EPA -660/3-73-015
September 1973
Ecological Research Series
Effect of Phosphorus
Removal Processes
On Algal Growth
Office of Research and Development
U.S. Environmental Protection Agency
Washington, D.C. 20460
-------
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
fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ECOLOGICAL
RESEARCH series. This series describes research
on the effects of pollution on humans, plant and
animal species, and materials. Problems are
assessed for their long- and short-term
influences. Investigations include formation,
transport, and pathway studies to determine the
fate of pollutants and their effects. This work
provides the technical basis for setting standards
to minimize undesirable changes in living
organisms in the aquatic, terrestrial and
atmospheric environments.
EPA REVIEW NOTICE
This report has been reviewed by the Office of Research and
Development, EPA, 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 consti-
tute endorsement or recommendation for use.
-------
EPA-660/3-73-015
September 1973
EFFECT OF PHOSPHORUS REMOVAL PROCESSES
ON ALGAL GROWTH
By
Jan Scherfig
Peter S. Dixon
Richard Appleman
Carol A. Justice
Project 16010 EJH
Program Element 1B1031
Project Officer
Thomas E. Maloney
Pacific Northwest Environmental Research Lab,
Environmental Protection Agency
Corvallis, Oregon 97330
Prepared for
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20460
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20102 - Price $1.20
-------
ABSTRACT
Laboratory studies were conducted to improve algal assay techniques
for use in evaluation of sewage treatment processes.
Laboratory studies (batch and continuous cultures) were conducted at
the Santee, California water reclamation plant to evaluate the effect
of tertiary waste treatment processes on the amount of algal growth
in the treated effluent.
Laboratory studies were also conducted to determine the growth limiting
nutrients in each type of tertiary effluent.
Field tests were conducted using special study ponds and the results
of the field tests were compared with the laboratory test results.
The laboratory and field tests showed the same relative ranking for
the treated effluents.
ii
-------
CONTENTS
Page
Abstract ii
List of Figures iv
List of Tables v
Acknowledgments vm
Sections
I Conclusions 1
II Recommendations 3
III Introduction 4
IV Batch Culture Assay Methods 5
V Continuous Culture Assay Methods 31
VI Field Test Systems 34
VII Evaluation of Treatment Processes 37
VIII References 73
IX List of Publications 74
X Glossary of Terms, Abbreviations and Symbols 75
XI Appendices 76
iii
-------
FIGURES
No.
1. Initial Experimental Batch Culture Systems 7
2. Growth of Selenastrum capricornutum in Unmodified
and Modified Medium 10
3. Effect of Iron and Manganese Addition to Unmodified
PAAP Medium 11
4. Composite Results of the Significant Factors
Affecting Algal Growth in Batch Tests. . 20
5. Effect of Plastic Flange Covers on Algal Growth
in Test Units 25
6. Typical Continuous Culture Unit 34
7. Typical Study Pond 36
8. Algae Growth Study Sampling Points 39
9. Final Cell Volume in Effluents Tested 57
10. Final Cell Number in Effluents Tested 58
11. Final Dry Weight in Effluents Tested 59
12. Dry Weight in Continuous Cultures during Steady State. 62
13. Biomass present in Study Ponds 65
iv
-------
TABLES
No.
1. Wash Solution Composition 7
2. Initial Medium Preparation Procedures 8
3. Initial Theoretical Medium Composition 9
4. Growth of Selenastrum capricornutum in modified and
unmodified PAAP medium 9
5. Effects of the addition of Iron and Manganese to
unmodified PAAP medium 12
6. Effects of CO , Light, Air, and Medium Concentration
on Algal Growth - Replicate 1 14
7. Effects of C0~, Light, Air and Medium Concentration
on Algal Growth - Replicate 2 15
4
8. Analysis of 2 Factorial Experiment Based on
Maximum Growth rate per Unit Volume per day 16
4
9. Analysis of 2 Factorial Experiment Based on
Maximum Growth rate per unit mass per day 17
10. Evaluation of the Significant Factors Affecting
Maximum Normalized Algal Growth Rates 18
11. Evaluation of the Significant Factors Affecting
Volumetric Algal Growth Rates 19
12. Evaluation of the Significant Factors Affecting
Algal Yields 19
13. Effects of CO- Enrichment on Nutrient Concentration ... 21
14. Comparison of the Effect of Autoclaving and of
Filtration on the measured growth potential of
Santee Secondary Effluent 23
15. Accuracy and Precision of Analytical Methods
at Different Concentration Levels 27
16. Effect of Soaked Materials on Algal Growth 28
17. Factorial Analysis of Combined Soaked and Non-
Soaked Material Experiment 29
-------
18. Effect of Non-Soaked Materials 30
19. Effect of Soaked Materials 30
20. Equipment for Eight Continuous Culture Systems 33
21. Reference Medium Testing of Nutrient Groups 43
22. Reference Medium Testing of Individual Nutrients 44
23. Chemical Composition of Secondary Effluents 45
24. Chemical Composition of Testing Effluents ........ 45
25. Testing of Nutrient Groups - Secondary Effluent
26 February 47
26. Testing of Nutrient Groups - Secondary Effluent
21-22 June 48
27. Testing of Individual Nutrients - Secondary
Effluent - 21-22 June 49
28. Testing of Nutrient Groups - Tertiary Effluent
26 February 50
29. Testing of Nutrient Groups - Tertiary Effluent
25 August 51
30. Testing of Individual Nutrients - Tertiary
Effluent - 25 August 52
31. Chemical Composition of Electrodialysis Effluent 53
32. Testing of Nutrient Groups - Electrodialysis
Effluent - 21-22 June 54
33. Testing of Individual Nutrients - Electrodialysis
Effluent - 21-22 June 55
34. Testing of Nutrient Groups - Ground Percolated
Effluent - 26 February. 56
35. Steady State Biomass Concentrations in Continuous
Culture at 10.5 days Residence Time . 63
36. Biomass in Study Ponds. 64
37. Secchi Disk Measurements in Study Ponds 66
38. Color Observations and Major Algal Components
Secondary Pond 67
vi
-------
39. Color Observations and Major Algal Components -
Tertiary Pond 68
40. Correlation between Biomass and Physical/Chemical
Parameters - Secondary Effluents 71
41. Correlation between Biomass and Physical/Chemical
Parameters - Tertiary Effluents .... 72
vii
-------
SECTION VIII
ACKNOWLEDGMENTS
The Santee County Water District acknowledges with thanks the
cooperation and assistance provided by many agencies, firms and
individuals throughout the long course of this project.
The support of the project by the Environmental Protection Agency,
and the help provided by Mr. Thomas Maloney, Project Officer, is
acknowledged with thanks.
The help and opportunities provided by Santee County Water District
for the field investigations are acknowledged with thanks.
viii
-------
SECTION I
CONCLUSIONS
1. This research project showed that algal assays are very useful tools
for evaluating waste treatment processes.
2. The batch algal assay can be used as a routine method at this time.
3. Continuous culture assays of very highly treated effluents such as
those resulting from electrodialysis processes are not yet practical
on a routine basis.
4. The results of the batch algal assays indicate that the nutrient which
limits growth of algae changes as a result of treatment. Thus, it is
very important in treatment plant evaluations to conduct parallel algal
assays and determine the actual limiting nutrient or nutrients after
each process step.
5. The batch algal assays performed in this study showed that phosphate
was not the only nutrient limiting growth of the test algae under
laboratory conditions. The results indicated that other nutrients
especially iron played a significant role in limiting algal growth.
6. Based on the laboratory results, it appears that the question of growth
limiting nutrients is very complex and that the concept of a simple
limiting nutrient is not valid. Instead, there appears to be several
strong positive and negative interactions among nutrients such as
nitrogen, phosphorus, iron, manganese, and some of the major cations
and anions. It appears necessary to provide further insight into
these interactions before a more rational approach to design of nutrient
removal processes can be made.
7. On a gross biomass basis, it was found that batch and continuous culture
laboratory assays provided the same ranking of effluents from different
treatment processes.
8. Using Santee secondary effluent as the basis, the continuous culture
algal investigations in the laboratory showed that the lime treatment
plus electrodialysis resulted in a 100 fold reduction in the algal
growth.
9. Comparison of electrodialysis and ground percolation indicated that at
Santee the two processes were approximately equally effective in reducing
algal growth as measured by the batch assay test.
10. Two study ponds were built and used to compare the secondary and tertiary
effluents. It was found that when the two types of effluent were exposed
to the same environment, the tertiary effluent (lime treatment) resulted
in less algal growth.
-------
11. The changes In agal growth in the study ponds due to changes in natural
conditions such as light and temperature were greater than the differ-
ence in growth of algae due to the difference in quality between secon-
dary and tertiary effluents. It is concluded that in future studies,
study ponds should preferably be operated over two full years to obtain
statistically better results.
-------
SECTION II
BECOMMENDATIONS
1, Evaluation of tertiary treatment processes should include algal assays
in addition to routine chemical effluent analyses.
2. Additional work should be done to understand better the nature of the
interactions occurring between different nutrients in treated effluents.
3. Much additional work should be conducted on the relationship between
the laboratory assay results and the growth of natural algal populations
in small study ponds. This will serve as a step towards understanding
the effect of testing effluents on large natural or artificial bodies
of water.
-------
SECTION III
INTRODUCTION
Increasing eutrophication of surface water combined with a growing need for
recreational water in arrid areas necessitates greater concern for the re-
moval of biostimulants including the classic nutrients from treated waste-
water. In the past, the major emphasis has been placed on methods for
reducing the phosphorous content in effluents by combinations of chemical
and biological processes. However, there is little scientific evidence
indicating that the efficiency of a treatment process in terms of the
removal of biostimulants, can be measured only by the degree of phosphorous
removal. Other nutrients may also be removed by the complex and not well
understood biological and chemical, phenomena occuring in phosphate removal
processes. On the other hand, organic compounds acting as growth stimulants
may be produced in significant quantities in traditional biological waste
treatment processes. Either of these phenomena can be expected to produce
a significant change in algal growth rates and yields when compared to phos-
phate removal alone.
Because of these complex and little understood phenomena there is a need for
a better method for evaluating the efficiency of both existing and proposed
phosphate removal processes with respect to the removal of other biostimulants.
Similarly there is a great need for a better understanding of the relative
efficiencies of individual steps in a sequence of treatment processes; to be
meaningful, the efficiencies must be measured in terms of actual reduced algal
growth. Such information can provide a more rational basis for the optimum
design of wastewater reclamation facilities.
The research program consisted of two overlapping parts; the development of
batch and continuous algal assay procedures as well as the field evaluation
of specific treatment processes.
The development of algal assay procedures was conducted in close cooperation
with several other groups of investigators coordinated under the National
Eutrophication Research Program, Pacific Northwest Water Laboratory, Corvallis
(U. S. Environmental Protection Agency).
The field evaluation of treatment methods for the removal of algal nutrients
was conducted in cooperation with the Santee County Water District, which was
conducting a demonstration project under partial sponsorship of the "U.S.
Environmental Protection Agency (Grant #WPRD5-01-67). The field evaluation
included comparison of algal growth in secondary effluents, tertiary effluants,
effluent from an electrodialysis process and effluent from a ground percolation
process. The laboratory evaluations were supplemented by evaluation of the
effluents in large scale study ponds located at the site of the Santee County
Water District's demonstration facility.
-------
SECTION IV
BATCH CULTURE ASSAY METHODS
Throughout the entire project sample preparation and test procedures were
under continuous development and evaluation. This resulted in a series
of changes in the detailed techniques used.
The evaluation of procedures were conducted in close coordination with other
laboratories doing related research. The evaluation of procedures was
coordinated by the Pacific Northwest Water Laboratory of the U.S. Environ-
mental Protection Agency. The results obtained in the work described here
have been incorporated in the Algal Assay Procedure Bottle Test (Reference
IV-1) and no additional recommendations are made with regard to the Bottle
Test.
The specific aspects of the test development which was conducted at UCI
included:
1. Medium Preparation Procedure
2. Effect of C02 Addition
3. Effect of Light Intensity
4. Method of Air Addition
5. Role of Medium Concentration
6. Glassware Preparation
7. Sterilization Techniques
8. Evaluation of Materials for Algal Culture Systems
INITIAL METHODS
A culture of the green alga Selenastrum capricornutum Printz was obtained
from the National Eutrophication Research Program; Pacific Northwest Water
Laboratory; Corvallis, Oregon, 97330. This species is one of three algae
selected as standard organisms for use in the Provisional Algal Assay Pro-
cedures . Cultures were grown at a temperature of 24°C+ 2°C in one liter
Pyrex Erlenmeyer flasks fitted with a neoprene rubber stopper and 1/8" I.D.
Pyrex tube in- and outlets, through which all gases were added. All glass-
ware and culture vessel components were acid cleaned and autoclaved according
to Provisional Algal Assay Procedures.
Air or a "C0~-enriched" air mixture was pumped continuously into each flask
and either bubbled through the medium ("aerated") or passed over the surface
of the medium ("ventilated") at a flow rate of 150-200 ml/min. Carbon dioxide
-------
was supplied from a 100% CO. compressed gas cylinder and released at low
pressure into a special gas mixing chamber (Matheson Model 665) by means of
a dual regulator system (Matheson Model 9 and Matheson Model 70A). Air was
provided by "Silent Giant" air pumps (Aquarium Pump Supply, Inc.) to achieve
the specified flow rate in each test flask. The concentration of (XL was
controlled by a needle valve at the C0« entrance into the gas mixing chamber.
All gases were humidified by bubbling through distilled water to stimulate
the effects of evaporation and then sterilized by passing through a Millipore
filter (0.45U) in a special Millipore filter holder (Millipore #YY30 142 00).
The concentration of carbon dioxide was varied throught the period of experi-
mentation so as to maintain the pH between 7.0 and 8.0 in all CO,, receiving
cultures. Each day pH measurements were taken and the necessary C0_ adjust-
ments made. Mixing was accomplished by a 2" -x 3/8" Teflon spin bar driven
by a magnetic stirrer at 300 + 50
Cultures were continuously illuminated by Sears 40W "Cool White" Fluorescent
lamps. Aluminum foil screens were used to ensure the selected light intensity
at the midpoint of each flask. Intensity was measured by a Weston illumination
meter (Model 703) and expressed as foot candles. The experimental arrangement
is shown in Figure 1.
Flasks were inoculated at the beginning of each experiment with a culture of
Selenastrum to a concentration of 10 cells/ml. Cells for inoculation were
separated by centrifugation from an actively growing culture (400 foot candles,
modified PAAP Medium) In order to minimize possible effects of nutrient carry-
over, the cells were rinsed in a special wash solution with a composition as
indicated in Table 1, and then resuspended for 48 hours in fresh wash solution
prior to inoculation. The wash solution containing the algal cells was then
added volumetrically (3-5 ml) so as to produce the prescribed number of cells
in each test flask.
Algal growth was measured gravimetrically by the vacuum filtration of a mea-
sured portion of algal suspension through a preweighted Millipore filter (0.45y)
and expressed as dry weight. The methods of handling, drying and weighing the
filters were essentially those outlined in the Provisional Algal Assay Pro-
cedures . Weights were measured on the fifth or sixth day after inoculation
and every other day thereafter until the seventeenth or eighteenth day. In
experiments where cultures were still actively growing on the eighteenth day,
as in the multiple concentrated medium tests, measurements were continued
every five to seven days until the experiment was terminated.
-------
INVESTIGATION OF MEDIUM PREPARATION PROCEDURE
The culture medium as outlined in the Provisional Algal Assay Procedures was
found initially to give relatively low algal growth rates and yields. It
appeared that trace metals were removed during filter sterilization, and that
the amounts removed varied from time to time. The method of preparation was
therefore modified (Table 2) and the effects of this modification determined.
Six replicates were prepared individually using both the procedure outlined in
the Provisional Algal Assay Procedures and the modified procedure. 800 m& of
each medium (within the theoretical composition shown in Table 3) was added
to each of six one liter flasks and the relative algal growth rates and yields
determined. Cultures were inoculated as previously described, grown at 400
foot candles and "aerated" with CCL-enriched" air mixture (0-2%) bubbled
through the culture in sufficient quantity to maintain the pH of each algal
culture between 7.0 and 8.0 throughout the period of experimentation. The
resulting growth of Selenastrum in the unmodified and the modified medium is
shown in Table 4 a and Figure 2.
FLOURESCENT LAMPS
CO
C02-AIR FILTER STERILIZATION MAGNETIC MIXERS
kJI VIM/2
MIXING
CHAMBER
FLASK WITH DISTILLED WATER
^TEFLON
SPIN BAR
FIGURE 1. INITIAL EXPERIMENTAL BATCH CULTURE SYSTEM
MgCl2.
MgSO.
CaCl,
41.20 mg/Jl
23.81 mg/£
11.32 mg/Jl
TABLE 1. WASH SOLUTION COMPOSITION
-------
TABLE 2. INITIAL MEDIUM PREPARATION PROCEDURES
Unmodified PAAP Procedure
Modified PAAP Procedure
Prepare 1 liter stock solutions
of individual salts at 1000 times
concentration.
2. Prepare a 1 liter combined trace
metal and Na2EDTA.2H20 stock
solutions at 1000 times concen-
tration.
1. FeCl-.BH-O and Na^CO are not
prepared as stock solutions but
instead are weighed and added
directly at each preparation.
2. H BO , MnCl ,4H20, ZnCl , CoCl
6H20; CuCl , NaMo04.2H 6 and
Na2EDTA. /H20 are each prepared
as individual 1 liter stock
stock solutions at 1000 times
concentration.
3. Add 1 ml of each stock solutions
to enough glass distilled water
to make 1 liter of culture medium,
adding K-HPO, last.
3. Add 1 ml per £ of each stock
solution to make glass distilled
water to make 1 £ in the follow-
ing order, adjusting the pH where
indicated:
NaNO_, MgCl0.6H_0, MgSO.,
J 2. 2. 4
Add 50 mg/£ of Na?CO
b J
Adjust pH to 7.5 with HC1
Add 1 ml per X, of B
stock
4. Filter sterilize at 0.5 atm.
through 0.45y Millipore filter.
Add 1 ml per £ of each of
the trace metal stock solutions
and the Na EDTA stock solution.
4. Filter sterilize under vacuum
through 0.45y Millipore filter.
5. Add 0.53 mg/£ of FeCl3.6H 0
directly from reagent bottle
immediately prior to use.
Medium pH: 10.1
Medium pH: 7.4-7.6
-------
TABLE 3. INITIAL THEORETICAL MEDIUM COMPOSITION
(Reference IV-2)
Major Salts
NaN03
K2HP03.3H20
MgCl2.6H20
MgS04
CaCl2
Na2C03
FeCl-.6H 0
85.00 mg/£
4.55 mg/£
41.20 mg/£
23.81 mg/£
11.32 mg/£
50.00 mg/£
0.53 mg/S,
H3B03
MnCl2.4H20
ZnCl2
CoCl2-6H20
CuCl2
Na_MoO. .2H_0
242
Na EDTA.1H 0
Trace Metal Solutions
618.40yg/2
1384.60yg/£
109.03yg/&
4.76yg/£
0.03yg/A
24.20yg/£
7.07 mg/£
Nutrients per 1 liter glass distilled water.
TABLE 4. GROWTH OF SELENASTRUM CAPRICORNUTUM IN
MODIFIED AND UNMODIFIED PAAP MEDIUM
Day
5
9
13
17
Unmodified Medium
Mean St. Dev.
.008 ± .004
.061 ± .028
.084 ± .071
Modified Medium
c *
V
.474
.458
.845
Mean
.140 ±
.414 ±
.486 ±
.475 ±
St. Dev.
.018
.036
.034
.032
C
V
.129
.087
.071
.067
Coefficient of Variation (C ) is defined as Standard Deviation
divided by mean.
9
-------
-
LU
.50
t .40
o
LJ
cr
t
.30
cr
Q
co 20
MODIRED
MEDIUM
UNMODIFIED
MEDIUM
20
FIGURE 2. GROWTH OF SELENASTRUM CAPRICORNUTUM IN UNMODIFIED AND
MODIFIED MEDIUM
Preliminary chemical analyses of the unmodified and the modified media
indicated that iron and manganese had been rranoved from the unmodified
PAAP media during filter sterilization. Consequently, an experiment was
10
-------
0
8 12 16' 20
Fe , Mn additions
DAYS
FIGURE 3. EFFECT OF IRON AND MANGANESE ADDITION TO
UNMODIFIED PAAP MEDIUM
11
-------
conducted in which 0.53 mg/& FeCl^.SHLO and 1.38 mg/A MnCl2.4H_0 were added
to the unmodified medium cultures on the seventeenth day or growth. The
results of this supplementation are presented in Table 4 and Figure 3.
They demonstrate a marked increase in the growth of Selenastrum Immediately
following the addition of iron and manganese as would be expected if iron
and manganese were being removed in the medium preparation procedure.
TABLE 5. EFFECTS OF IRON AND MANGANESE ADDITION TO
UNMODIFIED PAAP MEDIUM
Nutrients were added to growing cultures on the 17th
day. Each value is the mean for two cultures.
Fe added Mn added Fe, Mn added
Day 17th day 17th day 17th day
5 .007 .007 .011
13 .052 .045 .086
17 .048 .037 .167
21 .324 .117 .465
Growth of Selenastrum capricornutum in dry weight
EFFECT OF C02> LIGHT, AIR AND MEDIUM CONCENTRATION
Preliminary experimental work had indicated that certain other factors were
limiting both the rate of algal growth and total algal yield. The experi-
ments presented above showed that the modified method of medium preparation
minimized the effects of uncontrolled trace metal removal. Consequently,
this method of medium preparation was used in the remainder of the experi-
mental work reported here.
A2x2x2x2 (2) factorial experiment was designed to test the effects
of light intensity (350 foot candles vs. 500 foot candles), gas composition
("C02-enriched" air vs. non"C02-enriched" air), method of gas addition
("aeration" vs "ventilation") and medium concentration (100% vs 600%). The
six hundred per dent concentrated medium was included in the tests to ensure
that the range of algal concentrations encountered in nutrient rich samples
such as secondary effluents would be covered. Two replicate experiments were
run and the results are presented in Tables 6 and 7. The experiments were
analyzed statistically on the basis of both the maximum volumetric growth
rates (grams per liter per day) and the maximum normalized growth rates
(grams per gram of algae present per day). The statistical analyses were
performed with the use of the General Electric Mark II Computer Library and
12
-------
the results are presented in Tables 8 and 9 respectively. All of the
factors tested are shown to exert a statistically significant effect
using one of the above indices of algal growth.
Maximum normalized growth rates were obtained during the earlier stages
of exponential growth in all test cultures. Analyses using this index
showed that the selected light intensities, methods if air addition, and
"CO^-enrichment" affected Selenastrum growth (Table 10). The results show
that light at 500 foot candles decreases maximum normalized growth rates
while aeration and CO^-enrichment increases these values. Medium concen-
tration was shown to be insignificant in affecting maximum normalized growth.
This is explained by the high nutrient to cell ratio present in all cultures
during the early stages of growth. Analyses also indicated the presence
of an interaction between C0~ and light intensity using maximum normalized
growth rates.
Medium concentration and "C0?-enrichment" were found to be significant
factors affecting Selenastrum growth when maximum volumetric growth rates
were used for analyses (Table 11). Maximum volumetric growth was found
to occur during the middle to late stages of exponential growth prior to
the stationary growth phase. During this period nutrient availability
in the 100% concentrated medium cultures and C0~ in the non-CO~ enriched
cultures were shown to be limiting Selenastrum growth (Table II). An
interaction between CO^ and medium concentration was also found.
Algal yields were compared for all cultures using values obtained on the
15th and 17th-18th days of growth. Only the 100% concentrated medium
cultures were used for analysis on these days as total yield figures were
not obtained for the 600% concentrated medium cultures. As demonstrated
by the previous analyses of growth rates, "C02-enrichment" was effective
in accelerating Selenastrum growth on the days used for analysis, "CO-
enriched" cultures showed greater algal yields (Table 12). Figure 4~is a
composite representation of the significant factors affecting the growth
of Selenastrum per unit volume in the factorial experiments.
These results suggest that nutrient availability is markedly influenced
by medium pH. To test this hypothesis, medium samples from the 600%
concentrated medium cultures of Selenastrum were separated from the algal
cells by centrifugation at the end of the growth. Chemical analyses of
the medium showed a significantly larger concentration of iron, calcium
and manganese in the "C0_-enriched" samples compared with the non"C02-
enriched" samples, even though more algal growth had occurred in the
former (Table 13). The analyses also showed greater concentrations of
sodium, boron and silicon to be presented in the non"CO^-enriched" samples
(Table 13).
These findings both appear to be correlated with the effects of CO- on
medium pH. The pH values for the"C02-enriched" cultures ranged from 7.5 -
8.0 at the time of analysis while the values for the non"C02-enriched"
cultures ranged from 9.5 - 11.0.
13
-------
TABLE 6. EFFECTS OF C02> LIGHT, AIR AND
MEDIUM CONCENTRATION ON ALGAL GROWTH - REPLICATE 1
2 Factorial Experiment
CO. Enriched Air
ik>n-C02 Enriched Air
350 ft-c
500 ft-c
350 ft-c
600%
Medium
DAY
6
£ 8
10
12
13
15
18
Vent.
.056
.263
.568
.957
1.119
1.530
2.220
Aer.
.057
.278
.607
.984
1.178
1.545
2.311
100%
Medium
Vent.
.023
.136
.304
.409
.493
.530
.584
Aer.
.067
.258
.403
.483
.497
.513
.561
600%
Medium
Vent.
.113
.432
.812
1.232
1.514
1.976
2.616
Aer.
.137
.514
.881
1.453
1.711
2.048
2.459
100%
Medium
Vent.
.103
.318
.415
,514
.497
.525
.590
Aer.
.063
.295
.430
.489
.545
.532
.626
600%
Medium
Vent.
.043
.127
.219
.406
.490
.657
.967
Aer.
.043
.185
.321
.510
.556
.689
.924
100%
Medium
Vent.
.025
.101
.237
.262
.299
.358
.418
Aer.
.025
.159
.320
.418
.452
.503
.531
500 ft-c
600% 100%
Medium Medium
Vent. Aer. Vent. Aer.
.082 .106 .028
.129 .234 .075
.249
.438
.531
.544 .103
.698 .114
.798 .128
.592 1.041 .121
.767 1.426 .124
.070
.180
.296
.408
.461
.459
.494
Growth in dry weight (g/&) of Selenastrum capricornutum
-------
TABLE 7. EFFECTS OF CCL, LIGHT, AIR AND
MEDIUM CONCENTRATION ON ALGAL GROWTH - REPLICATE 2
2 Factorial Experiment
C00 Enriched Air
Non-CO™ Enriched Air
350 ft-c
600% 100%
Medium Medium
DAY Vent. Aer. Vent. Aer.
500 ft-c
600% 100%
Medium Medium
Vent. Aer. Vent. Aer.
350 ft--c
600% 100%
Medium Medium
Vent. Aer. Vent. -Aer.
500 ft-c
600% 100%
Medium Medium
Vent. Aer. Vent. Aer.
tn 5 .043 .040
7 .169 .165
9 .388 .399
11 .576 .584
13 .955 .911
15 1.220 1.150
17 1.560 1.434
21 2.032 2.003
26 2.460 2.241
33 2.728 2.492
040
132
276
363
451
508
532
-
-
_
.036
.182
.330
.436
.477
.510
.541
-
-
—
1
1
1
2
2
2
.084
.283
.554
.913
.240
.570
.814
.079
.404
.669
1
1
1
2
2
2
2
.097
.399
.737
.096
.454
.856
,123
.516
.667
.679
.069
.245
.350
.335
.441
.473
.481
-
-
_
.074
.308
.391
.496
.492
.517
.539
_
-
-
.032
.093
.117
.185
.223
.362
.536
.725
.960
1.440
.035
.135
.197
.335
.463
.545
.684
.875
1.115
1.488
.029
.055
.085
.124
.178
.260
.298
-
-
—
.034 .069 .073 .035 .052
.110 .094 .157 .058 .130
.165 .123 .252 .089 .190
.238 .182 .324 .138 .265
.356 .233 .503 .210 .396
.396 .308 .610 .257 .445
.455 .491 .756 .338 .499
.644 .968
.923 1.320
1.547 1.803
Growth in dry weight (g/&) of Selenastrum capricornutum
-------
TABLE 8. ANALYSIS OF 2 FACTORIAL EXPERIMENT
BASED UPON MAXIMUM GROWTH RATE PER UNIT VOLUME PER DAY
(g/A-day)
C02 Enriched Air
Non-C02 Enriched Air
350 ft-c
600% 100%
Medium Medium
500 ft-c
600% 100%
Medium Medium
350 ft-c
600% 100%
Medium Medium
500 ft-c
600% 100%
Medium Medium
Rep. Vent. Aer. Vert. Aer. Vert. Aer. Vert. Aer. Vert. Aer. Vert. Aer. Vert. Aer. Vert. Aer.
1 .230 .255 .084 .096 .282 .286 .108 .116 .103 .095 .068 .081 .095 .156 .024 .058
2 .190 .164 .072 .074 .188 ' .201 .088 .117 .087 .070 .041 .059 .092 .090 .041 .066
ANALYSIS OF VARIANCE
Main effects F Ratio
CO™ enrichment of air 89.72**
Light Intensity 2.92
Medium Concentration 98.88**
Method of air addition 1.86
Replicates 12.62**
Levels of significance
F(5%) =4.54 *Significant at 5% level
F(l%) - 8.68 **Significant at 1% level
Interactions
C02 - Light Int.
OT - Medium
C02 - Air add.
Light Int. - Medium
Light Int. - Air add.
Medium - Air add.
CO - Light-Medium
C02 - Light-Air add.
C02 - Medium-Air add.
Light-Medium-Air add.
CO™ - Light-Medium-Air add.
F Ratio
2.11
24.40**
0.17
,20
,20
0.42
0.77
0.26
0.02
0.13
0.19
1,
1.
-------
TABLE 9. ANALYSIS OF 2 FACTORIAL EXPERIMENT
BASED ON MAXIMUM GROWTH RATE PER UNIT MASS PER DAY
(g/g-day)
CO- Enriched Air
Non-C02 Enriched Air
350 ft-c
600% 100%
Medium Medium
500 ft-c
600% 100%
Medium Medium
350 ft-c
600% 100%
Medium Medium
500 ft-c
600% 100%
Medium Medium
Rep Vert. Aer. Vert. Aer. Vert. Aer. Vert. Aer. Vert. Aer. Vert. Aer. Vert. Aer. Vert. Aer.
1
2
.647
.595
.658
.613
.707
.535
.586
.670
.586
.544
.578
.609
.510
.561
.648
.613
.494
.484
.623 .603 .728 .318 .399 .452 .440
.588 .310 .528 .229 .365 .245 .429
ANALYSIS OF VARIANCE
Main effects F Ratio
C02 enrichment of air 35.10**
Light Intensity 20.28**
Medium Concentration 0.33
Method of air addition 9.40**
Replicates 6.70*
Levels of significance
F(5%) = 4.54 *Significant at 5% level
F(l%) = 8.68 ''^Significant at 1% level
Interactions F Ratio
CO - Light Int. 7.48*
CO^ - Medium 0.33
C02 - Air add. 2.72
Light Int. - Medium 0.56
Light Int. - Air add. 0.00
Medium - Air add. 0.20
CO - Light-Medium 0.36
CO^ - Light-Air add. 0.91
CO^ - Medium-Air add. 0.02
Light-Medium-Air add. 0.00
CO - Light-Medium-Air add. 0.55
-------
TABLE 10. EVALUATION OF THE SIGNIFICANT FACTORS AFFECTING
MAXIMUM NORMALIZED ALGAL GROWTH RATES
4
2 Factorial Experiment
(g/g-day)
Light Intensity
CO-Enriched Air
Non CO-Enriched
Total Effects
(Avg. of C0_ and
NonCO - AirJ
350 ft-c
500 ft-c
.626
.581
.544
.360
.585
.471
Total Effects
(Avg. of 350+500 ft-c)
.604
.452
Air Addition
C02Enriched Air
Non C02Enriched
Total Effects
(Avg. of CO- and
NonC02 - Air)
Ventilation
Aeration
.586
,622
.392
.513
.489
.568
Total Effects
(Avg. of vent.
and aer.)
.604
.452
Each value is the average of eight measurements (two replicates, four flasks
per replicate) except for the total effects which are averages or sixteen
measurements.
18
-------
TABLE 11. EVALUATION OF THE SIGNIFICANT FACTORS AFFECTING
VOLUMETRIC ALGAL GROWTH RATES
4
2 Factorial Experiment
(g/Jl-day)
Total Effects
(Avg. of C00 and
Medium CO-Enriched Air Non-C02 Enriched Nori-C02 Enriched
Concentration Air Air)
100% .094 .054 .074
600% .225 .099 .162
Total Effects .160 .077
(Avg, of 100% +
600% medium)
Each value is the average of eight measurements (two replicates, four
flasks per replicate) except for the total effects which are the average
of sixteen measurements.
TABLE 12. EVALUATION OF THE SIGNIFICANT FACTORS AFFECTING ALGAL
YIELDS
4
2 Factorial Experiment
CgA)
Days CO- Enriched Air Non-CO- Enriched Air
15 .514 .350
Each value is the average of eight measurements (two replicates, four
flasks per replicate). Values for cultures grown in 100% medium concen-
trations only.
19
-------
CO
o:
UJ
CD
UJ
£
g
CO
C02, 6X
16 20 24 28 32
FIGURE 4. COMPOSITE RESULTS OF THE SIGNIFICANT FACTORS AFFECTING
ALGAL GROWTH IN BATCH TESTS
Values expressed as dry weight (g/A). Data from replicate two used
for figure.
20
-------
TABLE 13. EFFECTS OF C0« ENRICHMENT ON
NUTRIENT CONCENTRATION
Element CO enriched Non-CO-enriched
Iron 0.34 0.05
Manganese 3.35 2.58
Calcium 45.75 19.25
Silicon 1.68 8.85
Boron 0.55 1.13
Sodium 168.00 187.50
Analyses were made on 600% concentrated medium cultures
of Selenastrum on the 33rd day after inoculation. Each
value is the mean for four cultures. All values in ppm.
GLASSWARE PREPARATION
Glassware and all other culture vessel components were acid washed during the
initial research period. In later experiments it was found that better repli-
cation was obtained if the wash water solution was modified to include 1-2% of
hydrofluoric acid. This modification was included in all tests of the treat-
ment plant effluents reported in this report.
STERILIZATION TECHNIQUES
All media for batch bioassays, either natural water, treated effluents or lab-
oratory synthesized media must be pretreated in some way to remove naturally
occuring organisms and ensure sterility. Several methods for accomplishing
this were considered and evaluated by laboratory experimentation. These
included autoclaving, ultra-violet irradation, exposure to ethylene oxide,
tyndallization and filtration.
Autoclaving
Autoclave sterilization at 121°C and 15 pounds pressure for 15-20 minutes
appears to be the method most suitable for most natural waters. Water samples
sterilized by autoclaving should be placed in a pre-heated autoclave and removed
as soon as the pressure is down in order to minimize heat damage to organic
materials which may be present.
21
-------
Ultraviolet Sterilization
The ultraviolet source used was enclosed in a wOoden box and a decastaltic pump
was used to vary the flow rate of an algal suspension (3 x 10^ cell/ml) past
the ultraviolet coils. A length of two meters of coiled tubing was in close
proximity to the ultraviolet source enclosed in the box. The lowest flow rate
was 8.25 ml per hr. The results indicated that this method was not suitable
for killing algae in a suspension of this density and further investigation
of this method was terminated.
Ethylene Oxide
Autoclaving is difficult to apply in certain cases. Complex apparatus can be
damaged by temperature changes such as occur during autoclaving or so large
as to be unsuitable for sterilization by this procedure. Other methods have
therefore to be applied for the elimination of contaminant organisms from
apparatus prior to use. Ethylene Oxide has been used extensively for this
purpose in medical bacteriology. Being gaseous permits adequate penetration
of complex apparatus to be achieved. There are many problems associated
with the use of Ethylene Oxide. The sterilization potential is reduced by
the presence of water vapor in the apparatus and by polymerization. Unless
removed the resulting powders and oily fluids are highly toxic to organisms
introduced subsequently. Despit such drawbacks, the use of Ethylene Oxide
as a means of sterilization is increasing steadily. Another serious problem
is a consequence of its high toxicity to humans and the need for great care
in handling. Even in a modern chemical laboratory, the fume hoods available
were unsuitable for work with material of such toxicity and further consider-
ation on this material for the sterilization of algal culture glassware had
to abandoned.
Tyndallization
The problems of autoclaving are largely a consequence of the temperatures which
have to be used. One way of partially circumventing these difficulties is to
use the procedure referred to as "Tyndallization." The medium or apparatus is
placed in water which is brought slowly to the boil and retained in a boiling
condition for a period of 30 minutes. This slow heating to 100°C and retention
at that temperature for 30 minutes is repeated for three successive days.
Since this procedure is more time consuming and in some cases, maximum algal
growth was not attained, it was not considered the best method for sterilizing
natural waters.
Filtration
Filter sterilization would appear to be the ideal method for the sterilization
of laboratory media in which all nutrients are in solution. Natural waters often
contain large amounts of particulate matter which would be removed by the fil-
tration process. Removal of particulate matter might be considered desirable
or even necessary in laboratories utilizing electronic particle counters to
monitor growth. Two types of filters were used in preliminary tests to deter-
mine to what extent the removal of the particulate material affected the algal
growth potential of the water. The water used in this test was a secondary
effluent from the Santee Treatment Plant. The two types of filters evaluated
were Whatman #1 and Millipore 0.45ym. Four replicates were made of effluent
filtered through Whatman #1, Millipore 0.45ym and growth compared with four
22
-------
replicates of nonfiltered effluent which were autoclave sterilized. Culture
vessels were 500 ml Erlenmeyer flasks filled to 250 ml with effluent. Flasks
which were to contain the filtered effluent were sterilized and the effluent
aseptically transferred from the filtration apparatus to these flasks.
Initial cell concentration of Selenastrum capricornutum was 1000 cell/ml
and Air - C0_ mixture used to contrpl pH. The results of this experiment
are shown in Table 14.
EVALUATION OF MATERIALS FOR ALGAL CULTURE SYSTEMS
Many different materials have been proposed and/or used to construct systems
for both batch and continuous algal cultures. During the early part of this
research it became clear that some materials could inhibit algal growth in
these systems and thus give biased results in evaluation of effluents. An
example of the effect of one such material is shown in Figure 5. Based on
these limited observations it was decided that all types of material intended
for the test units should be evaluated prior to use.
4
A modified 2 factorial experiment was designed to test the effects of rubber
stoppers (Neoprene vs. Cafe-au-lait (Rhoades Rubber Mfg. Co.)).
TABLE 14. COMPARISON OF THE EFFECT OF AUTOCLAVING AND OF FILTRATION
ON THE MEASURED GROWTH POTENTIAL OF SANTEE SECONDARY EFFLUENT
Growth
Day
NUMBER OF CELLS PER MJl
Autoclaved
Non-Filtered
Filtered
Millipore (.45y)
Filtered
Whatman #1
17
23
3.43 x 10
2.05 x 10'
6.81 x 10-
7.48 x 10-
9.55 x 10-
7.79 x 10-
Growth
Day
TOTAL CELL VOLUME ym per
Autoclaved
Non-Filtered
Filtered
Millipore (.45u)
Filtered
Whatman #
17
23
1.94 x 10
8
1.03 x 10
8
4.00 x 10
4.03 x
5.27 x
4.10 x 10
Each datum an average of 4 flasks.
23
-------
T-tests on this data indicate no significant difference between the two
types of filtration but a highly significant (0.1% level) difference
between filtered and nonfiltered effluent. The results indicate that
filtration removed significant quantities of nutrients, and it was
therefore decided not to use filtration as a means of sterilization when
a maximum biomass measurement is desired.
24
-------
3.0
t
2.0
UJ
O
CD
O
~ 1.0
NO FLANGE
COVERS
FLANGE COVERS
J I I I 1 L
2468 10 12
DAYS
FIGURE 5. THE EFFECTS OF PLASTIC FLANGE COVERS ON ALGAL GROWTH IN
TEST UNITS
25
-------
TABLE 14. EFFECT OF NONSOAKED MATERIALS ON ALGAL GROWTH
PYREX
NJ
Day
5
8
10
12
15
19
NEOPRENE STOPPERS
CAFE-AU-LAIT STOPPERS
Surgical
Tubing
Regular
Tubing
Surgical
Tubing
Regular
Tubing
Rep. Rep. Rep. Rep. Rep. Rep. Rep. Rep,
12121212
KIMAX
NEOPRENE STOPPERS CAFE-AU-LAIT STOPPERS
Surgical
Tubing
.052 .053 .042 .042 .026 .037 .021 .029 .063 .051
.222 .228 .240 .240 .027 .026 .023 .014 .228 .262
.226 .215 .294 .294 .005 .023 .002 .010 .228 .270
.333 .290 .366 .366 .042 .053 .040 .055 .281 .399
.416 .376 .461 .461 .088 .080 .168 .178 .391 .410
.401 .377 .482 .482 .050 .045 .200 .056 .405 .449
Regular
Tubing
Surgical
Tubing
Regular
Tubing
Rep. Rep. Rep. Rep. Rep. Rep. Rep. Rep.
12 121212
.056 .045 .027 .022 .026 .021
.240 .216 .040 .026 .081 .068
.335 .264 .025 .042 .155 .181
.373 .369 .090 .169 .294 .306
.480 .418 .269 .185 .427 .362
.510 .483 .257 .259 .414 .482
Growth in dry weight (g/£) of Selenastrum capricornutum
-------
TABLE 15
ACCURACY AND PRECISION OF ANALYTICAL METHODS
AT DIFFERENT CONCENTRATION LEVELS
COEFFICIENT OF VARIATION (%)
COMPOUND MEAN AT EACH CONC. LEVEL a)
Fe I 1 00
(yg/A) 5 20
31 II
Mn 2 30
(yg/A) 49 31
135 3
N0? .039 31
i i«\ -260 3
(mg/A) |>|4 2
NO, .030 80
P .112 21
(mg/£) .200 22
3.52 9
9.76 2
a) coefficient of variation = 100 (standard deviation)/(mean)
27
-------
TABLE 16. EFFECT OF SOAKED MATERIALS ON ALGAL GROWTH
NJ
00,
FYREX
NEOPRENE STOPPERS CAFE-AU-LAIT STOPPERS
KIMAX
NEOPRENE STOPPERS CAFE-AU-LAIT STOPPERS
Surgical
Tubing
Day
5
8
10
17
23
Rep.
1
.093
.253
.341
.572
.501
Rep.
2
. 141
.287
.357
.577
.501
Regular
Tubing
Rep.
1
.101
.246
.370
.607
.559
Rep.
2
.107
.290
.421
.605
.537
Surgical
Tubing
Rep.
1
.103
.212
.309
.534
.525
Rep.
2
.103
.212
.309
.534
.525
Regular
Tubing
Rep.
1
.096
.251
.369
.576
.517
Rep.
2
.096
.187
.244
.401
.369
Surgical
Tubing
Rep.
1
.123
.300
.379
.559
.517
Rep.
2
.129
.215
.343
.571
.417
Regular
Tubing
Rep.
1
.128
.314
.386
.598
.540
Rep.
2
.119
.242
.363
.562
.536
Surgical
Tubing
Rep.
I
.109
.282
.361
.540
.3*8
Rep.
2
.130
.261
.327
.524
.440
Regular
Tubing
Rep.
1
.114
.234
.331
.559
.449
Rep.
2
.111
.249
.374
.515
.385
Growth in dry weight (g/£) of Selenastrum capricornutum
-------
TABLE 17. FACTORIAL ANALYSIS OF COMBINED SOAKED
AND NONSOAKED MATERIAL EXPERIMENTS
(g/*)
NONSOAKED MATERIALS
PYREX GLASSWARE KIMAX GLASSWARE
SOAKED MATERIALS
PYREX GLASSWARE KIMAX GLASSWARE
NEOPRENE
STOPPERS
Replicates
Rep. 1
Rep. 2
Surg.
Tubing
.226
.215
Reg.
Tubing
.294
.294
CAFE-AU-LAIT
STOPPERS
Surg. Reg.
Tubing Tubing
.005 .002
.023 .010
NEOPRENE
STOPPERS
Surg. Reg.
Tubing Tubing
.228 .335
.270 .264
CAFE-AU-LAIT
STOPPERS
Surg.
Tubing
.025
.042
Reg.
Tubing
.155
.181
NEOPRENE CAFE-AU-LAIT
STOPPERS STOPPERS
Surg,
Tubing
.341
.357
Reg. Surg.
Tubing Tubing
.370 .309
.421 .309
Reg,
Tubing
.369
.299
NEOPRENE
STOPPERS
Surg. Reg.
Tubing Tubing
.379 .386
.343 .363
CAFE-AU-LAIT
STOPPERS
Surg. Reg.
Tubing Tubing
.361 .331
.327 .374
to
VD
Main Effects
Soaking Treatment
Glassware
Rubber Stoppers
Plastic Tubing
Replicates
Levels of Significance
F Ratio
424.45**
12.17**
173.59**
21.34**
0 02
F(5%) = 4.54* Significant at 5% level
F(l%) - 8.68** Significant at 1% level
ANALYSIS OF VARIANCE
Interactions
F Ratio
Soaking - Glassware
Soaking - Stoppers
Soaking - Tubing
Glassware - Stoppers
Glassware - Tubing
Stoppers - Tubing
Soaking-Glassware-Stoppers
Soaking-Glassware-Tubing
Soaking-Stoppers-Tubing
Glassware-Stoppers-Tubing
Soaking-Glassware-Stoppers-Tubing
4,
7.
5.29*
88.33**
.42
.86*
0.88
0.10
.28
.10*
.17
.94*
.95
1.
5,
0.
5.
3.
-------
TABLE 18. EFFECT OF NONSOAKED MATERIALS BASED ON
ALGAL YIELDS (g/£) ON THE TENTH DAY
Data Analyzed as a 2 Factorial Experiment
ANALYSIS OF VARIANCE
MAIN EFFECTS F RATIO INTERACTIONS F RATIO
Glassware 65.30** Glassware-stoppers 30.21**
Rubber Stopper 993.17** Glassware-tubing 19.88**
Plastic Tubing 88.07** Rubber stoppers-tubing 0.02
Replicates 0.15 Glassware-stoppers-tubing 38.21**
Levels of Significance
F(5%) = 5.59* Significant at 5% level
F(l%) - 12.25** Significant at 1% level
TABLE 19. EFFECT OF SOAKED MATERIALS BASED ON
ALGAL YIELDS (g/A) ON THE TENTH DAY
3
Data Analyzed as a 2 Factorial Experiment
ANALYSIS OF VARIANCE
MAIN EFFECTS F RATIO INTERACTIONS F RATIO
Glassware 1.81 Glassware-stoppers 3.58
Rubber Stoppers 18.31** Glassware-tubing 2.24
Plastic Tubing 8.12* Rubber stoppers-tubing 0.69
Replicates 0.32 Glassware-stoppers-tubing 0.26
Levels of Significance
F(5%) = 5.59* Significant at 5% level
F(l%) - 12.25** Significant at 1% level
30
-------
plastic tubing (Tygon Surgical Grade (S~50-HL) vs Tygon Regular Grade (R-3603)),
glass (Kimax vs Pyrex) and soaking treatment prior to use (pre-soaked material
vs nonsoaked materials). Substances were added as thin section to either
Kimax or Pyrex 1£ Erlenmeyer flasks. The results of these experiments are
presented in Tables 15 and 16. The experiments were analyzed statistically on
the basis of algal yields on the tenth day after inoculation. These analyses
were run in two ways:
4
1. combined as a 2 factorial experiment
3
2. separately as two 2 factorial experiments.
The results of these analyses are presented in Table 17 and 18, 19 respectively.
All of the nonsoaked materials were shown to exert significant effects on algal
growth (Table 18). These effects were inhibitory with the effects of Pyrex
> Kimax, Surgical tubing > Regular tubing, Cafe-au-lait > Neoprene. The soaking
pretreatment significantly reduced the overall inhibitory effects (Table 17)
and eliminated the difference between Pyrex and Kimax, but did not eliminate
the differences between the two types of stoppers or the two types of tubing
(Table 19). Interactions between the nonsoaked materials were shown to be
present (Tables 17, 18) but these effects were eliminated by the soaking
pretreatment (Table 19). Based on these results it was decided to use only
Pyrex glassware and surgical grade Tygon tubing in the algal assay units.
FINAL BATCH TEST PROCEDURE
Following the completion of the testing and procedure development described
above, the final batch procedure described in Reference IV-1 was adopted and
used in the testing of all treatment plant effluents.
As a part of this research, the UCI Laboratory also participated in compre-
hensive inter-laboratory evaluations of the procedure under the guidance
of the Pacific Northwest Water Laboratory, U.S. Environmental Protection
Agency,
31
-------
SECTION V
CONTINUOUS CULTUKE ASSAY METHODS
CHEMQSTAT DESIGN
The continuous culture units proposed in the Provisional Algal Assay Procedures
(Reference 2) were modified on the basis of the results obtained as described
in Section IV. The most important modifications included the use of C0?-air
for pH control and the redesign of the units such that the only three materials
in contact with the cultures were Pyrex glass, Surgical grade Tygon tubing and
Teflon. Soaked Neoprene stoppers were used to top the culture units but were
not in direct contact with the growing cultures.
The equipment needed for eight continuous culture tubes are listed in Table 20
and a typical set of four assembled units are shown on Figure 7.
CHEMOSTAT OPERATION
Before each new experiment was started, chemostats including feed and air tubes
were autoclaved at 121°C for 15 min. The chemostats were then filled with
sterilized media and inoculated to 10-* cells of jtelenastrum per ml. Flow was
initiated after significant algal growth was observed.
Because the secondary and tertiary effluents sometimes contained considerable
particulate matter, the feed pumps were set up to operate for a short time
each hour at a high flow rate to prevent clogging of feed tubes. The pumps
were calibrated to operate at 50 ml per hour per feed tube and the time was
set to have the pump on 1/12, 1/8 or 1/4 of an hour each hour. This resulted
in residence times of approximately 10, 6 and 3 days respectively. Throughout
the experiments no problems were found with this method.
Three parameters of biomass were observed during each experiment: dry weight,
cell volume and cell number. Samples needed for determination of cell volume
and number were taken directly from the chemostats through sampling ports at
the base of each unit. Effluents in the overflow flasks were used for dry
weight measurements in order to minimize disturbance of the systems.
A continuous record of the residence times was kept during the experiment.
This was done by weighing the feed flasks and overflow flasks everytime either
a dry weight sample was taken, or more feed medium was added. Residence
times at steady state were computed from the flow during steady state and the
time of steady state. Steady state was defined to have been reached when
chemostats maintained a constant cell mass for periods of time greater than
one residence time with only random fluctuations. Near the end of the steady
state period samples from the overflow flasks were collected for chemical
analysis.
From preliminary tests with chemostats it was found there was a problem of
algae sticking to the culture vessel, especially at the air/water interface.
A strong magnet was used to guide the magnetic spin bar inside the tubing
to scrap the inside walls. With this procedure it was never necessary to
remove the plug at the top of the chemostat.
32
-------
After some length of operation feed tubes and overflow tubes may become partly
clogged with participates, bacteria, and algae. The feed tubes were changed
twice during the 195 days of operation and the overflow tubes were changed
once. New feed tubes were autoclaved before being put into use. Feed tubes
from the pump to the chemostat have a tendency to get algae growing in them,
even though the medium is released 10 cm above the water level.
The overflow flasks may become coated with algae if overflow effluent is
allowed to remain too long in the flasks.
TABLE 20
EQUIPMENT FOR EIGHT CONTINUOUS CULTURE SYSTEMS
2 (or 1) pumps:
8 Culture Tubes:
Surgical Grade
TygonR Tubing
Pump Tubes:
Stoppers:
Nalgene:
8 Fluorescent lamps:
12 Magnetic Mixers:
2 Air Pumps:
30 1000 ml Erylmeyer
Flasks
1 Cam timer:
Buchler Dekastaltic Pump, Model No. 2-6500
Pyrex glass 5 cm i.d. 60 cm length with glass
tube near bottom and another for overflow at
a volume of 950 tnl or 50 cm from base.
Feed tubes 1/16 in (.16 cm) i.d.,
1/8 in (.32 cm) o.d.
Air lines 3/16 in (.48 cm) i.d.,
5/16 in (.79 cm) o.d.
R
Auto Analyzer Pumps .065 in (.165 cm) i.d.,
Technicon Corporation
Neoprene Size #11 for top of culture tube
Size #9 for top of overflow flasks
Tube connectors and stopcocks used as valves
30 watt "cool-white" G.E.
Thermolyne
Silent Giant
Pyrex, for feed storage, feed flasks, and overflow
flasks
Minarik Recycling Timer Model 5C BR-B-60
(one revolution per hour)
33
-------
FIGURE 6 - CONTINUOUS CULTURE UNIT
34
-------
SECTION VI
FIELD TEST SYSTEMS
Application of the chemostat, continuous culture concept to ponds of
much greater volume than a laboratory chemostat tube provide a transition
to those natural situations where effluents of different types are dis-
charged into natural bodies of water. In this way natural populations
of many algal species may be evaluated under realistic field conditions.
The algal flora in a pond receiving a specifically tn-.ated, and presumably
well defined, effluent might be expected to display uniform behavior,
particularly in an area such as Santee, California where atmospheric
variables are as stable and predictable as any to be found. Furthermore,
the flora of ponds receiving effluents from different treatments might
be expected to show certain specific differences.
Two study ponds were established in the location between the treatment
plant and the lakes. The operating parameters for the two ponds were
as follows:
POND VOLUME DEPTH FLOW RESIDENCE
NO. SOURCE (gal.) (ft.) (gal/min) TIME (days)
1 Secondary 116,000 2.5 10 8.02
Effluent
2 Tertiary 124,000 2.6 10 8.62
Effluent
A typical study pond is shown in Figure 8. Preliminary samples were obtained
from the study ponds during the spring and early summer months (1971) in
order to become familiar with algal floras. Samples were collected, generally
in the late morning, from the midpoint on the shore on each side of the pond
by means of a plastic container on a long rod. These four sub-samples
were then combined and examined microscopically in order to determine
the organisms present.
The organisms survey were limited to those suspended in the water because
filamentous material did not develop except on a few stones near the inflow
pipe. This was fortunate, in that assessment of the biomass of epiphytic
and epilithic peripbyton is notoriously difficult to undertake in any
meaningful manner.
Examination during the preliminary phase of monitoring the pond flora was
done with a compound microscope. Quantification was estimated by scanning
slides and judging whether the organism was "rare" - "occasional" -
"frequent" or "abundant." During the latter part of the investigation, the
number of organisms present were quantified more precisely by using a Nikon
inverted microscope, Model MS. A 5-ml aliquot of the undiluted pond sample
was pipetted into a Zeiss counting chamber and a second replicate made. The
organisms were settled by adding drops of iodine. Counting was accomplished
35
-------
FIGURE 7 - TYPICAL STUDY POND
36
-------
with Wild oculars containing a millimeter grid rules 10 squares to a
side. Ten strips of 10 squares were counted at random for the 5-ml
aliquot and the organisms recorded. The results were expressed in
numbers of organisms per liter. Determinations of total biomass based
on dry weight measurements were conducted in accordance with Standard
Methods. Estimates of color were made visually.
For measurement of turbidity a procedure equivalent to that of Secchi-
disc determination was used although the disc employed was not standard.
This was placed in the ponds and the depths at which it disappeared
noted. The measurements were made at mid-day, in sunlight, at the
middle of the south sides of the secondary and tertiary ponds.
37
-------
SECTION VII
EVALUATION OF TREATMENT PROCESSES
The original investigation plan called for evaluation of the tertiary
demonstration process for phosphate removal in a modified activated
sludge process at the Irvine Ranch Water District.
During the first phase of this investigation when the testing procedures
were being developed it became clear that the objective could not be
reached because of delays in the Irvine Ranch Water District's demonstra-
tion project. Consequently, it was decided instead to evaluate the
different tertiary effluents from the tertiary demonstration project at
Santee County Water District. In addition to the time advantage, this
change also was advantageous becuase it permitted the expansion of the
evaluation program to include effluents for an electrodialysis process
and effluents from a ground percolation process. In order to accomplish
this modified plan of investigation, an additional $9,000 was obtained
from the Santee County Water District's demonstration grant. At the same
time it was also decided that the data obtained and presented below would
also be made part of the report of the District's demonstration grant
report (Reference VII-1).
GENERAL OBJECTIVES
The general objectives of the treatment process evaluation were:
1. To determine the relative and absolute effectiveness of
the tertiary treatment processes with respect to removal
of algal nutrients, and
2. To determine the probable response of artificial lakes
and water reservoirs to the tertiary effluents from these
treatment processes.
SPECIFIC OBJECTIVES
The specific objectives of this investigation were:
1. To determine the growth limiting inorganic nutrient(s) in:
a. Secondary (act. sludge) Effluent.
b. Tertiary (lime treated) Effluent.
c. Electrodialysis Effluent.
d. Secondary Effluent after Ground Percolation.
2. To determine the sustained level of algal growth under standard
laboratory conditions in continuous culture for:
a. Secondary (act. sludge) Effluent.
b. Tertiary (lime treated) Effluent,
c. Electrodialysis Effluent.
3. To determine the level of algal growth in study ponds for the
secondary and tertiary effluents.
4. To compare the laboratory and study pond results and determine
if laboratory tests can be used to predict the response of an
artificial lake receiving a given effluent.
38
-------
NIGHT RECYCLE
o
LAGOON 'A'
BACKWASH
TERTIARY
EFFLUENT
SAMPLING POINT
SAN DIEGO RIVER
PRIMARY SEDIMENTATION
STANDARD RATE
ACTIVATED SLUDGE
REACTOR-CLARIFIER
RECARBONATION
DUAL-MEDIA FILTERS
LAGOON 'B1
SOIL PERCOLATION
SEVEN SANTEE
RECREATIONAL
LAKES
SECONDARY EFFLUENT
SAMPLING POINT
STUDY POND
NO. 2
^ POLYELECTROLYTE
NATURAL GAS
STUDY POND
NO. 3
Electrodialysis Effluej
Sampling Point
FIGURE 8 - ALGAE GROWTH STUDY SAMPLING POINTS
39
-------
METHODS AND PROCEDURES
Sampling
Samples were collected at the Santee Treatment Plant in February, June and
August 1971. The sample points are shown in Figure 9. The sample collection
corresponded to specific modes of plant operation as indicated in the
schedule shown below.
Secondary Effluent (1) Grab sample from inlet pipe to tertiary lime reactor-
clarifier on 26 February 171. A sample representative of normal secondary
effluent. (2) 24 hour composite sample from inlet pipe to tertiary lime
reactor-clarifier, on 21-22 June 1971. A sample representative of secondary
effluent and typical influent to the tertiary plant.
Tertiary Effluent (1) Grab sample of tertiary discharge after phosphate
removal, pH adjustment and sand filtration on 26 February 1971. (2) Com-
posite sample over 24 hour period from the lime reactor-clarifier. No pH
adjustment has been made at this point in the treatment process and pH of
this effluent was 9.8. (3) Grab sample after pH adjustment and filtration
The sand filters were back-washed for 1/2 hour before the sample was
collected. This sample representative of typical effluent from the tertiary
plant.
Electrodialysis Effluent (1) Composite sample collected after lime treatment
recarbonation, filtration, carbon adsorption and electrodialysis.
Ground Percolated Effluent (1) Grab sample after tertiary treatment, lagoon
detention and normal percolation of 400' in sand and gravel (on 21 June).
Samples were collected at Santee in Pyrex bottles, autoclaved and stored
in the dark at 4 ± 1°C until used. The four 5 gallom samples of each
effluent were thoroughly mixed before autoclaving.
Sample Pretreatment Samples were pretreated by autoclaving as discussed
in Section IV.
Continuous Culture Assays
The composite sample collected on 21-22 June were used throughout the entire
experiment. For purposes of comparison, the standard batch reference medium
(Section IV) was also used. The Santee samples were stored and used as
required while the reference medium was made up in batches from time to time
during the course of the experiment. The test alga, Selenastrum capricornutum
was inoculated into chemostats containing one of the Santee effluents or the
reference medium at an initial concentration of 10^ cells per ml. Duplicate
chemostats were set up for each effluent and the reference medium. The
chemostats were run until a steady state was achieved. (Steady state was
defined as less than 10% variation in cell concentration per week.) The
chemostats were maintained at a constant residence time of 10.5 days. This
residence time was approximately equal to the hydraulic residence time in the
study ponds described below under the field tests.
Determination of Growth Limiting Nutrients
The growth limiting nutrient(s) were determined by means of factorial spiking
experiments as described by Murray et al 1971 (VII-2) and in Reference IV-1).
"Factor" refers to the element (or combinations of elements added as a single
factor) as shown for each experiment. Two sets of spiking experiments were
conducted on each type of effluent.
40
-------
The first spiking experiments were designed to determine the group to
which the growth limiting nutrient belonged. The nutrients investigation
were classified in the following four groups:
1. Micronutrients: B, Zn, Co, Cu, Mo
2. Iron and Manganese
3. Phosphorus and Nitrogen
4. Macronutrients: Ca, Mg, Na, K, Cl, S, C
Data from the first spiking experiments were used to determine four indi-
vidual elements which appeared most significant and a second set of
factorial experiments were then designed to evaluate these four individual
growth limiting nutrients. The four elements selected for the second set
of experiments were:
1. Phosphorus
2. Iron
3. Nitrogen
4. Manganese
Spiking Concentrations
Spikings were performed by addition of specified amounts of concentrated
stock solutions of macro- and micronutrients such that the final spiking
after dilution into the effluent being tested corresponded to 30 percent
of the concentration of the reference medium described above. For example,
spiking with iron was done such that 11 yg Fe was added per liter of
effluent being tested.
Evaluation Parameters
Three different biomass parameters were used to determine algal growth:
cell count, total cell volume and dry weight.
Biomass can be expressed in various ways. Measurement of dry weight is
useful in terms of energy values, cell numbers are of significance from
a turbidity and analytical point of view, and total cell volume is a
useful parameters which may combine the advantages of cell counts and
dry weight. Measurements of cell volume correlate best with optical
density, what the eye actually sees, and this may be the most useful
assessment of algal growth, particularly by the nontechnical viewer.
CHEMICAL ANALYSES
The 21-22 June composite samples and reference medium were analyzed for
iron, manganese, nitrite, nitrate and phosphate.
Methods for analysis of nitrites, nitrates, and total phosphorus are those
given in FWPCA Methods for Chemical Analysis of Water and Wastes, 1969
(VII-3).
GROWTH LIMITING NUTRIENTS
Determination of the growth limiting nutrients was a key aspects of the
evaluation of the various treatment process effluents. This determination
was considered of special significance in this investigation because the
various treatment processes do remove different nutrients in unequal
41
-------
proportions. When considering process improvements, it is therefore
necessary to know which specific nutrient (or nutrients) are limiting
growth of algae. The batch assay evaluation in this investigation for
the Santee County Water District included only the limiting nutrients
as they affect the maximum algal biomass. This choir.e of approach
was based on the specific situation at Santee where all wastes are dis-
charged in lakes having hydraulic residence times in excess of ten days.
Basing the determination of growth limiting nutrients on continuous
culture experiments was considered. However, because limiting nutrients
change as a function of residence/growth time and the need to evaluate
and screen many potential limiting nutrients and their interactions, it
appeared that factorial multinutrient batch testing would provide a more
comprehensive evaluation.
Reference Medium - The results of the determination of groups of growth
limiting nutrients are summarized in Table 21. The corresponding summary
results of the evaluation of single nutrients are shown in Table 22.
The groups spiking experiment (Table 21) showed that addition of (Fe + Mn)
as a group resulted in an increase in the number of algal cells. The
increase amount of 1.12 10 cells per ml or of 23 percent and the increase
was statistically significant at the 95 percent level. However, the
addition of (Fe 4- Mn) did not result in a statistically significant
increase in either cell volume or dry weight despite the increase in
cell numbers.
Both (P + N) and macronutrients limit growth in the reference medium as
measured by all three parameters used, cell number, cell volume, and
dry weight. Addition of (P + N) shows a greater effect on the final
growth than does the addition of macronutrients.
The individual nutrient spiking experiment was based on the results
of the group spiking experiment and four individual nutrients were
tested. The four nutrients were Fe, Mn, P, N. The results in Table 22
show that of those four nutrients only phosphorus appears to limit growth
in the unspiked medium when all three biomass indicators are considered.
Spiking with iron results in a slight decrease in final biomass as
measured by both cell number and cell volume. This suggests that iron
might inhibit growth in the reference medium; however, more work would
be necessary prior to determining whether this is a supportable conclu-
sion.
Secondary Effluent
Secondary effluent samples collected on two different dates were evalu-
ated in the group spiking experiment. The chemical composition of these
effluents are shown in Table 23. The results for the first sample
(collected on 26 February 1971) are shown in Table 25. The only nutrient
group to have a significant effect was (Fe + Mn). The (Fe + Mn) addition
resulted in an increased biomass for all three parameters, thus indicating
that the growth of algae in that effluent sample was limited by either
iron or manganese.
42
-------
TABLE 21
REFERENCE MEDIUM TESTING OF NUTRIENT GROUPS
EFFECT
0 F
SPIKING
Cell Number Total Cell Volume
(No/m£) (mm3/*.)
Nutrient
Group With Without With Without
Spiked (a) (b) (a) (b)
Petals 5'67 x !°6 5J6 x '°6 323 293
Fe + Mn 5.97 x I06 4.85 x I06 322 294
P + N 6.57 x I06 5.07 x I06 369 247
Macro- 5.55 x- ,Q6 5.28 x I06 332 ' 284
nutrients
Dry Weight
(mg/£)
With Without
(a) (b)
197 177
1 90 1 84
223 151
201 172
SIGNIFICAiNT FACTORS ON FINAL DAY
Factor Cell Number Cell Volume Dry Weight
(Trace) * (c)
(Fe + Mn) **
(P + N) ** ** **
(MACRO) * ** *
Comparison (d)
+
+
+
+
a,b) Each value is the average of one-half of the 32 flasks in the factorial
experiment. For example, the value at (a) is the average of the 16 flasks
receiving the trace metal spike and the value at (b) is the average of the
16 flasks NOT receiving the trace metal spike.
(c) One star indicates that the addition of the group of nutrients resulted in
a change in final biomass which was significant at the 95$ level. Two stars
indicate a corresponding significance at the 99% level.
(d) A positive comparison indicates that addition of the nutrient group increased
the final biomass. A negative comparison indicates a decrease in final bio-
mass when the nutrient group was added.
-------
TABLE 22
REFERENCE MEDIUM TESTING OF INDIVIDUAL NUTRIENTS
EFFECT
0 F
SPIKING
Added
S i ng 1 e
Nutrient
P
Fe
N
Mn
Cell Number Total Cell Volume ' Dry Weight
(No/m£) (mmVJl) (mg/X)
With Without With Without With Without
(a) (b) (a) (b) (a) (b)
5.33 x I06 3.74 x I06 299 255 136 121
4.39 x I06 4.67 x I06 265 289 127 130
4.50 x I06 4.56 X I06 283 270 130 127
4.65 x I06 4.42 x (O6 287 266 127 130
SIGNIFICANT FACTORS ON FINAL DAY
FACTOR CELL NUMBER CELL VOLUME DRY -WEIGHT COMPARISON
p ** ** ** +
Fe
N
Mn ** +
a,b) Each value is the average of one-half of the 32 flasks in the factorial
experiment. For example, the value at (a) is the average of the 16 flasks
receiving the trace metal spike and the value at (b) is the average of the
16 flasks NOT receiving the trace metal spike.
(c) One star indicates that the addition of the group of nutrients resulted in
a change in final biomass which was significant at the 95% level. Two stars
indicate a corresponding significance at the 99% level.
(d) A posiMve comparison indicates that addition of the nutrient group increased
the final biomass. A negative comparison indicates a decrease in final bio-
mass when the nutrient group was added.
44
-------
TABLE 23
CHEMICAL COMPOSITION OF SECONDARY EFFLUENTS
Constituent
Phosphorus mg P/fc
Nitrate mg N/£
Nitrite mg N/£
Kjeldahl Nitrogen mg N/&
N/P ratio
Iron yg/£
Manganese ug/£
DATE OF
26 Feb 71
11.8
0.9
3.6
8.4
1.09
No data
No data
S A M P L I N
21 June
8.85
4.4
0.37
14.7
2.20
i ug/£ ±
7 g/SL ±
G
71
1
1
The results for the 21 June 1971 sample are shown in Table 26, also
showed an increased biomass as measured by cell number and total cell
volume (but not dry weight) with the addition of the (Fe + Mn) groups.
However, for this sample of secondary effluent the (P + N) spike resulted
in a significant increase for all three parameters of growth. These
results indicate that the secondary sample from 21 June has received a
different degree of treatment than the sample collected 26 February 1971.
TABLE 24
CHEMICAL COMPOSITION OF TERTIARY EFFLUENTS
Constituent
Phosphorus mgP/£
Nitrate mg N/£
Nitrite mg N/£
Kjeldahl Nitrogen mg N/£
N/P ratio
Iron yg/S-
Manganese yg/&
26 Feb 1971
0.77
1.10
0.16
4.6
7.61
No data
No data
21 June 1971
.280
4.3
< 1.0
7.82
46.86
< 1
1
25 Aug 1971
.21
< 1.0
< 1
1
The individual nutrient spiking was made only on the 21 June 1971 sample
and results as shown in Table 27 indicate that nitrogen is the limiting
nutrient for all three growth parameters. Spiking with the nutrients
P and Mn result in decreased biomass for all parameters. The Fe addition
has a slight but insignificant increase for cell number and volume on the
final day. However, analyses of the detailed results indicated that Fe
addition results in increased cell number and volume early in the experi-
ment, i.e. affecting growth rate but not final biomass. The number of
samples and the single source of effluent limit the conclusions which
can be drawn, it is therefore recommended to expand the investigations to
45
-------
include other secondary effluents and more samples of each effluent.
Tertiary Effluent
Three samples of tertiary effluent were collected under different treat-
ment plant operating conditions and the growth limiting nutrients deter-
mined. The chemical compositions of these effluents are shown in Table 24.
Results of the 26 February sample (Table 28) show that the (P + N) addition
results in an increased total cell volume but that the other parameters
are not affected significantly. At the same time, the (Fe + Mn) addition
results in a decrease in cell numbers. It should be noted that both
effects, although statistically significant, are numerically small.
The 21 June sample was taken directly from the lime reactor-clarifier tank
because of operating difficulties with the filters in the treatment plant.
The sample contained large amounts of flocculated material which appeared
to inhibit growth-of the test organism. Data from the chemostats described
later in the report indicate that batch experiments did not go to completion
and that maximum biomass was not attained even after 58 days. Also there
was a significant difference between the replicate flasks for the dry
weight parameter. Consequently the results have not been considered in
detail.
The results of the group and individual nutrient spiking experiments
for the 25 August sample are presented in Table 29 and 30. Group spiking
showed both (Fe + Mn) and (P + N) limiting for all three growth parameters.
Spiking with the single nutrients showed that P and Fe were both limiting
while N and Mn additions inhibited growth significantly.
Electrodialysis Effluent
The chemical composition of the electrodialysis effluent is given in
Table 31. Results of the nutrient group spiking experiment are shown in
Table 32. The addition of the (Fe + Mn) group resulted in a very signifi-
cant increase for all three growth parameters while the addition of the
macroelement group resulted in a decrease in the final total cell volume
and dry weight. This again indicates the potential importance of (Fe + Mn)
as growth limiting nutrients.
Results of spiking with the single nutrients are shown in Table 33.
Although addition of both Fe and P result in highly significant increases
for all biomass parameters, the significance for iron is much greater.
The N spike resulted in a significant decrease for all biomass parameters
while the Mn caused a decrease in dry weight only. Examinations of the
detailed factorial analysis indicated that the numerical effect of the P
and N additions are about the same, one increasing growth and the other
decreasing growth. Therefore, spiking with the combination of these two
nutrients as in the group testing experiments caused no significant
effect. This indicates the caution required in interpreting results both
from spiking with groups of nutrients and from evaluation of gross effects
of treatment process modifications. In both cases conclusions should be
based on overall results plus assessment of growth limiting nutrients.
46
-------
TABLE 25
TESTING OF NUTRIENT GROUPS - SECONDARY EFFLUENT - 26 Feb
EFFECT OF SPIKING
Cell Number Totaf Cell Volume
(No/mi) (mm3 /A)
Nutrient
Group With Without With Without
Spiked (a) (b) (a) (b)
Trace -^q x (Q- 3,99 x |n7 |o<^ 2I.°6
Meta Is
Fe + Mn 5 . 4.3 x 1 0 1 . 92 x 1 0? 25 1 8 I 5 1 P
P + N 3.98 x I07 3.38 x I07 20?5 2041
"nutrients4'08 x ^ 3'27 * ^ 2I41 15'5
Dry Weight
(mg/A)
With Without
(a) (b)
5.74 OQO
10?! 692
904 879
?32 850
SIGNIFICANT FACTORS ON FINAL DAY
Factor Cell Number Cell Volume Dry Weight
(Trace)
(Fe t Mn) ** ** **
(P + N)
(MACRO)
Comparison (d)
4-
a,b) Each value is the average of one-half of the 32 flasks in the factorial
experiment. For example, the value at (a) is the average of the 16 flasks
receiving the trace metal spike and the value at (b) is the average of the
16 flasks NOT receiving the trace metal spike.
(c) One star indicates that the addition of the group of nutrients resulted in
a change in final biomass which was significant at the 95% level. Two stars
indicate a corresponding significance at the 99% level.
(d) A positive comparison indicates that addition of the nutrient group increased
the final biomass. A negative comparison indicates a decrease in final bio-
mass when the nutrient group was added.
47
-------
TABLE 26
TESTING OF NUTRIENT GROUPS - SECONDARY EFFLUENT 21-22 June
EFFECT
0 F
SPIKING
Cell Number Total Cell Volume
(Nb/fflJl) (mm3 A)
Nutrient
Group With Without With Without
Spiked (a) (b) (a) (b)
I f"3f*^ ~? ~7
Metals 2-"'° x lo 2-i6x 10' 1326 1246
Fe + Mn 2.J?*; x 10'' 2.11 x 10' 1347 1224
P+N 2.33 x I07 2.IOx I0? 13^? 1 ?03
Mac^°7 , 2.08XI07 2.37 x !0? 123" 1332
nutrients
Dry Weight
(mg/A)
With Without
(a) (b)
464 44 1
474 433
4^7 440
435 472
SIGNIFICANT FACTORS ON FINAL DAY
Factor- Cell Number Cell Volume Dry Weight
(Trace)
(Fe + Mn) * *.*
(P + N) * ** *
(MACRO) ** *
Comparison (d)
+
+
a,b) Each value is the average of one-half of the 32 flasks in the factorial
experiment. For example, the value at (a) is the average of the 16 flasks
receiving the trace metal spike and the value at (b) is the average of the
16 flasks NOT receiving the trace metal spike.
(c) One star indicates that the addition of the group of nutrients resulted in
a change in final biomass which was significant at the 95% level. Two stars
Indicate a corresponding significance at the 99% level.
(d) -A positive comparison indicates that addition of the nutrient group increased
the final biomass. A negative comparison indicates a decrease in final bio-
mass when the nutrient group was added.
48
-------
TABLE 27:
TESTING OF INDIVIDUAL NUTRIENTS - SECONDARY EFFLUENT 21-22 June
Added
Single
Nutrient
P
Fe
N
Mn
EFFECT OF SPIKING
Cell Number Total Cell Volume Dry Weight
(No/mA) (mm3/*,) (mg/S,)
With Without With Without With Without
(a) (b) (a) (b) (a) (b)
1.43 x I07 1.59 x I07 820 913 415 457
1.53 x I07 1.49 x I07 867 866 432 440
1.56 x I07 1.46 x I07 908 325 451 421
1.37 x I07 1.65 x I07 775 958 377 495
SIGNIFICANT FACTORS ON FINAL DAY
FACTOR CELL NUMBER CELL VOLUME DRY* WEIGHT COMPARISON
p *x ## **
Fe
N * ** * +
Mn ** ** **
a,b) Each value is the average of one-half of the 32 flasks in the factorial
experiment. For example, the value at (a) is the average of the 16 flasks
receiving the trace metal spike and the value at Cb) is the average of the
16 flasks NOT receiving the trace metal spike.
(c) One star indicates that the addition of the group of nutrients resulted in
a change in final biomass which was significant at the 95% level. Two stars
Indicate a corresponding significance at the 99% level.
(d) A positive comparison indicates that addition of the nutrient group Increased
the final biomass. A negative comparison indicates a decrease in final bio-
mass when the nutrient group was added.
49
-------
TABLE 28
TESTING OF NUTRIENT GROUPS - TERTIARY EFFLUENT 26 Feb
EFJF ECT OF SPIKING
Cell Number Total Cell Volume
(No/mJl) (mm3/*)
Nutrient
Group With Without With Without
Spiked (a) (b) (a) (b)
Trace 4.37 x I07 4.31 x I07 1975 1956
Metals
Fe + Mn 4.24 x Id7 4.44 x I07 1950 1981
P + N 4.40 x I07 4.28 x I07 1989 1941
nutrients4'26 X '° 4.42 x I07 1946 1985
Dry Weight
(mg/A)
With Without
(a) (b)
888 914
915 886
891 911
880 922
SIGNIFICANT FACTORS ON FINAL DAY
Factor Cell Number Cell Volume Dry Weight
(Trace)
(Fe + Mn) *
(P + N) *
(MACRO)
Comparison (d)
+
a,b) Each value is the average of one-half of the 32 flasks in the factorial
experiment. For example, the value at (a) is the average of the 16 flasks
receiving the trace metal spike and the value at (b) is the average of the
16 flasks NOT receiving the trace metal spike.
(c) One star indicates that the addition of the group of nutrients resulted in
a change in final biomass which was significant at the 95% level. Two stars
Indicate a corresponding significance at the 99/6 level.
(d) A positive comparison indicates that addition of the nutrient group Increased
the final biomass. A negative comparison indicates a decrease in final bio-
mass when the nutrient group was added.
50
-------
TABLE 29
TESTING OF NUTRIENT GROUPS - TERTIARY EFFLUENT 25 Aug
EFFECT
0 F
SPIKING
Cell Number
(No/m£)
Nutrient
Group With Without
Spiked (a) (b)
Petals 3-l8x,06 3.46 x ,06
Fe + Mn 3.68 x I06 2:95 x I06
P + N 3.71 x I06 2.93 x I06
Mac:°7 3.07 x I06 3.56 x I06
nutr i enfs
Tota
With
(a)
234
283
276
223
1 Ce 1 1 Vo 1 ume
Without
(b)
266
217
224
277
Dry Weight
(mg/£)
With Without
(a) (b)
116 140
1 39 ||7
141 116
1 20 1 36
SIGNIFICANT
Factor Cell Number
(Trace)
-------
TABLE 30
TESTING OF INDIVIDUAL NUTRIENTS - TERTIARY EFFLUENT 25 AUG.
EFFECT
0 F
SPIKING
Added
S i ng 1 e
Nutrient
P
Fe
N
Mn
Cel 1 Number
(No/mA)
With Without
(a) (b)
3.16 x I06 2.75 x I06
3.53 x I05 2.38 x I06
2.78 x I06 3.14 x I06
2.48 x I06 3.44 x I06
Total Cel 1 Volume
(nrnVi)
With Without
(a) (b)
224 1 96
274 1 46
193 227
164 256
Dry Weight
(mg/£)
With Without
(a) (b) ,
1 14 95
151 58
92 1 16
73 135
SIGNIFICANT FACTORS ON FINAL
FACTOR CELL NUMBER
p **
Fe **
N **
Mn **
DAY
CELL VOLUME DRY WEIGHT COMPARISON
**
**
**
*#
** +
x* 4.
**
**
a,b) Each value is the average of one-half of the 32 flasks in the factorial
experiment. For example, the value at (a) is the average of the 16 flasks
receiving the trace metalspike and the value at (b) is the average of the
16 flasks NOT receiving the trace metal spike.
(c) One star indicates that the addition of the group of nutrients resulted in
a change in final biomass which was significant at the 95% level. Two stars
indicate a corresponding significance at the 99% level.
(d) A positive comparison indicates thet addition of the nutrient group increased
the final biomass. A negative comparison indicates a decrease in final bio-
mass when the nutrient group was added.
52
-------
TABLE 31
CHEMICAL COMPOSITION OF ELECTRODIALYSIS EFFLUENT
DATE OF SAMPLING
Constituent 21 June 1971
Phosphorus mg P/Ji .271
Nitrate mg N/& .390
Nitrite mg N/fc 0.27
Kjeldahl Nitrogen mg N/£ O.la
N/P ratio 2.77
Iron yg/& 1
Manganese yg/A 2
^
estimated value
Further, the reason for the negative effects of nitrogen on the algal
growth are not clear from the results obtained and further evaluation of
Electrodialysis Effluents should be made.
It should be noted that all biomass parameters are higher in individual
nutrient spiking experiments. Examination of the detailed results in the
appendix indicates that this is not caused by spiking alone, as control
flasks (Nos 31 and 32) with nothing added are also much greater (mean
cells/ml of 2.5 x 106 vs 4.5 x 105). It appears that the long storage
period had considerable effect upon electrodialysis effluent.
Ground Percolated Effluent
Results of nutrient group spiking are shown in Table 34. Addition of the
(P + N) group resulted in a highly significant increase in biomass for
all growth parameters while the macronutrient addition increased only cell
numbers. This data indicates that the (P + N) group is limiting in
ground percolated effluent.
DISCUSSION - BATCH ALGAL ASSAYS
The two secondary effluent samples which were evaluated in this work
exhibited very different chemical and algal growth characteristics.
Characteristic of both samples was the fact that phosphate was not growth
limiting (i.e. there was sufficient phosphate present); iron + manganese
limited growth in one sample and nitrogen in the other. It should be
recognized that the data here are based on only two samples and that broad
conclusions cannot be drawn. Nevertheless, both samples were collected
as 24 hour composite at a time when the activated sludge plant was
performing in a normal manner. Based on these observations, it appears
that more investigational work with a broad range of activated sludge
effluents should be made.
53
-------
TABLE 32
TESTING OF NUTRIENT GROUPS - ELECTRODIALYSIS EFFLUENT 21/22 June
EFFECT 0
Cell Number
(No/mA)
Nutrient
Group With Without
Spiked (a) (b)
Trace 2 94 x I06 2 52 x I06
Metals '
Fe + Mn 5.07 x I06 3.84 x I06
P + N 2.72 x 10° 2.74 x I06
Macr°7 +2.22 x I06 3.24 x I06
nutrients
F SPIKING
Tota 1 Ce 1 1 Vo 1 ume
(mm3/*)
With Without
(a) (b)
1 40 1 36
250 26
1 38 1 38
"103 173
Dry Weight
(mg/A)
With Without
(a) (b)
58 60
105 12
59 58
40 77
SIGNIFICANT
FACTORS ON FINAL DAY
Factor Cell Number Cell Volume Dry Weight
(Trace)
(Fe + Mn) **
(P + N)
(MACRO)
** *#
* #
Comparison (d)
+
-
a,b) Each value is the average of one-half of the 32 flasks in the factorial
experiment. For example, the value at (a) is the average of the 16 flasks
receiving the trace metal spike and the value at (b) is the average of the
16 flasks NOT receiving the trace metal spike.
(c) One star indicates that the addition of the group of nutrients resulted in
a change in final biomass which was significant at the 95^ level. Two stars
Indicate a corresponding significance at the 99% level.
(d) A positive comparison indicates that addition of the nutrient group increased
the final biomass. A negative comparison indicates a decrease in final bio-
mass when the nutrient group was added.
54
-------
TABLE 33
TESTING OF INDIVIDUAL NUTRIENTS - ELECTRODIALYSIS EFFLUENT 21/22 June
EFFECT
0 F
SPIKING
Added
S i ng 1 e
Nutrient
P
Fe
N
Mn
Cell Number Total Cell Volume Dry Weight
-------
TABLE 34
TESTING OF NUTRIENT GROUPS - GROUND PERCOLATED EFFLUENT 26 Feb.
EFFECT
0 F
SPIKING
Cell Number Total 'Cell Volume
(No/mJl) (mm3/*)
Nutrient
Group With Without With Without
Spiked (a) (b) (a) (b)
Trace fi fi
Metals 3.24 x 10 3.IOx 10 131 135
Fe + Mn 3.28 x I06 3.05 x I06 134 132
P + N 4.63 x I06 1.71 x I06 195 71
"nutrients3-34 x '°6 2.99 x 1C6 136 130
Dry Weight
(mg/A)
With Without
(a) (b)
95 92
96 90
121 66
97 89
S 1 GN 1 F 1 CANT FACTORS ON F 1 N AL DAY
Factor Cell Number Cell Volume Dry Weight
(Trace)
(Fe + Mn)
(P + N> ** ** **
(MACRO) **
Comparison (d)
+
+
a,b) Each value is the average of one-half of the 32 flasks in the factorial
experiment. For example, the value at (a) is the average of the 16 flasks
receiving the trace metal spike and the value at (b) is the average of the
16 flasks NOT receiving the trace metal spike.
(c) One star indicates that the addition of the group of nutrients resulted in
a change in final biomass which was significant at the 95% level. Two stars
Indicate a corresponding significance at the 99% level.
(d) A positive comparison indicates that addition of the nutrient group increased
the final biomass. A negative comparison indicates a decrease in final bio-
mass when the nutrient group was added.
56
-------
•••
i
10,000
LU
^
LU
O
1,000
100
10
liJ -
LJ S
6
Q
o
a
MEDIUM AND DATE OF COLLECTION OR PREPARATION
FIGURE 9. FINAL CELL VOLUME IN EFFLUENTS TESTED
-------
30
o:
UJ
CO
UJ
o
10
s
-L 10
Iff
ia
lo-*
UJ LU
cc 5
>
CO
CD
U.
GO
LU
Ll_
(0
CM
MEDIUM AND DATE OF COLLECTION OR PREPARATION
FIGURE 10. FINAL CELL NUMBER IN EFFLUENTS TESTED
-------
1,000
i n
£
«*•
h-
LU
cc
Q
100
10
IU =
U_ Q
LjJ UJ
o
•
• •
«
• I
• I
•
n:
o
Ed
CO
03
Lil
OJ
—>
OJ OJ
or:
QQ
B!
/
/
/
/
/
/
/
/
/
/
/
/
3
in
CM
LJ Q
C\J
otr
IT LJ
OQ.
CD
UJ
U.
(0
CVJ
MEDIUM AND DATE OF COLLECTION OR PREPARATION
FIGURE 11. FINAL DRY WEIGHT IN EFFLUENTS TESTED
-------
Of the two tertiary samples obtained, the one collected 25 August 1971
shows a much lower algal growth than the early sample collected 26 February
1971. These results are in agreement with the overall plant performance
during the two periods of operation represented by the two samples. During
the last period higher lime dosages were used and this resulted in better
phosphate removal as measured by chemical analyses.
The higher lime dosages also resulted in better iron removal, and both iron
and phosphate were limiting algal growth with iron being the most signifi-
cant of the two as indicated by the results in Table 30.
At the same time nitrogen and manganese appeared to be suppressing growth.
The reason for this effect is not understood at this time and further work
should be conducted to verify this finding.
Evaluation of the electrodialysis effluent indicated that iron and phosphate
were the growth limiting nutrients and that iron had the most significant
effect. This is similar to the results obtained with the tertiary effluent.
The results of the batch assays are summarized on Figure 10, 11 and 12.
.These data are in general agreement with those obtained with the continuous
culture units and signify to the relative improvement in effluent character-
istics resulting from the tertiary treatment processes investigated at
Santee.
t
CONTINUOUS CULTURE INVESTIGATIONS
Laboratory Investigations
Introduction
In order to evaluate a waste management program, or the quality of the
effluents from such a program, a procedure which will provide data on
different levels of algal growth at steady state may serve as a useful
management tool. A continuous-flow laboratory culture can serve as
such a tool because it simulates growth likely to occur in a body of water
receiving a given effluent.
In the research described here, one liter chemostates were used (IV-2,
VII-4, VII-5), and the results compared with the algal growths which
occurred in the field in test ponds receiving the same effluents. With
a residence time of 105 days, the laboratory chemostats required 99 days
to reach steady state. This is a surprising length of time, in view of the
consistency of the media and the conditions of culture. Steady state
conditions were maintained for the recommended two residence times or
21 days. Measurement of cell number, mass and volume were performed on
the effluents from the four chemostats on four or five occasions during
this 20 day period.
The biomass expressed as dry weight (mg/1), for each of the three types
of Santee effluents and the standard algal growth medium are shown in
Figure 13. The results show:
60
-------
1. The variation in biomass was insignificant over the
21 day period and that steady state conditions had,
in fact, been achieved and maintained; and
2. The growth of the three effluents and the reference
medium could be ranked in the following decending order,
Secondary > Tertiary > NAMM > Electrodialysis.
Biomass can be expressed in various ways. Measurement of dry weight is
useful in terms of energy values although in terms of practical appli-
cation cell number and cell volumes may be the most useful parameters.
Measurements of cell volume correlate best with optical density, what the
eye actually sees, and this is the most likely assessment of algal growth,
particularly by the nontechnical viewer. Average values for dry weight,
(expressed as mg/1), cell number (expressed as number/1) and cell volume
(expressed as mm3/!) are given in Table 35.
In every case, the three effluents can be ranked in the descending sequence,
secondary effluent > tertiary effluent > electrodialysis effluent.
The values obtained with the reference medium place it between tertiary
effluent and electrodialysis effluent, with values approximately half
those for the tertiary effluent in every feature measured. The results
are expressed graphically in Figure 13.
The major problems involved in these chemostat experiments were a consequence
of flocculant material present, particularly in the tertiary effluent. The
material appeared to be organic with possible nutrients chelated onto them.
Filtration would have removed these nutrients so that they had to be
retained in the chemostats despite the difficulties involved in using a
Coulter Counter on fluids containing such flocculant material. It might
well have been that the long time required for stability was consequence
of slow equilibration with these chelated nutrients. Examination of the
algae in the chemostats at the end of the experiment showed that after a
period of nearly four months, the levels of contamination were not excessive.
Bacterial browth was present in all the effluents and the reference medium,
with the highest levels in the tertiary effluent. A small quantity of
blue-green algal contaminant was also present in the chemostats with
tertiary effluent.
Field Investigations
The objectives of the field assay investigations were to determine:
1. The total biomass which will develop in continuous
culture, under field conditions, in the different
types of effluent.
2. The species composition of the biomass in the effluents
of different type.
3. The relative amounts of growth in the two ponds.
4. The comparison between absolute and relative amounts
of growth under field conditions with the corresponding
results determined in the laboratory continuous cultures.
61
-------
N5
1000
100
E
*>
H-
0
UJ
10
I
SECONDARY
TERTIARY
-
NAAM
ELECTRODIALYSIS
96 100 105
110 115
120
DAYS SINCE INNOCULATION
FIGURE 12. DRY WEIGHT IN CONTINUOUS FLOW CULTURES DURING STEADY STATE
125
-------
TABLE 35
STEADY STATE BIOMASS CONCENTRATIONS IN CONTINUOUS
CULTURES AT 10.5 DAYS RESIDENCE TIME
Biomass of chemostate continuous cultures of Santee secondary effluent,
electrodialysis effluent, and reference medium expressed in terms of
dry weight, cell number and total cell volume, using Selanastrum
capricornutum as the test organism. These figures are mean values for
the two chemostats established for each effluent and the reference medium.
a)
Effluent or
culture medium
Dry Weight
(mg/W
Cell Number
per m&
Total cell
volume
Santee 580
secondary
effluent
Santee 63
tertiary
effluent
Santee 2 . 7
electrodialysis
effluent
Reference 35 . 5
medium
144 x 10 1222
7.50 x 105 110
0.192 x 105 1.53
3.5 x 105 42
a)
All samples collected 21 June 1971
63
-------
TABLE 36
BIOMASS IN STUDY PONDS
Biomass expressed in mg/1
DATE POND 2 POND 3
1971 Secondary Effluent Tertiary effluent
19 Mar 60 30
1 Apr 142 104
15 Apr 36 36
19 May 62 20
21 June 62 12
22 July 63 17
25 Aug 109 21
23 Sept 139 28
AVERAGE 77 33
Standard 46 28
Deviations
Discussion
1. Algal biomass in ponds receiving secondary and tertiary effluents.
Considering the algal blooms present in the ponds receiving secondary and
tertiary effluent, the values for the tertiary pond were (with one exception)
lower than the values for secondary pond. Concurrent factors which varied
significantly between the two ponds and might be influencing or reflecting
these differences include:
1. Total phosphate was about 2 mg/1 greater in the
secondary pond than the tertiary.
2. Orthophosphate of the filtrate was also about 2 mg/1
greater than the tertiary.
3. Alkalinity of the secondary was about 100 mg/1
greater than the tertiary.
4. Ammonia filtrate of the secondary effluent was
about 1.5 mg/1 greater than the tertiary.
5. Suspended solids were about 35 mg/1 greater than
the tertiary.
Results
1. Estimates of total biomass in the ponds receiving secondary and tertiary
effluents.
64
-------
140
120
x
cr>
E 100
H-
31
CD
LU
80
60
40
20
0
Secondary (Study pond 2)
Tertiary (Study pond 3)
MAR
APR
MAY
JUN
1971
FIGURE 13. BIOMASS PRESENT IN STUDY PONDS
JUL
AUG
SEP
-------
The algal biomass, expressed in terms of mg per liter,
present in the ponds receiving secondary and tertiary
effluents, during the period of investigation are shown
in Table 36 and Figure 14.
2. Determinations of biomass by Secchi-disc measurements.
The depths at which the home-made Secchi-disc vanished
are indicated in Table 37.
3. Identification of species and relative abundance.
Summaries of the major algal species in the ponds
receiving secondary and tertiary effluents are
given in Tables 38 and 39.
4. Chemical oxygen demand of secondary effluent was about 30 mg/1 greater
than that of the tertiary effluent, and
5. COD filtrate was about 20 mg/1 greater than the tertiary.
DATE
5 Aug '71
20 Aug '71
27 Aug '71
14 Sept '71
AVERAGE
TABLE 37
SECCHI DISK MEASUREMENTS IN STUDY PONDS
POND 2 (secondary) POND 3 (tertiary)
10 inches
11.5 "
18 "
15
13.6 inches
22.5 inches
14
15
36
% better clarity
POND 2 POND 3
125%
21.7%
20%
21.9 inches
140%
61%
At the same time -
1. pH of the two was about equal.
2. Dissolved oxygen was about equal; and
3. nitrate filtrate was equal.
Ibwever, nitrate filtrate of the secondary pond was slightly lower than the
tertiary. The temperature during the entire study period climbed steadily
from a minimum in February 1971 of 16°C to a maximum of about 25°C in
August. Although the quality of tertiary effluent, as reflected in algal
pond growth, had made a good recovery by mid-august, the secondary effluent
did not show this rapid recovery and was continuing to deteriorate, in
terms of the quantity of algal growth, as the project was terminated.
66
-------
TABLE 38
COLOR OBSERVATIONS AND MAJOR ALGAL COMPONENTS - SECONDARY POND
DATE COLOR
1971
26 Feb. None recorded
19 Mar None recorded
Apr Lower end darker;
color not speci-
fied
ORGANISMS (abundant or I08)
navlculold diatom
Euglena
(navlculold diatom abundant
on rocks)
Chlamydomonas
Cryptomonas
Eutroptla T?)
ORGANISMS (frequent or 10 ;
Cryptomonas^
Ped1nomonas
Sphaerel Fop's Is (?)
6-mlcron flage!late
Eug[ena
Scendesmus acumlnatus
Osclllatorla
15 Apr
Yellowish-
green
19 May Yellow-green
21 June None recorded
22 July None recorded
None taken
Chlamydomonas
8
Osclllatorla, 1.8 x 10 /A g
coccold blue-green 3.78 x 10 /£
o
coccold green, 1.12 x 10 /& Q
coccotd blue-green 4,20_x 10 /£
Chlorogonlum, 2.27 x IO°/£
25 Aug Dark grassy green Chloj-oggnlum, 3.68 x 10 /£ „
coccoTdTiTue-green, 4.20 x 10 /£,
Q
23 Sept Dark grassy green Actlnastrum, 1.08 x 10 /$,
coccold blue-green, 3.28 x 10 /£
Chlorogonlum, 2.56 x I08/A
Chlamydomonas, 2.73 c IOe/A
None taken
Chlorogonfum
Osclllatorla
(Others, 10 )
Scenedesmus acumlnatur, 2^25 x 1
Merlsmopedla, eel Is 4.28 x lOVJl
flagellate, 1.87 x I07/A
Fed t nomonas, 1.75 x I07/Ji
coccotd green, 2.67 x I07/Jt -,
Merfsmopedla, cells 4.28 x 10 /I
coccold green, 3.13 x 10 /x,
OscfIlatorla, 1.25 x l07/£
-------
TABLE 39
COLOR OBSERVATIONS AND MAJOR ALGAL COMPONENTS - TERTIARY POND
CO
DATE
1971
26 Feb
19 Mar
I Apr
19 May
21 June
COLOR
ORGANISMS (abundant or I08)
25 Aug
23 Sept
None recorded coccold green
Chlaydomonas
None recorded (2 flagellates and 2 green
algae In 'occasional' amount)
Lower end 2-mIcron flagellate
darker; color
not specified
Brownish-green (none abundant, others 'occasional
to ra re ' )
None specified coccold blue-green, 1.6 x 10
Osclllatorta, 2.58 x IOB/£
Q
22 July None specified (10 only)
Bluish - to
IIght green
Bluish-green
coccold blue-green, 5.57 x 10
ActInastrum, 2.8 x 10 /£
Pedlastrum, 2.8 x l08/£
ORGANISMS (frequent or 10 )
Chloroccccum (?)
Euglena
navlculotd diatom
12-mIcron flagellate
3-mlcron coccold green
5-mIcron coccold green
Chlaymydomonas
Anklstrodesmus
Osclllatorla
Scenedesmus quadrlcauda. 1.0 x Iu
Chlamydomonas, 1.0 x I OVA
chrysophyte flagellate,
coccold green, 1.9 x 10 /£ _
small green fI age I I ate,0.9 x7IO//x
coccold blue-green, 4.6 x 10 /I
(others I06)
coccold green, 5.50 x 10/5,
Chlamydomonas,I.0 x !07/£ _
coccold blue-green, 7.25 x 10 /£
Merlsmopedla, cells 2.5 x !07/£
-------
Clarity of the pond waters was measured with the Secchi-disc. The
technique appears to be a simple method, highly suitable for assessing
the amount of algal growth which is supported by the effluents in each
of the study ponds. The major disadvantage is that particulate matter,
which might be present, also influences the readings. This is particularly
critical in any circumstances in which secondary effluent is likely to be
used. The disc procedure measures total turbidity, i.e. of both particulate
and biological origin, and the figures obtained correlate well with turbidity
measurements taken at the ponds by the plant personnel, (Appendix A).
Differences in disc measurements between secondary and tertiary effluents,
as they occur in the ponds, are generally quite marked, with the tertiary
pond registering greater clarity with one exception. Overall the tertiary
pond average 61% greater clarity over the period of these measurements,
taken during the hottest time of the year.
It is interesting to note that the tertiary pond, at the time when disc
measurements were less than those of the secondary (27 August) was
experiencing an abnormally high bloom of a coccoid blue-green alga.
The bloom caused the pond to appear uniformly bluish-to light green.
The pH at that time was on the decline to the point where it had just
fallen below that of the secondary pond. Total phosphate and alkalinity
were also low.
2. Floristic analysis of the algal growths in ponds receiving secondary
and tertiary effluents.
The algal flora in a pond receiving a specifically treated and presumably
well defined effluent might be expected to display uniform algal growth,
particularly in a region such as Santee where climatic fluctuation is
relatively limited. Unfortunately it was not possible to demonstrate
evidence in support of this expectation as indicated by the summary of
predominant species in Tables 38 and 39. The floristic changes, in terms
of species composition and quantity, are as dramatic as any which have
been found in any natural situation of an undefined nature (Tailing, VII-6).
Although the study ponds did not support permanent populations of ducks
or other waterfowl, transient visitors could be expected in almost any
outside pond. Early morning surveys would be needed to confirm this.
At Santee, bullfrogs were always apparent and more than once a large
bittern was flushed from a small cluster of cattails which grew in the
corner of one pond. By comparison with the nutrient status of the pond
water, the enrichment contribution from these animals was undoubtedly
negligible, but they act as a source of inoculation of the pond with new
algal components carried by feet or feathers and in the alimentary tract.
Atmospheric transportation of all manner of spores, cysts, and propagules
can be expected. Although it had been anticipated with the sources of
water available, that the flora of the ponds would be relatively uniform,
this was not the case. The composition of the algal flora was as varied
and completely unrelated between ponds as is usually the case with
freshwater environments.
69
-------
An effort was made to relate the color of the water in the different
ponds with the species composition. These data are included in Tables 38
and 39. In general the secondary study pond consistently appeared more
intensely green and more murky or turbid than the tertiary pond.
Color in the second pond varied from yellowish-green to dark grassy green.
The most abundant organism, when yellow-green was noted, was Chlamydomonas,
while another green flagellate (Chlorogonium), considerably larger than
Chlamydomonas, was present in large numbers. However, the second flagellate
was equalled in numbers by a blue-green filament which, by stieer size
and pigmentation differences, apparently did not contribute much to pond
hue. The presence of blue-green algae in less than bloom quantity is not
easily detected. The figures can be misleading until the exceedingly
small size of most blue-green algae is considered in comparison to the
larger green flagellates, such as Chlorogonium. When two green and two
blue-green organisms were present in almost equal numbers, the secondary
pond appeared dark grassy green. It is interesting that on the same day
(25 August) the tertiary pond a few feet away contained an excessive
amount of tiny coccoid blue-green alga and appeared bluish to light green.
Color is, therefore, not too relable a visual index of the constituents
of the population supported by the pond. A correlation appears between
turbidity and dry weight samples. It is suggested that Secchi-disc
measurements be taken as a rapid, simple indication of standing crop.
Unfortunately this was not appreciated until it was too late for meaning-
ful incorporation in the present study. It is also suggested that water
samples be taken more frequencly, preferably weekly, in order to monitor
more precisely the changes in biotic composition.
3. Correlation between Chemical Parameters and Algal Biomass.
Correlation by visual consideration of the data failed to disclose any
obvious relationship between the amount and kinds of organisms present
and the chemical analyses of the study ponds (Appendix A).
An attempt was therefore made to relate the various chemical and physical
parameters with the algal biomass over a series of periods throughout the
duration of testing. A correlation coefficient was claculated for both
secondary and tertiary effluents relating the algal biomass at the end
of the test periods with the average values for each chemial and physical
parameter during the period. The correlation coefficients are presented
in Tables 40 and 41. The highest correlation coefficient with secondary
effluent occurred with alkalinity and that was merely 0.52; no other
value exceeded 0.50. For tertiary effluent, the correlation coefficients
were slightly higher, but even so were not of any great significance.
The highest value occurred for the correlation between nitrate filtrate
and biomass and that was only .89. These data indicate that there is no
acceptable correlation between the biomass and any chemical parameters
for the two effluents when considered individually. However, the comparison
between the growth in the two ponds substantiates the laboratory results.
70
-------
TABLE 40 CORRELATION BETWEEN BIOMASS and PHYSICAL/CHEMICAL PARAMETERS - Secondary Effluent
Correlatfon, over 8 test periods, between the blomass on the last day of each period and
the average values for various chemical and physical parameters for each period, in ponds
receiving Santee secondary effluent.
Periods, indicated
in days of the year
51-78
78-91
91-105
I05-J39
139-172
1 72-203
203-237
CORRELATION
^ COEFFICIENT
Biomass, on last
day of period
(in mg/Jl)
60
142
36
62
2
63
109
139
Total Phosphate
Orthophosphate
Alkal inity
Ammonium f f It rate
Suspended sol ids
COD
COD f I It rate
pH
Nitrite filtrate
Nitrate fl Itrate
Turbidity, Avg.
Last day turbidity
Number of points1
1 1.4
10.6
175
4.2
33
54.8
44.9
7.4
2.50
1.4
6.5
4.0
8
10.8
10.3
148
2.5
37
51.6
39.1
7.3
0.94
1.8
6.8*
8.5
3
12.2
11.1
120
4.0*
46
55,0
37.3
7.3
0.52
1 .7
8.3
13.0
4
1 1.9
10.7
149*
l.73f
42
63.9*
45.5
7.3
0.56*
1.4
10.9
7.0
8
13.3
10.0
139
1.32
41*
45.8 x
34.8
7.4*
0.48
1.4
7.3*
7.0
9
15.1
12.3
175
3.38
51
54.3
38.7
7.4
0.54
I.I
9.5
10.0
8
14.7
13.4
260
44*
53.5
7.1
7.5
9.0
8
10. 5f
10. 5f
202
37f
64. 4f
7.3
9.0
14.0
21
"
.37
.16
.52
.12
.32
.42
.30
.50
.19
.42
.05
.32
^Number of points indicates number of days data taken in each period
* Number of data points less than indicated for particular period
t Outlier excluded from data
-------
TABLE 41 CORRELATION BETWEEN BIOMASS and PHYSICAL/CHEMICAL PARAMETERS - Tertiary Effluent
Correlation, over 7 test periods, between the bfomass on the last day of each period and
the average values for various chemical and physical parameters for each period, in ponds
receiving Santee tertiary effluent.
NJ
h'erioa, indicated
In days of the year
Blomass, on last
day of period
(in mg/JH
Total Phosphate
Orthophosphate
Alkalinity
Ammonium f 1 Itrate
Suspended sol ids
COD
COD filtrate
pH
Nitrite filtrate
Nitrate f i Itrate
Turbidity, Avg.
Last day turbidity
Number of points1
51-78
30
1.4
1.3*
214
3.6
17*
34.6
30.2
7.8
0.15
1.5
3.4
2.0
14
78-91
104
1.6*
1.7*
209
1.6*
22*
32.1
29.8*
7.8
0.38*
1.9*
2.1
1.0
7
9 1 - 1 05
36
1.8*
1.8*
160
3.8*
27
29.4
29.0*
7.9
0.38*
1.4*
2.5
3.3
8
105-139
20
2.0*
1.9*
198*
1.4*
24*
29.5*
27.6*
7.8
0.23*
1.5*
2.8
1.5
17
139-172
12
1.0*
0.9*
155*
2.7*
32*
23.0
21.1*
8.2
0.28*
1.4*
2.8
2.5
18
17? ?rn ?rn 9V7 7V7 9fifi CORRELATION
172-203 203-237 237-266 COEFF|C|ENT
17 21 28
0.6 0.2*
0.3 O.I*
128
4.1
27
24.7
8.2
0.26
1.2*
2.8
3.0
12
-
.30
.40
.52
.44
.39
.53
.53
.52
.57
.89
.70
.61
1 Number of points Indicates number of days data taken In each period
* Number of data points less than indicated for particular period
t Outlier excluded from data
-------
SECTION VIII
REFERENCES
IV-1 Algal Assay Procedure - Bottle Test
National Eutrophication Research Program
Environmental Protection Agency, August 1971
IV-2 Provisional Algal Assay Procedures - October 1971
VII-1 Almgren, Howard: Tertiary Treatment by Lime Addition at
Santee, California. Office of Research and Monitoring,
U.S. Environmental Protection Agency, 1972.
VII-2 Murray, S. et.al., Evaluation of Algal Assay Procedure -
PAAP Batch Test, Journal Water Pollution Control Federation
43, No. 10, pp 1991-2003 (1971)
VII-3 FWPCA Methods for Chemical Analysis of Water and Wastes,
Analytical Quality Control Lab., Cincinnati, Nov. 1969.
VII-4 Middlebrooks, E.G. et.al., Eutrophication of Surface Water-
Lake Tahoe, Journal Water Pollution Control Fed., 43, No. 2
pp 242-251 (1971).
VII-5 Miller, W.E. and Maloney, T.E., Effects of Secondary and
Tertiary Wastewater Effluent on Algal Growth in a Lake-
River System, Journal Water Pollution Control Fed., 43
No. 12, pp 2361-2365 (1971).
VII-6 Tailing, F.G., Element of Chance in Pond Populations,
Naturalist, pp 157-170 (1951).
73
-------
SECTION IX
PUBLICATIONS
1. Evaluation of Algal Assay Procedures-PAAP Batch Test
by Steven Murray, Jan Scherfig, and Peter S. Dixoii
Journal WPCF, October 1971.
2. Algal Assay Procedure Bottle Test, National Eutrophication
Research Program, Environmental Protection Agency, August
1971.
3. Evaluation of Materials for Use in Algal Culture Systems,
by, Carol Justice, Steven Murray, Peter S. Dixon, Jan
Scherfig, in Hydrobiologia. vol. 40-2, pp 215-221, 1972.
-------
SECTION X
GLOSSARY
No special terms, abbreviations, or symbols used.
75
-------
SECTION XI
APPENDICES
Page
A-l Chemical Tests on Study Pond Two, Part 1 77
A-l Chemical Tests on Study Pond Two, Part 2 78
A-2 Chemical Tests on Study Pond Three, Part 1 79
A-2 Chemical Tests on Study Pond Three, Part 2 80
A-3 Dissolved Oxygen Tests on the Study Ponds 81
76
-------
TABLE A-l CHEMICAL TESTS ON STUDY POND TWO, PART 1.
Date
Nov. 16, '70
Nov. 30
Dec. 7
Dec. 14
Dec. 28
Jan. 11, '71
Jan. 25
Feb. 8
Feb. 22
March 8
March 22
April 5
April 19
May 3
May 17
June 7
June 21
July 12
July 26
Aug. 9
Day of
Year
320
334
341
348
362
11
25
39
53
67
81
95
109
123
137
158
172
193
207
221
Ortho-phosphate
Filtrate
mg/l-P
0.96
8.8
-__
11.2
5.80
6.80
8.0
3.6
2.20
0.35
3.60
1.20
3.20
1.23
1.40
8.4
3.40
16.0
1.76
1.60
Total-
Phosphate
mg/l-P
0.98
12.0
-__
12.0-
6.60
7.20
9.0
5.0
_ •. »
1.00
4.80
1.76
3.80
24.0
3.8
10.0
5.80
16,4
4.60
3.8
Ammonia
Filtrate
mg/l-N
9.38
3.0
___
9.6
5.85
3.68
6.75
5.5
7.8
0.82
1.80
0.27
0.08
0.12
0.17
.92
2.90
0.08
0.22
2.20
Nitrite
Filtrate
mg/l-N
0.76
2.0
...
0.39
2.1
3.0
2.50
2.0
___
0.16
0.90
0.46
0.34
0.34
0.26
.32
0.44
0.19
0.13
0.29
Nitrate
Filtrate
mg/l-N
0.50
0.85
0.38
0.35
0.80
0.62
1.0
0.90
1.60
1.60
1.60
1.0
1.10
0.80
.90
3.5
2.20
0.60
0.22
Total
Kjeldahl
mg/l-N
13.2
6.0
_._
21.9
9.3
10.5
12.9
14.1
__-
2.9
10.2
0.78
6.36
8.4
4.83
8,19
8.19
6.03
4.52
7.81
-------
TABLE A-l CHEMICAL TESTS ON STUDY POND TWO, PART 2.
Day of
Year
320
334
341
348
362
11
25
39
53
67
81
95
109
123
137
158
172
193
207
221
pH
7.5
8.2
8.6
7.6
7.6
8.3
7.9
8.7
7.8
8.2
7.8
9.5
9.4
9.6
9.1
8.0
8.2
9.2
9.4
9.1
Suspended
Solids
mg/1
27
71
___
53
46
50
49
96
26
34
87
133
71
96
63
89
52
86
105
90
Turbidity
JTU
6.7
14
...
8
10
12
12
12
6
17
22.0
19
22.5
20
22
8.0
17.0
27.0
27
22
COD
mg/1
50.4
72.0
...
76.0
51.4
75.8
86.6
100.0
58.9
19.7
67.9
73.6
61.6
76.8
65.7
40.4
48.8
69.5
79.2
76.4
COD
Filtrate
mg/1
40.0
,44.'0
...
49.3
31.8
52.1
51.5
51.0
•• •.*
10.7
40.0
42.0
37,6
49.7
32.4
33.6
42.8
38.4
44.6
56.6
Color
40
60
60
40
35
60
70+
70+
40
30
25 '
40
30
40
30
25
25
40
40
40
Temp.
°C
16
15
...
15
13
12
14
18
16
17
18
20
16
18
1*9
23
22
24
23
24
Dissolved
Oxygen
' mg/1
10.6
8.7
...
9.6
13.5
15.0+
15+
15+
8.3
13.4
13.4
13.8
12.6
10.8
8.6
9.0
8.1
7.8
7.6
8.9
Alkalinity
mg/1
222.0
152.0
...
243.0
263.0
224.0
260.0
217.0
278.0
109.0
169.0
67.9
84.0
2.6
83.0
189.0
221.5
117.2
84.0
118.8
•vj
CXI
-------
TABLE A-2 CHEMICAL TESTS ON STUDY POND THREE, PART 1.
Date
Nov. 16, '70
Nov. 30
Dec. 7
Dec. 14
Dec. 28
Jan. 11, '71
Jan. 25
Feb. 8
Feb. 22
March 8
March 22
April 5
April 19
May 3
May 17
June 7
June 21
July 12
July 26
Aug. 9
• Day of
Year
320
334
341
348
362
11
25
39
53
67
81
95
109
123
137
158
172
193
207
221
Ortho-phosphate
Filtrate
mg/l-P
0.98
0.56
...
0.68
0.10
0.34
0.50
0.34
1.55
0,88
0.74
0.28
0.80
0.19
0.42
0.24
0,13
0.-88
0.23
0.05
Total
Phosphate
mg/l-P
1.0
0.68
...
0.70
0.46
0.39
0.66
0.70
...
1.65
1.25
0.62
1.55
0.88
1.17
0.63
0.52
1.35
0.79
0.40
Ammonia
Filtrate
mg/l-N
4.70
2.18
__.
6.6
5.40
3.0
5.33
2.9
5.2
0.25
1.55
2.2
0.30
0.07
0.13
0.01
1.55
0.86
0.05
0.42
Nitrite
Filtrate
mg/l-N
0.46
2.2
___
0.54
0.66
0.54
0.21
0.49
.--
0.16
0.15
0.50
0.32
0.36
0.37
0.50
0.25
0.14
0.14
0.28
Nitrate
Filtrate
mg/l-N
0.65
0.83
___
0.65
0.37
0.40
1.50'
1.2
1.0
2.0
1.80
1.60
1.4
1.30
0.85
1.0
0.15
1.10
0.60
0.50
Total
Kjeldahl
mg/l-N
5.7
3.9
___
10.2
6.0
9.6
10.5
3.01
— — .
1.5
8.7
3.73
4.62
7.5
4.53
6.33
2.61
1.89
12.82
3.72
-------
TABLE A-2 CHEMICAL TESTS ON STUDY POND THREE, PART 2.
Day of
Year
320
334
341
348
362
11
25
39
53
67
81
95
109
123
137
158
172
193
207
221
PH
8.6
9.7
9.3
9.2
9.3
9.4
9.3
9.8
8.1
9.4
8.6
10.0
9.7
10.0
9.8
10.0
9.4
9.2
9.4
9.0
Suspended
Solids
mg/1
12
25
...
35
41
27
28
57
27
30
78
79
67
82
50
95
59
46
54
47
Turbidity
JTU
2.2
3
...
5.8
12
3
8
12
11
13
15.0
12
12
15
13
13
10
11
6
12
COD
mg/1
30.0
30.4
...
47.9
41.0
43.9
70.8
68.3
60.7
32.8
59.1
78.4
52.0
69.1
69.0
66.0
58.6
47.7
32.2
46.6
.COD
Filtrate
rag/1
27.6
28.0
...
38.2
28.5
34.2
49.9
45.6
MOT M
28.3
40.9
44.0
27.0
41.2
39.1
38.0
39.3
38.4
27.5
36.1
Color
20
20
25
25
25
25
70+
60
45
50
45
35
25
40
30
45
30
20
25
40
Temp.
°C
15
15
w • «*
15
12
12
14
18
16
16
19
19
16
18
19
24
20
25
23
25
Dissolved
Oxygen
mg/1
11.2
8.5
...
10.2
11.8
15+
15+
15+
6.6
17.1
14.6
14.8
10.6
10.2
8.8
15.0+
8.7
7.2
7.4
9.2
Alkalinity
mg/1
223
128
___
185
207
167
154
165
212
197
•188
28
77.7
76,0
74.5
56.2
26.1
53.7
43.6
21.8
oo
o
-------
TABLE :A-3 DISSOLVED OXYGEN TESTS ON THE STUDY PONDS.
Time & Date
1500, Aug. 19, 71
1700
1900
2100
2300
0100, Aug., 20, 71
0300
0500
0700
0900
1100
1300
1500
Pond
D.O., mg/1
10.2
10.1
10.3
8.0
6.8
6.5
4.2
2.8
2.6
5.7
9.5
9.7
9.6
Two
Temp . , °C
30
27
26
26
25.5
25
25
24.5
24.25
25
27.5
30.5
30
Pond T
D.O., mg/1
9.8
9.6
9.9
7.1
6.6
5.9
4.3
3.3
3.4
5.3
5.9
8.5
9.2
tiree
Temp., °C
30
27
26
26
25.5
25
25
24.5
24.5
25
27
29.5
28
81
-------
1A . „ . 2_ , . . _. ,, . _ SELECTED WATER RESOURCES ABSTRACTS
Accession Number Subject Field & Group INPUT TRANSACTION FORM
Organization University of California, Irvine
-c
EFFECT OF PHOSPHORUS REMOVAL PROCESSES ON ALGAL GROWTH
Author (s) Jan Scherfig Project Designation
p WP-01446-01
Richard Appleman
Carol />. Justice 21,, .
Note
22
C-i tation
Environmental Protection Agency report number,
EPA-fifiO/l-71-m S. ScTil-pmhg
23 J
Descriptors (Starred First)
Algal assays, batch algal assays, continuous culture assays, tertiary waste
treatment, algae control, eutrophication, water reclamation, growth limiting
nutrients.
"^Identifiers (Starred First)
Algal assay procedures, growth limiting nutrients, algae growth control.
27Abstract
Laboratory studies were conducted to improve algal assay techniques for use in
evaluation of sewage treatment processes.
Laboratory studies (batch and continuous cultures) were conducted at the Santee
California water reclamation plant to evaluate the effect of tertiary waste
treatment processes on the amount of algal growth in the treated effluent.
Laboratory studies were also conducted to determine the growth limiting nutrients
in each type of tertiary effluent.
Field tests were conducted using special study ponds and the results of the field
tests were compared with the laboratory test results. The laboratory and field
tests showed the same relative ranking for the treated effluents.
Abstractor Institution
Jan Scherfig University of California, Irvine, CA 92664
------- |