WATER POLLUTION CONTROL RESEARCH SERIES
17O1ODRDO7/7O
A STUDY
OF
NITRIFICATION
AND
DENITRIFICATION
U.S. DEPARTMENT OF THE INTERIOR • FEDERAL WATER QUALITY ADMINISTRATION
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WATER POLLUTION CONTROL RESEARCH SERIES
The Water Pollution Control Research Reports describe the results
and progress in the control and abatement of pollution in our
Nation's waters. They provide a central source of information on
the research, development, and demonstration activities in the
Federal Water Quality Administration, in the U. S. Department of
the Interior, through inhouse research and grants and contracts
with Federal, State, and local agencies, research institutions,
and industrial organizations.
Inquiries pertaining to Water Pollution Control Research Reports
should be directed to the Head, Project Reports System, Planning
and Resources Office, Office of Research and Development,
Department of the Interior, Federal Water Quality Administration,
Room 1108, Washington, D. C. 20242.
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A STUDY OF NITRIFICATION AND DENITRIFICATION
by
B. J. Mechalas
P. M. Allen, III
W. W. Matyskiela
ENVIROGENICS
A division of
Aerojet-General Corporation
El Monte, California 91734
for the
FEDERAL WATER QUALITY ADMINISTRATION
DEPARTMENT OF THE INTERIOR
Program #17010 DRD
Contract #14-12-498
FWQA Project Officer, E. F. Barth
Advanced Waste Treatment Research Laboratory
Cincinnati, Ohio
July, 1970
For sale by the Superintendent ol Documents, U.S. Government Printing Office
Washington, D.C. 20402 - Price $1
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FWQA Review Notice
This report has been reviewed by the Federal
Water Quality Administration and approved for
publication. Approval does not signify that
the contents necessarily reflect the views
and policies of the Federal Water Quality
Administration, nor does mention of trade
names or commercial products constitute
endorsement or recommendation for use.
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ABSTRACT
A program to incorporate biological denitrification into a wastewater
treatment system was undertaken with the objective of developing a
process that depended exclusively on the carbon compounds contained
in the wastewater to supply metabolic energy to the microflora. A
unique treatment system incorporating a large microbial biomass
supported on submerged plastic rings was utilized as the laboratory
device. In the experimental program the incoming nitrogenous material
was oxidized to nitrate in an aerobic phase and reduced to nitrogen gas
in an anaerobic phase. This system accomplished a complete waste-
water treatment carbon oxidation in the aerobic phase, nitrogen
removal in the anaerobic phase.
The conditions for developing a nitrifying microflora were investigated
using a primary wastewater effluent as feed. The flow into the system
was varied to give a range of residence times, and the effluents from
the system were analyzed to determine the degree of oxidation.
Anaerobic batch experiments were carried out to determine if aerobic
reserves could support denitrification. These studies demonstrated
that under appropriate conditions almost 100% of the nitrates could be
reduced.
The final phase of the program was to couple the aerobic and anaerobic
stages. The effluent from the aerobic unit served as the feed for the
anaerobic process. At appropriate intervals this situation was reversed
by switching the airflows and feed sources. Over 95% of the wastewater
nitrogen in wastewater was removed. Nitrate-nitrogen removal rates
ranged from 0. 600 to 1. 00 mg/hr/g MLVS. A mathematical model was
developed which described the response to cycled aerobic-anaerobic
operation. The alternating cycle approach was shown to be an effective
method for removing nitrogen from wastewater.
This report was submitted in fulfillment of Program No. 17010 DRD,
Contract No. 14-12-498, between the Federal Water Quality Administra-
tion and Aerojet-General Corporation.
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CONTENTS
Section Page
I Conclusions v
II Recommendations vi
III Introduction 1
IV Materials and Methods 3
V Aerobic Oxidation in the Activated Bed Units 9
VI Denitrification 29
VII Cycled Operation of the Denitrification System 41
VIII References 81
IX Appendix I 83
Appendix II 85
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FIGURES
Page
1. Activated Bed Unit 4
?.. The Two Mirror Image - Activated Bed Unit 5
3. Effect of Organic Load on COD Removal Rates 11
4. Oxidation of Total Kjeldahl Nitrogen (TKN) 12
5. Dissolved Oxygen Utilization by the Microflora 14
6. Nitrogen Transformations in Unit 1 as Effected
by Residence Times 16
7. Nitrogen Transformations in Unit 2 as Effected
by Residence Times 17
8. Nitrogen Transformations in the Activated Bed
Units After Two Months of Adaptation 18
9. Ammonia Conversion to Nitrite 20
10. Affect of Residence Time on Rate and
Efficiency of Nitrification 22
11. Development of Ammonia Oxidizing Ability in
a Freshly Assembled Unit - Run 7869 25
12. Development of Nitrifying Ability in a Freshly
Assembled Unit - Run 7869 26
13. Development of COD Oxidizing Ability 27
14. Ability of Activated Bed Unit to Denitrify as
a Batch Process - 29°C 30
15. Principal Nitrogen Species Present During
Batch Denitrification - Unit 1 31
16. Principal Nitrogen Species Present During
Batch Denitrification - Unit 2 32
17. Decrease in NO^-N in Effluent as a Function of
Simultaneous Dilution and Denitrification
Unit 2, Run 111769, Phase c, 18°C 39
18. Decrease in NOo-N in Effluent as a Function of
Simultaneous Dilution and Denitrification
Unit 2, Run 1570, Phase d, 29°C 40
iii
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age
FIGURES
19. Schematic Illustrating Coupled Operation of the
Nitrification-Denitrification System 42
20. Aerobic-Anaerobic Cycling; A Comparison of
Experimental and Predicted Results
Unit 2, Run 111769, 18°C 45
21. Aerobic-Anaerobic Cycling; a Comparison of
Experimental and Predicted Results
Unit 1, Run 111769, 18°C 46
22. Influence of Temperature on Denitrification Rate 53
23. Oxygen Utilization with Units Connected in Series,
Run 91169 - 29°C 54
24. Continuous Denitrification with an Oxygen
Saturated Feed 56
25. Fraction of Total NO-j-N Removed Using an
Oxygen Containing Feed, Run 91169, 29°C 57
26. Continuous Denitrification at Increased
Residence Time 58
27. Chemical Changes Between Influent and Effluent
Flows During Denitrification 60
28. Oxidation of Residual COD entering the
Denitrification Unit, Unit 2, Run 1570 64
29. Oxidation of Residual COD Entering the
Denitrification Unit, Unit 1, Run 1570 65
30. Buildup of Ammonia During Continuous
Denitrification, Unit 2, Run 10269, 29°C 69
31. Graphical Illustration of the Nitrogen Transforma-
tions that Take Place in a Single Activated Bed Unit 70
32. Effects of Prolonged Anaerobiosis on Recovery
of Nitrifying Capacity 72
33. Graphical Representation of Possible Physiological
Consequences of Anaerobiosis 73
34. Recovery of Nitrifying Capability After Prolonged
Anaerobiosis, Unit 1 - Run 11769 74
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FIGURES
Page
35. Recovery of Nitrifying Capability After
Anaerobiosis, Unit 2 - Run 11769 75
36, Influence of Residence Time on Denitrification Rate
and Nitrogen Removal Efficiency ' 78
37. Graphical Representation of System Operation
Defined by the Mathematical Model 90
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TABLES
No. Page
1 Dimensions of Activated Bed Unit 6
2 Characteristics of Azusa Primary Effluent 8
3 Influence of Residence Time on Nitrification Rate 21
4 Dry Weight and Volatile Solids 23
5 Summary of Batch Denitrification Rates29°C 33
6 Summary of Batch Denitrification Rates 18°C 35
7 Continuous Run Denitrification Rates at 18°C 43
8 Changes in Ammonia Concentration During Cycled
Operation 47
9 Continuous Run Denitrification Rates at 29°C 49-50
10 Denitrification Rates vs. Temperature 52
11 Protein and Amino Acid Contents of Treatment
System Effluents - Run 1570 61
12 Summary of Protein and Amino Acid Contents of
Treatment System Feeds and Effluents
Run 1570 62
13 Oxidation of Endogenous and Exogenous Carbon
Sources During Denitrification 67
VI
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t. CONCLUSIONS
SIGNIFICANT FINDINGS OF THE PRESENT PROGRAM
This program achieved its initial objectives of demonstrating that
biological denitrification can take place with microbial stored food
reserves serving as the energy source for the process. A denitrifi-
cation process was developed that operated on a continuous feed of
primary effluent. The wastewater was carried through a complete
secondary treatment and discharged as a well oxidized and denitrified
polished effluent. The system includes a practical method of cycling
the microflora between aerobic and anaerobic modes without inter-
rupting continuous flow. The overall study was aimed at identifying
and evaluating some of the parameters that influence the systems ef-
ficiency and at developing the basic information that would permit
utilization of the process on an enlarged scale. Several of the more
significant findings of this program are presented.
Efficient nitrification must always precede denitrification to achieve
high overall nitrogen removal. The proportion of the incoming nitro-
gen oxidized to nitrate is strongly influenced by the organic loads.
There is an optimum loading of 1. 1 g COD/day / g MLVS which re-
sults in practically all of the incoming nitrogen being converted to
nitrate nitrogen.
The rate of oxygen uptake (Qy-v ) of the aerobic biomass is 10/^j2 O?/
hr / mg MLVS. This rate is Atypical of the endogenous respiration
of bacteria.
The residence time of sewage in the treatment units influences both
the types of nitrogen compounds present (NH. , NO_ , NO, ) at any
given time, and the rate of nitrogen oxidation. With a well developed
biomass, the Activated Bed units converted up to 95% of the incoming
NH--N, leaving NO. -N as the only oxidized nitrogen compound
present, in 71 minutes at a rate of 5.2 mg/hr / g MLVS.
The nitrification rate increases with decreasing residence time (in-
creased substrate flow). Efficiency of nitrogen oxidation remains
high until substrate excess is achieved.
The Activated bed units contained a MLVS that averaged 5500 mg/j?
for Unit 1 and 7100 mg/j? for Unit 2 or 6300 mg/jg for the combined
data from the two units.
A mathematical model was developed that accurately predicted the
performance of the system during coupled continuous operation and
cycling between aerobic and anaerobic modes. Corrections for tem-
perature and ammonia release were also included in the model.
VII
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Denitrification rates respond to changes in temperature in a manner
typical of biological systems - a doubling in rate for a 10 C rise in
temperature. During continuous operation the rates (R) averaged
0. 30 mg N removed/hr^g MLVS at 18°C and 0.68 mg N removed/hr/
g MLVS at 29 C.
The amount of dissolved oxygen entering with the nitrified feed ap-
pears to have no effect on the course of denitrification at residence
times of over 400 minutes. This is true even when the incoming feed
is oxygen saturated.
The bacterial cells making up the biomass do not appear to undergo
major physiological damage with anaerobic periods as long as 70 hours.
This is evidenced by the absence of large protein molecules and a
relatively low level of amino acids in the recirculating liquor. This
indicates that massive cell lysis has not occurred and the cells are
not losing their amino acid pools as would be expected under typical
anaerobic conditions. Some small nitrogen containing molecules
probably originating in the amino acid pools are released into the
medium from the cells as evidenced by the low level of ammonia
present in the circulating liquor. The rate of ammonia release is
0. 15 mg/hr/g. MLVS.
The anaerobic mode appears to be a physiologically "aerobic" enviro-
nment. This is evidenced by the biochemical data just discussed, the
oxidation of some of the residual COD entering the unit, and the rapid
recovery of oxidative metabolism when returned to the aerobic mode.
The energy derived from the oxidation of the residual COD entering
the denitrification unit is sufficient to account for only 12% of the
NO--N reduced. Thus the bacteria must be utilizing stored reserves
as fhe principal energy source for the denitrification process.
The stored carbon reserves appear to be capable of maintaining deni_
trification for up to 70 hours. Extended periods of anaerobiosis up to
165 hours indicate that part of the biomass may be lost, as evidenced
by the length of time required for nitrification to be restored.
The overall nitrogen removal efficiency under the test conditions of
the program was 70 - 90% of the total Kjeldahl nitrogen contained in
the primary effluent feed.
Vlll
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II. RECOMMENDATIONS
As a consequence of the above observations and the fact that the feasi-
bility of the concept was demonstrated using a municipal wastewater on
a continuous feed basis, several recommendations for future work can
be made. One is that the treatment units should be re-engineered to
take into account practical treatment plant operations. Hydraulic load-
ings should be balanced and pumping and aeration costs minimized.
Preliminary cost estimates based on the present program should be
made for a properly engineered system. It would be more appropriate
to design the prototype system on some intermediate scale and obtain
actual operating data for both costing and plant design purposes. As a
consequence it is recommended that the prototype system should have
a treatment capacity of 25, 000-50, 000 gallons wastewater per day.
The recommended program would consist of both the plant design and
operation, and a laboratory phase. The bulk of the laboratory effort
would be concentrated in the first part of the program. It would be
geared to providing inputs for the plant design such as methods of
aeration and balancing of hydraulic flows. The mathematical model
would also be modified to fit the enlarged and revised system. Other
experimental parameters would be checked to act as guidelines for
setting up an operational program for the plant.
The pilot plant phase should be designed to obtain actual operating data
for engineering purposes and accurate cost estimates. The overall
efficiency of the process should be evaluated and cost vs. removal ef-
ficiency estimates obtained. Operational modes should also be studied
to simplify control of the overall operation, The information generated
in this recommended program could then be used to design a full-
scale demonstration plant.
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III. INTRODUCTION
Numerous methods for accomplishing the removal from wastewaters of
the nutrients, nitrogen and phosphorus have been proposed. Chemical
processes for both nitrogen and phosphorus reductions are currently
under investigation at various installations. The control of phosphate
will probably continue to be dependent on a chemical system, but
nitrogen removal can be accomplished biologically and when properly
exploited has the potential of being both efficient and economical.
Nitrogen can be removed from a wastewater by two biological processes.
As ammonia it can be assimilated by living cells which incorporate it
into proteinaceous cell material, or as nitrate it can be the substrate
for denitrification, the biological conversion of inorganic nitrate to free
N2 or N£O gas. For the former process to be an effective mode of
removal the nitrogen contained in the wastewater would have to be the
limiting nutrient to assure that it is all incorporated into the cell
material. All cellular organic material must then be separated from the
waste effluent, and the generated cellular waste must be disposed of in
a manner such that it does not again become a source of pollution. The
second method is potentially more effective, since nitrates are volatil-
ized to nitrogen gas, which then escapes into the atmosphere.
In the course of biological conversion of nitrate ion to nitrogen gas,
carbon compounds are oxidized. Herein lies a current problem in
utilizing denitrification to remove nitrogen from wastewaters on a large
scale. In a typical wastewater treatment effluent which is amenable to
denitrification by virtue of the oxidized state of its inorganic nitrogen,
dissolved organic carbon has also been so oxidized that very little
remains to serve as a source of energy for the microorganisms. Hence,
in most biological denitrification processes under investigation (McCarty,
et al, 1969), an external source of organic carbon must be supplied.
Methanol is the favored compound.
The program described in this report explored the use of the stored
intracellular food reserves of the aerobic mlcroflora as the source of
oxidizable substrate. The principal goal of the study was to define the
conditions under which an aerobic, oxidizing microflora could be made
to denitrify, without the addition of supplementary carbon. An additional
aspect of this program is that all of the information generated is basic to
biological processes therefore applicable to other denitrification systems.
NITRIFICATION
True biological denitrification, or non-assimilative removal of nitrogen,
is performed only on the oxidized forms of nitrogen. The oxidation of
ammonia, or biological nitrification, is thus essential to nitrogen removal.
To assure complete denitrification, nitrification must also be complete.
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Nitrification is performed by chemoautotrophic bacteria, which fix CO2
as a source of carbon for cell material and obtain energy for the process
by oxidizing inorganic substrates. Two groups of the chemoautotrophs
are distinguished, each responsible for a specific phase of the nitrifi-
cation process. The first group, consisting of the genera Nitrosomonas,
Nitrosococcus, Nitrosospira, Nitrosocystis, and Nitrosogloea, can
oxidize ammonia to nitrite. The second group, comprised of the genera
Nitrobacter and Nitrocystis, is capable of oxidizing nitrite to nitrate.
Total oxidation of the nitrogen in wastewater to nitrate is therefore a
two-step process, each step being performed by different microflora.
Simple chemical descriptions of the two phases are:
(1) NH4+ + 1.502 ^ NO2" + 2H+ + H2O
(2) NO2" + 0.502 *- NO3"
The first phase of this program was directed towards an examination of
the behavior of the microflora during the aerobic oxidation of wastewater.
Principal emphasis was placed on defining the conditions that control
nitrification and on attaining complete oxidation of the inorganic nitrogen
in wastewater, as a preface to subsequent studies of denitrification.
DENITRIFICA TION
Biological denitrification is technically an anaerobic process wherein
the nitrate ion fulfills the role normally occupied by oxygen in aerobic
respiration--that of hydrogen ion acceptor in the electron transport
system. In other words, in the bacterial species which are capable of
denitrifying, the presence of nitrate ion permits the microbial cell to
maintain "aerobic" metabolism in the absence of oxygen. In the process,
nitrates are reduced to nitrogen gas and carbon compounds are oxidized.
This reaction is represented by the following equation:
4NO ~ + 4H* + 5CH7O *• SCO, + 2N, + 7H,O
J C* L. C. L.
Most of the denitrifying organisms are aerobes and are commonly found
in the wastewater treatment plant. These include species of Pseudomonas,
Achromobacter, Bacillus, and Micrococcus.
Since the growth of these microorganisms is not dependent on the reduc-
tion of nitrate, the presence of a large denitrifying population does not
in itself indicate that conditions are suitable for denitrification. The
existence of a large population, on the other hand, does point to a signifi-
cant denitrifying potential. It was the objective of the second phase of
this program to explore this potential and to define the conditions necessary
for maximum nitrogen removal.
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IV. MATERIALS AND METHODS
LABORATORY TREATMENT DEVICES
The sewage treatment system used throughout the program incorporated
flooded reaction chambers, inside of which the wastewater being treated
is continuously recirculated over a dense mass of microorganisms fixed
to the surface of plastic rings. Figure 1 diagrammatically shows a
laboratory scale treatment device employing this design. For aerobic
operation, air is injected at the discharge side of the recirculation pump
into the circulating liquid which then passes through a jacketed coil to
provide thorough mixing. Water from a controlled-temperature water
bath is circulated through the mixing coil jacket to maintain precise
operational temperatures for the entire activated bed unit. Wastewater
to be treated is metered into the recycle, and the treated effluent over-
flows at the same rate along with excess air at the separation chamber.
The experimental units employ an instrumentation shunt which allows
simultaneous measurement of recirculation flow, dissolved oxygen con-
tent of the liquid leaving the reaction chamber, and either pH or
oxidation-reduction potential, without disturbing the operation of the
unit. Recirculation flow is monitored by using a glass venturi flowmeter
which measures pressure drop as a junction of change in velocity of the
liquid as it passes through the venturi. The other measurements are
accomplished with electrodes and probes. Under normal operating
conditions, the liquid flows directly from the separation chamber to the
recirculation pump; the liquid is bypassed through the instrumented
shunt for measurement. The shunt allows easy maintenance of the probes
without shutdown of the unit, and allows unimpeded recirculation flow
under normal operation. Slime buildup due to biological growths in the
venturi flowmeter, which would change its hydraulic characteristics, is
also minimized. Figure 2 shows the two mirror-image activated bed
units, using a common measuring shunt with associated instrumentation,
as installed in our laboratory.
The bed chamber of each activated bed unit is filled with packing made
of 3/4-inch lengths of 3/4-inch O. D. , 1/2-inch I. D. PVC tubing.
Pertinent dimensions of the units are presented in Table 1. The volumes
quoted in the table represent the system under normal operation and do
not include the instrumented shunt.
The activated bed units as described are assembled for reasons of
laboratory convenience, and are not intended for strict scale-up to
plant size. By their design they allow precise control of experimental
conditions which is essential if reproducible results are to be obtained.
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Effluent
Ove rf low
Air-Liquid
Separation
pH or Redox
Electrode
Dissolved
Oxygen f
Electrode
Venturi
Flowmeter
Reaction
Chamber
and Bed
Temperature Control
and
Air-L-iquid Mixing
ACTIVATED BED UNIT
Figure 1
4
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Figure 2. The Two Mirror Image Activated Bed Units Used in the
Experimental Program. The interconnecting instrumen-
tation by-pass is visible between the units.
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TABLE 1
DIMENSIONS OF ACTIVATED BED UNIT
Total system volume, with packing, but without bed growth
Total system volume, with packing, and bed growth (air off)
rf
(air on)
Volume of bed growth and trapped "noncirculating" liquid
Volume of bed chamber with packing, without bed growth
Percent of system represented by bed chamber, by volume
Surface area of bed chamber and packing, without growth
Surface area of separation chamber, air mixing chamber,
and associated tubing, without growth
Total surface area, without growth
Percent of system represented by bed chamber, by
surface area
Average thickness of growth, total system
(1030 cm3/10330 cm2)
3740 ml
2710 ml*
2400 ml*
1030 ml
2500 ml
67. 0 %
8500 cm2
1830 cm2
10330 cm2
82.2 %
1. 0 mm
Average system volume used for calculations in
this report = 2500 ml.
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ANALYTICAL TECHNIQUES
The various analytical procedures used in gathering the data contained
in this report are described in Appendix I.
WASTEWATER FEED FOR THE STUDIES
The sewage used throughout this program was primary effluent obtained
from the Azusa, California, municipal sewage treatment plant. It was
procured three times weekly and stored in a 500-gallon, trailer-mounted
aluminum tank. A recirculation pump was installed in the tank to keep
the system from going anaerobic and the solids suspended. Pertinent
average analytical data is presented in Table 2. These analyses were
carried out on each tank of sewage when the actual data gathering experi-
ments were in progress, and represent about three analyses per month.
Composition of the sewage remained relatively constant throughout the
course of the year's study, as indicated by the standard deviations in
Table 2. This enabled valid comparison of data obtained early in the
year with that obtained later in the course of the work.
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TABLE 2
CHARACTERISTICS OF AZUSA PRIMARY EFFLUENT
(Average of 35 Samples) +
Characteristic
Chemical Oxygen Demand (COD)
Total Kjeldahl Nitrogen (TKN)
Ammonia Nitrogen (NH~-N)
Organic Nitrogen *
Nitrate Nitrogen (NO~-N)
Nitrite Nitrogen (NO2-N)
pH
Redox Potential
Average
Value
188 mg/1
49. 3 mg/1
40. 1 mg/1
9.2 mg/1
0.05 mg/1
0. 05 mg/1
7. 84
-246 mv
Standard
Deviation
j- 18 mg/1
_+ 4. 6 mg/1
jf 4. 2 mg/1
+_ 0. 05 mg/1
^0.09
+ 35 mv
+ Approximately three analyses per month.
* Obtained by subtracting NH3~N from TKN.
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V. AEROBIC OXIDATION IN THE ACTIVATED BED UNITS
RATIONALE OF THE EXPERIMENTAL PROGRAM
The activated bed unit is essentially a continuous flow microbial culture
device in which the growth rate of the microflora can be controlled.
As in other wastewater treatment processes, the ultimate objective of
the activated bed unit in continuous aerobic operation is to effect maxi-
mum oxidation of incoming wastes. Ideally, carbon compounds are
oxidized to carbon dioxide and nitrogenous compounds are oxidized to
inorganic nitrate.
Carbon oxidation and nitrogen oxidation, however, are performed by
two different types of bacteria. Heterotrophs represented by Zooglaeae,
Pseudomonas, and Chromobacter require preformed organic carbon as
a food and energy source. The chemotrophs of which the nitrifying groups
of Nitrosomonas, and Nitrobacter are representative are able to fix CO£
as their carbon source utilizing the oxidation of inorganic ions as a source
of energy. The fact that the two principal biological groups involved in
aerobic waste treatment have such divergent, nutritional needs, leads to
complications in attempting to simultaneously oxidize both the carbon
and the nitrogen.
As a generalization, the chemotrophs grow at a much slower rate than
do the heterotrophs. In a continuous culture system involving a mixed
microbial flora and a fixed feed rate, differences in growth rate have a
profound effect on the proportion of the total population a given group
may represent.
Thus, when a treatment system is operated at short residence (detention)
times, the heterotrophs predominate in the biomass. As residence
times are increased the slower growing organisms are able to remain
in the system and due to substrate limitation the growth rate of the hetero-
trophs is slowed down. With the increased availability of substrate,
i. e. , ammonia, there occurs a change in the relative growth rates of
the two groups and the proportion of nitrifiers increases. The overall
effect of lengthening residence times is to increase the sludge age.
Increasing the sludge age insures both oxidation of all of the incoming
carbon and complete nitrification of the incoming nitrogen.
The principles discussed above were tested in the activated bed units.
This was necessary to permit poising the system at the desired levels
of oxidation to provide a nitrified feed to the anaerobic system. The
stability of the biomass was an additional consideration since complete
recovery was desired after the anaerobic phase.
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EFFECTS OF ORGANIC LOAD AND RATE OF OXIDATION
Aerobic experimentation with the treatment units centered around defin-
ing the relationship between the average length of time an increment of
sewage feed spends in contact with the microflora and the degree of
£arbon or nitrogen oxidation achieved. Residence time is computed as
t = -p where v is the free liquid volume of the unit and f is the input
flow rate. Degree of carbon and nitrogen oxidation was also examined
as a function of organic loading, which is inversely proportional to resi-
dence time for a sewage feed containing a given concentration of organics.
Organic loading was calculated as
Organic load = ^/min)x COD (.fa x 1440 min/day
6 MLVS (g/j?) x L
where MLVS is the total volatile solids of the entire treatment unit
(MLVS data will be presented in a later section).
Figure 3 shows the relationship between organic load and rate of COD
removal in both of the treatment units. The data were obtained over a
three-month period by operation at various feed flows. Temperature was
maintained at 29° — 1°C, and recirculation rate within the units was held
constant at 2 1pm. Air flow into the units was 2. 1 slpm each. At this
air input and recirculation rate, the dissolved oxygen concentration in
the liquor leaving the reaction chamber of each unit was 7. 5 mg/j2. Since
the solubility of oxygen in air-saturated water at 29° at sea level is 7. 7
mg/JI, the liquid leaving the reaction chamber was essentially saturated.
The results plotted in Figure 3 show a rapid increase in rate of COD oxi-
dation as organic loading is increased, to the point (at an organic load
of 1. 1 g COD/g MLVS /day) where COD oxidation rate reaches a limit.
The relationship between metabolic rate and organic loading is a type of
saturation curve, as described by Herbert (1961) for the bacterial chemo-
stat. Growth rate is proportional to substrate concentration when this is
low but approaches a maximum value as the substrate concentration
increases. At maximum growth rate, substrate utilization should also
be maximal and- supplying substrate over and above this amount would
mean that only a portion would be oxidized and overall efficiency would
drop.
Figure 4 presents the overall nitrification results obtained in these first
aerobic runs as percent oxidation of total Kjeldahl nitrogen (TKN). TKN
vftlues are the sum of ammonia plus organically bound nitrogen, and do
not include inorganic nitrite and nitrate. In the instances of high oxidation
of TKN, all the ammonia and a small fraction of the organically bound
nitrogen were found to have been oxidized to nitrite and nitrate (see
Figures 6 and 7). Figure 4 shows that nitrification was complete for
organic loads up to 1.1 g COD/g MLVS /day. At loadings greater than
that level, degree of nitrification drops as a function of the increased
loading.
10
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32 _
28 .
24
ll
8?
IB-
S'
4-
2-
Unitl —O
Unit 2 — O
.2
i
.4
.8
1.0
1.2
1.4
i
1.6
DO
L8
I
2.0
r
2.2
"Organic
(gCOD/DAY/gMLVS)
Figure 3. Effect of Organic Load on COD Removal Rates - Aerobic
Mode. Units were operated over various flow fates over
a three -month period at Z9°C. Unit 1 = 5. 5 g MLVS;
Unit 2 = 7. 1 g MLVS {Table 4).
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Q — Unit 1
O — Unit 2
100
80 .
I
£60.
40 _
20 .
D
o o
.4
.6 .8 l.» 1.2 1.4 1.6
Organic 'Load (fCQD/gMLVS-Day)
1.8
2,0
Figure 4.
Oxidation of Total Kjeldahl Nitrogen (TKN^ as a Func-
tion of Organic Load. Aerobic mode - 29 C. Average
incoming nitrogen in Azusa feed - 49. 3 mg/1 (Table 2);
Unit 1 = 5. 5 g MLVS; Unit 2 = 7. 1 g MJ-VS (Table 4).
2.2
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UTILIZATION OF DISSOLVED OXYGEN IN INCOMING FEED
The rate of dissolved oxygen utilization in an actively nitrifying aerobic
unit was determined to establish the optimal oxygen requirement of the
microflora. The procedure used was to monitor the oxygen content of
the liquid flowing past an oxygen sensor placed in the circulating liquor.
At zero time the air flow to the unit was turned off and changes in the
oxygen level followed with respect to time. To check the base lines the
air was turned back on until the concentration returned to the original
starting (zero time) level. Various modes of operation were tested but
the one of significance was to maintain all conditions constant including
feed and recirculation and merely turn off the air. Feed rate was set
at 20 ml/min and temperature was 29°C.
A typical series is shown in Figure 5. The shape of the oxygen consump-
tion curve is highly reproducible and at least for this series, is indepen-
dent of the starting concentration at time zero. Because of difficulties
in calibrating the instrument while in the system, values below 0. 5 mg/i.
O2 cannot be considered as significant. By employing the initial maxi-
mum slope of this oxygen utilization curve, a rate of oxygen uptake by
the microflora can be obtained. In unit 2, 1.5 mg O2/liter/min is used.
Converted to p.t O2 /liter /hr:
1500 /ig 02 6Q m.n 22.4 nt 02 63,000 pi P.,
liter/min X hr X 32 ^g O2 ~ liter/hour
The oxygen utilization quotient (QQ ) can De obtained by dividing this
number by the biomass concentration of the bed:
63,000 u.1 O?
Q f/hr 8.9^02
2 7100 mg MLVS/I mg MLVS/hr
These O2 values can be compared with those in published bacterio-
logical literature. The Q-O2 for growing bacterial cultures is strongly
influenced by the age of the cells and the substrate being metabolized.
Cultures of E. coli when actively growing have been reported to have a
QO2 25 - 75717 O2/hr/mg dry bacterial weight (Spector, 1956). The QO2
for the biomass of the activated bed units is low when compared to cells
metabolizing in the logarithmic phase. Dawes (1963) reports values for
the endogenous ^O2 of E_. coli of from 15. 5 - 29. 3 ,u//hr/mg dry weight.
If one considers that the volatile solids of the activated bed biomass
averages 12% Unit 1; 87% Unit 2 (Table 4) and converts to a total dry
weight basis, the activated bed ^-O2 ranges from 12. 5 - 16, 2 ^f O2/hr/
mg dry weight. A *^O2 of 12. 5 is more typical of an endogenous
bacterial respiration and reflects the fact that the unit is poised at the
end of the log phase in the bacterial growth curve wherein the metabolism
of the organisms has started to slow down.
13
-------
O - Unit - 1
X - Unit - 2
17
Time
(minutes)
Figure 5. Dissolved Oxygen Utilization by the Microflora. Air
Feed into the units turned off at time = 0. Flow rate
of Azusa feed was maintained at 20 ml/min and tem-
perature at 29 C. Unit 1 = 5. 5 g MLVS; Unit 2 =
7. 1 c MLVS.
-------
NITRIFICATION AS A FUNCTION OF RESIDENCE TIME
A study of the relationship between degree of nitrification and influent
flow for primary effluent revealed that the residence time of sewage in
the treatment unit is the controlling parameter. Not only does resi-
dence time control the amount of ammonia oxidized but also the quanti-
tative mixture of the three principal nitrogen species, NH4 , NO2 and
NO3".
Figures 6 and 7 derived early in the program show the expected two-
phase pattern of ammonia to nitrite and then nitrite to nitrate conversions.
Differences in the two treatment units reflect different histories such as
time at various feed rates, temperatures, etc. At input flows that give
residence times, of from 30-120 minutes, significant levels of nitrite
appear. Only after about 120 minutes is all of the ammonia-nitrogen
present as nitrate. A simple rate calculation can be made for the over-
all conversion of NH4 to NO-j.
Given: NH4-N converted to NO3-N = 40 mg/£ (from Table 2)
Residence time = 2 hrs (5% NH^ remain-
ing, Figures 6 and 7)
MLVS = 6. 32 g /i (Table 4)
Therefore, the rate of conversion of ammonia to nitrate =
40 mg NO3-N/L 3.2mgNO3-N
2 hrs 6. 32 g / L MLVS ~ hr • g MLVS
The fact that NO?" accumulates as an intermediate indicates that under
these test conditions the NO£~ to NO3~ conversion is rate limiting for
residence times up to 60 minutes.
After two months of continuous operation, a similar set of experiments
was carried out monitoring the appearance of the oxidized nitrogen species
as a function of residence time. In this case only transient amounts of
nitrite were detected, as shown in Figure 8. The overall rate of con-
version of NH^ ^ NO3 also improved.
40 mg/L NOo-N 5.2mgNO,-N
R,
k(NH4 to NO3) 1.22 hrs • 6. 32 g /MLVS hr • g MLVS
The difference in the two rates of oxidation indicates a shift in the pro-
portion of the metabolic activity represented by the NO? oxidizing
organisms. Every indication is that the NH4 to nitrite conversion is now
the rate limiting step and that as soon as a molecule of nitrite is formed
it is immediately converted to the nitrate.
15
-------
•o
0)
8
D
O
A
% NH^-N remaining
% TKN oxidized to
% TKN oxidized to NO3~N
10
20
10 20 30" 40 50
Figure 6.
90 100
Residence time
*
{minutes)
Nitrogen Transformations in Unit 1 as Effected by
Residence Times. Aerobic mode - 29 C. Flow rates
of Azusa primary effluent were adjusted to give the
indicated residence times. One week of continuous
operation at each setting was used to stabilize the unit
prior to analyzing the effluents. MLVS = 5. 5 g/A.
-------
•o
^4
•o
1,
*y ft""1
I
100-
90 -
80.
70
60 -
50 -
40 .
30 -
20
10 -
D-
O-
A-
% NH,-N remaining
% TKN oxidized to NC^-N
% TKN oxidized to NOj-N
-100
- 90
» 80
-10
10
20
30 40
50
60 70
80
90 100 10 20 30 40 50 60 70
Residence time
(minutes)
Figure 7. Nitrogen Transformations in Unit 2 as Affected by
Residence Times. Aerobic mode - 29 C. Flow rates
of Azusa primary effluent were adjusted to give the in-
dicated residence times. One week of continuous opera-
tion at each setting was used to stabilize the unit prior
to analyzing the effluents. MLVS = 7. 1 git.
-------
loo
D NCVN
O NO2-N
200
300
Residence time - minutes
Figure 8. Nitrogen Transformations in the Activated Bed Units
After Two Months of Adaptation. System was opera-
ted continuously in the aerobic mode at 29 C. Flow
rates of incoming sewage were adjusted to give the
indicated residence times. Data from Unit 1 and Unit
were averaged together.
18
-------
The data for nitrite production presented in Figures 6 and 7 can be
averaged and presented as a function of residence time as in Figure 9.
Comparison of Figures 8 and 9 shows that while there was apparent
adaptation of the second stage nitrifiers or those that oxidize nitrite to
nitrate, there was no noticeable change in the rate of total ammonia
oxidation as shown in the nitrite curves of Figures 6 and 7 in the two
months of experimentation. The rate of production of nitrite from
ammonia remained stable, indicating early rapid acclimation of the cul-
ture to first stage nitrification. In the activated bed system, therefore,
nitrification is distinctly a two-stage process, with the microflora
responsible for each stage behaving as distinctly separate populations.
RATE OF NITRIFICATION
It is expected that the longer the residence time of the sewage in the
activated bed units the larger the proportion of the incoming nitrogen
that will be oxidized. However, as residence time is increased the rate
of nitrification is found to decrease. Table 3 presents a summary of
all of the aerobic runs carried out in the first six months of the program.
The rate versus time data is also presented in Figure 10. There is a
marked rate change at between 70-100 minutes which roughly correspond
with a total oxidation of 85-90% of the incoming nitrogen. In operation
of the activated bed units then, there appears to be a trade-off point
between a rapid rate and amount of organic nitrogen oxidized.
From these studies of residence time vs nitrate and nitrate in the aerobic
units presented in Table 3, it is possible to obtain a lower limit to the
rate of nitrification. For the residence time data at 74 minutes for Unit 1
and 71 minutes for Unit 2, a minimum rate of nitrification of 5.2 mg/hr/g
MLVS is calculated at 30°C for both units. The actual rate is probably
slightly higher, but residence time data was not collected for the period
30-70 minutes, for which times a better estimate of the nitrification rate
might have been calculated.
BIOMASS DEVELOPMENT IN THE ACTIVATED BED UNITS
As the critical nitrification and denitrification phases of the program were
completed, the units were disassembled at the end of these periods and
the biomass was removed from the packing rings of the bed for dry weight
and volatile solids determinations. Table 4 gives the results obtained at
these times. When the units were reassembled and placed in aerobic
operation again, it was observed that the biomass regrew to occupy the
clean reaction chambers more rapidly than during the start-up period at
the beginning of the program. This was undoubtedly due to the re-seeding
effect of small amounts of residual biomass not removed by the cleaning
operation. Past experience had shown that the initial start-up period of a
new bed is critical. At this stage the microflora is most susceptible to
predation by other organisms and changes in its external environment.
19
-------
100-.
o
80 -
60-
40-
20.
I
100
200
Residence Time
(minutes)
300
Figure 9. Ammonia Conversion to Nitrite. Nitrite production
data from Figures 6 and 7 were averaged together and
replotted as a function of residence time. This presents
the first-stage nitrification in a young biomass.
20
-------
TABLE 3
INFLUENCE OF
Residence NCX-N
Time formed*
(min) (mg/l)
360 0,0
228 0.8
139 1.9
93 0.8
74 0.4
228 0.3
167 3.0
100 0. 1
78 1. 1
71 0.4
RESIDENCE TIME ON NITRIFICATION RATE
Total
NO,-N Nitrification Nitrification
formed* NO2 + NO. %TKN** Rate (mg/hr/
(mg/^) fmg/f) Oxidized g MLVS)***
45.3
42.6
32.0
41.7
40.7
44.4
40.2
44. 1
39.8
39.5
* Each number is
given residence
** Unit 1 =
#** % Based
UNIT 1
45. 3
43.4
33.5
42.5
41. 1
UNIT 2
44.7
43.2
44.2
40.9
39.9
the average of five
time.
5. 5 g MLVS; Unit 2 - 7. 1
on avei
•age feed of 49. 3 m
92 1.4
87 2. 1
67 2. 7
87 5.0
85 6. 1
91 1.65
87 2. 2
89 3.7
83 4.5
81 4.8
separate runs at the
g MLVS.
g// TKN (Table 2).
21
-------
CM
6.0
5.0-
4.0-
£ w
3.0-
2.0.
1.0.
O - Unit 1
n - UnitZ
r 100
. 80
60
- 40
20
Closed symbols = nitrification {%)
Open symbols = rate
50
i
100
I
150
200
I
250
300
350
400
Residence Time
(minutes)
Figure 10. Affect of Residence Time on Rate and Efficiency of
Nitrification. Data from Table 3 is plotted to show
that at some limiting maximum nitrification rate, the
efficiency of the process starts to fall off.
-------
TABLE 4
DRY WEIGHT AND VOLATILE SOLIDS
Unit 1
Unit 2
Sampling
Date
6/13/69
7/2/69
12/18/69
2/17/70
Total Dry
Weight, g
19.6
29.5
19.4
8.2
Volatile
Mate rial
%
-
-
68. 3
74.8
Total Dry
Weight, g
22.3
9.7
22.8
26. 7
Volatile
Material
%
-
-
86.5
87.8
Ave rage:
19.2
71.6
20. 4
87. 2
Average Total
Volatile Solids
13.8
17.8
Average MLVS
5500 mg/f
7100
Average MLVS
Combined Units
6300 mg/j?
23
-------
These beds eventually recover. This effect can also appear if some
accident befalls the system such as a complete power failure for pro-
longed periods. These circumstances are reflected in some of the data
of Table 4 but it is presumed (through visual evaluation) that these low
levels were transitory and the average solids determinations reflect
the true picture. Figures 11, 12, and 13 show the response of the grow-
ing microflora during restart as changes in the NHo-N, TKN, NOz-N,
NO-j-N, and COD levels of the effluent. As noted previously, the frac-
tion of the microflora which oxidizes ammonia to nitrite grows at a
faster rate than the fraction which oxidizes nitrite to nitrate. The rate
of regrowth of the second group, however, is much more rapid than the
two months initially required for their development.
As shown in Figure 11, reduction of ammonia nitrogen in the effluent
developed quite rapidly, with maximum conversion occurring after the
first day of operation. This immediate reduction in ammonia level is
witnessed again as a rapid drop in total Kjeldahl nitrogen (TKN) level.
The capacity to oxidize the remaining organic nitrogenous components
also measured by the Kjeldahl analysis developed more slowly, as
evidenced by the gradual decrease in TKN after the first day of opera-
tion.
Nitrite (Figure 12) was the predominant product of ammonia oxidation
on the first day, accounting for 75% of influent nitrogen. By the second
day of operation, the microflora had oxidized more than half of this
nitrite to nitrate. In subsequent continuous operation, nitrate producers
presumably form the predominant nitrifying species, judging from the
fact that nitrite ultimately appears to be oxidized as rapidly as it is
produced.
Recovery of the capacity to remove COD, seen in Figure 13, occurred
rapidly until the third day of operation, when effluent quality became
stable at a level of 35% of influent COD remaining.
By the third day of operation, the effluent produced by Unit 1 was highly
oxidized. Essentially all of the ammonia had been converted to nitrate
and nitrite, and COD removal was maximal. In contrast, however, the
bed microflora at this point consisted of a relatively thin slime film
covering the packing rings. Development of a typically dense, luxuriant
bed growth required an additional 7 to 10 days of operation. It is
apparent then that the initial sparse flora which develops is capable of a
high rate of metabolism utilizing the majority of available nutrients.
The capacity of the bed to oxidize less available or more resistant com-
pounds develops more slowly. Maximum oxidation of organic nitrogen
required 6 to 8 days of operation, as did maximum levels of conversion
of nitrite to nitrate.
24
-------
100
X TKN in effluent
O NH3-N in effluent
Time frdm Startup
(days)
Figure 11. Development of Ammonia Oxidizing Ability in a Freshly
Assembled Unit - Run 7869. Sewagejlnfluent 15 ml/min
(mean residence time = 167 min); 29 C. Unit 1 had been
disassembled and most of the biomass rinsed off of the
walls and rings prior to the start of this run.
25
-------
O NO2-N
100
X N03 -N
90
Q 80.
W
N
§ TO.
g
3 60
§ 50,
W
30,
20
10.
Time from Startup
(day*)
Figure 12.
Development of Nitrifying Ability in a Freshly Assem-
bled Unit - Run 7869. Influent 15 ml/min (mean
residence time = 167 min); 29 C. Unit 1 had been
disassembled and most of the biomass rinsed off of
the walls and rings prior to the start of this run.
26
-------
•o—
Time from Startup
(days)
Figure 13. Development of COD Oxidizing Ability in a Freshly
Assembled Treatment Unit - Run 7^69. Influent 15 ml/
min (residence time = 167 min); 29 C. Unit 1 had been
disassembled and most of the biomass rinsed off of the
walls and rings prior to the start of this run.
27
-------
VI. DENITRIFICATION
BATCH PROCESSING OF NITRIFIED EFFLUENTS
With the conditions that control nitrification in the Activated Bed units
defined, there was now available a source of highly oxidized sewage
effluent for the denitrification experiments. The intent of this first
phase of the denitrification studies was to determine if the stored food
reserves of the microflora were indeed capable of supporting the reduc-
tion of the nitrates. To avoid the complications introduced by dilution
and overflow in a continuously flowing system, the initial studies were
carried out on a batch basis.
The two units were aerobically stabilized at a residence time (70 min)
that provided a completely nitrified effluent using Azusa primary
effluent as feed. Temperature was carefully controlled at 29. 0°C and
COD, NH^-N, NO^-N, and NO?-N were analyzed daily for a period of
14 days to insure that an equilibrium situation between feed and effluent
had been established and a stable biomass was present.
The denitrification experiments were carried out by turning off the
influent feed pumps and cutting off the air supply. The recirculating
liquor was then monitored versus time to determine the fate of the
various nitrogen compounds. Opportunity was also afforded, in these
experiments, to follow Q£ utilization and release of amino acids and
proteins into the medium. These latter results will be discussed in sub-
sequent sections.
Figure 14 presents one of the early experiments in this series. The
purpose was to determine if all of the nitrate contained in the circulating
liquor could be biologically converted. In a matter of less than 24 hrs
there was no detectable nitrate left in the system.
Figures 15 and 16 are representative of experiments intended to follow
changes in the nitrogen compounds of interest. Only traces of NO2~were
present and therefore these are not shown in the figures. An interesting
observation was the steady rise in ammonia nitrogen. This indicates
utilization of the amino acid pools of the microflora as anaerobiosis
progresses and does not originate from the NO3~N in the recirculating
liquor. This low level of ammonia was also observed in the continuous
experiments that will be discussed later.
The denitrification rates for the batch experiments at 29°C ranged from
0. 39 - 0. 89 mg/hr/"g ML/VS with a combined average rate for the two
units of 0. 58 mg/hr/g MLVS (Table 5). Low temperature data will be
discussed in the following section.
29
-------
e — Unit 1
x - Unit 2
Figure 14. Ability of Activated Bed unit to Denitrify as a Batch
Process - 29 C. Run 10969. Air supply and feed to
actively nitrifying units were both turned off at
time = 0. The decrease in NO ~in the recirculating
liquor was then followed. Denitrification rate, R =
0. 49 mg/hr / g MLVS.
-------
O — Expt. 102069 - 29°C
D -
Expt. 102869 - 18°C
OJ
7 1 1 T
18 19 20 21
T
22
24 25
Time
(hrs)
Figure 15.
Principal Nitrogen Spacies Present During Batch Deni-
trification - Effect of Temperature on Rates - Unit 1.
Air supply and sewage feed to actively nitrifying units
turned off at t = 0. Units acclimated to temperature
one week prior to start of run.
-------
I
Q - Expt. 11469 -29 C
D - Expt. 11669- 18°C
NO,-N
4
I
6
1
8
1
10
^ 1
12
I
14
16
18
20
22
24
Time
(hours)
Figure 16. Principal Nitrogen Spscies Present During Batch Deni-
trification - Effect of Temperature on Rates - Unit 2.
Air supply and feed to actively nitrifying units turned
off at t = 0. Units acclimated to temperature one week
prior to start of run.
-------
TABLE 5
SUMMARY OF BATCH DENITRIFICATION RATES
(29°C)
Experiment
J.1 Vj'., — m
Start
(mg/l )
!•>(%-/_ -m
End
(rag/I )
X line
Span
(hrs)
M.1 *-* •} ~ J.'
Removed
(mg/hr)
Removal Rate
(mg/hr / g MLVS)*
UNIT 1
10969
102069
102369
11469
44.0
40.8
39.2
48.8
17.0
6.4
24.0
26.4
10. 0
10. 0
6.0
7.8
2.7
3.4
2.5
3.4
0.49
0.62
0.45
0.62
UNIT 2
10969
102069
102369
11469
40. 0
40.4
38.0
46.4
28.0
15.2
11.2
14.4
10.0
4.0
6.0
7.8
2.8
6.3
4.4
4.1
0. 39
0.89
0.62
0.58
Average Rate (both Units) = 0.58 mg/hr/g MLVS
* MLVS - used for calculations were the average values from Table 4.
Unit 1 = 5. 5 mgli ; Unit 2 - 7. 1 mg/C.
33
-------
Temperature Effects on Denitrification Rate
A group of batch experiments were initiated to obtain some preliminary
information on the effects of temperature on denitrification rate. This
series was preceded by an acclimation of the microflora for a period of
one week at 18°C under aerobic conditions. Residence time was adjusted
to 83 min for the aerobic phase preceding the batch denitrification run,
to assure a well nitrified recirculating liquor.
Figures 15 and 16 are typical of these runs. It is quite apparent that
the rates are much slower than at 29°C. The 18°C runs are presented
in Table 6. The range for these runs is 0. 21 - 0. 34 with an average
rate for the combined units of 0. 26 mg/hr/g MLVS. These values will
be further refined and more fully discussed in the section dealing with
continuous operation.
CONTINUOUS FLOW-THROUGH AND DENITRIFICATION
The work to this point demonstrates that mature activated bed units are
capable of complete denitrification of secondary effluent without the
addition of external energy sources. Up until now only semi-batch
studies have been reported and it is necessary to demonstrate denitrifi-
cation during contimiuous operation. Information reported in this set of
experiments will then be used to predict overall denitrification when the
system was cycled in a coupled mode. To enable the prediction of the
equilibrium nitrate level, and to determine an optimum time for recy-
cling, a mathematical model of our system's performance was developed
and its predictions tested by operating the units under various test
conditions.
The Nitrification-Denitrification Model - A Mathematical Interpretation
A complete derivation of a model describing nitrification-denitrification
in which all parameters are considered to vary with time is not only
extremely difficult, but unnecessary at this point. An unnecessarily
complicated model also obviates easy comparison of actual experimental
data with the data predicted from the model.
In order to construct a model that would be useful in guiding the experi-
mental program, several simplifying assumptions were made. The
derivation assumes: (1) that the nitrogen composition of the primary
effluent feed to the Activated Bed units is constant and at 40-50 mg/f
ammonia nitrogen (NH_ rN), negligible nitrate nitrogen (NO., -N), and
negligible nitrite nitrogen (NO -N), and (2) that under anaerobic condi-
tions a given biomass in one unit denitrifies at a constant rate, as
ascertained in the batch denitrification studies. The final refined form
of the model takes into account all predominant inorganic species con-
taining nitrogen. The following symbols are used in the derivation and
model:
34
-------
TABLE 6
SUMMARY OF BATCH DENITRIFICATION RATES
Experiment
NO3-N NO3-N Time NO3-N
Start End Span Removed
(mg/l (mg/f) (hrs) (mg/hr)
Removal Rate
(mg/hr / g MLVS)*
UNIT 1
102869
11669
111769
46.4
44. 0
42.0
30.4
16.4
31.0
12. 7
24.0
7.5
1.26
1.15
1.47
0. 23
0.21
0. 27
UNIT 2
102869
11669
111769
40.4
45.6
44. 0
18. 0
4.8
3.0
12.7
24.0
17. 0
1.76
1. 70
2.40
0. 25
0.24
0. 34
Average Rate (both units) = 0.26 mg/hr / g MJLVS
* M.LVS - used lor calculation were the average values from Table 4.
Unit 1 - 5. 5 mg/f ; Unit 2 - 7. 1 mg/f.
35
-------
Y(T, t) = total inorganic nitrogen concentration (mg/ml)
(i. e. , NO3-N, NO2-N, NH3~N) in the anaerobic
effluent as a function of time and temperature
R = constant rate at which biomass removes inorganic
nitrogen (mg/min)
R(T) = RJ^Q (T) - R^ (T) = total rate of disappearance
of inorganic nitrogen as function of temperature
(mg/min)
N, = total inorganic nitrogen in aerobic feed (Azusa
primary effluent)
t = time (minutes)
T = temperature ( C)
N = total inorganic nitrogen concentration in anaerobic
unit at switching time, t
Q = anaerobic system volume (ml)
v = aerobic system volume (ml)
F = feed flow of nitrified liquor to an anaerobic
system (ml/min)
f = feed flow of sewage to aerobic system (ml/min)
c(t) = total inorganic nitrogen concentration in aerobic
system as a function of time
Also let F/Q = a, f/v = b.
From batch run denitrification results, as illustrated in Figures 14, 15,
and 16, rates of nitrate removal and ammonia buildup at 18 C and 29 C
were measured. Changes in nitrite nitrogen were found to be insignifi-
cant. For the Activated Bed system between 18°C and 29°C, experi-
mental rates were:
(T) = -0.40 + 0.035 T (mg NO3-N/min)
(T) = -0. 062 + 0. 0073 T (mg NH3-N/min)
R(T) = RNQ (T) - Rj^ (T) = -0. 34 + 0. 028 T (mg N/min)
describes the temperature dependence of the rate of disappearance of
all inorganic nitrogen during anaerobiosis.
36
-------
Appendix II gives a complete mathematical derivation of the model.
The resulting solutions are:
Y(t, T) = Nf -0.34 - O.OZ8T
^N1) [e-b(t-t,) _ e-
and
c(t) = (AN1) e'' + Nf (2)
The rate of nitrification in the aerobic Activated Bed units was as-
sumed to be very rapid and thus unimportant when compared to
nitrogen changes caused by simple dilution affects.
A computer program for solution of this equation was written and is
stored in Aero jet -General's computer Engineering Terminal System,
to facilitate rapid data analysis.
A closer look at the behavior of these solutions yields several inter-
esting features. If, for example, at to Unit 2 is switched to an
anaerobic mode, and the system is left in this mode (i.e. , no cycling),
the nitrate concentration in Unit 2 will theoretically stabilize at:
Lim y(t) = lim 1 . e- t Nf e
t - *• oo t — > oo from equation (1)
limy(t) = Nf - -| (3)
t — *-co
In fact, the stabilized level is the same for an anaerobic unit if the
cycling is stopped at some point, the unit remains anaerobic, and
nitrified feed continues at a steady rate. This assumes that the
microflora does not become energy deficient. Thus, if the experi-
mental nitrate concentration in the anaerobic unit is observed to be
changing with time at large t (i. e. , t oo, approx. ) and Nf and F are
held fixed, then R must be changing. Since, in particular, equation
(1) for y(t) is monotonically decreasing, any increase in nitrate con-
centration at large times can only be attributed to a decrease in R,
the denitrifying rate of the biomass. Thus,
limy(t) = Nf - j.
t — * CD
may be a useful tool in evaluating when and how the denitrifying ability
of the biomass fails with extended anaerobiosis. The discussions of
the experimantal results will demonstrate the usefulness of the model
in understanding the operation of the units.
37
-------
Coupled Operation and Control of Denitrification
The feed rate to the de nitrification unit could now be calculated to
give any desired denitrification efficiency. Using the equation de-
rived from the model:
(where Y is the desired equilibrium level in the anaerobic effluent).
Figures 17 and 18 show the results from the experiments in which the
above equation was used to set the flow rates for complete denitrifica-
tion. In these runs Activated Bed Unit 2 was in the anaerobic mode
and was receiving a highly nitrified feed from Activated Bed Unit 1.
The graphs show that the nitrified recirculating liquor contained in
the anaerobic Activated Bed units at time zero is simultaneously
denitrified and diluted. Nitrates in the liquor continue to decrease
until the equilibrium point between feed rate and denitrification rate
is reached. The apparent jump in NO, at the end of the experiment
in Figure 17 is not yet understood but it is indicative of a change in
denitrification rate late in the experiment.
38
-------
sO
10
30
40
50
60
70
80
90
100
110
120
Elapsed Time
(hours)
Figure 17n
Decrease in NO--N in Effluent as a Function of
Simultaneous Dilution and Denitrification - Unit 2,
Run 111769, Phase c, 18°C. Air supply and feed
to the actively nitrifying unit was turned off and
the oxidized effluent from Unit 1 was mete red in at
2. 5 ml/min (residence time - 1000 min).
-------
50
10 -
10
r
20
30
i
40
i
50
80
Figure 18.
60 70
Elapsed Time
(hours)
Decrease in NO--N in Effluent as a Function of
Simultaneous Dilution and Denitrification - Unit 2,
Run 1570, Phase d, 29 C. Air supply and feed to
the actively nitrifying unit was turned off and the
oxidized effluent from Unit 1 was mete red in at
2.5 ml/min (residence time - 1000 min).
90
-------
VII. CYCLED OPERATION OF THE DENITRIFICATION
SYSTEM
The system model proved to be extremely useful in understanding the
inorganic nitrogen levels in the Activated Bed units during coupled
aerobic-anaerobic cycling, which as a major objective of this study.
The model was shown to successfully predict nitrogen levels in both
units during coupled operation. It was useful in establishing physical
parameters of the system when a particular output of nitrogen was
desired in the anaerobic effluent. Moreover, the model pointed out
physiological features of the microflora, such as denitrification rate
changes during an extended anaerobic phase, and denitrification rate
dependence on temperature in cycled operation.
A series of cycling experiments were carried out to determine the
effects of alternating the microflora between the aerobic and anaerobic
modes. The units were kept at constant temperature and were fed
continuously throughout the experiments. A schematic description of
this mode of operation is provided in Figure 19. The assumptions
used in deriving the model were checked by calculating a predicted
result for each of the sets of experiments.
The first series of experiments was initiated by cutting off air and
feed flows to Activated Bed Unit 2 and letting it denitrify as in the
batch experiments. This was used to establish the rate of denitrifica-
tion - R. Once this rate was determined the oxidized effluent from
Unit 1 was fed into the anaerobic unit at a rate derived from the
model.
The initial aerobic-anaerobic cycling experiments were carried out
at 18 C. The reason for this was that the culture was well acclima-
ted to this low temperature having just been used in the 18 C batch
studies. At the time the air was turned off in Unit 2 at the start of
Run 111769, the recirculating liquor contained 44 mg/j8 of NO--N.
This decreased to 2. 4 mg/j8 in 17 hrs at which point the feed pump
supplying the nitrified effluent was restarted. All of this was done
to avoid the complication of dilution in determining denitrification
rate if the feed pump had not been turned off at the start. As experi-
ence was gained in using the model, this maneuver was no longer
necessary. Denitrification rates could be determined at any point
in time.
In each experiment the occurrence of a switch in modes (i. e. , aerobic
to anaerobic) was called a phase switch. Each phase was then timed
from the point of switching to enable readily monitoring the number of
consecutive hours a unit had been anaerobic.
Returning now to Run 111769i the denitrification rate was calculated
as 0. 340 mg/hr / g MLVS (Table 6; also Table 7, Phase a). The feed
pump at 17 hours was adjusted to a flow rate of 2. 5 ml/min which
gives a 1000 minute residence time in the Activated Bed units. On
a continuous feed basis, the rate of denitrification should stabilize
at some constant level of nitrogen removal. This point was established
41
-------
(a)
±-Timary
Effluent
, s
Feed '
Nf
Unit 1
Aerobic
>
Nitrified
Effluent p
c(t) 1
Unit 2
Aerobic
Effluent
^ Feed
Nf
Activated Bed Units prior to aerobic-anaerobic cycling.
(b)
Free
Nitrogen Gas
Primary
Effluent
Feed
Nf
Unit 1
Aerobic
Nitrified
\
Effluent '
c(t)
f
Unit 2
Anaerobic
Denitrified
Effluent
y(t)
Activated Bed Units during first cycle, between tQ and t^
(c)
Free
Nitrogen Gas
Denitrifj
Effluer
y(t)
>
Led
Lt
t
Unit 1
Anaerobic
Nitrified
f
^ Effluent
c(t)
>
f
Unit 2
Aerobic
Primary
Effluent
^
^ Feed
Nf
Activated Bed Units during second cycle, between tj and
Figure 19. Schematic Illustrating Coupled Operation of the
Nitrification-Denitrification System
42
-------
TABLE 7
CONTINUOUS RUN DENITRIFICATION RATES
AT 18°C
R = Rate
Elapsed Hours (mg/hr / MLVS)
Run No. Phase* from Phase Switch Unit 1 Unit 2
111769 a
121569
22.7
24.5
31.0
40. 7
13.2
24.2
28. 7
32.7
47.2
3.0
5.5
72.0
78.0
95.5
101.5
117.5
25.5
31.5
50.0
. 350
. 392
. 338
.280
.291
.282
.282
.218
.273
.651
.742
.323
. 338
.252
.216
.270
. 310
. 334
. 350
* Occurrence of a switch in modes (i.e. , aerobic to anaerobic) is
called a phase switch. Each phase was then timed from the point
of switching.
43
-------
at approximately 87% of the incoming nitrogen being removed resulting
in a electrification rate of 0. 28 mg/hr / g MLVS (Table 7, Phase a).
The sequence of events is illustrated in Figure 20.
The actual data as shown in Figure 20 obtained in Experiment 111769
is plotted along with the values predicted by the mathematical model.
It is observed that, except for one point (a), which will be discussed
later, there is excellent agreement between the two curves.
At 40. 7 hrs, Unit 2 was returned to the aerobic mode and its recovery
compared to that predicted by the model. The return to full nitrifying
capacity is rapid, but does not precisely fit the predicted curve.
Reference will be made again to this fact when we discuss Unit 1.
Also at the 40. 7 hr mark Unit 1 was switched from, the aerobic to the
anaerobic mode (Figure 21). In this case the batch mode was continued
only long enough to permit calculating the initial denitrification rate of
0.27 mg/hr / g MLVS (Table 6). At 49 hrs, nitrified effluent was fed
in at 2. 5 ml/min (1000 min residence time). In this mode (Table 7,
Phase b), the equilibrium rate was 0. 273 mg/hr / g MLSS. Although
the rates for the two units are very similar the actual % nitrate re-
movals differ (87%, Unit 2; 51%, Unit 1) as a consequence of the lower
MLVS in Unit 1.
As with Unit 2, except for an anomaly at (b), the experimental results
behaved as predicted by the model. Both anomalies (a) and (b) in
Figures 20 and 21 can be explained on the basis of the mechanics at
the moment of switching from batch to continuous anaerobic operation.
In Figure 20, the momentary increase in nitrate nitrogen at (a) cor-
responds exactly to an opposing decrease in the level of ammonia
nitrogen, which drops from 8. 7 mg/j0 at 20 hrs to 1. 0 mg/j2 at
22. 7 hrs (Table 8), then rises again to 8. 5 mg/j0 two hrs later. It is
probable that the increase in nitrate might have been due to a rise in
the dissolved oxygen level of the liquor in the anaerobic unit due to
air trapped in the hoses from the pumps, and resulting in either
oxidation of ammonia to nitrate or inhibition of denitrification.
At 49 hrs, the feed pump to Unit 1 (Figure 21) was similarly restarted
to continuous anaerobic operation. The anomaly at (b) is probably due
to similar circumstances. Whatever the real reason for the jump in
nitrate, it is apparent that a physiological disturbance occurred.
These experiments suggest that even though a low level of incoming
dissolved oxygen does not disturb the system - the recirculating oxy-
gen quickly dropping to below 0. 5 mg/j2 , a constant level of residual
oxygen may have a detrimental effect.
In figure 20 starting at 41 hrs and in Figure 21 starting at 90 hrs, the
behavior of anaerobic units when returned to aerobiosis and a waste-
water feed is plotted. Once the air supply is turned on there is an
almost immediate recovery in nitrifying ability. As the primary
44
-------
50
Continuous
Anaerobic
-X Experimental Results
• — Predicted Results from Model
Continuous
Aerobic
66
a
«»
1
••4
U
a
o
w>
o
*•
aa
E
10
40 50 60
Elapsed Time, hours
70
SO
90
Figure 20.
Aerobic-Anaerobic Cycling; A Comparison of Experi-
mental and Predicted Results. Unit 2, Run 111769;
18 C. Batch anaerobic run to obtain an initial denitri-
fication rate for use in the model and to eliminate NO-
in the recirculating liquor preceded continuous opera-
tion. Continuous feed flow set at 2.5 ml/min
(residence time = 1000 min).
-------
At
•3
u
U
a
-r4
a
o
5
«
-M
at
X X Experimental Results
Predicted Results from Model
50 ,
Continuous Aerobic.
Continuous Aerobic
Continuous Anaerobic
IQ .
10
20
30
40
50 60 70
Elapsed Time, hours
80
90
100
Figure 21.
Aerobic-Anaerobic Cycling; a Comparison of Experi-
mental and Predicted Results, Unit 1, Run 111769, 18 C.
Unit switched from aerobic to anaerobic mode at 40. 7
hrs. Brief batch phase preceded continuous operation
to determine an initial denitrification rate for use in the
model. This was later found unnecessary. Flow rate
= 2. 5 ml/min.
110
-------
TABLE 8
Hours
0
16.8
20.0
22. 7
24.5
31.0
40. 7
43. 0
45. 0
48. 0
54.0
65. 0
69.5
73. 5
88. 0
90.0
93.0
:HANGES IN AMMONIA CONCENTRATION DURING
CYCLED OPERATION
(Run 111769)
Elapsed Time
From Phase Phase
NH -N
(m|/l)
Switch (Table 7) Unit 1
22.
24.
31.
40.
13.
24.
28.
32.
47.
.3.
5.
a 2.
0.
1.
7 1.
5 1.
0 0.
7 0.
b 3.
3.
4.
2 1.
2 0.
7 0.
7 2.
2 3.
0 c 1.
5 1.
2
4
1
0
0
4
2
0
8
1 ^—
0
5
9
4
0
0
1
Unit 2
1.4
7.9
8. 7 -
1.0
0. 5
6.4
8.2
6.6
2.0
1. 5
0.8
0. 7
0. 7
4. 1
5.6
4.5
5.5
Blocked-in figures enclose the anaerobic stage of each run.
Arrows denote the times the pumps were switched on to begin
continuous operation.
47
-------
effluent feed continues there is an apparent decrease in the rate at
which nitrates are produced deviating from the amounts predicted by
the model. A preliminary assumption might be that it is due to,in-
complete nitrification, but since ammonia and nitrite levels in the
liquid at this time are negligible, this hypothesis can be rejected.
A more likely explanation to account for the missing nitrate is to at-
tribute it to nitrogen assimilation by the organisms of the biomass.
Once restored to anaerobic environment, the cells begin to synthesize
enzymes and replace depleted amino acid pools preparatory to resum-
ing cell growth. Although the cells completely recover their oxidative
abilities after anaerobiosis, the apparent decrease in nitrification is
due to part of the incoming nitrogen being shunted into the cells1
synthetic pathways.
The response of nitrifying beds to anaerobiosis can be qualitatively
described: after shutting off air and primary effluent feed, the dis-
solved oxygen is depleted and utilization of recirculating nitrate begins.
In batch operation, the nitrate is used up at an experimentally con-
stant rate, until it is ultimately depleted. In continuous operation,
nitrified feed is added at a steady rate and the nitrate concentration
in the circulating liquor of the anaerobic unit gradually decreases to
an equilibrium level which is a balance between the rate of nitrate in-
put and the rate of nitrate utilization. The concentration of nitrate
in the effluent of the unit equals that of the circulating liquor.
Since the feed to the anaerobic unit in continuous operation is the
highly oxidized effluent from an aerobic system, the anaerobic micro-
flora must rely on their stored reserve materials for a source of
energy-yield ing organic carbon. When these reserves approach
exhaustion, rate of utilization of nitrate is reduced. Ultimately,
under the stress of extended deprivation of available nutrients, the
microflora undergoes major physiological changes that jeopardize its
changes for survival when again placed in a favorable environment.
Thus, it is essential that the aerobic microflora be returned to aero-
biosis and a nutrient source of organic carbon before irreparable
damage to its oxidative capability occurs. In cycled operation, there-
fore, it is important to know the progress of denitrification during the
anaerobic phase. To enable the prediction of the equilibrium nitrate
level, and to determine an optimum time for recycling, the mathema-
tical model of our system was useful.
DENITRIFICATION RATE ASA FUNCTION OF TEMPERATURE
Tables 7 and 9 present continuous operating rate determinations, re-
sulting from cycled aerobic-anaerobic operation at 18 and 29 C, re-
spectively. The measurements of rate R during a given anaerobic
cycle were obtained by solving the following equation, for R:
48
-------
TABLE 9
CONTINUOUS RUN DENITRIFICATION RATES
AT 29°C
Elapsed Hours
Run No. Phase from Phase Switch
1570 a 7.5
26.0
50. 0
b 5.0
23. 3
48.0
c 5.0
25.2
50.2
74.7
94.4
d 6.5
24.2
51.5
75.2
e 68. 3
93.8
f 5.0
23.8
48.0
75.0
g 73.5
96.0
Rate
(mg/hr / g
Unit 1
0. 578
0. 523
0.632
0.519
0. 555
0. 555
0. 537
0.592
0. 373
0. 373
0.196
0. 050
MLVS)
Unit 2
0. 705
0. 580
0. 532
1.04
0.797
0. 757
0. 755
0.469
0. 584
0. 560
0.596
49
-------
TABLE 9 (Cont'd)
CONTINUOUS RUN DENITRIFICATION RATES
AT 29 C
Rate
Elapsed Hours (mg/hr / g MLVS)
Run No. Phase from Phase Switch Unit 1 Unit 2
1570 h 19.5 0.693
28.0 0.674
72.2 0.586
i 23.6 0.305
72.9 0.337
78.8 0.487
95.3 0.555
j 20.0 0.684
28.5 0.687
49.0 0.605
73.0 0.663
50
-------
FN£-FY(t)
R _ •*•
"
(5)
The results from these two tables plus the results of the batch experi-
ments previously reported were combined and averaged for each unit
at 18 and 29 C, and are presented in Table 10. Thus, each of the
four rate values in Table 10 is the result of averaging approximately
20 independently obtained rates. The standard deviations, obtained by
statistical analysis, indicate that the reliability of each value is high.
The combined rates for both units presented as a function of tempera-
ture in Figure 22 show a 2. 3-fold increase in denitrification for an
11 C temperature increase. This conforms remarkably well with the
maxim of chemistry and biology that a 10 increase in reaction tem-
perature can be expected to yield a doubling of reaction rate. Deni-
trification rate at 18 is 0. 30 mg/hr per gram volatile solids, and at
29 C is 0.68 mg/hr per gram of volatile solids.
DISSOLVED OXYGEN UTILIZATION AND CHEMICAL CHANGES
IN THE RE CIRCULATING LIQUORS
Oxygen Level and Denitrification
During continuous cycled operation there is a continuous level of 7. 5
8. 0 mg/JL of dissolved oxygen (DO) entering the anaerobic unit, along
with the nitrified feed. In order to evaluate the effects of this oxygen
on the course of denitrification, a series of experiments was carried
out to monitor the various parameters of interest.
Arrangements were made to include an oxygen probe in the system to
measure this parameter until a steady state DO level was achieved.
Ammonia, nitrite, and nitrate nitrogen levels were followed as well
as the COD at the beginning and the end of the run. In order to detect
subtle metabolic changes in the microflora during the course of the
runs, phosphates, amino acids (as tyrosine), and protein content of
the effluents were also followed.
Figure 23 presents the oxygen profile of two series of denitrification
runs in which the DO of the incoming nitrified effluent was close to
saturation at 8.0 ppm. These curves show the decline in the DO con-
centration of the re circulating liquor that was in the system at the time
the aeration was stopped. In the later portion of the curve the slope is
somewhat modified by the incoming DO of the influent feed and always
seems to taper off to a minimal value around 0. 5mg/j(?. The signifi-
cance of this low base-line is not yet understood but possible later
refinements in technique may permit restudying this effect.
Denitrification run 91169 was carried out at a residence time of 125
minutes. This meant that a relatively high input flow rate of 20 ml/
minute nitrified effluent was required. The high flow was used to
51
-------
TABLE 10
DENITRIFICATION RATES VS TEMPERATURE
Denitrification Rate*
Q (mg/hr / MLVS)
Temperature, C Unit 1 Unit 2
18 .29 + .04 .31 4-. 13
29 .70 + .13 .66 + .15
* Average includes both the batch and continuous electrification runs.
52
-------
o
3
*«•
r*
>1
n
P
§
l.G
8.
3
OQ
!T
•1
OQ
o
.6,
(0
O
Q.
in
10
15 20 25
Temperature, °C
30
35
Figure 22. Influence of temperature on denitrification rate. All
runs from both the batch and continuous experiments
are included. The values for Unit 1 and Unit 2 were
also averaged together.
53
-------
un
O
5-
tx>
ci
o
h
•H
O
V
PS
0! S"
•o
4)
Time
(minutes)
Figure 23. Oxygen utilization with units connected in series Run
91169 - 29 C. Unit was aerobic prior to start of the
experiment. At t = 0, the air and sewage flows were
turned off and the nitrified feed started at 20 ml /min.
The 0. 5 mg/1 residual DO remained constant throughout
the remainder of the run.
-------
insure that a metabolically significant amount of oxygen would be
entering the unit along with the feed. The amount of DO being made
available to the microflora in the "anaerobic" bed during this run was
calculated on the basis of a feed flow of 20 ml/min with a DO concen-
tration of 8. 0 mg/j0 .
The rate of O2 input is thus:
8. 0 mg/jd x 0.020 /min = 0. 16 mg O2/min
0. 16 mg O2/min = 0. 064 mg O9
2. 5j2 (volume of chamber)
jg/min
Assuming steady state conditions, this represents a QO2 °^ 0- 43
1 O2/mg MLVS / hr. This low Qo2 is about 5% of the oxygen re-
quired for maintaining aerobic respiration and is comparable to the
endogenous respiration rate of starved cells, in a Warburg vessel,
that are metabolizing their carbon reserves. It is important to de-
termine if during denitrification this low level of respiration makes
up a significant proportion of the biomass metabolism thus affecting
the rate of reduction of ~
Analyses of the effluents in run 9H69 during continuous denitrification
showed that the nitrites in the incoming feed were almost completely
removed (Figure 24) while the nitrate level decreased to its equili-
brium point of about 2 mg/j? being removed. Overall nitrogen re-
moval was about 10% of the total incoming nitrogen (Figure 25) which
represents a denitrification rate of 0. 75 mg/hr / g MLVS. This rate
is in agreement with the values previously reported using residence
times of 1000 minutes and achieving over 90% nitrogen removal. It
may be concluded that an oxygen input of 0.43/ij0 O2/mg MLVS/hr
has a negligible effect on the denitrification capacity of the micro-
flora. Since to achieve a 90% NO ~ to N2 conversion the denitrifica-
tion system is operated at about a 1000 minute residence times the
oxygen made available to the biomass would be only about 0. 005
/mg MLVS/hr. This would be quickly consumed through respira-
tion and would result in a dissolved oxygen concentration close to
zero in the recirculating liquor.
A continuous denitrification experiment wherein the residence was
doubled over the run presented in Figures 24 and 25 is illustrated in
Run 10269, Figure 26. The nitrite -nitrate values presented in this
figure have been corrected for the dilution of the incoming feed with
that already present in the unit at the start of the run. At 10 ml/min
it takes approximately 10 hrs to replace 90% of the liquid originally
present in the unit.
Once steady state is achieved, 40 mg/1 NO,-N is entering the unit and
26. 5 mg/1 NO--N is present in the effluent. This means that 13. 5
mg/1 or about 34% of the incoming NO^-N is being removed.
55
-------
40 .
O - influent
X effluent
NO,
. NO,
-------
-o o-
N removed
10 12 14 16
Hours
18
20
22
24
Figure 25. Fraction of total NO--N removed using an oxygen con-
taining feed Run 9Ho9, 29 C. Continuous denitrifica-
tion with an oxygen saturated feed. Run 91169, 29 C,
nitrified feed flow = 20 ml/min (residence time = 125
min). Dissolved oxygen content of feed - 7. 8 mg//.
R = 0. 75 mg/hr/g MLVS.
57
-------
50
Figure 26. Continuous denitrification at increased residence time,
Unit 2, Run 10269, 29 C. Flow rate of nitrified feed
set at 10 ml/min (residence time = 250 min). R =
0.455 mg/hr/g MLVS.
58
-------
At 10 ml/min the residence time in the system is 250 minutes or
4. 16 hrs. Then the rate of denitrification (R) is;
R = 13'5-mg/f = 0.455 mg/hr / g MLVS
4. 16 hrs - 7. 1 g MLVS
This rate compares rather favorably with the values for the batch
studies already presented.
The significance of these studies is yet to be fully developed. There
obviously is an upper limit to the amount of DO that can be tolerated
in a denitrification unit. What this maximum permissible level is and
whether denitrification is an all or none affair, that is, can O., and
NO ~be utilized simultaneously, will have to be answered in later
studies.
Protems, Amino Acids and Nitrogen in Feeds and Effluents
The chemical analypis of the runs used for the oxygen studies_is pre-
sented in Figure 27. There was very little change in the PO, , amino
acids and total protein in the effluent of the anaerobic unit as compared
to the influent indicating that the integrity of the microflora has not
been altered by test conditions - at least for the first 24 hours.
The presence of amino acids and protein in the supernatant liquor from
the various treatment units also offers some clues as to the physio-
logical state of the biomass. Accordingly, the data in Table 11 was
collected on a series of continuous runs up to 90 hours in duration.
There were no apparent trends with time from the start of a phase
switch and so the data presented in the table are averaged for the en-
tire series and presented in Table 12.
The incoming primary effluent, as might be expected, contains mate-
rial that reacts both with the Lowry protein analysis and the colori-
metric test for tyrosine. These materials are reduced by about 58%
during aerobic treatment and the amount of tyrosine material changes
by an additional 10% during the anaerobic phase. The protein like
material is reduced by another 60% during anaerobiosis giving an
overall removal of about 80%.
The significance of these observations is not clear. There are a
number of phenolic compounds and reducing agents that can interfere
with both of these tests. A more definitive resolution of the reactants
perhaps through chromatography is needed before they can be accepted
as proteins and amino acids.
The reactants appear to be materials that enter with the primary ef-
fluents, portions of which are refractory to biological attack. The
59
-------
O — Influent
D — Effluents
30 -
ZO J
9—©—e—o o
PO,
10 -
Protein
rj Protein
Tyrosine
10
12
14 16
18
20 22
24
Time
(hours)
Figure 27. Chemical changes between influent and effluent flows
during denitrification, Run 91169, 29 C. Flow rate
20 ml/min. Phosphates were determined using pro-
cedures described in standard methods; proteins were
determined by the JLowery procedure and the method of
Spies was used for the tyrosine analysis (see Appendix
I).
-------
TABLE 11
PROTEIN AND AMINO ACID CONTENTS OF TREATMENT SYSTEM
EFFLUENTS - RUN 1570
Total
Ami.no Acids
Proteins Tyrosine(Tyrosine x
Phase Mode mg// mg// 20) mg//
Elapsed Hours from
Time Phase Switch
529
529
534
553
577
604
604
529
534
553
577
604
700
700
720
728
773
773
700
720
728
773
UNIT 1
Azusa feed
0 f AER
5
24
48
75
Azusa feed
UNIT 2
0 AN
5
24
48
75
UNIT 1
Azusa feed
0 h AER
20
28
73
Azusa feed
UNIT 2
0 AN
20
28
73
25. 0
8.5
10.5
8. 5
9.0
8.0
15.5
8. 5
7. 0
6.5
4.5
10.5
24. 0
-
7. 0
4.5
-
8.0
9.5
4. 5
2.0
-
4.5
2.0
1.7
1.8
2.5
1.7
5.0
1.9
2.0
2.0
2.2
2.0
10.2
3.0
2.7
2.7
5.5
12.2
2.7
3. 0
3.0
3.5
90
40
35
37
50
35
100
37
40
40
45
40
204
60
55
55
110
245
55
60
60
70
61
-------
TABLE 12
SUMMARY
PROTEIN AND AMINO ACID CONTENTS OF TREATMENT SYSTEM
FEEDS AND EFFLUENTS - RUN 1570*
Total Amino Acid
Primary Effluent
Aerobic Effluent
Anaerobic Effluent
Flow rate into aerobic unit = 30 ml/min
Flow rate into anaerobic unit = 4. 0 ml/min
Ave MLVS = 6.3 g//
Proteins
mg/t
20. 00
6.65
3.95
Tyros ine
mg/f
8.80
2.85
2.63
(Tyros ine x
mg/jf
176.0
57. 0
52.6
20)
*lncludes phases f, h, i, and j (529-604 and 700-942 hours of
continuous coupled operation).
62
-------
small difference between the aerobic and anaerobic effluents shows
that both environments operate as metabolically aerobic systems
which would be the case if NO 'was the sole electron acceptor. Even
if the Lowery reactive materials were released as leakage products
by the sludge mass itself, they would represent only a very small
portion (-"0.2%) of the proteins contained in the biomass.
INFLUENCE OF EXTERNAL COD ON DENITRIFICATION RATES
Measurements of COD levels in the influent and effluent of the activa-
ted bed units during an anaerobic phase of cycled operation showed
that oxidation of some of the residual COD in the nitrified effluent
occurs during denitrification. Figures 28 and 29 illustrate this in
run 1570 during continuous operation. As a method for approximating
the rate of COD oxidation during anaerobiosis, as for example in the
phase depicted in Figure 28, we can use the fact that after 30 hours a
stable COD level in the effluent was achieved. This represents an
equilibrium balance between a constant rate of oxidation and a con-
stant input of nitrified influent. The amount of COD oxidized in this
case was 16 mg/1, and flow through the system was 5 ml/min. Thus,
the removal rate was:
16 mg 5 x 10"3L 60 min 1 n _,0 mg COD
„ ---i TT o v _ _ _._ y _________ V _ — — 0 /nQ o i
E min hr 17. 8 g MLVS * v hr-g MLVS
This rate of actual exogenous COD oxidation during anaerobiosis is
meaningful when compared to the potential for such oxidation provided
by nitrate reduction during denitrification. Average denitrification
rate for anaerobic phase d, Run 1570 above was 0. 755 mg NO -N/hr /
g MLVS. To convert this value to oxidative potential we need to con-
sider a balanced chemical equation describing denitrification.
A commonly used overall chemical description of denitrification is:
5 CH20 + 4 N03" 2 N2 + 5 CO2 + 3 H O + 4 OH"
An equivalent equation describing a similar oxidation by oxygen is:
5 CH2O + 5 O2 5 CO2 + 5 H2O
Clearly, the capacity of 4 moles of NO3 to oxidize 5 moles of CH^O
is equivalent to the capacity of 5 moles of O2- In other words, 1. DO
mg of nitrate expressed as nitrate nitrogen is the equivalent of 2. 85
mg of O-, in terms of oxidizing capacity in our system.
L*
To properly compare the potential to oxidize organic carbon with
actual oxidation of exogenous substrate we also need to consider the
state of metabolic activity of the microflora. A treatment unit is
essentially a continuous culture device with influent nutrient flow
rate adjusted to give minimum cell yield (wastage of produced cell
material in the effluent) and maximum oxidation of influent organic
materials. The culture as a whole resides in the late logarithmic
63
-------
O Nitrified influent
A Denitrified effluent
60-
50.
40.
a
o
o
30-
20'
10
10
20
30 40 50 60
Elapsed Hours of Anaerobiosis
70
30
Figure 28.
Oxidation of residual COD entering the denitrification
unit. Unit 2, Run 1570, phase d, 29 C. Flow of
nitrified feed = ml/min (residence time = 500 min).
Rate of removal of COD R = 0. 269 mg/hr/g ML VS.
-------
60
Ui
40
O 30
O
oo
£
20
10
O Nitrified influent
A Denitrified effluent
-£*-
10
20
30 40 50
Elapsed Hours of Anaerobiosis
60
70
80
Figure 29. Oxidation of residual COD entering the denitrification
unit. Unit 1, Run 1570, phase f, 29°C. Oxidation of
residual COD entering the denitrification unit. Unit 2,
Run 1570, phase d, 29°C. Flow of nitrified feed =
5 ml/min (residence time = 500 min). Rate of removal
of COD R = 0. 269 mg/hr/g MLVS.
-------
phase of the typical microbial growth curve. In this condition, the
flora, while still viable and exhibiting a small growth rate, uses most
of the available organic carbon in the external medium for mainten-
ance energy source. In the Activated Bed system, in other words,
most of the influent organic carbon "removed" by the microflora is
oxidized to CO?, with a very small fraction being converted to excess
cell material. Therefore, we can assume for purposes of discussion,
a simple stoichiometric balance between carbon removal and oxygen
consumption during aerobiosis, or between carbon oxidation and
nitrate reduction during anaerobiosis, without the complication of the
correction used by McCarty, Beck, and Amant (1969) for synthesis of
cell material.
The denitrification rate of 0. 755 mg NOj-N/hr / g MLVS, therefore,
represents a COD oxidation rate of 2. 2 mg O?/hr / g MLVS. Actual
oxidation of exogenous COD was 0. 269 mg O~/hr / g MLVS, hence
external COD accounted for 12. 0% of the total oxidation which must
have occurred in this denitrification phase. We can reasonably con-
clude that the remaining oxidation which must have taken place during
denitrification was performed on internal substrates, i.e., endogen-
ous reserves. Table 13 presents the results of similar calculations
performed on data obtained from earlier runs, verifying the consis-
tency of this behavior. This confirms that endogenous reserves are
the predominant source of carbon utilized during equilibrium anaero-
bic operation of the Activated Bed units.
THE EFFECT OF LENGTH OF ANAEROBIOSIS ON
DENITRIFICATION RATE
As in the previous batch studies, rate - R - appears to be roughly con-
stant for the duration of each anaerobic phase. This is especially
true after about the first 10 hours of operation when the units settle
down with minor fluctuations about some baseline level. In Table 9,
denitrification rate results of cycled operation at 29 C are presented.
Similarly, Table 7 presented the 18 C data. Relative stability in R
can be seen in samples taken after 10 hours of anaerobiosis. The 10
hours again reflect the time required to replace 90% of the NO- con-
taining liquor present at t = 0. The above data on utilization and
availability of exogenous and endogenous carbon correspond with the
hypothesized substrate effect. The stable low availability of bio-
logically usable exogenous carbon seen after about 10 hours of
anaerobic operation (Figures 28 and 29), forces the microflora to
rely on endogenous carbon reserves. The fact that denitrification
rate apparently remains stable after 10 hours in these runs implies
that the rate of mobilization of endogenous reserves is constant.
The reserves appear to be sufficient to furnish substrate for up to
75 hours without being exhausted.
66
-------
TABLE 13
OXIDATION OF ENDOGENOUS AND EXOGENOUS CARBON SOURCES DURING DENITRIFICATION
(all rates are per hr/g MLVS)
Denitrification Total Oxida-
Oxidation of
Actual Oxidation Exogenous^ Carbon^ ,(
Run No.
10269
10769
102069
102069
1570d
1570f
Unit
2
2
1
2
2
2
Rate
(mg NO,-Ny
0. 455
0.490
0. 830
0. 762
0. 755
0. 580
tion Rate
(as mg O2)
1.61
1.40
2. 37
2. 18
2. 15
1.65
Rate of Exogenous
COD (mg O2)
0. 162
0. 214
0. 180
0. 167
0.269
0.218
Total .Oxidation
10.0
15. 3
7.6
7.7
12.0
13. 0
-------
Although both the nitrification and the denitrification phase take place
in an aerobic environment, the denitrification rate is the slower of the
two (5. 2 mg/hr / g MLVS vs 0. 67 mg/hr / g MLVS at 29°C). This
apparent slowing of the metabolism of the microflora under anaerobic
conditions may be an effect of substrate concentration. Typically, in
metabolism and enzyme chemistry, as substrate concentration is re-
duced below a threshold level, reaction rate becomes substrate depen-
dent. The critical nutrient limiting denitrification rate in this case
may be internally or externally available carbon, or it may be the de-
creasing concentration of nitrate nitrogen itself. This will be discus-
sed further in a later part.
Nitrification proceeds from 5-8 times faster than denitrification in
these studies. If all of the effluents from a wastewater treatment plant
are to be denitrified, some provisions for balancing the resultant dif-
ference in hydraulic flows will have to be made. This would require
that the volume of the anaerobic tanks be 5-8 times the volume of the
aerobic tanks. Such a system could be developed using a series of
tanks wherein 1/5-1/8 of the tankage volume would be switched be-
tween aerobic and anaerobic modes at the appropriate time intervals.
Return of Denitrifying Capacity After Removal of the Biomass
The Activated Bed units were stripped down for an analysis of the bio-
mass on 12/18/69 as indicated in Table 4. The units were reassembled
with a small fraction of the microflora still clinging to the rings. They
were then operated aerobically for a period of 2 weeks at a 70 min
residence time prior to the start of run 1570.
The rate data for run 1570 presented in Table 9 shows that ther-n did
not occur any significant rate change through the entire series of
cycles - phase a through j. This indicates that the units had been
restored to their full denitrifying potential within two weeks after
start-up.
Distribution of Nitrogen Compounds During Denitrification
As previously reported, there occurs a continuous low-level release
of NH into the surrounding liquid during denitrification.
A statistical analysis of NH, production for all previous batch anaero-
bic runs was completed and the rate of NH3 appearance during
anaerobiosis was estimated. For periods of anaerobic operation of
less than 50 hours duration, the ammonia is produced at approxima-
tely a constant rate. There is some indication, however, that the
ammonia production rate drops significantly after about 50 hours.
The batch run experimental data gives a rate of 0. 15 _+ . 04 mg/hr /
g MLVS. Similar determinations carried out on continuously opera-
tine units calculated to the identical rate of 0. 15 mg/hr / g MLVS at
29 C for anaerobic phases less than 50 hours in duration. Figure 30
presents the NH--N concentration data on a typical 29 C anaerobic
68
-------
O Nitrified influent
& Denitrified effluent
c
,—I
53
W
C
DD
O
O
£
60
6
zo
30 40 50 60
Elapsed Hours of Anaeror-iosis
70
80
Figure 30. Buildup of ammonia during continuous denitrification.
Unit 2, Run 10269, 29 C. Ammonia nitrogen is pre-
sumed to be released by the cell biornaes rather than
through the reduction of nitrate.
-------
aerobic
anaerobic
O
ifl
£
40
20-
40-,
20-
eo
s
I
i
d
v
80
O
a
o
H
M
40.
Time
* Nit rite and organic nitrogen changes are insignificant at this scale.
Figure 31.
Graphical illustration of the nitrogen transformations
that take place in a single Activated Bed unit. The unit
is being cycled between aerobic and anaerobic modes
on the hypothetical time scale.
70
-------
run. At 18°C, the rate of NH, production is 0. 07 j- 0. 02 mg/hr / g
MLVS. A linear function of temperature fitted to these points gives:
RNH *T* = °* °62 + °' 0073T = rate of NH3-N appearance
This is the factor incorporated into the model.
Nitrite nitrogen was found to be of little significance throughout most
of the cycled operation of the units. In continuous operation of the
Activated Bed units with stable microbial ecology, i.e. , in units which
have equilibrated to cycled operation, nitrite concentration are never
greater than 1 mg/f NO?-N at any time in either aerobic or anaerobic
operation. Thus, nitrrte nitrogen was not considered in the model.
Figure 31 shows graphically the inorganic nitrogen distribution during
aerobic -anaerobic cycled operation.
Effects of Prolonged Starvation
Contrasted to the pattern of return to aerobics is by relatively healthy
beds, we see a different sort of behavior in Figure 32. In this case,
Unit 1 had been subjected to continuous anaerobic operation for 162. 5
hours, or approximately a week. Based on earlier data on the an-
aerobic performance of the bed, the level of nitrate nitrogen in the
liquor at time 0 in Figure 32 should have been about 19 mg/1. The
fact that about 33 mg/1 nitrate nitrogen were present indicates that a
change in the rate of denitrification, R, must have occurred; i.e.,
the bed had lost much of its initial capacity to denitrify.
At time 0 in Figure 32 inputs of air and sewage were reinstated to the
starved bed in Unit 1. The concentration of ammonia in the liquor
immediately begins to rise, and the residual nitrate nitrogen is washed
out of the system by simple dilution. Gradually the unit recovers,
oxidation of ammonia increases in rate until it exceeds the rate at
which ammonia is entering the system, ammonia level falls, and
nitrate builds up. The nitrogen balance of the data presented in
Figure 32 reveals ammonia, nitrate, and a trace of nitrite nitrogen
introduced by the primary effluent feed. Very little nitrogen is di-
verted to the synthesis of protein or cellular material, indicating a
lack of vitality in the bed, or a lagging response to a more favorable
environment.
Subjected to extended anaerobiosis, then, the starved aerobic flora
loses not only its denitrifying capacity, but also the ability to rapidly
revert to vigorous aerobic oxidation. A graphical illustration of this
loss of aerobic recovery capacity is presented in Figures 33, 34, and
35. Figure 33 shows as broken lines the hypothetical behavior of
nitrate level in the circulating liquor if all influent ammonia is im-
mediately converted to nitrate, Case 1, and if none of the influent
71
-------
to
*1
•H
i
K
•r4
U
.S
fl
U
O
eo
B
-X Nitrate - N
-O Ammonia - N
Nitrite - N
16 20 24 28 32 36
Elapsed Hours of Aerobioeis.
40
44
48
50
Figure 32. Effects of prolonged anaerobiosis on recovery of
nitrifying capacity. Unit 1, Run 11769 - 29°C. Unit
had been in the continuous denitrification mode for
162. 5 hours before being returned to aerobiosis and
primary effluent feed.
-------
I
\
U
to
o
z
Case /H) Hypothetical behavior if all influent
B immediately converted to NO.
"
Starting Level
(3) Typical experimental behaviors of
nitrate level in circulating liquor
Difference n3)-(2n is amount
Case \ f (2) Hypothetical behavior if none of influent
NH- is converted to NO,
• Anaerobic
Aerobic
Time
Figure 33. Graphical representation of possible physiological
consequences of anaerobiosis. Expected behavior
when the unit is returned to the aerobic mode.
-------
50
X X Actual Behavior
Expected Behavior
8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40 42 44 46 48
Elapsed Hours of Aerobiosie
Figure 34. Recovery of nitrifying capability after prolonged
anaerobiosis. Unit 1 - Run 11769. Experiment
shown in Figure 32 adjusted to show nitrate production.
-------
X Actual Behavior
or
&
'•-I
{H
M
V
,T3
1
.0
S
4)
ri
M
00
s
50
40
30
— — — — — Predicted Behavior
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Elap§«d Time of Aerobiosia (Hours)
Figure 35. Recovery of nitrifying capability after anaerobiosis,
Unit 2 - Run 11769; 29°C.
75
-------
ammonia is convertedto nitrate, Case 2, the nitrate decreasing by
dilution with the incoming feed. Actual experimental behavior falls
somewhere between these two limits, as indicated by the two solid
lines typical of observed behavior. The difference between the actual
experimental behavior and the behavior expected in Case 2 is the
actual amount of ammonia oxidized to nitrate by the bed.
Figure 34 shows this nitrate production by Unit 1 in the experiment
graphed in Figure 32, along with the plot for maximum possible con-
centration of nitrate. After prolonged starvation, the bed immediately
recovers about 35% of its potential nitrifying ability. This indicates
that at least a portion of the microflora survived the prolonged anaero-
biosis. Assuming continued linearity of the developing nitrifying
capacity, 4 to 5 days elapse before a state of complete nitrification is
restored. This rate of development is probably tied to the growth rate
of aerobic nitrifiers, as they replace numbers of their population
which were rendered non-viable by the extended anaerobiosis.
Figure 35 shows a similar presentation of nitrate production data for
Unit 2 after only a 40-hour period of anaerobiosis. A more vigorous
return to nitrification is evidenced.
Gases Generated During System Operation
An analysis was made of the gases given off by the activated bed unit
when operating in both the aerobic and anaerobic modes. Gas samples
consisted of 500 cc grab samples taken from the head space entrapped
above the liquid during anaerobic operation and from the discharge
vent in the aerobic unit. This sampling was carried out specifically
to determine if N?O (nitrous oxide) was one of the by-products of
denitrification.
A special analytical method was designed that utilized gas chromato-
graphy and mass spectrometry. This consisted of cryogenically con-
centrating each sample in a collection loop on the chromatograph,
inserting the condensate into a 20 ft Porapak column set for automatic
temperature programming, feeding the column effluent into a mass
spectrometer introduction system and continuously monitoring the
major ion fragment of N?O with the electron multiplier analyzer sys-
tem.
Standard samples of N7O and an inert diluent were prepared and ana-
lyzed to ppb levels. Extrapolated sensitivities were verified by mass
spectrometer calculations. With this procedure signals were observed
from standards containing approximately 1 ppb. Reproducible quanti-
tative measurements were achieved for samples containing 10 ppb of
N20.
Multiple analysis of both gas samples were carried out. No N?O was
observed in either sample nor was CO, CH^, or NO, present in a.ny
detectable quantities.
76
-------
Efficiency of Nitrate Removal
As in the aerobic nitrification experiments, there exists a relationship
in the anaerobic mode between denitrification rate, percent nitrogen
converted, and mean residence time in the treatment unit. Although
the experiments were not specifically designed to explore this aspect
due to the time required for each run, there is enough data available
to indicate trends and to arrive at tentative conclusions.
The most interesting relationship is the one between percent NO-j-N
remaining in the denitrification unit as a function of residence time.
This appears to be a more sensitive parameter than the rate-residence
time relationship. In Figure 36 both these relationships are displayed.
The denitrification rate appears to hold relatively constant over a
broad range of residence time; as a consequence of constant rate, there
is a dramatic response in process efficiency as residence time is
increased. A 90% nitrate removal is achieved at about 475 minutes (8
hours).
The efficiency of the overall process can be estimated from the infor-
mation in this report. In the aerobic stage about 85-95% of the incom-
ing nitrogen can be accounted for as NC>3. This is fed into the
denitrification stage wherein 85-95% can be reduced to nitrogen,
accounting for an overall removal by the denitrification process of from
70 to 90% of the incoming total Kjeldahl nitrogen.
It is very informative at this point to compare Figure 36 with Figure
10 which displays similar data for the nitrification experiments. In
Figure 10 we note that the rate of nitrification increases as residence
time decreases. At short residence times the feed flow, and as a
consequence the nutrient flow is increased. The microflora responds
to the increased nutrients by increasing its growth or metabolic rate
which results in all of the added nutrients being oxidized and as a
result the percent nitrification and nutrient utilization efficiency remains
high.
The events during denitrification present quite a different picture. As
displayed in Figure 36 decreasing the residence time down to ZOO
minutes has almost no effect on the metabolic rate as displayed by the
denitrification rate. Since the denitrification rate remains constant,
the continual addition of more NO-j-N with the decreasing residence
times means that there will be a marked sensitivity of process
efficiency to changes in residence as is actually the case.
Under the conditions of the anaerobic mode used in our experiments
altering the residence time would not effect the inflow of organic
carbon'- the carbon being already present in maximum concentra-
tions as stored reserves. Since the nitrates are in excess it must
77
-------
90
80 .
CO
60 -
50
"40
30
zo -
10 -
• — 7c remaining
O — denilrification rats
.0.8
—r~
50
-0.1
100
150
I—
200
—I—
250
300
Mean Residence Time
Figure 36. Influence of residence time on denitrification rate and
nitrogen removal efficiency. These points represent
a summary of the various phases of the 29 C denitrifi-
cation runs. These are all averaged values for both
units of the various runs discussed in this report.
Denitrification rate (R) = mg/hr/g MLVS.
-------
be concluded that the rate of denitrification is limited by the avail-
ability of the stored food reserves and in all probability this is
organic carbon. Below 200 minutes the residual COD in the nitrified
effluents may provide enough carbon to effect rates but the denitrifica-
tion efficiency is very low. Thus, carbon limitation is the controlling
parameter during denitrification. This shortcoming can be overcome
for a given volume of effluent either by supplementing the influent
with organic carbon or by increasing the mass (MLVS) of carbon con-
taining microbial cells.
The residence time, denitrification efficiency, denitrification rate
relationship needs further study. In this program, working with a
primary effluent of a relatively fixed nitrogen concentration meant
that there was a corresponding variation in organic load as flow rates
were changed.
79
-------
VIII. REFERENCES
1. Herbert, D., (1961), A Theoretical Analysis of Continuous Cul-
ture Systems. In "Continuous Culture of Microorganisms, "
SCI Monograph No. 12, Society of Chemical Industry, London.
2. Lowry, O. H., Rosebrough, N. J. , Farr, A. L. , and Randall,
R. J. (1951), Protein Measurement with the Folin Phenol Reagent,
J. Biol. Chem. , 193, 265.
3. McCarty, P. L. , Beck, L. , and Amant, P. (1969), Biological
Denitrification of Waste waters by Addition of Organic Materials.
Presented at the 24th Annual Purdue Industrial Waste Conference,
Lafayette, Indiana.
4. Rao, B. S. , and Gandy, A. F. , Jr. (1966), Effect of Sludge Con-
centration on Various Aspects of Biological Activity in Activated
Sludge, J. Water Poll. Cont. Fed. 38, 794.
5. Ribbons, D. W. , and Dawes, E. A. (1963), Environmental and
Growth Conditions Effecting the Endogenous Metabolism of
Bacteria. Annals N. Y. Acad. Sci. , 102, 564-586.
6. Spector, W. S. (1956), Editor, "Handbook of Biological Data, "
W. B. Saunders Co., Philadelphia.
7. Spies, J. R. (1957), Methods in Enzymology, Colorimetric
Procedure for Amino Acids, III, 467, Academic Press. New
York.
8. "Standard Methods for the Examination of Water and Sewage"
(1965), 12th edition, American Public Health Assoc. , New York.
81
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IX. APPENDIX I
Analytical Techniques
(1) Chemical Oxygen Demand (COD). This was the principal
test used to evaluate the efficiency of the oxidative process. The
chemical method using the alternate procedure for a 10 ml sample
size, as described in Standard Methods (1967), was used throughout
this work. Samples were permitted to settle for 30 minutes in the
cold before the 10 ml aliquot was removed.
(2) Total Kjeldahl Nitrogen (TKN). A micro Kjeldahl proce-
dure was used employing an Aminco **• Digestion and steam distilla-
tion system. Nitrogen was determined by nesslerization of the
distillate. NH.C1 was used as a standard.
4
(3) Ammonia Nitrogen. NaOH (18N) was added to the sample
prior to steam distillation into boric acid. Ammonia nitrogen was
determined by nesslerization of the distillate.
(4) Nitrates. The Brucine method, as described in Standard
Methods was used for this analysis. Potassium nitrate was used as
the standard.
(5) Nitrites. The packaged method of the Hach Chemical Co.
was employed for this analysis. Comparative assay with other tests
and the excellent reproducibility of the sodium nitrite standard showed
this to be a reliable test.
(6) Mixed Liquor Suspended Solids (MLSS). The Activated Bed
units were drained and the plastic rings containing the biomass were
removed. Each ring and the sides of the vessel were scraped and
rinsed with distilled water. The biomass suspension was placed in
an oven at 105 C and dried to constant weight (24-48 hrs). The total
dry weight represented the biological solids contained in the treat-
ment unit.
(7) Mixed Liquor Volatile Solids (MLVS). The dried solids re-
maining from the MLSS determination were carefully reweighed and
then placed in a 600 C oven for 24 hours. The loss in weight repre-
sents the volatile solids contained in the Activated Bed system.
(8) Phosphate. Phosphate was determined as the orthophos-
phate. The procedure Standard Methods (1967) was follwed except
that an amino acid solution (Hach Chemical Co. ) was used as the re-
ducing agent.
(9) Protein. The colorimetric method of Lowry, et al (1951)
was used. Bovine serum albumin was used as the standard.
83
-------
APPENDIX I
(10) Amino Acids. These were measured by determining the
tyrosine, tryptophane content using a modification of the colorimetric
method of Spiess (1957). Tyrosine was used as the standard. Pro-
teins were first removed by precipitation with 5% trichloracetic acid.
Total amino acids were calculated by assuming the tyrosine,
tryptophane content as 5% of the total amino acids present.
84
-------
APPENDIX II
DERIVATION OF THE MATHEMATICAL MODEL
This derivation assumes: (1) that the nitrogen composition of the
secondary effluent fed to the Activated bed units is rather constant
and consists of 40-50 mg/1 ammonia nitrogen (NH.,-N), and (2) that
under anaerobic conditions a given biomass in one unit denitrifies at
a constant rate, as ascertained in batch denitrification studies.
The following symbols are used:
y(t) = inorganic nitrogen in anaerobic unit (mg/ml)
R = constant rate at which biomass removes inorganic
nitrogen (mg/min)
Q = volume of anaerobic system (ml)
F - feed flow of nitrified effluent to an
anaerobic system (ml/min)
C(t) = |NO--NJ in aerobic unit (mg/ml)
V = aerobic system volume (ml)
f = feed flow of sewage to aerobic system (ml/min)
Also, let F/Q = a, f/v = b and Nf = inorganic nitrogen in the feed.
Consider a system consisting of two Activated bed units, both operated
in the aerobic mode (Figure 18 (a)). At time t = 0, one of the units
(Unit 2) is switched to the anaerobic mode by turning off the air and
feed flows, and simultaneously starting a metering pump to feed the
highly nutrified liquor from Unit 1 into Unit 2 (Figure 18 (b)).
Examination of the figure reveals that there is one input flow and two
exit flows of inorganic nitrogen from the anaerobic unit. The source
of nitrogen is the incoming nitrified liquor from Unit 1 and this liquor
contributes to the concentration of inorganic nitrogen in the system at
any given time an amount of:
85
-------
APPENDIX II
One source of nitrogen loss is the nitrate which leaves the unit via the
overflow. This contribution to inorganic nitrogen change in the
anaerobic unit is given by the term:
The principal loss of nitrogen in the anaerobic unit is by means of
denitrification. During continuous operation, this proceeds at some
constant rate, R. The contribution to the change in inorganic nitro-
gen due to this loss is represented by:
§ • dt
The combination of the terms for the nitrogen input with the two terms
for nitrogen output gives equation (1) which describes incremental
changes in nitrogen concentration in the anaerobic system.
- c(t) • F - dt-y(t) . F . dt-R • dt
Rearranging equation (1) gives us:
dy + F
dT Q; y
where F, Q, and R are constants.
c(t) F
Q
R
(1)
(2)
A solution for y(t) is obtained by noting that equation (2) fits the general
form of the first order linear differential equation:
p(t)y = g(t)
(3)
where p(t) and g(t) are any continuous functions of t. The general
solution to equation (3) is obtained by use of the integrating factor,
u(t), and is given by:
y(t) =
where
u(t) = exp
s)ds + const
(x) dx
(4)
(5)
86
-------
APPENDIX II
f
A comparison olLequations (3) and (2) identified p(t) as ^ a constant
and g(t) as c(t) ^ - fl f where F, Q, and Rare constants. The only
time dependence in g(r)r occurs as c(t), the nitrogen concentration in
the feed to the anaerobic unit, which is equal to the nitrogen concen-
tration in the aerobic unit. Two cases for the time behavior of c(t)
need to be considered. In the simplest case, c(t) is a constant, inde-
pendent of time. This is the case represented by Figure 18 (b), where
Unit 1, which has been aerobic for some time prior to the beginning of
the experiment, remains aerobic, while Unit 2 goes anaerobic. The
Unit 1 liquor maintains its steady-state nitrogen concentration, which
is just N, - NH_-N assuming the feed flow to the aerobic unit has been
judiciously chosen to give ~ 100% nitrification (which has been the case
in all experimental work to date). Assuming then that c(t) = Nf, a
constant for condition I8(b), the nitrogen concentration exiting the
anaerobic unit, y(t), is given by the solution to the differential equa-
tion (substituting c(t) = N, in equation 2):
dy F F ' Nf R
= '
ar
From equations (4), (5), and (6):
y(t) = , . . - + y(o) .
where y(o) is a constant of integration which must be evaluated from
the boundary condition at t = o. In this particular case, y(o) = N,,
since at t = o Unit 2 is switched from steady state aerobiosis to
anaerobiosis. Thus, y(t) for Unit 2 from t to t is:
y(t) =Nf |_l=e J . ^ |_I..
or simplifying and lettering F/Q = a
y(t) = Nf - f [l -e-atl (7)
Also, c(t) for Unit 1 from t to t, is:
1 » ' o I
c(t) = Nf (8)
87
-------
APPENDIX II
Now at time ^ the units are switched (see Figure 18(c)), and a more
complicated solution to the differential equation (equation 2) results
since c(t) is no longer constant. In Unit 2 just at t,, the liquor has
been denitrified to a concentration N 1. At t,, the air flow and feed
are resumed to Unit 2, and, assuming that thebiomass returns
instantaneously to its previous completely nitrifying capacity, all
available NH -N entering in the feed is immediately converted to
nitrate. As the liquor that was in Unit 2 is gradually replaced by
incoming primary effluent the nitrogen level rises to Nf. This Unit 2
liquor with nitrogen concentration rising to Nf provides the feed to
Unit 1 (i.e., c(t) at flow F into Unit 1). Thus, to find y(t) in Unit 1
(from equations 2, 3, 4, and 5) a mathematical description of the
nitrate concentration in Unit 2 (i.e., c(t)) is needed. A differential
equation, similar in form to equation (2) when solved, yields:
for t. s t s t2
c(t) = (NQ - Nf) e ^' VA" Ll' + Nf
The equation is easier to perceive if the simplifications below are
introduced.
f/V = b
'i.
(N0 -N£) = AN
Thus,
t
c(t) = ( A N 1 ) e "^"V + Nf (9)
Unit 1 at time t, is switched to anaerobiosis, with an initial concen-
tration of nitrogen, y(tj = Nf, and is fed at rate F with liquor from
Unit 2 at a concentration c(t) given by (9). Substituting (9) into (2)
gives the differential equation for y(t), the nitrogen concentration in
and exiting from Unit 1. This differential equation is solved in the
same manner as in the simpler case where c(t) is constant, and the
solution is, for t, st s't,, in Unit 1 t
ytt) - Nf - l - .
Further cycling of the units gives equations for c(t) and y(t) which are
identical to (9) and (10) except for shifts in the time axis (i.e. ,
88
-------
APPENDIX II
t~, t,. . . . are the switching times and appear in place of t, in
equation (9) and (10)). For example, in the case of t^ st st the
solutions are:
Unit 1 (aerobic):
c(t) = (AN2) e'b(t't2) + N,
Unit 2 (anaerobic):
y(t) = Nf - f Ti
Figure 37 illustrates the operations defined by these equations.
89
-------
fi
V
00
o
H .3
*
O
rt
4->
O
H
, Aerobic
(Primary Effluent)
.. Anaerobic
|Fed with Unit 2
1
TIME
ic J>L
: Effluent)T
Aerobic
(Primary Effluent)
vO
O
0)
oo
o
M ?
H o
o
A
I
Anaerobic
. Aerobic
(Fed with Unit 1 Effluent)^ (Primary Effluent)
i
Anaerobic-
with Unit 1 Effluent)
N
Figure 37. Graphical Representation of System Operation Defined
by the Mathematical Model. Cycled aerobic and
anaerobic operation, both units fed continuously.
-------
1
5
Accession Number
Q Subject Field & Group
05 D
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Organization ENVIROGENICS
Division of Aeroiet- General Cor-noratinn
9200 E. Flair Drive
El Monte. California 91734
Title
A STUDY OF NITRIFICATION AND DENITRIFICATION
1 Q Authors)
Allen, Paul H. Ill
Matyskiela, Walter W.
16
21
Project Designation
# 17010 DRD
Note
Citation Finaj report Contract No. DI 14-12-498,
37 figures, 13 tables, 90 pages
22
1970
23
Descriptors (Starred First)
^Denitrification, * Mathematical models, ^Nitrates, ^Nitrification,
#Rates, Aerobic conditions, Chemical oxygen demand, Evaluation,
Laboratory tests, Nutrients, Organic loadings, Oxygen requirements,
Wastewater treatment
25
Identifiers (Starred First)
*Tertiary treatment, ^Temperature, Residence Time,
Limiting substrate
27
Abstract
A program to incorporate biological denitrification into a wastewater treatment
system was undertaken with the objective of developing a process that depends
exclusively on the carbon compounds contained in the wastewater to supply meta-
bolic energy to the microflora. In the experimental program the incoming
nitrogenous material was oxidized to nitrate in an aerobic phase and reduced to
nitrogen gas in an anaerobic phase. Conditions for developing a nitrifying
microflora were investigated using a primary wastewater effluent as feed. Flows
into the system were varied to give a range of residence times. Anaerobic batch
experiments were carried out to determine if stored reserves could support
denitrification. Under appropriate conditions almost 100% of the nitrates could
be reduced. The effluent from the aerobic unit served as the feed for the anaerobic
process. At appropriate intervals this situation was reversed by switching the
airflows and feed sources. Over 95% of the wastewater nitrogen in wastewater
was removed. Nitrate-nitrogen removal rates ranged from 0.600 to 1.00 mg/hr/g
MLVS. A mathematical model was developed which described the response to
cycled aerobic-anaerobic operation. The alternating cycle approach was shown
to be an effective method for removing nitrogen from wastewater.
(Mechalas-Aerojet)
Abstractor
Byron J. Mechalas
institution
division oi
£1 Monte>
91734
WR:102 (REV JULY 1969)
WRSIC
SEND TO: WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U.S. DEPARTMENT OF THE INTERIOR
WASHINGTON, D. C. 20240
ft U.S. GOVERNMENT PRINTING OFFICE : 1910 O - 411-755
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