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 - 29C                               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,  18C                     39

18.     Decrease in NOo-N in  Effluent as a Function of
         Simultaneous Dilution and Denitrification
         Unit 2, Run 1570,  Phase d, 29C                      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, 18C                             45

21.     Aerobic-Anaerobic Cycling; a Comparison of
         Experimental and Predicted Results
         Unit 1, Run 111769, 18C                             46

22.     Influence of Temperature on Denitrification Rate        53

23.     Oxygen Utilization with Units  Connected in Series,
         Run 91169 - 29C                                    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, 29C            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, 29C             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 Rates29C            33

6      Summary of Batch Denitrification Rates 18C           35

7      Continuous Run Denitrification Rates at 18C           43

8      Changes in Ammonia  Concentration During  Cycled
               Operation                                      47

 9      Continuous Run Denitrification Rates at 29C           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 18C 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 NO 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  1C,  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

-------
    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 Z9C.   Unit 1 = 5. 5 g MLVS;
                          Unit 2 = 7. 1 g MLVS {Table 4).

-------
                                                         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

-------
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  29C.

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 30C  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. 0C 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 29C 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 - 29C
                                                   D  -
                  Expt. 102869 - 18C
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- 18C
                                                                                      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
                            (29C)
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  18C 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 29C.  The 18C 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 18C  and 29C,  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, 18C.  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 18C

                                                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 29C
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

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 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

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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

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                       O    Influent

                       D    Effluents
 30 -
 ZO J
    9eo	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).

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                          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

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                           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

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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, 29C.  Oxidation of
                                         residual COD entering the denitrification unit.  Unit 2,
                                         Run 1570,  phase d, 29C.  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

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                                   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

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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 29C).  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

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                                   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.

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                       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

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 run.  At 18C,  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

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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  - 29C.  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.

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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
                           Elapd Time of Aerobiosia (Hours)
Figure 35.    Recovery of nitrifying capability after anaerobiosis,

               Unit 2  - Run 11769; 29C.
                                 75

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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

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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

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                   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.

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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

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                        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

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                        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

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                          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

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                           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

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                           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

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                          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

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                         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

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               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.

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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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