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
Protection Agency, through inhouse research and grants
and contracts with Federal, State, and local agencies,
research institutions, and industrial organizations.

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 24C.
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  25C.  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 25C. 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
25C.  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 0C.  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 100C.  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 4C. 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)
(70C. 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
37C.  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

-------
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 24C. 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

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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 24C.

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
4C.

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 24C. 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
4C. 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

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7 n

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UJ
UJ
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Q.
O
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t-
tr
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                                       43
                                         Co
    60
    50
    40
z   30
    20
    10
                                	  ORTHOPHOSPHATE
        oooo-
                                                             60
                                                             50
                                                             40
     ID

     O
     O
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     UJ
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     O
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     cr
20
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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
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85
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-------
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
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    50
    40
    20
    10
                                           ---  ORTHOPHOSPHATE
                                                              50
                                                              40
                                                              30
                                                                 O
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                                                                 5
                                                                  o
                                                                  a:
                                                                  o

                                                                  a:

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                                                             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
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-------
                                       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
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                                                          < ffl 0
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-------
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 24C 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 24-37C.,
which would be characteristic of a biological rather than a
chemical phenomenon.

Figure 9A shows the effects of exposing Rilling sludge to
100C. 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 70C. 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

-------
                                                            o
                                                            to
                                                            o
                                                            to
                                                               O
                                                               O
                                                               UJ
                                                               o:
                                                               IE
                                                               UJ
                                                               Q_

                                                               2
                                                               UJ
                                                            8
 o
 o
o
0>
o
00
o
<>
o
in
   I	I

   O     O
   *     rO


1N3D d3d
o
C\J
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

-------
     UJ
     Q.
    CM
        50
        40
       30

       20
     UJ
     O  10
     OC
100

90

80

70
     UJ
     <  60
     O.
     3  50
     I-
     S  40
     O
       30
     Q.
        20

        10
                         TEMPERATURE CONSTANT (IOOC)
                                  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 (100C.) 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  24C.  for
         3 hr.
                              35

-------
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-------
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 24C. 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

-------
   Q.
  CM
  10
    100

     90

     80
     70
   UJ
   < 60
3 50

5 4
o
or 2
UJ
Q-
  20
      10
                                %
            100

             90

            , 80

             70
                                UJ
                                <60
                                0.
                                     40
                                   O
                                     20
                                  10
                                                          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 24C,  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  25C.  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, KHPO.

 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

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                                                 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.

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                                                      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

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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

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                      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

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   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

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                                                     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

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   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 24C.
                                 54

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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

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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

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                     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 100C. for 2 min.  (Fig. 9A)
or 70C. 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

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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

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                        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

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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

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     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 : 1972484-484/162
                        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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