WATER POLLUTION CONTROL RESEARCH SERIES 113O3O ELY O6/7I-I3
REC-R2-TI-I3
DWR NO. 174-16
BIO-ENGINEERING ASPECTS OF AGRICULTURAL DRAINAGE
SAN JOAQUIN VALLEY, CALIFORNIA
REMOVAL OF NITRATE BY AN ALGAL SYSTEM
PHASE n
JUNE 1971
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BIO-ENGINEERING ASPECTS 01 At ICULTURAL DRAINA
SAN JQA UIN VALLEY, CALIFORNIA
The Bio -Engineering Aspects of Agricultural Drainage
reports describe the results of a unique Interagency study
of the occurrence of nitrogen and nitrogen ren va1 treat-
ment of subsurface agricultural vastewaters of the San
Joaquin Valley, California.
The three principal agencies Involved In the study are
the Environmental Protection Agency, the United States
Bureau of Reclamation, and the California Department of
Water Resources.
Inquiries pertaining to the Blo-Etigineering Aspects of
Agricultural Drainage reports should be directed to the
author agency, but xxmy be directed to any one of the three
principal agencies.
THE REPORPS
The first, three-year phase of the interagency study is
to be reported upon in a series of twelve reports.
The second, one-year phase of the Interagency study was
limited to continued work on the two principal treatment
methods. The second phase work develops design criteria
and operational parameters for full-scale treatment
facilities.
This report, “R 4OVAL OF NITRATE BY AN ALGAL SY 1 ( --
PHASE II”, and the companion report, “DENITRIFICATION BY
ANAEROBIC FILTERS AND PONDS - - PHASE II”, contain the
results of the second phase of the interagency study.
These two reports are numbered sequentially, after the
first twelve, in the series entitled Bio-Engirieering
Aspects of Agricultural Drainage, Sari Joaquin Valley,
California”.
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BIO-ENGINEERING ASPECTS OF AGRICULTURAL DRAINAGE
SAN JOAQUIN VALLEY, CALIFORNIA
REMOVAL OF NITRATE
BY AN
ALGAL SYSTEM
PHASE II
Prepared by
California Department of Water Resources
William R. Gianelli, Director
The agricultural drainage study was conducted under the direction of:
Robert J. Pafford, Jr., Regional Director, Region 2
UNITED STATES BUREAU OF RECLAMATION
2800 Cottage Way, Sacramento, California 95825
Paul DeFalco, Jr., Regional Director, Pacific Southwest Region
WATER QUALITY OFFICE, ENVIRONMENTAL PROTECTION AGENCY
760 Market Street, San Francisco, California 94102
John R. Teerink, Deputy Director
CALIFORNIA DEPARTMENT OF WATER RESOURCES
1416 Ninth Street, Sacramento, California 95814
DWR-WQO Grant 13030ELY6/71-13
DWR-USBR Contract #14-06-200-3389A
June 1971
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REVIEW NOTICE
This report has been reviewed by
the Environmental Protection Agency
and the U. S. Bureau of Reclamation,
and has been approved for publica-
tion. Approval does not signify
that the contents necessarily
reflect the views and policies of
the Environmental Protection Agency
or the U. S. Bureau of Reclamation.
The mention of trade names or
commercial products does not
constitute endorsement or recom-
mendation for use by either of the
two federal agencies or the Cali-
fornia Department of Water Resources.
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ABSTRACT
Major findings are presented from a one-year operational
investigation conducted at the Interagency Agricultural
Wastewater Treatment Center (IAWTc) on the use of algae to
remove nitrogen from subsurface agricultural tile drainage
in the San Joaquin Valley of California. The objectives of
the study were to: (i) refine the design criteria, deter-
mined in a preliminary investigation, (2) develop operational
procedures, and (3) recommend a design for a prototype algal
nitrogen removal process.
The investigation demonstrated that the governing factors
affecting the algal nitrogen removal process are the total
amount of light available to the actively photosynthesizing
algae and the influent nitrogen loading. Accordingly, if
these two factors are known, the area required for maximum
nitrogen removal can be approximated.
Turbid conditions, resulting from the suspension of non-
photosynthesizing material during continuous or intermittent
mixing, were found to be detrimental to the prolonged opera-
tion of the system. Maximal nitrogen assimilation also
depended upon providing a completely balanced nutrient system,
and varying amounts of supplemental carbon, phosphorus, and
iron were required throughout the year.
Algal harvesting studies indicated that 90 percent or more
of the algae could be removed throughout the year, under con-
tinuous operation, using a chemical-flocculent-sedimentation
process but that the chemical additions required were
dependent upon a number of algal growth factors.
Continuous operation of algal test units during 1970 showed
the algal nitrogen removal process was capable of effectively
reducing the influent nitrate-nitrogen concentration and
other nutrients. The process reduced a vary-ing influerit
nitrogen concentration of from 15 to 30 mg/i NO -N to
1 to 4 mg/i soluble effluent nitrogen throughou € the year
using varying operating parameters.
Recommendations are also given for the design and testing of
“prototype” algal nitrogen removal plants using a modifica-
tion of the stirred reactor design, and a proposed ‘ slug-
flow” algal nitrogen removal system designed to correct
many of the inadequacies inherent in the first system.
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BACKGROUND
This report is one of a series which presents the findings of
intensive interagency investigations of practical means to
control the nitrate concentration In subsurface agricultural
wastewater prior to Its discharge Into other water. The pri-
mary participants In the program are the Water Quality Office
of the Environmental Protection Agency, the United States
Bureau of Reclamation, and the California Department of Water
Resources, but several other agencies also are cooperating in
the program. These three agencies Initiated the program
because they are responsible for providing a system for dis-
posing of subsurface agricultural wastewater from the San
Joaquin Valley of California and protecting water quality in
California’s water bodies. Other agencies cooperated in the
program by providing particular knowledge pertaining to
specific parts of the overall task.
The need to ultimately provide subsurface drainage for large
areas of agricultural land in the western and southern San
Joaquin Valley has been recognized for some time. In 1954,
the Bureau of Reclamation Included a drain in Its feasibility
report of the San Luls Unit. In 1957, the California
Department of Water Resources initiated an investigation to
assess the extent of salinity and high ground water problems
and to develop plans for drainage and export facilities. The
Burns-Porter Act, in 1960, authorized San Joaquin Valley
drainage facilities as part of the State Water Facilities.
The authorizing legislation for the San Luls Unit of the
Bureau of Reclamation’s Central Valley Project, Public Law
86-488, passed in June 1960, included drainage facilities to
serve project lands. This Act required tInt the Secretary of
Interior either provide for constructing the San Luis Drain
to the Delta or receive satisfactory assurance that the State
of California would provide a master drain for the San Joaquin
Valley that would adequately serve the San Luis Unit.
Investigations by the Bureau of Reclamation and the Department
of Water Resources revealed that serious drainage problems
already exist and that areas requiring subsurface drainage
would probably exceed 1,000,000 acres by the year 2020. Dis-
posal of the drainage into the Sacramento-San Joaquin Delta
near Antioch, California, was found to be the least costly
alternative plan.
Preliminary data indicated the drainage water would be rela-
tively high in nitrogen. The then Federal Water Quality
Administration conducted a study to determine the effect of
discharging such drainage water on the quality of water in the
San Francisco Bay and Delta. Upon completion of this study in
1967, the Administration’s report concluded that the nitrogen
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content of untreated drainage waters could have significant
adverse effects upon the fish and recreation values of the
receiving waters. The report recommended a three-year
research program to establish the economic feasibility of
nitrate-nitrogen removal.
As a consequence, the three agencies formed the Interagency
Agricultural Wastewater Study Group and developed a three-year
cooperative research program which assigned specific areas of
responsibility to each of the agencies. The scope of the
investigation included an inventory of nitrogen conditions in
the potential drainage areas, possible control of nitrates at
the source, prediction of drainage quality, changes in nitrogen
in transit, and methods of nitrogen removal from drain waters
Including biological-chemical processes and desalination.
vi
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C ONCLUS IONS . . . . . . . . . . . . . . . . . a
R.ECO!4ME.NDATIONS . . . . . . . . . . . . . . . .
I
THEORYANDRATIONALE.........
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Introduction . . . . , , . • , . . • •
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PhaselConclusions .........
PhasellObjectives •........
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ApproachRationale..........
Theory of Algal Nitrogen Assimilation
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Nitrogen Assimilation Mechanism . . .
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TypesofAlgalGrowth ........
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METHODSANDMATERIALS .........
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Experimental Procedures . . . . . . .
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Analytical Methods . . . . . . . . . . .
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Test Units . . . • , . . • . . , • • .
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II]
RESULTS AND DISCUSSION . . . . . . . .
Operational Procedures (Phase II, 1970)
Plant lnfluent . . . . • . . •
Factors Affecting Nitrogen Assimilation
Algal Species and Predation . . . . .
Cellular Nitrogen . . . . . . . . .
Effluent Soluble Organic Nitrogen . .
Operational Problems . . . . . . .
Pesticide Analysis . . . . . . . .
Evaporation During 1970 . . . . . .
DiurnalStudies ..
Operation Criteria -- 1970 . . . . .
IV ALGAL HARVESTING AND DISPOSAL . .
Algal Harvesting . . . . . . . . . .
AlgalDiaposal . .
V PROCESSEVALUATION...........
Summary of the Phase II Investigation
Costof Treatment
VI PROPOSED ALTERNATIVE TO THE MIXED-REACTOR
SYSTEM SThDIED IN PHASES I AND U . • . . . 115
A C1çNQWI.EDGEMEINTS . . . . . . . . • . • • , • •
H E ’ERE .ICES . . . . . . . . . . , . . . • • • •
PUBLICATIONS , . . . . . . • , • • • • • • • . .
CONTENTS
Ch ter
Page
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F IGURES
No. P e
1 Probable Nitrogen Pathways in Algal
Growth Unite . . . . . . . . . . 9
2 The Characteristic Pattern of Growth Shown
by a Unicellular Alga in a Culture of
LimitedVolume ..... .......... 11
3 ProJected Flow and Nitrogen Concentration of
San Joaquin Valley Agricultural Was tewaters
and Actual IAWTC Influent Nitrogen
Concentration................. 13
4 Schematic of IAWTC Facilities . . . . . . . . . . 15
5 Volatile Solids vs Absorbance . . . . . . . . . . 18
6 Algal Growth Units at IAWTC . . . . . . . . . . . 20
7 Monthly Variation in Total Dissolved Solids
and Total Alkalinity in Plant Influent
for 1970 . . . . . . . . . . . . . . . . 25
8 Seasonal Variation in Total Dissolved Solids
and Nitrate-Nitrogen in Treatment Plant
Influent . . , . . . . • . . . . . . . . . . 25
9 Effluent Nitrogen Concentration as Affected
by Depth at Mean Detention Time . . . . . . • • 29
10 Effect of Carbon Dioxide Addition on Effluent
Nitrogen Concentration at Three Depths . • • • 29
11 uantitat1ve Nitrogen Assimilation as Affected
by Depth - - Grams per Day per Unit . . . . . . 30
12 Light Energy Received During 1970 . . . . . . . . 31
13 Nitrogen Removal -- in Miniponds at Different
Depths and Light Energy Levels . . . . . . . . 32
14 Nitrogen Assimilation in One-quarter-acre Unit
at 2 1 4-inch Depth . . . . . . . . . . . . . . . 33
15 Remaining Light Due to Wall Shading . . . . . . . 34
16 Nitrogen Assimilation as Affected by
Temperature . . . . . . . . . . . . 36
viii
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FIGURES (Continued)
No, Page
17 Volatile Solids Increase with Time as Affected
byTemperature ................ 37
18 Changes In Nitrate, Algal Cell Counts and
Volatile Solids in Cultures of Different
Volumes . . . . . . . . , . . . . . . . • 38
19 Effect of Temperature on Algal Nitrogen
Assimilation . . . . . . . . . . a . . a . . 39
20 Effect of Day or Night Mixing on Nitrogen
Assimilation . . . . . . . . . . . . . . . .
21 Effect of Mixing and No Mixing With and Without
Addition of’ Carbon Dioxide on Nitrogen
Ass i 1ation . . . . . . . . . . . . . .
22 Suspension of Solids During Mixing . . . . . . 112
23 Effect of Detention on Nitrogen Assimilation
In 8-inch and 12-inch Units . . • . . . . . . • 113
211 NItrogen Removed at Different Detention Times
at 12-inch Depth with CO 2 . . . . . . . .
25 The Effect of Various Levels of Aeration on
Nitrate Reniova]. in the Light Box . . . a • . 118
26 Effect of Surface/Volume Relationship and
Various Types of Aeration on Cell Numbers
at Six Days, Average of Five Replications . . • 118
27 TypIcal Shift in pH and HC0 3 ’ in Algal
Test Units • • • • • , • • . . . • • , • • 149
28 Changes In Total Influent Nitrogen and
Effluent Total Soluble Nitrogen, Nitrite
and Soluble Organic Nitrogen -- Average
of All Units . e . . a a a a • • • • • a • • 50
29 Changes In Nitrite Concentration in Test Units
With and Without CO 2 Addition . a a a a a a • 51
30 Changes in Total Influent and Effluent Nitrogen
in Cntermed1ate Detention Time Units With and
Without Carbon Dioxide Addition , . a • a . • , 52
31 Carbon Dioxide Addition by Automatic
pH Control a a a a a a • a a a a a a a a a a a 53
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FIGURES (Continued)
No. Page
32 Phosphate Solubility as a Function of pH . . . . 55
33 Solubility of Ferric Hydroxide in Water
4.. # Of’I
O . ‘.1 a a a a e a . a • a a a a a a a a a a a
3)4 Solubility of Ferric Phosphate in Water
a C..) # • • . a a S S S • • • • • • • • • •
35 Effect of Varying Iron and Phosphorus
Concentrations on Nitrogen Assimilation --
LightBoxStudy.... ............ 59
36 Effect of Iron or Phosphorus Addition on
Nitrogen Assimilation with Carbon Dioxide
Addition . . . . . . . . a a . . . . . a • • • 61
37 Effect of Iron Addition on Nitrogen Assimilation
Without Carbon Dioxide Addition . . . . . . . . 61
38 Inf] .uent and Effluent Phosphorus Concentrations
In Units with Normal and Phosphorus-
Supplemented Tile Drainage . . . . . . . . . . 62
39 Typical Sludge Accumulation in Test Units • . . , 62
110 Iron and Phosphorus Solubility at Different
pH Values . • . . • . • . a • a a a a • a a a 614
41 Preferential Algal Uptake of Ammonia Over
Nitrate . . • . . . . . . . . . . . 67
112 Nitrogen Assimilation in Tile Drainage from
Three Sumps . . . . . . . . . . . . . . . . . 70
43 Comparison of Trace Metal Analysis of Filtered
and Unfiltered Tile Drainage . . . . . . . . . 73
41 Effluent Nitrogen Level in Soil Units . . . . . . 74
15 Effect of Blomass Control on Nitrogen
Assimilation . . . . a . a . • . . a . • • . a 77
46 Effect of a Nitrogen-deficient Medium
on Algal Nitrate Assimilation . . . . . . , . . 82
LIT Evaporation minus Precipitation --
EAWTC, 1970 . . . . . a a • • . . . . • a a • 87’
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FIGURES (Continued)
No. Page
48 Eight- and Twelve-inch Test Units with Lowest
Effluent Nitrogen Concentration During 1970 . . 90
49 Average Soluble Nitrogen Removed During 1970
as Related to Detention Time and Depth . . . . 92
50 Average Soluble Nitrogen Removal at Various
Depths in Intermediate Detention Time Units . . 93
51 ProJected Acres Required at Different Nitrogen
Concentration Necessary to Achieve a 2 - 4 mg/i
Effluent Concentration -- April Operating
Conditions and a Flow of 300 cfs . . . . . • • 914
52 Projected Acres Required at Different Nitrogen
Concentration Necessary to Achieve a 2 - 14 mg/i
Effluent Concentration -- August Operating
Conditions and a Flow of 300 cfs . . . . . . . 94
53 Chemical Cost Required for Algal Concentration. . 98
54 Chemical Cost to Remove Total Suspended Solids
tollOmg/lDuringl97O ............ 100
55 Effect of Bicarbonate Alkalinity on Iron Sulfate
Required to Reduce Suspended Solids to
40 mg/i . . . . . . . . . . . . . . . . . . . . 101
56 Cost of Separation in Shallow Depth Sedimentation
UnitDuringl97O ............... 101
57 Schematic of Flotation Chamber Tested at IAWTC. . 102
58 Theoretical Nutrient and Light Availability to
Algae in Two Types of Algal Reactors . . . . . 116
59 Theoretical Algal Growth Phases in Two Types
ofAlgalReactors . . . . . . . . . . . . . . . 116
60 Schematic Plan of Proposed Slug-Flow System . . 118
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TABLES
No. Page
1 operational Schedule for Phase II Studies,
1970 . . . . . * . . . . . . . . . . . . . . 1L
2 Chemical Analysis Schedule . . . . . . . . . . 16
3 Percent of Total Pond Materials Pound in Pond
Sludge - - During Mixing . . . . . . . . . . . . 63
Average Dissolved Effluent Phosphate in
Minipond During 1970 . . . . . . . . . . . . . 65
5 Summary of Micronutrient Trace Mineral
Requirements ................. 68
6 Trace Analysis -- 1970 . . . . . . • . • • . . 72
7a Nitrate in the Water of Opaque and Clear
Cylinders (Summer Test) . . . . . . . . . . . 75
7b Nitrate in the Water of Opaque and Clear
Cylinders (Fall Test) . . . . . . . . . . . . . 76
8 Predominant Algal Species During 1970 at IAWTC. . 79
9 Average Percent Algal Cellular Nitrogen Found
Under Different Growth Conditions in 1970 . . , 8].
10 Average Effluent Concentration of Organic
Nitrogen in Various Test Units During 1970 . . 83
11 Pesticide Analysis, 1970 . . . . . . . . . . . 85
12 Effect of Nutrient Addition and Mixing on
Nitrogen Removal, 1970 Studies . . . . . . 89
13 Average Total Soluble Nitrogen Removed, 1970 . . 91
114 Estimated Average Chemical Cost per Million
Gallons Separated (1970) (Jar Test Data) . . . 99
15 Estimated Algal Production by an Algal
Stripping Plant, 1975-2000 . . . . . . . a • io6
16 Amino Acid Analysis • • . a . a a a a a a a . 107
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CONCLUSIONS
Based on the results of this investigation, the following
may be concluded:
1. Removal of nitrogen from agricultural tile drainage
using the algal nitrogen removal process is a technically
feasible process on a year-round basis.
2. The total amount of soluble nitrogen in the plant
effluent will probably vary between 2 and 4. mg/i, depending
on the influent nitrogen loading and environmental
conditions. In addition, from 1 to 2 mg/i algal cellular
nitrogen can be expected in the plant effluent, depending
upon the degree of removal of suspended solids in the
separation process.
3. The principal factors affecting maximum algal nitrogen
assimilation are the total amount of light available to
the actively photosynthesizing algae and the influent
nitrogen loading.
A . Depending upon the total available light and nitrogen
loading, detention times required for maximum nitrogen
removal during 1970 varied between 5 and 16 days, and
operating depths varied between 8 and 12 inches.
5. Turbid conditions resulting from the suspension of
nonphotosynthesizing material during continuous or
intermittent mixing of an algal reactor are detrimental to
the prolonged operation of the system.
6. The optimal carbon-to-nitrogen nutrient ratio in the
system is approximately 5 to 1. If the carbon demand
exceeds the carbon available in the growth medium,
supplemental carbon can be added to the system as carbon
dioxide, or possibly as bicarbonate. In applying supple-
mental carbon, carbon dioxide should be added to the system
only during the two- or three-hour period of peak photo-
synthesis. Possibly bicarbonate may be effectively applied
by adding it continuously to the influent.
7. It appears from the data that a carbon deficiency in
the algal system partially blocks the cellular reduction
of nitrate to ammonia resulting in the excretion of nitrite
into the growth medium.
8. Phosphate is almost completely removed from the system
by algal phosphate assimilation and/or chemical precipita-
tion resulting from photosynthetically induced Increases
in pH.
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9. Iron is required in the algal system and affects both
nitrogen uptake and algal suspension.
10. More than 90 percent of the algae can be separated on
a year-round basis by the chemical-flocculation-sedimefltatiOfl
process.
11. Algae can be differentially separated to provide a
product of variable inorganic and organic composition.
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RECOMMENDAT IONS
1. At least two one-half to one million gallon per day
parallel “prototype” algal nitrogen removal plants should
be constructed and operated to permit investigation of
“plug flow” versus completely mixed systems; lined versus
unlined systems; and systems with biomass control versus no
biomass control. Such systems would be provided with
equipment for carbon, iron and phosphate addition in line
as needed, and for pH control to facilitate the avail-
ability of iron and phosphorus to the algae.
The prototype facility should be equipped for algal
harvesting so that the integration of growth and harvesting
may be further investigated by the promising harvesting
methods found in the recent investigation.
Operation of the prototype plants should be conducted for
at least two years to determine variations in climatic
conditions and waste compositions that may normally occur.
If possible, the prototype plants should be located so that
they could be used either for extended pilot investigations
to continuously update the technology of nutrient removal
or as integral parts of the algal nitrogen removal system.
2. Because even small changes in permissible nitrogen
discharge levels may profoundly affect process costs,
nitrogen discharge requirements in the western Delta-
Suisun Bay area should be studied further to determine
if seasonal or other changes in discharge requirements
could be made.
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CHAPTER I
ThEORY A1 D RATIONALE
Introduction
This report presents the major findings of a one-year opera-
tional investigation conducted at the Interagency Agricultural
Wastewater Treatment Center (IAWTC) at Firebaugh, California,
by the United States Bureau of Reclamation (USBR), the
Environmental Protection Agency (EPA), and the California
Department of Water Resources (i ffi) on the use of algae to
remove nitrogen from agricultural tile drainage in the San
Joaquiri Valley of California. This investigation, which
represents only one aspect of an overall program (1, 2, 3, Li),
was established to determine if a method(s) could be found
to reduce the total nitrogen content in agricultural tile
drainage (5, 6, 7, 8) to 2 mg/i or less, the level established
as a maximum for the San Francisco Bay-Delta area (9, 10).
Phase I Conclusions
The algal nitrogen removal investigation was conducted in
two phases. Phase I (ii) was designed to determine the
feasibility of using controlled algal growth and harvesting
to remove nitrate-nitrogen from agricultural tile drainage
(12, 13, 15, 16, 17). That study concluded:
1. Algal growth and harvesting is a technically feasible
method of removing nitrate-nitrogen from subsurface tile
drainage.
2. Preliminary costs of an a1 a1 system, as studied at the
IAWTC, would be approximately $135 per million gallons of
water treated.
3. Some nutrients (e.g., carbon, phosphorus, and iron) may
be limiting in tile drainage during portions of the year and
would have to be supplemented in an algal treatment plant to
achieve maximum nitrogen assimilation and removal.
t. No more than four hours of mixing (with velocities of
from 0.25 to 0.50 feet per second) would be required for
maximum nitrogen assimilation.
5. Theoretical detention time required for maximum nitrogen
assimilation would be from 5 to 16 days and appeared to be
inversely related to pond temperatures within the range of
12°C to 25°C, and independent of temperature within the
range of 25°C to 33°C.
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6. Optimum culture depth was 8 inches, but this depth could
be increased by lengthening the detention time. Comparison
of different depths (8 to 16 inches) showed that nitrogen
assimilation varied seasonally and was directly related to
available light.
7. A secondary study at the IAWTC indicated a symbiotic
process (algal-bacterial) removed substantial amounts of
nitrogen and should be considered as a potential nitrogen
removal process.
8. Under the study conditions, some mechanism to control
sludge accumulation appeared to be required during certain
times of the year.
9. Algae can be separated readily from agricultural tile
drainage by lower levels of floccuients (e.g., ferric sulfate,
5 mg/i; alum, 20 mgti; lime, êO mg/i; and cationic polyelec-
troiytes, 0.2 mg/i) than found necessary for harvesting
sewage-grown algae (18). This concentrated algae slurry can
then be dewatered and dried to produce a stable product.
10. Removal of nitrogen (by any method) from agricultural
tile drainage does reduce the stimulatory effect of drainage
waters on algal growth in the receiving waters (San Francisco
Bay-Delta).
11. If a market were developed for the algal product the
product would probably be worth approximately $80 to 4100 per
ton, an amount that could be subtracted from the overall
cost of treatment.
12. Phase II studies should be initiated to define opera-
tional criteria.
Phase II Objectives
At the completion of the Phase I studies in December 1969,
the Interagency Advisory Group authorized initiation of the
Phase II (operational) studies to be conducted at the IAWTC
from January 1970 through December 1970. The primary objec-
tives of the Phase II studies were to:
1. Ref me the design criteria.
2. Develop operational procedures.
3. Recommend a design for a prototype algal nitrogen
removal plant.
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Although this report, Phase II, deals specifically with the
1970 operational studies at the IAWTC, Phase I results have
been included where applicable.
p roach Rationale
The Phase I studies were designed to determine the feasibility
of using algae for the removal of nitrogen from agricultural
tile drainage. After preliminary studies were conducted to
define the basic operational factors that might be expected
to affect the efficiency of nitrogen assimilation by the
algae, a series of short-term investigations was conducted
to determine the effect of: (1) mixing, (2) detention time,
(3) depth, and (4) nutrient addition (carbon., phosphorus, arid
iron) on the operation of algal growth units. From the results
of these studies, which are presented in the introduction of
this report, it was concluded that algal nitrogen removal is
a feasible method of removing nitrogen from agricultural tile
drainage.
The Phase II studies were designed to follow the predicted
month-by-month nitrogen loading for San Joaquin Valley agri-
cultural wastewaters during one year’s operation. The goal
was to develop the basic techniques needed to determine the
seasonal operating criteria required in a full-scale plant,
rather than to maximize nitrogen assimilation in each unit.
Seasonal requirements were determined by monitoring algal
nitrogen assimilation over an entire year in growth units
operating on various combinations of growth regulating para-
meters (see Table 1). In working toward these goals, the
following questions had to be answered before an assessment
of the process could be obtained.
1. Could the operating criteria governing nitrogen assimila-
tion determined in the Phase I studies be applied effectively
to the Phase II studies?
2. Would any of the basic operating criteria determined in
Phase I change over an extended period of time?
3. Could sustained nitrate-nitrogen removal be obtained on
a long-term basis?
4. What levels of nitrogen assimilation could be expected
during different times of the year?
5. What are the optimal operational variables required for
maximum nitrogen removal throughout the year?
6. Would the algal harvesting criteria change?
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7. What type of algal plant design would be optimal for
maximum nitrogen removal?
The present investigation was designed to answer these and
other such questions pertaining to the continuous operation
of an algal nitrogen removal system.
Theory of Algal N1tro en Assimilation
Although algae have been used for a number of years in the
secondary treatment of domestic sewage In oxidation ponds,
only recently has their application to tertiary treatment
(nutrient removal) of wastewaters been considered. Shelef (19)
has stated that the major advantage of using algae in waste-
water treatment is the simultaneous accomplishment of biomass
production (by-product), oxygen production, carbon dioxide
adsorption, and nutrient assimilation. Furthermore, algae
are utilized in preference to higher plants (both would
achieve the same overall results) because they have a high
specific growth rate and continuous reproduction, and can be
grown In a continuous culture on a year-round basis.
Although there are numerous ways to repiesent the overall
stoichiometrics of algae photosynthesis and nutrient assimi-
lation, Equation [ 1] proposed by Jeweli and McCarty (20)
best seems to represent the algal nitrogen removal process
investigated at the IAWTC:
aCO 2 + cNO 3 + ePOj 3 + (c+3e)H + Eq. [ 1]
1/2 (b-c-3e)H 2 0 + sunlight CaHbNCOdPe +
(a + b/ 1 4 + 5c/4-d/2 + 5e/A4)0 2
This equation predicts that as plant photosynthesiB occurs,
a fixed ratio of major nutrients will be eliminated from the
growth media. For example, the following equation by
McCarty (21) represents the average stolchiometric incorpora-
tion of C, H, 0, N, and P into plant cell material:
106 CO 2 + 81 H 2 0 + 16 NO 3 + H.P0j 4 + 18 H + Eq. [ 2]
light — C 1 H 181 Oj N 16 P + 150 02
Equation [ 2] represents the algal composition typically
reported in the literature (22, 23, 2 , 25, 26, 27) for algae
8
-------
under good growing conditions and is usually about 6 to 11
percent nitrogen, 50 percent carbon, and 0.5 to 2 percent
phosphorus. However, the chemical composition of algae can
be extremely variable, differing with species and conditions
of growth (19, 26, 28, 29). For example, Foree and McCarty
(30) found that the cellular composition reported for
Chiorella pyrenoidosa varied from C6.2OHj.0.20 33 2N to
C 573 H 1032 O 100 N .
The extent of algal cellular production, Equation [ 1], is
proporti.onal to the concentration of the limiting nutrient
(22) and corresponds to “Lieber’g’s Law of the Minimum ’ and
Blackman’s concept of ItLimiting Factors”. The rate at which
this reaction will proceed is a function of available light
energy, which is the determining factor regulating algal
plant capacity, physical dimensions, operational parameters,
biomass concentration and production (19, 31, 32). Accord-
ingly, the ultimate goal of design in an algal nutrient
removal system should be the optimal utilization of light
with only the undesirable nutrient as the limiting factor.
The probable nitrogen removal pathways in the algal growth
units as studied at the IAWTC are presented in Figure 1.
LIGHT
CHEMICAL
ADDITION ALGAL NITROGEN ASSIMILATION
INFLUENT $ N 1 Oi— NO —-— N 2 02 — .NN 2 OW— NN —- 0
•1 POOL
I I I
(AEROBIC) I ALGAL
I PROTEIN
H I ___
NO 1 I N 1 (GAS)4NHNHI .P N NH 2 . 1
1- T I —
I (ANAEROBIC) I
INQ 02 _ Y! !_ — — A!GAL ( EF?LUEWI}
-NH4 ——SLUDGE
ALGAL GROWTH UNIT
FIGURE I - PROBABLE NITROGEN PATHWAYS IN ALGAL GROWTH UNITS
9
-------
If such a system is hydraulically balanced, the only pathway
utilized will be the algal uptake and assimilation of
nitrate-nitrogen, with subsequent conversion to cellular
material and the removal of the algae (and incorporated
nutrients) from the growth unit. This may not be the case
in large-scale units. Possible secondary nitrogen removal
pathways are presented in Figure 1 and their possible
significance will be presented in Chapter III of this report.
Nitrogen Assimilation Mechanism
In general, algae can utilize inorganic nitrogen as either
ammonia, nitrate, or nitrite. The reduction process by which
inorganic nitrogen is utilized requires energy in the form of
light (Equation [ 1]) and is temperature dependent. The level
of energy required depends upon the amount and type of
nitrogen reduction required.
The removal pathways of nitrate-nitrogen (the predominant
form in the tile drainage at the IAWTc) from the growth
mediumr via conversion into cellular material and subsequent
removal from the growth unit are illustrated in Figure 1.
The incorporation of nitrate-nitrogen into the cell involves
a chain of enzymatic reactions by which the nitrate ion is
first pumped into the cell, reduced to ammonia via several
reductase enzymes, and finally incorporated into chlorophyll,
nucleic acids, amino acids, and proteins (19, 21, 23, 24, 28,
33, 34, 35). Each step of the process is affected by a
number of physical and chemical factors which influence both
the rate and extent of assimilation.
Types of Algal Growth
Fogg (36) has defined two types of unicellular algal growth
systems. The first of these is generally referred to as the
t1 batch” culture and is characterized by the growth of uni-
cellular algae in cultures of limited volume (no nutrient
replenishment). In this type of culture, there are five
phases of algal growth: (1) the lag phase or period of
Initial adjustment to the growth medium; (2) the exponential
(log) phase, represented by a period of rapid cell division;
(3) the declining growth rate phase, in which nutrients or
light become limiting; (Li) the stationary phase, when
nutrients or light limit growth rate; and (5) the death
phase, where cell weight and cell numbers decrease. This
constitutes the normal pattern of growth for cultures of
limited volume; however, If the culture is not unialgal,
the original species may be replaced In the later phases of
10
-------
growth by algal species with slightly different nutritional
requirements. Figure 2 Illustrates the batch-culture type of
growth with unicellular algal cultures.
/ LEGEND
/
2- EXPONENTIAL PHASE
/ 3- PHASE OF DECLINING
/ RELATIVE GROWTH RATE
/ 4-STATIONARY PHASE
I 5-DEATH PHASE
/
AGE OF CULTURE
FIGURE 2-THE CHARACTERISTIC PATTERN
OF GROWTH SHOWN BY A UNICELLULAR ALGA
IN A CULTURE OF LiMITED VOLUME
The specific growth rate of cells during the exponential
phase Is a function of cell concentration and can be described
by the following Equation [ 31:
= KN Eq. [ 3]
Where K is the specific growth rate (day 1 ), N is the cell
concentration (in any applicable unit), and t Is the time in
days.
The second type of algal growth system encountered Is usually
referred to as “continuous flow” culture and is characterized
by maintenance of the exponential phase (previously described)
of algal growth by continual replenishment of the algal nu-
trients. In this system the algal population density is held
at a relatively constant (steady-state) level by manipulation
of the growth factors. This is the type of system most com-
monly used In experimental wastewater treatment plants utili-
zing algae and in investigations dealing with algal growth
kinetics (19, 25, 37, 38, 39). As will be demonstrated in a
11
-------
later section, the growth units utilized in the IAWTC
studies, thougri designed as continuous flow types, were in
actuality somewhere between batch and continuous flow.
12
-------
CHAPTER II
METHODS AND MATERIALS
The equipment arid methods used in the IAWTC studies were
described in detail in the Phase I report. A brief resume,
as pertains to the Phase II studies, is as follows.
Experimental Proce dures
The basic experimental test units utilized in the Phase II
Investigation along with the operating criteria of each unit
during 1970 are listed in Table 1. Although several units
were retained for special studies, the majority of experi-
mental test units were held on fixed operating schedules for
the entire year. The large lA-acre algal growth pond,
described in Phase I, was retained as a flexible unit,
serving as both a demonstration and algal production unit
with which algal separation could be studied on a seasonal
basis.
The predicted changes in nitrogen concentration for San
Joaquiri Valley agricultural wastewaters as well as the IAW’:rC
plant influent nitrogen levels are shown in Figure 3. An
910
FIGURE 3-PROJECTED FLOW AND NITROGEN CONCENTRATION OF SAN JOAQUIN VALLEY
AGRICULTURAL WASTEWATERS AND ACTUAL IAWTC INFLUENT NITROGEN CONCENTRATION
13
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TABLE 1
OPERATIONAL SCHELULE FOR PHASE II SflJDLES
1970
Unit
Investigation
(I hS) 1
Mixing Nutrient AdditionAl Det
g : 1 CO 2 P0 4 FeEl 3/25 15 /i
enti
5/18
on Ti
7/1
mes
9 / 1
(Days)
/18lio/19
11/17
1
2
Speeial ’
Special 1 ’
12
12
yes
yes
no/yes yes variaole 11.4 8
yes yes variable 11.4 8
5
5
3
3
3
3
3
8
8
10
10
3
No P0 4
12
yes
yes no yes 11.4 8
5
3
3
5
8
10
14
No Iron
12
yes
yes yes no 11.4 8
5
3
3
8
10
5
Detention time
12
yes
yes yes yes 8 5
3
1
2
8
12
15
6
Detention time
12
yes
yes yes yes 11.4 8
5
3
3
3
4
5
7
8
Detention time
Specia1 ’
12
12
yes
-
yes yes yes 16 11.4
yes/no yes yes 8 5
8
5
5
3
5
3
5
5
8
8
10
10
9
Biomass control
12
yes
yes yes ro 8 5
5
3
3
5
8
10
10
11
Biomass control—iron
No mix_speciall”
12
12
yes
no
yes yes yes 8 5
no yes yes 11.4 8
5
5
3
3
5
5
8
8
12
10
15
12
No mix
12
no
no yes yes 11.4 8
5
3
3
5
8
10
13
Depth
16
yes
yes yes yes 11.4 8
5
3
3
8
10
14
Depth
8
yes
yes yes yes 8 5
3
1
2
8
12
15
15
Depth
8
yes
yes yes yes 11.4 8
5
3
3
3
14
5
16
17
Depth
Special3l
B
12
yes
-
yes yes yes 16 11.4
variable yes yes 11.4 8
8
5
3
3
5
8
8
10
10
18
Detention time
12
yes
no yes yes 8 5
3
1
2
8
12
15
19
DetentIon time
12
yes
no yes yes 11.a 8
3
3
3
14
20
Detention time
12
yes
no yes yes 16 11.4
8
5
5
8
10
21
SoIl
12
no
no yes no 8 5
3
1
3
5
12
15
22 Soil
1/14-acre Demonstration -”
12
8-24
variable
no
variable
no yes no 11.4 8
yes yes 15 8
variable
5
5
3
5
3
7
3
8
8
10
10
15
1/ Nutrient Addition - Carbon added as CO 2 such that C/N 5/1
P0 4 added as !13P04 such that i/io
2/ Nutrient Addition — Iron added as FeC1 3 - 3 mg/l (3/25-5/31) 1 mg/i (5/31-12/31/70)
3/ Special Studies - Unit 1 Intensive-(3/21-5/31); No Fe or CO 2 (6/1-9/17); Temp. (9/18-12/31)
Unit 2 Intensive-(3/21-6/24); pH control (6/25—7/1); pH tlomass control (7/1-12/31)
Unit 8 Cyclic (3/21-5/31); bicarbonate addition (6/1-12/1)
Unit 11 Night nix (3/25-6/30); hydraulic study (7/1-8/31); no mix (9/1-12/31)
Unit 17 8-inch (3/25-5/31); no mix/CO 2 (6/1-8/1); no rnix/C02 biomass control
(8/i -12/31)
4/ 1/4—acre demonstration unit: detention time 12-inch (3/25-7/31); 24—inch (8/4-11/16);
8—inch (11/16—12/31)
14
-------
attempt was made to duplicate the predicted monthly changes
In influent nitrogen, although loading could only be approxi-
mated (detention times from the Phase I study were used as a
guide).
A schematic diagram of the IAWTC algal nitrogen removal faci-
lities is presented in Figure . As shown in this figure,
there were 22 experimental test units referred to as mini-
ponds, each with a surface area of 128 square feet and a
water volume of 1,000 gallons at a 12-inch depth, and a l/14_
acre demonstration unit. The plant influent came from a
common storage pond and the carbon dioxide was supplied to
all units through a common manifold system.
FIGURE 4-SCHEMATIC OF IAWTC FACILITIES
15
-------
Analytical Methods
Chemical Analysis
Chemical analyses (Table 2) of treated and untreated tile
drainage were made according to the various procedures out-
lined In Standard Methods (40) for the determination of the
chemical constituents considered pertinent to the Investiga-
tion.
TABLE 2
CHEMICAL ANALYSIS SCHEDULE
Constituent
Frequency
3 times/uk.
Method
Nitrate
Brucine, specific ion
elect rode
Nitrite
3 tImes/uk.
Dlazotization
Ammonia
Organic Nitrogen
Orthophosphate
Iron (total and
dissolved)
once/month
once/uk.
once/uk.
once/uk.
Kjeldahl-Dlsti ilatlon
Kjeldahl
Stannous chloride
Phenanthroline
Chemical Oxygen Demand
as required
Dichromate ref luxing
Dissolved Oxygen, DO
as required
Winkler-Azide
Modification
pH
Alkalinity
daily
twice/uk.
Glass electrode
Titration-pH meter
Electrical Conductivity
as required
Wheatstone Bridge
Total Dissolved Solids,
TDS
as required
Evaporation,
gravimetric
Samples for special analyse8, normally conducted each time
the storage pond was filled, were sent to the Department of
Water Resources’ laboratory at Bryte, California. In addi-
tion, samples for trace metal determinations were sent to
the U. S. Geological Survey laboratory in Sacramento for
analysis by emission spectrography.
Biological Analysis
The primary method used to determine changes In algal blomass
was measurement of volatile suspended solids on a Whatman GFA
16
-------
glass filter disc; in addition, a].]. units were examined at
least once a week to observe the condition and species of
algae present. At the same time as the species examination,
cell counts were determined by a microscope and a
hemacytometer.
In some light box studies, the progress of cell growth was
followed by measuring in vivo Chlorophyll fluorescence. A
Turner Model III fluoröiãeter was modified by adding a blue
light source and the proper combination of filters (Corning
CS5-60 primary and CS2-60 secondary) f or measurement of
chlorophyll a.
Physical Analysis
A continuous recording analyzer was used to monitor the l/1l _
acre demonstration unit for water temperature, pH, and sun-
light. Each of these parameters was also measured routinely
in the smaller experimental test units. In addition, a
weather station was located on the site to record daily
changes in air and water temperature, evaporation, precipita-
tion, and wind.
Quality Control
Quality control was conducted routinely and analytical tech-
niques were corrected, if not within the limits suggested in
Standard Methods (40). Because many of the results used In
the iñ ëst1.gation were based on changes in nitrate-nitrogen
as measured with the use of a specific ion electrode, special
mention should be made of this method of nitrate-nitrogen
analysis.
This Instrument was standardized against known concentrations
of nitrate in denitrified tile drainage as well as with ni-
trate standards. Usually, a plot of meter readings versus
concentration showed a straight line between 05 and 50 mg/i
nitrate-nitrogen. Although some problems were encountered
as a result of changes In total dissolved solids (TD8), as
well as some day-to-day variations in electrode response,
the specific ion electrode was considered rapid (up to 150
analyses per hour), simple, and reliable.
Data Analysis
In the following sections, many of the results are reported
as nitrogen assimilated, expressed either as mg/i or as per-
cent removed. In general, “nitrogen assimilatIon” Is ex-
pressed as the amount of soluble nitrogen disappearing from
17
-------
the medium as a result of assimilation and conversion to
algal cellular material. However, this method of expressing
nitrogen assimilation is a simplification of a very complex
system where, for example, algal cellular production and
decomposition occur simultaneously.
In certain instances, response of algal growth to nutrient
addition has been expressed as changes in volatile solids
(vs), in vivo fluorescence, absorbance, or changes in some
constituent other than nitrogen. A typical example is that
shown by the correlation of volatile solids to absorbance,
Figure 5. Similar correlations of algal biomass changes to
nitrogen assimilation have been determined for the other
items.
200
.
.
VOLATILE SOLIDS • T8(OD.). I I.S
• 99.1
ABSORBANCE (OPTICAL DENSITY)
FIGURE 5- VOLATILE SOLIDS Vi ABSQRBANCE
18
-------
Test Units
! 1 s1 Bloassays
Algal nutrient bioassays, as well as special studies, were
conducted routinely by batch culture techniques. These
studies were customarily conducted with duplicate or tripli-
cate 1,000 milliliter (ml) Erlenmeyer flasks containing
500 ml of the medium to be tested. Small concentrations of
algae (2,000 to 3,000 cells/ml), usually Scenedesmus
guadricauda , taken from the outdoor growth units were used
as the inoculum, The inoculum was not axenic nor even uni-
algal, although it normally contained 90 to 95 percent
Scenedesmus . The use of algae from the test units proved
to be an effective method of monitoring growth variables
seasonally, perhaps because the algae were acclimatized.
Lighting was usually continuous (300- to 400-foot candles
at the medium surface), and temperature, unless specifically
altered, was held at 22± 4°C. No means of automated inechani-
cal agitation was provided, but air (compressed only or
enriched with carbon dioxide) could be introduced to
individual flasks via a central manifold to provide agitation
and supplemental carbon. Air volume was regulated by short
sections of capillary tubing in each line and resulted in
approximately equal amounts of air being delivered to each
flask (4i).
Analyses similar to those previously described for the out-
door growth units (Table 2) were conducted during these
studies.
Operational Units
Miniponds . The primary experimental units utilized in the
Phases I and II investigations were 22 resin-coated plywood
growth units. Each minipond was 8 feet wide by 16 feet long,
with a surface of 128 square feet and a volume of 1,000
gallons at a 12-inch depth. Mixing pumps with 80-gallon-per-
minute capacity provided 0.25- to 0.5-foot-per-second (fps)
velocities to re-8uapend the algae (Figure 6). Three
pond depths were studied with these units -- 8, 12, and 16
inches. In addition, timers were placed in the electrical
circuits of the pumps to vary the hours of mixing during a
24-hour period. (The standard mixing schedule in Phase II
was from 8 to 8:30 a.m. -- sampling period -- arid from
12 noon to 3:30 p.m. -- peak photosynthetic period.)
19
-------
TYPICAL UNIT
3/4 PV.C . _____ S5 GAL
DRUM
010-MASS CONTROL DEVICE
EFFLUENT DETAIL
1000 GALLON UNITS
EFFLUENT
114 ACRE DEMONSTRATION POND
FIGURE 6 - ALGAL GROWTH UNITS AT IAWTC
-------
Tile drainage was individually metered from a storage pond
supply at rates to provide preselected detention times based
on the Phase I studies. This plastic-lined storage pond had
an 820,000-gallon capacity and was refilled from a tile
drainage field adjacent to the IAWTC site.
Nitrate-nitrogen was mixed into the storage pond as required
to build up the nitrogen level to the predicted concentration
(Figure 3). No adjustment was made when the wastewater
nitrogen level was in excess of predicted.
The effluent from the test units was drawn from near the
bottom of each unit (opposite the influent) and discharged
through a “broken” siphon arrangement (Figure 6). This
effluent tube was also used to maintain a constant depth in
the unit.
The ponds receiving supplemental carbon (as carbon dioxide)
had a mixture of atmospheric air and varying levels of carbon
dioxide metered into the intake side of the mixing pumps
during the afternoon mixing cycle. The carbon concentration
corresponded to the unitts nitrogen loading. To assure
complete addition to the test units, the bicarbonate forms
of carbon, as well as phosphorus and iron, were added to
individual test units daily, rather than to the central
storage pond supply.
An attempt was made to control algal blomass In several of
the test units by converting a 55-gallon drum into a settling
tank (Figure 6). The water from the growth unit was cycled
through the drum where some of the suspended material settled
out and then the supernatant was returned to the growth unit.
The sludge from each of these separation units was periodi-
cally collected, measured, and chemically analyzed.
One-quarter Acre Demonstration Unit . The l/ 4-acre unit was
an asphalt-lined pond with a 12.5-foot-wide folded raceway
channel approximately 800 feet long. The 1 4-foot center
baffles were constructed of aluminum siding attached to a
wooden upright frame. This unit could be operated at depths
varying from 0.5 to 3 feet. The effluent could be taken
from either the top or near the bottom of the mixing sump
(Figure 6). With its four available mixing pumps, operating
velocities of up to one foot per second were theoretically
possible at all operating depths. As with the smaller test
units, each pump had a timer which allowed for an almost
infinite variety of mixing schedules. To provide supplemen-
tary carbon, an air-carbon dioxide mixture could be metered
into the Intake side of the mixing pumps.
21
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CHAPTER III
RESULTS AND DISCUSSION
Operational Procedures (Phase II, 1970)
Operational Studies
The Phase II operational investigation was designed to deter-
mine the 8easonal effect of: (1) phosphate, (2) carbon, (3)
iron, (li.) mixin , (5) biomass regulation, (6) depth, (7) de-
tention time, (t ) soil, (9) light, and (io) temperature on
nitrogen assimilation by algae in tile drainage. The purpose
of’ the investigation was not to operate each unit at maximum
nitrogen removal efficiency but to determine which combina-
tion(s) of the above variables provided maximum nitrogen as-
similation under different environmental conditions. Good
experimental design dictated that only one combination of
variables be optimal over a given unit time. Consequently,
a decision was made early in the investigation to adhere to
the preplanned study design, except for special units, re-
gardless of the results in individual test units.
Flask Bioassays
During the IAWTC investigation (Phases I and II), a number of
light box algal bioassay studies were conducted which were
designed to determine factors that might alter the level or
extent of nitrogen assimilation by algae in agricultural tile
drainage. The results from these studies were then applied
to the operation of the miniponds. In general, the algal
bioassays proved to be a rapid and effective method of evalu-
ating nitrogen assimilation under different growth conditions.
Summaries of these studies, considered pertinent to an under-
standing of the algal process, are presented in the following
sections.
1970 Startup
Both the miniponds and the 1/LI_acre demonstration unit were
continuous flow (influent injected In one end, effluent re-
moved at the other end), stirred (semi-mixed) algal reactors.
At the termination of the Phase I studies in December 1969,
all of the miniponds except the units containing a layer of
soil were drained, cleaned, repaired, and refilled with tile
drainage containing a Scenedesmus inoculutn from the 1/k-acre
23
-------
demonstration unit. This procedure was completed in February
1970. The units were then operated on batch for several
weeks before placing them on the designated schedule. During
the next month and a half, there was a transition period
between Phase I and Phase II which resulted in a number of
operational items being neglected. By mid-March a large
error in the rate of Influent to many of the units was
detected. In addition, a volume measurement of the air-carbon
dioxide to each test unit showed that the flows were in error.
By the second week of April, these operational discrepancies
had been largely corrected and this resulted in a correspond-
ing improvement in nitrate assimilation.
From mid-April through December 1970, most of the miniponds
were operated continuously on the designated schedule.
Detention time was varied In an attempt to bracket seasonal
changes in optimal detention time. The remaining units were
retained for special studies. At the end of July, the
majority of the units were emptied and restarted with Inoculum
from the 1/11-acre demonstration unit.
Plant Influerit
Subsurface agricultural tile drainage (116) was pumped to an
820,000-gallon covered storage pond which provided the
influent for the algal nitrogen removal studies. Although
the main reason for having a storage pond was to assure a
source of influent, It was also intended to provide a
constant water quality to the test units, There were,
however, significant changes in T1 and general water quality
each time the pond was filled. The changes In TDL9 and total
alkalinity in the plant Inf].uent for 1970 are plotted in
Figure 7. The large change in Tt noted at the end of the
summer resulted from the crop rotation and water application
practices in the tile drainage field adjacent to the test
site, which coincided with changes In major nutrients that
had occurred over a three-year period at the IAWTC (Figure 8).
When the storage pond was filled with tile drainage, samples
were collected and analyzed for standard minerals trace
elements (thought to be required for algal growth pesti-
cides, and algal bioassay nutrient responses to carbon,
phosphorus, and iron addition.
Late in 1970, the aluminum and Iron levels in the plant
Influent increased noticeably. A check of the storage pond
roof (which was aluminum supported by iron girders) indicated
that there was a considerable amount of corrosion which was
probably responsible; however, It is not known whether this
had any effect on algal growth and nitrogen metabolism,
24
-------
8 ,00O
E
6,000
—I
4,000
o 2,000
-j
ALK -S r - --- - --.
In
A1 # 0
•-t’J, J ( )
0
0
U)
0
•3O0
E
•200 z
-J
4
-J
4
•Ioo -J
0 . I I I I I I I I
J F MA M J JASON D
FIGURE 7- MONTHLY VARIATION IN TOTAL DISSOLVED SOLIDS
AND TOTAL ALKALINITY IN PLANT INFLUENT FOR 1970
a
(I )
0
—I
0
U)
0
L U
0
(I)
C l)
0
-J
0
I -
FIGURE 8- SEASONAL VARIATION IN TOTAL DISSOLVED SOLIDS AND
NITRATE-NITROGEN IN TREATMENT PLANT INFLUENT
a
z
L J
0
I—
z
l U
I-
z
25
-------
Probably the corrosion of the storage pond roof caused a
buildup of iron in the growth units without iron addition
noted during the late summer of 1970.
Another item of importance was the accumulation of a sub-
stantial amount of silt, detritus, etc., on the bottom of
the storage pond. Analysis of this material for nitrogen
concentration indicated that it contained about 1,000 milli-
grams per liter of nitrogen; however, the total amount of
the material in the pond was unknown. It is possible that
this nitrogen recycled in the storage pond, which would
explain the fluctuations beyond analytical variation noted
in the influent nitrogen level between storage pond fillings.
A similar phenomenon attributed to nitrogen recycling from
dead algal material was noted in some special symbiotic
studies which will be discussed later.
Factors Affecting Nitrogen As imilatior3
According to Equation [ 1], carbon, nitrogen, hydrogen,
oxygen, and phosphorus are the major inorganic nutrients
required for production of algal cellular material. In
addition, boron, B; calcium, Ca; chlorine, Cl; cobalt, Co;
copper, Cu; iron, Fe; potassium, K; magnesium, Mg; manganese,
Mn; molybdenum, Mo; sodium, Na; sulfur, S; vanadium, V; and
zinc, Zn; are usually considered essential to normal algal
growth and metabolism of green algae (27, 42, 43, 414, 145),
The Phase I studies demonstrated that to achieve maximum
nitrate-nitrogen assimilation by the algae, the plant
inf’luent had to be supplemented at different times of the
year with varying amounts of carbon, phosphorus, and iron,
so that nitrate-nitrogen remained the limiting nutrient.
Furthermore, there were indications in the laboratory
studies that other trace elements might at times stimulate
nitrate-nitrogen uptake by the algae.
Algal growth rates and nutrient assimilation are governed
by the existing environmental conditions. To compensate for
changes in such factors as light and temperature, detention
time and depth were seasonally adjusted to provide conditions
conducive to maximum growth. Consequently, because all the
growth variables have important effects on nitrogen assimila-
tion, each will be discussed separately in the following
sections.
Effect of Light on Nitrogen Assimilation
Since algae used in wastewater treatment systems are exclu-
sively autotrophic, light can be considered to be their sole
26
-------
energy source (19, 32, 7). Light absorption kinetics, as
related to nitrogen assimilation, have been extensively
described by Shelef, Oswald, and Golueke (19), while the
practical application of light factors to algal wastewater
treatment systems has been described by Oswald In a number
of publications (i , 17, 31, 32, 48).
Nitrogen assimilation by algae is intimately linked to photo-
synthesis, and several workers have shown that light in the
blue wave1en ths is especially favorable for nitrate reduc-
tion (33, 34). In 1920, Warburg and Negelein In a classical
experiment (49) showed that light stimulated nitrate reduc-
tion by Ch1orella which is accompanied by oxygen evolution.
They considered that, both In the light and dark, nitrate
reduction is coupled with carbohydrate oxidation, but that
in the light the carbon dioxide which might be expected as
a product is assimilated by photosynthesis and replaced by
oxygen evolution. Light was thought to: (1) stimulate
nitrate reduction by increasing the permeability of cells to
nitrate; (2) through photosynthesis, produce organic compounds
available as electron donors for nitrate reduction;
(3) produce a photochemical reductant; and (4) through photo-
phosphorylation, stimulate nitrate reduction. Davis (50)
found little nitrate reduction by light-limited Chiorella in
the absence of carbon dioxide, unless glucose was added. He
suggested that carbohydrate metabolism was necessary to form
the reductant.
The absorption of light energy by dense algal cultures can
be approximated by the following modification of the Beer-
Lambert Law:
= I 0 e Eq. [ 14
Where 10 Is the incident light intensity, ‘d Is the Intensity
of light at any depth, d is depth in centimeters, c is the
algal,)concentration in mg/i, E is the extinction coefficient
in cmVmg, and e is the base of the natural logarithms. As
indicated in this equation, light penetration to I i
directly affected by Incident light and inversely affected
by depth and culture density. However, the Beer-Lambert Law
is only valid for true solutions (33) and does not apply
trict1y to algal suspensions (si). Furthermore, photosyn-
thetically linked reactions are time-intensity dependent,
rather than just intensity dependent, with light utilization
efficiency (growth rates) being highest at low cell densities.
Gates and Borchardt (47) found that the total available light
per functional cell was the Important factor and that the
optimal growth rate was dependent upon some minimal level of
light which, if surpassed, resulted in a decrease in
27
-------
efficiency; and that illuminating an algal cell with more
than saturating light intensities represented an inefficient
use of energy. Krause (33) found that excess light may even
be detrimental to chlorophyll production. Oswald (32)
stated: “The Bush equation dictates that efficiency of light
use increases with depth, ... but a limit exists where the
depth is so great that no light penetrates and losses, due
to algal respiration, exceed their gain to photosynthesis.”
The practical implication of applying light “input” to the
operation of algal growth units is that if light-limiting
conditions are to be avoided, the cell concentration must be
adjusted so that each actively growing cell will receive
optimum light. There are several ways this can be accom-
plished. First of all, depth and detention time can be
adjusted seasonally to maximize the available light penetra-
tion; for example, long detention time-shallow depth in the
winter and short detention time-deeper depths during the
summer. Secondly, cell concentration can be maintained at
a given level appropriate to the available light by regula-
tion of the blomass. Another possible method of increasing
the availability of light to individual cells is to move the
algae into the light path by induced turbulence (mixing);
however, mixing can be detrimental if noriphotosynthetic
material, for example, nonassimilating older algal cells and
suspended inorganic colloidal particles, interfere with light
penetration. If these older cells decompose and ammonia-
nitrogen becomes available under low-light conditions, it
will be assimilated instead of nitrate (28). The, signifi-
cance of algal decomposition on nitrate assimilation will be
brought out in a later section of this report.
Light availability to the algae and influent nitrogen loading
were found to be the most significant variables affecting
nitrogen assimilation during the Pha8e II investigation.
The effect of light on nitrogen assimilation was measured in
three miniponds of equal surface area which were operated at
different depths of 8, 12, and 16 inches, and equal detention
times, In addition, there were also some indications of the
effect of light (depth) on nitrogen assimilation In the 1/4-
acre demonstration unit, which was the only unit operated at
depths of over 16 inches. During the Phase II study, this
unit waS operated at depths ranging from 8 to 214 inches.
The changes in the influent and effluent nitrogen concentra-
tion In units which were operated at the three depths are
shown in Figure 9. From the unit startup In January until
mid-April, the nitrogen removal level in the 12-inch depth
unit was approximately 10 mg/I greater than in the corres-
ponding 8- and 16-inch depth units. This was considered to
be the result of insufficient light conditions, although none
of the units were operating efficiently. When the carbon
28
-------
E
2
w
0
z
-J
4
0
FIGURE tO- EFFECT OF CARBON DIOXIDE
ADDITION ON EFFLUENT NITROGEN
CONCENTRATION AT THREE DEPTHS
LESIND
£ o ssi oc o c vms i
I
P 1 . 1 . 1St 5- UYLLI€NT NITSOStN COWCLNmAflOU A$ AFPICTED I V OtP1Il
AT MIAN QITVITI I T1MI
15
MARCH
15
APRIL MAY
1970
29
-------
dioxide concentration to all the miniponds was corrected in
early April, the effluent nitrogen concentration at all
three depths decreased rapidly to comparable levels
(Figure 10).
Although light was undoubtedly the important factor, much of
the differences in nitrogen removal at the three depths are
now attributed to insufficient carbon concentrations during
the early period of the yea.r.
These units remained comparable in nitrogen removal until
the end of September, at which time the range broadened.
The larger differences at the three depths during time of
startup were thought to be the result of low biomass concen-
trations associated with startup because more light was
available during the spring than in the late fall.
The total nitrogen in grams per day per minipond assimilated
at the three depths Is shown in Figure 11. From April through
20
as
LEGEND
A CIIHNGE OF DE TT ,N TINE
14
I
II
I
IN INCTI UNIT
I
CO 1 CONPECIED
I I
12- INCH
JANJAflY FESRUAHY MARCaI
MAY
JIAIE
570
FiGURE lI-QUANTITATIVE NITROGEN ASSIMILATION AS AFFECTED BY DEPTH - GRAMS PER DAY PER UNIT
A*.Y AUGUST
NOVENSEB DECIMSEB
30
-------
September, the 16-inch depth unit assimilated nearly twice
the nitrogen as the comparable 8-inch depth unit. Light
energy ca1 ulated as total light energy per day in langleys
(gm ca1/cm /minute) is shown in Figure 12.
70 0
600
O’N’D
FIGURE 12- LIGHT ENERGY RECEIVED DURING 1970
Calculations of nitrogen assimilated from April through
September showed that the 16-, 12- and 8-inch depth niinlponds
were removing 111, 8, and 6 grams of nitrogen per day per
minipond, respectively. This corresponded to a period in
which light ranged from 1400 to above 700 langleys per day.
In October, the cultures n all three depths were removing
about 8 to 10 grams of nitrogen per day and light was at
about 200 to 1400 langleys per day. Finally, in December,
all three units averaged 5 to 6 grams nitrogen removed per
day per minipond at light levels of 200 larigleys or less per
day.
5O0
LU
Q.
40O
LU
-J
30G
4
-J
200
I-
I
C D
-J
00
G
I I I I I —
F M A M A S
MONTHS
31
-------
A detailed examination of the data plotted in Figure 11 and
Figure 12 shows an interesting relationship between light
availability and nitrogen assimilation. The average total
soluble nitrogen removed per day per minipond at the various
deptha during 1970 as related to the total light available
is shown in Figure 13. At about 300 langleya per day,
5 grams of nitrogen were removed per day per minipond,
regardless of depth. At 300 to 600 langleys per day, all of
the units removed increasing amounts of nitrogen, although
the deeper units were removing the greater amounts. At light
in excess of 600 langleys per day, the deeper units removed
increasing amounts of nitrogen proportional to increased
depth, with the 16-inch unit removing 14 grams per day.
However, the 8-inch depth unit decreased to about 5 grams
nitrogen removed per day at the higher light levels, indica-
ting light inhibition. The high light intensity in this unit
may have had a detrimental effect on chlorophyll production
(33). In the 16-inch unit, use of light energy was more
efficient because the same light was distributed through a
greater volume of culture.
0
(Li
>
0
C
Wa
Oa
-J
I-
0
I-
100-300 300600 600:750
LIGHT IN LANGLEYS PER DAY
FIGURE 13-NITROGEN REMOVAL-IN MINIPONDS
AT DIFFERENT DEPTHS AND LIGHT ENERGY LEVELS
II
16 N
32
-------
Nitrogen assimilation in the l/ 1 4-acre demonstration unit,
operated at a 2 2 4-inch depth, is shown in Figure l1 , t ir1ng
August, when this unit was first operated at a 214-inch depth,
there apparently was enough available light (500 to 600
langleys per day) to reduce the total nitrogen in the
effluent to less than 14 mg/i at a 10-day detention time.
By the middle of September, the available light had diminished
and the effluent nitrogen increased; however, this reduction
in removal came at a time when the detention time was
decreased to 8 days. Calculations indicated the effluent
nitrogen would probably have remained below 5 mg/i, even at
these lower light levels, if the detention time had remained
constant.
4O -800
35. .• -700
-SUNLIGHT(LANGLEYS PER DAY)
30- •.•. 600
.......... ....I
••• ‘•• F
10.5 DAY DETENTION .- .ft 7 9 DETENTION .. . — U,
2G I 4OO
__I
-EFFLUENT NITROGEN -
-j 5 \ I , IGO
————1
0
0 20 30 40 50 60
AUG I SEPT I OCT I
FIGURE 14—NITROGEN ASSIMILATION IN
ONE-QUARTER-ACRE UNIT AT 24-INCH DEPTH
33
-------
The effect of wall sh .ding on the actual light entering the
l/M-acre demonstration unit and a typical minipond is shown
in Figure l . The percent wall shading was calculated for a
one-foot deep unit; however, the percent shading varies
proportionally to the height of the divider wall and the
depth and width of the unit. This wall shading was thought
to be a significant factor in reducing unit efficiency,
particularly during the winter months when light was critical.
The effect of divider shading would, of course, become
negligible as algal growth units are enlarged and shading to
unit volume Is reduced.
Another factor affecting light availability was shading due
to algal flotation. Flotation occurred In a number of the
units during the latter part of the summer in both Phases I
and II , and In most cases this algal material was not removed
from the surface of the unit. In the last few months of’
Phase It, when it became obvious that light was extremely
critical, the floating algae were removed. The removal
usually was followed by an improvement in nitrogen
assimilation.
I .-
z
z
F-
x
-J
I-
E
a.
FIGURE I - REMAININ LIGHT DUE TO WALL SHADING
314
-------
Effect of Temperature on Nitrogen Assimilation
Since temperature depends to a large extent on light Inten-
sity, in nature any change in light will also affect the
growth of algae by affecting temperature (51). Accordingly,
the effect of temperature on algal growth rates normally
follows Van’t Hoff’s rule, namely a doubling for each 10 0 C
increase in temperature within the range of temperature
tolerance. Furthermore, temperature effects on growth rates
have beer’ found to be a function of light intensity; for
example, Krauss (33) found that a high temperature strain of
Chiorella is inhibited by light intensities above 1,000 foot-
candles when grown at 25°C, but not below 3,000-foot candles
at 39°C. According to Krauss, the high temperature appar-
ently permits a higher absorption of light energy without
damage. Conversely, Emerson (52) stated that the temperature
at which cells are grown appears to play little part ir’ the
efficiency of photosynthesis. He found cultures grown at
10°C showed only 0.7 percent lower efficiency than cultures
of corresponding density grown at 2000 and that highest
efficiencies were actually observed at around 10°C. However,
Oswald (53) found that the efficiency of light energy conver-
sion by Chiorella incSeased linearly between &I°C and 20°C
and declined above 20 C. It would then seem that though
there is little doubt that temperature and light interact in
their effects upon algal growth rate, the exact relationship
remains controversial.
To determine the effect of temperature on algal nitrate
assimilation in tile drainage, several studies were conducted
with the use of the light box using shallow trays of circu-
lating water to maintain the flask temperatures at 12.3,
21.6, and 28.0°C with a variance of ±30C. Three extensive
studies were conducted in a series during the early spring
of 1970. The first study determined, among other things,
that if algal cultures were started from a small inoculum,
the lag and early exponential phases of algal growth were
characterized by an increase in total biomass and nitrogen
assimilation. These increases were a function of increased
temperature, as would normally be expected; however, there
were indications that once these early stages of growth were
completed, the optimal temperatures for maximum nitrogen
uptake decreased.
In the second experiment of the series, cultures containing
an initial low cell concentration of the inoculum, mainly
Scenedesmus g dricauda, were incubated at the medium temper-
ature 21° ) until the exponential growth phase was reached,
at which time some of the cultures were slowly adjusted to
the extreme temperatures, 120 and 28°c, respectively. In
this particular study, the general pattern of growth and
35
-------
nitrogen assimilation was the reverse of that noted in the
first study, in that maximum nitrogen assimilation and blo-
mass production occurred at the lowest, Instead of at the
highest temperature.
Finally, a third study, designed to Incorporate the methods
used In the first two studies, was conducted at the three
temperatures (12.3, 21.6, and 28.0°c). As shown in Figure
i6, during the first phase of growth, the lag period became
shorter at the higher temperature. However, after this
Initial period, the time required for complete nitrogen as-
similation was about equal at all three temperatures tested..
This seems to correspond to the findings of Emerson (52).
There was also a change In the predominant algal species
after an extended period in he high temperature cultures
that was not noted in the l2C series. As shown In Figure 17,
the changes in volatile solids in this study corresponded to
changes in nitrogen assimilation rates (Figure 16).
Increases In volatile solids (FIgure 17) were found to
correspond directly to increases in nitrate assimilation.
Purthermore, If It is assumed that each culture received the
same level of light, the algae growing at the low temperatures
must have been more efficient than those cells grown at high
temperatures, inasmuch as the biomass production, volatile
solids, and nitrate assimilation of the former were greater.
I5o
a
—
0
>
0
LLI
z
0
0
5O
I- .
z — —I---
0 tO 20
TIME IN DAYS DAYS AFTER LAG PHASE
FIGURE 16-NITROGEN ASSIMILATION AS AFFECTED
BY TEMPERATURE
)00
EXPONENTIAL PHASE
LAG PHASE
20°C
30°C/ .,
30
36
-------
‘SOC
-a
-a
One other aspect of this temperature study considered signif-
icant to studies dealing with methods of analyzing algal
growth potential (AGP) was the use of in vivo fluorescence
as a measurement of biomass production Use of this method
of biomass measurement at the three temperatures indicated
that: (1) for a given level of growth,j vivo fluorescence
varied with temperature as predicted; and (2) after initial
correlation to nitrate assimilation in early growth stages,
in vivo fluorescence dropped off, although nitrate assimila-
tion (growth) continued at a steady rate.
The studies conducted at the IAWTC also Indicated that bio-
mass production is not necessarily synonymous with nitrogen
assimilation, within limits. Apparently, It Is the physio-
logical condition of the algae rather than the total biomass
produced that is significant In nitrogen assimilation. For
example, in many of the light box studies, algal cultures of
one-half the density of other cultures were round to assimi-
late more or equal amounts of nitrogen (Figure 18),
DAYS TO INCREASE FROM: 0 t 500 500 10 1000 ta /l
10C 32.
20C 22. 5.5
30C iT. 6.0
LAG PHASE
START OF CULTURES
10’ C
0
TIME IN DAYS
FIGURE 17-VOLATILE SOLIDS INCREASE WITH TIME
AS AFFECTED BY TEMPERATURE
37
-------
FIGURE 18- CHANGES IN NITRATE, ALGAL CELL COUNTS AND
VOLATILE SOLIDS IN CULTURES OF DIFFERENT VOLUMES
c’J
0
—0
00
UJj
0
2>
E
z
LU
C .,
0
I-
LU
A special temperature study conducted with the use of a
1,000-gallon minipond in the fall of 1970 (Figure 19)
indicated that light had a greater effect than temperature
on nitrogen assimilation. A comparison of nitrogen assimi-
latton by an algal culture grown in a unit at ambient temper-
ature and with that of one grown at sumeer temperatures of
25-30°C showed that the higher temperature had no beneficial
effect on nitrogen assimilation.
Flask bloassays seemed to Indicate that high temperature,
rather than excess light, was detrimental to the algal system
under study, although under certain circumstances both would
be equally Inhibitory. High temperature probably affected
the system In several ways: (1) by directly Inhibiting the
algae, (2) by increasing the nitrogen in the system via
speeded up a1ud e decomposition and resulting nitrogen regen-
eration, and (3) by adversely affecting nutrient solubility.
LEGEND
500 ml FLASKS
1000 ml FLASKS
TIME-DAYS (24 HOUR LIGHT)
38
-------
U
w
w
0
I -
SEPT (8
FIGURE 19- EFFECT OF TEMPERATURE ON ALGAL
NITROGEN ASSIMILATION
Effect of Mixing on Nitrogen Assimilation
100
DEC 30
The Phase I studies showed that mixing can affect algal
systems in a number of ways: (1) by moving the algae into
the light zone; (2) by reducing the extent of the anaerobic
areas; (3) by removing the solid material from the system by
keeping the solids in suspension and available for discharge
in the effluent; (ii) by replenishing carbon dioxide exchange
from the air to increase the surface area exposed to the
atmosphere; (5) by stirring the bottom deposits, thus making
more nutrients available; and (6) by preventing thermal
stratification. A study on the effect of duration of mixing
early in the Phase I investigation shoved that a four-hour
period of daylight mix was optimal for maximum nitrogen
assimilation. However, the study did not Include a strictly
night mix regime, although 2 1 4-hour mixing was included.
E
z
(U
o I0
0
I —
z
TIME IN DAYS
39
-------
A study was conducted early in Phase II to determine what
effect I hours of night mixing, as opposed to hours of day-
light mixing, would have on nitrogen assimilation. The study
showed that the benefits of night mixing were equal to those
of daylight mixing (Figure 20).
E
z
w
C,
0
I—
z
z
I&I
D
-J
I L .
La.
w
FIGURE 20-EFFECT OF DAY OR NIGHT MIXING
ON NITROGEN ASSIMILATION
When the performance of cultures under day mixing, with and
without the addition of carbon dioxide, was compared to that
of a nonmixed unit receiving no carbon addition, very little
difference in nitrogen assimilation was observed between
those mixed and nonmixed units which did not receive added
carbon dioxide. To test the hypothesis that carbon addition
and not mixing was the limiting factor affecting nitrogen
assimilation by the algae, one test unit was operated on a
nonniix schedule and carbon dioxide was injected by means of
a diffuser. Algal nitrogen assimilation in this unit was
then compared to that occurring in mixed units, with and
without carbon dioxide addition, and a nonmixed, no-carbon-
dioxide unit (Figure 21). During the first few months, the
only difference observed in nitrate assimilation between
90
TIME - DAYS
-------
*
these miniponds was caused by the addition of carbon dioxide.
Furthermore, when the influent nitrogen concentration
declined in June to the extent that carbon was no longer
limiting in the systems, nitrogen removal in all of the mixed
and nonmixed units receiving or not receiving additional
carbon dioxide was comparable, as is shown by the curves in
Figure 21.
Another aspect of mixing found to have been of importance
was the fact that since all the mixing units had the same
size pumps, the water velocity and solids removal became a
function of pond depth. A measurement of velocities within
the individual units showed a wide range between units. In
addition, since the effluent had to pass out of a riser-tube
arrangement and flow velocities were often negligible,
material of greater density than water tended to stay In the
rniniponds (Figure 6). The change in concentration of
volatile and suspended solids in the pond during a mixing
cycle is plotted in Figure 22, which shows that the suspended
solids began to settle even before the mixing pump had been
turned off. Mixing at velocities to 0.5 foot per second was
not found to be adequate to remove much of the settleable
solids in the unit. As a result, a sludge buildup occurred
as time progressed. These differences in pumping per unit-
volume could not be conveniently corrected during the study
and probably were a factor in reducing the efficiency of the
deeper units.
The results of Phase II studies, although indirect, indicate
that in spite of any beneficial effect it may have, mixing
of an algal growth unit containing large quantities of sludge
may be detrimental. One possibility Is that mixing brings
FIGU ( ZJ-EFF CT OF IXlNG AND NO MIXING WITh AND WITHOUT ADDITION Of CARSON DIOXIDE ON
NITROGEN ASSIMILATION
-------
$00
E
z
0
0
z
w
0
U,
U)
z
0
-J
0
U)
s:oo
FIGURE . SUSPENSION OF SOLIDS DURING MIXING
nonphotosynthesizing material into suspension, which reduces
the available light per active cell. A second reason is that
mixing can lead to the sludge becoming aerobic, which in turn
may result in bacterial nitrogen fixation, possibly adding
to the total nitrogen In the system. Conversely, if the
system Is not mixed, the sludge becomes anaerobic and as a
consequence some of the nitrogen in the algal system Is
removed through bacterial assimilation and/or denitrificatlon.
Thirdly, by stirring the sludge, nutrients become less avail-
able because of the precipitation of phosphate and iron which
usually occurs when the sludge becomes aerobic.
Effect of Detention Time on Nitrogen Assimilation
Figure 23 shows the change in the influent and effluent
total nitrogen during 1970 in 12- and 8-inch units operated
at three different detention times, with and without carbon
dioxide addition. From the early part of the year through
September, there was about 3-5 mg/i difference in nitrogen
assimilation at the three detention times tested, although
the longer detention time units did tend to be accompanied
by slightly lower (1-2 mg/i) effluent nitrogen concentration
MIXING PUMP ON
SUSPENDED SOLIDS
-. — —
SOLIDS
7:45M
830
8:45
s:oo
42
-------
I
I”
— I I
I
r
a usili WIThOUT CAR$Oh DIOXIOC
than were the intermediate and short detention times. From
the early part of the year through September, detention times
ranged from as long as 16 days in January to as short as
1 day in July. Usually, the spread between the short and
long detention time periods was on the order of 2 to 5
detention times, depending on time of the year. Because
detention time is a major operational parameter in algal
growth systems, some other factor must have limited nitrogen
assl,rnilation by the algae.
Late in the fall, the differences in nitrogen removal between
the three detention time units increased. During this
period, algal growth rates were quite low due to limited
light conditions and detention times apparently were adequate
to bracket the optimal flow required for maximum nitrogen
removal.
I C
j- INPLU(NT IAM( £5 TOP IC*P14
\ t \
“ %( ?,Jhl
I d E INFL.U(NT SAME £3 TOP G APw
LE I CND
SHOPT D(IEIdTIOH
INTEHMCDIAT( DETENTIQI*
1.0 116 DETUmopd
£ CNA IIG OP O€TEHTION
1 110
fl j ( 23 UflCT OF DETENTION ON NIThOGEN ASSIMILATION IN 6-INCH AND 12-INCH UNITS
143
-------
Figure 214 shows the total grams of nitrogen removed per day
per minipond in the 12-inch units enriched with carbon
dioxide and operated at the various detention times during
1970. Even though the effluent from the unit operated at
the longest detention time had a lower nitrogen content than
that from the units operated at shorter detention periods,
nitrogen removal in the latter units was greater in terms
of removal per unit per day. Influent loading also may have
had an indirect effect on unit operation, for example, on
sludge buildup and algae washout, etc.; however, these
relationships were not examined in detail at the time.
2
2(
W
>
0
WI
z
W
0
z
MONTHS
1970
FIGURE 24-NITROGEN REMOVED AT DIFFERENT DETENTION
TIMES AT 12 INCH DEPTH WITH CO 2
Effect of Carbon on Nitrogen Assimilation
Autotrophic photosynthesizing algae, unlike heterotrophic
bacteria and algae, use inorganic carbon as their carbon
source. Carbon dioxide is the most common form of carbon
used (33), although Sceriedesmus , the predominant species
studied at the IAWTC, and other algae supposedly can utilize
bicarbonate as readily as carbon dioxide (514). Regardless
of the carbon form used, there is considerable disagreement
as to the form in which it actually penetrates the cell or
chioroplast (33).
SHO DETENTION\ j
A
J F M A M J A S 0 N D
144
-------
Natural oligotrophic bodies of water probably contain an
almost limitless supply of inorganic carbon for algae growth
(147) in the form of alkalinity and from carbon dioxide absorp-
tion from the atmosphere. However, Gotaas et al (55) found
that the lack of sufficient inorganic carbon in raw sewage
can limit the production of algae. The same is probably true
of any water rich in nutrients but poor in carbon.
In poorly buffered systems, the assimilation of carbon dioxide
and bicarbonate by actively growing algae causes the equili-
brium indicated in the following equation to shift to the
right and the pH to rise.
002 ÷ H 2 0 .’— H 2 C0 3 -- -H ÷ HC0 3 H+ + CO 3 Eq. [ 5j
The concentration of any of the components of the carbon
dioxide-bicarbonate-carbonate buffer system is a function of
the temperature, pH, and TDS, as well as of the concentration
of the remaining nutrient components (56, 57). The equilib-
rium equation for the formation of hydrogen and bicarbonate
ions from carbonic acid is:
( Hco 3 )(H ) = K 1 Eq. [ 6]
(H 2 C0 3 )
In the Handbook of Chemistry and Physics (58), the dissocia-
tion constant, K 1 , is reported to be 3.5 x io- at 18°C. At
a pH of 8 the ratio of carbonic acid to bicarbonate ion is
0.0286, at a pH of 7 it is 0.286, and at a pH of 6 it is
2.86 (59). A similar equilibrium reaction between bi rbon-
ate and carbon dioxide has a reported K 2 of Ll.)4 x 10 - at
25 0 C (58); thus at pH 7, the ratio of bicarbonate to carbon-
ate ions would be 2,270 to 1, whereas at pH 11 the ratio
would be 1 to 14•4 (88).
At p11 values above 9, carbonate precipitates as calcium and
magnesium salts, thus decreasing the total alkalinity.
Furthermore, these precipitates also remove many algal nutri-
ents, especially phosphorus and heavy-metal trace elements.
This precipitation of nutrients with increase in pH was found
to be a significant factor in the operation of the IAWTC algal
test units, in which the pH levels were often over 10.
Nitrate reduction by algae is very dependent upon the products
of photosynthesis (34). Photosynthesis produces carbohydrates
which in turn provide the hydrogen donors required for
nitrate reduction. Consequently, green algae in the absence
of carbon dioxide are usually unable to reduce nitrate-
nitrogen at a high rate. Davis (5o) found that a ten-fold
145
-------
increase in nitrate reduction in the light would take place
after adding a carbon source. Bongers (214) was able to
inhibit the incorporation of ammonia (the final step in the
reduction process) into amino acids by limiting the supply
of carbon dioxide in the presence of light. Bongers also
found that cells grown in a complete nutrient medium (nitrogen
content 8 to 10 percent) in light, under carbon dioxide-
deficient conditions, excrete an amount of ammonia into the
medium equal to the amount of nitrate disappearing from the
medium. This was thought to be the result of a lack of
suitable carbon skeletons to function as ammonia acceptors.
Kessler (31i) found that as the carbohydrate reserves of a
cell are exhausted, there is a considerable increase in
nitrite accumulation in the medium, and presumably nitrate
reduction continues (50), even though nitrite and ammonia are
not assimilated. Furthermore, ammonia-nitrogen has been
reported to be toxic at pH values above 9 (214).
Several workers (53, 60) have reported that concentrations
of carbon dioxide much in excess of 0.5 to 10 percent are
either toxic to algae or are growth-rate limiting.
Conversely, Pew et al (38) and Gates and Borchardt (147) have
been able to grow algae at concentrations of carbon dioxide
as high as 100 percent. Pew et al concluded that it is
feasible to use highly concentrated carbon dioxide for
continuous algal growth, if the growth rate is balanced with
carbon dioxide addition rates. Since many of the earlier
workers did not account for a total balanced nutrient system
as a function of carbon dioxide addition, this might explain
their inability to use carbon dioxide levels higher than
10 percent.
One other facet of carbon dioxide addition pertinent to the
present study is the effect it has on the pH of the growth
medium. Changes of pH in the medium is a function of algal
cell growth (photosynthesis); the pH rises as carbon and
nitrate are assimilated by actively growing cells. As the
pH increases, many of the nutrients necessary for growth
precipitate out of solution (28). In the precipitated state,
their availability to algal growth is questionable. The
addition of supplemental carbon dioxide helps to stabilize
pH in an actively growing culture, acting both as a carbon
source and, by lowering the pH level, as a means of maintain-
ing nutrients in solution.
Since algae are approximately 50 percent carbon and 8 to 10
percent nitrogen, theoretically the ratio of carbon to
nitrogen in the growth medium should be 5:1. Comparison of
the influent total alkalinities with predicted nitrogen
concentration (Figures 3 and 7) indicates that carbon
probably would be a limiting nutrient during those times of
the year when the nitrogen content of the influent Is high;
146
-------
however, these calculations did not take into consideration
any air-water carbon dioxide exchange.
Because the relationship of carbon to nitrogen requirements
in tile drainage was unknown, a number of light box studies
were conducted to determine the effect of carbon addition on
nitrogen assimilation. In general, the studies showed that
the amount of carbon available to the algae during nitrogen
assimilation was an important factor. Data plotted In
Figures 25 and 26 from studies by Brown and Arthur (L 1)
Indicate the typical response of algal nitrogen assimilation
and biomass production to the addition of various conceritra-
tions of carbon.
In several other studies, the effect of carbon addition as
bicarbonate (a form thought to be utilized by Scenedesmus )
on nitrate assimilation was compared to that of L percent
carbon dioxide addition. The tests seemed to indicate that
bicarbonate-carbon, injected as sodium bicarbonate, was not
available for growth of Scenedesmus q dricauda . Studies
conducted later with the use of a minipond also indicated
that this form of carbon addition may not be as available as
that of carbon dioxide to Scenedesmus quadricauda . Initially,
when sodium bicarbonate was added to this minipond there was
a positive response by the Scenedesmus guadricauda culture
In both appearance and nitrogen assimilation. However, within
a 2- to 3-week period, the Scenedesmus culture was replaced
by a variety of green and blue-green algae. An examination
of the minipond showed a large amount (1-2 inches) of
sludge, which was assumed to be carbonate compounds. Never-
theless, even though there were different algae in this unit,
nitrogen removal continued to be comparable to a miriipond
which was receiving carbon dioxide for the remainder of the
year. The initial amount of sodium bicarbonate added to this
unit appears to have been too great.
Early in the Phase II study, It was decided to determine
whether the carbon-to-nitrogen ratio in the tile drainage
could be maintained by using the carbon available in the
alkalinity as a basis for calculating the carbon addition
required. Since the pH In the tile drainage was usually
about 7.0 to 7.5, most of the carbon was available as bicar-
bonate. The carbon addition was estimated as follows:
Ca = 5N 1 Cj Eq. [ 7]
Where Ca = carbon addition required, Nj = influent nitrogen
in mg/i-N, and C 1 = influent carbon from bicarbonate alkalin-
ity In mg/i-C.
-------
F )GU E 25-ThE OF RIOUS LEVELS OF AERATiON
ON NITRATE REMOWAL IN THE UGIIT BOX
bOO
to
0
S00 -.
S
200 -
I I I F T
I I I
250/ItS 250/250 300/250 500/500 1000/SOD
FLASK SIZE I CULTURE VOLUME
F1GIJRE 26-EFFECT OF SURFACE/VOUJ E RELATIONSHIP AND
VARIOUS TYPES OF AERATION ON CELL NUMBERS AT SIX DAYS,
AVERAGE OF FIVE REPLICATIONS
FROM BROWN AND ARTHUR (4))
148
-e
S
I
TIME—DAYS (24 140UR LIGHT)
q
AIR— \
o , \
‘%._ “
SWIRLED
A
-------
Although this method of estimating the required carbon did
not take Into account such things as (1) actual carbon
availability, (2) carbon dioxide air-water exchange, and
(3) calcium carbonate precipitation at high pH values, it
did serve as an effective way of estimating the amount of
carbon required in the units. The studies further showed
that carbon had to be injected only during afternoon periods
of peak photosynthesis. The data as plotted In Figure 27
depict the typical diurnal changes in pH and bicarbonate
levels accompanying varying rates of photosynthesis.
I . . ,
0
0
C
0
E
0
0
=
Figure 27- TYPICAL SHIFT IN pH AND HCO’ IN ALGALTEST UNITS
Actual operation of the units showed that there were more
problems to be encountered in injecting the carbon dioxide
into the system in the correct amount than in determining
the correct carbon concentration. Automatic carbon dioxide
injection with pH control would probably have eliminated
many of the operational problems encountered at the IAWTC.
49
-------
Figure 28 shows the average concentration of the total
influent and effluent nitrogen. It also depicts the average
effluent organic and nitrite-nitrogen concentrations during
1970. During the period January through May, the nitrite
concentration in most of the units was abnormally high, with
several miniponds over 5 m /l nitrite-nitrogen.
A DETENTION TIME CHANGE
t\
E FF. N
1%
1\1 t (1 ‘
S %
U % a
NITRITE -N 1, ‘ I P4 .J\
ORGANIC-N /. . /
— — — J— — —
J F M A M J A S 0 N D
MONTH
1970
FIGURE 28-CHANGES IN TOTAL INFLUENT NITROGEN AND EFFLUENT TOTAL
SOLUBLE NITROGEN, NITRITE AND SOLUBLE ORGANIC NITROGEN—
AVERAGE OF ALL UNITS
Since virtually no nitrite entered the system by way of the
influent, it appeared that nitrate was not being completely
reduced and, as a result, nitrite was being released into
the culture medium. A comparison of the performance of a
12-inch unit with and without carbon dioxide addition
(Figure 29) indicated that this nitrite release probably was
the result of a carbon deficiency for the amount of nitrogen
to be assimilated. This result agrees with the finding of
Krauss (33).
INF. N.
40
z
4
3o.
0
laJ
I—
z
-20
-J
0
5-
I’:
I’
/
‘I
50
-------
S.
I i i
U I
FiGURE 29. CHANGES IN NITRITE CONCENTRATION IN TEST UNITS
WITH AND WITHOUT CO ADDITION
As shown in Figure 29, when the carbon level was adjusted
in April, the nitrite concentration of the effluent from
the units receiving carbon dioxide declined immediately to
less than 1 mg/i, the normal level in the system. Further-
more, the concentration of nitrite in the effluent from the
unit not receiving carbon dioxide remained at a high level
until mid-May, at which time the nitrite decreased in
response to the decline in the carbon requirement. After
this time, the nitrite concentration of the effluent from
all the test units continued at a low level for the
remainder of the year (Figure 28).
The above data seem to indicate that, under conditions of
carbon deficiency, nitrate can be taken into the cell but
cannot be completely reduced to ammonia, and that nitrite
is then released from the cell at a level proportional to
the carbon deficiency. One other possibility is that
bacterial denitrification was occurring in the test units
at a fairly high rate and that the carbon deficiency
affected their rate of nitrate reduction. In any case, if
this carbon deficiency had been recognized earlier in the
year, measures could have been taken to improve the
nitrogen removal efficiencies of the test units (Figure 30).
0 APRIL 20
I9?O
51
-------
A CHANGE OF DETENTION
A ,-INFLUENT NITROGEN
All
:
CO 2
a
,‘
/
%1
V
c0 2 ADDITI0N7L /‘
V
ON
A
..
‘I
‘I
V
A A A
,
J F
I I — • 1 I
M A M J J A
F —
S
— — I
0 N
D
970
FIGURE
30-CHANGES IN TOTAL INFLUENT AND EFFLUENT
INTERMEDIATE DETENTION TIME UNiTS
WITH AND WITHOUT CARBON DIOXIDE ADDITION
NITROGEN
IN
3O
z
L I
0
I—
z 20
LI
-J
-J
0
U)
IG
0
3 1
I
I ‘a
I I
I
ii L j
A A
-------
In August and September 1970, the culture in a minipond was
maintained at a pH of 9.Ot.5 by injection of controlled
amounts of 100—percent carbon dioxide into the unit. The
carbon dioxide injection device consisted of an “in pond”
pH probe connected to a regulatory unit by which the
desired pH could be maintained by the opening and closing
of a solenoid valve on a 100 percent carbon dioxide cylinder.
The gas was injected into the culture through a diffuser
system placed on the bottom of the test unit containing the
culture. A clock was then connected to the solenoid to
indicate the time of day at which the pH exceeded 9.0 and
to determine the number of minutes per day carbon dioxide
was injected. Plotted in Figure 31 are the minutes during
which 100 percent carbon dioxide was injected each day,
and the changes in effluent alkalinity and nitrogen. With
this arrangement for automatic pH control, the carbon
dioxide came on early in the evening, much later than had
been expected. A hypothesis for the lateness of the hour
of high pH is that the stored cellular carbohydrates were
being used prior to peak photosynthesis. The carbon
addition which followed this peak served to replenish the
carbohydrate level. Thus, it is possible that carbon
dioxide was being injected prematurely in the rest of the
units during 1970 as they received carbon dioxide from
1:00 to 1I :00 p.m. each day.
400
0
U
U
U 300-
0
U
E
z: 200
1
4
-J
4
I-
z
(U
-J
(4-
w
100-
0
(I )
‘U
I —
z
z
(U
z
0
0
0
4
0 ’
U
FIGURE 31— CARBON DIOXIDE ADDITION BY AUTOMATIC p H CONTROL
MINUTES OF
-100% COa ADDITION
I
/ \
‘
/
ALKALINITY
0 tO
E
z
(U
C,
0
I-
z
I.-
z
‘U
-I
( 4-
(U
TIME IN DAYS
40
53
-------
The changes in length of time of 100 percent carbon dioxide
injection per day indicated the existence of a period of
carbon dioxide utilization which was always followed by a
recovery period during which the pH stayed below 90.
Apparently, the only thing saving the unit from complete
failure was the self-regulating pH mechanism. Possibly
because the gas was injected as very large bubbles, some
algal cells came into immediate contact with 100 percent
carbon dioxide gas which was toxic to the cells. The
possible toxicity of 100 percent carbon dioxide injection
to algae and its inhibition of nitrogen removal were
discussed in the Phase I report.
Later, the 100 percent carbon dioxide concentration was
reduced to 11. percent to determine whether carbon dioxide
concentration influenced algal response. However, the
unit was not operated at a time when the carbon supply was
inadequate. Consequently, the results are inconclusive in
terms of effect of carbon dioxide concentration.
Effect of Phosphorus and Iron on Nitrogen Assimilation
Phosphorus is on of the major nutrients required for the
normal growth of algae and is frequently a limiting factor
of algal growth in nature (22, 42, 61). In solution,
phosphorus is primarily present as inorganic orthophosphate,
a form used by all living organisms (62). Since it plays an
important role in photosynthesis, uptake is considerably
greater in the light than in the dark, particularly in the
absence of carbon dioxide (28). Phosphorus is indispensable
in energy transformation reactions, existing as adenosine
triphosphate (ATP) formed by photosynthetic phosphorylation
(37) via the esterification of inorganic phosphate (28).
Algae are also capable of storing phosphorus as condensed
polyphosphates to be used at times when phosphorus is
deficient (37). Azad and Borchardt (3?)f using Scenedesmus
and Chiorella , found the “critical level l of phosphorus to
be about 1 percent of the cell weight. At lower concentra-
tions, growth was proportional to the phosphorus concentra-
tion in the medium. They found that at the critical level,
growth was constant and independent of phosphorus and
defined the level of phosphorus incorporation into the cell
above the critical level as “luxury uptake”. They also
concluded that higher phosphorus concentrations were
required to grow the same concentration of algae at low
temperatures than at high temperatures.
Working with phosphorus-starved cells, Azad and Borchardt (37)
found that during “phosphorus dilution” the growth rate de-
clined to zero and the culture assumed the yellow-to--brownish
coloration typical of chiorosis. Indeed, the symptoms of
514
-------
phosphorus deficiency are the accumulation of fat, starch,
and cell wall substance which indicates some interference
with nitrogen metabolism (28).
Stumm and Leckie (22), discussing natural bodies of water,
stated that it is not possible to establish a critical level
of phosphorus because the rate of bioniass production is pri-
niarily influenced by the rate of supply of soluble phosphorus
to the algae. According to them, the rate is function of:
(1) regeneration of nutrients from the biota and detritus,
(2) the supply In the ln.fluent, (3) the exchange with the
sediments, and (14) the transport process (diffusion).
The pH of the medium may alter the rate of phosphorus uptake
either by a direct effect on permeability of the cell mem-
brane or by changing the ionic form of the phosphate (28).
As the pH level rises due to the activity of growing algal
cells, inorganic solid phases of phosphorus may form by
direct precipitation of phosphorus with calcium, aluminum,
and Iron compounds as well as with clays (21, 22, 26, 37,
51, 63, 64). Figure 32, taken from a paper by Stumm arid
Leckie (22), depicts the solubility of different phosphate
phases as a function of pH.
a.
(I )
0
a.
w
-J
-j
0
C ,,
a:
0
p 11
FIGURE 32-PHOSPHATE SOLUBILITY
AS A FUNCTION OF pH
FROM STUMM AND LECKIE (22)
55
-------
One of the most important reactions plotted In Figure 32 is
the exergonic interaction in the formation of bydroxyapatite:
10 CaCO (s) + 611POj 2 + 1120 + 21P
Eq. [ 8]
Ca 10 (P01 4 ) 6 (OR) 2 + 10 11C0 3
It is thought to be one of the principal control mechanisms
for the exchange of phosphates between the sediments and
overlying waters. Precipitates of metals, which are formed
at the pH normally encountered in algal systems, can also
cause some phosphate removal from the medium, either by pre-
cipitation as an insoluble salt or by adsorption upon some
insoluble substance (21, 22, 26, k3, 6i, 63, 6k). The extent
of phosphorus precipitation is increased under aerobic con-
ditions and conversely is decreased under anaerobic condi-
tions, as a result of oxidation-reduction changes. Bongers
(2k) and others also found that algae settling rates vary
according to the coagulation effect of the insoluble phos-
phate salts produced at high pH levels. This finding
responds to that by Steele and Yentech (28), who noted that
as cells aged or nutrients became depleted, the rate of algal
settling increased. Conversely, if for any reason the
concentration of soluble phosphate in solution is increased
beyond 5 to 20 mg/i (that is, by low pH conditions), phos-
phorus becomes toxic or inhibitory to the algae (27, 42).
Zabat et a]. (65), studying the kinetics of phosphorus assi-
milation by algae, found that the phosphorus content per unit
cell mass was higher under unfavorable conditions of pH and
temperature, although the cell yields were lower. Their
report presents a very comprehensive review of many aspects
of phosphorus, including its origin in lakes, its assimila-
tion by algae, and its removal in wastewater treatment plants.
The need for iron by actively growing algae is well substan-
tiated in the literature, although the form in which it can
be utilized Is highly debatable (26, 27, 28, 112, 66, 67).
An iron deficiency in the algal growth medium leads to a
reduction in the rate of growth because of a reduction of
photosynthesis brought about by a decline in chlorophyll
production. It has been postulated that the iron deficiency
reduces the synthesis of proteins within the chioroplast.
It was further demonstrated that an increase in temperature
caused a sharp increase in the iron requirement as well as
an increase in the requirement for magnesium, zinc, and
manganese (28).
-------
Excess iron can be toxic, depending upon algal species
(27, 28). According to Provasoli and Pinter (142), a given
concentration of iron can be in excess (toxic) at one pH
level and be deficient at a slightly different pH. The
change in iron solubility with pH and oxidizing-reducing
conditions has been well documented. Morgan and Stumm (68)
have made a comprehensive review of the chemistry of iron
and manganese in limnological cycles. In Figures 33 and 314,
taken from their paper on “The Role of Multivaler t Metal
Oxides in Limnological Transformations, as Exemplified by
Iron and Manganese”, the changes in iron and phosphorus
solubility are shown as a function of pH. As indicated
I A -
&
4
-J
0
I,
0
-j
FIGURE 33-SOLUBILITY OF FERRIC HYDROXIDE
IN WATER AT 25°c
FROM MORGAN AND STUMM(68)
-\
\ I”
“ s
/
“S •
Fe(OH4
2 4
pH
FIGURE 34 SOLUBILITY OF FERRIC PHOSPHATE
IN WATER AT 25c
FROM MORGAN AND STUMM(6
pH
a-
4
-j
0
57
-------
by the curves in Figures 33 and 3)4, very little Iron Is
soluble at the pH levels expected in systems with even
minimal algal growth. According to Hutchinson (67), ferric
iron can be present in excess of 0.01 ppm only as a suspen-
sion of oxides or hydroxides In aerated waters in which the
pH is above 5. Phosphate solubility In lakes has also been
correlated to that of Iron. Both of these important algal
nutrients are known to be released from the sediments under
anaerobic conditions and low pH levels,
The nitrogen-to-phosphorus ratio in the IAWTC tile drainage
was about 100 to 1 (based on a 20 mg/i nitrate-nitrogen
influent), as compared to about 10 to 1 in the algal cell.
As a result, phosphorus was the first nutrient studied in
Phase I, and was found to be insufficient In the tile
drainage for maximum nitrogen assimilation. Subsequently,
a general screening of known algal nutrients was made,
utilizing LAWTC tile drainage, Light box tests indicated
that the addition of Iron greatly Increased the level and
extent of nitrogen assimilation by the test organism,
Scenedesmus quadrlcauda . On this basis it was concluded
that iron as well as phosphorus was a limiting nutrient in
the IAWTC water. Additional tests with ferric chloride
(FeC ]. 3 ) and ferric sulfate (Fe 2 (SO)4) 3 ) indicated that both
forms were adequate sources of iron. However, iron applied
as ferric citrate (FeC6HcO) had to be applied at about twice
the concentration (as Pe7 of that required to give the same
effect on growth and nitrogen uptake as the other two forms.
The addition of a chelating agent the sodium salt of EDTA
(ethylenediarnine tetraacetic acidj, was found to decrease
the optimum concentration of Iron required for maximum
growth and nitrogen uptake 1 but the use of this material was
discontinued because of its high costs and the fact that it
contributes nitrogen to the system.
The beneficial effect of iron and phosphorus on nitrogen
assimilation is reiterated In this report because a reevalu-
ation of data Indicated that iron In addition to phosphorus
was an important rate-iImiting nutrient in many of the IAWTC
studies. Plotted In FIgure 35 are data obtained In one study
In which different concentrations of Iron and phosphorus
were tested for their effect on nitrogen assimilation by
Scenedesmus quadricauda . As shown In this figure, little
assimilation of nitrate-nitrogen took place In the absence
of Iron or phosphorus. The minimal effective level of each
of these two nutrients was found to be approximately 2 mg/i
(at 20 mg/i original nitrogen). At concentrations of iron
and phosphorus higher than 2 mg/I., a definite interaction
between phosphorus and iron as related to nitrogen assimila-
tion was observed to have taken place. Since high levels of
dissolved oxygen (DO) and pH (over 9) are associated with
high rates of nitrate assimilation during photosynthesis,
58
-------
/
/
/
/
/
/
/
4.0
I
1.5 3 4.5 € 1 5 9 10.5 2
PNOSPHORUS AS P0 4 (M9/l)
FIGURE 35-EFFECT OF VARYING IRON AND
PHOSPHORUS CONCENTRATIONS ON
NITROGEN ASSIt LATIOI4- LIGHIBOX STUD’r
undoubtedly this interaction corresponded to the coprecipita-
tion of iron and phosphorus under active growth conditions.
The maximum extent of nitrogen assimilation in the laboratory
cultures occurred when 2 to 3 mg/l of both iron and phosphorus
were added.
In the tests on the effect of adding iron, the units not
receiving carbon dioxide were operated from April through
September 18, 1970. After the latter date, the unit not
receiving iron was used in a temperature study. In this unit,
the addition of iron consistently improved the total nitrogen
assimilation by approximately 3 to 5 mg/l. Apparently,
carbon dioxide addition not only made up for the carbon
deficiency but, by lowering the pH level, also increased the
solubility of iron and possibly other nutrients in the tile
drainage.
In the Phase I investigation, iron had been added to the test
units at concentrations ranging from 3.0 to 6.0 mg/i FeC1 3
as Fe. During the four to six weeks duration of these
studies, upwards of 30 mg/i iron were found to accumulate in
the test units in a nonfilterable form. Iron in solution was
usually at concentrations less than 0.1 mg/i, the lower limit
of detection with the method used.
Because nonsoluble iron has been reported to be assimilable
by algae, the iron concentration in the Phase II investiga-
tion was decreased from 3.0 mg/l to 1.0 mg/i or less on the
—— ——
1.0
•/ ,/// 2.0
w
0
I-
w
I-
/
/
/
/
9.0
59
-------
assumptions that iron accumulated in the unit and that it
would be sufficient for the algal growth necessary for
maximum nitrogen assimilation. As with the phosphorus
addition, iron was added to individual miniponds rather
than to the storage pond.
Figures 36 and 37 illustrate the effect of adding iron and
phosphorus to the 1,000-gallon test units during 1970.
The data in Figure 36 are from ponds with the CO 2 addition
and show that phosphorus was necessary for maximum nitrogen
assimilation but that Iron (0.5 to 1 mg/i addition) had
no beneficial effect -- perhaps because the C02 increased
the availability of iron In the influent. In those ponds
not receiving C02, shown In Figure 37, iron did appear to
be a necessary addition. Phosphorus was added to both
units from which the data in Figure 37 were obtained.
The data plotted In Figure 38 show the changes In the
influent and the average effluent orthophosphate concentra-
tions characteristic of all of the ininiponds receiving
phosphorus. They also Indicate phosphate concentration of
the effluent from the unit which did not receive phosphate.
From these data, It was concluded that 80 to 90 percent
of the irifluent phosphate either was assimilated by the
algae or was precipitated out of solution. In addition,
the figure shows the average phosphate concentration In
the cultures in all of the test units as well as the
influent nitrogen and phosphate concentration. Apparently,
as the concentration of influent nitrogen decreased, excess
phosphate, that Is, that not assimilated or precipitated,
went into solution.
During the month of July, most miniponds were emptied,
repaired, cleaned, and restarted with a common Inoculum
from the 1/ 2 4-acre demonstration unit. Several months
later, samples of sludge from all the units were analyzed
for organic and Inorganic constituents. The results of
these analyses are plotted in Figure 39 and listed in
Table 3. They indicate that, even during mixing, up to
99 percent of the total solids In the units were present
as a sludge on the bottom of the unit in the form of:
(1) carbonates, (2) phosphates, (3) Iron, and (1 ) algal
material. Further calculations indicated that much of
the iron, phosphate, and carbon added during the two-to-
three-month period could be accounted for in the sludge.
This precipitation occurred to a larger extent than in
the Phase I studies and was probably accumulative through-
out the period.
60
-------
40
M J
MONTH
A DETENTION TIME CHANGE
A
FIGURE 36-EFFECT OF IRON OR PHOSPHORUS ADDITION ON NITROGEN
ASSIMILATION WITH CARBON DIOXIDE ADDITION
.4 .
E
z
w
0
0
z
-J
4
0
I—
FIGURE 37-EFFECT OF IRON ADDITION ON NITROGEN
WITHOUT CARBON DIOXIDE ADDITION
61
ASSIMILATION
N FLUENT NITROGEN
4-
E
z
w
z
-J
4
I-
3C
20
I0•
0•-
.\
PHOSPHORUS
NO I
1:
I.
I•j! _I
p.. ..
V.,
I’ . .
I 4-’u . ./
i I..
‘
.% I.
..‘I.
.4, A A A ______
J F M A 0 N 0
J A ‘S
41
A DETENTION TIME CHANGE
INFLUENT NITROGEN
NO IRON ADDED
IRON ADD
M
A
M
MONTH
J A
-------
10 20 30 0
AUGUST
20
SEPT EMB ER
1970
30
FiGURE 39-TYPICAL SLUDGE ACCUMULATION IN TEST UNITS
OCTOBER
20
00
50
30
I
N*’ OCTOSEft NOVIMSIS bSEt (m
7O
F4URE 3- INFUJENT *110 EFFUJENT PHOSPI4OSiIS CONCENTRATIONS IN UNITS WITH NORMAL AND PtoSpsORtj
IUP LE NTED TILE DRAINASE
800
! 600
0
-J
0
4’)
400
200-
lai
C,
0
U)
-J
0
U ,
IiJ
-J
-J
0
>
I-
z
C-)
C LI
0
62
-------
TABLE 3
PERCENT OF TOTAL POND MATERIALS FOUND
IN POND SLUDGE--DURING MIXING
Unit
No.
IF1].terable
I Solids j
CaCO 3 Total P
Ortho-P
Fe
1
87
8
1 4 .7
38
57
2
93
48
94
85
82
3
79
3
--
30
91
4
5
6
i 4
72
86
61
59
62
95
95
84
75
66
6i
66
70
83
7
8
9
10
11*
12*
13
14
15
16
17*
i8
19
20
21*
22*
86
8
76
81
99
99
89
87
74
88
92
91
92
98
99
99
69
45
32
32
83
77
97
26
66
74
40
8
86
91
8o
72
99
100
95
100
85
58
98
100
100
100
100
48
100
100
71
58
93
--
8i
100
68
49
87
100
100
100
100
92
82
99
27
50
86
71
77
72
--
65
74
68
33
85
82
80
90
--
--
*Not mixed.
Replicated samples of tile drainage containing 2.0 mg/l each
of iron and phosphate were adjusted in the laboratory with
sodium hydroxide to raise the pH level to that normally en-
countered in the algal units. The results of these tests,
Figure 40, showed that in tile drainage the amount of iron
and phosphate in solution is a function of pH, and that
between pH 8 and 9, levels that are lower than those normally
encountered in algal systems, very little phosphate or iron
is in solution. Therefore, it appears that: the actual
concentrations assimilated by the algae must be quite low;
the algae can assimilate some nutrients in the dark, that is,
when the pH is low; or they can use colloidal material.
63
-------
(TOTAL)
E
z
pH
FIGURE 40-IRON AND PHOSPHORUS SOLUBILITY
AT DIFFERENT pH VALUES
In Table k are listed the average phosphorus changes In the
effluent from the various unite during 1970. The data In-
dicate that: (1) mIxing tends to precipitate phosphate,
(2) the continual removal of sludge decreases the soluble
phosphate, and (3) phosphate loss is related to unit loading
and carbon availability. These conclusions agree with the
changes predicted for phosphate and iron solubility as shown
In FIgures 32, 33, and 311, specifIcally that, at high pH
levels and with the culture in an aerobic condition, phosphate
and iron will be In an insoluble form. The data led to the
conclusion that the actual availability of these nutrients
ifl the nonsoluble form was quite limited, and, hence, the
requirements of these nutrients must be quite low because the
nitrogen assimilation did not appear to be adversely affected.
In the last few months of Phase II, iron was added directly
to the blomass control settling tanks of the 1/4-acre demon-
stration unit and to several alniponds, although phosphate
was still added to the demonstration unit directly. This was
done to decrease the precipitation of phosphorus and iron.
Analysis of data from the units indicated that: (1) Iron was
being picked up by the algae as they passed through the
(SOLUBLE)
I.
9 (0 II
64
-------
TABLE 4
AVERAGE DISSOLVED EFFLUENT PHOSPHATE IN
MINIPOND DURING 1970
Unit
mg/i-p Unit J
mg/i-;
Mixed
Soil (no Fe)
Biomass Regulation
0.2
0.6
0.2
Nonmixed
Nonsoil (Fe)
No Biomass Regulation
0.4
0.3
0.5
Short Detention Time
0.5
0.25
Long Detention Time
0,2
0.1
CO 2
No CO 2
CO 2
No CO 2
biomass regulation device (any attempts to stop iron addition
during Phase II were found to be detrimental, apparently be-
cause iron was essential); (2) the volatile fraction of the
effluent solids was increased from the range 40 - 50 percent
to 60 - 70 percent; and (3) the ability of the algae to
remain in suspension was enhanced. An effect of directly
adding iron in this manner will be discussed In detail in
the chapter, “Algal Harvesting and Disposaltt.
It is postulated that the accumulation of inorganic and
organic material, as indicated by the data in Table 3 and
the curves in Figure 39, may be detrimental to maximum
nitrogen assimilation in that: (1) nutrients become unavail-
able to the algae because of precIpitation, (2) the
accumulation of Inorganic compounds flocculates much of the
viable photosynthesizing algae, (3) the suspended solids
present greatly interfere with light availability to the
algae in the mixed systems, (4) the re-solubilization of the
nutrients may bring about the production or release of toxic
substances, and (5) decomposition of the accumulated sludge
may release nitrogen which can add to the total soluble
nitrogen In the system.
From these and similar studies it was concluded that: (1)
iron and phosphate are essential to maximum nitrogen assimi-
lation in IAWTC tile drainage, (2) the method of nutrient
addition is very important, and (3) because of the factors
listed above, the type of algal reactor tested at the IAWTC
(with mixing) would probably not permit optimal algal growth
and maximum nitrogen assimilation on a long-term basis.
65
-------
Effect of Sludge Accumulation and Decom osltion
on Nitrogen Assimilation
Although not normally considered a growth regulatory factor,
sludge accumulation (algae, detritus, and inorganic nutrient
precipitates) can affect the nitrogen removal efficiency of
algal wastewater treatment systems in a number of ways.
According to Foree (29), the nature of the factors on which
the coefficients a, b, c, d and e in Equation [ 1) are based
determines the chemical composition of the algal matter syn-
thesized, and varies according to the species and age of the
algae, temperature, available nutrients, and other related
factors. Jewell and McCarty (20) and Foree and McCarty (30),
studying both the aerobic and anaerobic decomposition of algae,
found that a large fraction of the Initial particulate nitro-
gen and phosphorus was not regenerated (40 to 60 percent) but
remained in the undecomposed (refractory) particulate
material after active decomposition and regeneration appeared
complete. Conversely, Golterman (26) reported that nitrogen
Is rapidly re-mineralized after the death of algae and, in
the shallow waters of lakes, algae would probably be broken
down before they reached the mud.
Regardless of the extent of decomposition, the nutrients
released from algal decomposition have been found to be an
excellent source of nitrogen and other nutrients for algal
growth (31). As shown by the diagram in Figure 1, the only
nitrogen form directly resulting from the decomposition of
algal sludge is ammonia (21, 6)4.). Ammonia, in turn, is
readily utilized by the actively growing algae in preference
to other forms of inorganic nitrogen, such as nitrate or
nitrite (19, 23, 28, 34, 35). In the course of time, under
aerobic conditions, ammonia Is oxidized back to nitrate by
nitrifying bacteria. Hence, through algal accumulation and
decomposition, a variable amount of nitrogen will be recycled
in the system. Most likely, the extent of the sludge nutrient
recycle (decomposition) is a function of the degree of sludge
accumulation and temperature (30).
A test performed in January 1971, in which 16 mg/i of ammonia
was added to a ininipond unit which had been removing a fairly
constant level of nitrogen, Indicated that ammonia was assimi-
lated preferentially to nitrate. The data plotted in Figure
41 show that the ammonia concentration in the effluent
decreased faster than was predicted by dilution alone. The
observed difference was assumed to be caused by the preferen-
tial uptake of the ammonia-nitrogen by the algae, although
nitrification could have been partially responsible. It is
postulated that if this fact were not recognized, for example,
during periods of elevated decomposition, the assumption
could be made that the algae were assimilating less nitrogen
than the unit influent and effluent level would Indicate,
66
-------
30
UENT
EFFLUENT N0 3 -N
20 ..
z
w
EFFLUENT
N H 3 (THEORETICAL)
I0
—
—
\ A, EFFLUENT NH 3
S (MEASURED)
____ - I
0 10 15
TIME-DAYS
FIGURE 41-PREFERENTIAL ALGAL UPTAKE
OF AMMONIA OVER NITRATE
while in actuality they might have been utilizing more than
the influent level. Accordingly, it would seem that if the
system were not mixed (anaerobic), denitrification rather
than nitrificatlon would take place, which might benefit
total nitrogen removal. Although this test was performed
only one time during the winter at low growth rate levels,
what happened In the test appears to be a reasonable
explanation of what probably occurred during certain times
of the year in the test units.
Assimilation of ammonia by algae in preference to nitrate
is the result of the fact that a lesser amount of energy is
required for assimilating ammonia, and that ammonia can re-
press both the nitrate uptake mechanism and nitrate reductase
formation in the nitrogen-reducing reaction (34). Further-
more, the rate of ammonia assimilation is much more rapid
than that of nitrate assimilation or that of the subsequent
conversion of nitrogen to protein (35). Thus, the accumula-
tion and subsequent decomposition of algal sludge can affect
nitrate uptake.
Another beneficial effect of algal sludge decomposition re-
ported by Provasoli and Pinter (L4.2) to take place in nonmtxed
algal systems is the release of organic acids (especially
amino acids) which can act as trace-element chelators;
however, no attempt was made to determine whether this
phenomenon had any effect on the IA1 TC system.
ADDITION
.•
St
‘S
1
5
67
-------
The various effects of aerobic or anaerobic conditions
(mixing or noriniixing) on inorganic nutrient solubility in
the sludge were described in the discussion on iron and phos-
phate. A further detrimental effect of mixing is that the
suspension of accumulated inorganic precipitates and of non-
photosynthesizing algae will reduce light penetration into
the culture, to the extent that algal metabolic activity is
greatly retarded (31). Because of this decrease in available
lig ’it, Oswald and Golueke, working with facultative sewage
ponds, recommended that any mixing essential to the disruption
of any anaerobic layers that might form be carried out only
for 2 to A4. hours at night. Stumm and Leckie (22) also found
that disturbing the sediments can reduce the nutrient-binding
effect, expose large levels of nutrients to the water (ex-
cesses of some nutrients are toxic), and, most importantly,
reduce the buffering capacity of the sediments. Thus,
mixing must be carried out judiciously.
Effect of Water Quality on Nitrogen Assimilation
The trace elements (micronutrients) considered necessary for
sustained algal growth were listed in a previous section.
Their solubility as well as availability to algae are basic-
ally similar to those described previously for iron and
phosphorus. The amount of a specific trace element required
varies greatly according to algal species arid level of growth
(28). Summarized In Table 5 are the micronutrients normally
required for certain specific algal metabolic activities ( 43).
TABLE 5
SU)O4ARY OF MICRONUTRIENT
TRACE MINERAL REQUIREMENTS*
Process
Trace Element Req uIred
Photosynthesis
Manganese, iron, chloride,
vanadium
zinc,
Nitrogen Fixation
Iron, boron, molybdenum,
cobalt
Other Functions
Manganese, boron, cobalt,
copper, silicon
*Table from Fruh (1 13).
68
-------
As with all algal nutrients, the total concentration of a
particular niicronutrient in the growth medium is practically
meaningless, unless it is in a form capable of being assimi-
lated by the algae.
During the course of the Phase I and II investigations,
several micronutrients were evaluated for their potential
effect on nitrate assimilation. These studies were usually
only conducted once, with water of a specific quality. As
indicated in Figure 3, the quality of the water changed
greatly throughout the year, and, as a result, a variation
in response would be expected at different times of the year.
The micronutrients tested in the light box (at low concentra-
tions) were molybdenum (Mo), vanadium (V), manganese (Mn),
zinc (Zn), and potassium (K). Of these micronutrients, man-
ganese and potassium were found to improve assimilation of
nitrate by Scenedesmus guadricauda under the test conditions,
depending upon t level of carbon available (that is,
function of pH level). When K and Mn were added to several
miniponds, no measurable response in nitrogen uptake was
noted. Howeverd these tests were not definitive because, in
conducting them, consideration was not given to the general
constituents of the water or to the specific growth rate of
the algae which can affect the nutrient requirements. In
the light box studies, there was little response in improved
nitrogen uptake with Mo, V, or Zn addition; however, Zn was
found to appreciably increase the ability of the algae to
remain in suspension. Although these tests were very limited
and inconclusive, it is suggested that in any future work a
continuous nutrient check of the plant influent be made to
maintain a growth medium that will support extensive algal
growth and nitrogen assimilation.
During the course of this investigation, several light box
tests were conducted to determine if there would be any
differences in biological response to water from different
tile drainage systems in the San Joaquin Valley. In each of
these studies, the nitrogen, phosphorus, carbon, and iron
concentrations were brought to identical levels, and the
algae were cultured under similar light and temperature
conditions at the same low level (2,000 cells per ml) of
algal inoculum, Scenedesmus guadricauda . In other words,
all major growth factors were presumably equal. Therefore,
any difference in nitrogen assimilation was assumed to be
the result of some factor or factors other than those
named in this paragraph.
In the tests, the rate and amount of algal nitrogen assimi-
lation were usually noted to vary significantly according
to the tile drainage used in the medium. For example, in
69
-------
the last run of the series, tile drainage was collected
from 12 different areas in the San Joaquin Valley. Special
care was taken to assure that the tile drainage came from
fields with different agricultural crops and varying soil
composition. The tile drainages were then spiked with
nitrate-nitrogen to 50 mg/i, the level of the highest
drainage collected, and varying concentrations of phosphorus,
iron, and carbon were added to the flasks. Comparison of
nitrogen assimilation by Scenedesmus quadricauda over a
three-week period showed that there was a significant varia-
tion between the different tile drainages. Algae that were
cultured in sump B tile drainage (see Figure 142) assimilated
50 mg/i N0 3 -N in about 10 days, while at the other extreme
algae crown in sump A tile drainage had only assimilated
25 mg/i N0 3 -N in 25 days. The IAW C tile drainage, sump I,
along with the other nine tile drainages (which are not
included in Figure 142), were intermediate In level and time
necessary for complete nitrate assimilation.
6O
—5
1.
Is’
z 40 \
11.1
0 I.
o •
\•. SUMP A
20 I
S.
I—. S
z 10 rs. SLJMPI
‘I
SUMP 8
0
o to 20 30
TIME IN DAYS
FIGURE 42-NITROGEN ASSIMILATION
IN TILE DRAINAGE FROM THREE SUMPS
70
-------
A comparison of the water quality constituents (TDS,
alkalinity, trace elements, etc.) indicated that the only
apparent major difference between tile drainage in which
nitrogen removal was greatest and that in which it was least
was the level of molybdenum. The concentration of molyb-
denum in the former was 3/4 parts per trillion (ppt) and
that in the latter was 9 ppt. However, In view of the fact
that some precipitation of nutrients may have taken place,
these values do not necessarily represent the concentration
of molybdenum actually available to the algae.
On the basis of this study, it was concluded that the
combined San Joaquin Valley drainage would largely buffer
the effect of changes in water quality often noted at the
IAWTC, and that the chance of any minor nutrients limiting
growth and nitrogen assimilation would be slight.
To determine the effect of water quality changes on nitrogen
assimilation during Phases I and II, filtered samples of the
influent and growth unit effluent were analyzed for trace
elements each time the storage pond was filled during most
of 1970. The results of the analysis of the concentrations
of trace elements in filtered and unfiltered samples are
listed in Table 6.
Of the 17 trace elements monitored in 1970, Al, Cd, Cr, Cc,
Cu, Fe, Pb, Mn, Mo, NI, V, and Zn fluctuated during the
year. The concentrations of Be, BI, Ga, Ge, and Ti remained
below analytical detection levels. However, changes in
algal response after filling of the storage pond could not
be related to the fluctuations. Figure 43 illustrates the
effect of filtering (O. /4 q membrane filters) on the concen-
tration of six trace elements in influent and i/ 1 4-acre
demonstration unit water. These samples were collected and
filtered on October 27, 1970. The bar graphs show that in
most instances there were higher concentrations of the
elements in the pond than in the influent, perhaps because
of concentration by the algal cells. Except for aluminum
in the influent and molybdenum in both influent and pond
samples, there was little difference between the concentra-
tion in the filtered and unfiltered samples. The lack of
difference is rather surprising because the unfiltered
samples included algal cells and precipitates and presumably
more of the trace minerals. The form in which many of the
trace nutrients are available to algal cells, either
dissolved or particulate, Is not completely known, thus,
future studies should consider running analyses on both the
total sample arid the filtrate.
71
-------
TABLE 6
-4
TRACE ANALYSIS - 1970
(In micrograms /liter)
*Difference due to the presence of insoluble form and/or concentrated in the algae.
0 - Greater than.
Sample
Date
Al
e
BI. J
Cd I cr1
Co
[ Cu J
Ga
[ aej
Fe
Pb Mn 1 Mo
Ni Ti
V
( Zn
Influent
(filtered)
(unfiltered)
2/13
3/17
5/21
7/15
7/27
8/12
8/25
.10/27
11/20
10/27
L1. 1 1
11
L1. 4
60
63
20
1.1.4
01.4
27
46
L0.6
1.0.6
L0.6
1.0.6
1.0.6
Lo.6
1.0.6
1.0.6
1.0.6
1.0.6
1.0.3
1.0.3
1.0.3
1.0.3
1.0,3
1.0.3
1.0.3
1.0.3
L0.3
1.0.3
29
18
1.1.4
19
1.1.4
1.1.14
1.1,4
26
37
31
L] ..4
2.3
1.1.4
28
1.1,14
1.1.4
1.1,14
31
51
31
Ll.
1.1.4
Ia.4
1.1.4
629
20
1.1.4
1.1.4
1.1.4
1.1.4
31
34
L.1.4
1.l. 4
1.1,4
1.1.4
r .i.4
1.1.4
1.1.4
1.1.4
L5.7
1.5.7
1.5.7
1.5.7
L5 7
L5.7
1.5.7
1.5.7
1.5.7
1.5.7
1.0.3
1.0.3
1.0.3
1.0.3
L0.3
L0.3
1.0.3
1.0.3
1.0.3
1.0.3
7.3.
2.6
19
10
0100
20
8,9
2.9
7.1
4.9
1.1.4
1.1.14
L 1.4
1.1.4
1.1.4
L1.4
9.].
1.1.4
u.4
1.1.14
5 7
1].
1.1.4
7.1
13
4.6
7.4
14
6
12
54
51
71
14
37
1].
3.14
2
1 0
7.7
6.0
70.3
14.9
46
26
8.0
9.1
9.7
8.0
1.0.6
1.0.6
1.0.6
1.0.6
1.0.6
L0.6
L0.6
1.0.6
1.0.6
1.0.6
2.2
19
1.9
1. .
3.4
2.1
1.7
3.14
2.3
2.0
1.5.7
29
1.5.7
L5.7
1.5.7
1.5.7
1.5.7
1.5.7
1.5.7
1.5.7
Growth Unit
(filtered) No. 1
No. 17
acre
Mo. 17
No. 6
No. 6
No. 7
acre
(unfiltered)
2/13
3/17
5/21
6/2
7/21
8/21
11/20
10/27
10/27
L1. 4
L2.5
Ll. 1 4
1.1.4
23
21
1.1.4
220
i8o
1.0.6
L1.0
1.0.6
1.0.6
1.0.6
1.0.6
1.0.6
1.0.6
1.0.6
1.0.3
1.0.5
1.0.3
1.0.3
1.0.3
1.0.3
1.0.3
1.0.3
1.0.3
13
7
L1.14
1.1.4
19
1.1.4
49
544
514
1.1.14
L2.5
1.1.14
1.1.4
31
1.1.4
4.3
57
57
ti.4
1.2.5
1.1.14
1.1.4
1.1.4
1.1.14
L1.4
1.1.14
L1.4
15
1.2.5
57
1.1.4
1.1.4
1.1.4
1.1.4
1.1.14
1.1.4
1.5.7
1.10
1.5.7
1.5.7
1.5.7
1.5.7
1.5.7
1.5.7
1.5.7
1.0.3
1.0.5
1.0.3
1.0.3
1.0.3
1.0.3
1.0.3
1.0.3
1.0.3
9.4
3.2
26
31
9.1
7.4
1 1.6
060
60
1.1.14
1.2.5
1.1.4
1.1.4
29
L1. 4
1.1.14
1.1.4
1.1.4
3.4
1.2.5
L1.4
1.1.4
1.1.4
1.1.4
1.1.4
8.9
L1.4
140
25
514
37
214
20
290
37
89
14.0
1 ,0.5
1.0.3
4.3
14.3
14.0
7.1
11
13
3.0.6
1.0.5
1.0.6
1.0.6
1.0.6
1.0.6
1.0.6
1.0.6
1.0.6
2.
0. ’
1.1
1.4
2.5
2.3
1.0
2.3
1.8
L5.7
50
1.5.7
1.5.7
1.5.7
L5.7
230
1.5.7
1.5.7
Inf1uent
(unfiltered minus
filtered)
10/27
45
0
0
5
0
0
0
0
0
2.0
0
0
117
0
0
.3
0
Growth unite
(unfiltered minus
filtered)
10/27
193
0
0
40.9
55
0
0
0
0
147
0
3.6
23
18.5
0
.3
0
1. - Less than.
-------
INFLUENT POND
FILTERED _____ FILTERED rzzm,,n
UNFILTERED UNFILTERED
77
‘I
/
I
Cd
NI
P41ICRONUTRIENTS
FIGURE 43 COMPARISON OF TRACE METAL ANALYSIS OF FILTERED
AND UNFILTERED TILE DRAINAGE
Another aspect of the relation between change in water
quality arid nitrate assimilation was the accumulation of TDS,
resulting from evaporation from the test units. An examina-
tiori of the yearly average percent increase in the TDS in
the growth units shows that accumulation of TDS was a
function of flow and evaporation. There was a minimum of
8 percent Increase in TDS and a maximum of 31 percent
(shallow units with long detention times). Average
increases appear to have ranged between 15 and 20 percent.
There was a net TDS accumulation in all units, but there was
no obvious effect on algal growth response.
Effect of Soil on Nitrogen Assimilation
Several other studies considered pertinent to the Phase II
investigation were carried out by means of flask bioassays.
They were designed to determine whether or not the presence
or absence of soil had any influence on nitrate assimilation.
200
‘Ii
I .-
-j
150
U)
4
0
C.)
50
0
Mo
I
Cr
73
-------
In Phase I, results of tests with several of the miniponds
demonstrated that soil might be a significant factor. These
bioassays, although not conclusive, did indicate that the
presence of soil could be beneficial to nitrate assimilation.
Soil was thought to: (1) help buffer the system, (2) pro
vide chelating compounds, (3) provide micronutrients, and/or
(Li) help reduce nitrogen through bacterial denitrification.
Two miniponds containing soil were operated continuously
during Phases I and II. The performance of the units was
described in detail in the Phase I report. Figure U4 shows
changes in the total influent and effluent nitrogen in the
two units during 1970. From January through August, the
algae in the units were assimilating 50 to 90 percent of
the influent nitrogen, although some factor appeared to be
limiting the system. There was little difference in
nitrogen assimilation between the units, although they were
operated at different nitrogen loadings. After September,
a reduction took place in nitrogen removal. The reduction
was attributed to detention times too short for the avail
able light conditions.
E
w
z
-J
U
INFLUENT NITROGEN
A DETENTION TIME CHANGE
FIGURE 44-EFFLUENT NITROGEN LEVEL IN SOIL UNITS
7k
-------
A comparison of the performance of the units containing
soil with that of units that were not mixed and contained
no soil indicated that there was essentially no difference
in performance between the two systems. Consequently, it
was concluded that the accumulation of organic material,
and not soil specifically, was responsible for the
maintenance of nitrogen assimilation efficiencies in the
units. As will be shown later, the algae grown in the two
sets of units contained twice the amount of nitrogen found
in the cells of algae cultured in the units that were mixed.
Special studies were conducted in the summer and in the fall
of 1970. Four 10-inch diameter glass cylinders (two opaque
and two clear) were placed in a growth unit containing soil
which had been enriched with nitrate, phosphate, and iron.
The soil was removed from one opaque and one clear cylinder,
and the nitrate-nitrogen level was adjusted to 20 milligrams
per liter in each cylinder. The result of the summer and
fall studies are shown in Tables 7a and 7b.
TABLE 7a
NITRATE IN ThE WATER OF OPAQUE
AND CLEAR CYLINDERS (suMI€R TEST)
(expressed as nitrogen, in mg/i)
Date
Opaque Cylinders
Clear Cylinders
Without Soil
With Soil
Without Soil
With Soil
N0 3 -M NO -N
Level JAdded
N0 3 -N N0 3 -N
Level Added
N0 3 -N N0 -N
Level Added
N0 3 -N NOR-N
Level Added
7/31
8/o1
8/05
8/05
8/07
8/10
8/10
8/12
8/13
8/]J
8/15
20
9.2
7.8
-
6.7
7.0
-
6.0
5.5
6.8
6.1
20
5.6
3.0
-
0.7 +20
i6.
-
39.7
58.8
511
145
20
6.2
.6 ÷20
20
17
13
-
6.8
9.8
8.6
7.6
20
0 +20
21.1.8
1.8 +20
1.7 +20
21.8
12.2
38.9
30
20.2
8/17
8/18
8/19
8/21
5.7
14.8
3.6
14.3
142
40
32
30
3.6
2.1
1.2
0.8
7.4
2.7
1.4
0.8
7 ,5
-------
TABLE 7b
NITRATE IN THE WATER OF OPAQUE
AND CLEAR CYLINDERS (FALL TEST)
(expressed as nitrogen, in mg/i)
Date
Opaque Cylinders
Clear Qylinders -
Without Soil
With Soil
Without Soil
With Soil
- 3 - j- 110 3 -N
Level I Added
N0 3 -N N0 3 -N
Level Added
N0 3 -N N0 3 -N
Level Added
N0 3 -N NOR-N
Level Added
9/16
9/17
9/18
9/22
9/23
9/24
9/25
9/28
9/29
9/30
10/01
10/02
10/05
10/06
10/07
10/08
10/09
10/13
10/14
10/15
10/16
10/19
10/20
20.2
19.6
226
20.2
20.2
15.3
17.8
22.8
17.8
21.4
17.2
18.6
19.0
- +20
22.0
-
20.2
20.0
22.5
23.8
26.4
23.8
30.2
14.0
11.0
8.8
1.2 +20
19.0
19.2
11.2
10.6
7.2
7.2
3.7
2.8
1.0
-
0.8 +20
21.4
21.4
15.2
-
15.2
15.5
11.7
12.8
19.6
19.4
21.4
16.5
15.3
12.8
10.8
11.4
9.5
10.4
6.7
7.0
6.2
3.5 +20
24.1
24.5
21.4
17.2
19.6
10.3
20.0
17.5
22.0
14 4
4.3
4.0 +20
3.2
3.2
4.3
3.5
3.5
2.7
4.4
4.0
3.6
4.8
3.4
6.2
7.2
6.4
7.4
9.5
10.2
10.0
9.1
13.4
Analysis of the summer data in Table 7a demonstrates that
the system was primarily photosynthetic and that the organic
soil layer probably acted to buffer the system and provide
nutrients. Some bacterial denitrification undoubtedly
occurred.
The fall (low light conditions) test results in Table 7b
indicate that bacterial denitrification had increased and
that a substantial amount of nitrogen was being recycled
into the system from the organic layer. It was concluded
that this recycling may have occurred during the summer,
but was not detected because of the high levels of nitrogen
assimilated by the algae.
76
-------
Effect of Biomass Regulation
on Nitrogen Assimilation
The regulation of biomass during certain times of the year
was found to be an important factor in Phase I. During
Phase II, however, biomass regulation improved nitrogen
assimilation only slightly, as indicated in Figure $5. This
negligible effect of biomass regulation may have been due
to the method of nutrient addition which caused the removal
of more algae than inorganic sludge in the biomass
regulation system. This assumption is borne out by the
fact that when at the end of Phase II, iron was added by
way of the biomass regulatory chamber instead of directly
to the cultures, total nitrogen assimilation improved over
the control unit.
MONTH
1970
FIGURE 45-EFFECT OF BIOMASS CONTROL ON NITROGEN ASSIMILATION
77
3’
BIOMASS CONTROL-NO IRON
A DETENTION TIME
CHANGE
E
z
w
0
0
z
z
LU
-J
La
L
LU
-j
g
CONTROL WITH
ENT IRON ADDITION
J F
M
M
J
1
A
S
0
N
-------
Algal Species and Predation
The predominant algal species used in the Phase I investiga-
tion was the green alga, Scenedesmus guadricauda . Prelimi-
nary studies with this organism had indicated that it would
grow in tile drainage, and in fact, it was often detected
in the local water (though in small numbers). A ready
supply of fairly large quantities of Scenedesmus was avail-
able from the University of California’s Richmond Field
Station. This particular genus also had other features
that made it desirable as a test organism in tile drainage.
It has been studied extensively and a large amount of
information is available on its growth, nutrient require-
ments, separation properties, and potential use as a by-
product. Specifically, it is one of the few algae whose
nitrogen requirements have been studied. Scenedesmus
g uadricauda is known to use nitrate-nitrogen, the predomi-
nant form in tile drainage, as well as other nitrogen forms.
In addition, Scenedesmus has a high specific growth rate and
has been reported to use bicarbonate (the predominant form
of carbon in tile drainage), an ability not characteristic
of all algae.
During the Phase I study, 33 different species of plan.ktonlc
algae, considered capable of rapid growth, were evaluated on
the basis of their efficiency in assimilating nitrate and
the level of biomass obtainable in IAWTC tile drainage with
and without phosphorus or phosphorus and iron addition.
The rate of nitrate assimilation of each species was then
compared to that of Scenedesmus guadricauda cultured in the
growth units. In all cases, little growth took place with-
out phosphorus addition. Furthermore, the six sDecies
demonstrating the best nitrogen assimilation ana growth all
did significantly better with the addition of phosphorus and
iron, as compared to phosphorus addition only.
It was concluded from these studies that probably a number
of algae can be utilized effectively in the algal process
by simply adjusting the specific nutrient composition of
the growth medium. The problem, therefore, becomes a matter
of selecting a species that will assimilate the maximum
amount of nitrate in the shortest period of time and with a
minimum of nutrient alteration. In this particular case,
there was no apparent advantage in using a species other
than Scenedesmus and this aspect of the investigation was not
pursu d any further.
During the first six months of 1970, Scenedesmus was the pre-
dominant species In the miniponds, as indicated in Table 8.
-------
A tE 8
PBED(EtNANT ALGAL SPEC S DURING 1970 AT IAWTC
Algal ecies Present at 0 er Ten Percent of Total
i tinipond( T I
1janua yl Februar rj March April I May 1 June July A] ustISeptemberI October November December
1 S S S,D S S,D S,D S,D S S S,D S,D,U
2 5 S,D S S S S,D S,D S S S,U S,U
3 S S S,D S,D S,D D D S,D D,L D,U U
S S S,D S S S,N S,H S S,U S,U S,D,IJ
5 5 S S S S D D D S,D,U S,D,U S,D,U
6 s s S S S S,D S,D S,D S,D,U D,U D,U
7 S S S S S 5,D 5,0 5,0 S,0 S,D,U S,D,U
8 S S S S S,0 S,D,O S,O U U S,D,U S,U
9 S S S S S,O S S,H S,U S,O S,D,U S,D,U
10 S S S S S 5,0 S,H S,D S,D,0 S,D,U D,U
11 S S S,H,L 0 U S,U S,H S,H S,D,U
12 C C H,L H S,E S,D,U S,D,H S,D,U S,D,U H H
13 S S S S S S,D S,D S S,D S,D,U S,D,U
S S S S S S,D S,D,U S S S,D,U S,D,U
15 5 5 S S S S,D,O S,H S S,D S,D,U S,D,U
16 s s s s s S,D,0 S S S,H S,U S,D,U
17 S S,D S S,C,D S S,D,H S,D S,D,U D,U S,U S,D,H
18 S,D S,D S S S S,D D S,D S,D,H S,D,U S,D,U
19 S,C S S S S S S S S,D S,D,U S,D
20 S,C S S S S S S S S S,U S
21 P P P,H P,H,N H,U H H P,}t H H
22 H H H H,U H H H,U H,U U H H
-acre unit S S S S S S,D S S S,D S,U S D H
C - Carteria H - Heteromastix N - Ne.nnochloris
S - Scenedesmus D - Diatom L - Lagerheimia E - Euglena
P - Phacus 0 - Oscillatoria U - Unidentified
-------
On the other hand, during the same period, motile algae such
as Carteria, Phacus , or Heteromastix were commonly encount-
ere in the cultures not mixed. By mid-July and through the
remainder of the year, a change was noted in algal species
in the mixed units from predominantly Scenedesmus to a
mixture of Scenedesmus , diatoms, Oscillatoria , and/or motile
algae.
Since the species’ changes In the mixed cultures were not
observed until the units had been operated for some time, it
was 8uspected that the sludge accumulation and probable
decomposition had effected a change in general water quality
and nitrogen forms.
As discussed in the Phase I report, usually some algal pred-
ators were present in most of the units, although usually in
densities less than 500 per liter. It was generally observed
over the three-year investigation that these numbers increased
only after the unit had begun to fail for some reason. This
suggests that the decomposition of algal material after pond
failure releases cellular constituents which in turn stimu-
lates predator reproduction.
Cellular Nitrogen
As nitrogen deficiency develops, the nitrogen content of
Scenedesmus cells may decline from an original of 8 - 10
percent (dry weight) to as low as 2 percent (28). This
decrease In nitrogen content is paralleled by a drop in
chlorophyll (28). Syrett (35), working with nitrogen-
starved cells of Chiorella vul aris , found that (1) the
addition of nitrogen resulted in marked changes in the
respiration rate, (2) when both nitrate and ammonia-nitrogen
were added to the medium, ammonia was assimilated four to
five times faster than nitrate, and (3) assimilation of
ammonia-nitrogen ceased when the available cellular carbo-
hydrates were exhausted.
Richardson et al (70) found that with successive reductions
in the influentThttrogen the percent cellular nitrogen
dropped from 10 to 4 percent, while oxygen evolution,
carbon dioxide uptake, chlorophyll content and tissue pro-
duction were drastically reduced. They also found that the
percent cellular nitrogen must drop to approximately 3
percent of the dry weight before an Increase in lipid syn-
thesis can occur. They concluded that all the nitrogen was
bound In the essential cell constituents, and that the car-
bon fixed In photosynthesis was converted to lipids.
Krauss (33) and others reported that when the ratio of car-
bon to nitrogen is low, nitrite or ammonia may be released
So
-------
into the medium. Conversely, at high nutrient carbon-to-
nitrogen ratios, large amounts of intracellular carbon
accumulate as carbohydrates or lipids (28, 29, 33).
The percent cellular nitrogen was found to be a good indica-
tor of the nutrient balance in our system. The data plotted
in Table 9 represent the average percent cellular nitrogen
in algal cells grown under different operating conditions
during 1970 at the IAWTCS
TABLE 9
AVERAGE PERCENT ALGAL CELUJLAR NITROGEN FOUND
UNDER DIFFERENT GROWTH CONDITIONS IN 1970
Unit
Percent
Nitrogen
Unit
Percent
Nitrogen
-
Phosphate
added
7
No
phosphate
8
Mixed
7
Not
mixed
15
Soil
10
No
soil
15
Co 2
8
NO
CO 2
10
These data indicate that the algae not receiving phosphate
contained on the average of 1 percent more nitrogen than did
the algae which did receive phosphate. The algae in cultures
to which carbon was not added had 2 percent more cellular
nitrogen than did comparable cultures that received carbon
dioxide.
The data also show the difference between concentrations of
cellular nitrogen of algae grown in units containing soil
and those not containing soil, and between those in cultures
which were mixed and those not mixed. Cultures grown in
contact with soil consistently contained fairly low concen-
trations (50 to 100 mg/l) of motile green algae, such as
Carteria, Heteromastix, Euglena , and Goniurn , These algae had
a higher percentage of nitrogen than did the algae in the
control units. The algae were 75 percent protein, as opposed
to a normal 50-percent level of protein. The presence of
these particular types of algae, which are known to prefer
reduced forms of nitrogen, may be considered to be a further
indication that ammonia was being produced in the organic
sludge layer. Moreover, it was also possible that some
denitrification was taking place in the sludge-water inter-
face, with the decomposing algal material providing the
necessary carbon. However, the results of several analyses
of the dissolved oxygen content of this zone showed that
81
-------
rather than being anaerobic, it contained dissolved oxygen
at concentrations of 5 mg/i or more. A preliminary estimate
of the cost of treatment in these units (“symbiotic process”)
showed that it could be as little as one-third the cost of
the treatment processes described in Phase I. This low
estimate prompted the proposal of a research investigation
to define the mechanisms and costs involved.
Several studies were conducted which involved the use of the
light box during Phases I and II to determine whether sus-
tained low levels of nitrogen would be detrimental to the
algae. An example of the typical algal response obtained in
these studies is indicated by the series of curves in
Figure 46. Normally, in these studies, the algae were
cultured in tile drainage supplemented with phosphorus,
carbon, and iron. The nitrate level in the growth medium
was monitored and allowed to remain at zero concentration
for varying lengths of time, after which the cultured nedium
was re-spiked with nitrate, phosphorus, and iron. As can be
seen from the curves in Figure 46, the uptake of nitrogen by
the algae was usually immediate even after five days, with
24-hour light, in a nitrogen-deficient medium. However,
these tests do not necessarily represent what happened in
the large test units, where, for example, many nutrients may
be precipitated and settle out of the algal growth zone.
FIGURE 46.-EFFECT OF A NITROGEN—DEFICIENT MEDIUM
ON ALGAL NITRATE ASSIMILATION
S
zIl
U i
0
I-
z
RESPI KE
Q
S
S
S
S
.
S
S
S
S
S
S
S
S
it
I
I
I
•S..
—
0
0 10 20
TIME DAYS
30
82
-------
Effluent Soluble Organic Nitrogen
The average differences in soluble effluent organic nitrogen
during 1970 under various growth conditions are listed In
Table 10.
TABLE 10
AVERAGE EFFLUENT CONCENTRATION OF
ORGANIC NITROGEN IN VARIOUS TEST UNITS
DURING 1970
Type of Unit
1
Mixed
No CO 2
1.0
0.7
Not Mixed
0.7
1.2
No Co 2
Detention Time
short
0.8
0.9
1.2
IntermedIate
Long
Depth
8 inches
12 inches
16 inches
0.8
0.8
0.7
Temperature
Ambient
30 0 C
1.0
1.3
Inasmuch as the soluble organic nitrogen content of the ef-
fluent corresponds to changes in the influent nitrogen level
(see Figure 3) most, if riot all, of the soluble organic
nitrogen appears to represent a fraction that is not readily
biodegradable. Therefore, the small differences In average
soluble nitrogen, as given in Table 10, may or may not have
significance as applied to the overall system.
83
-------
Operational Problems
During the month of May and again during July 1.970 there was
a period in which the nitrogen concentration of the effluent
from the units increased as much as 10 mg/i in a few days.
In July many of the cultures turned from a lush green to a
dark brown color. The change in appearance was accompanied
by a succession of algal species from Scenedesmus to diatoms
and filamentous blue-green algae such as Oscillatoria . The
increase in nitrogen content of the effluent in July occurred
immediately after the ponds had been accidentally dosed with
the herbicide propanil carried by wind drift from an adjacent
field during a crop dusting operation. The herbicide was
actually observed entering the test units. Not only was the
herbicide actually seen, but also the weeds at the test site
area exhibited the typical response to herbicides. However,
a number of laboratory studies, as well as pesticide analyses
conducted shortly after this occurrence, indicated that the
pesticide was not responsible for the change in response, but
that the occurrence was simply coincidental. Further tests
indicated that neither light, temperature, change In deten-
tion time, water quality, nor the addition of carbon dioxide
could have been responsible for the apparent failure.
In retrospect, spring characteristically was a period in
which algal growth rates were at their maximum. The high
algal growth rates led to high pH levels and dissolved oxygen
concentrations which In turn were characterized by the accu-
mulation of considerable amounts of organic and Inorganic
sludge in the test units. Furthermore, temperatures are at
a maximum during the spring, with water temperatures often
reaching 30 0 C. Since the rate of algal decomposition in-
creases rapidly at high temperatures, the increase in algal
nutrient regeneration could have accounted for the change
in color as well as the increase in effluent nitrogen. To
support these conjectures is the fact that the Scenedesmus
cells were observed entering a zoospore stage, a stage
normally associated with “shock” changes in the environment.
In addition, the units left operating returned to their pre-
vious effluent nitrogen levels In from 2 to 15 days, also
indicating an adjustment to a shock in the system. Most
importantly, a comparison of the change In lnfluent and ef-
fluent nitrogen in all the units during 1970 indIcates that
this increase in effluent nitrogen often coincided with an
increase in the Influent nitrogen or an increase In nitrogen
loading as a result of detention time changes.
Pesticide Analysis
During 1970 when the storage pond was filled with tile drain-
age, samples were taken from t ie tile drainage Bump, the
storage pond and either the 1/4-acre demonstration pond or
B k
-------
one of the miniponds and were forwarded to the Department
of Water Resources water quality laboratory at Bryte,
California, for pesticide analysis. The tile drainage was
analyzed directly. Samples of algae from the test units
were concentrated to an 85 - 90 percent cake and/or were
oven dried before being analyzed for pesticide content.
The results of these analyses are presented in Table 11.
TABLE U.
PF TICIDE ANALYSIS
1910
Date
Sample
pesticide - parts per
trillion
(ppt)
DDT
Dieldri n BHC J Parathion
Influent
*
10 *
*
*
3/70
Teat Unit
---
--- ---
---
---
Algae
1 1,000
* *
*
*
Trnfluent
10
12
155
1 4 1 4
5/70
Test unit
27
* *
55
*
Algae
*
* *
*
*
Iat luent
- - -
-_ -
5--
7/6
Test unit
120
* *
*
9146
Algae
3140
* *
*
*
Influent
3
114
*
76
7/15
Test unit
---
--- ---
---
---
Algae
———
—“ an
as
as
In! luent
5
25 *
*
29ö
7/27
Test unit
n
as. 5 S
as S
a.
Algae
9
* *
*
*
Xnfl.uent
170
15 *
*
*
8/214
Test unit
2L 0
* *
*
*
Algae
as
as -a
a -a
a n
InZluent
260
10
*
*
9/14
Teat unit
*
* *
*
1200
Algae
---
--- ---
---
---
Influent
20
5 *
*
*
10/13
Test unit
4
* 2
*
*
Algae
31,000
* *
*
*
Inf luent
50
3 *
*
*
11/20
Test unit
Algae
125 /
(57O) !
* *
* *
*
*
*
*
* - None reported.
- No sample.
- Toxaphene.
8
-------
Because the pesticide level tended to be low in the IAWTC
tile drainage while that of the harvested algae at times
was higher, the algae apparently did concentrate some of
the pesticides, such as DDT. However, in several instances,
the amount of pesticide in the algae concentrate was less
than that in the influent water. It is possible that the
high temperatures (20 to 30°C) and the high pH levels (8 to
11) in the mixed cultures may have been conducive to the
degradation or volatilization of the pesticides. It Is
also possible that the efficiency of extracting pesticides
from algal cells was low and that the analytical results
do not reflect actual concentrations in the algae.
Evaporation Th.iring 1970
As previously described in the section on methods, a weather
station was located at the IAWTC site to monitor climatic
variations during 1970. Annual fluctuations in light and
water characteristics were discussed previously. The net
water loss by evaporation, which is evaporation minus
precipitation, Is shown for each month In Figure 47. As
that figure indicates, the net loss of water by evaporation
amounted to a significant total.
Data on net evaporation were not used in the interpretation,
in the evaluation of nitrogen assimilation efficiencies, or
in the Phase I estimates of the cost of treatment by the
algal nitrogen removal process.
Diurnal Studies
Several diurnal studies were conducted during Phases I and
II In which measurements were made of temperature, volatile
solids, nitrate, pH, alkalinity, cell counts, dissolved
oxygen (DO), and light penetration into the growth medium.
As might be expected, some of these parameters varied as a
function of the mixing regime. Variation in amounts of
solids in suspension as measured by volatile solids, cell
counts and packed cell volume, and In depth of light
penetration varied with the mixing regimes of the different
ponds. In those ponds with twice daily mixing, biomass in
suspension peaked during the mixing period and decreased to
a rather constant low level during the hours of darkness.
Nonmixed ponds had little diurnal variation in effluent
suspended solids. Cell counts at various depths in the
same culture did Indicate that some phototrophism occurred.
86
-------
I
FIGURE 47- EVAPORATION MINUS PRECIPITATION -IAWTC , 1970
Diurnal changes in caroon utilization (plotted as changes in
alkalinity bicarbonate in Figure 27), in pH level, and in
dissolved oxygen fluctuated with light intensity. Peak
photosynthetic activity measured as high pH, high DO’s, and
low bicarbonate levels usually occurred from 2 to k pm.
The magnitude of these various parameters varied with the
time of year and was a function of total light availability.
Determination of soluble orthophosphate during one diurnal
study showed that as the pH increased, the concentration of
orthophosphate decreased, probably because of precipitation.
The relation between soluble phosphate and pH was plotted
in Figure 32.
Another chemical constituent that was found to change over
relatively short intervals of time was total alkalinity.
The change should not have taken place when the influent
concentration remained constant. Analysis of the total
alkalinity in a completely mixed culture showed that the
1970
87
-------
diurnal shift in alkalinity probably resulted from the
precipitation and redissolving of calcium carbonate as a
function of the change in pH concurrent with carbon dioxide
addition. The amount of sludge buildup is indicated by the
data for percentage of solids in the form of sludge in the
units listed in Table 3. The breakdown of the data in
Table 3 shows that the sludge was composed of carbonates,
phosphates, Iron, decomposing algae, and other substances.
Because of this conglomeration, samples to be analyzed for
alkalinity had to be filtered to arrive at the true concen-
tration of bicarbonate available to the algae. Without
filtration, the addition of sulfuric acid used in titration
caused the precipitates to redissolve and, as a result,
total alkalinities were not Indicative of the actual carbon
available to the algae.
As discussed in the Phase I. report, several attempts were
made to measure the diurnal changes of nitrate-nitrogen
within the test units. The particular interest in this
parameter relates to the method of Influent injectIon (2)4
hours per day) and the time of daily sampling (8 a.m.).
Because algal systems are photosynthetic, most of the active
nutrient uptake and assimilation in theory should take place
during the daylight hours; and, therefore, the amount of
nutrients in the medium should be at their lowest shortly
after sunset. However, on two occasions, samples taken at
various times through a 36-hour period did not show any
measurable diurnal fluctuation In effluent nitrogen
concentration. Perhaps the detention times being applied
when these tests were made were sufficiently long to mask
any fluctuations in nitrogen.
Operation Criteria -- 1970
Early in the Phase II studies, specific constant operational
criteria were selected for the individual growth units
(depth, nutrient addition, mixing, etc.). Only detention
times were scheduled to be changed during the year. Only
those factors shown to be important to maximum nitrogen
assimilation in Phase I were included in the experiment
design. The proposed operating schedule had to be inter-
rupted in July 1970, when the algae in most of the outdoor
units died. The miniponds with dead cultures were drained,
cleaned, filled, and then Inoculated with algae from
those ponds still containing viable algal cells.
The effect of nutrient additions and mixing on maximum
nitrogen removal is shown In Table 12. In general, the
data show that additional phosphorus was required year-round,
88
-------
and that additional carbon was also required except during
the months of July, August and September. The inorganic
carbon requirement was apparently satisfied by either carbon
dioxide or bicarbonate, although the use of bicarbonate
caused a change in dominant algal species from those cultures
receiving carbon dioxide. The effect of additional iron on
maximum nitrogen removal was dependent on carbon addition.
When carbon dioxide was added to the growth units, iron did
not increase nitrogen removal, but was necessary when carbon
was not added. Mixing (four hours per day, either daylight
or night) was never necessary for maximum removal during the
1970 operational studies.
TABLE 12
EFFECT OF NUTRIENT ADDITION
AND MIXING ON NITROGEN REMOVAL
1970 STUDIES
E
Average
Influent
Nitrogen
(mg/i)
Nutrient Addition
Mixing
four
per
hours
— day
- — I(withoutl(with
I Fe 1 Fe
I carbon) Icarbon)
—
Jl/ 29.6 + + + - -
Fl! 29.5 ÷ + + - -
MT/ 29.5 + + ÷ - -
26.9 + + + - -
M 2k.7 + + ÷ - -
j 16.5 + + + - -
J 13.0 + - ÷ - -
A 13.1 + - + - -
S i4.4 ÷ - + - -
O 19.5 + + + - -
N 23.8 + + ÷ - -
D 20.6 ÷ + + - -
1/ Units not under operational control.
2/ Carbon added for three hours per day.
Beneficial to maximum N removal.
- Not beneficial to maximum N removal.
The effect of depth and detention time on maximum nitrogen
removal is illustrated in Figure 48. The influent nitrogen
levels were adjusted to approximate the concentrations
predicted for the San Luls Drain. The effluent values show
the soluble nitrogen only, and are average values for the
unit with maximum nitrogen assimilation during each period.
The low percentages of nitrogen removal during the months
89
-------
of January, February and March were probably caused by a
combination of factors including operational problems
associated with a change of study personnel and low available
light. Based on data from November and December, effluent
nitrogen levels should have been on the order of 3 to 5 mg/l.
Beginning in May, the effluent soluble nitrogen was consist-
ently in the 2 to 14 mg/i range. In addition to the soluble
portion, about 1 to 2 mg/i of particulate nitrogen (algal
cells) would remain in the effluent from the harvesting
processes
z
0
2
The detention times required for maximum nitrogen assimila-
tion were on the same order as found in Phase I, that is,
five days in the summer, and a maximum of about sixteen days
during the winter months.
During most of the year, the 8 - and 12-inch depth units
produced comparable levels of effluent nitrogen, although
from June through December, the effluent nitrogen concentra-
tions from the 5-inch units were always equal to or slightly
TH O ET)CAL MYD AULIC DETU4TIOId TIM(S
FIGURE 48 - EIGHT AND TWELVE INCH TEST UNITS WITH LOWEST EFFUENT NITROGEN CONCENTRATION
DURING 1970
90
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lower than those from the 12-inch units. From January
through May, the deeper units consistently produced an
effluent containing less nitrogen than the units operated
at 8-inch culture depth. These results are apparently
anomalous because any light inhibition of algae in the
shallow units should have occurred during the period of
maximum available light energy from June through July.
The 1970 results differ from those of the 1969 studies when
the shallow ponds had lower effluent nitrogen during the
entire year, and especially during the winter months.
In addition to looking at effluent nitrogen levels, the
factor determining the effectiveness of the removal systems
studied at the IAWTC, the amount of nitrogen removed per
unit of surface loading may be important in some applica-
tions of the algal process. The average number of grams
of nitrogen removed per day per unit are tabulated in
Table 13 (all units had equal surface areas). The data
from this table have been plotted in Figure 149 and indicate
that nitrogen removal per unit of surface area was greater
at short detention times and the deeper culture depths.
TABLE 13
AVERAGE TOTAL SOLUBLE NITROGEN REMOVED
1970
(lang1 ys/day) 100400
1400-600 600-800
6oo-4oo 40O-100
MONTH FJan.JYeb.
Mar.1 Apr. May I JuneIJ I
Aug.j Sept. Oct.INov.(Dec.
Total S
oluble N Removed (gm/day/u
nit) - Monthly Averages
De ten -
Depth tion
Time
1.3 2.5
1.6 2.14
2.5 2.5
2.8 2.0
3.8 3.5
3.6 3.2
3.8 14•5 14•5
5.9 7.5 8.3
7.0 10.6 18.7
5.0 6.7
7.4 6.)4
12.0 6.9
14 9 14.1 3.1
6.2 .6 3 9
6.14 5.5 3.9
8” LI!
I /
s /
Average
1.8 2.5
2.3 4.3
2.8 5.3
5.3 6.5
3•14 2.9
14.5 14•9
5.6 6.5
5.9 8.1
5.6 7.5 10.5
6.7 6.6 6.7
8.3 9.2 9.1
13.]. 14.7 14.1
8.1 6.7
7.5 8.
9.6 8.3
16.4 10.1
5.8 5.1 3.6
6.0 5.6 3.7
8.0 5.5 4.8
8.8 2.9 3.7
12” L
I
S
Average
3 .I 5.14
3.14 3.14
5.3 6.5
14.3 7.1
9.3 10.1 10.0
11.2 11.2 12.7
11.2 9.0
12.5 10.5
7.6 14.7 4.0
9.1 5.5 5.3
16” I
1/ Long detention time.
/ Intermediate detention time.
37 Short detention time.
91
-------
0
x
MINIPOND (MEAN DETENTION TIME)
X 8” DEPTH
2” DEPTH
A IS” DEPTH
10 I
AGURE 49-AVERAGE SOLUBLE NITR0G N M(Y IED WRING 1970 AS RELATED TO
DETENTION liME AND DEPTH
25-
— 20
C
S ..
5 -
Ew
‘5
ow
w I
10
l, o
I-
z
w
-J 5-
-I
0
The monthly changes in total nitrogen removal (gm/day/unit)
at the three culture depths -- 8, 12 and 16 inches -- are
plotted in Figure 50 and again illustrate that more nitrogen
Is removed per unit surface area in the deep ponds than in
the shallow units, especially during the months with maximum
available light energy. The practical implications of the
greater removal efficiency (in terms of quantity removed
per unit loading) In the deeper units can possibly be
realized In a two-step nitrogen removal system. The first
step involves the use of relatively deep (16 to 2Lt inches)
ponds operated at detention times of 2 to 5 days. The
effluent from these units could then be conveyed to
shallower algal ponds for any necessary final polishing.
Using the data from Figure 50, It is possible to estimate
the number of acres required to remove specified nitrate
levels from agricultural tile drainage. Figure 51 shows
such an estimate based on available light in the San Joaquin
Valley, and a drainage flow of 300 cubic feet per second,
the predicted peak flow for the San Luis Drain. For these
APPROX. AVERAGE 6”
X
X
X
X
4
X
X
e
DETENTiON TIME - DAYS
I
92
-------
•1.0
a
0
.5
E
0
I i i
>
0
Lii
z
‘U
C-,
0
z
‘ U
‘ U
U . ’
2
‘U
0
C
2
‘U
C)
‘U
0
a.
0
/
.75
S
U
0
J F M A M J J
1970
I — I I
#00-400 400 -600 600-800 400-600 #00 - 400
APPROX. LIGHT STRIP(ING IAWTC DURING 970 — LANGLEYS/ DAYS
FIGURE 50-AVERAGE SOLUBLE NITROGEN REMOVAL AT VARIOUS DEPTHS
IN INTERMEDIATE DETENTION TIME UNITS
93
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conditions an estimated 8,000 acres of 12-inch deep ponds
would be required to reduce 30 mg/i of nitrate-nitrogen to
2 to LI mg/i soluble nitrogen. The same estimate made.for
August conditions, Figure 52, shows that only 6,500 acres of
12-inch deep ponds would be necessary to achieve the same
level of nitrogen removal.
a
w
U i
U
4
0
U
0
Ui
U i
0
‘I
S.
INFLUENT NOt-N CONCENTRATION (sg/I)
FIGURE 51 - PROJECTED ACRES RE( JIRED AT
DIFFERENT NITROGEN CONCENTRATION
NECESSARY TO ACHIEVE A 2-4 mg/I EFFLUENT
CONCENTRATION- APRIL OPERATING CONDITIONS
AND A FLOW OF 300 cts
•I2 DEP’TH
INFLUENT NOt -N NCENTRATION (. FI)
FIGURE 52- PROJECTED ACRES REQUIRED AT
DIFFERENT NITROGEN CONCENTRATION
NECESSARY TO ACHIEVE A 2-4 mg/I EFFLUENT
CONCENTR*TK)N-IJGIZT OPERATING CONDITIONS
AND A FLOW OF 300 cfs
94
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CHAPTER IV
ALGAL HARVESTING AND DISPOSAL
a1 Harvesting
During Phase I, many physical and chemical algae separation
processes were screened and evaluated for their potential
use in the algal nitrogen removal process. The chemical-
flocculation-sedimentation method, along with several other
promising processes, was selected for testing in the Phase II
studies. The Phase II algae harvesting studies were designed
to determine whether the operating requirements and efficien-
cies of the chemical-flocculation-sedimentation process
varied seasonally during long-term operation and to further
evaluate other selected separation processes.
As described in the Phase I report, algal harvesting was
conducted in three stages based upon the percent algae
(solids) obtained. The first stage, concentration, involved
chemically or mechanically separating 90 percent of the
algae from the growth medium and concentrating them to 1 to
4 percent solids. This was followed by a second stage,
dewatering, which increased the slurry concentration to 8
to 20 percent solids. The dewatered algal mass was then
dried in the final stage to 85 to 92 percent solids by
weight. In this final stage, the dried algal product can
be stored without decomposition (31).
As applied to the algal nitrogen removal process, the
effluent from only the first stage of harvesting must be
free of algae, because this is the effluent that will be
discharged to the environment. If there were significant
amounts of algae in the effluent from the second and third
stages of harvesting, this effluent would then be recycled
through the separation system.
During the Phase I studies, algal concentration was accom-
plished either by the coagulation-flocculation—sedimentation
process or by rapid sand-filtration. The algal slurry was
dewatered with the use either of a vacuum filter or of a
self-cleaning centrifuge. ‘The concentrated algal slurry
was usually dried by air drying. (During the study an algal
sample was successfully spray dried by the De Laval
Separator Company at their spray-drying test facilities.)
Briefly, the processes (described in detail in Phase I) that
proved to be effective were:
95
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1. Shallow Depth Chemical-Flocculent Sedimentation
( Concentration ) - From 95 to 97 percent of the algae could
be removed by this process at detention times ranging from
40 to 65 minutes. The resulting concentrate was slurry
containing 1 to 2 percent algal solids. The amount of
chemical-flocculent (ferric sulfate and/or Cat-Floc) required
to bring about flocculation varied during Phase I. The
variation In requirement was due to changes in algal growth
as a result of operational or seasonal changes.
2. Rapid Sand Filter (Concentration ) - This unit was
operated on the basis of a design flow of 0.25 gpm per
square foot to produce a 1 to 3 percent solid concentrate
representing 95 percent or greater removal of algae.
3. Vacuum Filter (Dewatering ) - Ninety percent of the algae
from the effluent of the concentration process was removed
by the vacuum filter. The resulting concentrate contained
about 20 percent solids.
4. Self-cleaning Centrifuge (Dewatering ) - The self-cleaning
centrifuge dewatered the first stage concentrate to produce
a paste having a solids content of about 10 to 12 percent
(95 percent removal).
5. Algae Drying - Both air drying and spray drying proved
to be effective methods of producing a product that could be
stored without danger of deterioration.
The laboratory work, which was described in detail in the
Phase I report, consisted of jar tests to determine the
effectiveness of various mineral coagulants (lime, alum, and
ferric sulfate) and many polyelectrolytes on algal separa-
tion. The theory behind coagulation, flocculation, and the
use of polyeiectrolytes was also covered in the report.
In Phase I, the average requirement for 90 percent algal
removal, when ferric chloride had been added as a nutrient
to a culture, was approximately 140 mg/i for lime, 20 mg/i
for alum (aluminum sulfate), and 5 mg/i for ferric sulfate.
In addition, approximately 60 polyelectrolytes were tested,
singly and In conjunction with the three mineral coagulants.
Seventeen of the polyelectrolytes were found to be comparable
economically to the mineral coagulants. The cationic poly-
electrolyte Calgon’s “Cat-Floc” proved to be very effective.
Almost complete removal was accomplished at less than
0.2 mg/i.
Because Phase II was primarily concerned with long-term
operation, laboratory jar tests were routinely run to deter-
mine the seasonal variation of chemical requirements to
96
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obtaIn 90 percent removal of the algae. This included
testing the mineral coagulants and the more promising poly-
electrolytes to determine their economic potential under
continuous operation. All the harvesting methods used in
Phase II were described in detail in the Phase I report.
In addition to the laboratory evaluation of chemicals, the
following separation studies were initiated:
1. Separation of algae by sedimentation in a shallow-depth
sedimentation unit: the unit was operated continuously with
effluent from the 1/ 1 4-acre unit. In this separation unit,
chemical additions were applied as determined by the labora-
tory jar tests.
2. The effect of slurry depth on air drying: various bed
materials were used in this study.
3. FlotatIon as a means of algal concentration.
Laboratory-Jar Tests
Polyelectrolytes . Listed in the Phase I report were 17 of
the more promising of the polyelectrolytes tested, their
effective range of concentration, and the mineral coagulants
with which they were used. Because of the net negative
charge on algal cells, only catioriic polyelectrolytes proved
to be effective flocculents. The polyelectrolytes were
further evaluated in Phase II. In practice, because of the
high costs of poiyelectrolytes as compared to that of mineral
coagulants, it would be more economical to simply increase
the mineral coagulant concentration and omit the polyelectro-
lyte. The final evaluation of the polyelectrolyte-mineral-
coagulant combinations showed their use would be more costly
than with the mineral coagulants alone. However, it is
recommended that future studies on chemical coagulation-
flocculation and sedimentation should include an evaluation
of cationic polyelectrolytes to determine whether the
economics of their use would be competitive with the use of
mineral flocculents under various growth conditions. More-
over, care should be taken as to the type of materials used
as a coagulant with regard to the use the algae may be put.
For example, Calgon s Cat-Floc is approved by the U. S.
Public Health Service for potable water, but may not
be approved If concentrated in food. The nitrogen contents
of polyacrylamide and polyamide compounds were found not to
be significant due to the low concentrations used. However,
this could be an important factor in contributing nitrogen
to the effluent If higher concentrations were required.
97
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Mineral Coagulants . In 1970, routine jar tests were
performed to determine the variations in optimal chemical
requirements as a function of change in season. Figure 53
shows the average monthly cost (dollars per million gallons)
for separation to 90 percent solids removal and down to
30 mg/i, as determined on the basis of results obtained in
the tests. There were no determinations in July and no
L o mg/i studies in August. The values obtained for October
are not representative because changes were made in the
nutrient input to the culture. Table 1 4 presents the costs
of 90 percent algal solids removal.
12
I0
çSO% SCUDS REMOVED
z
0
-J
4
-J
-I
1;;
82 1.1 _ TED ‘
MONTH
1970
FIGURE 53-CHEMICAL COST REQUIRED FOR ALGAL CONCENTRATION
98
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TABLE 124
ESTIMATED AVERAGE CHEMICAL COST PER
MILLION GALLONS SEPARATED (1970)
(JAR TEST DATA)
Year Month
Dollars Per
Million
Gallons
Percent
of Plow
1969
December
13.80
2.5 4
1970
January
February
March
April
May
June
July
August
September
October
November
December
11.10
2.3 )4
0.914
1.03
0.56
0.98
1 17 1J
1.37
0.58 ,
i.6&d
3.16
11.41
2.514
2.30
8.146
13.55
114.00
13.55
i 14.oo
114.00
9.40
3.20
2.146
2.54
17 EstImated.
According to the data plotted in Figure 53, the cost to
remove solids to a residual of 40 mg/i is Independent of
original algal concentration. As discussed in the Phase I
report, a concentration of 140 mg/i in the effluent was found
to be about the maximum amount of allowable suspended solids
that could be discharged to maintain the suggested maximum
nitrogen content of 2 mg/i for discharge into the receiving
waters.
Ferric sulfate was found to be the most effective coagulant
tested in 1970. The only exception occurred when iron,
chloride or sulfate was not added to the culture as a
nutrient In December 1969, and in January and October of
1970. It was estimated from these data that the cost of
chemical separation would probably be five to ten times
greater when iron (i to 3 mg/i) Is not added to a culture as
a growth nutrient.
Figure 514 shows estimates of costs for removing algae at
optimum dosages of A1 2 (SO4) , of Ca(O I) 2 , and of Fe 2 (S02 4 ) 3
in terms of conditions prev i1ing during the 1970 runs.
99
-------
U)
z
-J
4
z
0
-j i
-J
(I)
4
-J
-J
8
I -.
U)
0
U
FIGURE 54 -CHEMICAL COST TO REMOVE TOTAL SUSPENDED SOLIDS
TO 40mg/I DURING 1970
Although removal did not appear to be related to biomass
concentration, some correlation was noted to degree of
alkalinity. Ferric sulfate concentrations necessary for
maximum solids removal were found to decrease with decreasing
bicarbonate concentration, as is shown by the curve in
Figure 55, in which required iron dosage is plotted as a
function of alkalinity.
Operational Studies
Chemical-FlocCUlerIt-SedimentatiOn Unit . The shallow-depth
sèdimentation unit was continuously operated with the require-
ments for chemical flocculent additions generally determined
by routine jar tests. The unit had a volume of about
130 gallons arid was operated at theoretical detention times
ranging from about one-half to one hour. In the jar test,
one hour was the standard settling time. However, since in
the sedimentation unit neither mixing velocity nor duration
of mix could be varied, the actual efficiency of removal
with chemical coagulation-flocculation and sedimentation
achieved in the tests did not meet expectations. The costs
in terms of dollars per million gallons of culture processed
as based on performance of the sedimentation unit are
plotted in Figure 6. The chemical costs alone usually
ranged from one to four dollars per million gallons treated.
1970
100
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CAT-I
FLOC
2
J • F • M A M
2
0
I-
4
w
0.
0
0
2
1 LIRON a CAT-FL0C
,-- • I I
J A S 0 N 0
FIGURE 56-COST OF SEFARA11ON IN SHALLOW DEPTH SEDIMENTATiON UNIT
DURING 1970
2.0
3.0
U)
0
2.0
I d
1.5!
I -.
10
z
Id
I-.
,‘z Id
..J.. a
>20G
z
-J
4
-J
4
uj 100
.
.
S
I
.
S
0
0 0.4 0.8 2 1.6
REQUIRED Fe(S0$) 3 DOSAGE (mg/I)
FIGURE 55- EFFECT OF BICARBONATE ALKALINITY ON IRON
SULFATE REQUIRED TO REDUCE SUSPENDED
SOLIDS TO 40 mg/I.
15
U)
2
0
-j
-J
4
z 10
0
-J
-J
U)
4 5.
-J
-j
g
I-
U)
0
U
I j
101
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Flotation . To determine the practicability of flotation as
a means of harvesting algae grown in agricultural wastewater,
a flotation device was constructed according to the design
diagrammed in Figure 57. The dispersed air flotation method
was chosen because the mixing cycle of the growth pond
limited the dissolved oxygen content to near saturation,
thus curtailing the effectiveness of the dissolved air
technique. As reported in the Phase I report, the suspended
material in the growth units contained about 50 percent algae
and 50 percent silt and precipitates of calcium, phosphorus,
magr esium and iron. Under appropriate conditions, these
precipitates could act as coagulants, since their specific
gravities are greater than the fluids. The high salinity of
tile drainage also affects the settleability of suspended
materials. In the flotation studies, when the compressed
air was injected at a volume equal to about 25 percent of
the hydraulic flow, the process worked quite well as a mixing
chamber for the coagulation-sedimentation process but did
not work well as a flotation device.
FIGURE 57—SCHEMATIC OF FLOTATION CHAMBER
TESTED AT IAWTC
POND
102
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From observations made in the study, the flotation-
separation process probably would not be practical unless
the percent of volatile solids could be increased in the
growth phase of the process.
Air Drying . Costs of drying bed construction, methods of
collecting the dried algae, and the use of the product
determine the type of material to be used in building a
drying bed. One of the procedures tested for processing
algal paste after its concentration and dewatering was air
drying. During Phase II, several studies were conducted to
determine whether sun drying would be economically compara-
ble to other methods -- as, for example, spray drying.
Four types of surfaces - - sand (wet and dry), cloth, black
plastic, and asphalt -- were tested as a substrate on which
to spread the algal paste. The main criterion was the
permissible loading rate of algal Blurry per unit surface
area. As is to be expected, increased radiation permitted
greater loadings than did increased initial solids concentra-
tion; that is, more energy input on the one hand, and on tI’ie
other, less water to evaporate. A dry sand bed and shallow
slurry depth proved to be a most effective method of drying,
but it resulted in a poorer grade product. Cloth as a
drying surface was the second in terms of efficiency. A
disadvantage in the use of cloth Is handling it in a large-
scale operation. Observations made in the tests gave reason
for concluding that an asphalt surface would be the most
practical, since the algal paste could be applied easily and
the dried product could be readily scraped off. Moreover,
the product would be a high grade one.
In terms of permissible loading rate per unit of surface
area (except for the sand bed), maximum efficiencies were
attained when the sludge layer was applied 1.5 inches deep,
However, the increase in efficiency was negated somewhat by
the longer time required for the necessary drying. At the
time of maximum drying efficiency, which corresponds to the
time of heaviest loading as based on the predicted drain
flow, the required slurry depth would be 1.25 inches, and
the drying time would be 3 days. In those operations, in
which preservation of vitamin and high protein quality is
essential, the deeper layer would have to be abandoned in
favor of a layer less than 0.5 inch deep to complete
drying in one day or less.
Differential Separation of Algae . The data on chemical
costs discussed in the preceding paragraphs were based on
results obtained in the shallow-depth, chemical-flocculent-
sedimentation unit normally operated on-line 2 4 hours per
day and from processing algae from the 1/ 4-acre demonstration
unit. The influent for the process was pumped from the
103
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mixing pump sump (Figure 6), an area where large quantities
of inorganic and organic sludge tended to accumulate.
Analyses of the effluent solids from the separation unit
showed that they usually were only about 50-percent volatile.
The nonvolatile portion mainly comprised precipitates of
carbonate, phosphate, and iron.
A 3,000-gallon capacity, incidental sedimentation unit was
operated. It was not preceded by a chemical flocculent. It
also was used with the 1/4-acre demonstration unit and was
used pr1m’rlly as a biomass regulation tank. The influent
to the tank also came from the sump for the mixing pumps.
It was passed through the tank at a rate which resulted in
theoretical detention times of from 2 to 3 hours. The
effluent line on this unit was modified with a U-tube device.
Effluent could be taken from the device at various depths.
The effluent device was installed after the algal portion of
the suspended solids was observed to settle differentially
according to percent volatile solids, presumably because cell
density increased with age. Depending on the depth from
which the effluent was drawn, up to 90 percent volatile
solids could be returned to the 1/ Il-acre demonstration unit.
The high volatile solids portion was thought to contain the
younger, actively assimilating cells, which are desirable
for algal high-rate growth systems. Several times each week
the flow to the biomass regulatory tank was stopped, the
supernatant was fed by gravity back to the l/ 1 4-acre unit,
and the settled sludge was removed. Analysis of the sludge
material showed that it was about 50 to 60 percent mineral
and clay material and 40 to 50 percent algae.
Sludge Accumulation in the l/ 1 4-acre Pond . Although the
biomass regulation tank helped to reduce the total solids in
the l/ 1 4-acre pond, much of the settleable material settled
within the pond and never reached the sedimentation tank.
It soon became apparent that sludge was rapidly accumulating
in the 1/4-acre demonstration unit. The accumulation led to
a reduction in the amount of light reaching the algal cells,
and hence to a deterioration in the performance of the pond.
An attempt was made to reduce the total nonassimilating
solids in the test unit by pumping out sludge from the areas
of greatest sludge accumulation. This proved to be only a
temporary solution. Because of the design of the pond system,
the problem of unwanted sludge accumulation proved to be a
difficult one to solve.
As mentioned earlier, iron normally was added as batch doses
to the Individual test units. Because iron is an excellent
algal flocculent (and was probably responsible for much of
the settling in the test units), its addition to the l/ 1 4-acre
unit was stopped. Within several days the color of the pond
culture turned from a lush green to a very peaked “washed-out”
l o u
-------
color, the amount of algae in suspension decreased, and the
amount of chemical required for separation increased. These
reactions were not expected, inasmuch as sludge in the pond
contained large amounts of insoluble Iron. Apparently, the
residual Iron in the pond was in a form unusable by the algae.
This must have been the case because, when iron addition was
resumed there was an immediate beneficial response in terms
of: (15 increased numbers of algae in suspension; (2) a
decrease in required chemical dosages for separation and,
consequently, a decrease in cost of separation; (3) a change
in color back to lush green; (4) an increase from 40 - 50
percent to 60 - 70 percent in the volatile solids content of
the pond effluent; and, most importantly (5) more nitrogen
assimilation.
When iron addition was resumed, the iron was added to the
biomass regulatory tank rather than directly to the growth
unit. Monitoring of the influent to and the effluent from
the biomass regulatory tank showed that although most of the
iron added to the tank remained in the tank and helped to
settle nonorganic solids, the amount, although small, of
soluble iron reaching the pond was sufficient to meet the
needs of the algae. The dissolved iron concentration in and
out of the biomass regulation unit leveled off at about
0.5 mg/i.
A similar addition of iron to the biomass regulatory tank of
one of the 1,000-gallon test units showed similar results,
particularly in terms of increased algal suspension.
Algal Disposal
No experimental work was done in either Phase I or II on the
disposal or potential use of algae as a by-product of the
removal of nitrogen from tile drainage through the culture
of algae. However, a discussion of these aspects is contained
in the Phase I report. The discussion was based on informa-
tion gained from a search of the literature. Briefly, because
of their high protein content, algae appear to have a poten-
tial use In this country as (1) an animal food supplement,
(2) a soil conditioner, (3) a food for the rearing of
organisms in commercial aquaculture, (4) a raw material for
certain drugs (5) a source of inorganic and organic
chemicals, (65 a raw material for adhesives, and (7) a food
for direct human consumption.
Work presently being carried out in underdeveloped countries
(71, 72, 73) indicates that there is a potential for the
immediate use of algae as a by-product of wastewater treatment.
In fact, in certain portions of Southeast Asia, both fresh
water and marine algae are presently being grown commercially
with artificial growth media for direct human consumption.
105
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Projections of algal production for the proposed algal-
nitrogen removal plant are shown in Table 15. As indicated,
up to 83,000 tons of dried algae would be produced annually.
If sold as a protein source, the monetary returns could
amount to eight million dollars per year, an amount which
could be credited to the cost of treatment.
TABLE 15
ESTIMATED ALGAL PROIXJCTION BY
AN ALGAL STRIPPING PLANT, 1975-2000
Year
Tons of Algae
per Year
Approximate
Substitute for
Value as
Soybean Meal
Maximum Minimum
Maximum
Minimum
1975
1980
1985
1990
1995
2000
13,300
27,200
44,300
62,000
75,300
82,610
8,41o
18,000
29,610
42,1100
54,100
65,510
$1,330,000
2,720,000
4,430,000
6,200,000
7,530,000
8,265,000
$ 8115,000
1,795,000
2,965,000
4,240,000
5,410,000
6,555,000
During Phase II, several samples or algae, soybean meal, and
cottonseed meal were analyzed for their amino acid content.
The results of the analyses are shown in Table 16. The algae
samples had a lower amino acid content than did either the
soybean or the cottonseed meal. However, the algae samples
contained only about 50 percent volatile material. A large
part of the algae samples was nonorganic. As stated earlier,
the percent volatile solids, as well as that of the cellular
nitrogen, can be varied by applying suitable operational
procedures.
106
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TABLE 16
AMII O ACID ANALYSIS
(micromoles/mg sample)
Amino Acid
Soybean
Meal
Cottonseed
Meal
Algae 1*
Algae 2*
Algae 3*
Algae 14*1
Lysine
.231
.131
.052
.107
.085
.033
Histidine
Arginine
.097
.205
.085
.281
.017
.01i2
.027
.110
.030
.093
.011
.0148
Aspartic Acid
.1465
.289
.099
.183
.177
.073
Threonine
.17
.117
.052
.086
.082
.01 11
Serine
.2149
.173
.055
.097
.092
.01414
Glutamic Acid
.668
.561
.100
.202
.161
.077
Proline
.237
.1 1 12
.055
.083
.082
.039
Glycine
Alanine
.295
.253
.230
.177
.108
.125
.209
.183
.189
.i6li.
.076
.071
Half Cystine
Valine
.032
.1914
.032
.142
.005
.056
.009
.1014
.0056
.091
Methionine
.038
.033
.015
.027
.026
.013
Isoleucine
.165
.0911
.0314
.0614
.058
.030
Leucine
.301
.186
.077
.131
.125
.052
Tyrosine
Phenylalanine
.072
.1119
.053
.129
.017
.032
.0145
.060
.0145
.059
.015
.032
* - Algae from
- Algae from
mixed unit.
noninixed unit.
107
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CHAPTER V
PROCESS EVALUATION
Sumrnary of the
Phase II Investigation
The Phase II operational studies confirmed the year-round
technical feasibility of stripping nitrogen from tile
drainage using algae. Several major changes in operating
conditions were observed during prolonged unit operation.
These changes were significant enough to warrant a reevalu-
ation of the process in terms of design and cost estimates.
Probably one of the most important changes from Phase I in
basic operating criteria was the discovery that mixing was
not required for maximum nitrogen removal. An analysis of
mixed and nonmixed cultures, both with and without carbon
supplementation, showed that the only difference in total
nitrogen assimilation between cultures was that which was
due to the adding of carbon dioxide by way of the mixing
pumps. Results of a special study involving a culture that
was not mixed but which received supplemental carbon dioxide
via a gas diffuser confirmed that mixing was not required.
Furthermore, at the end of May 1970, when the influent
nitrogen concentration decreased to a level at which carbon
supplementation was not required, all the comparable mixed
and unmixed cultures assimilated approximately equal amounts
of nitrogen.
A further comparison of mixed cultures with cultures not
mixed indicated that mixing could have been detrimental to
the system in that it: (1) reduced the amount of light
available to the algae because of the turbidity imparted by
the re-suspension of nonphotosynthesizing material,
(2) decreased nutrient solubility through reactions accom-
panying aeration, and (3) probably increased the recycling
of’ nitrogen into the system from the sludge. However,
because of the design of the growth units used in this study,
mixing was the only method of adequately removing solids
from the units.
The accumulation of’ large amounts of sludge under prolonged
unit operation was another major factor that was not evident
in the four- to six-week studies conducted during Phase I.
The units in Phase II were originally scheduled to operate
for one year but because of operational problems in July
1970, most of the units were drained, cleaned, and restarted;
thus there were two distinct runs, one of about six months
and one of about five months. Measurement of the sludge
accumulation in Phase II indicated that a substantial portion
109
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of the phosphorus, carbon, and iron became tied up in an
unassimilable form in the test units within a matter of
several months. This sludge buildup was thought to have
detrimental effects other than those previously mentioned.
As temperatures increased during the summer, algal decomposi-
tion was accelerated and there was a resulting increase in
release of ammonia from the sludge. Results obtained from
adding ammonia, the major nitrogen form released during
algal decomposition, to one culture, indicated that it was
preferentially assimilated with respect to nitrate-nitrogen.
It therefore seems likely that the algae could utilize
ammonia released via nitrogen regeneration in the test units
under periods of high temperatures.
During Phase II, the availability of light to the algae and
nitrogen loading was found to be the most important factor
affecting nitrogen assimilation. A comparison between the
effluent nitrogen concentrations from test units in which
the cultures had equal surface area but were maintained at
different depths showed that, although the effluent from
cultures operated at the 8-inch depth usually had a lower
nitrogen content than that from comparable ones held at a
16-inch depth, when compared in terms of total nitrogen
assimilated, the deeper unit was often much more efficient.
Relating total nitrogen removal to nitrogen loading and to
operating depths at different light energy levels indicated
that these two factors combined to determine the performance
of a culture. Extrapolation of all the 1970 operating data
for unit depth indicates that, when total light per day is
at 600 langleys, the minimum pond depth should be 2 4 inches;
at 300 lang].eys, it should be 16 inches; at light energy
levels less than 300 langleys, the recommended depth is
12 inches. In addition to looking at unit efficiency in
terms of grams nitrogen removed per unit surface, some
consideration must be given to effluent concentration since
waste discharge requirements often specify allowable limits
of individual components Using effluent nitrogen concentra-
tion as a criterion, the 8-inch ponds provided an effluent
which was most consistently close to the 2-mg/l limit
suggested in a 1967 Federal Water Pollution Control Adminis-
tration report (9).
The total nitrogen that could be removed by the algal system
per unit of time was found to be directly proportional to
the influent nitrogen loading and total light available to
the algae. The amount of light available to photosyrithe-
sizing algae was found to be reduced when the accumulation
of sludge reached a point at which Its re-suspension Imparted
an excessive turbidity to the culture medium.
110
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The shading of the cultures by pond walls and the resulting
light available to the algal cultures were not considered
in the Phase I studies because all but two of the units were
operated at the same depth. The difference in available
light between ponds with 18-inch walls as compared to those
with 10-inch walls was considered significant. Some theo-
retical wall shading estimates based on sun angle were made
in Phase II. These estimates showed that up to 30 percent
of the available direct sunlight may have been blocked from
the units during the winter months. No attempt was made to
estimate the effect of walls on diffused (scattered) light,
but the figures do Indicate that the small-unit data may be
conservative estimates of nitrogen removal rates in large
ponds where the effect of shading would be minimal.
Another important aspect of algal nitrogen assimilation noted
in the Phase II studies was that low operating temperatures
(10 to 15°C) were not necessarily growth limiting. A compar-
ison of the performance of a culture functioning at summer
temperatures (25 to 30°C) with that of a control culture
growing at the ambient temperatures prevailing during the
fall of 1970 showed that there was no difference in nitrogen
assimilation within the temperature range covered in the
comparison (15 to 30°C). The temperature effect noted in
the Phase I studies is now thought to be the result of
changes in total light energy reaching the cultures, rather
than of changes in temperature. Probably, temperatures
above 25 0 C were somewhat detrimental to the net nitrogen
removal efficiency of the system because of their acceler-
ating effect on the decomposition of bottom sludges and
subsequent release of algae nutrients tied up in the sludge.
The changes in detention times made In the Phase II studies
were based on Phase I data. After prolonged operation, they
were found not to bracket the optimal detention times neces-
sary for maximum nitrogen removal during most of the year.
In the Phase II studies, cultures operated for a period of
two weeks at detention times of two days during mid-summer
assimilated 90 percent of the influent nitrogen, whereas in
the Phase I studies a minimum of five days was indicated.
During the winter period in Phase II, when light was minimal,
a 15-day detention time was found to be adequate for the
production of an effluent having a soluble nitrogen content
of to 5 mg/i.
The nutrient studies during Phase II confirmed that some
supplemental carbon, phosphorus, and Iron would be required
throughout the year. In fact, all the studies indicated
that maintenance of maximum nitrogen assimilation depended
on a completely balanced nutrient system.
111
-------
Early in the spring, when carbon was the limiting nutrient
in the cultures, as much as 10 mg/i nitrite-nitrogen was
found in the pond effluent. When a sufficiently large
supplement of carbon was introduced into the cultures, an
immediate decrease in nitrite-nitrogen took place. The
decrease implies that although nitrate uptake or cellular
reduction to nitrite may not be affected by carbon deficiency,
the further reduction of nitrite to ammonia is strongly
dependent on an adequate supply of carbon.
The carbon-to-nitrogen ratio required in the growth medium
proved to be 5 to 1, a ratio which Is in agreement with the
ratio of the two major nutrients in the algal cells. It was
found that when the ratio of carbon to nitrogen was lower
than 5 to 1, the addition of either carbon dioxide or
bicarbonate was a satisfactory method of restoring the
correct rates and achieving the required nitrogen removal
efficiencies. The addition of bicarbonate did appear to
cause the algal species’ composition to change, but the
change did not appear to adversely affect nitrogen removal.
The total alkalinity of the influent was found to be a good
indicator of carbon availability, although neither air-water
exchange nor insoluble carbonate resolution was considered.
In a practical operation, at times of high nitrogen concen-
tration when supplemental carbon Is required, carbon dioxide
could be injected into the unit for two to three hours per
day or possibly bicarbonate could be mixed into the Influent.
Both phosphorus and Iron were found to be required at the
IAWTC throughout the year, although the exact amount of the
two elements available to the algae was not determined. The
phosphorus dosage to the cultures was based on a phosphorus-
nitrogen ratio of 1 to 10, which Is about that of the algal
cell. Usually a small amount of phosphorus was present in
the effluent. About 90 percent of the incoming phosphate
was removed by passage through the cultures, by way of algal
assimilation and/or precipitation.
The addition of iron to cultures receiving carbon dioxide
had no significant effect on nitrogen; however, the addition
of the same amount of iron to cultures not enriched with CO 2
caused a significant Increase in nitrogen removal. The CO 2
evidently enhanced the availability of iron to the algal
cultures. The method of adding Iron (as Fed 3 ) left much to
be desired and needs more study.
A comparison of the cultures grown In contact with soil with
one grown in the absence of soil under comparable conditions
(that Is, not mixed) indicated that the mechanism of nitrogen
removal was probably the same in both cultures. The results
obtained in several situ studies with cylinders from
which light was excluded from some and admitted to others
112
-------
indicated that an algal-bacterial symbiotic system probably
was involved in nitrogen removal. The extent to which each
of these groups of organisms contributed to the overall
system was found to vary with light availability. Tests
conducted in the spring and summer indicated that the system
was probably 90-percent photosynthetic, although the bacter-
ial portion probably was contributing to the overall process.
In the fall, nitrogen removal was mainly bacterial, with the
bacteria probably using the degradable carbon released from
the decomposing algae that had accumulated during the
previous portion of the year. Another point of interest
noted in the symbiotic studies was the recycling of nitrogen
from the sludge-organic layers back into solution. This
nitrogen recycling was thought to occur in all the algal and
symbiotic test units in varying degrees, and probably had a
significant effect on overall efficiency of operation during
1970.
The 1970 studies on the harvesting of algae demonstrated
that 90 percent or more of the algae in suspension could be
removed throughout the entire year in a continuous operation
by the chemical—flocculent-Sedlmentation process. However,
the level of chemical addition required to accomplish this
removal was found to depend on a number of algal growth
factors. In all cases, the cost of the chemicals would be
negligible in relation to overall treatment costs.
During the 1970 studies, algae assimilated enough nitrogen
to maintain a year-round effluent nitrogen concentration of
about 2 to 5 mg/i. This does not include the nitrogen
content of the algae not removed in the separation process
(about 5 percent), which would probably add 1 to 2 mg/i
nitrogen, although it does include the nondegradabie
dissolved organic portion. Projections using actual nitrogen
removal rates during Phase II indicate that it should be
possible to remove ‘vIrtually 100 percent of the influent-
soluble nitrogen when some of the operational problems are
resolved.
Cost of Treatment
As stated in the Phase I report, this was a preliminary
investigation and was not designed to provide definitive
costs data. However, analysis of the data collected during
the Phase II operational investigation indicated that the
two primary factors affecting the efficiency of the algal
system are: (1) the light available to the algae, and (2)
the influent nitrogen loading. If these two factors are
known, the area required for treatment can be derived, thus
providing a basis for calculation of treatment costs.
113
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CHAPTER VI
PROPOSED ALTERNATIVE TO THE MIXED-REACTOR
SYSTEM STUDIED IN PHASES I AND II
Logically, the next step in the nitrogen removal studies
will be to construct a pilot plant operating on the combined
flow of several tile drainage systems. The selection of the
specific processes to be studied, either algal or bacterial,
will depend on several factors including available time and
money. The purpose of this section is to provide some
details on an alternative algal system to that proposed in
the Phase I report. Although there are no experimental data
to verify the reliability of the proposed “slug-flow” system,
some theoretical considerations indicate that preliminary
testing would be beneficial.
The Phase I design proposed to meet the operating criteria
determined in the early stages of the investigation basically
considered of a number of high-rate growth units clustered
around a central pumping plant. Four hours of mixing per day
in each unit could be accomplished by directing the unit’s
flow to the central pumping plant via an elaborate network
of concrete pipes and automatic valves. In addition to the
pond structure, each unit was to have a 200 x 130 foot
sedimentation basin lined with concrete. The basin was to
be 10 feet deep at the Influent end and 15 feet deep at the
effluent end, and to be supplied with chain-driven scrapers
to remove the sludge. Basically, the proposed system was of
the same configuration as that of the test units investigated
at the LAWTC.
The Phase II investigation indicated that algae cultured in
a “stirred reactor” for any length of time probably would be
under unfavorable growth conditions, which could limit the
ultimate nitrogen removal potential of the process.
Probably the greatest hindrance to the nitrogen removal
activity of the algae would be the curtailment of light pene-
tration by the accumulation of a nonphotosynthetic sludge on
the bottom of the pond. As shown in Figure 58, it is
predicted that light and nutrients would remain above limit-
ing conditions until near the effluent end of a “slug -flow”
system. Light and nutrient limiting conditions to the algal
culture were the case in the stirred reactor used in the
IAWTC studies. As McCarty et al (70) have observed, algae
grown in the nutrient-defic3 nFconditioflS release organic
material into the medium. This material can affect algal
growth and aging. Conversely, the basic premise of a slug-
flow system is that all variables will be in excess until
near the effluent end of the unit, at which time one will
become limiting -- nitrogen, for example.
115
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-J
0
TIME
FiGURE 58-THEORETICAL NUTRIENT
AND LIGHT AVAILABILITY TO ALGAE
IN TWO TYPES OF ALGAL REACTORS
Another limitation of the stirred reactor system used at the
IAWTC is that the algal population is in all stages of
growth rather than entirely In exponential growth (Figure 59).
Because auto-flocculation as well as inorganic precipitate
buildup is primarily a function of culture age, high pH
values, and nutrient deficiency (and these conditions were
all encountered), a considerable buildup of sludge took place
In the test units. It Is postulated that in the proposed
system these factors could be largely controlled, except at
the effluent end of the growth channel. At that point,
provisions could be made for removing accumulating sludge.
C l )
-J
-j
w
C .,
0
I-
FIGURE 59-THEORETICAL ALGAL GROWTH PHASES
IN TWO TYPES OF ALGAl.. REACTORS
SLUG-FLOW REACTOR
LIMITING GROWTH LEVEL
STIRRED REACTOR IAWTC)
GROWTH PHASES
I -LAG
2- EXPONENTIAL
3-DECLINING
4- STATIONARY
5- DEATH
AGE OF CULTURE—TIME
116
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In Figure 60 is a diagrammatic sketch of a proposed algal
nitrogen removal system which Incorporates features that
should result in the correction of many of the apparent
inadequacies of the mixed system.
Basically, the influent tile drainage would enter a deep,
one- to two-day detention time regulatory pond. This unit
would provide: (1) buffering capacity, (2) a place for
settling of silt and detritus and thus enhance light penetra-
tion, (3) a uniform flow to the treatment units, and (14) a
means of isolating incoming flow when required. Algal
UreseedingIi probably would also be done after iron is added
in this unit. Although the algal inoculum would consist
mainly of actively growing cells (from the tailworks of the
plant), some settling of older cells would also probably
occur. A similar unit(s) would be provided to store flow
during high nitrogen loading-low light periods of the year
for more efficient treatment at a later time. Phosphorus
could be Injected into the overflow from the unit. The
amount added at this point would, of course, depend upon
influent nitrogen and alkalinity, arid whether or not treat-
ment was to be induced in the drain itself (15).
At this point, It might also be necessary to provide low-head
pumping into the distribution canal. In any case, tile
drainage would basically move by gravity flow through the
treatment units at the required depth and detention time.
(Head gates to individual units could be used selectively.)
Although gates would probably be provided between each
channel to allow series flow and reseeding after cleaning,
the normal treatment pattern would be slug flow.
Data available at this time indicate that because deeper
growth units are more efficient than shallower units of equal
surface area, the use of deeper ponds would be the most
economical approach in terms of land and construction costs.
Possibly, depth could be graduated throughout the units, that
is, deeper at the upper end and shallower near the outlet
where algal concentration would be maximum.
It is further postulated that by treating the water in a slug-
flow manner, auto-flocculation (due to high pH levels) and
sludge buildup would also be minimal throug1 .out most of the
units. Most of the precipitation would occur toward the end
of the channel, since amount of sedimentation is basically
a function of algal culture age. A small settling area
(approximately 100 x 100 x 8 feet) would be provided in each
unit. Supernatant from this area would then overflow into
a common tailwater car t]. which would carry the treated water
to the separation area. The algal inoculum for the plant
Influent could be recycled from this point or from the
separation unit itself.
117
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CHEMICAL
ADDITI ON
-j
4
2
4
U
2
0
I-
D
C l )
0
>1
1
ALGAE
FUTU R E
EXPANSION
0
I
0
I)
‘
0
I
I
INLET
0
I
0
I
I
0
6
J
I
0
p
0
I
I
0
6
!
INLET
)
I
/1
OUT
PONDS
‘I
“1
4:
LET1
(1
£T
0
4
0
.
GROWTH
-
-
.— — -
0111
LUDGE
-j
4
z
4
U
I-
z
LU
-J
I L .
-. J4
GULAT1OM. I CARBON I RECIRCUL ION LINE
POND ADDITION ]
4 1 1 N FLU
FiGURE 60-SCHEMATIC PLAN OF PROPOSED SLUG FLOW SYSTEM
LU
I-
4
0
EFFLUENTA
I TI
DITCH
I
0
LU
0
0
-J
U)
It’
ALGAE
0
4
0
EFFLU ENT
CANAL
DETAIL
-J
4
z
4
0
I-J
0
SEPARATI ON
FACILITIES
-------
At either end of the treatment channels would be an access
roadway to the individual units to allow for maintenance.
As mentioned earlier, individual units could be taken ‘off-
iine and cleaned when required.
An interesting feature about this design is the common tail-
water collection system at the end of the treatment units.
There would be a common roadway for either railed or rubber-
wheeled vehicles to the ‘in-line” sedimentation areas and to
the tajiwater ditch. (These units also would provide some
primary separation.) The roadway would have a common canal
in which settled material from these two areas could be
pumped for transport to the separation area. The primary
sludge product would undoubtedly have a much different
quality than that of the algal material remaining in suspen-
sion (tailwater canal). The settled material could be pumped
via a motorized unit on the road into the sludge canal on a
periodic basis, and/or when a unit was drained and taken out
of operation. Normal chemical or mechanical separation of
the algae-laden tailwater would then take place in the
separation area. It is postulated that this type of treat-
ment using projected operating criteria would be adequate to
meet the proposed discharge requirements.
119
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ACKNOWLEDGMENTS
This phase of the field investigation concerned with
nitrogen removal from tile drainage was performed under
the joint direction of Messrs. Donald G. Swain, Sanitary
Engineer, U. S. Bureau of Reclamation; Bryan R. Sword,
Sanitary Engineer, Environmental Protection Agency; and
Douglas L. Walker, Civil Engineer, California Department of
Water Resources.
The field work was under the direction of Messrs. James F,
Arthur, Research Aquatic Biologist, Environmental Protection
Agency; and Bruce A. Butterfield, Assistant Engineer,
California Department of Water Resources.
The cooperation and assistance given by the interagency
staff of the treatnient center was a major contribution to
the success of’ the field studies, These personnel were:
Robert G. Seals Chemist, Environmental Protection Agency
Norman W. Cederquist Technician, U. S. Bureau of Reclamation
Gary E. Keller Technician, U. S. Bureau of’ Reclamation
Dennis L. Salisbury Technician, California Department of
Water Resources
Elizabeth J. Boone Laboratory Aid, California Department of
Water Resources
Clara P. Hatcher . Laboratory Aid, California Department of
Water Resources
Consultants to the Project
Dr. William J. Oswald . University of California
Dr. Clarence G. Golueke University of California
Dr. Perry L. McCarty . Stanford University
Report Prepared by
James F. Arthur . . . Research Aquatic Biologist,
Environmental Protect ion Agency
Report Edited by
Randall L. Brown Associate Water uality Biologist,
California Department of Water Resources
Louis A. Beck Senior Sanitary Engineer,
California Department of Water Resources
G. Donald Meixner Supervising Engineer,
California Department of Water Resources
121
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REFER CES
1. California Department of Water Resources. “San Joaq uin
Master Drain”. Bulletin No. 127. January 1965.
2. California Department of Water Resources. “Waste Water
Quality, Treatment and Disposal”. Bulletin No. 127,
Appendix D. Apr11 1969.
3. San Joaquin Valley Drainage Advisory Group, “Final
Report”, January 1969.
14• Department of Water Resources, Bulletin No. 17’4-2,
“Progress Report, San Joaquin Valley Drainage Investiga-
tion Quality and Treatment Studies through December 31,
1967”, August 1968.
5. Beck, Louis A., and Percy P. St. Amant, Jr. “Is Treat-
ment of Agricultural Waste Water Possible?” Presented at
Fourth International Water Quality Symposium, San
Francisco, California; published in the proceedings of
the meeting; August iLl, 1968.
6. St. Amant, Jr., Percy P., and Louis A. Beck. “Nitrate
Removal Studies at the Interagency Agricultural Waste
Water Treatment Center, Firebaugh, California”. Pre-
sented at 1969 Conference, California Water Pollution
Control Association, Anaheim, California, and published
in the proceedings of the meeting; May 9, 1969.
7. St. Amant, Jr., Percy P., and Louis It. Beck. “Research
on Methods of Removing Excess Plant Nutrients from Water”.
Presented at 158th National Meeting and Chemical Exposi—
tion, American Chemical Society, New York, New York;
September 8, 1969.
8. St. Amant, Jr., Percy P., and Perry L. McCarty.
“Treatment of High Nitrate Waters”. Presented at Annual
Conference, American Water Works Association, San Diego,
California; May 21, 1969. American Water Works
Association Journal . Vol. 61. No. 12. pp. 659-662;
December 1969.
9. “Effects of the San Joaquin Master Drain on Water Quality
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Basins Comprehensive Water Pollution Control Project,
U. S. Department of the Interior, Federal Water Pollution
Control Administratic Southwest Region, January 1967.
123
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10. “Effects of the San Joaquin Master Drain on Water
Quality of the San Francisco Bay and Delta”, Central
Pacific Basins Comprehensive Water Pollution Control
Project, Appendix Part C, “Nutrients arid Biological
Response”, August 1968.
11. Brown, Randall L. “Removal of Nitrate by an Algal
System”, California Department of Water Resources
Bulletin No. 174-10. June 1970.
12. Beck, Louis A., Percy P. St. Aniant, Jr., and Thomas A.
Tamblyn. “Comparison of Nitrate Removal Methods”.
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Dallas, Texas. October 9, 1969.
13. Brown, Randall L., Richard C. Bain, Jr., and Milton 0.
Tunzi. “The Effects of Nitrogen Removal on the Algal
Growth Potential of’ San Joaquin Valley Agricultural
Tile Drainage Effluents” in Collected Papers Regarding
Nitrates in Agricultural Waste Water, U. S. 0overnmen
printing Office, 19b9 .
14. Butterfield, Bruce A., and James 11. Jones. “Harvesting
of Algae Grown in Agricultural Wastewatera” in Collected
Papers Regarding Nitrates in Agricultural Waste Water,
L Tëi nment Prin iri Orflce,19b9.
15. Goldman, Joel C., James F. Arthur, William J. Oswald,
and Louis A. Beck. “Combined Nutrient Removal and
Transport System for Tile Drainage from the San Joaquin
Valley” in Collected Papers Regardip Nitrates in
Agricultural Waste Water, U S. Government Printing
Office, 1969.
16. Arthur, James F., Randall L. Brown, Bruce A. Butterfield,
and Joel C. Goldman. “Algal Nutrient Responses in
Agricultural Wastewater” in Collected Papers Regarding
Nitrates in Agricultural Was e Water , U. S. Government
r1nt1ng Orfice, I9 .
17. Beck, Louis A., W. J. Oswald, and Joel C. Goldman.
“Nitrate Removal from Agricultural Tile Drainage by
Photosynthetic Systems”. Presented at the American
Society of Civil Engineers, Second National Symposium
on Sanitary Engineering Research, Development arid
Design, July 15, 1969, Cornell University, Ithaca,
New York.
18. Golueke, C. 0., W. J. Oswald, arid H. K. Gee. “Harvesting
and Processing Sewage-grown Planktonic Algae’. San.
Eng. Res. Lab., University of California, Berkeley.
SERL Report No 64-8. May 1968.
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19. Shelef, 0., W. J. Oswald, and C. 0. Golueke. ‘Kinetics
of Algal Systems in Waste Treatment; Light Intensity and
Nitrogen Concentration as Growth-limiting Factors”,
SERL Report No. 68-4, May 1968.
20. Jewell, William J., and Perry L. McCarty. “Aerobic
Decomposition of Algae and Nutrient Regerieratic,n .
Department of Civil Engineering, Stanford University,
Stanford, California, Technical Report No. 91, June 1968.
21. McCarty, Perry L. “Chemistry of Nitrogen arid Phosphorus
in Water”, Journal of American Water W iCc Association,
Vol. 62, No.
22. Stun m, W., and J. 0. Leckie. ‘ Phosphate Exchange With
Sediments; Its Role in the Productivit of furface
Waters”. Presented at the FIfth Intern tIoraI Water
Pollution Research Conference. (In print) Juiy
August 1970.
23. Cramer, Marian, and Jack Myers. ! Nitrate ReductIon and
Assimilation in Chiorella” . Journal of General
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32. Oswald, William J. “Growth Characteristics of Micro-
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37. Borchardt, Jack A., and Hardam S. Azad. “Biological
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38. Tew, R. L, J. 0. Sane, and R. P. Geck].er. “Hi ,hly
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43. Fruh, E. Gus. “The Overall Picture of Eutrophication”.
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44. Goldman, Charles R. (ed.). “Micronutrierit Limiting
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45. Hutner, S. H., L. Provaso].i, Albert Schatz, and
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December 1954.
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57. Ham, Robert K. “The Application of Some Fundamental
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61. Bogan, R. H., 0. E. Albertson, and 3. C. Pluntze. “Use
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62. Levin, Gilbert V., and Joseph Shapiro. “Metabolic
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65. Zabat, M., W. Oswald, C. Golueke, and H. Gee. “Kinetics
of Algae Systems in Waste Treatment, Phosphorus as a
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66. Shapiro, Joseph. “Iron Available to Algae: Preliminary
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67. Hutchinson, H. E. “Redox Potential and the Iron Cycle”,
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New York, 1015 pp. 1967.
68. Morgan, J. J., and Werner Stumm. “The Role of Multi-
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69. Deiwiche, C. C. “The Nitrogen Cycle”, Scientific
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129
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PUBLIC AT IONS
SAN JOAQUIN PROJECT, FIREBAUGH, CALIFORNIA
1968
“Is Treatment of Agricultural Waste Water Possible?”
Louis A. Beck and Percy P. St. Amant, Jr. Presented
at Fourth International Water Quality Symposium,
San Francisco, California, August 14, 1968; published
In the proceedings of the meeting.
1969
“Biological Denitrification of Wastewaters by Addition of
Organic Materials”
Perry L. McCarty, Louis A. Beck, and Percy P.
St. Amant, Jr. Presented at the 24th Annual Purdue
Industrial Waste Conference, Purdue University,
Lafayette, Indiana. May 6, 1969.
“Comparison of Nitrate Removal Methods”
Louis A. Beck, Percy P. St. Amant, Jr., and Thomas A.
Tamblyn. Presented at Water Pollution Control
Federation Meeting, Dallas, Texas. October 9, 1969.
“Effect of Surface/Volume Relationship, CO2 Addition,
Aeration, and Mixing on Nitrate Utilization by Scenedesmus
Cultures in Subsurface Agricultural Waste Wateri
Randall L. Brown and James F. Arthur. Proceedings
of the Eutrophication-Biostimulatlon Assessment
Workshop, Berkeley, California. June 19-21, 1969.
“Nitrate Removal Studies at the Interagency Agricultural
Waste Water Treatment Center, Pirebaugh, California”
Percy P. St. Amant, Jr., and Louis A. Beck. Presented
at 1969 Conference, California Water Pollution Control
Association, Anaheim, California, and published in the
proceedings of the meeting. May 9, 1969.
“Research on Methods of’ Removing Excess Plant Nutrients
from 1ater ”
Percy P. St. Amant, Jr., and Louis A. Beck. Presented
at 158th National Meeting and Chemical Exposition,
American Chemical Society, New York, New York.
September 8, 1969.
“The Anaerobic Filter for the Denitrification of Agricultural
Subsurface Drainage”
P. A. Tamblyn and B. R. Sword. Presented at the 24th
Purdue Industrial Waste Conference, Lafayette, Indiana.
May 5-8, 1969.
131
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PUBLICATIONS
(Continued)
1969
“Nutrients in Agricultural Tile Drainage”
W. H-. Pierce, L. A. Beck, and L. R. Glandon. Presented
at the 1969 Winter Meeting of the American Society of
Agricultural Engineers, Chicago, Illinois.
December 9-12, 1969.
“Treatment of High Nitrate Waters”
Percy P. St. Amant, Jr., and Perry L. McCarty.
Presented at Annual Conference, American Water Works
Association, San Diego, California. May 21, 1969.
American Water Works Association Journal . Vol. 61,
No. 12. December 19b9. pp. 659-662.
The following papers were presented at the National Fall
Meeting of the American Geophysical Union 4 Hydrology Section,
San Francisco, California. December 15-15, 1969. They are
published in Collected Papers Regarding Nitrates in
Agricultural Waste Water . USD1, FWQ.A, #13030 ELY,
December 1969.
“The Effects of Nitrogen Removal on the Algal Growth Potential
of San Joaqujn Valley Agricultural Tile Drainage Effluents”
Randall L. Brown, Richard C. Bain, Jr., and Milton G.
Tunzi.
“Harvesting of Algae Grown in Agricultural Wastewaters”
Bruce A. Butterfield and James R. Jones.
“Monitoring Nutrients and Pesticides In Subsurface
Agricultural Drainage”
Lawrence R. Glandon, Jr., and Louis A. Beck.
“Combined Nutrient Removal and Trans ort System for Tile
Drainage from the San Joaquin Valley
Joel C. Goldman, James F. Arthur, William J. Oswald,
and Louis A. Beck.
“Desalination of Irrigation Return Waters”
Bryan R. Sword.
“Bacterial Denitrification of Agricultural Tile Drainage”
Thomas A. Tamblyn, Perry L. McCarty, and Percy P.
St. Amant, Jr.
“Algal Nutrient esponses in Agricultural Was tewater”
James F. Arthur, Randall L. Brown, Bruce A. Butterfield,
and Joel C. Goldman.
132
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Organization
Department of Water Resources
San Joaquin District
Fresno. Cal I f’ornia
o Title
REMOVAL OF NITRATE BY AN ALGAL SYSTEM
10 1 4hor( ) 16 1 Project Designation
Arthur, James F. 13030 ELY 06/71-13
Note
Available from Department of Water Resources
Post Office Box 2385
Fresno, California 93723
22] Citation Bio-engifleeriflg Aspects of Agricultural Drainage - San Joaquin
Valley, California. Report No. 13030 ELY 06tT1-13
Pages 132, Figures 60, Tables 16, References 73
23 Descriptors (Starred First)
*Agricu].tura]. Wa8tee, •Water Pollution Control, Biological Treatment,
Nitrates, Treatment Facilities
25 Identifiers (Starred First)
*Algae Stripping, Scenedesmua , Algal Growth and Harvesting
27 Abstract
Major findinge are presented from a one-year operational investigation conducted at the Interagency Agrioul jra1
Wast.i,ster Trsat ren$ Center (IAW?C) on the use of algae to remove nit rosen from surface agricultural tile drainag, in
the San Joequin .1ley of California. The ob3eottves of the study were toz (i) refine the design criteria, determined
in a prslimimry Investigation, (2) develop operational procedures, and (3) recoimnend a design for a prototype algal
nitrogen removal proee$3.// The Investigation demonstrated thet the governing factors affecting the algal. nitrogen
removal prooeu ire the total amowrt of light autilabis to the actively photosyntheiizthg algae and the influent
nitrogen los4Ing. AceordingLy, it these two factors are la oiin 9 the area required for nitrogen removal can be
approximated.// ¶ rbid conditions, resulting from th. suspension of nonphotonynthesiilng satsrtal during continuous or
intermittent mixing, were found to be detrimental to the prolonged operation of the system. Msxita.ua nitrogen aaeimi1.*—
tion also depended upon providing a oompltel .y balanced nutrient system, and varying amounts of supplemental carbon,
rI osp}ores, and iron were required thr ghoui the yea.r.// Algae barvesting studies Indicated tbat 90 percent or more of
the alga. could be removed throughout the year, under continuous operation, using a ohemic*l t1oocU1eflt.3edimefltati0fl
prosess rt that the chemical a itiona required were depead.ent up t a number of algal growth factors .11 Contlnocus
operation of algal test units during 1970 showed the algal nitrogen removal process was ce4*ble of effectively reducing
ths Intlusmt nitrate-nitrogen concentration as well as other plant nutrients • l 1 he procesS reduced a varying influent
nitrogen concentration of from 1.5 to 30 mg4 N03- to 2 to 4 mg/i soluble effluent nitrogen throughout the year using
varying operating parsxneters.// Recomoendations are also given for the design end testing of “prototype” algal nitrogen
removal plants using a rnodifieatiefl of th. stirred reactor design, and a proposed “slug—flow” algal nitrogen renewal
system designed to correct tr z of the inadequacies inherent in the first system.
Abs tractor
Arthur
WR 102 )REV, JULY 1969)
CR 50
Instjt tion Department of Water Resources
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