plant
and then on a larger scale. Such studies might include the
effects of sewage from a system of lower phosphorus affinity
such as Tucson on a high affinity sludge such as Rilling over
a prolonged period of time. The sludge should be monitored
for gross changes in microbial population as well as for
phosphorus affinity. To successfully accomplish the former,
some population markers will have to be established.
The bacterial survey was inconclusive in pin pointing any
particular members of the bacterial population of Rilling
sludge as being responsible for the high uptake of phos-
phorus. One organism, Sphaerotilus natans, showed some
promise but not sufficient to account for the sludge's
superiority. It is possible that no single organism was
responsible but that the uptake was the result of synergistic
activity on the part of more than one genus, species, or
strain. The possibility also exists that the active organ-
ism was not isolated. Further experimentation should clarify
these points.
Too many experiments written into the literature have used
some form of synthetic sewage as 'a medium for sludge. Such
media containing glucose result in changes in the nature
of the sludge within a few hours. Coliform organisms which
are not usually the primary flora of sludge will prolifer-
ate. These organisms in the presence of glucose probably
will improve phosphorus uptake but will result in the sludge
losing other desirable characteristics.
Phosphorus uptake by Tucson sludge increased with starvation.
If provisions were made in the plant for sludge, before be-
ing returned, to dump its phosphorus in a tank containing
liquid relatively free of the element, uptake might be
improved. Also, effluent containing phosphate should not
be recirculated through the plant. Similar suggestions
have been made in the literature by other workers in the
field.
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The presence of filamentous organisms such as S_. natans has
generally been regarded as a nuisance that results in bulk-
ing sludge. However, the engineers at Rilling have reported
no difficulty in separating sludge from effluent. If in
pilot plant experiments the organism still shows some
efficiency in phosphorus removal, then conditions in low
affinity plants might be altered sufficiently to permit the
organism to grow on a limited scale.
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SECTION III
INTRODUCTION
The presence of large amounts of phosphorus containing
compounds in waste waters due to greater use of detergents
containing this element is thought to be partially respon-
sible for the nuisance growth of algae now observed in many
lakes and waterways throughout the United States that re-
ceive effluents from treatment plants (1). One objective
of waste water purification is to reduce the phosphorus
levels below 0.5 mg. of PO4 per liter which should aid in
the control of algal growth (2). Activated sludge treatment
of waste water, the most common method, usually is unable
to lower the amount of phosphorus in the effluent suffi-
ciently to prevent algal blooms when the element is the
limiting nutrient. However, a number of plants throughout
the country such as the Rilling Road plant at San Antonio,
Texas (3) and the Hyperion treatment plant at Los Angeles,
California (4) have reported sludges that have high phos-
phorus affinities and remove this element rapidly and com-
pletely when it occurs in their natural waste waters.
The mechanisms by which high affinity sludges remove phos-
phorus have not been fully elucidated. Waste waters are
usually low in utilizable sources of carbon so microbial
growth is relatively limited and slow. The rapid uptake of
phosphorus by biological mechanisms in excess of the meta-
bolic needs of the sludge cells is termed enhanced or luxury
uptake and implies that the microorganisms have the ability
to store the element in some form. Menar and Jenkins -(5)
concluded that the high phosphorus affinity shown by Rilling
sludge was not biological in nature. They believed that
excess removal, above that required for cell synthesis, was
controlled by joH and the presence of Ca in the waste water.
Under proper conditions of p_H, a precipitate of calcium
phosphate would form followed by an enmeshing into the acti-
vated sludge floe. Subsequent settling of the sludge would
result in apparent disappearance of the phosphate from the
supernatant fluid. Recent work suggests that removal by
Hyperion sludge is largely biological (4).
While it has been established that the microorganisms of
activated sludge play an important role in the stabilization
of organic waste waters (6), there is only limited
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information in the literature concerning the role played by
the various microbial components of activated sludge in
phosphate uptake. Among these is the work of Srinath et al.
(7). These workers investigated removal of radioactive
phosphorus (32P) from sewage by activated sludge, mixed
bacterial cultures isolated from sludge, Zoogloea, sp., and
the protozoan Epistylis sp. Based on their observations
the authors concluded that removal of P from sewage was
due largely to vorticellid protozoa such as Epistylis sp.
in sludge. The authors did demonstrate, however, that
bacteria were responsible for uptake of a considerable
amount of P but since the bacteria remained dispersed in
the medium it was concluded that bacterial efficiency of
removal was poor. Whether the protozoans play a primary
role in phosphorus removal or simply serve as a means for
concentrating phosphorus taken up by bacteria was unresolved,
In 1969 the FWPCA awarded a contract to The University of
Arizona to study the mechanisms of biological luxury phos-
phate uptake by sludge. The results of the investigation
will be reported under three headings: whole sludge exper-
iments, isolation of sludge bacteria, and volutin granules
in Z. ramigera.
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SECTION IV
MATERIALS AND METHODS
WHOLE SLUDGE EXPERIMENTS
Activated Sludge
Sludge from the Rilling Road plant at San Antonio, Texas was
concentrated by filtration at the plant and shipped to Tucson
overnight by surface carrier. Upon receipt the sludge was
stored at 4 C. until needed, usually within one week after
collection although its phosphorus uptake ability was not
impaired by 11 days of storage (see Table 1) . It was diluted
with tap water to the desired concentration just before use.
Return sludge from the Tucson plant was collected and allowed
to settle before use. Several experiments were conducted
with sludge from the Hyperion treatment plant located at Los
Angeles, California. The material was frozen and then ship-
ped by air and was used within one week after collection.
The freezing process did not seem to alter the sludge's phos-
phorus removal abilities.
Experimental Conditions
General procedure. Many of the experiments were conducted
by mixing 33-ml. of settled sludge in the desired concen-
tration (as determined by dry weights) with 66-ml. of liquid
contained in 38 X 200 mm. tubes. The desired amount of P
or Ca radioactivity, additional phosphate (as KH2PO4 and
when required, and any other inclusions were placed
in the tube prior to the addition of the sludge. The mix-
tures were aerated from the bottom of the tube at the rate
of 0.8 liter of prewet air per min. and incubated at 24°C.
Any sludge adhering to the sides of the vessel was removed
with a spatula and returned to the mixture prior to each
sampling. At the desired times, the aeration was stopped for
approximately 10 sec. and 10-ml. samples removed before the
sludge settled. The samples were centrifuged in the cold
at 27,000 X c£.' for 10 min. The supernatant fractions were
assayed for radioactivity (^2P or 45ca) and chemically for
orthophosphate and calcium hardness. The pellets were
extracted and the fractions assayed for radioactivity. The
usual experimental run consisted of a block of 12 tubes.
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Liquids used were: fresh raw sewage taken from the primary
clarifier at the Tucson plant; Tucson city water taken from
the tap; glass distilled water; and deionized water which
was distilled water passed through a Barnstead mixed bed
demineralizer.
When larger batch experiments were performed, increased
amounts of sludge and liquid (in proportions of 1/3 to 2/3)
were added to vessels of 1-liter, 2-liter, or 4-liter capac-
ity. The vessels were kept slightly less than half-full.
The aeration rate was increased to 3 liters of prewet air
per min. All other conditions were the same as above.
Prelabeled conditions. 133-ral. of sludge were placed in a
1-liter glass graduate cylinder with 266-ml. of tap water or
sewage and P radioactivity and aerated for 3 hr. (for
Rilling sludge) or 12 hr. (for Tucson sludge) at 25°C. After
aeration, 5-ml. samples were removed and assayed for radio-
activity and orthophosphate in the supernatant fractions and
total uptake of radioactivity in the cells. Samples (100-mL)
were transferred from the large aerator to 38 X 200 mm.
tubes and left undisturbed for 12 hr. After this time, 5-ml.
samples were taken to determine the amount of P radio-
activity and orthophosphate "dumped"; additional orthophos-
phate placed in some of the tubes, and aeration started.
The experiments were conducted as described under general
procedure except that 5-nil. samples were taken for analysis.
Starved conditions. Tucson sludge was prepared by adding
settled fresh return sludge to an equal volume of 0.85%
saline in distilled water and allowing it to stand undis-
turbed at 25°C. for 18 hr. After standing, the sludge was
mixed and allowed to resettle. The aqueous portion was drawn
j2 45
off and the sludge was added to sewage plus P or Ca con-
tained in cylinders; the experiments were conducted as de-
scribed under general procedure.
Determination of sludge mass. Dry weights were determined
for the normal condition experiments by filtering 100-ml.
samples taken from parallel larger batch experiments (4
liters) using predried and weighed 9-cm. circles of Whatman
no. 30 filter paper. The filter paper and sludge were
dried by heat to constant weight. The larger amounts were
used to minimize errors in sampling that occurred due to
cohesiveness of the sludge components when 10-ml. amounts
8
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were treated in this fashion.
Temperature effects studies. For studies concerned with the
effects of varying incubation temperatures, 33-ml. of sludge
and 66-ml. of tap water containing 32p radioactivity were
incubated separately for 30 min. at the desired temperature
to reach equilibrium. They were mixed together and the ex-
periments conducted as indicated under general procedure in
incubators set at the required temperatures.
For the constant temperature-varying time experiments, sludge
was heated in a boiling water bath for the desired time and
then cooled rapidly to 25 C. The experiments were conducted
as indicated under general procedure.
Optimum pH studies. The water used to dilute the sludge for
these studies was titrated with 10% hydrochloric acid (HCl.)
to establish acid ranges, with concentrated ammonium hydrox-
ide for ranges between ;p_H 7.0 to 10.0, and with 10% potassium
hydroxide for more extreme alkalinity. The experiments were
conducted as described under general procedure.
Manometric experiments. Sludge (0.8-ml.) was added to
1.2-ml. of tap water or sewage containing ^2P radioactivity,
and 2,4-dinitrophenol (DNP) when required, in the main por-
tion of a double sidearm 16-ml. reaction vessel containing
0.2-ml. of 20% potassium hydroxide. The flasks were placed
on a model GR-14 respirometer (Gilson Medical Electronics,
Middleton, Wis.) and equilibrated for 15 min. at 25 C.
with the vessels open to air. After this time the vessels
were closed and readings taken for 1 hr. When it was desired
to preincubate with DNP, the powdered inhibitor was added to
the sludge in sufficient amount to give the desired concen-
tration (10 M) when diluted in the reaction vessel. The
sludge and inhibitor mixture were incubated with shaking at
25°C. for 1 hr. prior to addition to the vessel.
Extraction Procedure
Three procedures were followed for extracting sludges during
the course of the experiments covered in this report. These
were: the Wiame method (8) as modified by Boughton (9), the
Ogur-Rosen method (10), and the method of Schmidt and
Thannhauser (11).
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Wiame method. This technique was employed as a rapid means
of obtaining the distribution of P or 45Ca radioactivity
among the various fractions resulting from the extraction of
the Tucson sludge. To the pelleted and washed sludge mate-
rial, 10-ml. of freshly prepared 10% trichloroacetic acid
(TCA) was added. The mixture was incubated for 30 min. at
4 C. The sample was centrifuged then for 20 min. at 17,300
X c[ at 0°C. The supernatant fraction was saved and desig-
nated the "cold acid soluble pool." According to work with
yeasts, this fraction should contain cellular orthophosphate,
free bases, nucleosides, nucleotides, and di-, tri-, and
polyphosphates. Any P or Ca radioactivity that was
adhering to the exterior of the sludge mass should register
in this fraction.
The residual pellet was extracted with 10-ml. of freshly
prepared ethanol-ether (3:1) for 30 min. at 45 C. The sample
was centrifuged as described above. The supernatant fraction
should contain lipids and phospholipids.
The residual material was extracted with 10-ml. of freshly
prepared 5% TCA for 30 min. at 100°C. The sample was centri-
fuged as described above. The supernatant fraction should
contain hydrolyzed UNA, DNA, long-chain polyphosphates, and
acid soluble protein. It was designated the "hot acid
fraction."
The residual material was extracted with 10-ml- of 0.1 N
potassium hydroxide for 30 min. at 70 C. The sample was
centrifuged as described above. The supernatant fraction
should contain alkaline soluble components.
The sum of the amount of radioactivity found in each of the
above fractions, as well as the residue remaining after the
potassium hydroxide treatment was taken as the total amount
of radioactivity fixed by the sludge. These agreed within
10% with the amount calculated as disappearing from the liquid
during the course of the experiments.
Ogur-Rosen method. This method was employed in the latter
experiments with Rilling sludge in an effort to obtain intact
RNA. The pellets of sludge cells were extracted with 20-ml.
of 70% ethanol for 30 min. at 4°C. and then centrifuged in
order to obtain alcohol soluble materials. The residue was
10
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then extracted with 20-ml. of 1 N hot perchloric acid (PCA)
(70°C. for 45 min.) which should extract hydrolyzed DNA
components as well as some polyphosphates.
Schmidt and Thannhauser method. This method was used for
measuring the amounts of DNA and RNA from sludge which could
not be done satisfactorily by the Ogur-Rosen procedure.
(See Results). The main differences in the modified proce-
dure, as compared to Ogur-Rosen, are substitutions of 10%
TCA for the cold ethanol and PCA steps in the latter proce-
dure and the use of 0.3 N potassium hydroxide for 60 min. at
37°C. instead of the hot PCA extraction. The use of dilute
warm base results in extraction of the nucleic acids. The
RNA is hydrolyzed into its component nucleotides. DNA is not
affected in the same way. The DNA may be separated from the
RNA components by precipitation with 1 N HC1.
Chemical and Radioactive Assays
Orthophosphate and total phosphate were determined by the
ammonium molybdate and Stanna Ver method as given in the 6th
edition of the manual of Hach Chemical Co. Ames, Iowa. The
amount of color developed was read in a Hach model 585 DC-DR
colorimeter. This method was found to have sufficient
accuracy as judged by the use of our own prepared standards.
Calcium and magnesium hardness in sewage was determined by
the ethylenediaminetetraacetate (EDTA) titration method of
Hach.
Biochemical oxygen demand (BOD) determinations were performed
according to standard methods (12). A Fieldlab Oxygen
Analyzer (Beckman Instruments, Inc., Fullerton, Calif.) was
used to measure the dissolved oxygen expressed in milligrams
per liter. Determinations of j)H were with a jpH meter (Leeds
and Northrup Co., Philadelphia, Pa.). Radioactive assays
were made in a Tri-Carb liquid scintillation counting system
(model 314 EX-2, Packard Instrument Co., Downers Grove, 111.)
using techniques that have been described (13).ooAH counts
were corrected for decay. Distribution of the P radio-
activity among the organic and inorganic phosphorus contain-
ing components of the various fractions was determined after
adsorption of the former by Norit A (14). Aqueous mixtures
of orthophosphate and polyphosphates were resolved in this
laboratory by the use of Sephadex G-50 (Pharmacia Fine
Chemicals, Piscataway, N.J.) columns which excluded poly-
phosphates and retained orthophosphate. Polyphosphate was
11
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measured by heating and then using the ammonium molybdate
technique. The chemical amounts of RNA present were deter-
mined by an orcinol colorimetric procedure (15). The amounts
of DNA were measured by the diphenylamine procedure of Dische
(16).
Chemicals
Carrier-free 32P (orthophosphoric acid in 0.2 N HCl) was
obtained from Schwarz BioResearch, Orangeburg, N.Y. New
England Nuclear Corp., Boston, Mass., was the supplier of
45CaCl2 in 0.5 N HCl, which was claimed to have a radiometric
purity of 99% and to contain 1.2 mg. of total solids per ml.
Chemicals used for determinations were obtained from Hach
Chemical Co. Sodium azide (Fisher), mercuric chloride
(Matheson, Coleman and Bell), and DNP (Mallinckrodt) were
obtained from local vendors. All other antimetabolites and
EDTA were obtained from Sigma Chemical Co., St. Louis, Mo.
All chemicals were of the highest purity commercially avail-
able .
ISOLATION OF SLUDGE BACTERIA
Media and Cultural Conditions
Trypticase soy agar (TSA) (Bioquest, Cockeyville, Maryland),
activated sludge extract agar (ASEA), and activated sludge
extract agar plus glucose (ASEAG) were used for the primary
isolation of bacteria from fresh return activated sludge
obtained from the municipal wastewater plant located at
Tucson, Arizona. Sludge samples ranged in temperatures from
22 to 24°C. and had a reaction of p_H 7.2 to 7.4 at time of
sampling. Sewage agar was used as the phosphorus uptake
medium.
ASEA was prepared by autoclaving return sludge for 15 min.
at 121 C. The suspension was filtered through cheesecloth
and Whatman No. 1 filter paper. Agar (1.5%) was added to the
filtrate and the mixture sterilized by autoclave. Where re-
quired, filter sterilized 0.5% glucose (final concentration)
was added aseptically to sterile ASEA. Sewage agar was pre-
pared by filtering effluent from the primary clarifier of the
Tucson plant through Gelman membrane filters (pore diameter
0.20u) and collecting in bottles. Agar (1.5%) was added and
the mixture sterilized by autoclave. When required the sew-
age agar was supplemented with 0.1% filter sterilized glucose
12
-------
added aseptically.
Viable counts were obtained by serially diluting sludge
following various treatments in order to disperse floes.
Sonication was done with a Bronwill Biosonic BP-1 sonifier
at a power setting of 60 for 3, 10, and 30 sec.; homogen-
ization with a Waring blender at high speed for 3, 10, and
30 sec.; and shaking in dilution bottles filled with Escher-
type stoppers were used. Isolated colonies were picked at
random from plates at the highest dilutions and subcultured
on ASEAG or TSA plates for pure culture. All cultures were
incubated aerobically for 96 hr. at 24°C.
In an attempt to enhance outgrowth of dominant sludge bac-
teria, samples of activated sludge were collected, homogen-
ized for 30 sec., and centrifuged for 5 min. at 500 X 3 to
remove large debris using a Sorvall RC-2B centrifuge set at
4°C.
Forty ml, of supernatant fluid was placed in each of five
sterile 150-ml. Erlenmeyer flasks and treated in the follow-
ing manner: one flask received no additions; a second flask
received 0.5% glucose (final concentration); a third received
0.5% glucose and 0.1% yeast extract; a fourth received 0.5%
glucose and 0.1% K2HP04; and a fifth received 0.5% glucose,
0.1% yeast extract, and 0.1% K2HPO . All additions were
presterilized and added aseptically. Flasks were aerated by
shaking at 250 rev./min. for 96 hr. on a New Brunswick gyro-
rotary shaker. Following incubation, samples were plated on
TSA, ASEA, and ASEAG plates which were then incubated at
24 C. for 96 hr. Colonies were picked at random and pure
cultures isolated.
Characterization of Isolates
Isolates were characterized on the basis of their morphology,
physiology, and biochemistry. Morphological characteristics
included cell size, shape, and arrangement; motility, deter-
mined by wet mount observations and reaction in motility
medium; type of flagellation using a Philips EM-75 electron
microscope; and colony size, form, margin, elevation, luster,
density, and pigmentation. Physiological and biochemical
tests employed were those described by Shewan et al. (17)
for the identification of genera of Gram-negative bacteria
and those set forth by the Manual of Microbiological Methods
of the Society of American Bacteriologists (18). These in-
cluded oxidase test; production of fluorescent pigment;
13
-------
2-ketogluconic acid formation; pigement production on skim
milk; gelatin liquefaction; nitrate reduction; and anaerobic
and aerobic dissimilation of glucose, lactose, and sucrose.
o
Cultures were incubated at 24 C.
Assay for Phosphorus Uptake
Qualitative Screening Procedure. Isolates were grown for 48
hr. on ASEAG slants at 24 C. The cells were suspended in a
small volume, 1 to 2-ml. of double distilled water. Samples
(0.5-ml.) were filtered through dried, tared 24 mm. 0.20 u
diam. Gelman membrane filters. The filters were placed on
sewage agar plates containing carrier free H^ PO (Schwarz
Bio-Research) at a final activity of 700,000 counts/min./ml.
or approximately 14,000,000 counts/min. per plate. A 64-mm.
petri dish accommodated five filters, two organisms in du-
plicate, and one control membrane. Plates were incubated
aerobically at 24 C. for 6 hr. After incubation, filters
were removed, dried to constant weight, weighed, placed in
vials and radioactivity measured.
Quantitative Measuring Procedure. The bacteria were streaked
from stock cultures onto sterile sewage-agar-glucose slants
(filter sterilized sewage, 2% agar, and 0.1% glucose). Two
slants were made per culture. The organisms were grown for
48 hr. at 24 C. The organisms were washed from each slant
using 1-ml. of sterile distilled water into an 8-ml. sterile
water blank and resuspended. Two ml. of each suspension
were then placed aseptically in each of four 500-ml. Erlen-
meyer flasks containing 100-ml. of filter sterilized raw
sewage and 0.1% glucose. The flasks were continuously
shaken at 200 rev./min. on a gyrorotary shaker at 24°C. for
48 hr. at which time the optical density of the suspension
in each flask was approximately 1 when measured with a Bausch
and Lomb Spectronic-20 colorimeter at 540 nanometers. The
contents of the four flasks were pooled and centrifuged at
4°C. at 27,000 X % for 10 min. The pellets were combined
and washed once with distilled water. The organisms were
resuspended in filtered sewage to an optical density corre-
sponding to a known dry weight of the bacteria. Fifty ml.
portions of the suspension plus P radioactivity were
added to 38 X 200 mm. Kimax tubes which were aerated and
sampled as previously described. Dry weights were determined
by collecting the cells on preweighed membrane filters (0.45
H pore size; Millipore Corp., Bedford, Mass.) and drying
at 70 C. overnight.
14
-------
VOLUTIN GRANULES IN ZOOGLOEA RAMIGERA
Experimental Conditions
Zoocfloea ramigera ATCC 19623, maintained on Trypticase soy
(Bioquest) agar slants supplemented with 0.25% glucose, was
employed in this work. Other media used included: activated
sludge broth prepared by coarsely filtering autoclaved acti-
vated sludge and adding varying amounts of glucose as supple-
ments; inoculating broth, a modification of Crabtree and
McCoy's arginine broth (19) containing 0.2 g. of K-HPO and
0.1 g. of KH^PO. per liter of medium; and arginine broth,
which was a modification of Crabtree and McCoy's broth con-
taining 4 mg. of KH2PC>4 per liter. The standard inoculum
for liquid media consisted of 0.01-ml. of stationary phase
bacteria. The organisms were grown for 120 hr. in 100-ml- of
liquid media gontained in 500-ml. Erlenmeyer flasks with
shaking at 24 C. prior to studying the effects of various
conditions on granule formation.
Staining Procedures
Neisser's stain was used to stain volutin granules. The
solution of methylene blue plus gentian violet stains the
granules deep blue and the chrysoidin solution stains the
cells yellow (20). Tandler's inorganic phosphate stain was
used to show the presence of inorganic phosphate in volutin
granules (21) . The cells were counter-stained red with
safranin.
Granule Counting
Smears stained for volutin were observed at 970 X by using
bright field microscopy. Photographs of some smears were
taken with a Leitz Orthomat Microscope Camera. An area near
the top of the slide was chosen for granule counting where
the yellow counter stain had thoroughly drained off. The
number of volutin granules in each of 30 cells was recorded.
The number of granules per cell approached a Poisson dis-
tribution and was treated as such in computing confidence
limits of means.
Chroma tography
Samples and standards (H3 PO^ and Na4P2O-y) were applied to
Whatman no. 4 chroma tography paper cut to 9 by 9 in. (23 by
23 cm.), developed in a mixture of isopropanol, concentrated
HCl, and water (170:41:39, v/v) (22), and sprayed with a
mixture of 60% PGA, in HCl, 4% ammonium molybdate, and water
15
-------
(5:10:25:60, v/v) (23). The paper was sprayed, air dried,
and exposed to ultraviolet light at 260 nanometers for 10
min., whereupon the inorganic phosphate appeared as blue
spots.
16
-------
SECTION V
RESULTS
WHOLE SLUDGE EXPERIMENTS
TABLE 1. EFFECT OF STORAGE TIME ON THE ABILITY OF RILLING
SLUDGE TO REMOVE PO4~P FROM TUCSON SEWAGE3
Storage timeb PO4-P in sewage
p.p.m. % removed
4
11
16
23
30
0
0
3.7
5.7
6.0
100
100
63
43
40
P04-P = 10 mg/1
Days after collection
Table 1 shows the effect of storage time at 4 C. on the
ability of Rilling sludge to remove PO.-P from Tucson sewage.
This material can be stored for at least 11 days after col-
lection and still remove all of the phosphorus from the waste
water.
Figure 1 shows the per cent uptake of radioactivity from
^ P or 45ca from the sewage by Tucson sludge under normal
experimental conditions. Zero time represents the interval
required to mix the sludge with the sewage, remove a sample
and separate the pellet from the liquid fraction by centri-
fugation. The total amount of radioactivity present was
determined prior to additions of the sludge. Chemical
orthophosphate could be determined only at zero time be-
cause the manipulation of the sludge contributed to total
phosphate. At zero time, approximately 2.5% of the P
radioactivity became.associated with the sludge as compared
to 12% of the total Ca activity. These fiqures represent
5% of the total P radioactivity and 67% of the total Ca
17
-------
I
Q.
8.5
8.0
7.5
7 n
_.• •-•
"
UJ
Q
UJ
UJ
O
X
Q.
O
X
t-
tr
o
43
Co
60
50
40
z 30
20
10
• • ORTHOPHOSPHATE
o—o—o—o-
60
50
40
ID
O
O
cr
UJ
a.
o
30 i2
O
O
cr
20
o
UJ
10
3 4 5 6 7 8 9 10 II 12
TIME (HOURS)
"3 O AC
FIG. 1. UPTAKE OF P, Ca, AND ORTHOPHOSPHATE FROM SEWAGE
BY TUCSON SLUDGE UNDER NORMAL CONDITIONS. Approximately
9,584,350 counts/min. of P radioactivity and approx-
imately 8,852,000 counts/min. of Ca radioactivity
were used per 10-nil. of mixture.
18
-------
TABLE 2. REMOVAL OF ORTHOPHOSPHATE FROM SEWAGE BY TUCSON
a
ACTIVATED SLUDGE
Sludge condition Time (hr.) % Removal
Normal
Starved
3
6
12
3
6
12
19
20
30
42
53
56
a
Based upon amount (milligrams per liter) present at zero time.
See Fig. 1 and Fig. 2 for chemical amounts of orthophosphate
and corresponding amounts of P.
radioactivity removed from the sewage in 12 hr. The
orthophosphate removed, as indicated by chemical methods
(Table 2), was 30% in 12 hr. as compared to 48% of P radio-
activity (Fig. 1).
Dry-weight determinations, using parallel experiments, in-
dicated that a sludge mass of 55.6 mg/100 ml. was present at
zero time. No increase in dry weight was observed by 3 or 6
hr. Determinations of BOD (not shown) indicated that the
sources of carbon were essentially consumed by 3 hr.
Figure 2 indicates the effect of sludge starvation on the
uptake of orthophosphate and radioactivity from P or Ca
from sewage. The process of starvation resulted in the dump-
ing or stripping of orthophosphate from the sludge which was
observed in this laboratory and by others (24, 25). Much
of this phosphate was present in the interstitial spaces of
the sludge mass and was not removed when the sludge was added
to sewage containing radioisotope. Therefore, the chemical
amount of orthophosphate present in the sludge-sewage mix-
ture was considerably higher (92.5 mg./liter) than that from
normal conditions.
19
-------
8.5
8.0
7.5
7.0
100
90
a:
uj 80
• • 32 p
o o 43Co
• • ORTHOPHOSPHATE
2 60
Q
UJ
50
UJ
£
I
a.
(/>
o
40
30
tr
O 20
10
12
100
90
80 5
UJ
70 o
£E
60
UJ
50 O
O
40 £
30
20
10
TIME (HOURS)
FIG. 2
32 4S
UPTAKE OF P, Ca, AND ORTHOPHOSPHATE FROM SEWAGE
BY STARVED TUCSON SLUDGE. Approximately 1,133,700
counts/min. of P radioactivity and approximately
626,300 counts/min. of Ca radioactivity were used
per 10-ral. of mixture.
20
-------
32
At zero time, approximately 4% of the P radioactivity and
18% of the 5Ca radioactivity were found to be associated
with the sludge. Despite the higher amount of orthophosphate
initially present as compared to the normal-condition exper-
iments (Fig. 1), this sludge was more efficient in removing
P radioactivity, taking up approximately 63% by 12 hr.
Orthophosphate removal from the sewage, as determined chemi-
cally, showed better agreement with the tracer results in
that about 56% disappeared (Table 2). Starvation enhanced
somewhat the uptake of Ca with approximately 30% becoming
associated with the sludge by about 12 hr. However, about
60% of the total taken up was removed at zero time. The
association of calcium with the sludge seemed mainly to be
confined to the radioactive ions added just prior to the
beginning of the experiment because no loss other than that
attributable to error in the method was found when calcium
hardness of the sewage (which was found to be approximately
130 mg./liter) was measured chemically during the treatment
of sludge.
Figure 3 shows the distribution of radioactivity of cell-
fixed P or Ca in various fractions. At zero time, only
09 J
33% of the P radioactivity in the normal sludge was asso-
ciated with the cold acid-soluble pool components or possibly
just adhering to the exterior of the sludge mass, perhaps as
calcium phosphate. The majority of the radioactivity already
was distributed among the various cell components. Most of
the radioactivity seemed to be associated with the fraction
that would contain nucleic acids and long-chain polyphos-
phates. The starved sludge had about 35% of its P radio-
activity in the soluble fraction and 42% in the nucleic
acid-polyphosphate fraction. The normal sludge had 92% of
its Ca radioactivity associated with the soluble fraction
and 6% with the nucleic acid fraction. This distribution
was unchanged by 12 hr. The starved sludge showed a lesser
amount, 81% of its Ca radioactivity associated with the
soluble fraction and more, 14%, associated with its nucleic
acid fraction. This distribution was unchanged by 12 hr.
21
-------
100
p 95
90
OJ
85
OJ M n ^ O «n
O (D X » H BU
rt vfl 1-3 3> CO
H- n> v n 1-3 7 «
< D !> 1-3 JO ' °
H- Q, O M M
rt en 1-3 O CO 70
L<^ M 2 C2
Hi O CO H 5 „ _
O a H O 6 5
t-1 CO O O H
CD S O en
M cj |_q JJ *> V
P- CO > O (K
lQ H M *T) n cm
• 125 2
O W » ^
I- O > ui 50
3 O ^
Q) O nj H 0 TIME 0 TIME 12 TIME
Qj (D W 1— 1 - — ^, — — ^
H- S
O CD
1 K '2D uDTAVC OV ^'/»
__ —
m_
1
0 TIME
"v-" ~~ _
IIDTAISC C
n
^
r- 1
m
x/
1
^
L
01
p
12 TIME 0 TIME
-^ <| ^ ^^^^^
av * 2 Q 1. n
-------
TABLE 3. RADIOACTIVITY (RA) RELEASED FROM 32P-LABELED TUCSON
SLUDGE DURING THE UPTAKE OF ORTHOSPHOSPHATE
a.
Time
(hr)
0
0.5
3
6
RA in liquid phase
Counts/min
712,200
770,000
975,200
1,173,600
Per cent of
total fixed
19
21
27
33
Orthophosphate
in liquid phase
pH
Mg/
liter
27.5
25.0
23.0
21.6
%
removed
8.10
9 8.15
16 8.20
21 8.20
a 32
Approximately 3,671,500 counts/min. of P radioactivity were
fixed per 10-ml. sample of sludge-sewage mixture.
Amount of radioactivity found in supernatant fraction of 10-ml
sample of mixture.
23
-------
32
Sludge prelabeled with P was placed in fresh raw sewage to
examine the possibility that the apparent discrepancy ob-
served when the data obtained by measuring P uptake into
normal cells was compared to chemical orthophosphate remain-
ing in the liquid might be due to phosphate turnover (Table
3). A considerable portion of the radioactivity from the
sludge was found in the liquid phase at zero time (19%).
About 33% of the radioactivity was in the liquid phase by 6
hr. Approximately 21% of the orthophosphate in the mixture,
as determined chemically, was removed from the liquid phase.
This compares to the figure of 20% observed for the normal
experiments (Fig. 1).
Figure 4 shows the effects of DNP on the ability of sludge to
take up P, Ca. and orthophosphate. Under our experimen-
tal conditions, P uptake was inhibited approximately 83%
and 45Ca uptake was inhibited approximately 34%. Some dump-
ing of orthophosphate from the sludge cells into the liquid
phase was observed
Figure 5 shows the distribution of P or Ca radioactivity
among the various fractions of the sludge cells subjected to
2,4-DNP treatment for 3 hr. The most striking feature is
the inhibition of P incorporation into the nucleic acid-
polyphosphate fraction. The distribution of Ca radio-
activity was essentially unchanged from that of normal cells.
The next sludge to be examined was obtained from San Antonio
(Rilling). The Rilling sludge removed all P added to
Tucson sewage and its phosphorus content (about 10 mg. per
liter PO.-P) by 3 hr. (Fig. 6). Tucson sludge removed about
26% of the radioactivity and about 2 mg. per liter of phos-
phorus (not shown) by 6 hr. The presence of DNP (10 M)
resulted, in this case, in approximately 44% inhibition of
uptake by Rilling sludge after 3 hr.
Figure 7 shows the percentage of the P radioactivity
taken up by the sludges among the fractions obtained by the
use of the Ogur-Rosen extraction procedure. Orthophosphate
seems to predominate. Although there is some apparent in-
crease in the amount of labeled polyphosphate isolated from
the Rilling sludge, this does not seem to be sufficient to
be responsible for the high phosphorus affinity shown by this
sludge. The presence of DNP inhibited the uptake of 32P
radioactivity into organic phosphate compounds. No signifi-
cant amounts of radioactivity were found in the ethanol and
ethanol-ether fractions so they were not included in the
24
-------
8.5
8.0
7.S
7.0
(T
UJ
O
Q
UJ
UJ
d.
CO
O
X
a.
o
x
cr
o
50
40
20
10
• --- • ORTHOPHOSPHATE
50
40
30
O
UJ
5
o
a:
o
a:
o
o
20
10
TIME (HOURS)
FIG. 4. THE EFFECT OF 2,4-DINITROPHENOL (2,4-DNP) ON THE UPTAKE
OF 32P, 45Ca, AND ORTHOPHOSPHATE FROM SEWAGE BY TUCSON
SLUDGE. Approximately 5,925,100 counts/min. of 32p
radioactivity and about 7,853,200 counts/min. of Ca
radioactivity were used per 10-ml. of mixture.
Approximately 10~3 M final concentration of 2,4-DNP
was employed.
25
-------
hr) 100
M
P 95
01 90
85
to to 'rj O
CD tT1 » H _n
m q > to 8O
H-i /™N i
D O >-3
*^3 O t-3 /O 75
(-•• W M H
ua o ro
• w 53 a 70
>-< to 1-3
*" H 2 c •
WOO 0 65
M X ro 2; r
5 g>o y 60
0) > H ^ <
302! jf 55
H O > ^
i-h o 6 uj 50
0 25 t H ^
H M§^ < 45
DJ q 3 o a
9 en H ^40
O H M H
13 O J^ H ^ 3 5
rt 1 1-3
cn 3 D H?
OH 30
OOiz;^
hh H H 33 , «
^ i-3 O * 3
n M ;ri s
OJ W O 20
Qj O h3 Co
M- JE N)
o s; w na 15
QJ H 2
o :> o o . n
rt 2 f 33 10
H- W
I-1- 1-3 33 Ln '
rt W W O
< O > QJ o
^
^T^m
U777\
PJ
Mt:;M
^ji
W%
[ 1 10% TCA
INMIIlllllllll ETOH-ETHER
V////^7/A 5% TCA (HOT)
••O..N KOH
E^
§gH TIME 0 TIME 3 MRS TIME 0 TIME 3 MRS
H O 2; _____^^ _ ^___^^^ _.
m~~*
•"- -^- •!
w 32p UPTAKE 2, 4 DNP 45Ca UPTAKE 2, 4 DNP
-------
STORED TUCSON
STORED S.A.
2,4-DNP S.A.
036
TIME (HOURS)
FIG. 6.
A COMPARISON OF UPTAKES OF 32P RADIOACTIVITY FROM
SEWAGE BY TUCSON AND RILLING SLUDGES. Approximately
130 mg. (dry weight) of Tucson sludge and 265 mg.
(dry weight) of Rilling sludge were aerated in
Tucson sewage containing approximately 180,000,000
counts per min. per 100-ml. of P radioactivity
for up to 6 hr.
27
-------
STORED TUCSON
STORED S.A.
2,4-DNP S.A.
A=ORTHOPHOSPHATE
B=POLYPHOSPHATE
C = ORGANIC PHOSPHATE
3HR
O.IN COLD PCA
6HR
0 3HR 6HR
IN COLD PCA
0 3HR 6HR
IN HOT PCA
W 4->
u a -H
z P >
o -H
S >< -P
< ffl 0
(0
>H CO O
EH W -H
HOT)
> Q (0
M t> M
E-i A
U W 4-1
O Q
H W
Q H
< •
P CP
W -H
U fa
O
OS (1)
ft QJ
W
2
U -
LO a
o ^
Di
I C
OS QJ
D X
O fO
O -P
CO
(N
o
-------
figure.
Table 4 shows that no external sources of energy or addition
of specific ions are necessary for phosphorus removal by
Rilling sludge. Various ions were detected in sewage and
tap water but no Ca++ or Mg++ were detected in the distilled
waters by the analytical methods employed. Hyperion sludge
(data not shown) gave identical results.
Table 5 shows the effect of diluting Rilling sludge with
various concentrations of salts in distilled water on the
uptake of 32p radioactivity. A concentration of 1% NaCl is
almost totally inhibitory. The inhibitory ion appears to
be Na+ as NaHC03 is quite effective against uptake and KCl
is relatively ineffective. Similar results were obtained
when orthophosphate uptake was measured (data not shown).
The salt effects were reversible as activity was restored
when the sludge was washed in tap water (data not shown).
Rilling, like Tucson sludge, will dump phosphate into its
suspending medium under conditions of storage. Under our
experimental conditions, this phosphate represents an addi-
tion to the amount that is already present in the waste
water prior to the addition of the sludge. Table .6 shows
the results of an experiment designed to discover how much
phosphate Rilling sludge will contribute to its suspending
medium after overnight storage and whether this phosphate
is preferentially removed by the sludge. The data in Table
6 indicate that approximately 29% of the 32P radioactivity
appears in the liquid.phase after 12 hr. of storage. This
radioactivity and orthophosphate is almost completely re-
moved by 1 hr. after aeration is resumed (Sample 1). Samples
2 and 3 represent experiments in which dumped orthophosphate
was supplemented with additional KH2P04-K2HP04. A comparison
of the rates at which radioactivity and chemical orthophos-
phate were removed by 3 hr. indicates that the P radio-
activity which was derived from the sludge cells was not
removed preferentially to added orthophosphate. Table 6 also
shows that this quantity of Rilling sludge can remove approx-
imately 100 mg. per liter of added orthophosphate from tap
water in 3 hr. after periods of storage if the sludge is not
removed from its suspending medium. Hyperion sludge was
found to have the same capability by similar experiments con-
ducted in this laboratory.
29
-------
TABLE 4. EFFECT OF SUSPENDING MEDIUM ON REMOVAL OF ORTHO-
32
PHOSPHATE AND P RADIOACTIVITY (RA) BY RILLING
a
ACTIVATED SLUDGE
Orthophosphate
Medium
Tucson
Sewage
Tucson
Effluent
Distilled
H2°
Tap H2O
Deionized
H20
Initial
(mg/1.)
77
110
110
110
110
Final
c
ND
ND
ND
ND
ND
o/
/o
Removed
100
100
100
100
100
RA in
Initial
102,480
177,700
166,100
167,160
176,800
Medium
J Finalb
BKD
BKD
BKD
BKD
BKD
Removed
100
100
100
100
100
b
Approximately 250 mg. (dry weight) of sludge contained in a
final volume of 100-ml. were used per experiment. All ex-
periments were aerated at 24 C for 3 hr.
Counts/min./ml.
'ND = None Detectable
BKD = Background
30
-------
TABLE 5. EFFECT OF VARIOUS SALT CONCENTRATIONS ON THE UPTAKE
32
OF P RADIOACTIVITY (RA) BY RILLING ACTIVATED
SLUDGE
Salt
NaCl
NaCl
NaCl
NaHCO
KC1
RA in Medium
Concentration (%) , %
(Final) Final Uptake
0.01 BKD° 100
0.10 BKD 100
1.00 80,570 3
1.00 99,350 13
1.00 42,270 63
pH
(Final)
8.2
7.9
7.7
9.1
8.2
Approximately 250 mg (Dry Weight) of sludge was diluted to
100-ml. with tap water containing indicated salt concentration
and aerated at 24°C for 3 hr. The P radioactivity used in
the NaCl experiments was approximately 83,460 counts/min/ml.;
the initial radioactivity in the other experiments was about
113,220 counts/min/ml.
Counts/min/ml.
°BKD = background
31
-------
TABLE 6. UPTAKE OF RADIOACTIVITY (JRA) AND ORTHOPHOSPHATE
RELEASED FROM 32P LABELED RILLING SLUDGE3
RA in liquid phase
Orthophosphate in
liquid phase
J. -Lllie
(Hr.) Sample
0 1
2
3
0.5 1
2
3
1 1
2
3
3 1
2
3
Counts/min/ml .
94,780
103,300
102,440
7,330
37,460
51,130
730
13,780
34,820
BKDd
3,500
18,280
%
Removed
--
--
--
92
64
50
99
87
66
100
97
82
mg/liter
105
206
350
6
84
166
NDC
25
103
ND
4
42
%
Removed
--
--
--
94
59
53
100
88
71
100
98
88
a 32
Approximately 346,990 counts/min. of P were fixed per ml. of
mixture.
Sample 1 represents orthophosphate dumped from sludge after 12
hr. Samples 2 and 3 represent dumped + additional orthophosphate
ND = none detectable
BKD = background
32
-------
Figure 8 shows the effect of incubation temperature on the
uptake of 32p radioactivity by Rilling sludge. The optimum
temperature appears to be in the range between 24-37°C.,
which would be characteristic of a biological rather than a
chemical phenomenon.
Figure 9A shows the effects of exposing Rilling sludge to
100°C. for varying lengths of time up to 20 min. An exposure
time of only 2 min. resulted in a loss of more than 50% in
ability to remove 32P radioactivity. Figure 9B shows the
effects of varying the temperature for a constant time (30
min.) on the sludge's ability to remove 32p. Rilling sludge
can withstand a wide temperature variation (between 5 to
50 C.) for a half-hour under our laboratory conditions with-
out affecting its phosphorus removal capabilities. A drop
to 48% in uptake capability resulted when the sludge cells
were exposed to 70°C. for 30 min. Autoclaving for 30 min.
resulted in complete loss of ability to remove radioactive
phosphate.
The effects of varying the JDH of the diluting fluid on 32P
uptake is seen in Fig. 10. The values represent final JDH.
A p_H range between 7.7 to 9.7 appears to be optimal for
phosphorus removal by Rilling sludge in the laboratory.
Table 7 shows the effects of various antimetabolites on the
uptake of 32p radioactivity by Rilling sludge. Of those
listed, the antimetabolites containing heavy metals such as
p-chloromercuribenzoic acid (PCMB) and HgCl2 were effective
against phosphorus uptake. Inhibition occurred when the
32P radioactivity was in either tap water or sewage (Table 7,
Fig. 11B) . No inhibition was observed when 10~3]y[ EDTA was
added to the diluting fluid (data not shown).
Figure 11 shows the effects of various concentrations of
09
four antimetabolites on the uptake of J^P radioactivity by
Rilling sludge. These compounds, which act on enzymes in-
volved in energy yielding reactions and ATP formation, were
quite effective against phosphorus uptake by the sludge.
Phosphorus uptake by Hyperion sludge was inhibited by DNP
(10~3M). No other antimetabolite experiments were conducted
with this sludge.
33
-------
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FIG. 8. EFFECT OF INCUBATION TEMPERATURE ON THE UPTAKE OF P
RADIOACTIVITY BY RILLING ACTIVATED SLUDGE. Approximately
250 ing. (dry weight) of sludge in a final volume of 100-
ml. of tap water were aerated for 3 hr. at the indicated
temperatures in the presence of approximately 130,000
counts/min/ml. of P radioactivity.
34
-------
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Q.
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£ 30
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TEMPERATURE CONSTANT (IOO°C)
TIME VARIED
6 8 10 12 14 16 18 20
TIME (MINUTES)
B
TIME CONSTANT (3OMINUTES)
TEMPERATURE VARIED
10 20 30 40 50 60 70 80 90 100 110 120
TEMPERATURE CO AUT°>CLAVE
FIG. 9. EFFECT OF VARYING TEMPERATURES AND TIMES ON THE UPTAKE
OF 32P RADIOACTIVITY BY RILLING ACTIVATED SLUDGE. (A)
temperature constant (100°C.) time varied; (B) time
constant (30 min) temperature varied. Approximately
250 mg (dry weight) of sludge in a final volume of
100-ml. with tap water containing approximately 261,000
counts/min./ml. of P radioactivity were subjected to
the indicated conditions and then aerated at 24°C. for
3 hr.
35
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PH
-------
TABLE 7. EFFECT OF VARIOUS ANTIMETABOLITES ON THE UPTAKE OF
32P RADIOACTIVITY (RA) BY RILLING SLUDGE3
Experiment
Number
1
2
3
4
5
6
7
8
9
Antimetabolite
Final
Name cone .
None 0
p-Chloromer-
curibenzoic
acid (PCMB) 10~
PCMB 10~4
Gramicidin 10
Rotenone 10
Oligomycin 10
Antimycin A
Type III 10~
HgCl2 10~
-3
PCMB 10
RA in medium
c
Final
BKDd
103,480
41,100
BKD
160
BKD
BKD
180,220
108,800
uptake
100
52
81
100
99
100
100
16
49
pH
(final)
8.4
8.2
8.3
8.5
8.4
8.2
8.4
8.1
8.2
aApproximately 250 mg (dry weight) of sludge contained in a final
volume of 100-nil. were used per experiment. All experiments were
aerated at 24°C. for 3 hr. Tap water was medium for experiments
1-7 and Tucson sewage was medium for experiments 8 and 9.
Approximately 213,320 counts/min/ml• of ^2P radioactivity were
added initially for all experiments.
^moles/liter
ccounts/min/ml•
_q
BKD = background
37
-------
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CM
10
100
90
80
70
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Q-
20
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90
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70
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<60
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20
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B
IO"
IO"5
IO
IO
^4~
FT-
FINAL CONC. IODOACETIC ACID
(MOLES/LITER)
" I015 IO"* 10'
FINAL CONC. HgCI2 (MOLES/LITER)
10
IO'4 IO'3 I0~2
FINAL CONC. 2,4 DNP (MOLES/LITER)
FIG. 11.
IO'4 I0~3 IO"2
FINAL CONC. NaN3 (MOLES/LITER)
EFFECT OF VARIOUS CONCENTRATIONS OF METABOLIC
INHIBITORS ON THE PER CENT UPTAKE OF 32P RADIO-
ACTIVITY BY RILLING ACTIVATED SLUDGE. (A) iodoacetic
acid-initial radioactivity approximately 83,460 counts/
min'ml.; (B) HgC^ - initial radioactivity approximately
213,200 counts/min/ral.; (C) 2,4-dinitrophenol-initial
radioactivity approximately 318,900 counts/min/ml.;
(D) NaN^ - initial radioactivity approximately 180,000
counts min/cc. Approximately 250 mg (dry weight) of
sludqe in a final volume of 100-ml. with tap water were
aerated at 24°C, for 3 hr. in the presence of inhibitor
and
32
P.
38
-------
Table 8 shows that oxygen is utilized by the Rilling sludge
in the presence of either Tucson sewage or tap water. Some
increase in Qo2values occurred in the presence of sewage,
however. When DNP was added to the sludge just prior to the
start of the experiment, the uptake of ^2p radioactivity was
inhibited but little effect was seen on the Qo2 values.
When the sludge was preincubated for 1 hr. with DNP prior to
the addition of the water containing the ^2P radioactivity,
considerable inhibition of both oxygen utilization and uptake
of radioactivity occurred. This indicates that the Qo2 was
affected by a higher initial concentration of DNP than has
been used for most of the experiments shown in Table 8.
The nucleic acid content of the sludges were investigated.
The orcinol procedure indicated that the 1 N cold PCA ex-
traction (Ogur-Rosen) was not removing all of the RNA from
sludge and that a large amount was present in the hot PCA
fraction which should contain hydrolyzed DNA. The Schmidt
and Thannhauser method was used in order to get a better
separation and a more accurate recovery of the nucleic acids.
Table 9 gives the per cent dry weight of the nucleic acids
extracted from Rilling and Tucson sludges. This table in-
dicates that no net synthesis of DNA and little if any net
synthesis of RNA occurs in the sludges by 6 hr. Some RNA
must be synthesized as indicated by the action of DNP (see
Fig. 7) which inhibits the incorporation of 32p radioactivity
into organic phosphate. However, almost equal amounts seem
to be broken down.
32
Table 10 shows the specific activities of the P labeled
RNA isolated from the sludges. Radioactivity is incorporated
into the nucleic acid. However, when this data is examined
in conjunction with that shown in Table 9, turnover of the
components seems to be the mechanism of incorporation rather
than net synthesis.
39
-------
TABLE 8. EFFECT OF 2,4-DINITROPHENOL (DNP) ON RESPIRATION AND
UPTAKE OF 32P RADIOACTIVITY (RA) BY RILLING SLUDGE3
Flask Contents
Q
°2C
Per Cent Uptake
RA
Sludge, 0.8-ml
HO, 1.2-ml.
13.4
93
Sludge, 0.8-ml
Sewage, 1.2-ml
15.0
87
Sewage, 1.2-ml.
HO, 0.8-ml.
None
None
Sludge, 0.8-ml.
Sewage, 1.2-ml. + DNP
15.6
66
Sludge, 0.8-ml.
H20, 1.2-ml. + DNP
11.4
35
Sludge, 0.8-ml.
H0, 1.2-ml. + DNPC
2.6
21
a
Experiments represent averages of duplicate flasks on Gilson
respirometer incubated with shaking at 25°C. for 1 hr.
b
Flask contents included the variables listed in the table plus
0.2-ml. of 20% KOH in center well. Sludge used =5.75 mg./ml.
dry wt. Radioactivity introduced = 18,000 counts/min/ml. of H
P04- 2,4-Dinitrophenol used = 10~^lfinal concentration.
CQ-02 = ul 02/hr/mg. dry wt. sludge.
d 0-5
Sludge preincubated with DNP for 1 hr. prior to adding P RA.
40
-------
TABLE a. NUCLEIC ACID CONTENT OF ACTIVATED SLUDGES
Sludge source
Time
(Hr.)
Per cent dry weight
Total nucleic
acid
RNA
DNA
San Antonio
(Rilling)
0
3
6
14.6
16.5
16.1
12.2 2.4
14.2 2.3
13.8 2.3
Tucson
0
3
6
15.3
15.9
16.2
11.8 3.5
12.5 3.4
12.7 3.5
41
-------
TABLE 10. SPECIFIC ACTIVITIES (COUNTS/MIN./MG.) OF 32P LABELED
RIBONUCLEIC ACID (RNA) ISOLATED FROM ACTIVATED SLUDGES
Sludge source Time RNA
(hr.) (spec. act.)
0 6,150
San Antonio 3 90^200
(Rilling)
6 102,700
0 2,230
Tucson 3 75,800
6 85,400
42
-------
ISOLATION OF 'SLUDGE BACTERIA
Viable bacterial counts of Tucson sludge obtained on
successive days were determined using TSA, ASEA, and
ASEAG plating media following various treatments to dis-
perse floes. Results of typical experiments are presented
in Table 11. As indicated, viable counts were approximately
two- to three-fold higher on ASEAG medium than on TSA
irrespective of the nature of the method of dispersal used.
In addition, sonication and homogenization proved to be
far more effective than conventional shaking for recovery
of bacteria from sludge. Four trials were made for each
condition except that ASEA was used as a plating medium
in only one trial. Approximately 5 to 10 colonies were
picked from plates at the highest dilution and streaked
for pure culture.
Eighty-five pure cultures have been isolated from Tucson
sludge. The majority, 78, were Gram-negative, nonspore-
forming, rod-shaped bacteria. Five isolates were Gram-
positive rods or cocci including 3 bacilli, 1 micrococcus,
and 1 streptococcus. Two isolates proved to be yeasts.
The Gram-negative rod shaped bacteria were further char-
acterized according to previously described methods. The
results of these tests are summarized in Table 12. All
Gram-negative isolates have been tentatively placed in five
groups. The Pseudomonas-Xanthomonas (type 1) was the
largest comprising 38% of the total Gram-negative bacteria
isolated. These organisms were characterized primarily on
the basis of polar flagellation, production in some cases
of fluorescent pigments, positive cytochrome oxidase test,
and their predominantly oxidative metabolism of sugars.
Members of the second largest group, Alcaligenes (type II),
comprising 22% of the Gram-negative isolates were char-
acterized primarily on the basis of lack of pigmentation
and motility, positive cytochrome oxidase test, and their
inability to metabolize sugars. The Escherichia-Aero-
bacter group (type III) , comprising about 17% of the total,
were characterized primarily on the basis of peritrichous
flagellation and their predominantly fermentative metab-
olism of sugars particularly lactose. Members of the
Flavobacterium group (type IV), approximately 12% of the
isolates, were so designated owing to the possession of
a nondiffusible yellow pigment and a positive cytochrome
oxidase reaction. Nine of the isolates, approximately 12%
of the total, were placed in the Achromobacter group (type
V). This group was characterized primarily on the basis
of morphology and their limited biochemical activity. No
attempt was made to accurately speciate each individual
isolate since we were primarily interested in biological
43
-------
TABLE 11. EFFECT OF VARIOUS TREATMENTS ON THE RECOVERY
OF BACTERIA FROM TUCSON ACTIVATED SLUDGE.
Treatment
Shaking
Sonication
3 Sec.
10 Sec.
30 Sec.
Viable counts X 10 on:
TSA1 ASEA1 ASEAG1
1.52 5.3 7.5
5.5 - 18.5
9.0 - 25.0
10.1 - 30.2
Homogenization
3 Sec.
10 Sec.
30 Sec.
7.5
8.5
11.7
19.5
27.2
29.1
TSA, Trypticase soy agar; ASEA, activated sludge extract
agar; ASEAG, activated sludge extract agar + 0.5% glucose,
I
"Mean value derived from counts of three replicate plates.
44
-------
TABLE 12. PHYSIOLOGICAL PROPERTIES OF BACTERIA ISOLATED FROM TUCSON ACTIVATED SLUDGE,
No. Gel-
of Mo- Cyto- Ni- atin
iso- til- chrome trite hydrol- Glu- Lac-
Type lates ity Pigment oxidase test ysis cose tose
I 30 + fluores- + + vari- A
cent or able
achromo-
genic
T T 1 "7 4- -U —
-LJ. JL / T . T
I'll1 13 + + - AG AG
IV 9 - yellow + vari- - - -
able
Vq
Su- Tentative
crose Group
A Pseudomonas
Xanthomonas
- Alcaliqenes
AG Escherichia
Aerobacter
- Flavobacter-
ium
- Achromobacter
in
LType 1 - Polar flagella; type 3-peritrichous flagella
-------
TABLE 13. SOURCE AND DISTRIBUTION OF GRAM-NEGATIVE
ISOLATES FROM VIABLE PLATE COUNTS
Number Type
Plating Medium of Isolates I II III IV V
TSA1
1
ASEA
ASEAG1
26
14
8
12
6
6
5
5
0
2
1
0
2
2
0
5
0
2
TSA, trypticase soy agar; ASEA, activated sludge extract
agar; ASEAG, activated sludge extract agar plus 0.5%
glucose.
activity such as phosphorus uptake rather than identity.
For this reason we relied to a large extent on the deter-
minative scheme for the identification of Gram-negative
bacteria proposed by Shewan, et al. (17) and were accord-
ingly able to place our isolates into five major groups
based on their gross morphological and physiological
properties.
The source of the Gram-negative isolates studied as well
as their distribution according to type are shown in Table
13 and Table 14. Thirty-four of 48 isolates obtained from
the plating series were types I and II, Pseudomonas-
Xanthomonas and Alcaligenes respectively. Moreover, since
tnese isolates were picked from plates at highest dilutions,
these data suggest that members of the Pseudomonas-
Xanthomonas group are representative of predominant aerobic
Ffeterotrbphic bacteria of sludge. Source and distribution
of isolates from the enrichment series are shown in Table 14.
Types of bacteria appear to be randomly distributed and
because of limited numbers few generalizations can be made.
It is of interest, however, that 10 of 25 isolates obtained
from enrichment media containing glucose were of type III,
Escherichia-Aerobacter. Thus addition of glucose to activated
sludge favors development of lactose fermenting bacteria.
All Gram-negative isolates were assayed for their ability
to remove P from sewage. A summary of the data obtained
46
-------
TABLE 14. SOURCE AND DISTRIBUTION OF GRAM-NEGATIVE
ISOLATES FROM ENRICHMENT MEDIA1
Enrichment
Medium
ASE2
2
ASEG
2
ASEGP
ASEGY2
ASEGPY2
Number
of isolates
5
7
5
8
5
I
2
2
1
1
0
II
1
1
1
4
0
Type
III
0
4
3
2
1
IV
1
0
0
1
3
V
1
0
0
0
1
Primary isolation media TSA and ASEG agar.
2
ASE, activated sludge extract; G, glucose 0.5%; P, K»HPO.
0.1%; Y, yeast extract 0.1%.
rs shown in Table 15. Zoogloea ramigeraf ATCC 19623 and a
laboratory strain qf E. coli were also assayed as to their
ability to remove P radioactivity. The former had a
specific activity of 150,000 counts per min. per mg. of dry
wt. The data indicate that the Pseudomonas-Xanthomonas,
Alcaligenes, and Achromobacter take up the largest amounts
of radioactivity from phosphorus. Analysis of variance of
data indicated that the Pseudomonas-Xanthomonas group,
which includes Z_. ramigera, take up significantly (within
99.9% confidence" limits) more P radioactivity than the
members of the Escherichia-Aerobacter group which includes
E. coli.
Since the addition of glucose to sludge appears to favor
the development of a predominantly fermentative microflora,
it was of interest to determine whether or not2addition
of glucose to sewage affected the removal of P radio-
activity by sludge bacteria. Eight isolates were, £ selected
for study including four with high affinity for P and four
with low affinity. Z_. ramigera and E. coli were included
for comparative purposes. Results are shown in Table 16.
The presence of glucose in sewage markedly enhanced the
uptake of P radioactivity from sewage. Indeed there was
47
-------
32
TABLE 15. REMOVAL OF RADIOACTIVE PHOSPHORUS ( P) FROM SEWAGE BY ACTIVATED
SLUDGE BACTERIA.
Group
Pseudomonas
-Xanthomonas
Alcaligenes
Achromobacter
Flavobacter ium
Escher ichia
-Aerobacter
Number of
Isolates
30
17
9
9
13
Specific Activity Log Mean Standard
Low High Mean Specific Activity Error
9,000 232,000 49,680 4.697 ±0.239
5 nnn ^nnnnn T^^on /I^/IP n^/i/i
_^_
8,000 83,000 24,380 4.387 ±0.458
1,000 41,000 10,570 4.024 -0.368
Counts per min. per mg. dry wt. cells; radioactivity used approximately
14,000,000 counts per min. per plate.
-------
32
TABLE 16. THE EFFECT OF GLUCOSE ON THE UPTAKE OF P
RADIOACTIVITY FROM SEWAGE BY SELECTED
SLUDGE BACTERIA.
Organism
High Group 50
6
93
8
Z. ramiqera
Low Group 36
46
70
30
E. coli
Specific
Plus 0.1% Glucose
536,000
256,000
588,000
200,000
306,000
616,000
544,000
497,000
541,000
365,000
Activity
Minus Glucose
281,000
171,000
141,000
45,000
71,000
26,000
17,000
14,000
13,000
11,000
1
Counts per min. per mg. dry wt. cells; corrected for
controls; radioactivity used approximately 14,000,000
counts per min. per plate.
49
-------
very little difference between representatives of both
groups when glucose was present. In the absence of
glucose, however, representatives of the high group took
up P radioactivity in significantly greater quantities.
For example in the presence of glucose E_. coli and Z_.
ramigera had specific activities of 3657000 and 306,000
counts per min. per mg. dry wt. respectively. In the
absence of glucose Z_. ramigera and E. coli had specific
activities of 71,000 and 11,000 counts per min. per mg.
respectively in the experiment shown in Table 16.
Twelve of_these organisms were picked for their "high"
or "low" P uptake ability and subjected to quantitative
assays. Data from some representative experiments are
given in Table 17. The amount of radioactivity in the cells
was obtained by extracting them with hot 4N HCl and measur-
ing the fraction using a scintillation counting system. In
all cases the readings were the same as that obtained for
disappearance of the radioactivity from the medium.
Of the organisms listed in Table 17, No. 6 and No. 8
represent the "high" uptake group, as shown by the screen-
ing procedure, and were members of the Pseudomonas-
Xanthomonas group. Zoogloea ramigera was used as a "high"
uptake control. This organism has been found in many
sludges although it was not isolated from Tucson material.
Organism No. 100 represents the "low" category and is a
member of the Escherichia-Aerobacter group. Escherichia-
coli served as a control.
The data from all the bacteria tested indicated that the
"high" uptake organisms did take up more P than did the
"low" uptake organisms and that generally there was a
corresponding drop in the amount of PO.-P in the medium.
The next step in the study was a comparison of organisms
isolated from high phosphorus affinity sludges and those
from Tucson sludge. Cultures were isolated from Rilling
sludge and that from a Houston, Texas plant which was
reported to be an effective phosphorus remover. The total
number of cultures isolated were 151, 98% of which were
Qram negative. All of the isolates were subjected to the
P screening procedure. Table 18 shows a comparison (as
per cent) of the P affinities of the 229 different cultures
isolated from the Houston, Rilling, and Tucson plants.
On the basis of the percentage of isolated organisms fall-
ing into each category of P affinity, the data in Table
18 does not account for the high uptakes of phosphorus
shown by the Rilling sludge as compared to Tucson sludge.
50
-------
32
TABLE 17. UPTAKE OF P RADIOACTIVITY (RA) AND P04~P FROM TUCSON
SEWAGE BY BACTERIA ISOLATED FROM TUCSON SLUDGE AND
KNOWN ORGANISMS.3
Organism
Zoogloea
ramiqera
(ATCC
19623)
No. 6
No. 8
Escherichia
coli
(lab strain)
No. 100
Time
(Hr.)
0
2
4
6
0
2
4
6
0
2
4
6
0
2
4
6
0
2
4
6
RA in
Cts . /min
10,000,000
8,200,000
6,210,000
5,550,000
9,950,000
8,457,500
6,467,500
5,870,000
10,100,000
8,585,000
6,565,000
6,000,000
10,000,000
9,050,000
8,900,000
8,100,000
10,500,000
9,500,000
8,500,000
8,085,000
sewage
% of
total fixed
18
38
45
__
15
35
41
_^ __
15
35
41
__
10
11
19
_ _
11
19
23
P04~P in
Mg. /liter
8.0
7.0
5.8
5.2
8.0
7.2
6.0
5.4
8.0
7.6
6.4
6.0
8.0
7.8
7.6
6.4
8.0
7.2
6.6
6.4
sewage
o/
/a
removed
12
27
35
__
10
25
32
__
5
20
25
__
3
5
20
__ __
10
18
20
1 Approximately 20 mg. (dry wt.) of the organism was aerated in
"3 O /~\
50-ml. of Tucson sewage containing P at 24 C.
51
-------
TABLE 18. A COMPARISON OF 32P AFFINITY RANGES FOR BACTERIAL
TYPES ISOLATED FROM VARIOUS ACTIVATED SLUDGES
Sludge source
% of bacterial
types isolated
Range
Tucson
Houston
San Antonio (Rilling)
17
13
22
High
105-106
Tucson
Houston
San Antonio (Rilling)
70
48
44
Moderate
104-105
Tucson
Houston
San Antonio (Rilling)
12
26
19
Low
103-104
Tucson
Houston
San Antonio (Rilling)
1
13
15
Very low
Less than 10'
Counts/min./imj. of organism
52
-------
32
There is a slight increase in the percentage of high P
affinity organisms isolated from Rilling sludge. However,
there is a considerably higher percentage of organisms in
the moderate affinity range from Tucson than from Rilling
(70% vs 44%). The distribution of organisms isolated from
Houston sludge resembles that of Rilling sludge.
A filamentous organism, S>. natans, was isolated from Rilling
but not from Tucson sludge which appeared superior to the
other microorganisms in its ability to remove P radio-
activity and phosphorus from Tucson sewage. This data may
be seen in Table 19. Table 20 compares the uptake of
phosphorus by this organism to those listed in Table 17
which confirms the superior phosphorus removing ability
of the filamentous organism.
53
-------
TABLE 19. UPTAKE OF P RADIOACTIVITY (RA) AND PO.-P FROM
TUCSON SEWAGE BY SPHAEROTILUS NATANS ISOLATED FROM
RILLING SLUDGE3
Time
(Hr.)
RA in sewage PO.-P in sewage
counts/min. % of total Mg./liter % removed
fixed
0
3
5
18
6
4
,326
,767
,364
,000
,000
,100
--
63
76
5.
2.
1.
8
5
5
--
57
74
3 mg. (dry wt) of bacteria were aerated in 10-ml. of Tucson
sewage containing 32p radioactivity at 24°C.
54
-------
TABLE 20. AMOUNT OF P04~P REMOVED FROM TUCSON SEWAGE PER MG.
(DRY WEIGHT) OF ORGANISM
Organism
Zoogloea ramigera
No. 6a
No. 8a
Escherichia colia
No. 100a
Sphaerotilus natans
Amount PO.-P (mg./l.) removed
0.14
0.13
0.10
0.08
0.08
1.43
total amounts of PO.-P removed, see Table 17. Exposure time
of organisms to sewage was 6 hr.
3For total amounts of PO.-P removed, see Table 19. Exposure time
of organism to sewage was 5 hr.
55
-------
VOLUTIN GRANULES IN ZOOGLOEA RAMIGERA
Figure 12 shows that the organism was in the stationary
phase in both arginine broth and the inoculating broth
approximately from 72 hr. until 120 hr. after inoculation.
Its doubling time was 6 hr. in arginine broth and 8 hr. in
the inoculating broth. Cell growth was decreased or
stopped if glucose, initial phosphate, or magnesium were
deleted from the medium.
Arginine broth contained 3 mg. per liter of phosphate
ion; therefore, cultures grown in this medium were phosphate
starved by 120 hr. Abundant volutin granules were formed
within 4 hr. following the addition of 1.8 g. per liter
of phosphate ion to these cultures (Fig. 13A). These
granules apparently contained inorganic phosphate as they
stained with Tandler's inorganic phosphate stain (21).
If the addition of excess orthophosphate was withheld from
a culture in arginine broth, granulation never occurred.
32
When cells labeled with H PO. under phosphate starved
conditions in arginine broth were extracted by the Ogur-
Rosen procedure, most of the radioactivity appeared in
the 1 N cold PCA fraction. Norit A removed the nucleic
acids and left most of the activity with the unadsorbed
inorganic phosphate. Chromatograms revealed that most
of the radioactivity resided in the 1 N PCA fraction as
orthophosphate and pyrophosphate rather than as nucleo-
tides. Microscopically the granules remained distinct
but completely disappeared when treated with 1 N PCA.
Glucose variation.
In the absence of glucose essentially no granules were
formed in arginine broth by the cultures despite the
presence of excess phosphate. The presence of 0-1 g.
per liter of the monosaccharide gave abundant granules
which were too faint to count. The presence of 2 g. per
liter of glucose gave abundant dark granules. A further
increase in glucose concentration gave darker and more
numerous granules; the optimum of 2.5 granules per cell
was reached at 10 g. per liter of the sugar after which
increased carbohydrate caused a gradual decrease in count
(Fig. 14).
Granule counts at 0 g. and 10 g. per liter amounts of the
sugar were correlated by assaying media for loss of radio-
activity. Carrier free H., PO. was added to 120 hr.
cultures of Z. ramigera in arginine broth containing either
no glucose or" 10 g. peT" liter. In 30 min. the cells in the
56
-------
o
«•
10
-------
***»
» y
* •• I
- I '' ' \
iff I
- ' s' A -I I
/ ~ ' .
\ ' %
N
B
FIG. 13. PHOTOMICROGRAPHS OF VOLUTIN GRANULES IN ZOOGLOEA
RAMIGERA. Smears, prepared 24 hr. after adding 1.8
g/liter orthophosphate to phosphate-starved cultures
of J5. ramiqera grown for 120 hr. in arginine broth,
were stained by Neisser's procedure. Magnification
is 1250X. (A) Unmodified arginine broth; (B)
Arginine broth with 1 mg./liter magnesium ion; (C)
Arginine broth with 80 mg./liter magnesium ion.
58
-------
o
2.0
1.8
« 1.6
0
V)
UJ
2
i.o
0.8
0.6
10
20
30
40
50
60
70
GLUCOSE (G/LITER)
FIG. 14. GRANULATION IN ZOOGLOEA RAMIGERA AT DIFFERENT GLUCOSE
CONCENTRATIONS. Granule counts were made 24 hr. after
adding 1.8 g./liter orthophosphate to phosphate-starved
cultures in arginine broth. The brackets indicate the
95% confidence limits for each mean.
59
-------
medium containing the sugar removed fivefold more P from
the medium per mg. dry weight than did the cells in the
medium lacking the hexose.
Initial phosphate variation. With only 0.6 mg. per liter of
initial phosphate in the arginine broth the granule yield
was low. The yield rapidly increased to abundant granulation
at a level of 3 mg. per liter; then the level rapidly de-
creased to a low level by 12 mg. per liter (Fig. 15) .
Magnesium variation. No granules were formed in arginine
broth in the absence of magnesium ion; but a level of 1 mg.
per liter gave large, dark, abundant granules which often
filled the whole cell (Fig. 13B). The maximum number of
granules occurred when magnesium was present in a range of
from 1 mg. to 20 mg. per liter, but at 20 mg. per liter the
extra large granules associated with 1 mg. per liter were
absent. Further increase in magnesium gave a decreased
yield (Fig. 16). At a level of 80 mg. per liter the gran-
ules were faint (Fig. 13C).
Interactions. A factorial design was used to find inter-
actions between glucose, initial phosphate, and magnesium in
arginine broth. The concentrations of glucose used were 2
g. per liter and 10 g. per liter; the concentrations of
initial phosphate ion used were 3 mg. per liter and 18 mg.
per liter; and the concentrations of magnesium ion used were
2 mg. per liter and 20 mg. per liter. The organism was
grown in arginine broth with the eight possible combinations
of the above three nutrients. As usual, 1.8 g. per liter of
phosphate were added at 120 hr. and the smears made 24 hr.
later.
The results were illustrated with three-dimensional co-
ordinates by drawing two planes, one representing 2 g. per
liter of glucose and the other 10 g. per liter of glucose
(Fig. 17). No significant difference was found between the
two planes at three of the corners, but one corner of the 2
g. per liter of glucose plane was irregularly elevated at 18
mg. per liter of initial phosphate and 20 mg. per liter of
magnesium. This represented increased granule production
due to interaction between low glucose, high phosphate, and
high magnesium concentration.
60
-------
1.8
in
6
UJ
o
^
CO
UJ
<
cr
1.6
1.4
1.2
0.8
0.6
- x
0
1
6
1
12
1
18
1
24
INITIAL PHOSPHATE (MG/LITER)
FIG. 15. GRANULATION IN ZOOGLOEA RAMIGERA AT DIFFERENT INITIAL
PHOSPHATE CONCENTRATIONS. Granule counts were made
24 hr. after adding 1.8g./liter orthophosphate to
phosphate-starved cultures in arginine broth. The
brackets indicate the 95% confidence limits for each
mean.
61
-------
o
\
to
cc
2.0
8
1.6
1.4
1.2
1.0
0.8
0.6
X
_L
0
20
40
60
80
100
120
MAGNESIUM (MG/LITER)
FIG. 16. GRANULATION IN ZOOGLOEA RAMIGERA AT DIFFERENT
MAGNESIUM CONCENTRATIONS. Granule counts were
made 24 hr. after adding 1.8g./liter orthophosphate
to phosphate-starved cultures in arginine broth.
The brackets indicate the 95% confidence limits for
each mean.
62
-------
in
6
UJ
o
UJ
D
Z
<
tr
0.8 -
0.6
03 18
INITIAL PHOSPHATE (MG/LITER)
FIG. 17. GRANULATION IN ZOOGLOEA RAMIGERA AT DIFFERENT
CONCENTRATIONS OF GLUCOSE, INITIAL PHOSPHATE, AND
MAGNESIUM. Granule counts were made 24 hr. after
adding 1.8 g./liter orthophosphate to phosphate-
starved cultures in arginine broth. The brackets
indicate the 95% confidence limits for each mean.
63
-------
SECTION VI
DISCUSSION
Enhanced phosphorus uptake by Rilling sludge appears to be
biological in nature with no specific requirements for exo-
genous sources of carbon or ions. The uptake is character-
ized by an optimum temperature range, an optimum p_H range,
and is inhibited by several antimetabolites that are active
against enzymes that result in the ultimate synthesis of
adenosine triphosphate.
The experiments shown in Fig. 9 indicate that at least two
types of enzyme systems or microbial populations exist that
participate in this uptake. One is heat labile and seems
to be inactivated by heating at 100°C. for 2 min. (Fig. 9A)
or 70°C. for 30 min. (Fig. 9B). The second is very stable
and is not fully inactivated until the sludge is autoclaved.
Calcium phosphate precipitation as advocated by Menar and
Jenkins (5) seems to play a negligible role under our exper-
imental conditions. The optimum precipitation of phosphates
from waste waters by calcium oxide seems to occur at pH 11
(26). According to Fig. 10 pH 11 is approximately 90% in-
hibitory for the uptake of 32p radioactivity by Rilling
sludge. In addition, the glass distilled water contained
little if any Ca++ and yet the uptake of phosphate from it
was not affected (Table 4). The presence of EDTA in tap
water did not affect uptake despite the fact that the com-
pound is a chelating agent for calcium (The Merck Index).
The uptake of 45ca radioactivity by the Tucson sludge
seemed to have little relationship to the uptake of 32p_
Figure 3 shows that a maximum of 33 to 35% of the approxi-
mately 4% of the 32p associated with the sludge at zero time
could be in the form of an inorganic calcium precipitate.
The uptake of phosphorus by sludge was inhibited by DNP,
which is a well known uncoupler of oxidative phosphorylation.
Tucson sludge had both uptake and retention of the element
affected by the inhibitor (Fig. 4). Uptake of 32P radio-
activity by Rilling sludge is totally inhibited by heavy
concentrations of DNP (Fig. 11C) and 90% inhibited by NaN3
(Fig. 11D). This antimetabolite affected the incorporation
of 32p into organic compounds (Fig. 7).
The other antimetabolites tested are reported to affect mem-
brane function and in some cases other enzyme systems in
various organisms. Gramicidin, Rotenone, Oligomycin, and
Antimycin A had no effect on phosphate uptake (Table 7) at
65
-------
the concentrations tested. With the exception of Gramicidin,
the other compounds are claimed to have little effect on
bacteria. Both Oligomycin (27) and Antimycin (28) are effec-
tive against fungi. Rotenone seems to affect phosphorylation
in higher plants (29). The mercurials (PCMB and HgCl2) are
effective against a variety of organisms. They are reported
to interfere with membrane function and respiration primarily
by combining with sulfhydryl enzymes (30). They are effec-
tive against Rilling sludge in tap water and Tucson sewage
(Table 7, Fig. 11B). lodoacetate effectively inhibits uptake
of 32p radioactivity (Fig. 11A). This compound affects some
sulfhydryl enzymes but seems to inhibit phosphorylation
mainly by acting against the Embden-Meyerhoff pathway (31).
Hotchkiss (32) reported that a related compound (lodoaceta-
mide) as well as DNP, HgCl2, and Gramicidin were inhibitors
of phosphorus uptake by Staphylococcus aureus.
The luxury nature of the phosphorus uptake by Rilling sludge
has been confirmed by reports that it can attain total phos-
phate compositions of 6 to 8% P on a dry weight basis (25) .
Tables 9 and 10 show that the high phosphorus activity is
not related to increased synthesis of nucleic acids. The per
cent dry weight of nucleic acids and their specific activi-
ties for both Rilling and Tucson sludges are quite similar.
A number of different bacteria were successfully isolated
from several sludges by the use of media containing ASE.
Similar successes using this type of medium have been re-
ported by other laboratories (33, 34). Activated sludge
contains heterogenous populations of bacteria. Species of
Pseudomonas-Xanthomonas and Alcaligenes groups appear to
predominate when no supplemental sources of carbon such as
glucose are added to sewage. The development of a predom-
inantly fermentative microflora from sludge, as well as
increased affinity for 32p, j_s observed in the presence of
supplemental glucose.
A filamentous form, S_. natans seems to have the best phos-
phorus affinity of those bacteria isolated (Tables 19 and
20). On the basis of the amount of phosphorus it removes
from sewage, it is probably not the primary organism as the
intact Rilling sludge can remove about 10 fold as much. It
is possible that the primary remover will be this organism
in combination with others.
The volutin granules in Z_. ramigera appeared to be composed
of long chain polyphosphate: They formed immediately after
adding excess orthophosphate to a phosphate-starved culture.
They were metachromatic, a characteristic of long chain poly-
phosphate but not of orthophosphate, pyrophosphate,
66
-------
metaphosphate, or polyphosphate of less than eight phosphate
units in length (35). They stained with Tandler's technique
which is specific for inorganic phosphate. Most of the
radioactivity of 3 P labeled orthophosphate taken up by
phosphate-starved cells was extracted with the nucleic acids
but not subsequently adsorbed by activated charcoal, indi-
cating insoluble polyphosphate, the polyphosphates of high
chain length (36). Chromatographically this label was mostly
in the orthophosphate and pyrophosphate position, probably
due to considerable degradation during extraction. This may
be the reason why so much orthophosphate is apparently pre-
sent in Rilling sludge.
The apparent accumulation of polyphosphate by Z_. ramigera
when excess orthophosphate was added to a phosphate-starved
culture was similar to the polyphosphate overplus phenomenon
in A. aerogenes (37). The enzyme involved was probably
the same as polyphosphate kinase found in Escherichia coli
which takes the terminal phosphate from ATP and builds the
polyphosphate polymer (38); in A. aerogenes this enzyme
mediates the only route of polyphosphate biosynthesis (37).
Optimum concentrations for glucose, initial phosphate, and
magnesium were found for granulation in arginine broth. Too
little glucose apparently caused a shortage of ATP, the
intracellular phosphate source for polyphosphate biosynthe-
sis; too much glucose caused typical catabolic repression.
Too much ini.tial phosphate apparently caused repression of
polyphosphate kinase such as occurs in A. aerogenes (37).
The requirement for magnesium ion was similar to the mag-
nesium requirement by purified polyphosphate kinase from E_.
coli (38). Too much magnesium caused typical cationic in-
hibition.
Granulation occurred in activated sludge in the presence of
2 g. per liter glucose. Granulation in arginine broth
showed an unexpected rise in the factorial design with the
combination of 18 mg. per liter initial phosphate, 20 mg./
liter magnesium, and 2 g. per liter glucose. Tucson raw
sewage contains about 34 mg. per liter orthophosphate
19 mg. per liter magnesium ion (personal communication).
The carbohydrate level of whole sewage in one study was
about 44 mg. per liter carbon: most of this was glucose and
sucrose (39). Thus the phosphate and magnesium levels used
in the factorial design were close to sewage levels; how-
ever;/ the glucose concentration was 18 times that found in
sewage.
67
-------
SECTION VII
ACKNOWLEDGMENTS
Dr. Irving Yall, Professor of Microbiology and Medical
Technology at The University of Arizona was the Project
Director and Principal Investigator and Dr. Norval A.
Sinclair, Associate Professor was Co-Investigator. Both
were the authors of this Report. Dr. William H. Boughton
was Research Associate. Richard J. Gottfried, Richard
C. Knudsen, William C. Lafferty, and Frank A. Roinestad
were Research Assistants.
We are grateful to Mr. W. N. Wells, Waste Disposal Engineer
for the City of San Antonio, Texas for supplying us with
many samples of Rilling sludge. We also wish to thank
Mr. W. F. Garber and Dr. C. H. Connell for providing samples
of Hyperion and Houston sludges and Mr. N. 0. Dye and Mr.
E. J. Trueblood of the City of Tucson Sewage Division for
their cooperation. Dr. Franklin M. Harold of the National
Jewish Hospital, Denver, Colorado kindly provided samples
of various polyphosphates which were used as chromatographic
standards.
The support of the project by the Environmental Protection
Agency and the help provided by Dr. Robert L. Bunch, the
Project Officer, is acknowledged with thanks.
We are grateful to Mr. Robert A. Day, Managing Editor,
American Society for Microbiology and Mr. Lawrence K.
Cecil, Editor of Water for permission to use previously
published material.
We wish to express our sincere appreciation to Mr.
Lawrence K. Cecil, Consulting Engineer without whose
counsel this project would not have been undertaken.
69
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SECTION VIII
REFERENCES
1. Hammond, A. E., "Phosphate Replacements; Problems
With the Washday Miracle," Science, 172, pp 361-364
(1971).
2. Dryden, F. D., and Stern, G., "Renovated Waste Water
Creates Recreational Lake," Environmental Science
and Technology, 2_, pp 268-278 (1968) .
3. Vacker, D., Connell, C. H., and Wells, W. N., "Phosphate
Removal Through Municipal Waste Water Treatment at San
Antonio, Texas," Journal Water Pollution Control Feder-
ation, 3j>, PP 750-771 (1967).
4. Bargman, R. J., Betz, J. M., and Garber, W. F., "Con-
tinuing Studies in the Removal of Phosphorus by the
Activated Sludge Process," Water-1970. Chemical
Engineering Progress Symposium Series, 67, No. 107,
pp 117-121 (1971).
5. Menar, A. B., and Jenkins, D., "The Fate of Phosphorus
in Sewage Treatment Processes. II. Mechanisms of
Enhanced Phosphate Removal by Activated Sludge; "SERL
Report 6j^-6_, University of California, Berkley (1968) .
6. Gaudy, A. F., Jr., and Gaudy, E. T., "Microbiology of
Waste Waters," Annual Reviews of Microbiology, 20,
pp 319-336 (19667";
7. Srinath, E. G., Meera Bai, B., and Pillai, S. C. ,
"Removal of Radioactive Phosphorus From Sewage by
Activated Sludge," Water and Waste Treatment, 11,
pp 410-416 (1967). —
8. Wiame, J. M., "The Occurrence and Physiological Behavior
of Two Metaphosphate Reactions in Yeasts;" Journal of
Biological Chemistry, 178, pp 919-929 (1948).
9. Boughton, W. H., "Phosphate Metabolism by Zoogloeal
Organisms From Activated Sludge," Ph.D. Thesis, The
University of Arizona, Tucson (1969).
10. Ogur, M., and Rosen, G. "The Nucleic Acids of Plant
Tissues. I. The Extraction and Estimation of Desoxy-
pentose Nucleic Acid and Pentose Nucleic" Acid,"
Archives of Biochemistry, 25, pp 262-276 (1950).
71
-------
11. Schmidt, G., and Thannhauser, S. j., "A Method for the
Determination of Desoxyribonucleic Acid, Ribonucleic
Acid, and Phosphoproteins in Animal Tissues, "Journal
of_ Biological Chemistry, 161, pp 83-89 (1945) .
12. American Public Health Association, Standard Methods
for the Examination of Water and Wastewater, 12th Edition,
American Public Health Association, Inc., New York
(1965).
13. Yall, I., Norrell, S.A., Joseph R., and Knudsen, R. C.,
"Effect of L-Methionine and S-Adenosylmethionine on
Growth of an Adenine Mutant of Saccharomyces cervesiae,"
Journal of_ Bacteriology, 93, pp 1551-1558 (1967) .
14. Crane, R. K., "Use of Charcoal to Separate Mixtures of
Inorganic, Ester and Nucleotide Phosphates," Science 127,
pp 285-286 (1958).
15. Ashwell, G., in Methods in Enzymology, Volume III,
Colowick, S. P-, and Kaplan, N. 0., Editors, p 87,
Academic Press, New York (1957).
16. Dische, Z., in The Nucleic Acids, Volume !_, p 285,
Chargaff, E., and Davidson, J. N. , Editors', Academic
Press, New York (1955).
17. Shewan, J. M., Hobbs, G., and Hodgkiss, W., "A Deter-
minative Scheme for the Identification of Certain
Genera of Gram-Negative Bacteria, With Special Refer-
ence to the Pseudomonadaceae," Journal of Applied
Bacteriology, 2_3, PP 379-390 (1960) .
18. Conn, H. J., Jennison, M. W., and Weeks, O. B.,
"Routine Tests for the Identification of Bacteria,"
Manual of Microbiological Methods, Conn, H. J. ,
editor,~~pp 140-168 McGraw-Hill, New York (1957).
19. Crabtree, K., and McCoy, E. "Zoogloea ramigera Itzigson,
Identification and Description," International Journal
of_ Systematic Bacteriology, 17, pp 1-17) (1967) .
20. Conn, H. J., Bartholomew, J. W. , and Jennison, M. W. ,
"Staining Methods," Manual of Microbiological Methods,
Conn, H. J., Editor, pp 10-36, McGraw-Hill, New York
(1957).
21. Tandler, C. J., "A Chemically Specific Technique for
the Intracellular Localization of Inorganic Phosphate,"
72
-------
Journal of Histochemistry and Cytochemistry, 5_,
pp 489-49¥ (1957).
22. Wyatt, G. R., and Cohen, S. S., "The'Bases of the
Nucleic Acids of Some Bacterial and Animal Viruses:
the Occurrence of 5-Hydroxymethyl Cytosine," Bio-
chemical Journal, 55 pp 774-782 (1953).
23. Hanes, C. S., and Isherwood, F. A., "Separation of
the Phosphoric Esters of the Filter Paper Chromatogram,"
Nature (London), 164, pp 1107-1112 (1949).
24. Levin, G. V., and Shapiro, J., "Metabolic Uptake of
Phosphorus by Wastewater Organisms," Journal Water
Pollution Control Federation, 37, pp 800-821 (1965).
25. Wells, W. N., "Differences in Phosphate Uptake Rates
Exhibited by Activated Sludges," Journal Water Pollution
Control Federation, 41, pp 765-771 (1969) .
26. Owen, R., "Removal of Phosphorus From Sewage Plant
Effluent With Lime, "Sewage and Industrial Wastes, 25,
pp 548-556 (1953).
27. Marty, E. W., Jr., and McCoy, E., "The Chromatographic
Separation and Biological Properties of the Oligomycins,"
Antibiotics and Chemotherapy, 9, pp 286-293 (1959).
28. Leben, C., and Keitt, G. W., "An Antibiotic Substance
Active Against Certain Phytopathogens," Phytopathology,
38_, pp 899-906 (1948) .
29. Harold, F. M. , "Antimicrobial Agents and Membrane Function,
"Advances in_ Microbial Physiology, 4_, pp 45-104 (1970) .
30. Passow, H., Rothstein, A., and Clarkson, T. W., "The
General Pharmacology of the Heavy Metals," Pharmacol-
ogical Reviews, 13, pp 185-224 (1961).
31. Webb, J. L., Enzyme and Metabolic Inhibitors, Volume
III, pp 1-281 Academic Press, New York (1966).
32. Hotchkiss, R. D., "The Assimilation of Amino Acids
by Respiring Washed Staphylococci," Archives of
Biochemistry and Biophysics, 65, pp 302-318 (1956) .
33. Lighthart, B., and Oglesby, R. T. , "Bacteriology of an
73
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Activated Sludge Wastewater Treatment Plant- a Guide
to Methodology," Journal Water Pollution Control
Federation, 41, pp R267-R281 (1969) .
34. Prakasam, T. B. S., and Dondero, N. C., "Aerobic
Heterotrophic Bacterial Populations of Sewage and
Activated Sludge," Applied Microbiology, 15, pp 1122-
1127 (1967).
35. Ebel, J. P., Colas, J., and Muller, S., "Recherches
Cytochimiques sur les Polyphosphates Inorganiques
Contenus dans les Organismes Vivants," Experimental
Cell Research, 15_, pp 21-42 (1958) .
36. Harold, F. M., "Inorganic Polyphosphates in Biology,
Structure, Metabolism, and Function," Bacteriological
Reviews, 30, 772-794 (1966).
37. Harold, F. M., "Enzymic and Genetic Control of Poly-
phosphate Accumulation in Aerobacter aerogenes,"
Journal of General Microbiology, 35, pp 81-90 (1964) .
38. Kornberg, A., Kornberg, S. R., and Simms, E. S.,
"Metaphosphate Synthesis by an Enzyme From Escherichia
coli," Biochimica et Biophysica Acta, 20. pp 215-227
7T9T6) .
39- Painter, H. A., and Viney, M., "Composition of a
Domestic Sewage," Journal of Biochemical Micro-
biological Technology and Engineering,T, pp 143-162
(1959r
74
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SECTION IX
LIST OF PUBLICATIONS
Roinestad, F. A., and Yall, I., "Volutin Granules in
Zoogloea ramigera," Applied Microbiology, 19, pp 973-
979 (1970T
Yall, I., Boughton, W. H., Knudsen, R. C., and Sinclair,
N. A., "Biological Uptake of Phosphorus by Activated
Sludge," Applied Microbiology, 20, pp 145-150 (1970).
Yall, I., Sinclair, N. A., Boughton, W. H., Knudsen,
R. C., and Lafferty, W. C., "Phosphorus Utilization
by the Microorganisms of Activated Sludge, "Water-
1970, Chemical Engineering Progress Symposium Series,
6T_, No. 107, pp 95-99 (1971) .
Boughton, W. H. , Gottfried, R. J., Sinclair, N. A., and
Yall, I., "Metabolic Factors Affecting Enhanced Phosphorus
Uptake by Activated Sludge," Applied Microbiology, 22,
pp 571-577 (1971).
Boughton, W. H. , Gottfried, R. J-. , Sinclair, N. A., and
Yall, I., "Metabolic Comparisons of High and Low Phos-
phorus Removing Sludges," Water-1971 (in press).
75
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SECTION X
GLOSSARY
Antimetabolite- A substance that prevents metabolism-see
Inhibitor.
Adenosine triphosphate-ATP- A compound composed of adenine
(a purine base), ribose (a 5-carbon sugar) and three phosp-
horus atoms as esters on the 5" carbon of the sugar.
Aseptic- Precaution to exclude undesired bacteria.
Deoxyribonucleic acid-DNA- A molecule of high molecular
weight composed of subunits of nucleotides containing
deoxyribose as the sugar, frequently found in cell nucleus.
Equivalent- That amount of a substance (measured in grams)
numerically equal to the formula weight divided by the
valence.
Inhibitor- An agent which slows or interferes with growth
of bacteria.
Metabolism- The sum of the processes concerned in the build-
ing up of protoplasm.
Molarity-M- The number of moles of solute per liter of
solution.
Mole- The formula weight of a substance expressed in grams.
Normality-N- The number of equivalences of a substance
(solute) per liter of solution.
Nucleotide- 5'-Phosphate ester composed of a purine or
pyrimidine base, pentose ( 5-carbon sugar) and an atom of
phosphorus.
Polyphosphates- Inorganic compounds containing more than
three atoms of phosphorus (joined in straight chain).
Ribonucleic acid-RNA- Molecule of high molecular weight
composed of subunits of nucleotides containing ribose as
the sugar, frequently found in cell cytoplasm.
Synergism- The total effect is greater than the sum of two
or more effects taken independently.
Volutin- A chromatoid substance, occurring as metachromatic
granules in the cytoplasm of various cells.
U. S. GOVERNMENT PRINTING OFFICE : 1972—484-484/162
77
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SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
1. Report No.
2.
4. Title
Mechanisms of Biological Luxury Phosphate Uptake
7. Authoi(s)
3. Accession No.
w
5. Report Date
6.
S. Performing Organization
Report No.
Yall, I. and Sinclair, N. A.
9. Organization Arizona University, Tucson,
Microbiology and Medical Technology Dept.
12. Sponsoring Organization
15. Supplementary Notes
10. Project No.
EPA,
17010 DDQ
11. Contract/ Grant No.
13. Type of Report and
Period Covered
16. Abstract Activated sludges obtained from the Rilling Road plant located
at San Antonio, Texas and from the Hyperion treatment plant located at
Los Angeles, California have the ability to remove large amounts of
phosphorus from Tucson sewage and other liquors by means of biological
mechanisms. Most of the phosphorus seems to accumulate within the
sludge cells as orthophosphate. Tucson sludge seems to take up phos-
phorus by biological mechanisms but removes considerably less from its
medium than does Rilling sludge. However, phosphorus uptake by Tucson
sludge is improved if the sludge is starved prior to the addition of
sewage. The bacteria isolated from Rilling sludge do not individually
seem to account for a high phosphorus affinity when compared to those
from Tucson sludge. A culture of Sphaerotilus natans was isolated from
Rilling but not from Tucson sludge"! This organism had a higher affinity
for phosphorus than others tested but not sufficient to account for the
superior removal properties exhibited by the Texas sludge. A known
sludge bacterium, Zoogloea ramigera formed volutin granules when excess
orthophosphate was added to a phosphate starved culture. However, the
conditions necessary to produce these granules in this organism probably
do not exist in normal sewage.
na.Descriptors Phosphates*, Activated Sludge*, Bacteria*, Pseudomonas,
Enteric Bacteria
nb.identifiers Tucson, (Ariz.) *, San Antonio, (Texas)*, Los Angeles, (Calif.)*
Houston, (Texas), Sewage Treatment Plants, Bacterial Isolation*
17c. COWRR Field & Group
18. Availability
05D
19. Security Class.
(Report)
20. Security Class.
(Page)
21. No. of
Pages
22. Price
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, D. C. 20240
Abstractor Irving Yall
[ institution Arizona University
WRSICI02 (REV. JUNE 1971)
GP 0 9 13.261
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