EPA-R2-72-031
NOVEMBER 1972 Environmental Protection Technology Series
An Investigation of Phosphorus
Removal Mechanisms
in Activated Sludge Systems
Office of Research and Monitoring
U.S. Environmental Protection Agency
Washington, DC 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were'established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fieJ.ds. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
U. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL
PROTECTION TECHNOLOGY series. This series
describes research performed to develop and
demonstrate instrumentation, equipment and
methodology to repair or prevent environmental
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pollution. This work provides the new or improved
technology required for the control and treatment
of pollution sources to meet envixonmental quality
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EPA-R2-72-031
November 1972
AN INVESTIGATION OF PHOSPHORUS REMOVAL MECHANISMS
IN ACTIVATED SLUDGE SYSTEMS
By
W. E. Morgan
and.
E. Gus Fruh
Project 17010 DUX
Project Officer
Dr. R. L. Bunch
Biological Treatment Research Program, AWTRL
Environmental Protection Agency
National Environmental Research Center
Cincinnati, Ohio ^5268
Prepared for
OFFICE OF RESEARCH AND MONITORING
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C.
For sale by the Superintendent of Documents, U.S. Government Printing Office
Washington, B.C., 20402 - Price $2
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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 necessarily 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.
ii
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ABSTRACT
The magnitude of two phosphorus removal mechanisms, metabolic
uptake and chemical precipitation with calcium, in activated sludge
systems was investigated using synthetics substrates representative of
actual wastewaters. Using completely mixed continuous flow laboratory
activated sludge units with operating conditions that precluded signi-
ficant precipitation of phosphorus , normal growth defined as constant
85 to 90 percent carbon removal occurred above 0.9 to 1 .'0 percent sludge
phosphorus (influent COD:P ratio of 670:1). Between 1.0 and 1.6
percent (influent COD:P ratio of 220:1) a storage zone existed with
all phosphorus present utilized, and above 1.6 percent a variable
saturation zone occurred with an upper limit near 3.0 percent.
An alkaline phosphatase bioassay verified qualitatively the normal
growth phosphorus requirement and storage zone, but did not define
the upper limit of the saturation zone.
An acclimated activated sludge unit with a substrate containing 2 mM
calcium, 0.4 mM phosphorus , 0.8 mM magnesium and 2 .5 mM
bicarbonate attained a maximum of 3.7 percent sludge phosphorus
after 39 days of operation at pH 7.6. A similar system with the
addition of 1 mg/1 fluoride attained 4.6 percent sludge phosphorus.
An increase in magnesium to 2 .0 mM had little effect on phosphorus
precipitation. Alkalinity was implicated to exert both a kinetic
effect as well as an effect on residual soluble phosphorus in calcium-
phosphorus systems. The presence of soluble organics also was
shown to be inhibiting with increasing concentrations.
This report was submitted in fulfillment of Project Number 17010 DUX,
under the sponsorship of the Environmental Protection Agency.
111
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CONTENTS
Section Page
ABSTRACT iii
CONTENTS v
FIGURES viii
TABLES xi
I CONCLUSIONS 1
II RECOMMENDATIONS 3
III PURPOSE AND NEED OF THE STUDY 5
Biological Removal of Phosphorus 6
Metabolic Phosphorus Requirements 6
An Enzymatic Technique to Detect Surplus Phos-
phorus Uptake 10
Chemical Removal of Phosphorus 12
Dibasic Calcium Phosphate 14
Apatites 15
Kinetics of Solid Formation 15
Fluoroapatite 17
Beta-Tricalcium Phosphate 20
X-ray and Infrared Adsorption Measurements 20
Effect of Operational Characteristics on Phosphorus
Removal 23
Evaluation of Phosphorus Removal Measurements ... 24
Effects of Dissolved Oxygen 25
Organic Loading and Aeration Solids 29
Calcium, pH and Other Ionic Constituents 31
Literature Evaluation 33
Objective and Scope 34
IV EXPERIMENTAL PROCEDURES 37
Experimental Studies 37
Batch Units for Biological Studies 37
Completely Mixed Continuous Flow Units for Bio-
logical and Chemical Studies 37
Inoculum 38
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Contents (Continued)
Section Pa9e
Synthetic Wastewater Substrates 40
Automatic pH Control System ^
Batch Units for Chemical Precipitation Studies 41
'Analytical Techniques 41
Alkaline Phosphatase Enzymatic Assay 41
" Phosphorus Measurements 44
Other Measurements 44
Precision of Analyses 46
V METABOLIC UPTAKE OF PHOSPHORUS 47
Determination of Operating Conditions to Preclude
Phosphorus Precipitation 47
General Procedures 48
Calcium-Phosphorus Systems 49
Effect of Magnesium 53
Nucleation Sites 53
Synopsis 55
Minimum Required Sludge Phosphorus for Normal
Growth 55
General Procedures 56
Results 56
Synop sis 58
Metabolic Incorporation of Phosphorus with Controlled
Chemical Precipitation 60
General Procedures 60
Initial High Sludge Phosphorus Inoculum 61
Initial Low Sludge Phosphorus Inoculum 63
Addition of Solid Forms of Calcium and Phosphorus . . 65
Synopsis 68
Evaluation of Alkaline Phosphatase Enzymatic Assay ... 68
General Procedures 69
Results 70
Synopsis 76
Summary 77
VI CONCURRENT CHEMICAL AND METABOLIC
INCORPORATION OF PHOSPHORUS 79
Operating Conditions to Induce Chemical Precipitation. . 79
General Procedures 80
Experimental Results 81
VI
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Contents (Continued)
Section Page
Calcium-Phosphorus Systems 81
Effect of Magnesium 81
Effect of Fluoride 84
Effect of Inorganic Substrate Components 84
Effect of Organic Substrate Components 88
Synopsis 90
Calcium Precipitation of Phosphorus in Completely
Mixed Continuous Flow Laboratory Activated Sludge
Units 91
General Procedures 91
Control Unit 93
Normal Calcium : Magnesium Ratio Unit 93
Low Calcium : Magnesium Ratio Unit 96
Fluoride Unit 96
Synopsis 97
Effect of Induced Upset on Activated Sludge Systems
Exhibiting Calcium-Phosphorus Precipitation 102
Normal Calcium : Magnesium Ratio Unit 102
Low Calcium : Magnesium Ratio Unit 103
Fluoride Unit 103
Effect of Added Solid Hydroxyapatite 110
Synopsis Ill
Summary 112
VII DISCUSSION 115
Metabolic Phosphorus Incorporation " 115
Calcium-Phosphorus Precipitation 119
VIII ACKNOWLEDGEMENTS 127
K REFERENCES 129
X LIST OF PUBLICATIONS 137
IX APPENDICES 139
vii
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FIGURES
No.
1 Zones of Sludge Phosphorus Content for Algae 8
2 Relationship of Enzyme Activity and Cellular Phos-
phorus Content in Dilute Bacterial Cultures 13
3 Predicted Soluble Phosphate at Equilibrium for Systems
with High and Low Magnesium Concentrations 15
4 Effect of Time on Phosphate Removal at pH 7.6 with
Seeded and Unseeded Solutions 18
5 Effect of Magnesium Concentration and pH on
Soluble Phosphate, Calcium and Magnesium 19
6 Infrared Spectra of Hydroxyapatite Precipitates 22
7 Infrared Spectra for Samples Described in Table
Precipitated for 24 Hours at 29°C 22
8 Soluble Phosphate Uptake as a Function of Dissolved
Oxygen Concentration 28
9 Sketch of Completely Mixed Continuous Flow Unit. ... 39
10 Influent Phosphorus , Effluent Phosphorus , Percent
Sludge Phosphorus Content, and Percent Carbon
Removal for Laboratory Completely Mixed Contin-
uous Flow Activated Sludge Units 57
11 Influent Phosphorus , Effluent Phosphorus , Percent
Sludge Phosphorus Content, and Percent Carbon
Removal for Laboratory Completely Mixed Contin-
uous Flow Activated Sludge Units 59
12 Results of Alkaline Phosphatase Bioassay-
Minimum Sludge Phosphorus Content Study,
First Test Series 71
13 Results of Alkaline Phosphatase Bioassay-
Minimum Sludge Phosphorus Content Study,
Second Test Series 72
14 Results of Alkaline Phosphatase Bioassay-
Metabolic Incorporation of Phosphorus with
Controlled Chemical Precipitation Study 73
15 Results of Alkaline Phosphatase Bioassay-
Batch Tests 75
16 Batch Chemical Precipitation Tests; 2 mM Calcium,
and 0.33 mM Phosphorus g2
17 Batch Chemical Precipitation Tests; 2 mM Calcium,
0.33 mM Phosphorus, and 0.8 mM Magnesium .. g3
viii
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FIGURES (Continued)
No. Page
18 Batch Chemical Precipitation Tests; 2 mM Calcium,
0.33 mM Phosphorus, and Inorganic Components
of Substrate C Less Magnesium 86
19 Batch Chemical Precipitation Tests; 2 mM Calcium,
0.33 mM Phosphorus, and All Inorganic
Components of Substrate C 87
20 Percent Sludge Phosphorus Content for Six Week
Acclimated Laboratory Activated Sludge Unit:
Normal Ca:Mg Ratio of 2.5 94
21 Effluent Calcium and Phosphorus Concentrations for
Six Week Acclimated Laboratory Activated Sludge
Unit: Normal Ca:Mg Ratio of 2 . 5 95
22 Percent Sludge Phosphorus Content for Six Week
Acclimated Laboratory Activated Sludge Unit:
Low Ca:Mg Ratio of 1 98
23 Effluent Calcium and Phosphorus Concentrations for
Six Week Acclimated Laboratory Activated Sludge
Unit: Low Ca:Mg Ratio of 1 99
24 Percent Sludge Phosphorus Content for Six Week
Acclimated Laboratory Activated Sludge Unit:
Fluoride Unit 100
25 Effluent Calcium and Phosphorus Concentrations for
Six Week Laboratory Activated Sludge Unit:
Fluoride Unit 101
26 Effect of pH Upset on Percent Sludge Phosphorus
in Laboratory Activated Sludge Unit: Ca:Mg
Ratio of 2.5 104
27 Effect of pH Upset on Laboratory Activated Sludge
Unit: Ca:Mg Ratio of 2.5 105
28 Effect of pH Upset and Addition of Apatite Spike on
Percent Sludge Phosphorus in Laboratory Activated
Sludge Unit: Low Ca:Mg Ratio of 1; Increased to
2.5 for Apatite Spike 106
29 Effect of pH Upset and Addition of Apatite on
Laboratory Activated Sludge Unit: Low Ca:Mg
Ratio of 1; Increased to 2 .5 for Apatite Spike 107
30 Effect of pH Upset on Percent Sludge Phosphorus
in Laboratory Activated Sludge Unit: Fluoride Unit. . 108
31 Effect of pH Upset on Laboratory Activated Sludge
Unit; Fluoride Unit 109
IX
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FIGURES (Continued)
Nip. Page
32 Influent Phosphorus, Effluent Phosphorus, Percent
Sludge Phosphorus Content, and Percent Carbon
Removal for Laboratofy Completely Mixed
Continuous Flow Activated Sludge Units 116
33 Qualitative Summary of Phosphorus Precipitation
found in Batch Tests With 2 .0 mM Calcium 120
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TABLES
No. Page
1 Phosphorus Uptake by Activated Sludge at San
Antonio , Texa s 11
2 Composition of Solutions Used in Infrared Analyses . . 21
3 Chemical Analyses of Fresh Water Supplies of Some
Municipalities Reporting High Sludge Phosphorus
Content 33
4 Effect of Increased Sludge Mass on Enzymatic
Activity 43
5 Effect of Filtration Time on Enzymatic Activity 43
6 Check Procedure 43
7 Comparison of Alkaline Ash Method to Persulfate
Method for Determination of Total Phosphorus 45
8 Precision of Analyses 45
9 Calcium-Phosphorus Systems in Inorganic Medium
Less Magnesium, Ammonia, and Bicarbonate 50
10 Calcium-Phosphorus Systems in Distilled Water
(add pH 6.5, 7.3, and 8.0) 50
11 Calcium-Phosphorus Systems in Distilled Water
(add pH6.6, 7.4, 8.0, 8.5) 51
12 Calcium-Phosphorus Systems With Trace Elements ... 52
13 Calcium-Phosphorus Systems With One Millimole
Magnesium 54
14 Effect of Nucleation Sites (8 mg/1 Kaolinite) on
Calcium-Phosphorus Systems With and Without
Magnesium, 0.8 mM/1, Containing all Components
of the Biological Substrate 54
15 Initial High Phosphorus Sludge Content After 5 Days
of Operation With Controlled and Uncontrolled pH . . 62
16 Initial Low and High Sludge Phosphorus Inoculum;
10 mg/1 Phosphorus and 40 mg/1 Calcium Influent
Concentration 64
17 Initial Low Sludge Phosphorus With CaHPO4 and
CaCO3 Spikes; 10 mg/1 Phosphorus and 40 mg/1
Calcium Influent Concentrations 67
18 Batch Tests 74
19 Batch Chemical Precipitation Tests 85
XI
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TABLES (Continued)
No. Page
20 Batch Chemical Precipitation Tests; 2.0 mM
Calcium, 0.33 mM Phosphorus, and 50 mg/1
COD of Organic Components of Substrate C 89
21 Comparison of Sewage from San Ramon, California
and Synthetic Wastewater Substrate 121
22 Relationship Between Soluble Phosphorus, Dissolved
Oxygen and Soluble Organics in an Activated
Sludge System 125
XII
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SECTION I
CONCLUSIONS
1. The limits of metabolic incorporation of phosphorus in
completely mixed continuous flow laboratory activated
sludge systems are:
a. the minimum required sludge phosphorus content for
normal growth, i.e. , a constant 85 to 90 percent
carbon removal, is approximately 1 percent and occurs
at an influent COD:phosphorus ratio of 670:1 (100:0.15);
b. sludge phosphorus content greater than 1 percent repre-
sents "luxury uptake" and is the rule and not the
exception in activated sludge systems;
c. a storage zone exists between 1 and 1.6 percent sludge
phosphorus content where all phosphorus present in the
influent is utilized;
d. all phosphorus in the influent is utilized and a sludge
phosphorus content of 1.6 percent occurs with an
influent COD:phosphorus ratio of 220:1 (100:0.45);
e. an increase in influent phosphorus beyond the optimum
influent COD:phosphorus ratio of 220:1 will result in
an increase in sludge phosphorus content but not all
the phosphorus present in the influent will be utilized;
and,
f. the maximum sludge phosphorus content obtained by
metabolic incorporation is approximately 3 percent.
2. The minimum required, storage, and saturation zones for
metabolic incorporation of phosphorus were qualitatively
defined by the alkaline phosphatase bioassay; however,
a. the bioassay did not define the upper limit of metabolic
incorporation of phosphorus , and
b. a quantitative relationship between alkaline phosphatase
activity and percent sludge phosphorus content was not
obtained, thereby precluding an engineering application
as an operating tool.
3. Phosphorus incorporation in activated sludge greater than the
maximum possible by metabolic mechanisms was obtained
at pH 7.6 in six week acclimated completely mixed continuous
flow laboratory activated sludge systems with precipitation
of phosphorus restricted to calcium-phosphorus forms.
1
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4. A sludge phosphorus content of 3.7 percent was obtained with
calcium-phosphorus precipitation which is much lower than
v • the sludge phosphorus contents reported, 6 to 7 percent, in
activated sludge plants exhibiting high phosphorus removals, e.g.
San Antonio Rilling, Baltimore.
5. High influent magnesium concentrations (Ca:Mg ratio/^ 1) did
not affect the sludge phosphorus content, but did delay the
start of a calcium-phosphorus precipitation in an activated
sludge system.
6. The addition of 1 mg/1 fluoride increased the sludge phosphorus
content to 4.6 pe/cent removing over twice the phosphorus
required for normal'metabolic requirements.
7. .Calcium-phosphorus systems are sensitive to upsets in
operating pH conditions and the presence of fluoride did not
affect a calcium-phosphorus system within 50 days after
upset.
8. Other factors were found that affect calcium-phosphorus systems
in activated sludge:
a. organic concentration has a substantial negative effect
on calcium-phosphorus precipitation;
b. the normal events of high dissolved oxygen and increase
in pH of plug flow systems can be described as symptoms
• of removal of an organic poisoning effect of calcium-
phosphorus precipitates by microbial incorporation of
biologically degradeable carbon; and,
c. alkalinity affects dilute calcium-phosphorus systems and
appears to exert a significant effect at high concentrations.
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SECTION II
RECOMMENDATIONS
During the course of this laboratory investigation of metabolic
incorporation of phosphorus and chemical precipitation of phosphorus
with calcium,two areas with potential as phosphorus removal methods
were noted and should be further investigated.
The storage zone defined for activated sludge, between 1 and 1.6
percent, exhibits an uptake of essentially all phosphorus present
in the influent. Studies should be undertaken to evaluate techniques
for rapid stripping of phosphorus from sludge to obtain the low sludge
phosphorus contents required. The characteristics of such low
phosphorus content sludges should be investigated, particularly of
solids-liquid separation and organic uptake within existing system
designs. If efficient operation within the storage zone could be
attained, relatively low effluent phosphorus concentrations would
be possible using a metabolic removal mechanism. Such a method
could be appropriate for municipal wastewaters with below average
influent phosphorus concentrations, less than 8 mg/1 P.
Precipitation of phosphorus with calcium, though not sufficient to
account for the high removals reported by some activated sludge
plants, does indicate that a chemical removal mechanism can occur
in activated sludge. Additional studies are indicated to investigate
the presence of small quantities , less than 3 to 5 mg/1, of other
cations such as Fe III and Al III that have been reported to be
reduced in concentration passing through activated sludge plants.
Separate and combined cation studies could identify the optimum
cation(s) and concentration^) to induce significant chemical removal
of phosphorus .
Also, a possible organic poisoning effect on such chemical precipi-
tation should be investigated. Such an effect could be present in
some plants exhibiting marginal organic and poor phosphorus removal
characteristics. Improved organic removal could possibly enhance
phosphorus removal with no additional changes in a system.
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SECTION HI
PURPOSE AND NEED OF THE STUDY
The quantity of phosphorus incorporated by activated sludge has been
reported by several investigators to range between 2 and 3 percent
based on volatile suspended solids present in the sludge (1,2,3).
While this range is considered "normal" and is found in most activated
sludge systems, there have been some activated sludge systems
reported to exhibit higher percentages of incorporated phosphorus, up
to 7 percent in some cases (4,5). This unusual and unaccounted for
incorporation of phosphorus has led to several investigations to
determine the mechanism(s) responsible for this phenomenon.
Two hypotheses for this abnormally high sludge phosphorus are:
1) a biological incorporation in excess of required metabolic uptake
by the biomass present in the sludge, so called "luxury uptake";
and
2) a physical-chemical formation of a solid specie that subsequently
is sorbed or becomes enmeshed in the sludge mass.
These two viewpoints have caused considerable discussion in the liter-
ature and both hypotheses can be at least partially validated. However,
insufficient field and laboratory data exist to substantiate clearly the
mechanism(s) responsible for the high phosphorus content of some
sludges.
Biological incorporation of phosphorus occurs with the synthesis of
new cells, but the magnitude of phosphorus incorporation beyond that
necessary for new growth is not well-defined. Some means of isolating
biological uptake and evaluating the magnitude of this phosphorus removal
mechanism quantitatively could aid in determining if other mechanism(s)
are involved significantly in the removal of phosphorus from domestic
wastewaters.
In this section a thorough evaluation of the literature is conducted,
including research on the biological uptak^ of phosphorus, various
chemical mechanisms of phosphorus removal and the field studies
which have attempted to define the operating conditions that could have
induced the high sludge phosphorus content observed at several
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activated sludge plants. Subsequently, the specific objectives and
scope of this investigation are outlined.
Biological Removal of Phosphorus
A major phosphorus removal mechanism found in activated sludge
systems is the biochemical uptake of phosphorus to satisfy the
metabolic requirements of the biomass. For normal activated sludge
systems which remove a small percentage of the influent phosphorus,
the accepted range of cellular phosphorus content is between two
and three percent of the volatile solids. However, several investi-
gators (4,5) reported activated sludges with significantly higher sludge
phosphorus contents, and the capability of removing much higher
percentages of the influent phosphorus which they have attributed to
"luxury uptake" of phosphorus (6,7,8). These differences have led
to some discussion as to the amount of phosphorus that is meta-
bolically required, or that can be assimilated by the biomass.
Metabolic Phosphorus Requirements
The phosphorus assimilation for specific microorganisms is variable.
The presence of volatin granules reported to contain polyphosphate
has been observed in some bacteria (9).
Varma and Stonefield (10) reported different phosphorus assimilation
through a growth cycle for E_. coli and P_. vulgaris using P^2 techniques.
Subsequent tests also exhibited significant differences between these
pure cultures and mixed activated sludge cultures. Yall, et al. (11)
using P^2 techniques reported the results of 88 bacterial isolates
from an activated sludge exhibited variable phosphorus assimilation.
A discussion of the biological assimilation of phosphorus to fulfill
the metabolic requirements of the mixed bacterial cultures in activated
sludge systems should be prefaced by a short discussion of the para-
meters required to define this removal mechanism. A number of past
studies (1,12,13,14) have reported required ratios of carbon, as bio-
chemical oxygen demand (BOD), to phosphorus in the influent waste,
to sustain efficient operation of activated sludge systems, i.e. BOD
removal and rapid solid-liquid separation. The reported ratios of
BOD to phosphorus, 100 to 1, satisfy these factors and preclude
phosphorus from being a limiting nutrient in domestic wastewater
treatment systems. However, these ratios do not define the limits of
the metabolic uptake of phosphorus by bacteria, i.e. the minimum
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phosphorus required for normal growth and the maximum phosphorus
that can be biologically incorporated. The metabolic uptake of
phosphorus by bacteria should be defined within these limits.
Greenberg, et_al_. (15) investigated the minimum required sludge
phosphorus content for good growth using "fill and draw" laboratory
units. The substrate used had a BOD:P ratio of 100:0.42 and an ionic
composition which precluded any significant phosphorus removal by
chemical precipitation. They reported that BOD removals greater than
90 percent occurred when the sludge phosphorus content was one
percent or greater, but below this value BOD removal declined. As
they increased the phosphorus in the substrate (BOD:P ratios of
100:0.95 and 100:2.6), the sludge phosphorus content increased to
a high of 1. 8 percent.
Hattingh (16) also investigated the effect of low sludge phosphorus
content using "fill and draw" laboratory units. During these experi-
ments the sludge phosphorus content was reduced to less than 1.0
percent after 32 days with a substrate having a BOD:P ratio of 100:0.24,
but the biomass still removed greater than 95 percent of the influent
BOD. Using a substrate with a BOD:P ratio of 100:1.65 a maximum
sludge phosphorus content of 2.2 percent was obtained. As with
Greenberg's study, the composition of the substrates precluded any
significant phosphorus removal by chemical precipitation.
Greenberg reported that the biomass in the low phosphorus cultures
was primarily filamentous with poor solid-liquid separation character-
istics. Hattingh reported no marked differences in protozoal or bacterial
population between the high and low phosphorus cultures; however,
some tendency to bulk occurred in the low phosphorus cultures. The
low phosphorus content of the bacteria did not affect the BOD removal
efficiency, but did affect the solid-liquid separation characteristics
of the sludges.
Borchardt and Azad (17) conducted experiments to determine the lower
limit of phosphorus content in algae to maintain good growth. Using
cultures of ChloreUa and Scenedesmus they reported three cellular
phosphorus cdntent regimes under steady state growth conditions:
the storage zone, one to three percent cellular phosphorus; the growth
dependent zone, zero to one percent cellular phosphorus; and the
saturation zone, greater than three percent (see Figure 1.). Within
the one to three percent cellular phosphorus regime, maximum growth
occurred with the removal of all phosphorus from the substrate. Above
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Growth
Dependent
Growth
ndependent
Excess P04
Zone H
Zone
2000 Foot Candles, 20°C, 1500 rpm
pH = 8.3 ± O.I, Algae Dry Wt. 50 mg/l
Luxury Uptake
ro
1 sj-
O
Q_
0123456789 10
Steady State Phosphate Supplied ( mg/PO^/nr)
Figure 1 . Zones of Sludge Phosphorus Content for Algae (after Borchardt and Azad).
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three percent cellular phosphorus additional phosphorus in the sub-
strate above that required to maintain the three percent cellular
phosphorus level appeared in the effluent. Moore and Fruh (18)
reported similar results with various algal cultures. The lower limit
of cellular phosphorus content for maximum growth in these algal
experiments was approximately one percent and agreed with the
conclusions of Greenberg, _e;t aK and Hattingh based on BOD removal
by mixed cultures of activated sludge microorganisms.
Other investigations have been conducted to determine only the
maximum cellular phosphorus content of activated sludge and the
results have indicated that the upper limit of cellular phosphorus
approaches three percent (19,20). A recent extensive study of sludge
phosphorus content by Jenkins and Menar (3) covering a wide range of
organic loading rates, 0.2 to 13.8 pounds of BOD per pound of volatile
suspended solids, reported a minimum sludge phosphorus content of
2 .45 percent and a maximum of 3.03 percent with a weighted mean of
2.62 percent.
There are instances reported in the literature that have alluded to
"luxury uptake" occurring during laboratory tests. Randall, et al.
(21) conducted tests to investigate phosphorus release by activated
sludge. They observed that phosphorus uptake was remarkably high,
an average of 9 mg/1 P associated with approximately 250 mg/1 COD
removal. They implied that this high phosphorus removal suggested
"luxury uptake. " However, their substrate medium did not preclude
phosphorus precipitation.
Following 9 mg/1 P removal, the increase in the percent sludge phos-
phorus based on the original 2000 mg/1 suspended solids was calcu-
lated from other data presented by Randall,_et_a_l. to be 0.45 percent.
No data were available on the initial percent sludge phosphorus for
comparison, but an increase of 0.45 percent would not be unusual in
a batch test. Additionally, the difference between the phosphorus
uptake and phosphorus subsequently released during the leaching phase
of the experiments averaged 2.5 mg/1. If this is assumed to be
incorporated in new growth (approximately 90 mg/1 for 250 mg/1 COD
removed), the percent sludge phosphorus content was approximately
2.7 percent and not indicative of unusual phosphorus incorporation.
Wells (22) reported the results from several batch type experiments at
the San Antonio Treatment Plants using raw wastewater and hence also
did not preclude phosphorus precipitation. To show the effect of
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vigorous aeration on phosphorus uptake, samples from the return
sludge of the Rilling, East and West Plants were mixed with the same
sample of raw wastewater and aerated vigorously. The Rilling plant
sludge rapidly removed 14 mg/1 P in approximately one hour. The
East Plant sludge removed approximately 2 mg/1 P in eight hours,
while the West Plant removed 12 mg/1 P during the same eight hour
aeration period. In similar previous aeration batch tests the East and
West sludges had removed little phosphorus. Thus, Wells indicated
the possibility of the West Plant sludge becoming somewhat more
"active" in phosphorus removal.
Using the data reported, the initial phosphorus contents of the sludges
were computed and are presented in Table 1. Though initially
different, the final sludge phosphorus content of the East and West
Plant sludges during the above test series are essentially the same,
and are not indicative of unusual phosphorus removal. The Rilling
plant sludge phosphorus content was initially high, 3.9 percent, and
did increase to 4.35 percent. However, this sludge previously had
been exhibiting such phosphorus removal characteristics and the
mechanism responsible has not been determined. (The Rilling Plant's
phosphorus removal is discussed further in subsequent sections of
this review.)
Similar results have been reported for batcji type experiments (8,23),
but the unusual phosphorus removals or percent sludge phosphorus
contents have been associated only with the lag phase of the growth
cycle. This phase of the growth cycle normally is associated with the
uptake of nutrients without a significant increase in the number of cells
present. These phenomena can be observed by measuring this increase
in cellular content of several nutrients, including phosphorus (24).
An Enzymatic Technique to Detect Surplus Phosphorus Uptake
The requirement for a quantitative measurement of phosphorus assimi-
lated by the microorganisms is provided by the alkaline phosphatase
enzymes utilizing p-nitrophenylphosphate as a sole source of phos-
phorus. This compound is colorless, but upon hydrolysis of the
phosphate group the yellow salt, p-nitrophenol, is liberated. The
reaction occurs as follows:
alkaline
phosphatase
p-nitrophenylphosphate + H^O -> p-nitrophenol +
(colorless) , 35°C ' (yellow)
10
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Table 1
Phosphorus Uptake by Activated Sludge at San Antonio, Texas
(after Wells)
Plant
East
West
Rilling
Total
phosphorus
ma /I
80
60
128
Initial
Soluble
P
mg/1
8
12
14
Conditions
Sludge
P
mg/1
72
48
114
Suspended
solids
mg/1
3290
2760
2940
Percent
P
2
1
3
.2
.75
.9
Final Conditions
Soluble
P
mg/1
6
0
0
.0
.0
.0
Sludge
P
mg/1
74
60
128
Percent *
P
2.
2.
4.
25
2
35
* No data supplied on final suspended solids; therefore, sludge phosphorus based on initial
solids.
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Thus, if p-nitrophenylphosphate is the sole source of phosphorus,
the alkaline phosphatase activity can be directly related to the
quantity of p-nitrophenol present. The rate of the reaction can be
determined photometrically (absorption maximum, 410 nya) measuring
the increase in p-nitrophenol with time.
Fitzgerald and Nelson (25) developed an alkaline phosphatase bioassay
technique for detecting surplus metabolic uptake of phosphorus by
algae. In their work they found significant differences in the alkaline
phosphatase activity of algae that were phosphorus deficient when
compared to algae that were not phosphorus-limited.
Using the technique developed by Fitzgerald and Nelson with modi-
fications reported by Fruh and Pessoney (26), Moore,_et_aj^. (23)
evaluated the alkaline phosphatase bioassay to detect surplus phos-
phorus uptake by bacteria. Their experiments were conducted using
batch units inoculated with mixed bacterial culture. During the
experiments bacteria grown in phosphorus-limiting substrates and
containing varying amounts of cellular phosphorus were placed in
batch units containing different concentrations of phosphorus. At .
different phases in the growth cycle an alkaline phosphatase assay
was conducted and phosphorus content of the sludge was measured.
These investigators reported that there was an apparent correlation
between the cellular phosphorus content and the enzymatic activity
exhibited by the bacteria. Some of their results are presented in
Figure 2. However, these results are based on batch unit tests and
do not include all the variables possible in a continuously fed system,
i.e. , constant nutrient supply and relatively constant quantity of
bacteria present. In addition, the high phosphorus sludge concentra-
tions only were found in the lag phase of the growth cycle.
Chemical Removal of Phosphorus
Domestic wastewaters generally contain all of the cations necessary
for the formation of insoluble forms of phosphorus. The concentrations
of these cations are a function of the water supply, types of industrial
wastes discharged into the system, and the characteristics of the soil
associated with surface runoff and infiltration. Though phosphorus
will combine with many species,the three aqueous metal ions Al III,
Fe III and Ca++ are generally considered to be the primary cations
involved in the formation of insoluble forms of phosphorus. Investi-
gations at the Baltimore Back River Wastewater Treatment Plant (27)
and the Los Angeles Hyperion Treatment Plant (28) have reported
apparent balances of phosphorus removed with these and other cations.
12
-------
• - Normal Conditions
A - Shortly After P Spiking
2 3 4 5 6 7 8 9 10 II 12 13 14 15 16
Cellular P Content (% of VSS)
Figure 2 . Relationship of Enzyme Activity and Cellular Phosphorus Content in Dilute
Bacterial Cultures (after Moore, et al.).
-------
In laboratory and field investigations the precipitation of inorganic
phosphorus with Fe III and Al III generally conform to the solubility
diagrams for FePC>4 and A1PC>4 (29,30,31,32). However, solubility
predictions for precipitation of phosphorus with calcium are difficult to
corroborate experimentally, particularly in short-term experiments (33).
In general the concentrations of Fe III and Al III in domestic wastewaters
are much smaller than calcium. If phosphorus removal by chemical
precipitation is occurring naturally, i.e. no addition of chemical
coagulant to the activated sludge, it would be reasonable to assume
that the major cation involved would be calcium. This assumption was
made in this research and chemical removal of phosphorus was limited
to calcium-phosphorus systems.
In discussing solid forms of calcium-phosphorus systems three compounds
have been considered the major forms of interest in wastewaters:
1) dibasic calcium phosphate , CaHPC>4;
2) the apatites; and
3) beta-tricalcium phosphate.
Other forms exist (see Appendix A), but are not considered to contribute
significantly to removal of phosphorus from natural aquatic environments
or wastewaters. This review of the literature on the solubility of
calcium-phosphorus compounds is intended to present the possibilities
that may occur in calcium-phosphorus systems, such as found in
activated sludge systems, and not as a detailed study of these forms.
Variables and parameters that can influence the formation of these
solid forms, whether in the kinetics involved or in interference or
enhancement from the presence of other species, have been noted to
indicate the complex nature of the calcium-phosphorus system.
Difficulties of corroboration using X-ray diffraction and infrared ab-
sorption techniques also are noted.
Dibasic Calcium Phosphate
i -
Dibasic calcium phosphate has been postulated to occur in activated
sludge systems, but little evidence supports this premise. Although
the theoretical solubility of CaHPC>4 is exceeded in domestic waste-
waters, the theoretical solubilities of the apatites and beta-tricalcium
phosphate are more than an order of magnitude lower within the pH
range encountered. Leckie and Stumm (33) could not confirm CaHPC>4
precipitation in their laboratory using solutions comparable to sewage
(e.g. [Ca++] = 2 x 10~3 M, [HCO3~] - KT3M, Ptotal = 10~3M, pH= 7.7).
The resulting precipitate, although poorly crystalized, gave X-ray
patterns of hydroxyapatite.
14
-------
Apatites
Apatites, Ca^g (PO^ (X)2 , are the least soluble of the calcium-phos-
phorus precipitates. Hydroxyapatite, CaiQ (PO^s (OH.)2 > is
representative of this group, has been studied extensively, and will
be used to describe this group.
Hydroxyapatite has certain well-defined characteristics which can be
identified by infrared absorption or X-ray diffraction. It theoretically
has a Ca:P ratio of 1.67, but has been observed to form with ratios
from 1.3 to 2.0. Controversy exists as to whether or not equilibrium
is attained, but it is generally agreed that it is a slow process at
normal temperatures (33). These properties have led to some confusion
in determining the existence, or nonexistence of apatites in activated
sludge systems. The variable molar ratio and the inability to obtain
X-ray or infrared verification of apatites in some activated sludge studies
(34,35) have precluded definitive identification in these heterogeneous
solutions.
Kinetics of Solid Formation: Ferguson and McCarty (35) investigated
the formation of solid phases from calcium-phosphorus systems. They
derived a theoretical model based on six components (total concentra-
tions of Ca++, PO4=, CO3=, Mg++, H+ and H2O) to predict the type
of solid phases precipitated (see Figure 3). In systems with no
magnesium they predicted a minimum soluble phosphorus residual
between pH 7 and 8 with calcium to phosphorus ratios greater than two.
This low solubility was attributed to the precipitation of hydroxyapatite.
Above pH 8 the soluble phosphorus increased due to competitive precipi-
tation of calcite.
In the experimental tests they confirmed their model for the low magnesium
system, but did not attain the low soluble phosphorus residuals predicted
by the model. They obtained/soluble phosphorus residuals between pH 7
and 8 in the 10~4 molar region and above pH 8 the residuals increased
to 10~3 molar. The soluble phosphorus residual did not decrease until
above pH 10. X-ray diffraction and infrared absorption measurements
indicated the precipitates contained apatite. The time period of the
tests was 24 hours.
These residuals are higher than predicted from theoretical solubility
equilibria, but can be explained partially by the crystal forming kinetics
of hydroxyapatite. Corsaro, _et al_. (36) investigated the formation of
15
-------
-3
CT1
Q_
o>
_o
ZJ
~o
en
en
o
-4
-5
-6
-7
I !I T
Beta-Tricalcium Phosphate
and Calcium Carbonate
( High Mg System )
Hydroxypatite and
Calcite
( Low Mg System)
8
10
II
pH
Figure 3. Predicted Soluble Phosphate at Equilibrium for Systems with High and Low
Magnesium Concentrations (PO4t= 0.003 M, CO3t= 0.05 M, Cat= 0.07 M,
T= 25°C) (after Ferguson and McCarty).
-------
hydroxyapatite from solutions containing solid calcium phosphate and
calcium and phosphate ions. In both cases the hydroxyapatite crystals
formed were very small and did not grow. Reflux of the precipitates did
not result in increased perfection of the crystals and growth was
negligible after 90 hours. Leckie and Stumm (1970) reported that
formation of hydroxyapatite occurs in two steps:
1) the formation of nuclei; and
2) the two dimensional growth of crystals on the surface.
The second step is time-consuming, but can be reduced greatly if
apatite nuclei are present. Calcite, silica and undefined solids in
domestic wastewater also can act in this capacity (33).
The slow growth of crystals experienced by Corsaro, _et al_. and Leckie
and Stumm can explain the higher soluble residuals found by Ferguson
and McCarty in short-term tests. Neither Corsaro or Ferguson and
McCarty had any apatite nuclei available and the precipitates formed
were subject to the slow two dimensional growth described by Leckie
and Stumm.
Ferguson, _e;t al_. (37) investigated the precipitation of calcium-phosphorus
compounds under conditions of pH and concentration similar to those
found in domestic wastewaters. Figure 4 depicts the results of one
series of tests, and shows the time lag, or induction period, described
by Leckie and Stumm at pH 7.6. In another experiment they added the
precipitate formed in a previous test to observe the effect of "seed"
being present. The presence of the previously formed solid caused
immediate precipitation with approximately first order kinetics and
there was no induction period.
Fluoroapatite: There are still other factors that can affect the rate of
precipitation. Leckie and Stumm (33) reported that fluoride, even in
trace concentrations (less than 1 mg/1), enhances the rate of precipi-
tation, presumably because fluoroapatite, which is less soluble than
hydroxyapatite, is being formed. More significantly, because F~ can
substitute for OH~ in the apatite structure, adding F~ has an effect
similar to raising the pH. Within the pH range normally encountered
in activated sludge systems, 7.0 to 8.5, the solubility is an order
of magnitude lower than hydroxyapatite.
Fluoroapatite could be a significant factor in phosphorus removal due
to the natural presence of fluoride,as well as to its addition to many
water supplies for dental health in concentrations that can enhance
precipitation (1 mg/1).
17
-------
Cat = 0.0020 M
C03t = 0.0038 M
T = 26° ± I°C
~ 0.20
Cat= O.OOI95M
C03f= 0.0038M
T = 26°±
Unseeded
0
0
40 60
Time (hours )
100
Figure 4. Effect of Time on Phosphate Removal at pH 7.6 with Seeded and Unseeded Solutions
(after Ferguson, et al.).
-------
-2
o>
o
-4
-5
SOLUBLE PHOSPHATE
— A aO
.0001M
0.001 M
0.003 M
1111
-2
-3
en
o
-4
-5
i i I I
_AOO» SOLUBLE CALCIUM
-2
78 9 10
PH
II 12
7 8 9 10 II 2
SOLUBLE MAGNESIUM
— . 0.003 M
-oo—o-,Q.OOI M
^^ /^
n0.0.0001 M
~*"~"
7 8 9 10
pH
Figure 5. Effect of Magnesium Concentration and pH on Soluble Phosphate, Calcium anc
Magnesium (CO3t= 0.03 M, PO4t= 0.002 M, Cat= 0.005 M, T= 27°C, time at 24 hrs
(after Ferguson and McCarty).
-------
Beta-Tricalcium Phosphate
In their theoretical study Ferguson and McCarty (35) predicted that
beta-tricalcium phosphate would precipitate in solutions containing
magnesium. This compound has received little attention in past studies
of suspected solid calcium-phosphorus phases in wastewaters. Beta-
tricalcium phosphate (B-Ca3[PO4]2), has been found to precipitate,
but the presence of other ions is needed to stabilize its formation (38).
Magnesium stabilizes the formation of beta-tricalcium phosphate, and
is often included about 6 to 8 percent. Other impurities also stabilize
it, such as manganese and ferrous iron. From their theoretical model,
Ferguson and McCarty (35) predicted that the calcium-phosphorus pre-
cipitate could be beta-tricalcium phosphate in preference to hydroxyapatite
for the Ca:Mg ratio found in most fresh and wastewaters (1.5 to 6)
(see Figure 2).
In their experimental tests magr^esium did alter the phosphorus residual
below pH 9, and the minimum phosphorus solubility reported for the
hydroxyapatite system between pH 7 and 8 did not exist (see Figure 5).
With no magnesium present this minimum was near 10 M, but with
magnesium ([Ca++] = 5 x 10~^M;initial conditions) was near 5 x 10
The lowest values for Ca:P ratios in the samples without magnesium
approached 1.67, the value for hydroxyapatite; while in the magnesium
samples, the Ca:P ratio approached 1.5, the value for beta-tricalcium
phosphate.
X-ray and Infrared Absorption Measurements
Yu and Mark (39) used infrared absorption to analyze precipitates
obtained from solutions of CaCl2 and (NH^HPC^ in pure solution, and
in the presence of a surface active agent (p-nitrophenol) and blue-
green algae. The results are shown in Figure 6. The precipitates from
several tests, including some using samples of recirculating sludge from
anaerobic digesters, also were analyzed by Ferguson and McCarty (35)
using infrared absorption and the spectra obtained are reproduced in
Figure 7. Table 2 contains the characteristics of these samples. The
965 cm"1 band is of interest. Studies have shown that this peak in the
spectra of hydroxyapatite can be used to measure the degree of crystal-
Unity (40).
Ferguson and McCarty1 s samples 1,2 and 3 (without magnesium) in Figure
7 show the same 965 cm"1 peak1 as that of Yu and Mark's sample in pure
solution in Figure 6. The samples of Yu and Mark with p-nitrophenol and
blue-green algae present show the shoulder only indicating no ordered
20
-------
Table 2
Composition of Solutions Used in Infrared Analyses
After Yu and Mark (see Figure 6)
1. Equal Parts of 0.75 M CaCl2 and 0.25 M (NH4)2 HPO4
2. 500 ml 0.75 M CaCl2, 500 ml 0.25 M (NH4)2 HPO4 , and 700 ml 0.02 M p-nitrophenol
3. 500 ml 0.75 M CaCl2 , 500 ml 0.25 M (NH4)2 HPO4 , and 700 ml of an algae solution
After Ferguson and McCarty
Sample Ca
1 . pure sys .
2.
3.
4.
5.
6.
7.
8.
9.
pure
pure
pure
pure
pure
M.P
M.P
M.P
sys.
sys .
sys .
sys .
sys .
.S.L.*
.S.L.
.S.L.
5.0
5.0
5.0
5.0
5.0
5.0
6.0
6.0
6.0
(see Figure 7)
P Mg
2.0
2.0
2.0
2.0
2.0
2.0
2.2
2.2
2.2
0
0
0
2.0
2.0
2.0
2.7
2.7
2.7
co3
30
30
30
30
30
30
28
28
28
COD
pH (mg/D
7.91
10.06
11.46 --
8.11 —
10.06
11.45 —
7.35 214
9.94 99
11.25 186
Precipitated
uu3
1.65
2.69
2.44
1.35
2.74
2.96
0.1
3.65
4.25
Ca
3.81
4.48
4.66
3.52
4.55
4.95
2.21
5.20
5.41
P
2.03
1.72
1.95
2.57
2.95
2.45
1.29
2.06
2.08
Mg
—
--
.29
.88
1.92
.23
.92
2.47
* Menlo Park Supernatant Liquor; concentrations in mMoles per Liter.
-------
965 965 965
Frequency ( cm"1)
Figure 6. Infrared Spectra of Hydroxyapatite Precipitates. Left,
formed in pure H9O: A, 18 hrs .; B, 3 days; C, 4 days;
Zj
D, 7 days; E, 9 days. Center, effect of p-nitrophenol:
A, 1 day; B, 3 days; C, 6 days; D, 9 days. Right, effect
of blue-green algae: A, 1 day; B, 5 days; C, 10 days;
D, 20 days (after Yu and Mark).
8
965 965 965
965 965 965 965
Frequency ( cm"1)
965 965
Figure 7. Infrared Spectra for Samples Precipitated for 24 hrs. See
Table 2 for Composition (after Ferguson and McCarty).
22
-------
crystals, but amorphous hydroxyapatite. Samples 4,5 and 6 with mag-
nesium show: a small peak, a shoulder only, and little, if any, shoulder.
These samples have a Ca:Mg ratio of 2.5. From these results it would
appear that Ferguson and McCarty's sample 4,5 and 6 did not have well
ordered crystaline apatite but an amorphous apatite. Samples 7 and 8
(from anaerobic sludge) show a small shoulder indicating an amorphous
form. Sample 8 (Ca:Mg ratio of 2.2) and sample 9 (Ca:Mg of 2.2) show
no shoulder and could indicate no apatite present.
Martens and Harris (41) studied the effect of magnesium on the direct
precipitation of apatites in marine environments. They reported that
magnesium inhibits the formation of apatite, and that the critical Ca:Mg
ratio where this occurs is between 4.5 and 5.2. Above this value they
obtained X-ray diffraction patterns with strong apatite peaks, and below
they obtained no indications of apatite. The pH range was maintained
between 7.5 and 8.0. They did not report the Ca:P ratios of the precipi-
tate above the critical Ca:Mg ratio, but reported a ratio of 1.35 below;
Ferguson and McCarty defined a magnesium influenced system when the
Ca:Mg ratio is 5 or less, and Martens,and Harris defined the ratio be-
tween 4.5 and 5.2 or less. Both investigators obtained Ca:P ratios that
indicate a solid form other than apatite. The theoretical and actual
increase in residual phosphorus solubility formed by Ferguson and
McCarty would seem to point to beta-tricalcium phosphate.
Effect of Operational Characteristics on Phosphorus Removal
Several prototype and pilot plant as well as laboratory studies have been
conducted to investigate the abnormal phosphorus removal capabilities
and sludge phosphorus content found in several activated sludge plants.
Some of these studies have involved evaluation of the operational charac-
teristics of plants enjoying high removals of phosphorus and subsequent
attempts to duplicate these characteristics of plants in other plants having
normal phosphorus removal (42-49). Other studies were pilot plants with
associated specific laboratory experiments to verify the significance of
these operational characteristics (3,34). The major implicated charac-
teristics are:
1) dissolved oxygen (DO) levels along and at the effluent end of
the aeration tanks;
2) the organic loading, pounds of applied BOD per pound of solids
under aeration; and
3) calcium, pH and other,ionic constituents.
Prior to evaluating the results of these field studies, a discussion of the
parameters used to define the removal of phosphorus from wastewaters
would allow a more meaningful interpretation of the results of these studies,
23
-------
Evaluation of Phosphorus Removal Measurements
The reporting of influent phosphorus as soluble ortho-phosphate is
misleading. The quantity of condensed and organically bound forms
of phosphorus may equal the quantity of orthophosphate present in
the influent. The biomass can hydrolyze the condensed forms rapidly
as well as release the bound forms of phosphorus for metabolic
purposes during the treatment cycle. As a result the influent phos-
phorus concentrations reported as ortho-phosphate can be significantly
less than that actually available to the biomass. Erroneous percent
phosphorus removal values also can occur from reporting the effluent
phosphorus concentrations as soluble ortho-phosphate. The reporting
of all forms of phosphorus (total P) present in the waste stream and
all soluble forms (total filtrate P) in the effluent of the plant would
better describe the quantity of phosphorus removed from the waste-
water.
The practice of reporting phosphorus removal on a percentage basis
is questionable. A treatment plant experiencing 80% P removal having
an influent containing 10 mg/1 P cannot be compared to other plants
with 80% P removal having influents cycling from 5 to 15 mg/1 P
where the phosphorus removed from the waste streams would range
from 4 to 12 mg/1 P.
Moreover, the percent phosphorus removed from a waste input does
not describe the phosphorus removal capability of the sludge beyond
that particular treatment plant and wastewater characteristics. Assuming
only biological uptake of phosphorus, part of the removal of phosphorus
would be to satisfy the metabolic requirements of the biomass. This
removal might be directly related to the removal of other nutrients,
i.e. organic carbon expressed as BOD or COD. Some investigators
(15 ,3) have related the uptake of phosphorus to the BOD or COD
removed to define the phosphorus removal capabilities of sludges on
a common basis (pounds phosphorus removed per 100 pounds of BOD
or COD removed).
The last area of possible confusion is the lack of reporting or neglecting
sludge phosphorus content. Without a knowledge of initial and final
sludge phosphorus content the results of most studies, and in particular
batch-type systems, have little meaning.
24
-------
Effects of Dissolved Oxygen
The operational characteristic receiving the most attention in both
field and laboratory studies has been the level of dissolved oxygen
(DO). Without particular reference to the biological uptake of
phosphorus,this is indeed an important characteristic of any aerobic
biological system. The reports of several field investigations on
activated sludge plants with abnormal phosphorus removal capa-
bilities have assigned major significance to the maintenance of DO
levels in the aeration tank and effluent much in excess of those
previously reported for oxygen to be non-limiting.
Vacker, _e;t a_L (4) attributed this increased phosphorus removal
enjoyed by the Rilling plant above the East and West plants at San
Antonio to several factors, but noted that DO levels were a major
difference in the operating characteristics between the plants. These
three plug-flow activated sludge plants are at a common site and
receive the same wastewater for treatment. The Rilling plant exhibited
DO levels of 2 mg/1 or greater during the last half of the aeration tank
length, and usually had a DO in the aeration tank effluent near 5 mg/1.
The East plant seldom exceeded DO levels of 3.0 mg/1 in the effluent
and usually dropped to a level of 1.0 to 2.0 mg/1 during peak loading
periods. The West plant was similar, but usually fell to lower levels
than in the East plant. The East and West plants contained an average
of 3.7 to 6.0 mg/1 O-PO4 as P opposed to 1.9 mg/1 O-PO4 as P in
the Rilling plant effluent against a common influent containing 9.1
mg/1 O-PO4 as P- In the report of average daily operation data for
1965 the phosphorus removed at Rilling averaged 7.3 mg/1 O-PO4 as
P, East averaged 5.5 mg/1 O-PO4 as P, and West averaged 3.3 mg/1
O-PO4 as P- The calculated percent sludge phosphorus reported for
the plants was 6.2, 3.7, and 2.8 percent respectively.
Witherow (5) reported the results of incorporating the operating
characteristics of the Rilling plant at the West plant in an attempt
to induce high phosphorus removals. High phosphorus removals were
obtained, but required the removal of two tanks with inefficient air
diffusers to obtain DO levels similar to Rilling. During the test the
operational changes affected the aeration solids level (increase from
900 to 1800 mg/1 MLSS), control of organic and hydraulic loading,
solid-liquid separation (2 to 4 mg/1 polyelectrolyte added to prevent
bulking), and the DO level in the aeration tanks.
While these changes closely duplicated the characteristics of the
Rilling plant, similar uptake of phosphorus at the West plant had been
25
-------
noted by Vacker,e_t al_. (4) to occur with changes in the DO level only.
They reported the results of increased aeration efficiency during a
periodic cleaning of the diffusers at the West plant. The cleaning
of the diffusers was accomplished in a manner that permitted the
approximation of tapered aeration and reversed tapered aeration, as
well as one tank with all diffusers cleaned. Loadings and solids level
were th;e same as normally present, solids varied between 1510 mg/1
at low flow and 810 mg/1 at peak flow. Normal removal occurred in
the one tank with no diffusers cleaned, but increased removal occurred
in the three other partially and fully cleaned tanks with effluents
containing 1.5 mg/1 O-PC>4 as P or less. Comparing these two tests
would seem to indicate the significance of DO levels as the primary
characteristic influencing :phosphorus removal capability of the West
plant sludge. It also should be noted that the pattern of air distri-
bution was not highly significant, only the maintenance of DO levels
exceeding 2 mg/1 along most of the aeration tank length.
A study was conducted at the Baltimore Back River Sewage Works (42)
to evaluate the high phosphorus removal obtained in the activated
sludge plant under operating conditions similar to the San Antonio
Rilling plant. The effect of DO levels again was noted. In a test,
three jugs containing 15 liters of mixed liquor from the influent end
of the aeration tank were aerated at three levels of air flow, 5, 10,
and 15 liters per minute. Simultaneously, a slug of waste was
monitored through the aeration tank on the basis of the five hour
detention time determined by dye tracer studies. The jugs and the
slug of waste exhibited the same phosphorus removal, approximately
24 mg/1 O-PO^ as P, and had residual O-PO4 concentrations less
than 0.1 mg/1 P. Again the rapid uptake of phosphorus appeared to
occur when the DO levels began to increase above the initial values,
although not as clearly as at San Antonio.
The results of these field evaluations of activated sludge plants
enjoying high phosphorus removals implicate DO levels as a signi-
ficant operational characteristic. However, attempts to verify this
in other field and laboratory tests have not met with success.
Menar and Jenkins (34) conducted laboratory and pilot plant studies
of operating conditions that enhance phosphorus removal. Of particular
interest in their study was the effect of DO levels. They concluded
that the DO leveljDer se_ did not enhance the removal of phosphorus,
but is fortuitously correlated to conditions indicating a rise in pH,
i.e. the stripping of CO2, and that enhanced phosphorus removal occurs
as the result of chemical precipitation.
26
-------
A pilot plant was operated that incorporated the operational character-
istics reported as significant in the San Antonio studies. Influent to
the San Ramon, California, Valley Community Services District was
used in the study. This waste had characteristics similar to the San
Antonio waste with the exception of magnesium, 44 mg/1 versus 17
mg/1 and alkalinity, 405 mg/1 versus 225 mg/1 as CaCO3. (These
higher values at San Ramon may have exerted an effect and will be
discussed later.) The physical layout of the pilot plant closely
resembled the two-pass aeration basin of the Rilling plant.
During the six week test the DO level was maintained consistently
above 1.5 mg/1. The phosphorus removed during this time was a
function of the amount of carbon removed as defined by Sawyer (1),
Helmers,^t aj_. (12,13,14) and Jenkins and Menar (3), i.e., approxi-
mately 0.9 pounds phosphorus per 100 pounds of COD removed. The
maximum sludge phosphorus content was 2.9 percent and averaged
2.3 percent (based on volatile solids, VSS), which agrees with the
above that phosphorus removal is a function of carbon removal.
The operating characteristics then were changed to include a
preaeration basin. In addition to a higher DO, the pH was raised
allowing the condensed phosphates to hydrolyze and release the
bound calcium. The preaeration time was 4.3 hours and normal aeration
time was 4.4 hours. During preaeration the pH rose from 7.7 to 8.3
and maintained this level through normal aeration. The DO level of
the mixed liquor in the preaeration basin increased to 5.4 mg/1, and
subsequent normal aeration increased this to 6.2 mg/1 at the effluent
end.
These conditions produced enhanced phosphorus removal, approximately
double that expected from normal biological incorporation. The sludge
phosphorus content reflected this increase and averaged 4.5 percent.
Calcium also was removed from the wastewater, 8.0 mg/1. Menar and
Jenkins concluded that chemical precipitation was the cause of the
enhanced phosphorus removal. Attempts to define the characteristics
by X-ray diffraction were not successful.
Other studies were conducted with an effluent solids level (from 2000
to 5000 mg/1 MLSS). These subsequent tests were conducted under
conditions typical of the extended aeration process. The mean
residence times of the sludges exceeded 12 days and substrate removal'
rates were 0.2 to 0.25 pounds COD per pound of VSS. With
preaeration and its effect on pH, two to three times the expected
phosphorus removal occurred with sludge phosphorus content reflecting
27
-------
to
co
Cone, mg/l
0.0
OA to 0.5
" to
to
0
234
Aeration Time ( hours )
Figure 8. Soluble Phosphate Uptake as a Function of Dissolved Oxygen
Concentration (after Hall and Englebrecht).
-------
the increased removal. Calcium also was removed from the waste-
water and the sludge calcium content increased in relation to the
increased phosphorus removal. An average of 6.8 percent phosphorus
and 10 percent calcium was reported for the sludge.
Hall and Englebrecht (20) used fill and draw laboratory units to observe
the effect of DO on the uptake of phosphorus . They used mixtures of
nitrogen and oxygen to provide varying DO levels, but with the same
turbulence in each unit. Their results are presented in Figure 8. With
DO levels below 2 mg/1 there is a lag period; however, after six
hours there is little difference in the amount of phosphorus removed.
A recent investigation of the Baltimore plant by Milbury, _ert aL (50)
evaluated the effects of DO levels in the aeration tanks. They
reported that the DO levels in the aeration tanks did exert some effect
on phosphorus removal, but at lower levels than previously reported.
They concluded that a DO of 1.0 mg/1 at the two-thirds point along
the aeration tank length and an effluent with 2.0 mg/1 DO, no impair-
ment of phosphorus uptake would occur. The rapid uptake of phos-
phorus did not always occur concurrently with an increase in DO
or an abrupt increase in pH. The pH range varied from 6.8 to 7.3
and always increased through the aeration tanks, even when little
phosphorus removal occurred. This high phosphorus removal at
approximate neutral pH conditions indicated to these investigators
either excess biological uptake or a solid species of phosphorus not
as pH dependent as those postulated by Jenkins and Menar.
Furthermore, a batch test was conducted to observe the effects of
DO level under laboratory conditions. They reported no impairment
of phosphorus uptake at DO levels above 0.5 mg/1.
Organic Loading and Aeration Solids
Vacker,_et_al.. (4) implicated the organic loading as exerting an
effect on the phosphorus removal capability of activated sludge as a
result of their study at the San Antonio plants. Significance was
attached to a loading rate around 0.5 pounds of applied BOD per pound
of MLSS; however, this was apparently as much towards maintaining
a balance between sludge wasting, MLSS, and rates of sludge return
to keep aerobic digestion of solids to a minimum, thus preventing
phosphorus return to the mixed liquor.
Witherow ( 5) obtained the organic loading rates presented from the
rates existing in the high phosphorus removal plants when abnormal
29
-------
removals were occurring. The loading rates from normal phosphorus
removal plants investigated were generally lower. There was some
deviation from the optimum range, 0.26 to 0.35 pound BOD per pound
MLSS, in the high removal plants when they were experiencing low
removals.
Hall and Englebrecht (20) used fill and draw laboratory units to
investigate the effects of organic loading and MLSS on phosphorus
removal. Their tests spanned a range of loading rates from 0.33 to
4.0 and MLSS from 750 to 8000 mg/1. These variations were ob-
tained by maintaining a constant substrate concentration and varying
MLSS during one series and by maintaining a constant MLSS and
varying the substrate concentration in another series. During both
series they maintained a constant COD:P ratio of approximately
75:1 so that the rate of phosphorus uptake was not affected. No
effect from either the organic loading or MLSS was noted.
Hall and Englebrecht maintained a stock culture that was fed a sub-
strate composed of glucose and yeast extract for carbon sources and
the normal mineral content found in laboratory media . Chemical
precipitation was precluded as a significant phosphorus removal
mechanism based on substrate content. From the data available it
was possible to compute a yield coefficient for the stock culture,
O.S3, and this is within the range expected for such a medium.
Using this yield coefficient and the data on phosphorus and COD
removal, it was possible to compute a cellular phosphorus content
that would be close to the actual value reported. This was done for
both series and wqs found to range between 2.35 to 2.5 percent for
all tests. The ratio of phosphorus removed to COD removed was 1.2
pounds P per 100 pounds COD removed.
These tests didtnot indicate any effects from varying organic loading
or MLSS over a range that would represent most conditions found in
activated sludge plants. The sludge phosphorus contents obtained
were within the normal range for activated sludge.
Jenkins and Menar (3) investigated organic loadings over a large
range, 0.2 to 13.8 pounds BOD per pound VSS . in a pilot plant study.
The sludge phosphorus content did not vary significantly over this
rather large range and had a weighted average of 2.62 percent for
all loading rates. From 0.2 to 3.0 pounds BOD per pound VSS, the
ratio of phosphorus removed to COD removed was 0.86. Ab^ve
these loadings the ratio increased to 1.7 at the highest loadings.
30
-------
These investigators concluded that organic loading in the range found
in most activated sludge plants, 0.2 to 3.0, did not exert an effect
on phosphorus removal.
Witherow (5) reported the results of field evaluations of several
activated sludge plants. There was an optimum solids level indicated,
between 2000 and 4000 mg/1 MLSS, but the results were based on
changes in MLSS only and were not related to the changes in organic
loading resulting from these changes in aeration solids. The decline
in removal at high MLSS levels probably reflected the low organic
loading present which would induce a loss in VSS with a release of
phosphorus. This was noted by Vacker, _ej^ a_l_. (4) at San Antonib
and by Jenkins and Menar (3) to occur at loading levels below 0.2
pounds BOD per pound MLSS.
Calcium, pH and Other Ionic Constituents
Vacker,et al_. (4) assigned major significance to a biological removal
of phosphorus at San Antonio, but did report that a removal of hardness
i.e. calcium,occurred through the aeration tank. This removal was
small, and it was not assigned major significance.
It is interesting to note the stoichiometric requirements in the con-
centration realm of interest in activated sludge systems. The
quantity of phosphorus to be removed is usually between 6 to 12
mg/1 P (2 x 10~4M to 4 x 10~4M). Considering the two main suspected
solid forms and their Ca:P ratios: hydroxyapatite, 1.67, and beta-
tricalcium phosphate, 1.5; the required amount of calcium is between
12 and 27 mg/1 Ca (3 x 10~4M and 7 x 10~4M). Vacker, _et al_. reported
the calcium content of San Antonio fresh water varied between 59 and
90 mg/1 (1.5 x 10~3M and 2.25 x 10~3M). These reported values do
not account for the insoluble or bound forms of calcium that are re-
leased to solution in the treatment plant. The small amount of calcium
required for significant removal of phosphorus could be within the
normal variation encountered and be masked by this variation.
Menar and Jenkins (34) reported chemical precipitation in their pilot
plant study using San Ramon wastewater, where the Rilling plant
exhibited abnormal phosphorus uptake. The enhanced phosphorus
removal, up to three times that required for the carbon removed,
was directly tied to the removal of a precipitating cation, calcium.
But the pH level required to induce the chemical precipitation was
far in excess of the values reported for San Antonio and other plants
31
-------
with abnormal phosphorus removal capability. The pH levels attained
at San Ramon were greater than 8 while San Antonio achieved abnormal
phosphorus removals at pH 7.6 and Baltimore (50) at a pH near 7.0.
Recent reports by Ferguson and McCarty (35), Ferguson,_et_§JL (37)
and Leckie and Stumm (33) have provided possible explanations for
the dilemma occurring with the wide pH range encountered in reports
of enhanced phosphorus removal. Ferguson and McCarty reported
that two solid forms would precipitate from calcium-phosphorus
systems; apatite and beta-tricalcium phosphate. The condition
influencing the form precipitated was a function of other ions, notably
magnesium. If magnesium was present in sufficient concentration
so that the calcium : magnesium ratio was less than five, the
precipitation of beta-tricalcium phosphate would be favored. Should
the conditions for the precipitation of beta-tricalcium phosphate
occur, the resulting soluble fractions of calcium-phosphorus systems
that can exist in the pH range from 7.0 to 9.0 provide possible
explanations for the observations at several activated sludge plants.
As pointed out by Ferguson and McCarty the formation of beta-
tricalcium phosphate changes the residual soluble phosphorus,
particularly between pH 7.0 and 8.0 where the residual soluble
increases significantly over that found where apatite is the solid
form. This effect could have caused the results obtained by Menar
and Jenkins (34) in their pilot plant study at San Ramon, California.
In duplicating the characteristics and conditions found at the San
Antonio Rilling plant they did not attach any significance to two
differences in the composition of the wastewaters: the magnesium
content of the San Ramon wastewater was close to three times that
of San Antonio wastewater and the alkalinity was almost double.
Should this high magnesium content have inhibited apatite and favored
the formation of beta-tricalcium phosphate, this would have caused
higher soluble phosphorus residuals than predicted for an apatite system
and could have precluded any significant precipitation at the calcium
and phosphorus concentrations present. To obtain a significant
calcium-phosphorus precipitation the pH had to be increased to levels
above 8.0. While they did cause precipitation to occur, by increasing
the pH through CO2 stripping via increased aeration rates (thereby
raising the dissolved oxygen) and raising the sludge phosphorus
content to a high of 6.8 percent, the soluble phosphorus residual
was still quite high, almost 0.3 x 10~4M (o/10mg/l P). This high
residual could have been influenced by not only the effect of magnesium,
but also by the competitive precipitation of calcite (35) which was
significantly greater at San Ramon at the pH encountered.
32
-------
Secondly, the implication of fluoride in the enhanced precipitation
of apatites as pointed out by Leckie and Stumm can lower the pH
range for significant removal by precipitation. The concentration
found in municipal water supplies adding fluoride for dental health
as well as that occurring naturally in the range of significance
(/»! mg/1 F~) reported by Leckie and Stumm. Several cities reported
having abnormal phosphorus removal containing fluoride at this
concentration according to the Geological Survey Water-Supply Paper
1812 (1962). Table 3 is a listing of several activated sludge plants
reporting high phosphorus removal. These include Baltimore, 1.0
mg F~/l and Amarillo, 2 .0 mg F~/l. The presence of fluoride in the
Baltimore plant could account for the high phosphorus removal at the
low pH range, 6. 8 to 7.2, reported by Milbury, _ejt al_. (50).
Table 3
Chemical Analyses of Fresh Water Supplies of Some
Municipalities Reporting High Sludge Phosphorus Content:
Values are in Milligrams per Liter
(Taken from Geological Survey Water-Supply Paper 1812, [62]).
Amarillo
Baltimore
Fort Worth
Los Angeles
San Antonio
Ca
38
18
45
42
62
Mg_
30
3
8
12
16
275
45
146
135
240
F
1.9
1.0
.3
.6
.3
Literature Evaluation
In the previous sections the two viewpoints postulated to identify the
cause of abnormal uptake of phosphorus by some activated sludge
plants beyond the amounts described in nutrient requirement studies
(1,12,13,14) have been reviewed. There is still insufficient evidence
to positively determine the primary mechanism(s) responsible; a
biological uptake termed "luxury uptake", or direct chemical precipi-
tation and subsequent sorption or enmeshment in the activated sludge
mass .
33
-------
The result of field and laboratory studies can partially validate both
mechanisms and the following preliminary conclusions are postulated.
1) The lower limit of sludge phosphorus content is approximately
one percent for good operation of activated sludge systems
based on carbon removal. The cellular phosphorus content
that normally can be expected due to biological uptake when
excess phosphorus is available to a biomass is between two
and three percent and appears related to improved solid-liquid
separation. The upper limit of biological assimilation of
phosphorus has not been defined, though, and has been post-
ulated to exceed this range by as much as twice in some
activated sludge plants.
2) A possible means of determining the extent of biological phos-
phorus assimilation is the alkaline phosphatase bioassay.
3) The two main suspected calcium phosphate precipitates are
apatites and beta-tricalcium phosphate.
4) The possibility of a chemical removal mechanism exerting a
significant effect on the quantity of phosphorus removal in
activated sludge plants cannot be refuted, but the wide pH
range reported presents a more complex problem in describing
the existence and forms of this removal mechanism. The
nature of the precipitate formed, a colloid, plus the small
concentrations of ions involved pose problems in definitively
describing its existence by physical means. The inability to
identify the presence of precipitates in sludges by X-ray or
infrared techniques has not aided the proponents of this removal
mechanism, even though the presence of contaminants, such
as found in activated sludge, has been shown to obscure the
presence of precipitates.
5) The complexities of extraneous cations purported to remove
phosphorus by chemical precipitation,as well as other un-
controllable conditions (such as pH, variable ionic concen-
trations in cyclic flow, etc.) preclude the use of field
experiments to quantitatively evaluate metabolic incorporation
of phosphorus and the chemical precipitation of phosphorus
with calcium.
Objective and Scope
The objectives of this research are to evaluate the range of metabolic
phosphorus removal, and to investigate the significance of calcium
removal of phosphorus at ionic concentrations and pH generally
34
-------
encountered in domestic wastewater treatment plants. To accomplish
this purpose it will be necessary to:
1) define the range of cellular phosphorus content; and
2) define the chemical conditions that induce phosphorus removal
by precipitation with calcium.
The complexities of contributing factors that can exert a significant
effect have been delineated previously. Control of all these factors
is not possible in field tests; therefore, only laboratory experiments
were conducted in order to permit their inclusion and evaluation in
this investigation.
The laboratory experiments were designed to provide a means of
differentiation and evaluation of the magnitude of phosphorus removed
by each mechanism. The scope of the study included the following
components.
1) Investigation of the range of metabolic incorporation of
phosphorus in bench scale completely mixed continuous flow
activated sludge systems using a synthetic substrate
representative of actual wastewater under conditions precluding
significant chemical removal.
2) Concurrent with determining the magnitude of metabolic incorp-
oration of phosphorus as described above, evaluating the
alkaline phosphatase bioassay technique as a means of defining
the range of biological phosphorus uptake and the saturation
value of cellular phosphorus content in a laboratory activated
sludge system.
3) Simultaneous operation of bench scale completely mixed continuou
flow activated sludge systems under pH, ionic concentrations
and chemical conditions that both favor and preclude significant
chemical removal of phosphorus with calcium as the precipi-
tating cation. The effect of high and low Ca:Mg ratios, the
presence of 1 mg/1 F in the influent, and the addition of
nucleation sites were studied.
35
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SECTION IV
EXPERIMENTAL PROCEDURES
The first part of this section describes the apparatus and procedures
employed during the experimental studies. The analytical techniques
are described in the second section.
Experimental Studies
Both batch and completely mixed continuous flow activated sludge
units were utilized for the biological studies. Differentiation
between metabolic incorporation and chemical precipitation of
phosphorus within the sludge was accomplished using synthetic
substrates with defined calcium and phosphorus concentrations.
Appropriate pH ranges were maintained by using an automatic pH
control system, so that significant calcium precipitation of phos-
phorus did, or did not, occur. The concentration of calcium and
phosphorus and the pH ranges that precluded or favored calcium
precipitation of phosphorus were determined by batch chemical pre-
cipitation tests.
Batch Units for Biological Studies
A plexiglass unit was constructed providing five separate compartments.
Each compartment was 3 x 5 x 15 inches with a sampling port one inch
from the bottom. Aeration was provided by a one inch diffuser stone
permanently mounted one inch from the bottom opposite the sampling
port. The initial test volume was 2.5 liters.
The desired concentration of substrate for a particular experiment
was added to a unit partially filled with distilled water. Subsequently
a concentrated inoculum of activated sludge was added, and the unit was
brought to the final test volume with distilled water. High aeration
rates were used to maintain the solids in suspension and to maintain
saturated dissolved oxygen conditions.
Completely Mixed Continuous Flow Units for Biological and
Chemical Studies
A sketch of the plexiglass completely mixed continuous flow unit is
shown in Figure 9. The units provided aeration and settling sections
37
-------
separated by a moveable baffle with an overall test volume of 8 liters.
Solid-liquid separation was accomplished by gravity settling and sub-
sequent return of the solids to the aeration section by hydraulic action.
A diffuser stone was suspended from a moveable platform over the
aeration section. This was used primarily to provide oxygen, although
the positioning of the stone also was used in conjunction with the
moveable baffle to provide the necessary hydraulic action to return
the solids from the settling section. No difficulties were encountered
in providing saturated dissolved oxygen conditions, maintaining
suspension of solids, and returning settled solids during normal
operation. During some tests sludge bulking presented problems
in returning solids to the aeration section. These occurrences are
reported in experimental results.
A variable speed tubing pump (Cole-Palmer Masterflex) was used to
deliver the substrate to the units. Initially an 18 liter plastic feed
tank was used to provide 2 days of feed; however, during the first
series of biological tests contamination was noted in several feed
tanks. This was corrected by changing to a twice daily feed schedule
using 4 liter aspirator bottles.
Prior to sampling or wasting of sludge, the baffle was removed and the
contents of the aeration and settling sections allowed to mix. The
variability in the quantity of solids on the settling section necessi-
tated this action to maintain a common reference for solids measure-
ments in the units.
Inoculum
The source of inoculum for the biological studies was the Govalle
Waste water Treatment Plant, Austin, Texas. The sludge was screened
to remove large discrete particles, elutriated with distilled water
to remove grit and nutrients present in the occluded wastewater,
then concentrated by gravity settling.
When low phosphorus content sludge was required, the sludge was
placed under anaerobic conditions for 24 hours subsequent to the
above pretreatment. Sludge phosphorus content was approximately
2 percent. After this period 750 mg/1 of carbon in the form of glucose
was added. At the end of an additional 12 hour anaerobic digestion
period the sludge was elutriated and concentrated. This provided
sludge with a phosphorus content between 1 and 1.5 percent.
38
-------
t
r
*
L
r
1 "
-3j-
~
o
\- — Moveable Diffuser
\ Support Platform
\
in" ».
Top View
Moveable
Baffle^
Section —.
L
VJ
r
V_oJ._
A Aeration
Section
^1
^ 3"
^ C4
Volume
~5L
Air .
Diff user-^-
L_^
D
%.
Air
Air
Effluent Standpipe
Cross Section
Figure 9. Sketch of Completely Mixed Continuous Flow Unit.
39
-------
Synthetic Wastewater Substrates
During the course of the experimental studies several substrates
were required (see Appendix B). The substrate used by Higgins (51)
during previous studies in this laboratory evaluating the alkaline
phosphatase enzymatic assay with batch tests was utilized in the
preliminary phases of this study. However, the carbon source was
changed from a single source, glucose, to a multi-carbon source
to provide a diverse carbon source for the short term (less than one
biological solids retention time) completely mixed continuous flow
studies. This medium, designated as "Substrate A", provided a
suitable substrate for use during the minimum sludge phosphorus
content study; i.e. , chemical precipitation was avoided as only
trace quantities of alkaline or heavy metals necessary for biological
metabolism were present. The yeast extract and peptone provided
the minimum phosphorus concentration, 0.3 mg/1, during this study.
Additional phosphorus as monobasic potassium phosphate was added
to obtain higher phosphorus concentrations.
The subsequent short term biological tests combining metabolic and
chemical removal of phosphorus required several changes. The carbon
concentration was reduced and calcium also was removed from the
basic substrate. This medium was designated "Substrate B".
The final biological study was a long term series (greater than 100
days) and a different organic source was used. The multi-carbon
source was replaced by glucose, and since minimum phosphorus
concentrations were no longer required, the yeast extract concentra-
tion was increased and Sego was added to the medium. This medium
was designated as "Substrate C".
Automatic pH Control System
During the biological studies it was necessary to maintain a pH
range with either a maximum or minimum allowable pH. The control
system was composed of two sets of pH electrodes (Corning), a
titration pump for each set of electrodes; an electrical circuit that
shifted between these sets at six minute intervals; a locally constructed,
battery operated expanded scale pH meter; and a recorder (Minneapolis
Honeywell) that provided a signal to the titration pump when the preset
pH limit was not maintained within the biological unit. The recorder
was adjusted to provide a full scale (10 inch) travel for one pH unit.
No temperature compensation was provided.
40
-------
The system pH meter initially was calibrated using a Corning Model
12 pH meter. The electrodes were calibrated using a standard pH 7
buffer solution at least three times daily and at other times when
adjustments were made to the units. At regular intervals the probes
were cleaned and serviced according to the directions supplied by the
manufacturer. Prior to each series of tests the pH meter was calibrated
To evaluate the performance of the system a 24 hour test was run using
chart paper to record the pH variation. With the system set to maintain
a maximum pH of 7.05, the maximum pH recorded was 7.05 and the
minimum was 6.9. The value below the preset limit was a function
of the normality of the titrant and the associated overshoot in titrating
to maintain the preset limit. This was controlled in subsequent use
by using low normality titrant. Although the results of this evaluation
are good, the normal drift of pH electrodes and the occurrence of
low battery voltage caused additional drift for short periods upon
infrequent occasions. Continuous recording of the pH was not
possible due to economic considerations.
Batch Units for Chemical Precipitation Studies
The procedures used for the chemical precipitation tests are described
as they occurred during the experimental studies.
Analytical Techniques
The analytical techniques used in this study that are not described
in Standard Methods for the Examination of Water and Waste Water,
Twelfth Edition (52),are described in this section.
Alkaline Phosphatase Enzymatic Assay
The initial intent was to duplicate the analytical techniques used by
Higgins (51) and Moore, _e_t a_l_. (23) to measure the alkaline phospha-
tase enzymatic activity, thereby maintaining continuity with their
batch studies. However, several problems arose from the higher
solids content and different characteristics of the sludges used during
this study. The changes required to alleviate these problems did not
have a significant effect, and are included for evaluation in this
respect. The basic technique to measure alkaline phosphatase activity
is described below. Following this description the problems encount-
ered during this study are described.
41
-------
To measure the enzymatic activity a sample was taken from a unit,
filtered through a prewashed 0.45 p. membrane filter, placed in1 a
beaker containing 100 milliliters of a p-nitrophenylphosphate (PNP)
medium at pH 9.0 (51), and incubated at 35°C. The components of
the PNP medium were the same as the synthetic substrate (less the
yeast extract and peptone) from which the sample was taken except
that the sole source of phosphorus was the PNP. Following various
incubation periods the concentration of p-nitrophenol (PN) liberated
by the action of alkaline phosphatase was measured with a Bausch
and Lomb Spectronic 20 spectrophotometer (wavelength 410 m^.) using
a calibration curve of known standards of a p-nitrophenol salt (53).
Concurrent with the above, a volatile suspended solids (VSS)
measurement was made of the sample and this was used to report the
enzymatic activity as ju-moles PN/1 per milligram VSS per hour.
During the preliminary experiments the enzymatic activity of continuous
flow units could not be replicated. Agglomerated masses of sludge
were noted on the filter membrane that had not been present at the
lower concentrations used in batch tests. This difficulty was corrected
by using a blender at low speed for one minute to disperse the bacteria
prior to filtering the sample.
Tests also were carried out to observe the effects of increased solids
in the continuous flow units on the enzymatic activity. The data
are presented in Table 4. It is apparent that accurate solids measure-
ments (1250 mg/1) could not be attained using sample volumes less
than four milliliters. The data in Table 4 also indicate that there is
a suppression of the enzymatic activity at higher sample volumes.
However, the time from start of filtration to placement of the sample
in the incubation chamber also was significantly greater for the larger
sample volumes. The effect of filtration time on the enzymatic activity
for two different samples is presented in Table 5.
A PNP concentration of 500 mg/1 was used during early batch tests, but
was increased to 1000 mg/1 to avoid any limitation of substrate concen-
tration during the continuous unit tests. As a check that the PNP
concentration did not become limiting, random samples were diluted
and the enzymatic activity for diluted and undiluted samples was
measured (see Table 6). These data indicate that the substrate con-
centration was adequate, but also indicate that the filtrability of the
sludge is more important than the actual quantity of sludge.
This problem was solved by using dilutions when necessary to prevent
excessive filtration time. Also, the distilled water washing used
42
-------
Table 4
Effect of Increased Sludge Mass on Enzymatic Activity
Using Sludge with 1250 mg/1 VSS
Sample
Volume
(ml)
1.0
2.0
4.0
5.0
Sample
Mass
(mg)
1.66
2.79
5.13
6.20
Computed
Concentration
(mg VSS/1)
1660
1395
1282
1240
Filter
Time
(min)
2
3
14
23
Enzymatic
Activity
(uM/l/mg VSS/hr)
8.9
9.1
8.6
6.7
Table 5
Effect of Filtration Time on Enzymatic Activity
Test
1
2
Filter
Time
(min)
3
23
56
3
50
100
Enzymatic
Activity
QaM/l/mg VSS/hr)
4.2
3.9
3.7
4.7
4.4
3.8
Table 6
Check Procedure
Sample
Undiluted
1/2 Dilution
Sample
Mass
(mg)
9.39
4.70
Filter
Time
(min)
10
5
Enzymatic
Activity
(uM/l/mg VSS/hr)
7.0
6.6
43
-------
during the batch tests by Higgins (51) and Moore, _et.a_L (23) was
omitted to reduce filtration time.
Phosphorus Measurements
The forms of phosphorus measured were limited to total phosphorus
and total soluble phosphorus. Total soluble phosphorus was obtained
by first filtering samples through a 0.45 p. membrane filter and sub-
sequent measurement of phosphorus in the filtrate. From these measure-
ments the sludge phosphorus content was computed by subtracting
the total soluble phosphorus from the total phosphorus in the aeration
tank. This value was used to compute the percent sludge phosphorus
content based on volatile suspended solids. Phosphorus removal
within the tank was computed by subtracting the total soluble phos-
phorus in the substrate.
Initially the alkaline ash method reported by Jenkins (56) was used,
but difficulties arose with the random occurrence of apparent fusion
of phosphorus to the porcelain crucibles used during the ashing
procedure. This led to the use of a modified form of the persulfate
method of Murphy and Riley (54), which is only slightly different than
the method appearing in the FWPCA Methods for Chemical Analyses
of Water and Wastes (55) under Phosphorus, Total. Appendix C con-
tains the total phosphorus method used during this study and differences
between this method and the above FWPCA method are noted.
Jenkins (57) reported the results of several methods for determination
of total phosphorus. Complete recovery was difficult and required
drastic digestion procedures, usually a nitric/perchloric or nitric/
sulfuric acid digestion yielding the best results. Another method,
the alkaline ash, was reported by Jenkins (56) to provide identical
results with the nitric/sulfuric acid digestion of the American
Association of Soap and Glycerine Producers (58). This alkaline ash
method was chosen for recovery comparison with the persulfate method
used in this study. The results are shown in Table 7. From these
tests a recovery factor of 0.92 was obtained and is applied to the
measured sludge phosphorus content during the metabolic uptake
experiments.
Other Measurements
Calcium was measured using a Perkin-Elmer Atomic Absorption Spectro-
photometer (Model 303). The method described in the Perjcin-Elmer
44
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Table 7
Comparison of Alkaline Ash Method to Persulfate Method
For Determination of Total Phosphorus
Mean
S.D.
Recovery F
Persulfate
(mg/1 P)
9.75
10.50
10.50
10.75
11.00
11.25
11.90
10.80
0.68
10.80 „ „„
aCt°r 11.70 °'92
Alkaline Ash
(mg/1 P)
10.50
11.12
11.50
11.88
11.90
12.25
12.75
11.70
0.73
Analysis
Table 8
Precision of Analyses
No. of Minimum Mean Maximum S.D. Variance
Samples _ __ _ (%)
Enzymatic Assay - 9
juM PN/l/mg VSS/hr
Phosphorus , Total - 7
mg/1
Phosphorus, Total 10
soluble - mg/1
Calcium - mg/1
10
3.4 4.7 5.6 0.7 15.0
9.75 10.81 11.90 0.68 6.3
5.72 5.85 5.98 0.07 1.2
70.0 70.8 73.5 1.0 1.4
45
-------
Analytical Methods for Atomic Absorption Spectrophotometer (59),
including the use of lanthanum as a suppressor of anionic interferences,
was used.
Suspended solids were measured using glass fiber filters with a Millipore
membrane filter apparatus. This method appears in the FWPCA Methods
for Chemical Analyses of Water and Wastes (55) under Solids , Non-
filterable. Reeve Angel 934 AH 5.5 cm filters were used.
Soluble total organic carbon (TOG) was measured with a Beckman
Total Carbon Analyzer using techniques recommended by the manu-
facturer. Samples were filtered through a 0.45/j membrane filter,
acidified, and inorganic carbon purged with nitrogen gas.
Fluoride was measured using the Orion Research Fluoride Electrode
(Model 94-09) and Orion Research Specific Ion Meter (Model 401).
This method appears in the FWPCA Methods for Chemical Analyses of
Water and Wastes (55) under Fluoride, Specific Electrode.
Measurements of pH were made with either a Corning Model 12 pH
Meter or an Orion Research Specific Ion Meter (Model 401). The pH
meters were standardized with buffer solutions prior to each use.
Gravimetric measurements were made using a semi-analytical balance
(Mettler Model H16). The readability of this balance is 10~5 gram.
Precision of Analyses
The reproducibility of major analytical procedures was determined to
establish the precision of a particular analytical technique. These
results are presented in Table 8.
46
-------
SECTION V
r,
METABOLIC UPTAKE OF PHOSPHORUS
This part of the study was conducted to define metabolic uptake of
phosphorus by activated sludge systems. To accomplish this, synthetic
substrates were developed using concentrations of calcium and phos-
phorus and apH range which precluded significant chemical precipitation
of phosphorus. Next, it was necessary to define the minimum cellular
phosphorus content required for the normal growth rate of bacteria in
activated sludge. This minimum cellular phosphorus level was assumed
to be the cell phosphorus content at which normal carbon removal
(approximately 85-90 percent) from a substrate begins. Also, it was
necessary to define the range of cellular phosphorus content above
this minimum value in systems that restrict significant phosphorus removal
to a metabolic mechanism. As discussed in the literature review, the
cellular phosphorus content above this critical level would be defined
as "luxury uptake. " Concurrently with these experiments the alkaline
phosphatase bio-assay was evaluated as a method to detect "luxury
uptake" of phosphorus.
Determination of Operating Conditions
To Preclude Phosphorus Precipitation
The objective of these experiments was to design a synthetic wastewater
in which significant phosphorus precipitation would not occur. How-
ever, in developing this substrate, consideration was given to the
following:
1) the pH range for good growth of the biomass; and
2) the requirements of the^biomass for calcium, magnesium and
trace metals.
As discussed in the literature review the laboratory experiments
emphasized the calcium-phosphorus system. Control of precipitation
was accomplished by restricting the presence and concentrations of
cations 4n tne synthetic wastewater substrates to the biologically
required quantities.
Although calcium plays a minor role in the growth of microorganisms
(24) it was desired to maintain calcium to phosphorus ratios represent-
ative of actual domestic wastewaters, between 1.5 and 10 to 1. In
47
-------
the experimental study to define the minimum required sludge phosphorus
content for normal growth the concentrations of phosphorus in the sub-
strate would be much smaller (0.01 to 0.15 mM/1) than actual domestic
wastewaters (0.25 to 0.5 mM/1). This allowed the calcium concen-
tration of the substrate for this study to be reduced to a minimal value,
0.15 mM/1. (See Appendix B Substrate A.) These low concentrations of
calcium and phosphorus precluded significant precipitation in the pH
range of interest, 6.5 to 8.5.
The experimental study to evaluate the range of sludge phosphorus
content beyond the minimum required for normal growth required a
higher phosphorus concentration representative of actual domestic
wastewaters. This necessitated the experimental determination of
calcium and phosphorus concentrations and the pH range such that
significant precipitation of calcium-phosphorus compounds did not
occur, yet provided a substrate representative of actual wastewaters.
The effects of pH, magnesium, trace metals and nucleation sites were
all considered. The effects of these components in the synthetic
wastewater substrates only were evaluated in defining the pH and
concentrations of calcium and phosphorus to preclude significant
precipitation of phosphorus.
General Procedures
Chemical precipitation tests were required to determine operating
conditions for the metabolic uptake of phosphorus in excess of minimum
required sludge phosphorus content study. During this study Substrate
B (see Appendix B) was used, and the stock components of this substrate
were used to prepare the test solutions for the precipitation tests.
The components for each test were added to two liter erlenmeyer flasks
containing distilled water and the final sample volume brought to 1
liter. The pH was immediately adjusted to the desired value using
sulfuric acid or sodium hydroxide. The flasks were not sealed to the
atmosphere, but were cotton plugged to prevent the entry of foreign
matter into the solution. All calcium and phosphorus concentrations,
including initial values were measured analytically as described in
Chapter 4, Experimental Procedures.
The basic time period of these tests was 24 hours; however, several
tests were continued for longer periods.
48
-------
Calcium-Phosphorus Systems
A series of tests were conducted to induce significant precipitation of
calcium-phosphorus compounds. This was accomplished using relatively
high calcium and phosphorus concentrations compared to concentrations
normally encountered in domestic wastewaters. This was necessary
to cause significant precipitation and still remain within the pH range
of biological systems.
Two tests were run using 10"^ M phosphorus with 10"^ M and 10
calcium at pH 8.5. With the exception of magnesium, bicarbonate,
and nitrogen, the solutions contained the inorganic components and
EDTA as used in Substrate B. The soluble calcium and phosphorus
concentrations remaining in solution were measured after one day
and the results are presented in Table 9. As expected the 10:1 Caip/PT
ratio precipitated essentially all the phosphorus, and the 1:1 Ga^/P^
ratio precipitated approximately half of the calcium and phosphorus.
The next series of tests were conducted using 0. 1 mM and 0.33 mM
phosphorus added to 0. 5 mM and 1 . 0 mM calcium at pH 6.5, 7.25,
and 8.0. No other inorganic components of the substrate were added
in this series to remove any complexing effects and to reduce the ionic
strength to a minimum. The flasks were agitated intermittently during
the first 24 hours and left quiescent for 22 additional days. The results
of the 0.33 mM phosphorus and the 1.0 mM calcium concentrations
after 1 and 23 days are presented in Table 10. No significant phosphorus
removal occurred in any test. All of these solutions exceed the theo-
retical solubility product for hydroxyapatite and the theoretical solubility
product for the dicalcium phosphate is exceeded at pH 7.25 and 8.0
in solutions containing 1.0 mM calcium.
The failure of phosphorus to precipitate at the lower pH's prompted a
similar series using only 0.33 mM phosphorus and 1.0 mM calcium
and including a test at pH 8.5. The presence of small concentrations
of inorganic components may exert an effect on the solubility of
phosphorus . These potential effects were included in this series by
duplicate solutions containing the trace elements present in the sub-
strate. The concentrations of the components were small: boron
2 x 10~4 M, zinc 3 x 10 M, manganese 7 x 10~6 M, molybdenum
5 x 10~6 M, copper 6 x 10~6 M, and cobalt 2 x 10~6 M. The flasks
were agitated intermittently during the first 24 hours and left quiescent
for 3 additional days of the test. The results after 1 and 4 days are
presented in Tables 11 and 12. Again there was no significant precipi-
tation at pH 8 or lower. Precipitation did occur in the solutions at
49
-------
en
O
Table 9
Calcium-Phosphorus Systems in Inorganic Medium
Less Magnesium, Ammonia, and Bicarbonate
Day pH
0 8.5
1
0 8.5
1
P
Total
0.976
0.937
0.937
a. 995
P
Soluble
0.412
0.033
P
Precip . .,
0.525
0.962
Ca
Total
1.003
1.000
9.850
9.750
Ca
Soluble
0.413
8.280
Ca Ca:P
Precip . Precipitate
0.587 1.12
1.470 1.53
(Concentrations in millimoles per liter.)
Table 10
Calcium-Phosphorus
(add pH 6.5
Day
0
1
23
0
1
23
0
1
23
PH
6.5
6.3
7.3
7.0
8.0
7.7
P
Total
0.355
0.371
0.361
0.322
0.336
0.329
0.329
0.332
0.329
P
Soluble
0.355
0.361
0.322
0.336
0.332
0.329
Systems in
,7.3, and
P
Precip .
-
-
-
-
-
-
Distilled
8.0)
Ca
Total
0.956
1.000
0.988
0.949
0.962
0.949
0.921
0.926
0.934
Water
Ca Ca
Soluble Precip .
0.988
0.988
0.912
0.937
0.921
0.913
(Concentrations in millimoles per liter.'
-------
Table 11
Day
0
1
4
0
1
4
0
1
4
0
1
4
PH
6.6
6.7
7.4
7.5
8.0
8.0
8.5
8.1
P
Total
0.319
0.313
0.326
0.319
0.313
0.319
0.309
0.309
0.319
0.294
0.319
0.355
Calcium-Phosphorus Systems in Distilled Water
(add pH 6.6, 7.4, 8.0, 8.5)
P P Ca Ca Ca Ca:P
Soluble Precip. Total Soluble Precip. Precipitate
0.300
0.300
0.313
0.313
0.313
0.306
0.110 0.209
0.077 0.278
1.038
1.019
1.041
1.038
1.000
1.000
0.975
1.000
0.988
1.000
1.000
0.988
0.988
1.000
0.975
0.962
0.958
0.962
0.687 0.313 1.50
0.588 0.400 1.44
(Concentrations in millimoles per liter.)
-------
en
to
Table 12
Calcium-Phosphorus Systems With Trace Elements
Day
0
1
4
0
1
4
0
1
4
0
1
4
PH
6.6
6.8
7.4
7.6
8.0
8.1
8.5
8.2
(Concentrations
P
Total
0.326
0.319
0.326
0.319
0.303
0.329
0.309
0.306
0.300
0.313
0.313
0.291
P P
Soluble Precip .
0.313
0.326
0.294
0.311
0.294
0.303
0.206 0.107
0.139 0.152
Ca
Total
0.520
0.520
0.487
1.023
0.988
0.988
1.423
1.396
1.430
1.038
0.980
1.000
Ca
Soluble
0.520
0.475
0.962
0.962
1.324
1.353
0.812
0.700
Ca Ca:P
Precip. Precipitate
-
- -
-
- -
-
-
0.168 1.57
0.300 1.97
in millimoles per liter.)
-------
pH 8.5, both with and without the trace elements present; however,
the solutions with the trace elements did exhibit an increase in phos-
phorus solubility.
Effect of Magnesium
Ferguson and McCarty (35) reported that magnesium exerts a significant
effect in calcium-phosphorus systems, increasing the phosphorus
solubility between pH 7 and 9. They reported that the effect of mag-
nesium as a minor component requires a Ca:Mg ratio greater than 1
and vanishes as the ratio exceeds 50. Such an effect is of interest in
that this pH range is the same as biological systems and the Ca:Mg
ratio in many activated sludge systems fall in the required range.
In general synthetic waste substrates contain magnesium concentrations
from 0.75 to 1.0 mM • If 1 mM of magnesium is used, 1 mM of
calcium would provide the required Ca:Mg ratio reported by Ferguson
and McCarty and the requirement of a biological substrate. Also, this
concentration of calcium with 0.33 mM of phosphorus has been
used in previous tests and no significant precipitation occurred at
pH 8 or lower, but did occur at pH 8.5. To observe the possible effect
of magnesium at pH 8.5 a test was conducted using these concentra-
tions of calcium, phosphorus, and magnesium at pH 6.5, 7.4, and 8.5.
No other inorganic components of the biological substrate were included
(but 3 mg/1 copper was added as an algicide). The flasks were agitated
intermittently for 24 hours. The pH 8.5 test was left quiescent for an
additional 17 days.
The results after 1 day are presented in Table 13. A precipitation of
phosphorus in the flask with magnesium occured at pH 8.5, but the
precipitate was small compared to the test without magnesium. After
18 days no further increase in the precipitated phosphorus occurred.
The test without magnesium precipitated approximatley 85 percent of
the phosphorus in 4 days, but the presence of magnesium reduced
this to approximately 20 percent. These results indicate that magnesium
increased the solubility of phosphorus at pH 8.5.
Nucleation Sites
All test series have indicated that significant precipitation of calcium-
phosphorus without nucleation sites present did not occur with 10~3
M calcium and 3.3 x 10"^ M phosphorus concentrations at pH 8 or
lower. Although nucleation sites are not to be included in the substrate,
there is the probability that such sites may inadvertently be included
53
-------
Table 13
Calcium-Phosphorus Systems With One Millimole Per Liter Magnesium
Day
0
1
0
1
0
1
18
PH
6.5
7.4
8.5
P
Total
0.326
0.326
0.334
0.329
0.334
0.321
0.336
P
Soluble
0.326
0.329
0.255
0.258
(Concentrations in millimoles per
P
Precip.
-
-
(0.066)
(0.078)
liter.)
Ca
Total
1.030
1.050
1.003
1.045
1.019
1.038
-
Ca
Soluble
1.045
1.038
0.997
0.800
Ca
Precip .
-
-
(0.041)
(0.230)
Ca:P
Precipitate
-
-
(0.710)
(3.000)
Table 14
Effect of Nucleation Sites (8 mg/1 Kaolinite) on Calcium-Phosphorus Systems With and
Without Magnesium, 0.8 mM/1, Containing All Components of the Biological Substrate
Day
0 '
1
0
1
0
1
0
1
PH
8.2
8.1
8.2
8.1
8.2
8.2
8.2
8.3
Mg
No
i\ \J
No
Yes
Vpo
J. C- O
(Concentrations in
Kaolinite
VPS
_L c; o
No
Yes
No
J.^ ^
millimoles per
P
Total
0.316
0.322
0.322
0.316
0.322
0.316
0.300
0.303
liter.)
P P
Soluble Precip.
0.313
0.306
0.313
0.313
0.322
0.313
0.291
0.300
Ca
Total
0.990
0.988
1.000
1.000
1.000
1.023
0.980
1.030
Ca Ca
Soluble Precip.
0.975
0.975
0.980
1.000
0.975
1.005
0.975
0.975
-------
in the inoculum taken from the treatment plant. Should this occur the
quantity would be small and their size would be near or in the colloidal
range. One of the many likely sources would be some form of clay.
To investigate the effect of such an occurrence a series of tests were
conducted with 8 mg/1 of kaolinite present in the solutions. The solutions
contained all the components of Substrate B, but magnesium was not
included in a duplicate set. Duplicates also were run with no kaolinite
present. The pH was adjusted to 8.2, a value between observed
precipitation at 8.5 and no precipitation at 8.0. Mechanical mixing
of the solutions was accomplished using magnetic stirrers. To preclude
the possibility of biological growth the flasks were intermittently
exposed to ultraviolet light. Other procedures were not changed.
The results of these tests are presented in Table 14. No significant
removal of phosphorus occurred in 1 day.
Synopsis
The objective of the tests was to define operating conditions for bio-
logical systems with respect to calcium and phosphorus concentrations
and the pH range where chemical removal of phosphorus would not be
significant. The substrates with 1 mM/1 calcium and 0.33 mM/1
phosphorus concentrations at pH 8.0 or lower did not cause chemical
precipitation of phosphorus. The presence of inorganic components of
the biological substrate increased the solubility of phosphorus, part-
icularly magnesium which had been reported to exert a significant
effect in the pH range of interest. Small concentrations of nucleation
sites in the form of kaolinite did not induce precipitation of phosphorus
at pH 8.2 within one day. Should some nucleation sites inadvertent-
ly enter the biological system with the inoculum the effect probably
would not be significant.
The operation of activated sludge systems under these conditions will
provide a synthetic substrate that is representative of actual waste-
waters and provide control of chemical precipitation as a significant
phosphorus removal mechanism.
Minimum Required Sludge Phosphorus for Normal Growth
This phase of the study was undertaken to define the minimum required
cellular phosphorus for normal growth of bacteria as found in activated
sludge systems. The experimental units were completely mixed continuous
flow laboratory activated sludge units operated for less than one bio-
logical solids retention time (mean cell retention time - days).
55
-------
General Procedures
Low sludge phosphorus content inoculum was used with initial phos-
phorus contents between 1 and 1.5 percent. The low sludge phosphorus
content inoculum was obtained as described in Section 4. Substrate A
provided a carbon concentration of 330 mg/1 TOG (900 mg/1 COD).
Influent COD to phosphorus ratios were from 0.03 to 1.5. Influent
calcium was 0.15 mM/1 and a buffer of 2 .5 mM/1 carbonate was included
in Substrate A. With an average initial inoculum of 500 mg/1 volatile
suspended solids (VSS) the initial organic loading was approximately
2 pounds COD per pound VSS per day. No sludge was wasted and as
the solids increased this organic loading decreased to less than 1
pound COD per pound VSS per day at the end of the series. The
hydraulic residence time was 24 hours. The initial conditions in the
continuous units were adjusted to approximately steady-state operating
conditions; i.e. , nutrient levels were adjusted to provide 50 percent
of the inorganic and 10 percent of the organic components of the substrate
The substrate was modified in the second series of experiments; calcium
was removed entirely from the substrate and 10 mg/1 phenol was added.
Total organic carbon (TOG) was used to report soluble carbon. The
relative removal of carbon from the substrate was the primary parameter
defining normal growth, and the simplicity of obtaining TOC data
prompted the use of this means to define carbon removal in the
experiments.
Results
Figure 10 presents the results of the first test series after 72 , 96, and
120 hours of operation. The percent TOC removal did not approach
a constant 85 to 90 percent until the percent sludge phosphorus
increased approximately 1 percent. Below this point carbon removal
was not constant, indicating that phosphorus was limiting. Although
phosphorus is not limiting above 1 percent sludge phosphorus content,
the effluent does not contain significant phosphorus until the
sludge phosphorus content exceeds approximately 1.6 percent.
Beyond 1.6 percent sludge phosphorus content, phosphorus appeared
in the effluent and the rate of the increase in sludge phosphorus
content declined rapidly. The maximum sludge phosphorus content
obtained during these experiments was 2.5 percent.
56
-------
100
95
o 90
e
0)
o
.0
o>
o
\_
O)
Q_
85
80
75
70
65 L 0.0
Phosphorus
Content
Effluent
Phosphorus
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
Influent Phosphorus ( Ib P/ 100 Ib COD )
0
Figure 10. Influent Phosphorus, Effluent Phosphorus, Percent Sludge Phosphorus Content,
and Percent Carbon Removal for Laboratory Completely Mixed Continuous Flow
Activated Sludge Units (First Test Series).
-------
These results indicate that the minimum cellular phosphorus required
for normal growth is between 0.9 and 1 percent. The COD:phosphorus
ratio of the substrate at 1 percent sludge phosphorus content is
100:0.15. No significant phosphorus appeared in the effluent until
the influent CODrphosphorus ratio exceeded 100:0.45.
The results of the second test series using Substrate A but with no
Calcium and 10 mg/1 phenol after 72 and 96 hours of operation are
presented in Figure 11. The relationship between percent sludge
phosphorus and TOO removal is essentially the same as that depicted
in the first test series. A phosphorus limiting condition occurred
below 1 percent sludge phosphorus, and no significant phosphorus
appeared in the effluent until the sludge phosphorus content was
approximately 1.6 percent. The COD:phosphorus ratios atthese
points are the same as the first test series , 100:0.15 and 100: 0.45 ,
respectively. The maximum sludge phosphorus attained was 1.75
percent.
Synopsis
The results of both test series indicate that the minimum sludge
phosphorus content for normal growth, i.e. , a constant 85 to 90
percent TOG removal, is between 0.9 and 1 percent. In these short
term experiments an influent COD:phosphorus ratio of approximately
100:0.15 was required for this minimum sludge phosphorus content.
Between 1 and 1.6 percent sludge phosphorus content no significant
phosphorus appeared in the effluent. The influent COD:phosphorus
ratio at 1.6 percent sludge phosphorus content was 100:0.45. Above
1.6 percent sludge phosphorus content, phosphorus appeared in the
effluent in both test series .
Although the general relationships between percent sludge phosphorus ,
TOG removal and effluent phosphorus for the second test series were
the same as the first test series, a difference was noted in the maxi-
mum percent sludge phosphorus attained. The maximum sludge
phosphorus content attained, 2.5 percent, occurred during the first
test series at an influent COD:phosphorus ratio of 100:1.63. The
maximum sludge phosphorus content during the second test series was
1.75. Above an influent COD:phosphorus ratio of 100:0.6 little in-
crease in sludge phosphorus content occurred up to the maximum ratio
of the test series, 100:1.0. This could be attributed to the presence
of the phenol, or the lack of calcium in the second test series influent.
58
-------
en
CD
100
95
o 90
E -
o>
QL
c 85
O
-Q
i_
<3 80
3.5
3.0
2.5
2.0
T3
^ 1.5
- 2 1.0
o
l_
0>
a.
0.5
65 L 0.0
TOC
Sludge Phosphorus Content
Effluent
Phosphorus
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
Influent Phosphorus ( Ib P/IOO Ib COD)
14
12
CT>
10 e
8
6
4
o
.c
Q.
Q_
C
0
1.6
Figure 11. Influent Phosphorus, Effluent Phosphorus, Percent Sludge Phosphorus Content,
and Percent Carbon Removal for Laboratory Completely Mixed Continuous Flow
Activated Sludge Units (Second Test Series).
-------
However, calcium was being released by the sludge throughout the test;
the concentration in the effluent was 8.5 mg/1 in the unit with an
influent COD:phosphorus ratio of 100:1.0.
Metabolic Incorporation of Phosphorus
With Controlled Chemical Precipitation
The sludge phosphorus content of normal activated sludge has been
reported to vary between two and three percent. The purpose of this
phase of the study was to observe the reaction of sludge phosphorus
content in short term completely mixed continuous flow systems inoculated
with activated sludges having initial sludge phosphorus contents at the
extremes of these limits. Subsequent operation under conditions repre-
sentative of actual wastewaters, but with significant chemical precipi-
tation controlled as described in the previous section, would indicate
the trend of metabolic incorporation of phosphorus. Should the initial
high phosphorus content sludge contain any solid phase phosphorus,
the controlled conditions should solubilize this phosphorus with a
reduction in the sludge phosphorus content. Under controlled conditions
any increase in the low initial phosphorus content sludge should reflect
only metabolic incorporation of phosphorus.
General Procedures
Inoculum for the high sludge phosphorus content experiment was obtained
by using activated sludge as taken from the treatment plant. Only
elutriation with distilled water was done to remove nutrients and soluble
calcium and phosphorus. The initial sludge phosphorus content was
approximately 3.4 percent. The low sludge phosphorus content inoculum
was obtained as described in Chapter 4, to provide an initial sludge
phosphorus content of approximately 2.0 percent.
Substrate B provided a carbon concentration of 350 mg/1 COD (130 mg/1
TOC). Sludge was wasted to maintain an organic loading between 0.3
and 0.4 pounds COD per pound VSS per day with a hydraulic residence
time of 24 hours. The initial conditions in the continuous units were
adjusted to approximately steady-state operating conditions; i.e. ,
nutrient levels were adjusted to provide 50 percent of the inorganic and
10 percent of the organic components of the substrate. TOC again was
used to measure carbon removal.
60
-------
Initial High Sludge Phosphorus Content Inoculum
This series of experiments involved 9 continuous flow units with three
having identical influent phosphorus concentrations. Influent concen-
trations of 5 , 7.5, and 10 mg/T phosphorus were used providing COD:
phosphorus ratios greater than 100:1 for all substrates. Two of the
three units were controlled at pH 7 and the third unit allowed to seek
its own operating pH. One controlled pH unit and the uncontrolled pH
unit received 40 mg/1 calcium in the substrate. The remaining controlled
pH unit received no calcium in the substrate.
Table 15 presents the results after 5 days of operation. The effluent
TOC of all units indicate normal operation with respect to carbon removal.
The controlled pH units all decreased in sludge phosphorus content.
The 7.5 and 10 mg/1 phosphorus units were quite close at 2.65 percent
and the 5 mg/1 somewhat lower at 2.2 percent. The effluent phosphorus
concentrations of the controlled pH units were all greater than the
influent concentration. Normal carbon removal with excess phosphorus
in the effluent indicated that the sludge was slowly releasing phosphorus.
The units with no calcium in the substrate did have calcium in the
effluent; however, the units with calcium in the substrate did not show
an increase in the calcium concentration of the effluent to accompany the
increased effluent phosphorus concentration.
The uncontrolled pH units remained above 3 percent sludge phosphorus
content with the 5 and 7.5 mg/1 phosphorus units near the initial value
of 3.4 percent. The effluent of these two units showed a small reduc-
tion in the influent phosphorus concentration. The 10 mg/1 phosphorus
unit had a lower sludge phosphorus content, 3.1 percent, and the effluent
phosphorus concentration exceeded that of the influent. Again the
calcium concentration in the effluent was essentially the same as the
influent.
After the 5 days of operation the 5 mg/1 phosphorus units were reversed
in the pH control, i.e. , the uncontrolled pH unit was controlled at pH
7 and the previously controlled units were uncontrolled. After an
additional 5 days of operation the initially controlled pH unit with no
calcium in the influent showed no significant change in sludge phos-
phorus content. The previously controlled pH unit with calcium showed
a small increase in sludge phosphorus content, 2.2 to 2.6 percent.
The initially uncontrolled unit with calcium did change significantly
in sludge phosphorus content. This unit dropped from 3.4 to 1.7 percent
61
-------
Table IS"
Initial High Phosphorus Sludge Content After 5 Days Of Operation With Controlled And
Uncontrolled pH
PH ,T,
Control
Yes
Yes
No
Influent
P, mg/1
5
5
5
Effluent
P , mq/1
5.28
5.82
4.72
% Sludge
P
2.17
2.24
3.43
Influent
Ca , mg/1
40
0 -
40
Effluent
Ca , mg/1
38.0
5.4
39.0
vss
mg/1
907
959
1162
% VSS
86
86
78
Effluent
TOG
20
20
18
PH
7.0
7.0
7.7
en
to
Yes
Yes
No
Yes
Yes
No
7.5
7.5
7.5
9.00
10.00
7.12
2.68
2.65
3.35
10
10
10
11.35
11.35
11.35
2.64
2.67
3.12
40
0
40
40
0
40
40.7
8.4
38.2
38.2
6.0
34.4
1057
1131
1083
920
920
1010
89
89
79
89
90
81
27
23
21
14
15
14
No
No
Yes
5
5
5
4.50
4.65
6.40
2.59
2.19
1.69
40
0
40
39.5
3.1
52.5
1050
1160
1318
86
88
87
22
22
23
7.0
7.0
7.7
7.0
7.0
7.7
(After 5 days of operation pH control of 5 mg/1 influent phosphorus units was reversed.
an additional 5 days, the below data was taken.)
After
7.6
7.9
7.0
-------
sludge phosphorus content. This was accompanied by an increase in
the effluent phosphorus content, 4. 7 to 6.4 mg/1, and a large increase
in the effluent calcium concentration, 39.0 to 52.5 mg/1. Table 15
presents the results of this pH reversal experiment.
Initial Low Sludge Phosphorus Content Inoculum
This series of experiments were similar to the initial high sludge
phosphorus content previously run, but used inoculum that was low
in phosphorus content. During the previous series no significant
differences were noted with the different influent phosphorus concen-
trations, or the presence or absence of calcium in the substrate.
Therefore, this series used only two units with the 10 mg/1 phosphorus
and 40 mg/1 calcium substrate. To maintain correlation between the
series two units with initial high sludge phosphorus content were
included that duplicated the 10 mg/1 phosphorus and 40 mg/1 calcium
units from the previous series. As in the previous series one continuous
unit of each set was pH controlled and the other unit allowed to seek
its own operating pH. No other changes to the general procedures
were made.
The results of this series of experiments are presented in Table 16.
The effluent TOG values for all units indicated normal carbon removal
during the experiments.
The controlled and uncontrolled pH units with initial low sludge phos-
phorus content were not significantly different in sludge phosphorus
content after 5 days of operation, 2.35 and 2.55 percent respectively.
These values are close to those found in the previous series, and
show an increase in sludge phosphorus content form the initial 2
percent. Phosphorus removal from the influent was occurring in both
units.
However, unusual results were noted in the effluent calcium concen-
trations . The uncontrolled pH unit did not show any change in calcium
within the unit, effluent calcium was equal to influent calcium. But,
the controlled pH unit had a high effluent calcium concentration,
approximately 40 percent above the influent concentration. This
release of calcium from the sludge was not balanced by phosphorus.
Even though the inoculum was subjected to anaerobic treatment to
reduce the phosphorus content, it is apparent that this treatment did
not remove all the solid forms of calcium.
63
-------
Table 16
Initial Low and High Sludge Phosphorus Inoculum; 10 mg/1 Phosphorus and 40 mg/1
Calcium Influent Concentration
0-1
Unit
Description
Controlled pH;
Low Phosphorus
Sludge
Uncontrolled pH;
Low Phosphorus
Sludge
Controlled pH;
High Phosphorus
Sludge
Uncontrolled pH;
High Phosphorus
Sludge
Day
5
6
7
8
9
5
5
6
7
8
9
5
6
7
8
9
Effluent
P, mg/1
8.75
10.00
10.12
10.00
9.25
8.88
10.75
12.00
14.00
10.75
9.50
11.62
12.00
13.38
11.12
10.38
% Sludge
P
2.57
2.35
2.47
2.28
2.85
2.36
3.44
2.96
2.31
2.18
2.55
3.22
2.93
2.27
2.68
2.46
Effluent
Ca , mg/1
57.5
55.0
55.0
42.5
32.0
40.5
54.0
48.5
46.0
41.0
40.0
39.0
40.5
39.0
49.0
44.5
VSS
mg/1
989
1028
1042
977
878
996
945
1023
1055
1070
967
980
1051
1107
1119
984
% VSS
81
84
82
83
84
76
83
86
87
87
89
65
75
76
83
82
Effluent
TOG
19
20
20
20
24
19
19
20
19
19
25
20
20
21
20
21
PH
7.0
7.0
7.2
7.1
7.1
7.6
7.0
7.0
7.0
7.0
7.0
7.6
7.5
7.0
7.0
7.0
-------
Unusual results also occurred with the initial high sludge phosphorus
content units. Both the controlled and uncontrolled units maintained
high sludge phosphorus contents with the controlled unit unchanged
from the initial conditions, 3.45 percent, and the uncontrolled unit
down to 3.2 percent. Excess phosphorus was being released by both
sludges.
Again the effluent calcium results are unusual. The uncontrolled pH
unit was not releasing calcium to the effluent, yet excess phosphorus
was being released. The controlled pH unit was releasing calcium
to the effluent, and at a concentration similar to the low initial
phosphorus content unit. It appears that solid forms of both calcium and
phosphorus were present, but calcium-phosphorus solids were not the
sole major form.
Operation of the controlled initial low sludge phosphorus content unit
and the two initial high sludge phosphorus units was continued for four
additional days. During this period the controlled low initial sludge
phosphorus content unit and the uncontrolled high initial sludge phos-
phorus content unit were subjected to a pH reversal on day 7.
As seen in Table 16 small changes in sludge phosphorus content occurred
in the initial low sludge phosphorus unit before and after the pH
reversal. The effluent phosphorus concentration indicated phosphorus was
being released. The effluent calcium remained above the influent
calcium concentration, but decreased after the pH reversal. Day 9
showed the highest sludge phosphorus content, 2.85 percent. This
was accompanied by a small removal of phosphorus from the influent,
less than 1 mg/1, and a removal of calcium from the influent, 8 mg/1.
The sludge of the controlled initial high phosphorus unit continued to
release both calcium and phosphorus through day 8. Effluent phosphorus
increased to a high on day 7, then decreased. During this period the
sludge phosphorus content decreased to 2.18 percent. On day 9
effluent calcium was. the same as the influent and a small removal of
phosphorus from the influent occurred. At this time the sludge phos-
phorus content increased to 2.55 percent.
The sludge of the uncontrolled initial high phosphorus unit continued
to release phosphorus through day 6. Before the pH reversal no calcium
was released by the sludge. After the pH reversal on day 7 release of
calcium occurred, but the release was not as great as either of the
previously controlled units in this series. The release of calcium
continued through day 9.
65
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Addition of Solid Forms of Calcium and Phosphorus
During the previous series it was noted that solid forms of calcium
and phosphorus may have remained within the sludge under uncontrolled
pH conditions. To investigate this possibility two uncontrolled pH
units were spiked with solid forms of calcium and phosphorus: one
with 350 mg/1 calcium carbonate and the other unit with 170 mg/1
dibasic calcium phosphate. The low initial sludge phosphorus inoculum
was used. If these addditions remained in the solid form, sludge
phosphorus content in the dibasic calcium phosphate unit and the
percent volatile suspended solids for both units would change in relation
to the other units. On a basis of 1050 mg/1 VSS the solid phosphorus
spike (42 mg/lP) would increase the initial sludge phosphorus content
by 4 percent. The percent VSS would be decreased approximately 9
percent by the calcium phosphate and 18 percent by the calcium carbon-
ate based on a "normal" percent VSS of 84 percent.
The results of the uncontrolled low initial sludge phosphorus unit
with solid calcium carbonate and dibasic calcium phosphate added are
presented in Table 17. The effluent TOG values for all units indicated
normal carbon removal during the experiments.
During the 9 days of operation both units removed phosphorus from the
influent. No calcium release from the calcium carbonate unit occurred
and a small release from the calcium phosphate unit only occurred on
day 8. The percent VSS showed little variation in the calcium carbonate
unit and only slightly more in the calcium phosphate unit. Both units
were apparently holding the added solids.
The sludge phosphorus content of the calcium carbonate unit varied
slightly with a minimum of 2.35 and a maximum of 2.9 percent, and
this was in the range expected from the previous series. The 2.9
percent sludge phosphorus content occurred on day 9. The calcium
phosphate unit exhibited a slow decrease in sludge phosphorus content
from a high of 6.25 to a low of 5.15 percent sludge phosphorus on day
8. This decline could be primarily attributed to the daily wasting
necessary to maintain a constant VSS. On day 9 the sludge phosphorus
content increased to 5.75 percent. Although the effluent calcium
decreased, the effluent phosphorus did not indicate an increase in
phosphorus uptake concurrent with the increased sludge phosphorus
content.
66
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Table 17
Initial Low Sludge Phosphorus With CaHPC>4 and CaCC>3 Spikes; 10 mg/1 Phosphorus
and 40 mg/1 Calcium Influent Concentrations
Unit
Description
Uncontrolled ph;
Low Phosphorus
Sludge with
170 mg/1 CaHPO4
Uncontrolled pH;
Low Phosphorus
Sludge with
350 mg/1 CaCOs
Day
5
6
7
8
9
5
6
7
8
9
Effluent
P, mg/1
8.75
8.38
9.12
8.88
9.25
8.75
8.75
9.50
9.50
9.50
% Sludge
P
5.73
5.42
5.25
5.15
5.75
2.59
-
2.33
2.41
2.90
Effluent
Ca , mg/1
39.5
40.0
40.0
48.5
34.5
40.0
39.5
39.0
39.0
39.5
vss
mg/1
993
1080
1097
1168
1007
903
-
1131
1070
917
% VSS
76
72
71
65
72
60
-
65
65
65
Effluent
TOC
21
19
20
20
21
18
20
20
19
21
pH
7.8
-
7.5
7.3
7.2
7.6
-
7.5
7.3
7.2
-------
Synopsis
Under operating conditions precluding significant chemical precipitation
of phosphorus,the high initial sludge phosphorus units all decreased
in sludge phosphorus content. The initial low sludge phosphorus
content inoculum units increased in sludge phosphorus from the initial
2 percent, but did not exceed 3 percent. The high sludge phosphorus
contents maintained by the uncontrolled units indicate that solid forms
of phosphorus may remain within the sludge until slowly removed by
wasting to maintain a constant solids content; or, as in the pH reversal
with both the 5 mg/1 and the 10 mg/1 phosphorus units, subjected to
a pH that will solubilize the solid forms. The addition of solid
calcium carbonate and dibasic calcium phosphate to two experimental
units substantiated this, and the solid forms remained in the sludge
as the pH slowly decreased to a minimum of 7.2 after 9 days of opera-
tion. The concentrations of calcium (1 mM/1) and phosphorus (0.33
mM/1) do exceed the theoretical solubility product for dibasic calcium
phosphate at this pH. These results indicate that activated sludge
can hold solid forms of phosphorus at pH's encountered in many
activated sludge plants.
Because of the disparity of correlation between the leaching of phos-
phorus and calcium from the sludges during these experiments, the
high sludge phosphorus contents cannot be assigned to a solid form
of calcium-phosphorus being slowly released to solution. Other
cations present originally in the inoculum from the wastewater treat-
ment plant can hold phosphorus in solid form and may have caused the
results in these tests. Long term (in excess of four biological solids
retention time) experiments using laboratory activated sludge systems
with calcium as the sole significant precipitating cation should be
conducted under conditions that both favor and preclude significant
chemical precipitation of phosphorus. This should provide a means of
defining an upper limit of metabolic incorporation of phosphorus under
laboratory conditions.
Evaluation of Alkaline Phosphatase Enzymatic Assay
To Detect "Surplus Uptake" of Phosphorus
Fitzgerald and Nelson (25) developed an alkaline phosphatase bio-assa
technique for detection of "surplus uptake" of phosphorus by algae.
Using the modification of Fruh and Pessoney (26), Higgins (51) and
Moore,e_t aJL (23) evaluated this technique to detect "surplus uptake"
by activated sludge. Their experiments were conducted with batch
68
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units. The purpose of this chapter is to present the results of an
evaluation of the alkaline phosphatase assay as a method to detect
"surplus uptake" of phosphorus using completely mixed continuous
flow laboratory activated sludge systems.
During the experimental studies defining the limits of metabolic
incorporation of phosphorus by activated sludge, the percent sludge
phosphorus content was the primary parameter (coupled with carbon
removal by the sludge) used to define the minimum sludge phosphorus
requirement for normal growth, and in conjunction with effluent phos-
phorus was used to evaluate the range of "surplus uptake" of phos-
phorus. Concurrently with these studies the alkaline phosphatase
activity of the activated sludges was measured. This gave a direct
comparison of the results from the two methods to evaluate the alkaline
phosphatase bio-assay to detect "luxury uptake" of phosphorus.
General Procedures
The procedures used to measure the alkaline phosphatase activity are
outlined in Chapter 4.
The enzymatic activity study using completely mixed continuous flow
activated sludge units was concurrent with the metabolic studies,
and the general operating procedures for the activated sludge units are
described in the appropriate sections of those studies. The alkaline
phosphatase activity was measured at the same time from samples
used to obtain the data presented in the previous sections. The assay
medium was the same as the substrate in use during a particular
experiment (less yeast extract and peptone).
Inoculum for the batch tests was obtained from the completely mixed
continuous flow activated sludge systems used during the metabolic
uptake of phosphorus with controlled chemical precipitation study. /
Although the substrate of the continuous flow units contained 40 mg/1
of calcium, the calcium concentration was reduced to 6 mg/1 for the
batch test substrates to duplicate that of Moore, _e_t a_L (23). Also,
the pH of the batch units increased during the test period (up to 8<.5)
and this low calcium concentration was necessary to preclude the
possibility of chemical precipitation of phosphorus. The phosphorus
concentration varied from 0.2 mg/1 to 6.35 mg/1. The time period of
the tests was 22 hours.
69
-------
Results
The relationship between the alkaline phosphatase activity and the
percent sludge phosphorus content for the first test series of the
minimum required sludge phosphorus content for normal growth study
is depicted in Figure 12. The data presented were obtained after
3,4, and 5 days of operation. The percent sludge phosphorus content
data corresponds to that previously presented in Figure 10.
There is an apparent relationship between the enzymatic activity and
the percent sludge phosphorus content. At sludge phosphorus contents
of 1 percent and lower relatively high enzymatic activities were
exhibited by the sludges. This corresponds to the minimum required
sludge phosphorus content for normal growth, i.e. below which
phosphorus is limiting. No sludge phosphorus contents between 1
and 1.6 percent occurred in this test series, but the results of sludge
phosphorus contents above 1.6 percent indicate a relatively constant
enzymatic activity. The relationship depicted between 1 and 1.6
percent could be assumed. This assumption would follow the experi-
mental results described in the section defining minimum required sludge
phosphorus content, i.e. , a transition from a limiting nutrient condition
to a condition of available surplus phosphorus.
The results of the second test series of the minimum required sludge
phosphorus content study are presented in Figure 13. The only difference
between the two test series was in the substrates; calcium was removed
and 10 mg/1 of phenol was added to the substrates in the second test
series. The percent sludge phosphorus content data corresponds to
that previously presented in Figure 11. The relationship between enzy-
matic activity and percent sludge phosphorus from the first test series
also is shown.
A significant suppression of the enzymatic activity apparently occurred
with the changes in the substrate. As seen in Figure 13 the maximum
enzymatic activity measured did not exceed the minimum enzymatic
activity measured in the first test series. The minimum enzymatic
activities also were correspondingly lower. Although the enzymatic
activities are suppressed, the relationship between enzymatic activity
and percent sludge phosphorus content between 1 and 1.6 percent is
depicted as a transition zone. Such a transition zone assumed in the
1 to 1.6 percent range in the first test series appears to be valid.
Figure 14 presents the results of the metabolic incorporation of
phosphorus with controlled chemical precipitation study. Again, the
70
-------
8
o
o
fsl
a
14
E 12
10
8
o
E 4
0
AB
I
0 1.0 2.0
Percent Sludge Phosphorus
3.0
Figure 12. Results of Alkaline Phosphatase Bioassay - Minimum
Sludge Phosphorus Content Study, First Test Series.
71
-------
8
CO
14
E 12
\
10
8
o
< 6
o
o
E 4
>^
M
C
LU
0
0
Results of First
Test Series
\
1.0 2.0
Percent Sludge Phosphorus
3.0
Figure 13. Results of Alkaline Phosphatase Bioassay - Minimum
Sludge Phosphorus Content Study, Second Test Series.
72
-------
18
\
oo
^^
EI2
10
>» 8
o
< 6
o
E 4
M
0
0
Figure 14,
o - Unit spiked with
solid phosphorus
Results of First
Test Series
\
\
1
1
i
1
1.0 2.0 3.0 4.0 5.0
Percent Sludge Phosphorus
6.0
Results of Alkaline Phosphatase Bioacsay - Metabolic
Incorporation of Phosphorus with Controlled Chemical
Precipitation .Study,
73
-------
Table 18
Batch Tests
TSS
mg/1
139
140
141
149
137
138
130
128
131
133
Initial
TOG
mg/1
135
118
118
120
125
135
135
118
135
135
Conditions
P
Soluble
mg/1
0.20
0.27
0.77
0.76
0.72
1.29
2.42
3.12
4.75
6.35
Final Conditions
Sludge
P
1.73
1.75
1.77
1.78
1.80
1.76
2.12
2.07
2.04
1.76
TSS
mg/1
240
231
225
228
234
255
289
247
272
259
TOG
mg/1
42
14
15
15
17
25
25
-
25
25
P
Soluble
mg/1
0.04
0.02
0.04
0.04
0.04
0.10
0.30
0.67
2.36
4.25
K>
Sludge
P
0.99
1.12
1.19
1.28
1.19
1.53
1.67
1.56
1.98
1.95
Enz.
Act.
8.3
8.0
7.8
7.8
7.3
6.0
2.5
2.7
2.5
2.4
A
TSS
mg/1
101
91
84
79
97
117
159
119
141
126
Phosphorus
utilized
0.16
0.25
0.73
0.72
0.68
1.19
2.12
2.45
2.39
2.10
-------
1 O
_ 16
-C
v> 14
CO
E 12
5 10
r 8
;>
0
< 6
0
O
E 4
M
^"
LU
2
0
1 i
— —
— —
— —
X.
r*
i •
i
i
V
**•— -^_
"~" ^™"
1 1
0 1.0 2.0 3.0
Percent Sludge Phosphorus
Figure 15. Results of Alkaline Phosphatase Bioassay - Batch
Tests.
75
-------
relationship between enzymatic activity and percent sludge content
obatined in the first test series of the minimum sludge phosphorus
content study is shown.
These data appear to fit the relationship previously demonstrated.
Although the enzymatic activities appear to be slightly lower, a rela-
tively constant enzymatic activity occurred at these higher percent
sludge phosphorus contents.
The results of the batch tests are presented in Table 18. Figure 15
depicts the relationship between enzymatic activity and percent sludge
phosphorus. Carbon removal was essentially complete in all units
indicating that the activated sludges were in, or close to the stationary
phase of the growth cycle. As a result no high percent sludge phos-
phorus contents were obtained similar to the results of Moore, et al.
(see Figure 2); the high percent sludge phosphorus contents of their
study only occurred during the lag phase of the growth cycle.
The low enzymatic activities in this series occurred at much lower
percent sludge phosphorus contents compared to the results of
Moore ,_e_t_a_l. The results indicate that a much sharper transition
occurred in this test series between the high and low enzymatic
activities exhibited by the batch units, but still depict a relationship
between enzymatic activity and percent sludge phosphorus content
similar to the continuous flow tests.
Synopsis
In previous sections of this chapter two zones of percent sludge phos-
phorus content were defined: the growth dependent zone, less than
1 percent sludge phosphorus; and the storage zone, between 1 and 1.6
percent sludge phosphorus content. The relationship between alkaline
phosphatase activity and percent sludge phosphorus content demon-
strated in these tests defines similar zones.
In the study on the minimum required sludge phosphorus content
for normal growth, phosphorus was shown to be limiting below 1 percent
sludge phosphorus content. The enzymatic activities measured in
this zone were relatively high, indicating that the microorganisms were
exerting a phosphorus demand. Between 1 and 1.6 percent sludge
phosphorus, normal growth occurred but all phosphorus in the substrate
was utilized. The enzymatic activities measured in this zone depicted
a transition from the relatively high enzymatic activities of the phos-
76
-------
phorus limiting zone to the relatively constant enzymatic activities
obtained for sludges having a phosphorus content above 1 .6 percent.
Above 1.6 percent sludge phosphorus content, phosphorus appeared in
the effluent, and concurrently with this the enzymatic activities
approached a relatively low constant value. The higher sludge phos-
phorus contents of the experimental study with controlled chemical
precipitation also demonstrated a relatively constant enzymatic activ-
ity above 1.6 percent sludge phosphorus content.
The results of this batch test series generally agree with the results
reported by Moore, _e;t al_. with respect to the conditions necessary
for high enzymatic activities. However, the enzymatic data from the
batch tests compared with those from the continuous flow experiments
indicate that a quantitative evaluation of phosphorus demand using
this technique is not possible.
In previous and current batch tests with similar substrate and environ-
mental conditions, the enzymatic activity was quantitatively related.
However, for microorganisms fed with different substrates and cultured
under different environmental conditions, the enzymatic activities
cannot be quantitatively correlated although each is qualitatively high
or low in relation to the sludge phosphorus content within its own
test series. For example, fora sludge phosphorus content of 1.75,
enzymatic activity ranged from above 4.0 (Figure 14) to less than
1.0 (Figure 13). This variation in enzymatic activity greatly exceeds
the variance of the test.
Summary
With a synthetic substrate that precluded chemical precipitation of
phosphorus, the minimum required sludge phosphorus content for normal
growth, i.e. , a constant 85 to 90 percent carbon removal, was found
to be between 0.9 and 1.0 percent (based on volatile suspended
solids) in short term (less than 1 biological solids retention time)
completely mixed continuous flow laboratory activated sludge units.
Additionally, two zones of sludge phosphorus content for activated
sludge were defined:
1) below 1 percent, a growth dependent zone; and,
2) between 1 and 1.6 percent, a storage zone.
Beyond 1.6 percent sludge phosphorus content additional phosphorus
was incorporated by the sludge, but at a constantly decreasing rate.
The maximum sludge phosphorus content attained was 2.5 percent.
77
-------
The range of "luxury uptake" of phosphorus during these experiments was
1.5 percent, from 1 to 2.5 percent sludge phosphorus content.
Operating conditions of pH (7.0) and concentrations of calcium (1 mM/1)
and phosphorus (0.33 mM/1) were determined to provide a synthetic
substrate representative of actual wastewaters that precluded significant
chemical precipitation of phosphorus content at the extremes of the
"normal" sludge phosphorus content of 2 to 3 percent, the maximum
phosphorus content attained in short term completely mixed continuous
flow laboratory activated sludge units was less than 3 percent. Under
conditions of no pH control (7.8 to 7.2) solid forms of calcium and
phosphorus added as calcium carbonate and dibasic calcium phosphate
remained within the sludge until removed by sludge wasting.
The alkaline phosphatase enzymatic activity bio-assay was evaluated
as a method to detect "luxury uptake" of phosphorus by activated
sludge. The results obtained using the enzymatic assay served as
substantiating evidence of the defined zones of sludge phosphorus
content.
78
-------
SECTION VI
CONCURRENT CHEMICAL AND METABOLIC
INCORPORATION OF PHOSPHORUS
In the preceding section the incorporation of phosphorus in the sludge
was restricted to metabolic uptake by operating conditions of pH
and concentrations of calcium, phosphorus and other cations such
that significant precipitation of phosphorus did not occur. The purpose
of the experimental studies reported in this section was to determine
the magnitude of phosphorus incorporation in the sludge mass by
precipitation of phosphorus with calcium.
To accomplish this it was necessary to define operating conditions
representative of actual wastewaters, but such that chemical preci-
pitation of calcium and phosphorus would be induced. Simultaneous
operation of completely mixed continuous flow activated sludge systems
with operating conditions that favor and preclude significant calcium-
phosphorus precipitation would provide a means of separating the
quantity of phosphorus incorporation in the sludge by each mechanism.
Additionally, two ions, magnesium and fluoride, have been reported
to exert an effect on the calcium-phosphorus system; and, a concurrent
investigation of possible effects was included in the experimental
studies.
Operating Conditions to Induce Chemical Precipitation
In determining the operating conditions to induce chemical precipitation
of phosphorus with calcium, the results of several studies discussed in
the literature review, and the operating conditions of some activated
sludge plants reporting high phosphorus removals were considered:
1) magnesium exerts an effect on the solubility of phosphorus
within the concentration and pH ranges encountered in most
activated sludge plants (35,37);
2) there is a kinetic effect at these concentrations and pH's (33,37);
3) fluoride may exert a significant effect on the residual soluble
phosphorus (33);
4) the pH range of several activated sludge plants reporting high
removals of phosphorus is below 8 (4,27,28); and
5) the concentrations of calcium and phosphorus are less than 2.5
mM and 0.5 mM, respectively, at these plants, and fluoride is
present in the influent wastewaters at concentrations between
0.5 and 2.0 mg/1.
79
-------
These areas of consideration present a relatively large latitude in
choosing operating conditions to induce chemical precipitation in
calcium-phosphorus systems in activated sludge. However, the choice
of conditions was based on the availability of a study by Ferguson, _et
al_. (37).
Ferguson, _et al_. investigated calcium-phosphorus systems containing
2.0 mM of calcium and 0.24 mM of phosphorus in chemically
defined solutions containing only calcium, phosphorus and carbonate
at pH's of 7.6 and 8.0. Their results showed significant phosphorus
precipitation occurred at both pH 7.6 and 8.0. Although the residual
soluble phosphorus was the same at both pH's after 5 days, the presence
of an induction period noted was a function of pH.
The effect of varying concentrations of magnesium exerted an effect
on the residual soluble phosphorus, increasing the residual soluble
phosphorus as the calcium to magnesium ratio decreased to 3 to 1 at
pH 8.0. An induction period was noted at a calcium to magnesium
ratio of 1.4 to 1, with a further increase in the residual soluble phos-
phorus .
The concentrations of calcium and phosphorus and the pH ranges in
the study of Ferguson, jrt al_. satisfy the limits already described;
thus, their results would provide a basis for comparison with the
results obtained during this study in solutions containing the components
of the biological substrate . Therefore , concentrations of 2 . 0 mM
of calcium and 0.33 mM of phosphorus were chosen for this study.
The slightly higher phosphorus concentration maintained continuity with
the preceding experimental studies presented in Chapter 5. A pH of
7.6 was chosen to approximate the range of some activated sludge
plants reporting unaccounted for high phosphorus removals.
As in the experimental study to determine operating conditions to
preclude a significant calcium precipitation, the effects of the compo-
nents in the synthetic wastewater substrate were evaluated only to
define the conditions that induce calcium precipitation of phosphorus.
The quantitative effects on the solubility of calcium-phosphorus
systems were not investigated.
General Procedures
Batch tests were conducted in 500 milliliter Erlenmeyer flasks with
ground glass stoppers. Continuous mechanical mixing was provided
80
-------
by magnetic stirrers. The substrate to be used during the concurrent
metabolic and chemical removal study was Substrate C and stock
solutions of the components of this substrate were used to prepare the
test solutions. Substrate C contained the same ratios of inorganic
components used in Substrate B. The buffer system also was composed
of 2.5 mM of bicarbonate. Trace elements were present in the same
concentrations used in previous continuous flow experiments (see
Chapter 5). Sterile techniques were employed to prevent contamination
of the test solutions containing the organic component of the substrate.
The pH of the test solutions was initially adjusted to 7.6. The pH was
checked twice daily thereafter, and adjusted to this value if required.
The pH of the test solution containing the organic component was
initially adjusted to 7.6; and, as the flasks were kept sealed during
the tests to prevent contamination, no further pH adjustments were
made. The flasks containing only inorganic components also were
sealed except during pH adjustments.
Experimental Results
Calcium-Phosphorus Systems: The results of a batch test containing
only calcium and phosphorus are presented in Figure 16. Tyndall
light scattering was observed 8 hours after the start of the test. The
presence of solids became visible shortly thereafter, and the turbidity
of the solution increased rapidly. At 15 hours, almost half of the
phosphorus had been precipitated. Unfortunately, the pH decreased
to 6.8 overnight. After adjustment to pH 7.6 the precipitation of
phosphorus continued as before and at 41 hours 8.0 mg/1 of phos-
phorus had been precipitated.
Effect of Magnesium: Another batch test contained the basic calcium-
phosphorus concentrations but also the concentration of magnesium,
0.8 mM/1 (Ca:Mg ratio of 2.5:1), to be used in the substrate during
the continuous unit studies. The results are presented in Figure 17.
These results are very similar to those obtained in the calcium-
phosphorus only system; the curves are almost identical up to the
41 hour sample. The same pH decrease noted in the calcium-phos-
phorus only test also occurred during this test. The residual soluble
phosphorus concentration was 0.043 mM (1.33 mg/1) after 120 hours.
81
-------
00
0.30
^ 0.25'
\
S
£ 0.20
(A
1 0.15
JC
0.
1 o.io
Q_
0.05
0.00
-~"^
l^^Light Scattering Noted
^••"•O^c 3 3
V \ Calcium
X
V
\Phosphorus
^s***« -
_L_ 1 1 1 I 1 1 1 1 1 1 1 1
i:. I
2.0
1.9
1 O «— '
l.8\
^
1.7 E
1.6 E
3
"-5 1
O
1.4
1.3
1.2
0
18 27 36 45 54 63 72 81 90 99 108 117 126
Time ( hours )
Figure 16. Batch Chemical Precipitation Tests; 2 mM Calcium, and 0.33 mM Phosphorus.
-------
oo
co
in
ZJ
0.30
0.25
0.20
O.I 5
CL
i o.io
0.05
0.00
Light Scattering Noted
2.1
2.0
1.9
I.8
1.7
6
o
o
1.4
1.3
1.2
0 9 18 27 36 45 54 63 72 81 90 99 108 117 126
Time ( hours)
Figure 17. Batch Chemical Precipitation Tests; 2 mM Calcium, 0.33 mM Phosphorus, and
0.8 mM Magnesium.
-------
The results of the next batch test containing a high magnesium
concentration, 2.0 mM (Ca:Mg ratio of 1:1), are presented in Table
19. The high magnesium concentration test solutions did not exhibit
any measureable reduction in soluble phosphorus after 60 hours.
Effect of Fluoride: A test containing only the basic calcium-phosphorus
concentrations with 1 mg/1 fluoride added was conducted to observe
the effect of fluoride on residual soluble phosphorus. Another test
solution containing 1 mg/1 fluoride with a Ca:Mg ratio of 1 was also
studied. Results are presented in Table 19.
Fluoride did reduce the residual soluble phosphorus compared to the
calcium-phosphorus solution with no fluoride. After 27 hours there
was a 0.024 mM (0.74 mg/1 P) difference between the test solutions
with and without fluoride. The fluoride concentration was reduced
to less than 0.1 mg/1 indicating that fluoride did enter into the reaction.
There was approximately a 4 hour increase between the time that light
scattering was noted in the unit without fluoride and when light
scattering in the fluoride unit was observed.
The test solution containing magnesium and fluoride lost some
fluoride, but no calcium or phosphorus. The use of glass flasks and
the adsorption of fluoride by glass could account for this small loss.
Effect of Inorganic Substrate Components: The effect of the inorganic
components of the substrate, including carbonate but not magnesium,
was observed in a batch test. The concentrations of these inorganic
components were the same as would be used in the synthetic substrate
for the concurrent metabolic and chemical removal study. The EDTA
was included in the inorganic stock solution and was present in this
test solution. The results are presented in Figure 18.
The results of this test were again similar to the experiment containing
only calcium-phosphorus except that a much longer induction period
occurred; more than twice the time period previously observed. The
residual soluble phosphorus was slightly lower, 0.032 mM (1.0 mg/1).
The presence of the inorganic components apparently exerted only a
kinetic effect, with little effect on the final residual soluble phosphorus.
A similar batch test contained all the inorganic components (including
carbonate and magnesium) of the substrate and the EDTA. These
results are presented in Figure 19. There was a significant increase
84
-------
CO
on
Table 19
Batch Chemical Precipitation Tests
Time
(hrs)
0
60
0
60
0 (1)
27
0 (2)
27
Calcium
(mM/1)
2.0
2.0
2.0
2.0
2.0
1.5
2.0
1.4
Phosphorus
(mM/1)
0.310
0.310
0.310
0.310
0.310
0.071
0.310
0.047
Magnesium
(mM/1)
2.0
2.0
2.0
2.0
-
-
-
-
Fluoride
(ma/1)
-
1.0
0.85
_
-
1.0
<0.1
(1) - Light scattering noted at 10 hours
(2) - Light scattering noted at 14 hours
-------
CO
CD
Scattering
Noted
0.00
2.1
2.0
1.9
l.8
1.7
1.6
O
.4
.3
.2
0 9 18 27 36 45 54 63 72 81 90 99 108 117 126
Time ( hours )
Figure 18. Batch Chemical Precipitation Tests; 2 mM Calcium, 0.33 mM Phosphorus, a-nci
Inorganic Components of Substrate C Less Magnesium.
-------
0.30'-
Phosphorus
Calcium
Light
Scattering
Noted
i i
2.1
2.0
1.8 \
1.7 £
1.6 §
1.5
1.4
o
o
CJ>
1.3
1.2
18 27 36 45 54 63 72 81 90 99 108 117 126
Time ( hours )
Figure 19. Batch Chemical Precipitation Tests; 2.0 mM Calcium, 0.33 mM Phosphorus,
•and latt -Intrrgarrfic' CoWpo'frsnrs* of "Sub's trate"C.~ """' --*----—' "•
-------
in the induction period, almost 3 times that of the test with the
inorganic components containing carbonate but not magnesium.
Although the induction period was much greater, the residual soluble
calcium and phosphorus were not significantly different from the previous
tests, and the effect of the inorganic components such as carbonate
and magnesium at these concentrations again appeared to be kinetic.
After 114 hours, 20 mg/1 of hydroxyapatite was added to the solution.
A comparison of results obtained before and six hours after the apatite
spike does not indicate significant additional phosphorus removal,
although the lowest soluble phosphorus residuals did occur in this test.
Effect of Organic Substrate Components: The effect of the presence
of the organic components of the substrate was investigated in the next
batch series. The organic components of the substrate were added at
a concentration of 50 mg/1 COD. (The theoretical carbon concentration
in the aeration basin of a completely mixed continuous flow activated
sludge system is the effluent carbon concentration. Assuming 90
percent COD removal of a substrate with a COD concentration of
500 mg/1 during later experiments, 50 mg/1 COD would duplicate the
expected carbon concentration during the biological experiments.)
The effects of the inorganic components of the substrate were investi-
gated as done in the previous batch tests. Table 20 presents the
results after 120 hours.
A calcium-phosphorus precipitation occurred in all the tests. As
noted in Table 20, the pH of the batch tests containing all the compo-
nents of the substrate and all the components less magnesium initially
went to 9. Turbidity was noted in the solutions, but appeared to
solubilize when the pH was adjusted to 7.6. Apparently some solid
form remained in the colloidal state providing nucleation sites , lowering
the residual soluble calcium and phosphorus.
An additional batch series was conducted at varying COD concentrations,
0, 50, 100 and 150 mg/1, in solutions containing 2.0 mM calcium,
0.33 mM phosphorus, and all inorganic components (including
carbonate and magnesium) of Substrate C. After 120 hours the solution
containing no organic component had a residual soluble phosphorus of
0.05 mM (1.55 mg/1), and the solution with 50 mg/1 COD had a
residual soluble phosphorus of 0.185 mM (5 . 75 mg/1). However,
the solutions with 100 and 150 mg/1 COD did not have any measure'able
precipitation.
88
-------
Table 20
Batch Chemical Precipitation Tests; 2.0 mM/1 Calcium,
0.33 mM/1 Phosphorus, and 50 mg/1 COD of Organic Components of Substrate
Additional Initial Final Calcium
Components pH pH Initial Final
mM/1 mM/1
7.6 6.4 1.95 1.72
Magnesium (1) 7.6 6.4 1.97 1.71
Inorganic Compo-
nents less 7.6 (2) 6.2 1.98 1.47
magnesium
Phosphorus
Precipitated Initial Final Precipitated
mM/1 mM/1 mM/1 mM/1
0.30 0.319 0.196 0.111
0.26 0.306 0.190 0.116
0.51 0.316 0.061 0.255
10 All Components 7.6 (2) 6.15 1.98 1.59 0.39 0.309 0.061 0.248
of Substrate
(1) - 0.8 mM/1; Ca:Mgof2.5
(2) - Increased to pH 9 during initial pH adjustment. Immediately readjusted to 7.6.
-------
Synopsis
The objective of these tests was to define operating conditions for
biological systems with respect to concentrations of calcium and
phosphorus and pH representative of actual wastewaters such that a
chemical precipitation of phosphorus with calcium would occur. The
results from field tests of activated sludge plants reporting unusually
high sludge phosphorus contents and laboratory studies of calcium-
phosphorus systems led to the investigation of a system with 2.0 mM
(80 mg/1) calcium and 0.33 mM (10 mg/1) phosphorus at pH 7.6.
Significant precipitation of calcium and phosphorus occurred at these
concentrations in a calcium-phosphorus only system. The addition
of 0.8 mM magnesium (a Ca:Mg ratio of 2.5) did not exert a kinetic
effect, nor change the residual soluble phosphorus after 41 hours. The
residual soluble phosphorus after 41 hours was 0.071 mM (2.21 mg/1)
in the calcium-phosphorus system and 0.079 mM (2.45 mg/1) in the
system with a Ca:Mg ratio of 2.5.
A calcium- phosphorus system with 2.0 mM magnesium (a Ca:Mg
ratio of 1) did not exhibit any measureable precipitation after 60 hours.
A batch test of a calcium-phosphorus system with 1 mg/1 fluoride induced
precipitation with a lower residual soluble phosphorus, 0.047 mM
(1.47 mg/1) than a similar test with no fluoride, 0.071 mM (2.21 mg/1):
a difference of 0.024 mM (0 . 74 mg/1) after 27 hours . Less than
0.1 mg/1 fluoride remained in solution indicating that fluoride did
enter into the reaction.
A similar system with 1 mg/1 fluoride and 2.0 mM magnesium did
not induce precipitation. Fluoride apparently did not induce the
formation of fluoroapatite.
Batch tests with the inorganic components of the substrate (including
carbonate) indicated that only a kinetic effect was exerted in solutions
at a Ca:Mg ratio of 2.5, but no phosphorus precipitated at a Ca:Mg
ratio of 1.
The addition of organic components of the substrate at a concentration
of 50 mg/1 COD increased the residual soluble phosphorus, but
significant precipitation still occurred. A concentration of 100 mg/1
did prevent precipitation.
90
-------
With the exception of the microorganisms in the activated sludge all
the components of the completely mixed continuous flow activated
sludge units were present in the test Solutions in which significant
phosphorus precipitation with calcium'occurred. Operation under
these conditions should induce precipitation in the concurrent chemical
and metabolic incorporation experiments.
Calcium Precipitation of Phosphorus in Completely Mixed
Continuous Flow Laboratory Activated Sludge Units
With the operating conditions previously determined, phosphorus
incorporation in the sludge by chemical precipitation with calcium
could be induced or precluded in a synthetic wastewater substrate.
Comparison between concurrently operated units, one favoring and
the other precluding significant chemical precipitation, would define
the sludge phosphorus content incorporated by a chemical removal
mechanism. Also, concurrent operation of two additional units favor-
ing chemical precipitation, one with a Ca:Mg ratio of 1 and one with
1 mg/1 fluoride in the substrate, was included to evaluate their
effect at these concentrations in an activated sludge system.
General Procedures
Four completely mixed continuous flow laboratory activated sludge
units were used during the experimental tests. All four test units
utilized a phosphorus concentration of 0.41 mM (12.7 mg/1). The
increase in yeast extract and additions of Sego to the substrate
increased the total phosphorus concentration in the basic substrate.
To maintain continuity with previous experimental tests 10 mg/1 of
orthophosphate also were added to the substrate in this test series,
and this increased the initial total phosphorus concentration to 12.7
mg/1. This increase should not have had a significant effect on the
calcium-phosphorus system as most of this 2.7 mg/1 increase was in
combined forms of phosphorus, i.e. , growth factors present in the
yeast extract and Sego. Again, the composition of the substrate with
respect to minor trace metals precluded significant precipitation of
phosphorus with these cations (see Chapter 5). The units were:
1) a "control unit" with 1.0 mM (40 mg/1) calcium similar to
the continuous flow tests described in Chapter 5;
2) a "normal Ca:Mg ratio unit" with a Ca:Mg ratio of 2.5
containing 2.0 mM (80 mg/1) calcium;
91
-------
3) a "low Ca:Mg ratio unit" with a Ca:Mg ratio of 1.0 containing
2.0 mM (80 mg/1) calcium; and,
4) a "fluoride unit" with 1 mg/1 fluoride added to the substrate,
and with a Ca:Mg ratio of 2.5 containing 2.0 mM (80 mg/1)
calcium.
Inoculum for these tests was obtained by using activated sludge as
taken from the treatment plant. The sludge was screened and elutriated
to remove grit and discrete particles. The units were operated for
6 weeks, approximately 4 biological solids retention time, with the
control unit substrate. This removed any solid forms of calcium or
phosphorus that may have been occluded within the inoculum, and
also acclimated the sludge to the substrate. Prior to beginning the
experimental tests the sludges from the 4 units were mixed together
and then reapportioned, thus assuring a common inoculum that con-
tained no solid forms of calcium or phosphorus and was acclimated to
the substrate.
Substrate C provided a carbon concentration of 500 mg/1 COD. During
the first part of the tests solids were wasted to maintain 1200 mg/1
VSS. This required small variations in the quantity wasted daily,
particularly during periods of sludge bulking. When no bulking was
occurring the quantity wasted varied from 0.7 to 1.0 liter. In the
second part of the tests daily sludge wasting was constant, 0.8 liter
per day, and the VSS remained within 1200± 200 mg/1. This wasting
schedule provided a maximum biological solids retention time of 10
days. An average effluent suspended solids of 20 mg/1 would reduce
this to approximately 9 days. The organic loading varied between
0.35 and 0.5 pounds COD per pound VSS per day with a hydraulic
residence time of 24 hours.
As in previous tests the substrate contained 2.5 mM bicarbonate.
This quantity provided a substrate with an alkalinity similar to that
reported for activated sludge systems with unexplained high phos-
phorus removal.
The pH during the tests was to be maintained at 7.6 in all units. The
automatic pH control system only had two sets of probes , and pH
control for 4 units was required. During the acclimation period the
pH was monitored closely and it was determined that manual control
of two units was possible. The operating conditions favored a
"natural" pH close to 7.6 and very little titrant was used by the
automatic system to maintain this value.
92
-------
In previous experimental tests the percent sludge phosphorus was
computed applying a 0.92 recovery factor (see Chapter 4). The presence
of inorganic solid phosphorus within the sludge mass as a result of the
precipitation of phosphorus with calcium produced sludge phosphorus
contents exceeding the normal "metabolic" phosphorus content. There
is no way to quantitatively differentiate between the portion of sludge
phosphorus content attributable to metabolic incorporation, which
would have the recovery factor applied, and that portion due to inorganic
solid phosphorus which would not have the recovery factor applied.
The normal variation of sludge phosphorus content precluded applying
the recovery factor to the sludge phosphorus content of the control
unit and using this value for all units. Less relative error would be
expected in not applying the recovery factor to any unit, and this
was done.
To provide a means of comparing metabolic incorporation (required
plus storage as defined in Chapter 5) of phosphorus to another removal
mechanism, the phosphorus required to remove 100 pounds of COD,
0.86 pounds of phosphorus, determined by Menar and Jenkins (34)
was used. The quantity of phosphorus required, 4.0 mg/1, for a 92
percent reduction of COD in the test substrate is shown on the graph
depicting phosphorus removal from the substrate.
Calcium data between days 8 and 22 was not obtained because of
equipment malfunction.
Control Unit
During the 39 days of the test, sludge phosphorus content of the control
unit varied from a low of 2 .0 percent to a high of 3.07 percent on day
29. However, the sludge phosphorus content decreased to 2.8 percent
on day 33 and did not exceed 2 .8 percent for the remainder of the test.
With the exception of the first week of operation the phosphorus
removed from the substrate was within 1 mg/1 of the expected 0.86
pounds phosphorus per 100 pounds COD removed. No significant
(greater than 1 mg/1) removal of calcium occurred during the test.
Normal Calcium : Magnesium Ratio Unit
Figures 20 and 21 present the results of the test unit with 2 mM/1
calcium compared to the control unit. Initially, the units were similar,
no unusual sludge phosphorus content was noted. After one week of
93
-------
5.0
en 4.5
13
i_
O
"I 4.0
O
Q_
3.5
(75 3.0
o>
o
\_
CD
Q_
2.5
2.0
0
10 15 20 25 30
Time (days)
35 40 45
Figure 20. Percent Sludge Phosphorus Content for Six Week Acclimated Laboratory
Activated Sludge Unit: Normal Ca:Mg Ratio of 2.5.
-------
82
- 80
\
£ 78
I 76
o
5
74
72
Influent
~ 12
^ 10
3 8
t_
O
"I 6
o
f 4
Influent
Control
Normal
0
10 15 20 25 30 35 40 45 50
Time (days)
Figure 21. Effluent Calcium and Phosphorus Concentrations for
Six Week Acclimated Laboratory Activated Sludge Unit:
Normal Ca:Mg Ratio of 2.5. ,
95
-------
operation the test unit began to increase in sludge phosphorus content,
and this increase was accompanied by increased removal of phosphorus
from the substrate. After 16 days a difference of 0.75 percent in the
sludge phosphorus content between the test and control units existed.
On day 17 the pH dropped to 7.3, and then increased to 8.4 over a
6 hour period. The pH was slowly readjusted to 7.6 over a 2 hour
period. During the third week of operation the sludge in the test
unit began to bulk. The bulking lasted for five days.
After 22 days the sludge phosphorus content had increased to 3.4 percent
and appeared to be leveling off. The test unit had removed more phos-
phorus from the substrate than the control except during the period
of bulking. Calcium removal was also occurring. After 39 days
(~4 biological solids retention time) the sludge phosphorus content of
the test unit increased to 3.7 percent. Approximately 2.5 mg/1 of
phosphorus in excess of "normal" metabolic requirements was removed
from the substrate, which was accompanied by a removal of 6 mg/1
(0.15 mM/1) calcium. The sludge phosphorus content of the control
unit was 2 .8 percent at this time.
Low Calcium : Magnesium Ratio Unit
Figures 22 and 23 compare the results of the low Ca:Mg ratio unit to
the control unit. The relationship of this test unit to the control unit
is similar to the results obtained in the test unit with the normal
substrate Ca:Mg ratio except that a longer lag period occurred. The
phosphorus removed from the substrate in the test unit always exceeded
that removed by the control unit, although the maximum difference
was 1.4 mg/1 and was generally less than 1 mg/1.
A comparison of the calcium and phosphorus removed from the substrate
in this test does not indicate a relationship similar to the normal
Ca:Mg ratio unit. The percent sludge phosphorus appeared to level
off at the same time as the normal Ca:Mg ratio unit and the maximum
sludge phosphorus content attained after 39 days, 3.7 percent, was
the same as the normal Ca:Mg ratio unit. No significant bulking
occurred in this unit.
Fluoride Unit
Figures 24 and 25 compare the test unit with 1 mg/1 fluoride in the
substrate to the control unit. The relationship to the control unit
96
-------
again was similar to the other two test units until the third week.
Then, when the other units appeared to be leveling off in percent
sludge phosphorus content, the fluoride unit began a steady increase
in percent sludge phosphorus content. After 36 days of operation
the sludge phosphorus content exceeded 4 percent, and after 39 days
was 4.6 percent. This was 1.8 percent more than the control unit .
No significant bulking occurred in this unit.
The phosphorus removed from the substrate for the first 22 days was
similar to the other test units, except the normal Ca:Mg ratio unit
during the period of bulking. As seen in Figure 25 phosphorus removal
from the substrate after this time, and particularly during the period of
steady increase in percent sludge phosphorus content, began to increase
in relation to the other units. Calcium removal occurred at the same
time. The quantity removed varied, but appeared to be related to the
removal of phosphorus from the substrate. On day 39 the increase of
sludge phosphorus content to 4.6 percent was accompanied by both
increased phosphorus and calcium removal, approximately 4.3 mg/1
(0.14 mM) phosphorus above "normal" metabolic requirements and
7.3 mg/1 (0.183 mM) calcium.
During the early period of operation the fluoride concentration in the
mixed liquor remained relatively constant at 0.9 mg/1, but did begin
to decrease slightly after 29 days. By day 36, 0.25 mg/1 fluoride
was removed. On day 39, a 0.25 mg/1 removal also occurred.
Though some fluoride was being removed, it did not exceed 0.25 mg/1
at any time.
Synopsis
A six week acclimation period operating under the control unit conditions
preceded the actual test period. This assured that any solid forms
of phosphorus present in the initial inoculum were removed during
sludge wasting, and any subsequent solid forms of phosphorus could
be attributed to a precipitation with calcium.
The results of the normal Ca:Mg ratio test unit indicated that a
calcium-phosphorus solid had precipitated. The percent sludge phos-
phorus was higher than the control unit, a maximum of 0.9 percent.
No further effect from a low Ca:Mg ratio was observed. The normal
and low ratio units differed only in an apparent kinetic effect. The
maximum sludge phosphorus content attained by both units was 3.7
percent.
97
-------
CD
CO
5.0
en 4.5
3
\_
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Q- 4.0
o
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CL
CD
3.5
-------
82
^ 80
\
en
E 78
o
o
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76
74
72
14
12
10
8
A:
\
to
O
6
4
Influent
Control
Normal
I I J
0 5 10 15 20 25 30 35 40 45 50
Time (days)
Figure 23. Effluent Calcium and Phosphorus Concentrations for
Six Week Acclimated Laboratory Activated Sludge Unit
Low CarMg Ratio of 1.
99
-------
5.0 i-
4.5
s-
o
O>
CP
o
o
c
o>
O)
Q_
3.5
3.0
2.5
2.0
0
Control
10 15 20 25 30 35
Time (days)
40 45
Figure 24. Percent Sludge Phosphorus Content for Six Week Acclimated Laboratory
Activated Sludge Unit: Fluoride Unit.
-------
82
80
en
.= 76
-------
The unit with 1 mg/1 fluoride attained the highest sludge phosphorus
content, 4.6 percent. The removal of calcium and phosphorus from
the substrate attest to the formation of a calcium-phosphorus compound.
Fluoride did apparently enter into the reaction with 0.25 mg/1 removed
from the substrate during this period of high sludge phosphorus content.
Effect of an Induced Upset on Activated Sludge Systems
Exhibiting Calcium-Phosphorus Precipitation
During the previous test period a calcium-phosphorus precipitation
was induced in the three test units after four biological solids retention
time. At this time the three test units were subjected to a temporary
upset in operating conditions such that chemical precipitation of
phosphorus with calcium was inhibited. This change was in the form
of the pH reversal used during the metabolic incorporation of phosphorus
with controlled chemical precipitation study in Chapter 5. The pH of
the three test units was decreased to 7, held at this pH for 4 hours,
then slowly readjusted to pH 7.6 over an additional four hour period.
No other operational changes occurred and the general procedures
remained the same.
Control Unit
The control unit was not subjected to the upset. Normal operation
of this unit occurred until approximately day 60. At that time severe
bulking occurred, and the results became somewhat erratic. However,
the percent sludge phosphorus remained between 2.2 and 2.7. Phos-
phorus removal was erratic and less than the "normal" required
phosphorus for metabolic uses during this test period.
The reduction in phosphorus removal from the substrate occurred during
the periods when the sludge phosphorus content declined. The reduction
in sludge phosphorus content was accompanied by a release of phos-
phorus, and this release increased the phosphorus available for
metabolic uses. Calcium effluent was comparable to influent at all
times.
Normal Calcium: Magnesium Ratio Unit
At the end of the previous test period, day 39, the normal Ca:Mg
ratio unit had 3.7 percent sludge phosphorus content, an effluent
phosphorus concentration of 6 mg/1, and an effluent calcium concentra-
tion of 74 mg/1. On day 48, 9 days after the pH adjustment the percent
sludge phosphorus had decreased to 3.4 percent, effluent phosphorus
102
-------
had increased to 8.1 mg/1, and the calcium effluent had increased
to 78 mg/1. Figures 26 and 27 present the results of this test unit.
On day 55, a slight decrease to 3.3 percent sludge phosphorus
content occurred. Phosphorus removal from the substrate had increased
slightly.- but the effluent calcium had increased to approximately the
influent concentration. The unit was still removing more phosphorus
than required for "normal" metabolic requirement; however, recovery
from the upset had not occured in 16 days.
Because the major difference between the 3 previous test units had
occurred with the addition of fluoride to the substrate, fluoride was
added at this time to this unit and the results will be discussed
subsequently.
Low Calcium : Magnesium Ratio Unit
Figures 28 and 29 present the results of the low Ca:Mg ratio unit. At
the end of the previous test period, this unit had 3.7 percent sludge
phosphorus content, an effluent phosphorus concentration of 7.95 mg/1,
and an effluent calcium concentration of 80.8 mg/1. On day 48, the
sludge phosphorus content had decreased to 3.25 percent, effluent
phosphorus concentration had increased to 8.6 mg/1, and the effluent
calcium had increased slightly to 81.6 mg/1. No significant difference
in sludge phosphorus content was noted between the different Ca:Mg
ratio units with no apparent recovery occurring in this unit.
(At this time the concentration of magnesium was reduced in this unit
to the normal ratio to acclimate the unit for an experiment with the
addition of a solid calcium-phosphorus spike similar to that done in
Chapter 5. The results will be discussed in a subsequent section.)
Fluoride Unit
Figures 30 and 31 present the results of the unit with 1 mg/1 of fluoride
in the substrate. This unit had the highest sludge phosphorus content,
4.6 percent, and the maximum removals of calcium and phosphorus,
7.3 mg/1 and 8.3 mg/1 respectively, in the previous test. The phos-
phorus removed from the substrate was over twice that required for
"normal" metabolic uptake.
This unit had the largest reduction in sludge phosphorus content, a
decrease of almost 1 percent, after the upset. This unit experienced
103
-------
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-C
Q.
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-------
82
- 80
\
E 78
| 76
o
5 74
72
14
_ 12
? 10
Influent
10
Q.
to
O
.C
Q_
8
4
Influent
Control
Normal
(On day 55 I mg/l F~ was
added to the substrate )
i
i
J
40 45 50 55 60 65 70 75 80 85 90
Time (days)
Figure 27. Effect of pH Upset on Laboratory Activated Sludge Unit:
Ca:Mg Ratio of 2 .5 .
105
-------
o
0,
5.0
4.5
S- 4.0
a
-C
CL
a* 3.5
3.0
CD
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2.5
2.0
..Apatite Spike
ro
o
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c.
o
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in
Q.
CO
cu
I-
Control
40 45 50 55
60 65 70
Time (days)
75 80 85 90
Figure 28 . Effect of pH Upset and Addition of Apatite Spike on Percent Sludge Phosphorus in
Laboratory Activated Sludge Unit: Low Ca:Mg Ratio of 1; Increased to 2.5 for
Apatite Spike.
-------
\
en
l
CJ
o
O
en
JE
to
82
80
78
76
74
72
14
12
10
8
II 86.1
Influent
I _ I
I L J I _ L
Q.
C/l
O
Influent
Control
Normal
( On day 48 Ca:Mg ratio
increased to 2.5 )
40 45 50 55 60 65 70 75 80 85 90
Time (days)
Figure 29. Effect of pH Upset and Addition of Apatite on Laboratory
Activated Sludge Unit: Low Ca:Mg Ratio of 1; Increased
to 2.5 for Apatite Spike.
107
-------
5.0 i-
CD
oo
40 45 50 55 60 65 70 75 80 85 90
Time (days) -
Figure 30. Effect of pH Upset on Percent Sludge Phosphorus in Laboratory Activated Sludge
Unit: Fluoride Unit,
-------
82
80
78
I 76
O
5 74
72
Influent
Influent
in
t_
O
_C
Q.
10
O
40 45 50 55 60 65 70 75 80 85 90
Time (days)
Figure 31. Effect of pH Upset on Laboratory Activated Sludge Unit:
Fluoride Unit.
109
-------
a slow decrease in sludge phosphorus content, from 3.65 on day 48
to a low of 2.9 on day 83. During this period calcium was removed
from the influent, but this was not matched by phosphorus removals
significantly different from "normal" metabolic requirements. This
unit experienced severe bulking between day 65 and 80. At the
completion of the test run on day 89 , this unit had a sludge phos-
phorus content of 2.9 percent.
The residual soluble fluoride did not decrease below 0.9 mg/1
following the upset.
On day 55 fluoride (1 mg/1) was added to the substrate of the normal
Ca:Mg ratio unit. This gave two units with fluoride and provided
duplicate units for observation.
On day 62 , the sludge phosphorus content in this new fluoride unit
decreased to 3.1 percent which was matched by an increase in
effluent phosphorus . This unit experienced severe bulking after day
65. On day 70 the sludge phosphorus again increased to 3.3 percent
and the effluent phosphorus decreased slightly. From this time on the
results were erratic; however, the percent sludge phosphorus content
exceeded that of the control unit by at least 0.6 percent during the
last 20 days of operation and was 0.75 percent greater at the comple-
tion of the test run on day 89. Calcium was being removed from the
influent, but phosphorus uptake was at or above the "normal" meta-
bolic requirement during this period.
The residual soluble fluoride also did not decrease below 0.9 mg/1
for this new fluoride unit.
The removals of calcium, phosphorus and fluoride and the percent
sludge phosphorus content were approximately the same in the two
fluoride units from day 70 to the end of the test period.
Effect of Added Solid Hydroxyapatite
On day 62, the solid spike, hydroxyapatite (tri-basic calcium
phosphate) was added to the previous low Ca:Mg ratio unit. Prior
to the spike the unit had 3.05 percent sludge phosphorus, and after
the spike, 80 mg/1 Ca10 (PO4)6 (OH)2 , the percent sludge phosphorus
content should have increased to 4.55 percent.
Fifteen minutes after the addition of the spike the sludge phosphorus
content had increased to 4.5 percent and the effluent phosphorus
110
-------
increased to 8.45 mg/1, an increase of only 0.2 mg/1. The effluent
calcium increased to 86.1 mg/1, an increase of 5.8 mg/1. Although
the calcium effluent concentration increased, it was apparent that
the spike did not significantly solubilize. See Figures 26 and 27.
Daily analyses were performed for the next 5 days. During this time,
no unusual calcium or phosphorus release occurred to the effluent
and the percent sludge phosphorus decreased as a function of sludge
wasting. On day 70, the sludge phosphorus content appeared to
stabilize near 3.85 percent and this was accompanied by an increase
in the removals of calcium and phosphorus from the substrate.
However, further sludge phosphorus reduction occurred on days 73
and 76 with decreased removals of calcium and phosphorus from the
substrate.
The solid spike was not solubilized, but it also did not induce
increased phosphorus incorporation by the sludge. This experimental
unit was terminated at this time.
Synopsis
The temporary upset of the three test units caused a significant
reduction in the sludge phosphorus contents. In the fluoride unit the
sludge phosphorus content decreased almost 1 percent and in the other
units decreased approximately 0.4 percent. Recovery did not occur
in any unit after 2 biological solids retention times. However, in
the test units the sludge phosphorus content did continue to exceed
that of the control unit by 0.8 percent.
The effect of fluoride that occurred during the previous test period
ceased. No significant fluoride removal from the substrate occurred
for the remainder of the test period, 50 days.
Fluoride was added to the substrate of the normal Ca:Mg ratio unit
on day 55 , giving two test units with fluoride for comparison. By
day 70 the results of both units were almost identical and continued
in this manner for the duration of the test. Neither unit removed any
significant fluoride from the substrate.
The effect of a solid spike was observed by the addition of hydroxy-
apatite to a test unit. The addition of this solid did not induce
additional calcium-phosphorus removal from the substrate. The added
111
-------
sludge phosphorus content from the spike decreased during sludge
wasting and was no longer evident after one biological solids retention
time.
The control unit was not subjected to the upset and continued to operate
normally. The sludge phosphorus content varied between 2.2 and
2.85 percent.
The test units did maintain a sludge phosphorus content above the nor-
mal range for activated sludge, i.e. greater than 3 percent, during
most of the test period. A low of 2 .9 percent occurred in the fluoride
unit on day 89 , but the control unit sludge phosphorus content was
2.3 percent at this time. Although higher sludge phosphorus contents
were maintained by the three test units compared with control, the
much higher sludge phosphorus contents attained during the earlier
tests were not regained after the upset.
Summary
A synthetic wastewater substrate was developed that induced signi-
ficant chemical precipitation of phosphorus with calcium. A concurrent
chemical and metabolic incorporation of phosphorus test series was
conducted to evaluate the magnitude of this chemical removal mechanism
No significant phosphorus was precipitated in the control unit. The
substrate of this unit contained calcium and phosphorus concentrations
similar to the substrates used during the metabolic incorporation of
phosphorus precluding chemical precipitation, but at a pH of 7.6.
These results substantiate the results obtained during that study
with respect to precluding significant phosphorus precipitation at
pH 7,0.
A chemical precipitation of phosphorus did occur at pH 7.6 and
concentrations of 2 mM calcium and 0.33 mM phosphorus. The
quantity of phosphorus precipitated in the continuous flow experiments
was less than indicated by batch chemical precipitation tests con-
taining all components of the substrate. However, the absence of
microorganisms during the batch chemical precipitation tests limited
evaluation of all components of the activated sludge system. The
presence of the microorganisms probably can exert an effect on the
formation of calcium-phosphorus solids , precluding ordered crystals ,
and a possibility exists that metabolic by-products can act as
112
-------
crystal poisons. This was substantiated in part by the batch experi-
ments showing that increasing COD concentrations inhibited phos-
phorus removal.
A Ca:Mg ratio of 1 did not exert a long term effect on percent sludge
phosphorus content. Both the normal Ca:Mg ratio of 2.5 and the Ca:
Mg ratio of 1 units attained 3.7 percent sludge phosphorus content
after 39 days of operation.
The test unit with 1 mg/1 fluoride obtained the highest sludge phos-
phorus content, 4.6 percent. The removal of fluoride, 0.25 mg/1,
from the substrate indicated that fluoride did enter the reaction.
However, after the upset of the units the presence of fluoride in the
substrate was not significant. Two test units had 1 mg/1 fluoride
during a 34 day test period, and no significant fluoride removal
occurred in either unit.
The addition of a solid form of calcium-phosphorus, hydroxyapatite,
did not induce additional phosphorus incorporation in the sludge mass,
Although phosphorus was precipitated by calcium during these tests
at pH 7.6, it is apparent that this removal mechanism is sensitive
to upset.
113
-------
SECTION VIT
DISCUSSION
The objectives of this research were to determine the mechanisms
of high phosphorus removal from some wastewaters with subsequent
high sludge phosphorus contents. The research scope was limited
to an evaluation of two commonly proposed mechanisms: metabolic
phosphorus incorporation (both required and "luxury uptake") by
activated sludge cultures, and calcium-phosphorus precipitation. In
addition an alkaline phosphatase bioassay was evaluated to define
"luxury uptake" of phosphorus.
Metabolic Phosphorus Incorporation
The results of short term (less than one biological solids retention
time) completely mixed continuous flow laboratory activated sludge
systems operated under saturated dissolved oxygen conditions with
substrates that precluded chemical precipitation are compared to the
results of Hattingh (16) in Figure 32. Hattingh reported an influent
COD:phosphorus ratio of 250:1 (100:0.4) as "optimum"; i.e., the
concentration of influent phosphorus such that phosphorus just begins
to appear in the effluent. Sawyer (60) reported an influent optimum
BOD:phosphorus ratio of 150:1 (
-------
100
cr>
I- 3.5
(Hattingh: Carbon removal based on COD)
A A
- 3.0
Percent Carbon Removal
Effluent
Phosphorus
Phosphorus
Content
Hattinah(l963
UJ
65 L 0.0
0.0 0.2 0.4
0
0.6 0.8 1.0 1.2 1.4
nfluent Phosphorus ( Ib P/IOO Ib COD)
.6
Figure 32. Influent Phosphorus, Effluent Phosphorus, Percent Sludge Phosphorus Content, and
Percent Carbon Removal for Laboratory Completely Mixed Continuous Flow
Activated Sludge Units,
-------
beyond 1 percent would be termed "luxury uptake." The range
between 1 and 1.6 percent sludge phosphorus content would be the
storage zone for activated sludge; all phosphorus present in the
influent would be incorporated in the sludge. A saturation zone as
described by Borchardt and Azad for specific algae cannot be finitely
defined for activated sludge because of the heterogeneous nature of
the biomass, but will vary from 1.6 to approximately 3 percent.
Referring to the conditions depicted in Figure 32 , relatively high
alkaline phosphatase activity was exhibited in the phosphorus limiting
condition below 1 percent sludge phosphorus content. A transition
occurred with gradually reduced enzymatic activity exhibited as the
sludge phosphorus content increased towards 1.6 percent. Above
1.6 percent the alkaline phosphatase activity remained relatively
low and constant. The enzymatic bioassay did define the general
range of storage between 1 and 1.6 percent sludge phosphorus content.
However, above 1.6 percent sludge phosphorus content the enzymatic
activity did not define the upper limit of the saturation zone.
The maximum sludge phosphorus content obtained in any test defining
the limits of metabolic incorporation of phosphorus was approximately
3 percent. The minimum sludge phosphorus content tests, and also
substantiated by the alkaline phosphatase bioassay, indicate that a
"luxury uptake" of phosphorus by activated sludge is not the exception
but the rule.
In evaluating these results from an engineering aspect a primary
consideration is that above 1.6 percent sludge phosphorus content
phosphorus appears in the effluent in the absence of any chemical
precipitation. In general almost all activated sludge systems treating
municipal wastes operate in this area. However, if operation could
be achieved below 1.6 percent sludge phosphorus content, a major
benefit would occur: no significant phosphorus in the effluent. The
addition of carbon as shown by Sawyer (1) to obtain the "optimum"
carbon to phosphorus ratio depicted in Figure 32 is not feasible.
Without regard to the influent carbon to phosphorus ratio a feasible
operation could occur by reducing the phosphorus content of the
return sludge to some value below 1.6 percent. The minimum value
would be approximately 1 percent, that required for normal growth.
Neglecting new growth an increase in sludge phosphorus content
from 1 to 1.6 percent in an activated sludge system with 2000 mg/1
117
-------
volatile suspended solids represents the incorporation of 12 mg/1
phosphorus. This quantity of phosphorus is typical of the influent
concentration found in many municipal wastewaters.
Such a scheme was first proposed by Levin and Shapiro (8). The
stripping of phosphorus from activated sludge can be accomplished
using a period of anaerobiosis or slightly acidifying the sludge. The
sludge is returned to the system after sufficient phosphorus is
stripped and the concentrated supernatant receives further treatment
prior to ultimate disposal. Additionally, digester supernatant must
not be returned to the activated sludge system. Although significant
phosphorus removal is possible using this scheme, two areas of concern
appear. Initially, the sludge must be stripped of phosphorus. The
use of short time anaerobic stripping does not appear feasible. The
City of Trenton, Michigan (61), investigated the use of anaerobic stripping
and found that significant phosphorus release required up to 2 days.
During the course of the present research a similar time period was
required to reduce the phosphorus content to levels below 1.5 percent
(see Section IV ). Adequate short time stripping can be achieved
using acidification, but imposes both a chemical requirement and the
need for a suitable pH control and monitoring system to avoid toxic
pH conditions.
A "normal" activated sludge with 2.6 percent sludge phosphorus
content could be reduced to 2 percent; however, a return to 2 .6
percent with no significant phosphorus appearing in the effluent
would not be assured. The results of this research and past investi-
gators indicate that beyond 1.6 percent sludge phosphorus content
phosphorus will appear in the effluent. This imposes an upper limit
of 1.6 percent for such a scheme.
Consideration of these two areas would be necessary in evaluating
possible use of this phosphorus removal scheme. The cost of
additional treatment for stripping with adequate safeguards and
the range of phosphorus removal possible would dictate a comparison
with other phosphorus removal methods for a specific wastewater.
Additionally, the effect of a phosphorus stripping scheme on normal
operating characteristics of activated sludge systems, with particular
reference to adequate and rapid solids-liquid separation, is not
known. No general conclusion as to the effectiveness or practicality
of this removal method can be made.
118
-------
Calcium-Phosphorus Precipitation
Using six week acclimated completely mixed continuous flow lab-
oratory activated sludge units with a synthetic wastewater substrate
representative of actual municipal wastewaters, a sludge phosphorus
content of 3.7 percent was obtained. The substrate composition
restricted chemical precipitation to calcium-phosphorus forms and
contained 0.41 mM (12 . 7 mg/1) phosphorus with 2 .5 mM (150
mg/1) bicarbonate. The pH of the continuous flow units was controlled
at 7.6. The sludge phosphorus content of the unit with 2.0 mM
(80 mg/1) calcium was 0.9 percent higher than an identical unit with
only 1.0 mM (40 mg/1) calcium which restricted significant calcium-
phosphorus precipitation.
A comparison of the continuous flow activated sludge units with a
"normal" Ca:Mg ratio of 2 .5 and a low Ca:Mg ratio of 1 revealed no
significant differences after 39 days of operation. Three weeks of
operation were required to produce a significant calcium-phosphorus
precipitation in the unit with a Ca:Mg ratio of 1, but this was only
5 to 7 days more than required for the unit with a Ca:Mg ratio of
2.5. Both units had sludge phosphorus contents of 3. 7 percent after
39 days.
Ferguson,et aL (37) reported that an uncertainty exists as to the
effect of low CarMg ratios on dilute calcium-phosphorus system.
The effect of magnesium is hypothesized to be either mainly kinetic,
(a delay in initiation of the precipitation) or to have a significant
effect on the type of calcium-phosphorus solid that is formed,
(^tri-calcium phosphate vice an apatite). The results of batch
chemical precipitation tests for several calcium-phosphrous systems
are presented in Figure 33. A calcium-phosphorus system with 2.5
mM magnesium (Ca:Mg of 2.5) did not show any kinetic effect,
nor indicate a solid form with a different Ca:P ratio as a result of the
presence of magnesium. However, a calcium-phosphorus system
with a CarMg ratio of 1 did not exhibit any measureable precipitation
after 60 hours.
The addition of the inorganic components of Substrate C, less mag-
nesium, to a calcium phosphorus system did exert a kinetic effect,
tripling the time required for precipitation. This substrate contained
2.5 mM/1 bicarbonate. This result is similar to that reported by
Ferguson, _et aL, for a calcium-phosphorus system with 3.8 mM
119
-------
i 1 r—i 1
Light Scattering Noted
CA- P only
MG = 2.5
CA: MG =
CA-P with Inorganic
Components of Substrate C
(2.5mM/l HC03")
CA= MG = 2.5 with
norganic Components
of Substrate C
(2.5mM/l HC03~)
0.00
0
18 27 36 45 54 63 72
Time (hours)
81 90 99 108 117 120
Figure 33. Qualitative Summary of Phosphorus Precipitation found in Batch Tests with 2.0
mM Calcium.
-------
bicarbonate. Addition of magnesium, Ca:Mg ratio of 2.5, to a test
solution with the inorganic components of the substrate (including
2.5 mM bicarbonate) did cause a kinetic effect, extending the time
required for precipitation by a factor of 3. No significant effect was
noted in residual soluble phosphorus between test solutions.
Menar and Jenkins (34) reported the results of an extensive pilot-
plant study using a wastewater with a Ca:Mg ratio of 0.9. Phosphorus
incorporation in the activated sludge in excess of normal metabolic
requirements was obtained; sludge phosphorus contents up to a
maximum of 6.8 percent were obtained, but a pH in excess of 8.0
was required. A continuous flow activated sludge unit in the present
research with a Ca:Mg ratio of 1 exhibited phosphorus incorporation
in the sludge in excess of normal metabolic requirements with a
maximum sludge phosphorus content of 3.7 percent. Although the
sludge phosphorus content of this research is significantly less
than reported by Menar and Jenkins, this was obtained at pH 7.6
and produced a lower effluent phosphorus concentration.
Table 21 compares the two influents. A major difference in operating
conditions was alkalinity, 2.5 mM bicarbonate in this research and
405 mg/1 as CaCC>3 (/v 8.0 mM bicarbonate) in the wastewater used
by Menar and Jenkins. A comparison of the calcium and magnesium
concentrations to the total hardness does not indicate the presence
of other significant cations .
Thus, it is indicated that magnesium exerts only a kinetic effect in
dilute calcium-phosphorus systems, and alkalinity probably affects both
the magnitude and kinetics of the precipitation of calcium phosphorus
forms.
Table 21
Comparison of Sewage from San Ramon, California
and Synthetic Wastewater Substrate
Constituent
Calcium
Magnesium
Alkalinity as CaCC>3
pH in
pH out
Total Hardness as CaCOS
San Ramon (1)
(mg/1)
66 (1.65 mM)
44 (1.85 mM)
405
7.7
>8.0
346
Synthetic Waste-
water (mg/1)
80 (2.0 mM)
48 (2.0 mM)
125
6.9
7.6
(1) Mean of analytical data collected during study.
121
-------
Addition of 1 mg/1 fluoride to the substrate with a Ca:Mg ratio of
2.5 and 2.5 mM bicarbonate further increased the sludge phosphorus
content to 4.6 percent, 0.9 percent greater than the unit without
fluoride, after 39 days of operation. Over twice the required phos-
phorus for normal metabolic incorporation was removed and the
effluent phosphorus decreased to 4.1 mg/1, the lowest obtained in
any experiment. As pointed out by Leckie and Stumm (33) a lower
residual soluble phosphorus was obtained with fluoride present in
trace quantities. The time required for significant precipitation to
occur was slightly longer than the unit without fluoride, but the
same as the low Ca:Mg unit.
Though calcium was shown to precipitate phosphorus, it was also
shown to be sensitive to upset. Sludge phosphorus contents higher
than the control unit were exhibited after a pH upset, but previous
levels of sludge phosphorus content were not regained after
18 days of additional operation. The presence of fluoride did not
effect the quantity of phosphorus precipitated after the pH upset
and continued operation for 50 days did not indicate any effect from
the presence of fluoride. The addition of fluoride did enhance phos-
phorus removal with calcium, but the failure of fluoride to regain a
high sludge phosphorus content after upset reduces the engineering
significance of fluoride addition to water supplies.
In further assessing the results, the amount of phosphorus incorp-
orated from chemical precipitation in the completely mixed continuous
flow activated sludge systems was less than expected from the
batch chemical precipitation tests. During the batch tests there
were organic substances present, but no microorganisms. In the
presence of 50 mg/1 COD there was less phosphorus precipitated
than in systems containing only inorganics. Also, the presence of
organics at twice the concentration in the activated sludge units
(100 mg/1 COD) prevented any precipitation after 5 days during the
batch tests.
Leckie and Stumm (33) and Yu and Mark (39) offer partial explanation
for these results . These investigators reported that organic sub-
stances can act as crystal poisons. This effect could be a poisoning
of nuclei formed in the early stages of precipitation, thereby preventing
growth and restricting the precipitation of a solid form to the formation
of nuclei. Also, the possibility exists that a more soluble metastable
amorphous form is precipitated and the presence of organics prevents
122
-------
the formation of a less soluble stable form (62). Yu and Mark
reported that the presence of blue-green algae, acting as a surface
active agent, prohibited the formation of ordered crystals of apatite,
and an amorphous form developed. The presence of microorganisms
could have exerted an effect, both by their presence, as well as from
the by-products of their metabolism which, may have added organic .
substances not found in the substrate that were effective crystal
poisons.
An organic poisoning effect could be exhibited in plug flow systems
in the following manner. At the inlet end of the aeration tank a
high carbon concentration exists which is reduced as the flow
proceeds down the tank. When the carbon concentration is reduced
sufficiently to alleviate the poisoning effect, calcium-phosphorus
precipitation could occur.
As pointed out by Menar and Jenkins (34) in discussing the requirement
for high dissolved oxygen postulated as enhancing "luxury uptake"
of phosphorus (4,5,8), a dissolved oxygen concentration increase
fortuitously coincides with a reduction in dissolved and gaseous CC>2
content. Thus an increase in dissolved oxygen should be accom-
panied by an increase in pH, thereby favoring a chemical precipi-
tation of phosphorus. This point in the aeration tank also coincides
generally with the reduction of organic matter, thereby alleviating
any organic poisoning effect which could have inhibited chemical
precipitation of phosphorus.
The results implicating an organic poisoning of calcium-phosphorus
precipitation indicate a re-evaluation of operating conditions reported
to enhance phosphorus removal. The conditions observed at the
San Antonio Rilling Plant (4) do show an increase in dissolved oxygen
with a rise in pH up to 7.6 - 7.8 occurring at the point where
high phosphorus removal begins . However, Milbury ,_et_aJL. (50)
report no correlation between pH level and the point of rapid phos-
phorus removal, although a pH rise did occur across the plug flow
aeration tank. This rise was almost linear with no sharp increases
and the effluent varied between 6.8 and 7.3. The dissolved oxygen
level also was not related to the rapid phosphorus removal, provided
the system was not oxygen limited. Oxygen limiting conditions
always were associated with phosphorus releases and an accompanying
reduction in organic removal. Though the results of several monitor-
ing runs at Baltimore (28) do show a correlation between an increase
123
-------
in dissolved oxygen and the rapid uptake of phosphorus, several
runs did not and were presented to show this lack of correlation.
Table 22 presents two of these monitoring runs with the inclusion
of organic removal parameters. These data do implicate the occurrence
of an organic poisoning effect; a phosphorus removal was associated
with a reduction in soluble organic concentration independent of the
dissolved oxygen concentration. The 8/27/69 Control data show a
drop in COD and TOG through secondary clarification that was
accompanied by a removal of 12 mg/1 phosphorus. Additional runs
presented to show the lack of correlation between dissolved oxygen
content and high phosphorus removal were not as widely separated
as these two events, but also show a similar relationship.
The results of Milbury,et a_l_. (50) at Baltimore and the inhibitory
effect on calcium-phosphorus precipitation found with increasing
organic concentrations in the chemical precipitation batch tests
would seem to indicate that an organic poisoning effect does exist.
Further consideration of the results obtained in this research using
completely mixed systems, a system not normally associated with
high phosphorus removal, lend support to this hypothesis. Menar
and Jenkins (34) pointed out that an average lower pH in the aeration
tank and the residence time distribution are not favorable to calcium-
phosphorus precipitation. In this research a pH of 7.6 was maintained;
however, a soluble organic concentration, 30 to 55 mg/1 COD, was
achieved in the aeration tank: a low soluble organic concentration.
Such a low soluble organic concentration would have reduced the
poisoning effect allowing a calcium-phosphorus precipitation to
occur.
In summary calcium was shown to be capable of precipitating phos-
phorus in the pH range and concentrations of calcium and phosphorus
encountered in municipal wastewaters. However, the additional
phosphorus removed and incorporated in the sludge by calcium in
these experiments would not account for the high removals and sludge
phosphorus contents reported for several activated sludge plants.
It is apparent that other cations are involved in chemical precipitation
of phosphorus in these systems. Those plants reporting cation con-
centrations through the system do show sufficient removals of several
suspected cations, Fe III, Al III, Ca, Zn, to account for the phos-
phorus removals obtained when taken together. A single cation is
not responsible.
124
-------
ND
cn
Table 22
Relationship Between Soluble Phosphorus, Dissolved Oxygen and Soluble Organics in an
Activated Sludge System (after City of Baltimore)
Position Along Aeration Tank
Primary
9/17/69 - Test Effluent
Ortho P , mg P/l 8.7
D . O . , mg/1
Soluble BOD, mg/1 210
Soluble COD, mg/1 240
pH 6.5
8/27/69 - Control
Ortho P, mg P/l 4.8
D . O . , mg/1
Soluble COD mg/1 130
Soluble TOG mg/1 110
pH 6.4
Inlet
10.2
0.0
120.0
80.0
6.8
50.8
0.0
70.0
65.0
6.8
1/6
1.3
0.1
60.0
65.0
6.9
14.1 i
-
60.0
60.0
-
1
0
1
28
55
6
13
-
60
60
-
/3
.6
.2
.0
.0
.9
.9
.0
.0
1
i 0
! 3
,17
',45
• 6
*
13
6
50
50
6
/2
.3
.6
.0
.0
.9
.7
.0
.0
.0
.7
2/3
0.2
5.4
14.0
50.0
6.9
-
-
55
50
-
5/6
0.3
5.6
13.0
45.0
7.0
-
-
40
50
-
Exit
0.2
6.6
11.0
45.0
7.0
12.5
4.9
45.0
50.0
6.7
Return
Sludge
20.6
-
-
90.0
6.6
78.0
-
-
-
6.7
Secondary
Effluent
0.3
1.6
-
50.0
7.2
0.5
1.3
35.0
25.0
6.8
Note: "| " designates maximum P removal and | designates maximum D.O. increase.
-------
ACKNOWLEDGEMENTS
Research support was provided by the Office of Water Programs ,
Environmental Protection Agency Research Grant 17010 DUX.
Acknowledgements are also extended to Dr. J.F. Malina , Professor
of Environmental Health Engineering, Dr. R.E. Speece, Professor
of Environmental Health Engineering, and Dr. Allen J. Bard,
Professor of Chemistry, all of The University of Texas at Austin for
their suggestions and aid during this investigation.
Thanks are also extended to all those persons who had a part in this
work: Mr. Frank R. Hulsey for his technical assistance and partic-
ularly for the design and construction of an automatic pH control
system; Miss Margaret Lynn Shahan and Mrs. Gretchen L. Morgan
for analyses of samples and assistance during the course of the
experimental studies.
Thanks are extended to Beatrice Mladenka for her assistance in
preparing this report.
January 1972
127
-------
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19. Sekikawa , Y. , Nishikawa , M., Okazaki, andKato, K. ,
"Release of Soluble Phosphate in the Activated Sludge Process, "
Third International Conference on Water Pollution Research,
Munich, Germany (September 1966).
20. Hall, M.W., and Engelbrecht, R. , "Uptake of Soluble
Phosphate by Activated Sludge: Parameters of Influence,"
Proceedings of the 7 th Industrial Water and Waste Conference,
The University of Texas, Austin, Texas, p. II-8 (1967).
21. Randall, C.W., Marshall, D.W., and King, P.H., "Phosphate
Release in Activated Sludge Process," Journal of the Sanitary
Engineering Division, ASCE, Vol. 96, p. 395 (1970).
22. Wells, W.N. , "Differences in Phosphate Uptake Rates Exhibited
by Activated Sludges, " Journal of the Water Pollution Control
Federation, Vol. 41, p. 765 (1969).
23. Moore, H.G., Higgins, R.B., andFruh, E.G., "Surplus
Phosphorus Uptake by Microorganisms - Batch Tests with
Diluted Activated Sludge Cultures , " Center for Research in
Water Resources Report No. 41, Environmental Health
Engineering Program, The University of Texas, Austin, Texas
(1969).
24. Thiman, K.V. , The Life of Bacteria, 2 nd edition, The Macmillan
Company, New York (1963).
25. Fitzgerald, G.P., and Nelson, T.C., "Extractive and
Enzymatic Analyses for Limiting or Surplus Phosphorus Algae,"
Tournal of Phycology, Vol. 2, p. 32 (1966).
26. Fruh, E. G., andPessoney, G., "Investigation of Procedures
to Measure Surplus Phosphorus Uptake by Algae," to be
published.
27. Bargman, R.D., Betz, J.M., and Garber, W.F., "Continuing
Studies in the Removal of Phosphorus by the Activated Sludge
Process , " Presented at the 3 rd Joint Meeting of The American
Institute of Chemical Engineers and Institute Mexicano De
Ingenieros Quimicos, Denver, Colorado (August 30-September 2,
1970).
131
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28. City of Baltimore, Maryland, "Phosphate Study at the Baltimore
Back River Wastewater Treatment Plant, " Water Pollution
Control Research Series, Program No. 17010 DFV, Advanced
Waste Treatment Research Laboratory, Cincinnati, Ohio (1970).
29. Stumm, W- , "Chemical Precipitation of Phosphates, "
Discussion in Advances in Water Pollution Research, Vol. 2,
p. 220, The Macmillan Company, New York (1964).
30. Tenney, M.W. , and Stumm, W. , "Chemical Flocculation of
Microorganisms in Biological Waste Treatment," Journal of
the Water Pollution Control Federation, Vol. 37, p. 1370 (1965).
31. Wuhrmann, K. , "Objectives, Technology, and Results of
Nitrogen and Phosphorus Removal Processes," in Advances in
Water Quality Improvement, Water Resources Symposium No. 1,
edited byE.F. Gloyna and W.W. Eckenfelder, Jr., The
University of Texas Press, Austin, Texas (1968).
32. Eberhardt, W.A. , and Nesbitt, J.B., "Chemical Precipitation
of Phosphorus in a High-Rate Activated Sludge System, "
Journal of the Water Pollution Control Federation, Vol. 40,
p. 1239 (1968).
33. Leckie, J. , and Stumm, W. , "Phosphate Precipitation," in
Water Quality Improvement by Physical and Chemical Processes,
Water Resources Symposium No. 3, edited by E.F. Gloyna
and W.W. Eckenfelder, Jr. , The University of Texas Press,
Austin, Texas (1970).
34. Menar, A.B. , and Jenkins, D. , "The Fate of Phosphorus in
Sewage Treatment Processes. II. Mechanism of Enhanced
Phosphate Removal by Activated Sludge, " SERL Report
No. 68-6, University of California, Berkeley, California (1968).
35. Ferguson, J.F., and McCarty, P.L., "The Precipitation of
Phosphates from Fresh Waters and Waste Waters, " Technical
Report No. 120, Department of Civil Engineering, Stanford
University, Stanford, California (1969).
132
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36. Corsaro, G., Lauderbach, P., and Schwantje, H., "Formation
and Behavior of Hydroxyapatite," Journal of the American
Water Works Association, Vol. 56, p. 355 (1964).
37. Ferguson, J.F.Jenkins, D. , and Stumm, W. , "Calcium
Phosphate Precipitation in Wastewater Treatment," Presented
at the 3 rd Joint Meeting of the American Institute of Chemical
Engineers and Institute Mexicano De Ingenieros Quimicos,
Denver, Colorado (August 30-September 2 , 1970).
38. Mooney, R.W. , and Aia , M.A. , "Alkaline Earth Phosphates,"
Chemical Reviews , p. 433 (1961).
39. Yu, T.C. , and Mark, H.B. , Jr. , "The Effect of Surface
Adsorbents on the Rate of Crystal Formation of Hydroxyapatite
Precipitates," Environmental Letters, Vol. 1, No. 1, p. 55
(1971).
40. Brudevold, F., Steadman, L.T., and Smith, F.A., "Inorganic
and Organic Components of Tooth Structure," Annals of the
New York Academy of Science, Vol. 85,p. 110 (1960).
41. Martens, C.S., andHarriss, R.C., "Inhibition of Apatite
Precipitation in the Marine Environment by Magnesium Ions,"
to be published.
42. Scalf, M.R., Pfeffer, P.M., Lively, L.D., Witherow, J.L.,
and Priesing, C.P. , "Phosphate Removal by Activated Sludge.
Amenability Studies at Baltimore, Maryland," Robert S. Kerr
Water Research Center, Federal Water Pollution Control
Administration, Ada, Oklahoma (1968).
43. Myers, L.H., DePrater, B. L. , Lively, L.D., Witherow, J.L.
and Priesing, C.P. , "Phosphate Removal by Activated Sludge.
Amenability Studies at Washington, B.C. , " Robert S. Kerr
Water Research Center, Federal Water Pollution Control
Administration, Ada, Oklahoma (1968).
44. Scalf, M.R., DePrater, B.L., Pfeffer, F.M., Lively, L.D.,
Witherow, J.L. , and Priesing, C.P. , "Phosphate Removal by
Activated Sludge. Amenability Studies at Mansfield, Ohio,"
Robert S. Kerr Water Research Center, Federal Water Pollution
Control Administration, Ada, Oklahoma (1968).
133
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45. Myers, L.H., Horn, J.A. , Lively, L.D., Witherow, J.L.,
and Priesing, C.P. , "Phosphate Removal by Activated Sludge.
Amenability Studies at Indianapolis, Indiana," Robert S. Ken-
Water Research Center, Federal Water Pollution Control
Administration, Ada, Oklahoma (1968).
46. Myers, L.H., DePrater, B.L., Lively, L.D., Witherow, J.L.,
and Priesing, C.P. , "Phosphate Removal by Activated Sludge.
Amenability Studies at Smithfield, North Carolina," Robert
S. Kerr Water Research Center, Federal Water Pollution Control
Administration, Ada, Oklahoma (1968).
47. Horn, J.A. , DePrater, B.L., and Witherow, J.L., "Phosphate
Removal at Fort Worth, " Robert S. Kerr Water Research Center,
Federal Water Pollution Control Administration, Ada, Oklahoma
(1968).
48. Pfeffer, P.M., Scalf, M.R., DePrater, B.L. , Lively, L.D.,
Witherow, J.L. , and Priesing, C.P. , "Phosphate Removal
by Activated Sludge. Amenability Studies at Pontiac,
Michigan," Roberts. Kerr Water Research Center, Federal
Water Pollution Control Administration, Ada, Oklahoma (1968).
49. Lively, L.D., Horn, J.A. , Scalf, M.R., Pfeffer, F.M.,
Witherow, J.L. , and Priesing, C.P. , "Phosphate Removal by
Activated Sludge. Amenability Studies at Cleveland, Ohio,"
Robert S. Kerr Water Research Center, Federal Water Pollution
Control Administration, Ada, Oklahoma (1968).
50. Milbury, W.F., Stack, V.T., Jr., and Bhatla, M.N., "Effect
of Dissolved Oxygen on Phosphorus Removal in Municipal
Activated Sludge Treatment, " Presented at the 3 rd Joint
Meeting of the American Institute of Chemical Engineers and
Institute Mexicano De Ingenieros Quimicos , Denver, Colorado,
(August 30-September 2, 1970).
51. Higgins , R.B. , "Enzymatic Method for the Detection of Luxury
Phosphorus Uptake," Master's Thesis, The University of
Texas, Austin, Texas (1969).
134
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52. Standard Methods for the Examination of Water and Wastewater,
12 th edition, American Public Health Association, American
Water Works Association, Water Pollution Control Federation
(1965).
53. Sigma Technical Bulletin No. 104, "The Colorimetric Determination
of Phosphatase , " Sigma Chemical Company, St. Louis (1963).
54. Murphy. J. , and Riley, J. , "A Modified Single Solution Method
for the Determination of Phosphate in Natural Waters, "
Analytica Chimica Acta , Vol. 27, p. 31 (1962).
55. FWPCA Methods for Chemical Analyses of Water and Wastes,
Federal Water Pollution Control Administration, U.S. Department
of the Interior (1969).
56. Jenkins, D. , "A Study of the Sources of Phosphorus in the
Sewage of a Small Residential Community," SERL Report
No. 65-6, University of California , Berkeley, California (1967).
57. Jenkins, D. , "Analysis of Estuarine Waters," Journal of
Water Pollution Control Federation, Vol. 39, p. 159 (1967).
58. Subcommittee on Phosphates, Technical Advisory Committee,
Association of American Soap and Glycerine Producers, Inc. ,
"The Determination of Orthophosphate, Hydrolyzable Phosphate,
and Total Phosphate in Surface Water, " Journal of American
Water Works Association, Vol. 50, p. 1563 (1958).
59. Perkin-Elmer Analytical Methods for Atomic Absorption
Spectrophotometer, Perkin-Elmer Norwalk, Connecticut (1968).
60. Sawyer, C.N., "Bacterial Nutrition and Synthesis ," in
Biological Treatment of Sewage and Industrial Wastes, Vol. 1,
edited by J. McCabe and W.W. Eckenfelder, Jr. , Reinhold
Publishing Corporation, New York (1956).
61 . City of Trenton, Michigan, "Phosphate Removal by Biological
Process, " Final Report of Demonstration Project WPD 173, Federal
Water Pollution Control Administration (1969).
135
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62 . Public Water Supplies of the 100 Largest Cities in the United
States, 1962 , Geological Survey Water-Supply Paper 1812,
U.S. Department of the Interior, 1965.
136
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PUBLICATIONS
Fruh, E. G., Morgan, W.E., Moore, E.G., Higgins , R.B.,
Shahan, M.L. , "Enzymatic Technique to Detect Surplus Phosphorus
Uptake by Activated Sludge" Presented at the Fifth International
Waste Conference, San Francisco, 1970 (Pergarnon, in press).
137
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SECTION X
APPENDICES
Appendix
Calcium-Phosphorus Solids
Table A-l: Calcium-Phosphorus Solids
Substrates ,
Table B-l: Substrate A ..,
Table B-2: Substrate B
Table B-3: Substrate C . . ,
Table B-4: Trace Elements
Analytical Method for Phosphorus Determination
139
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Table A-l
Calcium-Phosporus Solids
Chemical Formula
Ca(H2P04)2
Ca HP04
Ca HPO4-2H2O
Ca10(P04)6(OH)2
Ca10(P04)6(F2)
Ca4H(P04)3-3H20
/-Ca3(P04)2
Common Name
monocalcium phosphate
dicalcium phosphate
dicalcium phosphate
dihydrate
hydroxyapatite
fluoroapatite
octacalcium phosphate
beta-tricalcium phosphate
Solubility Product
pKgp (298° K)
1.14
6.90
6.60
115.6
118.0
46.9
27.0
140
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Table B-l
Substrate A
Element Concentration Added as
(mg/1)
Carbon
Nitrogen
Potassium
Magnesium
Sulfur
Calcium
Iron
Bicarbonate
EDTA
330 (TOG)
30
20
18
10
6
0.4
150
50
* Multi-Carbon Source
NH4C1
KC1
MgCl2-6H2O
Na2SO4
CaCl2-2H2O
FeCl3
NaHCO3
Na9CinH1.OflN?.2H90
Trace Elements - 1 ml for each liter of substrate (see Table B-4)
* Multi-Carbon Source
Compound Concentration - mg/1
Glucose 111
Sucrose 167
Lactose 190
Sodium Citrate 21
Succinic Acid 139
Yeast Extract 62
Peptone 75
Phosphorus - added as KH2PO4
141
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Table B-2
Substrate B
Element
Carbon
Nitrogen
Potassium
Magnesium
Sulfur
Iron
Bicarbonate
EDTA
Concentration
(rng/1)
130 (TOG)
15
20
18
10
0.4
150
50
Added as
* Multi- Carbon
Source
NH4C1
KC1
MgCl2'6H2O
Na2SO4
FeCl3
NaHCOo
O
Na,ClnH1/,OoN9-2H9(
Trace Elements - 1 ml for each liter of substrate (see Table B-4)
* Multi-Carbon Source
Compound Concentration - mg/1
Glucose 44
Sucrose 65
Lactose 75
Sodium Citrate 8 . 5
Succinic Acid 55
Yeast Extract 2.5
Peptone 30
Phosphorus - added as
Calcium - added as CaCl2
142
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Element
Carbon
Nitrogen
Potassium
Sulfur
Iron
Bicarbonate
EDTA
Table B-3
Substrate C
Concentration (mg/1)
500 (COD)
50
35
15
0.4
150
50
Added as
* Multi-Carbon
Source
NH4C1
KC1
Na2SO4
FeCl3
NaHCO3
Na2CinHi40oN2-2
Trace Elements - 1 ml for each liter of substrate (see Table B-4)
*Multi-Carbon Source
Compound
Glucose
Yeast Extract
Sego (1)
Concentration - mg/1
290
72
0.5 (ml/1)
(1) Product of Pet Milk Company
Phosphorus - added as
Calcium - added as CaCl2
Magnesium - added as MgCl2
Fluoride - added as NaF
143
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Element
Boron
Zinc
Manganese
Molybdenum
Copper
Cobalt
Table B-4
Trace Elements
Concentration
(g/1)
2.00
2.00
0.40
0.47
0.40
0.10
Added as
H3B03
ZnSO4'7H2O
MnCl2-4H2O
MoO3
CuSO4-5H2O
Co(NO3)2-6H2O
144
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APPENDIX C
Analytical Method for Phosphorus Determination
The analytical procedures used for total phosphorus determination
are described below. Variations from procedures listed in FWPCA
Methods for Chemical Analysis of Water and Wastes (55) are shown
in parentheses.
Reagents
1. Sulfuric acid solution, 5N: Dilute 70 ml of concentrated H2SO4
with distilled water to 500 ml.
2. Potassium antimonyl tartrate solution: Weigh 1.3715 g.
K(SbO) C4H4O6 • 1/2 H2O, dissolve in 400 ml distilled water
in 500 ml volumetric flask, dilute to volume. Store in glass-
stoppered bottle.
3. Ammonium molybdate solution: Dissolve 20 g (NH^ MoyC^-
^H2O in 500 ml distilled water. Store in plastic bottle at
4° C.
4. Ascorbic acid, 0.1 M: Dissolve 1.76 g. of ascorbic acid in
100 ml of distilled water. The solution is stable for about a
week if stored at 4°C.
5 . Combined reagent: Mix the above reagents in the following
proportions for 100 ml of the mixed reagent: 50 ml of 5 N
H2SC>4, 5 ml of potassium antimonyl tartrate solution, 15 ml
of ammonium molybdate solution, and 30 ml of ascorbic acid
solution. Mix after addition of each reagent.
6. Strong-acid solution: Slowly add 310 ml cone. H2SO4 to 600
ml distilled water. When cool, dilute to 1 liter.
7. Potassium persulfate. (ammonium persulfate.)
Procedures
1. Add 1 ml of strong-acid solution to 50 ml of sample in a 250
ml Erlenmeyer flask.
145
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2. Add 0.8 gram of potassium persulfate. (0.4 gram ammonium
persulfate.)
3. Autoclave for 1 hour (30 minutes) at 121°C and 15-20 psi.
4. Add phenolphthalein and adjust sample to pink with 1 N
112804. Bring back to colorless with one drop strong-acid
solution, and allow to cool.
5. Transfer to a 100 ml volumetric flask, bring to volume with
distilled water.
6. Add 8 ml of combined reagent to sample and mix thoroughly.
After a minimum of ten minutes, but no longer than thirty
minutes, measure the color absorbance of each sample at 880
m/i with a spectrophotometer, using the reagent blank as the
reference solution.
146 4U.S. GOVERNMENT PRINTING OFFICE: 1972 514-149/1011-3
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SELECTED WATER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
/. Report No.
3. Accession No.
w
4. Title s. Report Date
An Investigation of Phosphorus Removal Mechanisms 6.
in Activated Sludge Systems 8. Performing Organization
7. Author(s)
W.E. Morgan, E.G. Fruh
9. Organization Texas University
Environmental Health Engineering Program
Department of Civil Engineering
12. Sponsoring Organization
Report No.
10. Project No.
17010 DUX
11. Contract/Grant No.
17010 DUX
13. Type of Report and
Period Covered
15. Supplementary Notes
Environmental Protection Agency report
number EPA-R2-72-031, November 1972.
16. Abstract The magnitude of two phosphorus removal mechanisms, metabolic uptake and
chemical precipitation with calcium, in activated sludge systems were investigated using
synthetic substrates representative of actual wastewaters. Using completely mixed con-
tinuous flow laboratory activated sludge units with operating conditions that precluded
significant precipitation of phosphorus, normal growth defined as constant 85 to 90 per-
cent carbon removal occurred above 0.9 to 1.0 percent sludge phosphorus (influent COD:P
ratio of 670:1). Between 1.0 and 1.6 percent (influent COD:P ratio of 220:1) a storage zone
existed with all phosphorus present utilized, and above 1.6 percent a variable saturation
zone occurred with an upper limit near 3.0 percent. An alkaline phosphatase bioassay
verified qualitatively the normal growth phosphorus requirement and storage zone, but did
not define the upper limit of the saturation zone. An acclimated activated sludge unit witt
a substrate containing 2 mM calcium, 0.4 mM phosphorus, 0.8 mM magnesium and 2.5
mM bicarbonate attained a maximum of 3.7 percent sludge phosphorus after 39 days of
operation at pH 7.6. A similar system with the addition of 1 mg/1 fluoride attained 4.6
percent sludge phosphorus. An increase in magnesium to 2.0 mM had little effect on phos-
phorus precipitation. Alkalinity was implicated to exert both a kinetic effect as well as
an effect on residual soluble phosphorus in calcium-phosphorus systems. The presence o
soluble organics also was shown to be inhibiting with increasing concentrations. (Morgan
17a. Descriptors Texa S )
*Wastewater Treatment, ^Phosphorus, Nutrient Requirements, *Enzyme, *Bioassay,
*Chemical Precipitation, *Calcium, Magnesium, Fluoride,Alkalinity
176. Identifiers
Phosphorus Removal, Metabolic Uptake, Luxury Uptake, Calcium-Phosphorus
Precipitation
17c. CO WRR Field & Group Q 5 D
18. Availability
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 W.E. Morgan
institution The University of Texas at Austin, Texas
WRSIC 102 (REV. JUNE 1971)
GP 0 91 3.26 !
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