17090 FQJ 09/71

The Water Pollution Control Research Series describes the re-
sults 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
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Inquiries pertaining to Water Pollution Control Research Reports
should be directed to the Chief, Publications Branch (Water),
Research Information Division, R&M, Environmental Protection
Agency, Washington, D.C.  20460.


    Anthony F.  Gaudy, Jr.,  and Elizabeth  T.  Gaudy
      Bioenvironmental Engineering Laboratories
  School Civil  Engineering  & Microbiology Department
                Oklahoma State University
                Stillwater,  Oklahoma
                        for  the

          Office of Research and Monitoring

                  Project  #17090 FQJ
                    September 1971
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price $1.25

                   EPA Review Notice
This report has been reviewed by the Environmental
Protection Agency and approved for publication.
Approval does not signify that the contents necessarily
reflect the views and policies of the Environmental
Protection Agency, nor does mention of trade names or
commercial products constitute endorsement or recommenda-
tion for use.


Over the past decade, many of the thousands of experiments accomplished
in the principal investigators' laboratories were designed to permit
interrelated analyses and correlation of the 10-year research effort
into a general body of conceptual principles applicable to design and
operation of activated sludge processes.  The aim of this report is to
present these biological concepts in terms readily understood by engi-
neering professionals.  The document is by no means a design and oper-
ational manual, although some recommendations for design and operational
procedures are included.

Generalized concepts of BOD exertion, the use of ACOD as a design and
operational tool, the stoichiometry and mass balance concepts of treat-
ment, and kinetic,equations for microbial growth.-are. presented.   Design
models are discussed, and a mode,! for completely mixed reactors  holding
recycle solids, XR, constant is,recommended.  Some guidelines for
accommodation ofLvarious types of shock .loadings.are included.  Con-
cepts of oxidative assimilation and the multiple effects of solids con-
centration, nitrogen concentration, and detention time are related; a
new activated sludge process (continuous oxidative assimilation) for
nitrogen-deficient wastes is presented.  Data supporting the concept
of total oxidation are presented, and a modification of the extended
aeration process incorporating chemical hydrolysis of portions of the
sludge is recommended.  In the final chapter, some possible flow dia-
grams for complete aerobic treatment (purification and sludge disposal)
of metabolizable organic wastes are presented.
The report was submitted in fulfillment of project 17090 FQJ under the
sponsorship of the Environmental Protection Agency.
Key words:  activated sludge, kinetic models, shock loads, nitrogen-
     deficient wastes, ultimate disposal, oxygen demand, oxidative
     assimilation, endogenous phase, purification efficiency.

Abstract                                                           ill
Contents                                                             v
List of Figures                                                   viii
List of Tables                                                     xii
Conclusions                                                          l
Recommendations                                                      3
  I.  Introduction                                                   5
         The Perspective                                             5
         The "Technological  Gap"                                      6
         The Nature of the Report                                    7
 II.  Measurement of Purification                                    9
         The Concept of Biochemical  Oxygen Demand                     9
         Exertion of Biochemical  Oxygen Demand                      10
         Theoretical COD and BOD                                    12
         ACOD as a Parameter of Purification                        14
         Parameters for Design and Operation                        16
         Additional Information                                    17
         Summary and Conclusions                                    is
III. 'Fundamental Stoichiometry of Biological Treatment             19
         "Equation" for Purification                               19
         Partition of Substrate Between Respiration  and  Synthesis   20
         Energy Coupling Through  ATP                               21
         Energy Balance                                            22

         Summary and Conclusions                                   26
 IV.  Kinetics of Microbial  Growth                                 29
         Exponential Growth                                         29
         Relationship Between u and  S                              32
         Sludge Yield                                              40
         Summary and Conclusions                                   46
  V.  Kinetics of Completely Mixed Reactors                         49
         Criteria for Complete Mixing                              49
         Growth in the "Steady State"                              54
         Cell Recycle with Constant  Concentration  Ratio             58
         Cell Recycle with Constant  Return Solids  Concentration     70
         Summary and Conclusions                                   80
 VI.  Factors Tending to Disrupt the Steady  State  -  Shock  Loadings 83
         Quantitative and Qualitative Changes  in Substrate         84
         Qualitative Shock Loads                                   90
         Changes in Chemical  Composition  of  the  Inflowing  Waste
            Other than the Organic Substrates                       92
         Changes in Environmental  Conditions not Involving
            Chemical Composition of  the Waste                       94
         Summary                                                   96
VII.  Process Modifications  for Nitrogen-deficient Wastes           99
         Nitrogen Supplementation for Biological Treatment         99
         Oxidative Assimilation                                   105
         Proposed New Process for Treatment  of Nitrogen-
            deficient Wastes                                      108

          Summary and Conclusions                                  H4
VIII.  Extended Aeration Process                                  115
          Recent Investigations of Extended Aeration               116
          Engineering Control  of Total  Oxidation                   124
          Summary                                                 129
  IX.  Summary and Recommendations                                131
Acknowledgments                                                   141
References                                                        143
Patents and Publications                                          149,
Glossary of Terms, Abbreviations,  and Symbols


Number                                                          Page

   1.  Generalized plot of substrate concentration,  bio-
       logical  solids concentration, and oxygen utilization
       during exertion of biochemical  oxygen demand.
       Circles  mark inflection points.                             il

   2.  Arithmetic and semi-logarithmic  plots of microbial
       growth during the substrate removal  phase in  a
       batch system.                                              31

   3.  Effect of initial  substrate concentration,  Sg, on  the
       rate and total amount of microbial growth.  The ver-
       tical lines indicate the end of  the  exponential  growth
       phase.                                                     34

   4.  Hyperbolic plot of the relationship  between specific
       growth rate, y, and initial  substrate concentration,
       S0 (upper graph),  and a straight line plot, S0/y vs.
       SQ, of the same data (lower graph).                         35

   5.  Effect of the numerical  value of the saturation con-
       stant, IG, upon the degree  of curvature  of  the hyper-
       bolic relation between y and S0.   Curves were  calculated
       using equation 18 with ym.Y = 0.4 hr"' and  the values of
       ..   u       J.L.  j-       ma A
       KS shown on the figure.                                     37

   6.  Hyperbolic plot of the relationship  between specific
       growth rate, y, and initial  substrate concentration,
       S0, for  a heterogeneous microbial  population of sewage
       origin growing on  a concentrate  prepared from  the
       soluble  portion of municipal  sewage.                        41

   7.  Constancy of sludge yield throughout the substrate
       removal  period during growth on  glycerol  of a  hetero-
       geneous  microbial  population of  municipal sewage origin.    43

   8.  Flow diagram for a continuous flow,  completely mixed
       reactor  of the once-through type.                           so

   9.  Theoretical  dilute-in and dilute-out curves for a  com-
       pletely  mixed reactor calculated  for a dilution rate
       of 0.125 hr-1.                                             52

  10.  Comparison of theoretical and experimental  dilute-in
       curves for a completely mixed laboratory reactor
       operated at various detention times.                        53

Number                                                          Page

  11.  "Steady state" parameters measured in a completely
       mixed laboratory reactor of the once-through type,
       operated at a dilution rate of 0.33 hr"1 with nom-
       inal influent substrate concentration, S<, of
       1060 mg/1 glucose COD.                                     59

  12.  Flow diagram for a continuous flow, completely mixed
       reactor employing cell recycle at a constant ratio,
       c, of recycle solids, XD, to aeration solids, X\
       Diagram represents the kinetic model proposed by
       Herbert.                                                   61

  13.  Comparison of predicted levels of X and S at various
       dilution rates in a once-through reactor (broken
       lines) and a reactor employing cell recycle concen-
       tration ratio, c, as a system constant in accordance
       with the kinetic model equations of Herbert.  Values
       used for calculation were:  l^max* 0.5 hr~l, Ks, 75
       mg/1; Y, 0.60;  a, 0.25; c, 4.0, Sn-, 1000 mg/1.            63

  14.  Operational data for a laboratory reactor operated
       according to the kinetic model of Herbert with cell
       recycle at a constant recycle ratio, c, of 1.5.
       S-j = 1060 mg/1 glucose COD, « = 0.25, aerator deten-
       tion time = 3 hr.                                          66

  15.  Comparison of observed (solid lines) values of X  and
       S with values predicted (broken line curves) by the
       kinetic model equations of Herbert using experimen-
       tally determined values of the biological constants
       (see text for details).                                    67

  16.  Flow diagram for a continuous flow, completely mixed
       reactor employing cell recycle at a constant recycle
       solids concentration, XR.  Diagram represents the
       kinetic model proposed by Ramanathan and Gaudy.            71

  17.  Comparison of values for X and S at various dilu-
       tion rates predicted by the two kinetic models for
       cell recycle.  Curves A are those predicted by the
       model equations of Ramanathan and Gaudy.  Curves  B
       are those predicted by the model equations of
       Herbert.                                                   74


  18.  Predicted levels of aeration solids,  X,  and  effluent
       COD, S, for a range of organic  loadings, S-j,  at  var-
       ious dilution rates.  Curves were computed according
       to the model equations of Ramanathan  and Gaudy using
       the following values for system constants:    ymax>
       0.5 hr-1; KC, 75 mg/1; Y, 0.60; a, 0.25; XR,
       10,000 mg/1.                                                76

  19.  Expanded scale plot of the curves from Figure 18 for
       organic loadings up to 1000 mg/1  COD.                        77

  20.  Response of a continuous flow,  completely mixed
       reactor of the once-through type to a severe  quanti-
       tative shock load consisting of a change in  S-j from
       450 mg/1 to 1450 mg/1  COD.   The dilution rate was
       0.244 hr-1.                                                 87

  21.  Response of a continuous flow,  completely mixed
       system of the once-through type to a  quantitative
       shock load consisting  of a two-fold increase  in  S^.
       The dilution rate was  0.125 hr*1.                           89

  22.  Relation between effluent substrate concentration, S,
       and NH3 - N concentration in the feed at three COD:N
       ratios for dilution rates ranging from 1 to  1/12 hr-1.
       Feed COD was 1060 mg/1.                                     101

  23.  Relation between effluent and influent concentrations
       of NH3 - N at three COD:N ratios for  dilution rates
       ranging from 1 to 1/12 hr~!.                               102

  24.  Relation between purification efficiency, nitrogen
       content of the sludge, dilution rate, and feed COD:N
       ratio.                                                     103

  25.  Substrate removal, sludge accumulation,  and  sludge
       composition in the absence of protein synthesis  in
       a batch reactor.                                           107

  26.  Proposed flow sheet for the treatment of nitrogen-
       deficient industrial wastes by the continuous oxida-
       tive assimilation modification of the activated
       sludge process.                                            109

  27.  Laboratory-scale pilot plant employed in operational
       feasibility studies for the continuous oxidative
       assimilation process.                                       ill

Number                                                          Page

 28.  Operational data for treatment of nitrogen-free
      acetic acid waste by the continuous oxidative assim-
      ilation process with nitrogen supplementation in the
      endogenous aerator at a COD:N ratio of 70:1.               H3

 29.  Performance data for a continuous flow extended
      aeration activated sludge in a laboratory pilot
      plant operated with total solids recycle, showing a
      period of accelerated autodigestion.                       119

 30.  COD removal capability of extended aeration activated
      sludge after the number of days of continuous oper-
      ation with total solids recycle indicated on each
      graph.                                                     120

 31.  Effect of aging on endogenous oxygen uptake by an
      extended aeration activated sludge during long-term
      pilot plant studies employing total solids recycle.        122

 32.  Total oxidation, during the endogenous phase, of the
      biological solids synthesized during the substrate
      removal phase in a batch system using a heterogeneous
      population developed from a sewage seed.                   123

 33.  Metabolism of hydrolyzed activated sludge by an
      extended aeration activated sludge.                        126

 34.  Proposed extended aeration activated sludge process
      incorporating chemical hydrolysis for control of
      sludge concentration.                                      i27

 35.  Possible flow diagram for activated sludge process
      incorporating suggested modifications for operational
      control of purification efficiency and including
      sludge disposal by aerobic autodigestion aided by
      chemical hydrolysis.  This diagram is based on the
      conceptual principles and research findings presented
      in this report.                                            135

 36.  One of several  possible modifications of the flow
      diagram shown in Figure 35.   See text for details.          138


Number                                                            Page

   I.   Values of ymax and Ks  for Heterogeneous  Populations of
       Sewage Origin Growing  on Glucose                            39

  II.   Statistical  Summary of Sludge  Yield  Values  for Heter-
       ogeneous  Populations of Sewage Origin  Grown on Various
       Carbon Sources                                              45

 III.   Operational  Parameters for a Completely  Mixed  Contin-
       uous Flow Reactor  With Cell Recycle                          68


The general conclusion which emerges from the decade of research inves-
tigations briefly summarized in this report is that activated sludge
processes must be designed and operated with greater consideration of
the biological nature of the process.  Concepts based on microbial phys-
iology can be adapted to systems employing heterogeneous populations,
and application of biological principles can lead to innovative improve-
ments in the basic process.  Design of treatment processes must include
provision for operational controls which will ensure reliability of per-
formance, and operation must be based on daily assessment of purifica-
tion efficiency.  Both design planning and operational procedures should
attempt to minimize disruptions of the process.  Each treatment facility
should be designed, not according to a standard flow sheet, but as a
combination of unit processes chosen to accomplish specific objectives
in purifying a specific waste.  The activated sludge process can and
should deliver reliably a high degree of purification of organic wastes,
but this requires both good design and careful, intelligent operation.

Some of the specific conclusions discussed in the body of the report
are listed below with reference to the pertinent chapter.

1.  Insofar as design and operation of activated sludge processes are
concerned, the BOD test should no longer be used as the parameter of
pollutional potential or as a measure of purification efficiency.  For
design and for daily assessment of operation, the ACOD measurement
should replace the BOD test (Chapter II).

2.  Treatability studies should precede design and a stoichiometric
balance based on measurements of ACOD, oxygen uptake, and ACOD of the
biological solids is recommended for use in these studies (Chapter III).

3.  Kinetic equations for microbial growth may be employed with heter-
ogeneous populations, but no precise  values for the biological con-
stants can be expected.  The values of these constants should be deter-
mined in treatability studies.  In general, the following ranges seem
reasonable:  ymax, 0.4 to 0.6 hr"1; Ks, 50 to 125 mg/1; Y, 0.4 to 0.6
(Chapter IV).

4.  A kinetic model was developed specifically for use in designing
activated sludge processes.  The model employs recycle solids concen-
tration, XR, as a system constant, and it is designed to maximize puri-
fication efficiency by maintaining a "steady state" as nearly as is
possible with a heterogeneous microbial population and with a waste
which may vary quantitatively and qualitatively.  The model requires use
of an aerated sludge consistency tank and, for wastes subject to con-
siderable variability, a surge basin is recommended (Chapter V).

5.  Allowable limits for several types of shocks commonly encountered

in waste treatment were suggested.   In general,  it was recommended
that for aeration periods of 6 to 8 hours,  it should be attempted
through both design and operation to prevent fluctuations in flow rate
of greater than 100% of the design flow,  fluctuations in concentration
of metabolizable organic material in the  waste of greater than 100%,
fluctuations in pH of greater than one unit and  rapid changes in tem-
perature of greater than IQOC (Chapter VI).

6.  The standard level  of nitrogen supplementation based on a BOD:N
ratio of 20:1 should be reconsidered in design of treatment facili-
ties for wastes deficient in nitrogen. To  avoid leakage of nitrogen
in the effluent, treatability studies should determine the optimum com-
bination of detention time, purification  efficiency, nitrogen supple-
mentation and nitrogen concentration in the effluent, since all  of
these factors are related.   For treatment of wastes containing little
or no nitrogen, a new modification of the activated sludge process was
developed, and it is recommended that these wastes be treated by con-
tinuous oxidative assimilation (Chapter VII).

7.  The extended aeration process was shown to be theoretically  sound
and a modification designed to allow engineering control  of solids
levels by chemical  hydrolysis of a  portion  of the sludge was developed
(Chapter VIII).

8.  Two possible flow sheets for the activated sludge process, incor-
porating the kinetic model  discussed in Chapter  V and the hydrolytic
assist to total oxidation described in Chapter VIII, were presented.
These flow sheets summarize the biological  principles and research
findings presented in the report (Chapter IX).


One of the inevitable recommendations resulting from the past year of
data analysis and the past decade of intensive research on activated
sludge processes  is that those designing activated sludge processes
should pay greater attention to the biochemical and biological prin-
ciples responsible for the behavior, i.e., the success or the failure,
of the process.  It has become apparent to us, and to some other
researchers as well, that the potential for biological processing of
waste organic substrates has barely been tapped, and that such proc-
esses will, as time goes by, become increasingly important for pollu-
tion control and possibly in the future for direct recycle of organic
materials.  Also, in the ultimate sense, the rivers, reservoirs,
lakes, and oceans which environmental engineers and scientists must
learn to control are indeed biological reactors.  Thus, no matter what
alternatives to biological treatment we may seek for the immediate
future (and there are some depending upon the nature of the organic
matter in a waste water) we eventually must face up to the need for
biological engineering since the environment of which we may claim to
be custodians is a biological one.  And thus, as the reader scruti-
nizes this report and the principles, process modifications and
approaches to design outlined herein, he may also employ the report
in conjunction with, or use it as a springboard to, the basic micro-
bial and biochemical literature.  It is also recommended that the
report be used as an aid in assessing the research literature in the
environmental field.

Concerning the technological biochemical concepts and process modi-
fications presented in the report and briefly stated in the preceding
conclusions, it is recommended that the next step include larger
scale developmental research relative to the design model herein sug-
gested (Chapter V), as well as pilot plant research or field-scale
testing of the continuous oxidative assimilation process (Chapter
VII).  Also, developmental research on the hydrolytically assisted
extended aeration process (Chapter VIII), and pilot plant research on
the total process flow sheets (Chapter IX) would seem a fruitful
extension of the research.  Also it is felt that the continued anal-
ysis of the experimental data from which tentative rough guideline
limits for various shock loadings were suggested (Chapter VI) can
considerably refine these guidelines.

In addition to the developmental research on principles and processes
recommended in this report and further conceptual analysis of the
shock load investigations we have already accomplished, there is, in
general, a great need for definitive, fundamental biological research
on the biomechanics of the bio-mass, i.e., heterogeneous or natural
microbial populations, and on the ecology of heterogeneous microbial
systems.   Paradoxically, these are areas which have been shunned by
most basic biological scientists as being too applied, and by most

engineers as being too basic and beyond the scope of the engineer's
background knowledge.   Clearly, if we are to make successful inroads
toward technological  control and protection of the environment, this
situation must be corrected.  The recommendation to correct the sit-
uation by performing  the needed research is made to those readers
who have a proclaimed  professional  dedication to pollution control,
particularly to those  now beginning their training for this field
and who will be active in it well into the 21st century.


The Perspective

The activated sludge process has been increasingly employed for secondary
treatment of municipal and industrial wastes containing significant
amounts of organic material.  The process has many "modifications," and
various flow diagrams and hydraulic regimes (e.g., completely mixed as
compared to plug flow), but its chief distinguishing characteristic is
that the heterogeneous microbial populations (biomass) of which the
sludge is largely composed, are grown in fluidized culture with provi-
sion for maintenance of the organisms in suspension by turbulent mixing
and for introduction into solution of sufficient oxygen for the respira-
tory requirements of the microorganisms.  In these characteristics the
process is distinct from other secondary treatment processes, e.g.,
fixed bed processes such as trickling filters.

As with other secondary treatment processes, the objective of the acti-
vated sludge process is the removal of organic compounds from the waste
water.  The term "secondary treatment" is used here simply because it is
one which has a specific significance to engineers, and it implies the
traditional employment of activated sludge as a "second line of defense"
regarding removal of organic matter from waste waters; primary treatment
signifies a first line of defense embodying removal of initially settle-
able organic matter in raw waste.  The term "tertiary treatment," as
herein employed, is used to signify a third line of defense for removal
of organic matter not removed by secondary treatment.  The terms "pri-
mary," "secondary," and "tertiary" are rather meaningless unless one
specifies that they are being employed to describe processes for removal
of biologically usable organic substrates.  As employed herein, they
apply only to processes for removing organic waste materials which exert
a "demand" on the dissolved oxygen resource in natural waters into which
waste may be discharged.  Even within this specified boundary, one must
impose some qualifications or exceptions as to the subject matter which
will be herein discussed, since it will be readily recalled that, as it
was originally introduced, the so-called extended aeration (total oxida-
tion) process not only combined secondary treatment with the sludge
disposal process, but also combined primary and secondary treatments as
well.  Also, it should be recognized that fluidized biological processes
(as well as fixed beds) can be employed to remove inorganic pollutants
such as ammonia, which can cause a drain on the dissolved oxygen
resources in the receiving stream, and that the process may also remove
from waste water some organic matter which does not exert significant
biochemical oxygen demand; for example, cellulose particles, which are
not readily metabolized, may become entrapped in the sludge, etc.  At
the outset it should also be recognized that the biological processes
are by no means the only ones which can be considered for removal of

biologically available organic compounds from waste waters.   Chemical
coagulation has been employed for years to remove colloidal  organic
material, and in sequential  combination with other processes, e.g.,
adsorption on activated carbon, many types of soluble organic material
can be removed as well.  Such a combination of processes may become a
useful alternative to biological treatment, especially for relatively
low strength municipal waste waters.

The point to be emphasized here is that there is no one universally
recommendable group of unit processes for accomplishment of removal of
organic substrates.  Water reclamation plants of the future will not be
describable by any sort of "standard" flow diagrams such as have been
used in the past, but by groups of unit processes joined in such a man-
ner as to accomplish rejuvenation of a specific waste water in accord-
ance with the purity requirements of a specific environmental situation.
Thus, while much stricter standards of effluent quality will undoubtedly
evolve, the required effluent quality will be attained by less_"standard"
arrangements of unit processes to treat the waste waters.  It is inno-
vativeness rather than standardization which is needed, and it is inno-
vativeness which is the essence of engineering.

While it is highly desirable to seek alternatives to such biological
processes as the activated sludge process, it may be an even more fruit-
ful endeavor to seek innovativeness within the biological process.
It does seem fair to say that, in many situations, if biological treat-
ment processes could be designed and operated so as to ensure reliable
delivery of the degree of treatment for which they are "designed," much
of the need for treatment beyond the secondary stage might be obviated.
Some engineers, scientists, and technologists in the area of pollution
control would welcome a replacement for biological treatment as  a means
of removing organic substrates from used waters.  If the reasons for
seeking such replacement involve frustration in attempts to understand
the process, or the feeling that it is, perhaps, a process too compli-
cated to control, these reasons are not at all soundly based.  It is
important for us to recognize that engineers will, in fact,  be forced to
gain a better understanding of the kinetics and mechanism of biological
treatment systems because, regardless of how we decide to prepare water
for re-use, or regardless of who makes that decision, it must be remem-
bered that our natural receiving bodies are "in-stream" biological
reactors.  Thus, if pollution control engineers are to play a leading
role in overall management of water quality control systems, they must
lead in understanding the systems, since understanding is the most
essential ingredient for technological leadership.

The'Technological Gap"

The first aim of researchers in any profession is to gain insight (under-
standing) regarding a natural process or phenomenon.  In engineering
practice, the ultimate aim is to employ such understanding to accomplish
a practical goal, to satisfy a social need.  When the distance between

understanding and practice becomes large, there is said to be a techno-
logical gap, and it is said that there is currently such a gap between
research and practice in the water pollution control field, especially
with regard to activated sludge.  Such gaps are natural; they always
exist, and they are helpful.  They are caused as often by the fact that
practice gets out ahead of understanding as by the fact that understand-
ing gets out ahead of practice.  Most seem to attribute the cause (blame
or credit) to the somewhat banal belief that engineers are simply
appliers of bodies of known scientific information.  In many regards,
this is surely not always the case.  For example, often because of a
pressing need of society, the "appliers" must devise processes and
employ them prior to the unveiling of the basic facts which permit full
understanding of the processes.  Such was the case with the activated
sludge process.  The trouble arises when the apparent success of a proc-
ess engenders a lassitude with regard to seeking understanding and when
theory or hypothesis (a product of the mind), often offered without the
benefit of penetrating experimental proof, becomes technological dogma
which is often overly simplified half-truth.

Thus, while causes for the technological gap cannot be laid on any par-
ticular doorstep, its existence is well recognized.  It should be equal-
ly well recognized that researchers and practitioners have a joint
responsibility to lessen (but not to completely close) the gap.   Fortun-
ately, its complete closure seems impossible to attain, and this is a
desirable situation because it is the gap which provides the driving
force for progress in the profession.  The pressure for progress will
come sometimes from the practicing and sometimes from the researching
segment of the profession.  While both groups share the responsibility
for lessening the gap, perhaps the greatest weight of responsibility
resides with researchers.  They should not be content with presenting
their results, arguments, conclusions, and recommendations for the scru-
tiny and criticism of other researchers in their own and allied fields,
but should from time to time communicate some of the things they think
they understand (and can defend with sound practical data) to the wider
and ultimately more vital technological audience which includes both
researchers and practitioners, the total investigational level of pro-
fessional activity.  It is to this aim which the present communication
is dedicated.

The Nature of the Report

In the following pages we will attempt to bring into focus some major
biological concepts and insights applicable to design and operation of
activated sludge processes and to further understanding of biological
treatment processes in general.  This report is not intended to be a
manual of practice for functional design and/or operation of activated
sludge treatment plants.  The aim is to transfer some of the concepts
developed through laboratory research investigations to practicing engi-
neers.  Whether the concepts become accepted and incorporated into tech-
nological practice is, in large measure, dependent upon how well we

communicate and defend them, and how well  they are received by those
who scrutinize them.   We present them for  the scrutiny of fellow pro-
fessionals (both researchers and practitioners) in the interest of
advancing, but surely not of writing "finis"  to, development of con-
cepts in a field of professional activity  which is only now emerging.
The ideas and concepts herein presented have  been developed with the
aid of a decade of intensive experimental  activity and observation
relative to activated sludge processes in  our laboratories.  The pri-
mary purpose for undertaking the present research project, which is
culminating with the  preparation of the following report, was to draw
together, correlate and analyze information obtained  from various
individual, but related, research projects which we have conducted
over the past ten years, in an effort to derive practical  unifying
biological concepts.   We shall not attempt to cover all  aspects of
the work in this short report, but we shall draw upon all  of this
information in presenting and clarifying "Biological  Concepts for
Design and Operation  of the Activated Sludge  Process."



The Concept of Biochemical Oxygen Demand

The most widely employed measurement of effluent purification or effi-
ciency of activated sludge processes is the amount of biochemical oxygen
demanding material (BOD) which has been removed by the process.  Bio-
chemical oxygen demand is also employed in assessing the "pollutional
potential" of a raw or treated waste before discharge to a receiving
stream.  The assets and liabilities of employment of this parameter of
measurement have been argued for years by engineers, but the fact
remains that the concept of BOD is valid and usable, and certainly it
is used (and misused) in all segments of the water pollution control
field.  Today it is nearly impossible and surely unwise to neglect con-
sideration of the idea of biochemical oxygen demand.  It seems far bet-
ter for pollution control professionals to clarify understanding of BOD
to help ensure its enlightened application, rather than to join the
chorus decrying use of the concept because of the inadequacy of the BOD
test.  It is necessary to make a distinction between the concept and
the test.  While we can defend and recommend the concept, we shall not
defend the test for all of its present applications, but will recommend
a more satisfactory measurement for assessing efficiency of treatment
by the activated sludge process.

Through many years of expansion of population and industry, study and
observation have shown that most, if not all, of the many natural and
technological uses to which the water resource is put are seriously
impaired or totally negated if the dissolved oxygen (DO) in surface
waters is seriously depleted.  It has also become widely known that the
prime reason for depletion of DO is its utilization in aerobic meta-
bolic processes by microorganisms feeding upon the organic food (i.e.,
carbon source or substrate) contained in municipal wastes and various
industrial wastes discharged to the stream.  Thus a conceptual principle
has been established which recommends that the "pollutional load" should
be assessed, not by measuring its amount (i.e., amount of metabolizable
organic matter in the waste) but by estimating the magnitude of its
effect (i.e., the amount of oxygen that will be used because of the
presence of the organic matter).  Such a principle is defensible in an
engineering sense, because it goes directly to the heart of the matter,
potential  depletion of the DO resource in the receiving stream, and
because it measures a colligative effect of that Organic matter in a
waste water which is readily available as organic carbon source to
microorganisms without requiring determination of either the total
amount or types of that organic matter.

On this basis, the use of the standard BOD test for estimating the
"pdllutional  potential" of a waste, either raw or treated, which is to

be discharged to a receiving stream can be defended to some extent.
However, even for this application of the concept, other techniques
may be preferable because of the time required for standard BOD incu-
bation and because of inaccuracies introduced by the dilution tech-
nique itself (1).

The use of the BOD test in measuring purification efficiency cannot be
defended, and it is primarily this application for which we shall
recommend a different approach.   It should be remembered that the pur-
pose of biological treatment is  removal of the biologically available
organic matter (carbon source) and that any means of measuring the
amount removed during treatment  can be employed.  The choice of a
method for this measurement should be based upon two criteria: (1) the
method used should actually measure the percentage of metabolizable
(biochemical oxygen-demanding) organic matter which has been removed
by treatment, (2) the method used should be sufficiently rapid so that
results may be available when needed, i.e., while the opportunity
exists to exert operational control over a malfunction leading to
decreased purification efficiency.  The standard 5-day BOD dilution
bottle technique meets neither of these requirements (2).

Exertion of Biochemical  Oxygen Demand

As stated above, the concept, or principle, of biochemical oxygen demand
is a vitally important one and much research effort in our laboratories
and elsewhere has been devoted to study of the details of BOD exertion.
An examination of the sequence of events which make up the total
course of BOD exertion is pertinent to a discussion of the proper appli-
cation of the concept.

Figure 1 is a generalized plot based on data obtained in many experi-
ments of various types in our laboratory.   This figure shows the  course
of microbial growth (increase in biological solids, X) which would be
observed if one placed a small inoculum (seed) of microorganisms  in a
vessel in the presence of a biologically available source of carbon
(substrate, S) to which the microorganisms were acclimated.   The  "ves-
sel" might be a BOD bottle, a batch reactor, a reach of stream, or a
pond or lake.  The figure also shows a plot of accumulated oxygen
uptake (BOD, y), and a plot of the total amount of organic matter
remaining in solution measured by a general chemical test involving the
digestion of organic matter to C02 and HpO in the presence of potassium
dichromate under acid conditions, i.e., the chemical oxygen demand (COD)
test.  The total amount of substrate removed is A COD.  This will be
discussed in more detail below.

The type of data diagrammed here can be generated experimentally  by
plotting results of analyses of  samples taken at various times and
analyzed for biological  solids,  X (using the membrane filter technique),
and for COD, S, determined on the filtrate.  The course of accumulated


                                         2 UPTAKE, y
 Figure  1.  Generalized plot of substrate concentration, biological
 solids  concentration, and oxygen utilization during  exertion of
 biochemical oxygen demand.   Circles mark inflection points.

0? uptake, BOD exertion or y, can be measured in a number of ways,
efg., by a manometric technique, or if the system is very dilute, by
DO depletion in BOD bottles.  Thus we have three very important para-
meters :

     1.   The amount of biological solids, X, existing in the
         system at any time.

     2.   The amount of organic substrate (COD)  in the system
         at any time.

     3.   The amount of oxygen used (y), the BOD expressed at any

As an aid to analysis, we will assume that the  system contains no nitri-
fying bacteria (or any other aerobic autotrophs), and therefore all  of
the oxygen uptake is due to the respiration of  aerobic microorganisms
which require organic carbon (aerobic organotrophs); thus the oxygen
uptake represents only carbonaceous BOD.   The experiment is terminated
at the point where 02 uptake may be continuing  very slowly but has for
all practical purposes stopped, and therefore all of the carbonaceous
BOD has  been expressed.  This point along the "BOD curve" has been
known for some time as the so-called ultimate BOD, and is designated by
the familiar symbol, L .

As shown in the figure, Q£ uptake (BOD exertion) goes on long after the
organic  matter (COD) has been removed.  It is apparent from the plot of
biological solids, X, that Op uptake, after the substrate removal phase
has ended, goes on at the expense of the biological  solids which were
produced during that phase.  The figure also shows a decided break or
pause in the curve at the termination of substrate removal.  This pause
or "plateau" is not always manifested; the Q£ uptake sometimes continues
on a smooth ever-declining curve (see dotted lines).  Also, it has been
demonstrated that "plateaus" exist which do not coincide with the ter-
mination of the substrate removal phase (3). However, in most cases
where the plateau is observed, it serves as a "marker" for the termina-
tion of  the substrate removal phase, and its significance as such was
first recognized by A. W. Busch (4).  Subsequent research by Wilson and
Harrison (5), and Gaudy and his co-workers (6-10) has helped to clarify
understanding of BOD exertion.  Much of this work has been brought
together and analyzed in recent literature (2,  10, 11), and its inclu-
sion here would add unnecessary length to this  report.

Theoretical COD and BOD

In the research literature one encounters the terms "theoretical COD"
and "theoretical  BOD."  These are useful  terms, but have been subject
to considerable misinterpretation and misuse.  The term "theoretical
COD" is  synonymous with "calculated COD."  It represents the


stoichiometric amount of oxygen which would be required to oxidize
(chemically) all of the organic matter to carbon dioxide and water.
This value can be calculated if one knows which organic compound(s)
are being oxidized or if one knows or can estimate the composite empir-
ical formula for this organic matter, and if the concentrations are
known.  For example, if one wished to know the theoretical COD of a
sample which contained 200 mg/1 of sucrose (table sugar), it could be
easily calculated from the following balanced equation:

      C12H22°11 + 12 °2 	>~12 C02 + 1] H2°                 (1)
        (342)     (384)

The theoretical COD is 200 (384/342) = 224 mg/1.

Such calculated values are useful as checks against the values obtained
by the COD test, and they can be important for some research investiga-
tions.  The term is not a useful one for the engineer dealing solely
with whole waste problems, however, since the determination of an
empirical formula for a whole waste is indeed an exercise in futility
and a wasteful expenditure of investigational efforts.   ;

The term "theoretical BOD" is sometimes employed to signify the total
amount of oxygen which would be required to oxidize (biologically) to
C02 and F^O all of the organic substrate which is available to the
organisms as food material if it were possible for organisms to do this.
In accordance with this definition and with reference to Figure 1^ the
theoretical BOD (i.e., theoretical L ) would be equal to A.COD.  For
this to be so would require total biological  oxidation of the organic
substrate in the waste.  Oddly enough, some who have used this defini-
tion for theoretical BOD would also argue that it is theoretically
impossible for total oxidation to occur.. Total oxidation of the orig-
inal organic matter in the waste>would require that all of the biolog-
ical solids produced during the substrate removal phase (Figure 1) were
autodigested during the subsequent "endogenous" phase; i.e., the bio-
logical solids returned to the initial concentration.  Such an occur-
rence is not a theoretical impossibility, as will be discussed in a
later chapter, but the chance of its occurrence in any ecosystem in a
short-term experiment (e.g., incubation of BOD's) is not very high.
The degree of autodigestion of the biological solids can be expected to
vary; the relative decrease in the solids level may at times be greater
and at other times be less than that shown in Figure 1.  The term
"theoretical BOD," like "theoretical COD," is not a particularly useful
one to incorporate into the practicing jargon of the field; however,
it is important to know what these terms mean and what they imply when
one analyzes research information (a responsibility shared equally by
practitioners and researchers).

ACOD as a Parameter of Purification

The curves shown in Figure 1  could also be considered as representing
the course of waste purification rather than BOD exertion, i.e., the
emphasis may be placed upon substrate removal  rather than 02 uptake.
If the waste were being purified in an activated sludge aerator, the
initial solids would, of course, be much higher and the kinetics of
substrate removal and solids  accumulation would be affected by this dif-
ference, as will be discussed in a later chapter.   However, the same
reactions would be involved;  carbon source would be removed, biological
solids concentration would increase, and oxygen would be taken up.

In an activated sludge system of any type other than extended aeration,
however, only the reactions at the left of Figure  1 are important.  In
the usual activated sludge aerator, the reactions  occurring during the
"endogenous" phase (right-hand portion of Figure 1) do not occur or, if
the system approaches plug flow, may occur only briefly toward the exit
end of the aerator, because the solids are separated in the clarifier.
Depending upon the efficiency of flocculation  and  settling, the effluent
contains the non-metabolized  organic material  (COD) which remains in the
water, but little of the biochemical products  of purification (biolog-
ical solids).  At any rate, this is the aim:  retention of the sludge
at the treatment plant.

Thus, at the end of the substrate removal phase the plant has accom-
plished its primary purpose,  removal of organic carbon source which
would have caused an oxygen uptake (BOD exertion)  in the receiving body
if this organic carbon source had not been removed from the waste.  The
amount of organic matter removed in the aerator is the difference between
initial (influent) and terminal (effluent) COD values:

      CODi - CODg -  ACOD '                                         (2)

The term " ACOD" may be more  generally defined as  the amount of COD
removed at any time, i.e., the difference between  the COD present at the
time of measurement and the COD initially present.  When the amount
remaining cannot be further reduced biologically,   ACOD is a measure of
the amount of organic matter  in the waste sample which was available to
the microorganisms.  This is  precisely the interpretation which many
people place on BOD but, as can be seen in Figure  1, the BOD of the
waste sample, L0, is not equal to  ACOD.  It is loosely related to it,
since both result from the presence of the same amount of organic mat-
ter, but the relationship can vary because the measurement of LQ
requires that one consider not just removal  of the carbon source, but
the subsequent fate of the biomass which was produced from the organic
carbon source.  A significant portion of L0 is exerted after the orig-
inal carbon source of the waste has served as  microbial food, and the

extent of this oxygen uptake is dependent upon the degree of aerobic
digestion of the biological solids produced.  The degree of digestion
is dependent upon many factors, some of which will be discussed in a
later chapter.  For now it is sufficient to state that the degree of
aerobic digestion will vary, and because of this, L  can be expected
to be more variable than  ACOD.

For applications in which BOD is measured (either as BOD5 or L0) and
is interpreted as a measure of usable organic carbon source, it seems
reasonable to use a more direct, rapid, and reliable test for this
parameter, i.e.,  ACOD.  This recommended concept is a simple and
straightforward one, but can lead to some confusion unless it is
clearly delineated that we are not comparing the COD test and the BOD
test.  We are concerned here with  ACOD, and the COD test is to  ACOD
what the DO test is to BOD.  It should also be emphasized that inso-
far as testing for the amount of organic carbon source available to
microorganisms is concerned, the COD remaining after the growth
period (i.e., CODe) is not really significant from the standpoint of
assessing the amount of biochemical oxygen-demanding material present.
The determination of  ACOD is a measurement of only that portion of
the COD of the waste which is available as biological substrate for
acclimated microorganisms (or which may be otherwise removed due to
the presence of the biomass, e.g., the small amount of colloidal COD
which could be adsorbed on the cell surfaces, etc.).  The residual
COD, if sufficient aeration time has been allowed, is composed of
material not utilizable by a microbial population.  Thus one can employ
ACOD to assess the amount of biochemical oxygen-demanding organic mat-
ter present, regardless of the presence of some non-biodegradable COD
in the waste.  For example, a waste may contain a significant amount
of lignin, which is subject to chemical attack in the COD test but is
not attacked to any extent biologically.  Thus COD  would be high, but
would not represent a serious stream liability with regard to deple-
tion of dissolved oxygen.  It may be a detriment to the stream because
of color, but not because it will be used by aerobic microorganisms at
an appreciable rate.  It is important to re-emphasize here that in the
biological treatment process, the concern is primarily with removal of
the carbon source in the waste that is used as "food" for the growth
of microorganisms which are in turn removed by sedimentation (i.e., up
to the time when the substrate removal phase is ended), and that  ACOD
measures this purification.  On the other hand, in the BOD bottle,
oxygen uptake proceeds well beyond this phase; in fact, if the curves
of Figure 1 are applied to the BOD bottle, the substrate removal phase
ends in one or two days.  While it is a simple matter to standardize
the time of incubation of BOD's, e.g., BOD5, the actual rate of 02
uptake is not subject to such arbitrary standardization, and use of
the 'standard 5-day BOD test" as a measure of plant efficiency is there-
fore inferior to the  ACOD method.

Parameters for Design and Operation

The use of  ACOD in design and operation of activated sludge processes
is rather straightforward.  In the experimental  investigation which
should precede design calculations, one can acclimate a heterogeneous
population of microorganisms (e.g., from a sewage seed) by repetitive
feeding cycles of the organic waste whose treatability is being inves-
tigated.  During this time one can determine the extent to which the
organic matter in the waste serves as microbial  food (i.e., COD-j -
CODe =  ACOD).  Through repetitive runs a usable base level for CODe
can be established, and the fraction of the total organic matter which
is amenable to biological attack can be determined.   This fraction,
ACOD/COD-j, can be employed as a useful  parameter, efficiency of COD
removal.  This term is not equivalent to efficiency  of purification
since, depending upon the waste water under consideration, CODp may
include some organic matter which does not serve as  usable food mater-
ial for microorganisms.  Efficiency of treatment, based upon quality
of the effluent in terms of its effect on the receiving stream, would
be much higher than efficiency of COD removal  for such a waste.

In day-to-day operation of the process, the efficiency of COD removal
can be used as a guide to assessing performance.  Also, the CODe is an
important operational parameter which should be  continually compared
to the base line CODg established in the treatability study which was
made in the design stage.  This routine check can help detect possible
changes in the nature of the waste between design and operational  per-
iods and throughout the life of the plant.

The use of  ACOD as the primary operational parameter should do much to
enhance intelligent and careful daily control  of the process, since
information on plant efficiency is available immediately rather than 5
days later as it would be if BOD^ is used.  If CODe  remains relatively
constant, there is little possibility that the actual treatment effi-
ciency, i.e., percent removal of metabolizable organic matter, has
changed significantly.  However, the COD test run on the effluent does
not distinguish between metabolizable and non-metabolizable organic
matter remaining in the effluent, and it is therefore absolutely neces-
sary that frequent checks on the metabolizable organic content of the
effluent be made.  A hypothetical example may illustrate a possible
operational problem which would not be detected  by use of  ACOD as the
sole criterion of efficiency.  A waste containing 2000 mg/1 COD, of
which 1000 mg/1 was not biologically metabolizable,  would be completely
"purified," i.e., would exhibit no further oxygen demand on discharge
if CODe were 1000 mg/1.  A  ACOD of 1000 mg/1, or a  50% efficiency of
COD removal, would be established as the baseline for 100% removal of
biochemical oxygen-demanding material.   However, a subsequent change in
the character of the waste, reducing the content of  non-metabolizable
material and proportionately increasing the metabolizable organics,
e.g., 2000 mg/1 total COD including only 500 mg/1 non-metabolizable COD,
might overload the plant.  If the same total COD removal were continued,


i.e.,  ACOD = 1000 mg/1, the COD of the effluent would remain the same
but the plant would now be discharging 500 mg/1 of utilizable, BOD-
exerting organic matter, and the actual purification efficiency would
be 1000/1500 = 67%.  Such a series of coincidental alterations, yield-
ing exactly the same results for  ACOD and CODp is, of course,
extremely unlikely, but such a possibility emphasizes the necessity
for biological testing of the effluent.  For this purpose, the stand-
ard BOD test could serve to detect significant changes in purification
efficiency.  However, from the standpoint of rapidity of measurement
and of obtaining a more credible estimate of the oxygen demand which
will be exerted by the treated effluent in the receiving stream, meas-
urement of oxygen uptake in an open, stirred jar reactor is preferable
(1, 2).  In addition, the plant operating engineer can and should
periodically determine the  ACOD of a sample of the waste; i.e., a
sample of aeration tank influent.  This can be done in an aerated
batch laboratory reactor using settled activated sludge from the plant
as a source of initial biological solids.  Aeration of this reaction
liquor should be continued until the COD of filtered samples becomes
essentially constant.  Comparison of the  ACOD obtained in the labora-
tory experiment with the current  ACOD through the activated sludge
tank allows a direct assessment of the actual purification being
achieved in the treatment plant aeration tank.  A record of the values
of  ACOD and CODe obtained in these periodic laboratory experiments and
in similar experiments which were made during the treatability studies
preceding design will allow detection of significant changes in the
nature of the waste or in the operational efficiency of the plant.

Design engineers and plant operating engineers can work more as a team
in devising changes and modifications to keep the plant "alive" if
close operational supervision is initiated and maintained on a daily
basis.  Such a view may seem somewhat naive to some design engineers
because of past and present experiences both on the design and opera-
tional sides of the coin.  However, when one addresses himself to the
future, it is becoming more and more apparent that design and opera-
tional technologies must merge.  From the standpoint of prolonging his
career, the rapidity with which pollution control  plants are being
designed today should be enough proof to convince any consulting engi-
neer that his consulting capacity should include operational consider-
ations as well as design.

Additional  Information

We have in this chapter attempted to present a useful conceptual prin-
ciple for measurement of purification.  Much productive thinking and
experimental  investigation which amplify this concept have been accom-
plished by various researchers.  Some articles in the research liter-
ature which will  be useful for further study of the information pre-
sented here are listed in the reference section.  Symons, et al.  (12)
described the use of  ACOD as a means of measuring the efficiency of
treatment,  and also outlined some procedures for making treatability


 studies.   The  concept  of  ACOD was  also described  in  an  article  by
 Hiser and  Busch  (13).  More  recently,  the  concept  of  ACOD  as well  as
 overall consideration  of  biochemical oxygen demand as applied to the
 treatment  plant  and  the DO profile  in  receiving  streams  have been
 treated extensively  by Gaudy (2).   Exertion of BOD and methods for  the
 estimation of  assimilating capacity of the receiving  stream have been
 discussed  by  Isaacs  and Gaudy  (14,  15).  Jennelle  and Gaudy have
 studied the effect of  dilution of the  sample upon  exertion  of BOD,  and
 have recommended alternative procedures for measurement  of  BOD (1).

 Summary and Conclusions

 As. a parameter for measuring organic loading or  the degree  of purifi-
 cation in  treatability studies for  the design of biological treatment
 facilities, ACOD, i.e.,  CODi - CODe,  represents the most straightfor-
 ward estimate  of the amount  of biologically available organic matter.
 Since the  BOD  test has been  used to estimate what  ACOD  actually meas-
 ures, use  of  the standard BOD test  as  a design and operational para-
 meter does not seem warranted.  Furthermore, since L0 (ultimate  BOD)
 can only approach ACOD as an upper limit, the  ACOD parameter gives a
 more conservative estimate of the ultimate biochemical oxygen demand of
 the waste  sample.  For daily operation,  ACOD is recommended as  the
 primary Operational  parameter for assessing plant  efficiency.  Periodic
vdeterminations of ACOD of the waste in a laboratory reactor allow
 detection  of changes in the  nature  of  the waste or in operational
 efficiency of  the plant.


"Equation" for Purification

the, stoichiometry of purification is often represented by "pseudo-
chemical" equations in which the organic matter in the waste is shown as
the prime reactant being converted to another form of organic matter
(microorganisms) plus C02 and [-LO.  Most engineers are familiar with
this representation:

   Omanir      Microorganisms
   matter  + °2	3*" Microorganisms + C02 + H20         (3)

The above equation is a gross simplification of a "chemical" equation
(and of microbial growth); it is only qualitative, since no numerical
coefficients are included, and significant elements have been omitted.
For example, sources of nitrogen and phosphorus and small  amounts of
many other elements are needed on the left side of the equation, because
they are part of the microorganisms produced and are needed both in
reactions which form new cellular components and in the reactions by
which the waste is degraded.  The organic matter in the waste serves pri-
marily as the carbon source, but depending upon the waste in question,  it
may also contain some nitrogen and phosphorus which are metabolically
available to the cells.  The microorganisms shown above the arrow are the
biocatalysts required to make the reaction proceed.

The reaction is irreversible, although the microorganisms produced are
themselves organic matter and could conceivably be used as a source of
organic carbon for other microorganisms.  Thus, the product can be
recycled through the forward reaction but the process cannot be reversed.
Reversal of the reaction can be brought about with respect to C02 and
HpO as reactants and organic matter (e.g., algae) and 02 as products
tnrough the process of photosynthesis (photo-autotrophic metabolism).
This is essentially a reversal of the purpose of biological treatment,
and it is now being realized that because of this fact the problems one
causes are perhaps equal to or worse than the problems one solves by
attempting to rely on photosynthetic oxygen production in biological
waste water treatment (e.g., quiescent oxidation ponds).  The "swapoff"
in organic matter actually conserves organic carbon rather than dissi-
pating it.  Conservation of organic matter from waste water sources by
such direct recycle may become important as a source of food supply for
human beings and lower animals in the long range, but in the intermediate
future, at any rate, all modes of disposal of organic carbon in wastes
involve processes which in the main lead ultimately to C02 and H20, which
are recycled eventually through the carbon and the hydrologic cycles.


Returning again to the irreversible equation 3, it could be made more
mathematically precise if the elements and their combining weights
(empirical formulas) for "organic matter" and "microorganisms" (or
activated sludge) were known.  The chemical  composition of the organic
matter in the sample is seldom known to such an extent that it can be
reduced to a representative chemical formula.  There are various empir-
ical formulas which describe the approximate elemental composition of
bacterial cells with regard to C, H, 0, and  N,  and these have proven of
some use to researchers in analyzing data from  investigations wherein
readily metabolizable specific compounds of  known chemical composition
comprised the organic matter being treated.   Such a simple stoichiometric
chemical equation for purification does represent a simplifying prin-
ciple; however, it is not easily applied, as a  chemical  equation, to
practical concepts for design of treatment processes.   The point to be
kept in mind is that the equation represents (as does  any other chemical
equation) a mass and/or energy balance, and  this principle, as we shall
see shortly, can be usefully employed to provide quantitative information.

Partition of Substrate Between Respiration and  Synthesis

Another representation of purification of wastes is often employed and is
perhaps more familiar to water pollution control  engineers than is the
equation given above.  The diagram shown below  actually  represents the
same reactions shown in equation 3, but shows separately the two general
types of reactions in which the organic matter  participates.   That is,
it represents the partition of the waste into (1) energy-yielding, C^-
consuming reactions (respiration), and (2) energy-requiring biosynthetic
reactions (synthesis of new cells).
      Organic matter
                                         utilization  (respiration)
                                      Sludge  production  (synthesis)
The portion of the organic  matter which  is  used  for  respiration  is  oxi-
dized to C02 and HpO;  i.e., this portion of the  organic  matter accounts
for essentially all  of the  CL  shown  on the  left-hand side  of  equation 3
as a reactant and the  C02 ana  hLO shown  on  the right-hand  side as  prod-
ucts.  Thus, this diagram might be more  easily reconciled  with equation
3 in the form shown  below.
      Organic  matter
                                          +  HO  (respiration)
                                      New  cells  (synthesis)

The amount of Op which would be required for complete oxidation of the
organic material varies with the nature of the compounds present.  One
quantitative measure of the relative amounts of oxygen required is the
respiratory quotient (RQ) which is based upon the relative amounts of
C02 (totally oxidized carbon) which can be produced from different
classes of organic compounds by use of the same amount of 0?.  The res-
.piratory quotient, moles C02 produced per mole Q£ used, is 1.0 for car-
bohydrates, 0.8 for proteins, and 0.7 for fats.  Thus, for a waste con-
taining primarily carbohydrates, which may be represented as C^O, the
balanced equation for oxidation (RQ =? 1.0) can be written as

     CH0 + 0  - 5»-C0  + H0 (+ xATP)                       (4)
For proteins or fats, a proportionally greater amount of $2 would be
required for that fraction which was oxidized.

Another term, ATP (adenosine tri phosphate) has been introduced on the
right-hand side of equation 4 to represent the amount of chemical energy
which was trapped from that released during the oxidation.  There are
other compounds which organisms can use to trap chemical energy, but ATP
is the major one, and the others can be expressed in equivalents of ATP,
in any event.  It is this trapped chemical energy which now enables the
microorganisms to use some of the organic matter (Ch^O, as one example)
to make the cellular components needed for new cells:

     CHJD + ATP - s—New microorganisms                      (5)

Thus, the diagram and the equation may be combined:

                                C00 + H00 + ATP (respiration)
                                  2    2                           (6)
                                 New cells (synthesis)

The number of moles of 02 used is approximately equal to the number of
moles of C02 produced (for carbohydrates) and the 02 which is used is
involved in the complex process of trapping some of the energy released
during the oxidation.

Energy Coupling Through ATP

We cannot, in this brief presentation, attempt to explain in simplified
terms the mechanics of the processes of biological energy generation and
utilization, but a few facts concerning the role of ATP should be men-
tioned briefly.  The precursor of ATP is ADP  (adenosine diphosphate).
The ATP is thus formed by adding inorganic phosphate, iP, to ADP.


      ADP + IP 	5»-ATP                                      (7)

When the free energy which is contained in ATP is used to drive (to
"power") a biochemical reaction requiring energy (a synthesis), the
reaction is reversed and ADP and iP are regenerated.  It is because of
this regeneration of ADP and iP that the phosphorus requirement for cell
growth is very low compared to the concentration of carbon source in the
waste, and that phosphorus is said to be reused in the system.  The ATP
thus acts as a "shuttle" which couples oxidation of the waste to synthe-
sis of biological solids.

The efficiency with which microbial cells can use ATP to power synthetic
reactions is only approximately known, and it can be expected to vary
from species to species.  Therefore, a numerical coefficient could not be
given for the ATP produced in equation 4.  It should be remembered that
the reaction by which ATP is made from ADP and iP is a synthesis that
requires energy (which is provided from that energy released by the oxi-
dation of the organic matter in the waste).   An oxidation of an organic
compound in the waste is represented by a loss of an electron.  This
electron thus "donated"  by the carbon source is "accepted" by another
compound which in turn passes it along to another until  in the final
transfer, oxygen accepts the electron and water is formed.  A certain
amount of dissolved oxygen thus disappears from solution, or one can say
that a certain amount of oxygen uptake has occurred.  In the metabolic
process of passing each  electron along from each temporary donor-acceptor
system, energy is released in a stepwise fashion and at various points
along the way ATP is produced from ADP and iP.  The ATP harvest from each
passage of an electron from carbon source to oxygen is not the same for
all species of microorganisms, nor can it be said with certainty that
each time CU is used ATP is generated; i.e., oxidation is not necessarily
always coupled to ATP generation.  Thus the efficiency of the process is
not 100%, and efficiency of ATP generation can be expected to vary some-
what from species to species.  For this reason, exact numerical coeffi-
cients cannot be assigned to any of the terms in the equations given
above.  This is true even when known pure compounds are metabolized by a
heterogeneous population of microorganisms.   For wastes of unknown chem-
ical composition, it is  even more futile to attempt to theorize regarding
quantitative relationships to fit the oversimplified equations used to
represent the stoichiometry of treatment.  The practical approach to use
of these relationships lies in treatability studies, in small-scale pilot
plants, in which the quantitative estimates may be based upon actual
measurements.  Equation  3 then becomes useful as a check on the methods
used for measurement.

Energy Balance

Although equation 3 is not suitable for direct use with the majority of
wastes (because the chemical  formulas are unknown), the sum of the
amounts of carbon in the COz and cells produced should balance the amount

of organic carbon which has disappeared from the waste due to the growth
process  (except for wastes containing compounds sufficiently volatile to
be stripped in the aerator).  Thus, we can make use of the mass and/or
energy balance which the equation  (or any other chemical equation) rep-
resents.  One approach requires the capture of all of the C02 produced
and use  of a carbon analyzer to determine the organic carbon content of
the waste and of the cells produced.  A carbon balance can then be made
in which the sum of the carbon produced should be equal to that which
has been used.

However, a more readily facilitated method for determining the stoichio-
metric balance for any specific waste is the energy balance, in which all
components of the balance are expressed in terms of oxygen, i.e., as oxy-
gen uptake and COD.  If a certain amount of oxygen would be required to
completely oxidize soluble organic matter in the waste, and only part of
it is oxidized, then the remaining chemical oxygen demand should be equal
to the original minus that which has been expressed as 0- uptake, or:

      COD  + COD.  = 09 uptake + CODQ + COD,                         (8)
         W      1 S    L.             6      TS

where CODW, CODis, CODe, and CODfs are the COD's of the waste, the ini-
tial biological solids, the effluent, and the final biological solids,
respectively.  Since CODW - CODe = ACOD as defined in Chapter II, and
CODfs ~  cODjs represents the increase in biological solids expressed as
COD, or ACOD, . ,  ,.0-iids» equation 8 may be written as:
      ACOD =02 uptake + ACODbioli SQlids                            (9)
This balance represents the partition of the substrate between respir-
ation and synthesis, in accordance with the diagram shown previously
and in accordance with Figure 1, Chapter II.

By returning to Figure 1, we may see the stoichiometric balance depicted
graphically.  At the end of the purification phase, the amount of or-
ganic matter removed is represented as ACOD in accordance with concepts
presented in Chapter II.  The amount of COD which has been oxidized is
represented by the accumulated 02 uptake at this point.  This is the
amount of "BOD," y, which has been expressed.  All of the so-called
"BOD" has now been removed from solution.  The metabolizable organic
matter which has not been oxidized (i.e., expressed as 02 uptake or BOD)
has been incorporated into new biological solids.

By using ACOD as the measure of biologically available organic matter in
the waste, we have expressed this quantity in useful terms; i.e., in
equivalent units of oxygen.  Also, D£ uptake is measured directly in such
terms.  The increase in biological solids (A cells) can also be expressed
in terms of oxygen equivalents, either by a calculation or by direct


measurement.   Using an empirical  formula for the cells, the 02 required
to oxidize them completely to C02 and H20 could be calculated, thus
yielding a "theoretical  COD" of the cells.   One such empirical formula
is employed below in a balanced equation for total oxidation of the
cells to C02 and

      C5H?N02 + 5 02 -- 2— 5C02
       (113)    (160)

The ratio of combining weights for cells and 02 is 160/113 = 1.42;  thus
each mg of biological solids is equivalent to 1.42 mg 02, or the calcu-
lated COD of the cells = 1.42 x the dry weight of the cells.  Alter-
natively, one can measure the COD of the cells directly by employing
the COD test (16).

The mass balance inherent in equation 6, in the diagrams showing par-
tition of substrate, and in the graph of Figure 1, can now be very
usefully employed in treatability studies to determine whether the
measurements are adequate, i.e., whether all of the organic matter
removed from the waste can be accounted for.  From equation 9

                         Accum. 0? uptake + ACOD, , ,     -, -d
        %n          T r\r\           £              DlOI.  SO II Qb       / 1 i \
        Recovery = 100 x - -    -       (11)
All measurements may be expressed as mg/1 .

There are also other ways in which one may  approach the calculation of
the materials balance.  Various methods, including that described above,
have been discussed by Gaudy, et al . (16).   However, the energy balance
method, equation 11, employing the experimental  determination of ACOD
of the biological solids is straightforward and  readily facilitated and
is applicable to whole organic wastes of complex and largely unknown
chemical composition.  Thus there is a relatively easy way to check on
the validity of the substrate partition between  that which has been
dissipated as inorganic carbon (C02) in the reactor and that which has
been converted to microorganisms (biological sludge).  Since these
organisms are to be separated in the clarifier,  the "BOD" has at this
point on the curve of Figure 1 been "purified."

The balance is not absolutely complete, since some of the substrate car-
bon may be physically stripped from the aerator, depending upon the
nature of the original compounds in the waste or the compounds the
microorganisms may make during metabolism (17,18).   Also, a small
amount of the C02 produced may be reincorporated into the microorgan-
isms since there are some biochemical syntheses  which require a "one-
carbon fragment" derived from C02> but these possible causes for dis-
turbances in the simple proportion described by  equation 9 are not
usually of sufficient magnitude to negate the fundamental validity of


the principle set forth in the stoichiometric equation or the diagram.
The materials and energy balances they imply and the method of making
such a balance herein recommended provide an engineer with a rela-
tively easy "bookkeeping" procedure to check the validity of experi-
mental data obtained on the waste under study.

If one is willing to assume that none of the carbon in the waste is
unaccounted for, the energy balance given above, equation 9, can be
used to estimate oxygen requirements for metabolism.  By measuring
ACOD of the waste and of the sludge, 62 uptake during the purification
phase can be approximated as  ACOD minus  ACOD of the biological solids.

The sludge yield, Y, is an important parameter which is usually calcu-
lated at the termination of the purification phase.  It is best defined
as the ratio of the amount of biological solids produced (Abiol. solids
= final - initial) to  ACOD:

      Y _ Abiol. solids _ AX                                       ,,9x
               ACOD       AS                                       u '

The importance of this parameter is obvious in regard to considerations
for subsequent handling of the sludge.  The sludge or cell yield will
be considered in greater detail in subsequent chapters.

The materials or energy balance could be applied at any point along the
curve, either before or after the peak in biological solids and level-
ing off of the substrate removal curve.  The ratio between that which
has been respired and that which has been channeled into synthesis
(i.e., y/AX) at any time may change; for example, it can be seen that
to the right of the purification phase in Figure 1 the ratio y/AX
increases.  However, the balance would still be expected to apply; i.e.,
ACOD is still accountable for largely as 0^ uptake plus the oxygen equi-
valent of the net amount of biological solids synthesis.

Occurrences which follow the purification phase can be identified as
occurrences in the autodigestive or endogenous phase, or the phase of
aerobic digestion, and a term accounting for this phase is sometimes
included in stoichiometric equations.  The term "endogenous respira-
tion" is one which has been used quite often, and it has been subjected
to various interpretations.  In a strict microbiological sense, it is
employed to signify the metabolism of its internal carbon stores by a
microbial  cell.  This type of metabolism may proceed concurrently with
metabolism of the external (exogenous) carbon source or after the
exogenous substrate is exhausted, or both, but if measured, rather than
theoretical, values are used in stoichiometric calculations, this
metabolism is included and need not be considered separately.

The most practical  way to define "endogenous metabolism" insofar as
biological  treatment is concerned, is as that metabolism which occurs
after removal  of the primary substrate, i.e., after the purification


phase has ended.  It represents respiration (C»2 uptake) at the expense
of the accumulated biological  solids (see Figure 1);  i.e., a net
decrease in biological  solids  concentration.   This decrease may be due
to oxidation of internal  carbon resource in individual  cells but, more
importantly, with natural  populations in waste treatment processes it
represents also cannibal ization of the microorganisms previously syn-
thesized by predator cells (other bacteria or higher  microorganisms such
as protozoa, etc.).  To the organisms performing the  cannibal ization,
the cells on which they are feeding represent an external, not an inter-
nal, carbon source.  The stoichiometry of this phase  can be written as
a grossly simplified chemical  equation in which the organic carbon of
the cells is oxidized to C02 and water:
             + 02  Cannibalizing ^^ + ^  + m1cro0rganisms       (13)

Since the cells contain considerable protein  which is  deaminated during
the autodigestive process, some inorganic nitrogen (NH-)  will  also
appear on the right side of the equation as well  as some  inorganic

In this phase, cells of one type are converted to cells of another type
and to C02 and H20.  The similarity of this equation and  equation 3 is
obvious, and as stated previously this amounts simply  to  recycling of
the products through the forward reaction with the result that the net
solids production is decreased and 02 utilization and  CO? production
are increased.  In short, as the endogenous phase is prolonged, the
partition of the initial substrate between utilization for energy pro-
duction and for synthesis is shifted further  toward total utilization
for energy.  Whether this reaction can approach completion depends upon
the ecology of the microbial system, i.e., the right feeding organisms
must be present for sequential cannibal ization to occur  (proper food
chain).  This important aspect will be discussed in a  later chapter on
the extended aeration process.  It is important here to note that the
stoichiometry of this phase is more variable  than that of the  purifi-
cation phase; it is less well  defined and is, in any event for all but
the extended aeration process, a minor consideration in activated sludge

It is important next to consider description  of the course of  growth
during the purification phase to develop useful relationships  between
growth and substrate removal,  since these are phenomena which  proceed
in the activated sludge reactor and accomplish the real purpose of the
activated sludge process.

Summary and Conclusions

Purification proceeds by utilization of the organic matter in  the waste


for two general types of reactions:  (1) oxidatipn, or respiration,
which degrades the organic material to C0£ and HjjO by oxidaitive reac-
tions which produce energy in the form of ATP, (2) synthesis i>f new
cells from the organic material by reactions which require -energy
supplied by ATP.  Determination of the fractions of the organic matter
utilized for these two purposes is important in treatability studies
carried out prior to designing treatment facilities.  Ati energy bal-
ance based upon expression of organic matter in the waste and the syn-
thesized sludge in equivalents of oxygen and requiring only measure-
ments of COD and of 02 uptake is the most readily facilitated method
for all types of organic wastes.



In the previous chapter, the growth of microorganisms, or synthesis of
new cells, was treated from a thermodynamic point of view.  That is,
the primary concern was determination of the distribution of substrate
between energy-producing and energy-consuming reactions, since this
distribution corresponds to the portion of organic matter which is
effectively removed from the waste (as C02 and HpO) and that which
remains as synthesized sludge requiring further disposal.  Because
sludge production is a primary concern in both design and operation of
biological treatment systems, it is important to examine in some detail
the kinetics of microbial growth and the factors which control growth
and the concomitant removal of organic substrates from the waste.

There are two primary relationships between growth and substrate re-
moval .  One relationship concerns the amount of growth (i.e., biolog-
ical solids accumulation) which one could expect from the utilization
of a given amount of substrate.  This relationship was defined as the
cell or sludge yield in the previous chapter.  The second relationship
is one between the rate of growth and the concentration of substrate.
It should be noted that growth rate can be affected by many experimen-
tal or operational factors.  When the supply of any required microbial
nutrient is limited, it will become the critical factor determining
rate of growth or total amount of growth, or both.  For example, nitro-
gen and phosphorus are well known essential nutrients, and when either
of these is present in insufficient amount it, rather than the organic
carbon source, becomes the growth-limiting component of the waste.
Nitrogen and phosphorus are usually added to the waste in sufficient
amounts so that they are not factors which exert a determinative effect
on growth rate.  In short, pollution control engineers usually aim to
make the carbon source the growth-limiting nutrient.  This principle
of design is based upon the use of biological treatment primarily for
removal of compounds which exert a biochemical oxygen demand.  Consid-
eration of other limiting factors in biological treatment may be neces-
sary to avoid addition of other nutrients to the receiving water, and
this aspect will be discussed in Chapter VII.  For most organic wastes,
however, the carbon source is the limiting nutrient and the most
important factor in determining the kinetics of microbial growth dur-
ing the purification phase.  The following discussion will therefore
be directed toward the relationship between growth and removal of
organic substrates.  First, it is important to define exponential growth.

Exponential Growth

In Figure 1 it may be observed that the course of substrate removal
takes the approximate form of an inverted S-shaped curve and that the
curves for 02 uptake and biological soltds concentration Cwhich depict


the "fate" of the removed substrate) also follow S-shaped curves.
The S-curves are not necessarily symmetrical,  and in most cases they
are not.  For example, the inflection point in the biological  solids
(X) curve usually will be located (as shown in Figure 1)  somewhat
higher than the mid-point of the curve.   The inflection point  in the
62 uptake curve may or may not come at about the same relative posi-
tion as that for X.  Generally, an inflection  point can be observed
in the substrate curve in a position approximately directly above  the
one in X.  The precise locations of all  of these inflection points
are not overly important, but it is emphasized that when  such  an
experiment as shown in Figure 1 is run in the  laboratory  or in nature,
both the disappearance of the initial growth resource (in this case,
the carbon source) and the increase in biological  solids  usually pro-
ceed at an increasing rate, followed by a period of decreasing rate,
until  the resource is depleted.  That is, in such an experiment as
that shown, one kinetic order does not prevail  throughout the  course
of the experiment whether one examines substrate removal, 02 uptake,
or cell growth.

The period of increasing rate of growth can very often be described
mathematically as one in which X increases exponentially.  Such a
kinetic situation can be expressed in the form of a first order dif-
ferential equation:

         =  WX                                                  (14)
The rate of increase in X,  i.e.,  dX/dt,  is  proportional  to  the  amount
of X present at any instant.   The proportionality  constant,  u,  is
known as the specific growth  rate,  or the exponential  growth  rate

Equation 14 is a useful  description of growth,  but is  more  valuable
in its integrated forms:

      Xt = XQeyt                                                (15)

      In Xt - In XQ =  ut                                       (16)

From the latter form it can  be seen  that  a  plot  of  In  X  vs.  t would
form a straight line of slope   y.

In Figure 2, the biological  solids  (X)  curve  of  Figure 1,  up to  the
point of maximum solids accumulation,  is  replotted  on  both arithmetic
and semi-logarithmic coordinates.   It  can be  seen that the extent


_ r\j -fc- — ro w 4^
^ ooooooooo



1 f







TIAL — ^-j





) 2 4 6

8 1C
Figure 2.  Arithmetic and semi-logarithmic plots of
microbial growth during the substrate removal  phase
in a batch system.

(duration) of the exponential  phase of growth can be assessed by
noting the extent of the straight line portion of the semi-log plot.
The end of the exponential  phase corresponds to the point of inflec-
tion on the arithmetic plot.   The numerical  value of y can be deter-
mined as the slope of the line in accordance with equation 16, but
is most easily computed by determining from the semi-log plot the
time required for X to double  in value (i.e., tj) by extrapolation, if
necessary, since the exponential phase may be shorter than one doub-
ling time.  Substituting these values in equation 16, y is defined as:

      y = 1n 2 = 0.693                                          (17)
           *d      td

It is important to examine the concept of exponential (logarithmic)
growth, since various interpretations and significances have been
assigned to "log" growth by basic microbiologists and by engineers as
well.  It is important to remember that, regardless of any particular
physiological significance of  this phase of growth, it is unequivo-
cally, by definition, growth which is mathematically describable by
equations 14 through 17.  It  is that growth for which the doubling
time t^, is constant regardless of the magnitude of tj or of y .  It
is that period of growth for which one's data will  be described by a
straight line on a semi-logarithmic plot of X vs. t.   In short, log-
arithmic or exponential growth is simply a mathematical description,
and the symbol y is a proportionality factor which  is empirically
determined from experimental data.  The importance  of the mathematical
definition of exponential growth will become apparent when continuous
flow cultures are considered  (Chapter V).

Relationship Between y and S

We have defined y simply as a  mathematical descriptor which is a rate
constant allowing one to assign a numerical  value to the rate of
increase in X when the doubling time for X is constant.  There are
some workers in both the fields of microbiology and biological  engi-
neering who maintain that the  doubling time cannot  be constant, and
therefore logarithmic or exponential  growth cannot  exist, unless the
substrate, and all other required nutrients, are present in excess
concentration (e.g., see reference 19).  This is an old concept which
has been passed along as an axiom for so many years that it has become
dogma.  We state here that the concept is false, and we will not bur-
den the reader with all of the proven reasons for our statement.  A
more detailed discussion is provided in the research literature (20,
21).   We will state here simply that the weight of  experimental data
indicates that exponential  growth identifiable by specific values for
y , its descriptor, can occur  at concentrations of  substrate substan-
tially below those for which  y approaches some maximum value at which
addition of higher concentrations of substrate causes no further
increase in  y.

This statement can be verified by a relatively simple experiment.  If
one were to run a series of growth experiments concurrently in separ-
ate reaction flasks into which he placed small inocula of cells accli-
mated to a particular substrate, and in each reaction flask he had
placed increasing concentrations of the substrate covering a wide
range of concentrations, he would observe growth curves (arithmetic
plots of X vs. t) which would be different in two respects.  First, he
would find that the more substrate he had in the flask, the more total
growth (i.e., higher concentration of X) he would have at the end of
the substrate removal phase, i.e., the increase in biological  solids
should vary with initial substrate and the proportionality factor, Y,
should be constant.  He would also observe that the rate at which X
attained its final value would be lower for the systems in which he
had placed the lower substrate concentrations.  He would also observe
that the growth curves would become more alike and would become prac-
tically superimposable for the flasks into which he had placed higher
and higher substrate concentrations.

If one now plotted the growth curves on semi-logarithmic coordinates
(i.e., In X vs. t), the data for the period up to the inflection point
on the arithmetic curves would plot as straight lines, the slope
increasing with increasing initial substrate concentration, until a
maximum slope was attained.  Such an hypothetical experiment is shown
in Figure 3.  From this figure, one observes an apparent dependence of
the value of y on the initial substrate concentration in which the y
was generated.  The mathematical description of this apparent rela-
tionship may be examined initially by plotting the values of y against
the corresponding values of initial substrate concentration, S .  Such
a plot is shown in the upper portion of Figure 4.  It is seen that as
the initial substrate concentration approaches higher and higher
values, y approaches a maximum value ( Vpiax^ anc' father increases in
initial substrate concentration cause no further increase in y; i.e.,
the value for y eventually becomes independent of substrate concentra-

It has been observed that most growth data of this type can be satis-
factorily fitted with a hyperbolic function, as given below:
        - "max
This equation (the "Monod equation") has appeared in innumerable places
in both the microbiological and the pollution control literature with
the symbol S instead of S0, and such "familiarity" is in large measure
responsible for the varying "theoretical" interpretations which have
been placed upon equation 18.   These aspects are described elsewhere
(20, 21).  It is important here to note that the equation is a useful
relationship which was derived empirically and is simply an analytical


  Figure 3.  Effect of initial substrate concen-
  tration, S0, on the rate and total amount of
  microbial growth.  The vertical lines indicate
  the  end of the exponential growth phase.



So _ So KS
LL Mrr,nv Mrr,«v
' ' max /max

J ^max











= 0.6 hr~'
= 125m



                    S0 x  10'
Figure 4.  Hyperbolic plot of the relationship
between specific growth rate, y,  and  initial
substrate concentration, S0 (upper graph), and
a straight line plot, S0/y vs. S0, of the same
data (lower graph).

expression which has been found to fit a large amount of experimental
data.  Like ymax, the term Ks is a constant in the equation.   Whereas
ymax represents the constant value of y which is  not exceeded in the
presence of higher substrate concentrations,  Ks (the "saturation" con-
stant) determines the sharpness of curvature  of a plot of V-  vs S0-
The higher the value of Ks, the flatter will  be the curve and the more
slowly will y become asymptotic to ymax as  substrate concentration is

This behavior of the expression is shown in Figure 5 (taken  from ref-
erence 22) in which are plotted curves for  y  calculated from equation
18 for a range of values of SQ.  For each curve,  the indicated value
was used for Ks and ymax for all curves was 0.4 hr"1.   It can be seen
from the figure and equation 18 that if K$  were infinitely small, there
would be very little dependence of y on S,  and for all  practical  pur-
poses only one exponential  growth rate constant (umax) would describe
growth.  When Ks is very small, the curve rises very sharply  and breaks
to the right very sharply.   In this instance,  y can be  said  to be very
sensitive to a small change in substrate concentration  at very low
concentrations only.  For systems which exhibit a very  low value of  KS,
the sharply rising portion of the curve could  possibly  be fitted to  a
straight line which intersected the horizontal  asymptote, ymax-   How-
ever, there are ample data in the literature  (22-26)  to indicate that
the relationship between specific growth rate  and substrate  concen-
tration is such that it can be most easily  fitted to the continuous
hyperbolic function, equation 18.

It can be seen from equation 18 that the substrate concentration at
which  yis equal to half the value of yma  is  the numerical  value of
1C.  Thus from a plot of the data (y vs.  S  )  one  could  estimate  ymax,
then locate the intersection of 0.5 y    with  the curve and  read tne KS
value on the abscissa (see Figure 4).  A better procedure is  to  arrange
equation 18 in one of its straight line forms.  Two of  these  forms are
given below:

      ir = ir- + ii—V                                        09)
           max    max  o
      S     S      K

      IT = y-9" + y~^~                                          (2°)
            max    max

In the lower portion of Figure 4,  the y  and  S0  values  shown  in  the  upper
portion are plotted in accordance  with equation 20.  These forms  of
equation 18 simply allow one to make a more  accurate estimate of  the
values of the kinetic "constants"  y    and Ks because  one usually has
only a small number of values of y in the  low substrate  ranges, and by
using a straight line form one is  not required  to make a graphical
estimate of curvature.


Equation 18 has been employed by biochemical  (fermentation) engineers
and microbiologists, most of whom deal  with pure cultures of micro-
organisms.  For any specific pure culture, ymax» Ks> and Y are con"
stants for that organism under constant cultural conditions.  In the
activated sludge process, natural microbial populations exhibiting a
rather high degree of heterogeneity and variability are used (anything
but "pure" cultures).  Values for the kinetic constants have been
shown with pure cultures to vary with species, substrates, tempera-
ture, pH, etc.; therefore, in the pollution control field one must
evaluate these constants in repetitive  experimentation in order to
determine if there is a reasonably narrow range of variation for engi-
neering use.  Also, there could be some valid doubt as to whether
equation 18 is the most logical one to  employ for relating y and SQ for
systems such as activated sludge.

For these reasons, we carried out many  experiments over a considerable
period of time using different heterogeneous  populations developed from
various sewage seeds.  All experiments  were run at 25 ± 2°C and at
neutral pH.  A synthetic waste was used in all experiments, and a num-
ber of different carbon sources of different  types were used as sub-
strates.  However, in order to determine the  variability in the con-
stants due only to differences in the populations used in different
experiments, a large number of repetitive runs were made with a single
substrate.  Glucose was used in these experiments, since it is easily
metabolized by most bacteria and should allow the greatest heterogen-
eity possible in a microbial population growing on a single organic
compound.  Values for ymax and Ks determined  in twenty-two separate
series of growth experiments using glucose as carbon source are shown
in Table I.

As these data show, even under constant conditions and with a simple
synthetic waste, there is no single value which can be used as  "the"
kinetic constant for a heterogeneous population.   Values obtained for
these constants with other substrates were also variable (26),  but
only a few experiments were made with each and the range obtained with
glucose is probably more representative of the range to be expected
with heterogeneous populations at moderate temperatures and neutral pH.
Based upon the values given in Table I  and upon other experiments, it
would appear that ranges of 0.4 to 0.6  hr-1 for ymax and 50 to  125 mg/1
for Kg might be chosen as representing  reasonable estimates for use in
applying these kinetic constants to heterogeneous populations such as
activated sludge.

In all  of these studies in which ym,x and K$  were evaluated for differ-
ent heterogeneous populations, the fit  of tne data to the hyperbolic
equation of Monod (equation 18) was also determined.  This equation
(using initial substrate concentration, S0) was found to provide a
more satisfactory fit of the experimental data than did other equa-
tions and relationships tested with the same  data (22, 27).

Municipal sewage represents, perhaps, one of  the most complex mixtures


                              TABLE I


            3       S    GROWING ON GLUCOSE.
0.38 :
. 11
' 0.46
     Averages:  ym,v = 0.53 hr  , K  = 74 mg/1.
                 IN a X               S

of carbon sources found in nature.   It is such a weak substrate that it
is difficult to use in growth experiments requiring a gradation of
initial substrate concentration.  Therefore, the soluble portion of
a municipal sewage was concentrated in a flash evaporator, and growth
on various dilutions of this carbon source was studied (26).   Values of
y  were determined from semi-logarithmic plots of optical  density vs.
time, and values for one such experiment are plotted vs.  the  correspond-
ing substrate concentrations (measured as COD) in Figure 6.   Average
values for sewage were umax = 0.46  hr"'  and K§ = 52 mg/1.   These are
in general accord with the range previously given for glucose.  Data
such as these provide some assurance that these relationships indeed do
exist for complex populations and for complex substrates,  and that there
is a reasonably narrow range of expected values.

In the pure culture area, very little experimentation regarding the
curvature factor (Ks) of equation 18 has been done.  It is generally
believed that the value of Ks will  be much below 50 mg/1  of substrate,
largely because in the early work by Monod (23) low values were found.
It is often overlooked that these studies involved only two bacterial
species and that other early studies yielded a very high  value for K$
(24).  These aspects and their ramifications for use of equation 18 in
research need not be dealt with here but are covered elsewhere (20,
21).  However, before passing on to another important "kinetic con-
stant" it is appropriate to mention that the analytical  geometry of
equation 18 is the same as that of  the Langmuir adsorption isotherm
and that of the Michaelis-Menten equation for dependence  of enzyme
velocity on substrate concentration.  It is necessary to  point out that
similarity in kinetic form does not imply similar mechanism.

The point to be emphasized here is  that equation 18 is usable as a
kinetic description for determining the effect of initial  substrate
concentration on y.  It is an entirely empirical one which can be
recommended for use because it provides a good fit to growth  data.  It
will be used in a later chapter in  the development of a kinetic model
for describing biological behavior  in a continuous flow reactor, i.e.,
an activated sludge tank.

Sludge Yield

In the previous chapter, sludge yield was defined as the  amount of bio-
logical solids produced per unit of organic material  used. Within the
context of the mass balance equation, it is therefore most commonly
determined at the completion of the reactions, i.e., at the point of
maximum biological  solids accumulation, which normally corresponds to
the point of termination of substrate removal.  It is important to note
that while its determination at the end of the substrate  removal phase
is valid, the factor Y is applicable throughout the growth curve, i.e.,
during the exponential  and the declining phases of growth. Thus Y is
useful as a kinetic constant, and we can write


        Pm=  0.46  h
        Ks = 55
            200      400     600    800
    Figure 6.  Hyperbolic plot of the relationship between
    specific growth rate, y, and initial substrate concen-
    tration, S0, for a  heterogeneous microbial population
    of sewage origin growing on a concentrate  prepared
    from the soluble portion of municipal sewage (26).

In large measure, the above statement is true and can be shown to
apply to heterogeneous microbial  populations.  The relative constancy
of sludge yield throughout the substrate removal  period is demonstrated
in Figure 7.  These results were obtained in an experiment in which
glycerol at relatively high initial  concentration was fed to a hetero-
geneous population at low initial  solids concentration (28).  Biolog-
ical solids and filtrate COD concentrations were measured and consti-
tute the primary (raw) data shown in Figure 7A.  The substrate removal
curve was constructed by subtracting the COD curve from the initial
COD.  In Figure 7B, this curve and the biological solids curve are
plotted on semi-logarithmic coordinates, and in Figure 7C, the sub-
strate removal curve is plotted vs.  the biological solids curve.   It
is apparent that the ratio of solids produced to substrate removed,
i.e., AX/AS, is relatively constant  throughout the substrate removal
period.  The calculated correlation  coefficient,  r, is 0.975.  The
sludge yield determined in the usual way at the end of the substrate
removal period is thus also applicable to the exponential phase of
growth; i.e., Y is a constant during any particular experiment.

It should be emphasized that this constancy of Y does not imply that
one would obtain precisely the same  value for the next experiment.
Sludge yields, likeM.,,^ ancl KS,  can be expected to vary, depending
largely on the species of cells present and upon the substrate.  The
substrates one encounters in waste waters are, except for certain
highly specific industrial wastes, not so selective that one could
expect the same species to predominate all  of the time; therefore, Y
will vary not only from waste to  waste (substrate to substrate) but
even for the same substrate at different times, and we must seek to
determine a range of expectable values.  Thus it is important to
orient our thinking, not to precise  constants based upon theoretical
reasoning (energy trapping, thermodynamics, energetics, etc.), but to
a probable range of values which  will be observed for naturally
developed populations.  This is,  incidentally, the type of thinking
which engineers should be readily able to perceive.  It may be more
convenient to use one value, but it  is unrealistic to do so for the
natural biological populations with  which pollution control engineers
must deal.  For an engineer to accept a certain value for a biological
"constant" to two significant places is as  much of a mistake on his
part as is the insistence on the part of some basic microbiologists
that valid conclusions can be drawn  only from studies in which pure
cultures were used.  The engineering mechanics of the bio-mass, which
is indeed a rather new engineering material, are just now beginning
to evolve.  At this stage of the development of this vital engineer-
ing field, one cannot afford to replace empiricism and experimentation
with rather weakly based simplifying theory.  The importance of micro-
bial ecology and the fact that shifts in predominance continually


                      (COD) SUBSTRATE
                       REMOVED (S,
                      12     16

                   TIME, hrs.

p - 0 3^ hr~ '

8 A
.A I
/ ,
/ /
J /





~> hr'1

                                                          TIME, hrs
 . 800
<" 600
                                                 END LOG—•
                                             GROWTH PHASE
g 400


                                          Y  X/Sr -- 0 48

                                          r  0.975
    '0     200   400   600   800    1000    1200   1400   1600   1800   2000   2200  2400

                            (COD)  SUBSTRATE REMOVED , mg/M
      Figure 7.   Constancy of sludge yield throughout the sub-
      strate removal period during growth  on glycerol of  a
      heterogeneous microbial population of municipal sewage
      origin (28).

occur, mean that there is an inbuilt variability in the properties of
the bio-mass, and the engineering profession has to gain some defin-
itive insight regarding the range of variability and its effect on
working theories for design and operation before it can embrace any

Statistical ranges for cell yields on a variety of carbon sources are
given in Table II.  For more in-depth discussion of cell yield in het-
erogeneous microbial populations, the reader is referred to recent
articles in which values obtained in our laboratories during the last
decade are summarized (28, 29).  It is important to note that most of
the sole carbon sources shown in Table II do not exert a very selec-
tive effect on the population.   Many types of organisms can metabolize
these compounds.  All of the organisms employed in the many studies
for which Y was determined were grown up (acclimated) on the compound
tested from original sewage seeds all taken from the same municipal
sewage treatment plant (Stillwater, Oklahoma).   All  values were obtain-
ed under the same operational (experimental) conditions.  All  values
were measured in the same manner, at the end of the substrate removal
period (see Figure 1).  In cases where eight or more experiments were
run, the standard deviation, coefficient of variance (CV), and 95%
confidence limit (CL) were calculated.  For example, the average
yield, 7, in twelve experiments employing lactose was 47.1%.   The
lowest yield obtained was 30, and the highest,  61%.   The standard
deviation of the mean was 7.5%, i.e., two-thirds of the Y values were
between 39.6 (47.1 - 7.5) and 54.6 (47.1 + 7.5).  Expressed as a per-
centage of the mean Y value (i.e., expressed as the coefficient of
variance, CV), two-thirds of the values fell within ± 15.9% of the
mean Y of 47.1, and there is a  95% chance that  if the experiments were
run again, the ? would fall between 42.4 and 51.8% (95% CL).

The fact that a wide range of values can be obtained for any given sub-
strate attests to the variability of the bio-mass and to the engineer-
ing need to work within a range of reasonably expected values, i.e., a
statistical range.  Data such as these and other data as well  (28, 29)
indicate that a reasonable range of ? for engineering calculation is
40 to 60% for most carbon sources consisting largely of sugars and
sugar alcohols and combinations thereof.

The cell yield can also be obtained from continuous flow reactor data
and although we have detected some differences  in average values (27,
29) for batch and continuous flow systems, most fall within the range
cited above.  Also, while there is some evidence that reactor deten-
tion time in continuous flow processes may exert some effect on Y
(i.e., somewhat lower values may be observed at very high detention
times), the data available indicate that at reactor detention times
below 24 hours, the effect is not readily observable.

                              TABLE II

Carbon Source
11 .4
Y is expressed as percent substrate used for synthesis,  i.e.,
100 x AX/AS.

Summary and Conclusions

In this chapter we have considered the relationship between substrate
removal, the desirable result of biological  treatment, and microbial
growth, which accompanies substrate removal .  Each species of micro-
organism is characterized by an absolute maximum growth rate which can
be achieved under optimum conditions with all  nutrients in excess.
Under these conditions, growth is exponential  and is described mathe-
matically as

      g-=yX                                                   (14)

At maximum growth rate, y = y   .

Growth rate may be limited at values below y,,,^ by sub-optimal  con-
ditions of temperature, pH, aeration, etc.,  or by limiting concentra-
tion of any required nutrient.  Since the objective of biological
treatment is removal of the organic carbon source with the greatest
possible efficiency, the process is normally designed so that the  car-
bon source is the limiting nutrient, e.g., by addition of nitrogen and
phosphorus, if necessary.  Therefore, the specific growth rate, y,
becomes a function of substrate concentration, i.e., y = f(S).   With
substrate concentrations below those which allow growth at the maximum
rate for the cultural conditions used (pH, temperature, etc.),  expon-
ential growth occurs, i.e., growth which plots as a straight line  on
semi -logarithmic coordinates.  The proportionality constants (y) for
exponential growth can be evaluated from the slopes of such plots  of X
vs. t for various initial substrate concentrations using equation  17


where t. is the time required for a doubling in mass.  A plot of these
values of y vs. initial substrate concentration for heterogeneous
microbial populations has been shown to fit a hyperbolic function,  the
"Monod equation" (equation 18).

             + S
           s    o
Evaluation of the kinetic constants, y    and K ,  may be made using
one of the straight-line forms of the hyperbolic equation (e.g.,
equation 19 or 20).

For heterogeneous populations, affected by ecological  shifts which
cause changes in predominating species, precise values for the kin-
etic "constants" cannot be expected.  However, repeated determina-
tions of these constants for populations selected from municipal  sew-
age have demonstrated that a usable range of values can be defined
for such populations.  For heterogeneous populations of sewage origin,
a reasonable range for ]J    (at neutral pH and 25 + 2°C) appears  to
lie between 0.4 and 0.6 nr-1 and KS values are generally between  50
and 125 mg/1 .  The fact that values within the same range were found
for growth on the soluble portion of municipal sewage lends added cre-
dence to these results.  Values outside these ranges may be observed,
but the ranges appear adequate to cover most situations in which  a
readily metabolized carbon source is the limiting nutrient for natural
populations at approximately neutral pH and moderate temperatures.

The rate of growth, dX/dt, is also related to substrate concentration
by the following expression:
       _      .
      dt   dt

The kinetic "constant," sludge (cell) yield, Y, applies in general
throughout the growth cycle and substrate removal  period,  i.e.,  the
exponential, or logarithmic, phase and the declining phase of growth.
Its determination is usually most easily facilitated by determining
the amount of biological solids produced and substrate (COD)  removed
at the end of the substrate removal period:

      Y=§                                                    (12)

Generally, expectable values for Y will lie between 0.40 and  0.60

Thus, during exponential growth, both the rate of  accumulation of
solids and the total amount of biological solids synthesized  are
relatable to the concentration of carbon source in definable  fashion,
which can be quantitatively estimated by three biological  systems
constants, Vmax> Ks> and Y.
In the following chapter, these biological  kinetic constants which are
determinable in batch systems, are to be related to physical and
hydraulic characteristics of continuous flow systems, e.g., activated


It is a well known fact that throughout much of its history, the acti-
vated sludge tank has been designed as a long, narrow tubular reactor
through which the waste passes under aeration.  The reactor has usually
been considered as a plug, or piston, flow system into which a waste
containing a relatively high concentration of organic carbon enters,
and from which a low concentration of organic carbon exits at the
effluent end.  Thus X and S are envisioned as changing, X increasing
and S decreasing, from influent to effluent ends.

It has long been known also that this does not actually happen, because
such perfect piston flow is extremely difficult to obtain.  The longer
and narrower the tank, and the less turbulent the mixing, the closer
will such a hydraulic regime be approached.  It is roughly approximated
in some rivers.  It is difficult to attain in laboratory growth react-
ors, and seldom, if ever, does it obtain in activated sludge tanks.

At the other end of the spectrum of hydraulic regimes which are theo-
retically possible in biological reactors is one in which the inflowing
material is instantaneously and homogeneously, i.e., completely, mixed
with the liquid and suspended particles in the reactor.   While perfect
plug flow is very difficult to attain, conditions approaching perfect
complete mixing are less difficult to achieve.  Since even activated
sludge tanks which are not designed as such are in the main closer to
complete mixing than to plug flow, the kinetic aspects of the former
are of most critical importance in considering the activated sludge

We can say with considerable certainty that growth of heterogeneous
microbial populations in batch systems does, in general, follow the
"rules" covered in the previous chapters.  In this chapter we will
examine the behavior of microbial populations in a completely mixed
reactor under continuous flow conditions.

Criteria for Complete Mixing

A simple reactor of the continuous flow, once-through type is diagram-
med in Figure 8.  The batch reactor has been converted to continuous
flow by the continuous pumping of medium into the tank at a constant
rate of flow, F, and concurrent withdrawal by overflow at the same
rate so that the volume of mixed liquor, V, is constant.  The inflowing
concentration of carbon source is S-j.  The concentrations of substrate
and cells in the reactor and in the reactor effluent are S and X,
respectively.  If the reactor is completely mixed, the concentrations
of substrate and cells are the same in all parts of the reactor;
therefore, these concentrations in the mixed liquor exiting the reactor






                      D  = F/V  =  l/t
     Figure  8.   Flow diagram for a continuous flow, com-
                pletely mixed reactor of  the once-through
are also  the  same.  Also, if the reactor  is  completely mixed, the
incoming  substrate is "instantaneously" mixed;  i.e., diluted in the
reactor,  the  dilution factor being the ratio of the volumetric rate
of inflow,  F, to  the volume of reaction liquor  in the aeration tank,
V.  Thus  the  dilution factor, or_the dilution rate, D, is equal to
F/V, and  D  is the reciprocal of t, the mean  residence time  (detention
time) in  the  reactor.  This hydraulic parameter is an extremely impor-
tant one, as  will be shown below.

Before the  Mnetics of completely mixed systems are discussed, it is
necessary to  consider ways to ascertain whether a reactor is, or is
close to  being, completely mixed.  The criterion of the sameness of X
(biological solids concentration) in the  reactor and the reactor
effluent  can  be checked  (approximated) by measuring the transmission
of light  (OD, optical density) by the mixed  liquor in the reactor and
in the reactor effluent.  The optical density provides a quick estimate
of biological  solids concentration.  In cases where X is so  high that

correlation between optical density and biological solids is not pos-
sible, the samples of reactor mixed liquor and effluent can be equally
diluted before the comparison is made.  Also, for best results the floe
must be vigorously dispersed before reading optical density.  The cri-
terion of complete mixing with respect to substrate could be checked by
sampling at the same places, but this is more time-consuming and, in
any event, if the mixing in the reactor is vigorous enough to satisfy
the criterion with respect to suspended particles (X), it can be
assumed that the criterion will also be satisfied with respect to sub-
strate molecules (S).

Complete and instantaneous mixing characteristics of the reactor can
also be checked by performing either dilute-in or dilute-out (washout)
studies.  A washout study could be performed by filling the reactor
tank to the operating volume with water containing a known concentra-
tion, C0, of dye (for example), or suspended particles (e.g., clay).
If clear water is now pumped into the tank and the concentration in the
effluent is measured, it can be seen that, if complete mixing obtains,
the concentration will be continually decreased (diluted) by the factor
F/V, i.e., D.  Thus we may write

      £ - -DC                                                  (22)

or, after integration:
      Ct - Coe-                                                 (23)

If, on the other hand, the reactor had been filled with clear water
and at zero time water containing dye, clay, or another marker material
at C0 had been pumped into the tank, the concentration Cj. in the efflu-
ent at any time thereafter would be given by the dilute-in equation:

      Ct - CQ(1 - e~Dt)                                         (24)

Theoretical dilute-out and dilute-in curves calculated for a dilution
rate of 0.125 hr~' are shown in Figure 9.

A comparison of theoretical and observed dilute- in curves obtained for
one of the laboratory-scale continuous flow reactors employed in
research in our laboratories is shown in Figure 10.  Experimental  val-
ues for two dilution rates, 1/4 and 1/8 hr  , are shown.  Circular,
well-agitated tanks of considerable depth in relation to surface area
appear to possess the geometry favorable to complete mixing.

 8       10

TIME, hr.
                  Figure  9.   Theoretical  dilute-in and dilute-out curves for a completely
                  mixed reactor  calculated  for a dilution rate of 0.125 hr-1.

                        HEORETICAL  EQUATION

                             = c0(.-e-D<)
                              A	A	  8-HOUR
                       6      8     10
                       TIME, HOURS
 Figure 10.   Comparison of theoretical and experimental
 dilute-in curves for a completely mixed laboratory
 reactor operated at various detention times (41).

Growth In the "Steady State"

Growth under continuous flow conditions  may be studied in a  reaction
vessel fulfilling the requirements of complete mixing  as  outlined above,
i.e., growth in a reactor such as shown  in Figure 8.   To  start the sys-
tem, one may fill the reactor with waste,  add  seed from a source such
as sewage, and allow the cells to grow under batch conditions.  The
pumps may be started approximately when  the solids have reached the
maximum level (i.e., at the end of the substrate removal  phase) or be-
fore.  There are a number of ways to initiate  continuous  flow opera-
tion.  The essential fact is that the system will, after  a time, attain
a condition (provided F is not exceedingly high or low) wherein values
for X and S will remain essentially constant.   The concentrations
attained will be close to those exhibited  in batch systems at the end
of the substrate removal  phase in instances in which SQ in the batch
reactor was equal to S-j in the continuous  flow reactor.  However,
unlike the batch system (Figure 1), the  biological  solids concentra-
tion, X, would not decrease below this point;  it would remain relative-
ly steady, as would S.  A continual feed stream allows the cells dis-
placed from the reactor (by the factor F/V or  D) to be replenished and
X remains approximately constant.  The substrate concentration remains
approximately constant because cell yield, Y,  remains  approximately
constant.  In general, then, the system  "approaches" a steady state
with respect to X and S.

It is not necessary to ascribe a physiological  significance  to the
term "steady state" since, like exponential growth, it may be defined
on a strictly mathematical basis; i.e.,  dX/dt  = 0, and dS/dt = 0.  It
has been experimentally demonstrated for pure  cultures, where there is
no chance for change in the predominating  species except  by  mutation
and, therefore, no change in the biological constants, that  relative
steadiness in X and S can be attained.  Also,  after considerable expen-
diture of experimental effort (22), it has been shown  that a "pseudo"
steadiness in X and S can be observed with heterogeneous  populations
of microorganisms, where there is much chance  for changes in the pre-
dominating species, and therefore opportunity  for change  in  the bio-
logical constants.

Having established the possibility of steady state growth experimen-
tally, we may consider the mathematics of  such growth. In Chapter IV
it was stated that equation 14 describes the behavior  of  X in an expon-
ential phase of growth characterized by  a  constant proportionality
factor, y, the specific growth rate.  We can determine whether, in the
steady state continuous growth situation,  the  cells are in the expon-
ential phase by removing this constraint from  v as we  have defined it
and by assuming that the specific growth rate, u, is dX/dt)(l/X) in
any phase of growth, exponential or otherwise.  Considering  X in the
reactor, there is a continual pressure for biological  solids to
increase due to growth (dX/dt = uX) and  to decrease due to cells being
diluted out of the reactor (dX/dt = -DX).   Since the  total weight of


cells is the volume multiplied by the concentration, the change in
mass can be expressed in terms of a mass balance;

      Rate of change     Rate of          Rate of
      in biological   =  increase due  -  decrease due
      solids             to growth        to outflow

      V ^| = VyX - FX                                           (25)

Since  - = D» dividing through by V and substituting D yields:
         = yx - DX                                              (26)

However, we know from experimental observation that a state is attain-
ed wherein dX/dt =0, or at least approximately so.  Thus,

       y = D                                                    (27)

The identity above is a very important one.  It states that the spe-
cific growth rate is equal to the dilution rate, D, i.e.,  F/V; there-
fore, it is subject to hydraulic control.  The incoming flow rate to
the reactor is then a very important physical parameter which can
exert a considerable effect on the biological system.  This fact has
obvious engineering significance, since F is a system parameter which
is more subject to engineering control than are the biological para-
meters Umax5 KsJ ancl Y<

Equation 27 has another significance which may not be generally appre-
ciated.  It states in essence that the specific growth rate, u, is in
fact a proportionality factor during exponential growth, and that
completely mixed systems operating under conditions approaching a
steady state with respect to X (i.e., dX/dt = 0) are operating in an
exponential phase of growth.  This conclusion follows from  the fact
that D was held constant; therefore y is constant, and if y is con-
stant, by definition, growth is exponential.  The biological solids
concentration could not be held steady (dX/dt = 0) unless  the cells
were replenished at a constant specific rate, y = D = In 2/td,  Again,
this condition satisfies the previous definition of exponential growth,
and if a once-through system such as that shown in Figure  8 is said to
be in steady state (dX/dt =0), it must also be said that  exponential
growth obtains.

It is now possible to write a mass balance for the reactor  with
respect to change' in substrate concentration, S, and then  to let
dS/dt approach zero in the steady state:


      Rate of       Rate of          Rate of          Rate of decrease
      change in  =  increase due  -  decrease due  -   due to consump-
      substrate     to inflow        to outflow       tion in reactor
As with the balance for biological  solids,  the rate  of change  in  total
mass of substrate, (V)(dS/dt),  in the reactor is  the sum  of  the fac-
tors causing an increase and a  decrease.  The term for substrate  con-
sumption, i.e., yX/Y, comes directly from the equation

      dS/dt = }(dx/dt)                                          (21)

and the equation

      4  = yX                                                   (14)
As in the case of the mass balance for  biological  solids,  we  can  sim-
plify the substrate balance by dividing by V  and  substituting D for

      «= DS. - DS -^                                        (29)

Also, since y = D, the dilution rate can be substituted  in the con-
sumption term, and letting dS/dt = 0 as steady  state  is  approached
provides an equation for the average steady state value  of X  (i.e., X),
which can be written in terms of the average  steady state  value of
S (i.e., S)

      X = Y(S. - S)                                             (30)

This equation for the steady state value of X indicates  that  the  sol-
ids concentration is dependent on the biological  constant, Y, and on
ACOD, wherein ACOD = S. - S.  This is very similar to the  expression
for X in a batch system.

The equation for X is not really useful as a  predictor for X  unless
5 is known or can be predicted by an independent  relationship not con-
taining X.  Again, equation 27 (y = D)  provides a useful  relationship.
Combining equations 27 and 18 (the Monod equation), but  using the
steady state concentration of S, i.e.,  S, instead of  S ,  we can write

from which

      5 = Ks  iT-~T                                          (32)

an equation for S in terms of the biological system constants, Ks and
^max> anc' the selected value of D (i.e., F/V) which is subject to engi-
neering control^  It is seen by equation 32 that the effluent substrate
concentration, S, is dependent, not on X, but on D and the biological
constants, in the simple once-through (no cell recycle) system shown
in Figure 8.

Again, it is emphasized that equations for X and for S can be shown
experimentally to provide a reasonable assessment of the biological
solids and substrate concentrations exiting the completely mixed re-
actor.  These equations were presented as theory as early as 1950 by
Monod (30).  They were further developed and tested by Herbert, et al.
(19)  [also see Herbert (31)].  In all of these cases, the equations
evolved from the assumption that very small changes in S caused essen-
tially instantaneous changes in y; i.e., that S in the Monod equation
(equation 18) was not S0 in batch or S, the steady state substrate con-
centration in a completely mixed continuous flow reactor, but S at any
time and place.  It can easily be shown that y is actually not imme-
diately sensitive to small changes in S (20, 21).  For example, in a
batch system, a considerable amount of substrate is removed during the
time y is constant, i.e., during exponential growth.  This happens at
initial substrate concentrations below those which will allow develop-
ment of ymax but it is readily observable only in systems for which Ks
is not extremely small.  Therefore, when equation 18 is written using
S, rather than SQ, to describe growth in a batch system, the equation
is not really correct.  Our studies with both pure cultures and a. heter-
ogeneous population have shown that y and S are not tightly coupled and
that "slippage" in the equation may amount to 50% (20, 21).  The workers
cited above observed very low values of KS in their studies with pure
cultures, and this contributed to a theoretical development based on
some incorrect assumptions which led to some misleading interpretations
by subsequent researchers.  Thus, there is an obvious inadequacy in the
"theoretical" basis of equations 30 and 32.  For this reason we have
derived them as approximate empirical models for depicting "steady
state" values of X and S.  In brief, we have taken a practical approach
which should be more appreciated by engineers than by biological kin-
etics researchers.  Experimentally, a relatively steady state develops,
and these equations can be used to model it and to predict levels of X
and S; the steady state does not inevitably develop because of these

equations or because of any theory on which they are based.   The fact
that a process can be described quantitatively with formulas of pre-
dictive value is not necessarily ascribable to correctness  of theory
or hypothesis.  The equations are best looked  upon as  an outcome of
enlightened empiricism.  They are important equations  because they
are basic to description of the behavior in X  and S of completely
mixed, continuous culture reactors.

For further discussion of once-through reactors, demonstration of the
pseudo-steadiness in X and S which develops using heterogeneous popu-
lations of sewage origin, and levels  of X and  S at various  dilution
rates, the reader is referred to an  article by Gaudy,  Ramanathan, and
Rao in the research literature (22).   Figure 11 is reproduced from
this publication and shows the typical degree  of steadiness  developed
in the laboratory reactor.  It is apparent that S approaches a steady
state value much more closely than does X.  Using equations  30 and 32,
it is possible to select values for ymax, KS,  and Y (e.g.,  0.5 hr  ,
75 mg/1, and 0.6, respectively) for  any value  of S^ (e.g.,  1000 mg/1)
and to calculate values for X and S  at various dilution rates
(beginning with 1/24 and progressing  toward D  = ymax).   These calcu-
lations have been made and will be shown in Figure 13  in the follow-
ing section.  On a plot of X and S vs. D it will be seen that X and 5
remain about the same over a fairly  wide spread of D values  (or deten-
tion times, D = 1/t); the substrate  concentration will  remain rather
low and will sweep upward sharply as  D approaches ymax-  The biologi-
cal solids concentration will fall rapidly (wash out).   Comparison of
such calculated values with those observed for heterogeneous popula-
tions in laboratory reactors validates use of these equations for once-
through continuous flow systems (22).

Cell Recycle with Constant Concentration Ratio

If cells (sludge) are recycled to the reactor  after being separated
from the mixed liquor, as is the general practice with activated
sludge processes (see Figure 12), there are, in addition to the engi-
neering control possible through control of F  and V, i.e.,  D, two
other important parameters which are subject to engineering  control.
These are the hydraulic rate or ratio of sludge recycle and  the con-
centration or concentration ratio of biological solids in the recycle
flow.  Mass balance equations for steady state operation of such a
system have, as with the once-through reactor, been developed by
workers in the field of microbial kinetics for pure cultures and, as
with the once-through system, much research effort in our laboratories
has been expended to determine their applicability to systems in
which heterogeneous populations are  employed.

The kinetic model to be discussed first is one which has been devel-
oped by Herbert (31).  This model is  useful, but we have not found it
well adapted for use with activated  sludge systems, and will present



          ^  400
                                                BIOLOGICAL  SOLIDS
                                             EFFLUENT COD
                                                   16       20      24

                                                    TIME,  DAYS
              Figure 11.  "Steady  state" parameters measured  in a completely mixed laboratory reactor
              of the once-through  type, operated at a dilution rate of 0.33 hr-1 with nominal influent
              substrate concentration, S-j, of 1060 mg/1 glucose COD (22).

a modification of it.   First, it is important to show the equations of
Herbert, since they provide a direct and simple expression of kinetic
behavior in systems employing cell  recycle.

Figure 12 shows a flow sheet for a  reactor with recycle.   After leaving
the reactor, the mixed liquor is passed through a concentration step
(clarifier, centrifuge, etc.); in Herbert's  case, a  centrifuge was used.
Cells are concentrated to the desired degree for recycle.   The parameter
which is employed as a systems constant in Herbert's model  is the sludge
concentration factor,  c, which is the ratio  of cell  concentration, XR, in
the recycle to cell concentration,  X, in the reactor.   The parameter for
recycle flow is termed the recycle  ratio,  <*, which is  the ratio of recycle
flow rate to the rate of flow, F, of the incoming medium.   Exiting the
system is the flow, F, containing the excess or waste  cell  concentration,
Xe.  The dilution rate, D, for the  system  as a whole is as  before, F/V,
but the dilution rate  for the reactor,  Dr, is now increased (detention
time decreased) because of the recycle  flow  and is equal  to (1  + a)D.

The two new system controls (constants), a  and c, effect changes in the
mass balance for the reactor.   The  materials balance for  the rate of
change of biological solids in the  reactor is:

      Rate of change in  _  recycle  +   growth  _ outflow
      biological solids  ~  rate        rate      rate

      v    = aFcX + Vy X " F(1 + a)X
The materials balance for rate of change  of  substrate  in  the  reactor  is:

      Rate of change  _  inflow  +  recycle   _   outflow   _  consumption
      in substrate       rate       rate      "   rate      "  rate

      V at = FSi  +aFS  "F(1  +a)S "  V T                           (34)

Dividing both equations by V, substituting D for F/V,  letting dX/dt
and dS/dt equal  zero (i .e. ,_steady state  conditions) and  substituting
the Monod relationship for S, equations 33 and  34 can  be  solved simul-
taneously, yielding the following equations  for steady state  concen-
tration of biological solids, X,  and substrate  concentration  S:
             K [)(1  + a -  uc)

      S - .    -   Dd + a -  -.c)                                    (36)
           ma x




— ^


c ^


r inr




                       XD= CX
Figure  12.  Flow diagram for a continuous flow,  completely mixed reactor
employing cell recycle at a constant ratio, c, of recycle solids, XD, to
aeration solids, X.   Diagram represents the kinetic model proposed by
Herbert (31).

Letting the factor (1 + a - ac)  = A

      X = I (S1 - S)                                               (37)

             K AD

      S ' r-^AD                                               (38)
           max   ttu

Equations 37 and 38 are those developed by Herbert (31).   They provide
a fairly accurate description of the steady state levels  of biological
solids and substrate  concentrations for completely mixed  continuous  cul-
ture devices employing cell recycle; they help delineate  important kin-
etic principles, and  extend those developed in arriving at equations 30
and 32 for once-through systems.  Selecting the same  values for the  bio-
logical constants as  those used  in making calculations for the once-
through system and selecting <* = 0.25 and c =  4.0, the calculated levels
of X and S for S^ = 1000 mg/1 are plotted for  various values of D in
Figure 13.  The values of X and  S for the once-through system of Figure
8 are plotted in broken lines on the same figure to illustrate the
effect of recycling solids.  The values of 0.25 for a and 4.0 for c
were chosen because they are values which are  close to those typical of
many field installations.  Design hydraulic recycle flow  rates of 0.25
times the design flow are commonly used, and it is not uncommon to
observe recycle sludge concentrations at least four times the concen-
tration of biological solids in  the aeration tank. Thus, the values
chosen are realistic  ones (as are those for the biological  constants).
The term A in equations 37 and 38 permits selection of a  wide combina-
tion of values of c and a.  It is obvious that selection  should be such
that A does not go to zero.  For example, one  could not select a = 0.25
and c = 5.  If one were to concentrate the return solids  much beyond
c = 4, a could be reduced.

As seen from the figure, cell recycle permits  the existence in the re-
actor of considerably higher concentrations of cells, X,  than is pos-
sible in a once-through reactor, and consequently allows  greater utili-
zation of substrate at lower detention times (higher  dilution rates).
In both cases, the biological constants are the same, but with cell
recycle the engineering or physical constants, a and  c, exercise a con-
trol over the system  over and above that in a  once-through system which
seeks the natural steady state substrate and solids levels governed
solely by the biological constants, Y, KS, and vm    and  the dilution
rate, D.  It was shown previously that for a once-through reactor,
P = D (equation 27).   However, for a system employing cell recycle,
V is less than D.  According to  Herbert's derivation  of  the equations
for this kinetic model (31), n is related to D by a factor depending
upon the values chosen for a and c, i.e., u =  AD.  From  this it fol-
lows that the specific growth rate,  u, in a system employing cell
recycle can be considerably lower than that in a once-through system




                                               DILUTION  RATE, hr
                         Figure 13.  Comparison  of predicted levels of X and S  at  various
                         dilution rates in a  once-through reactor (broken lines) and  a
                         reactor employing cell  recycle concentration ratio, c, as a  system
                         constant in accordance  with the kinetic model equations of Herbert.
                         Values used for calculation were:  umax> °-5 hr~l,  Ks, 75 mg/1;
                         Y, 0.60; a, 0.25; c, 4.0, Si =1000 mg/1.

with the same system dilution rate.   For  example,  in  the system shown
in Figure 13 where 1 + a - ac = 0.25,  u = 0.25D.   Thus,  at  a  D  of 0.2,
y is only 0.05 hr"1 rather than 0.2  hr'1  as  in  the once-through sys-
tem.  Therefore, on the average, the probability  that a  cell  will Divide
during a pass through the reactor with recycle  is  much less than in  the
once-through system.  However, as pointed out previously, according  to
the mathematical definition of exponential growth  (y  is  constant), if A
and D are kept constant and if dX/dt  = 0, growth  is  exponential  even
at the very slow specific growth rates of recycle  systems.

We have in Herbert's model, a simple and  direct, albeit  empirical, pic-
turization of completely mixed continuous microbial growth  systems in
steady state conditions.  One could  also  use these model  equations in
X and S to assess the effect on effluent  substrate levels of  various
values of the biological constants as  was done  for values of  D  in Fig-
ure 13.  It was shown in Figure 13 that S remains  fairly low  over a
wide range of dilution rates; i.e.,  that  these  systems are  capable of
delivering rather low effluent concentrations of S at high  dilution
rates.  However, operation at very low detention times cannot now be
recommended because of problems with sludge  flocculation and  settle-
ability at high specific growth rates, and because in this  area even
more than in other engineering areas of functional  design and opera-
tion, safety factors are needed.  In any  event, it can be seen  through
study of these model equations and in  charting  the behavior of  X  and  S
that biological treatment systems of this type  possess potential  cap-
abilities for removing substrate loadings much  in  excess of present
day loadings.

One cannot expect the experimental data (lab or field) to trace a per-
fect fit to such a model for the simple reason  that for  heterogeneous
populations the biological constants vary somewhat even  if  one  could,
as in the lab, operate with S-j, D, a,  and c  steady.   Herein lies
an important point, or engineering concept,  which  deserves  emphasis.
One could use these equations in design with the assumption of  constant
values of Sj, D, a, and c, in accordance  with the  model.  But if the
design provided no means for attempting to hold these physical  (engi-
neering) parameters constant, the operator could not  hope to  attain
the designer's goal.  In brief, if designers use a model, the design
must attempt to accommodate the model; this  is  a major requirement of
"engineering control" of processes.  An equalization  or  surge basin
could attenuate swings in Sj and D.   If it were aerated, it could also
provide some degree of treatment as  an unsteady once-through  reactor.
The recycle flow is fairly easily controlled.   Control of c requires
sensing for X in both the reactor mixed liquor  and the clarifier under-
flow, and a means of adjusting XR to hold c  approximately constant at
the chosen value of Xp>/X.  This latter requirement is not very  easily
attained, especially if X is subject to much variation.   As with the
once-through model, the recycle model  was originally  proposed on the
assumption that pure cultures would  be employed, for  which  Ks,  ymax,
and Y are constants, and we have undertaken  experimental studies to


determine the applicability of this recycle model for heterogeneous
(natural) populations.

Experimentation with the once-through model had indicated some varia-
tion  in S and somewhat more variation in X (e.g., Figure 11), but
average values of S and X during operation at various values of D did
produce dilute-out patterns in S and X in general accord with equa-
tions 30 and 32 (22).  To test the applicability of Herbert's recycle
model to heterogeneous populations, a laboratory pilot plant was set
up in accordance with the flow diagram for the model which was shown
in Figure 12 (27).  The biological sludge was not recirculated direct-
ly from the bottom of the clarifier, but was channelled to another
tank in which the recycle sludge concentration, XR, was adjusted in
accordance with c = XR/X.  Data obtained for one set of values of the
operational constants are shown in Figure 14 (taken from reference 27).
The concentration factor, c, was purposely held low (1.5) in these
experiments in order to ensure that we could always obtain the required
XR by diluting underflow.  The sludge recirculation ratio,  a, was
maintained at 0.25.  Figure 14 shows the variation in X and XR for S-j
of 1000 mg/1 glucose (1060 mg/1 COD)(top portion) and in substrate,
S, in terms of COD and carbohydrate (lower graph).  In the example
shown, the detention time in the activated sludge tank was three
hours; i.e., Dr = 1/3 hr-1, and the overall dilution rate, D, was
1/3.75 hr-1  i.e., Dr/(l + a ) .  It is seen that in general, c was
maintained close to the selected 1.5 ratio.  Also, it is apparent that
X varied; the reactor did not attain a "steady state."  The values for
S (COD) also varied considerably, but the scale is somewhat expanded
(8 to 1, as compared with the top graphs), and the effluent substrate
concentration, S, could be adjudged as fairly steady over the two-
month period of operation at this detention time.  The values of S
measured as carbohydrate (using the anthrone test) showed only slight
variation.  Both the COD and carbohydrate measures for S shown in the
figure were obtained by analysis of the membrane filtrate.  However,
it should be noted that during this run, had COD been plotted for the
clarifier supernatant (actual effluent), the values would be only
slightly higher because the sludge settling characteristics were rather
good.  In general, the supernatant COD was 25-30 mg/1 higher than the
filtrate COD.  The average value of aeration solids during this period
was 753 mg/1, and the average effluent COD was 79 mg/1.  These values
along with other parameters for operation at this and other values of
D are given in Table III.  In Figure 15, the average experimental
values for X and S from Table III are plotted as the "steady state"
levels (X and S) for the four dilution rates examined.  The heavy
lines through these experimental points describe a portion of a dilute-
out in X and rise in S as D increases.  This experimental behavior
agrees with the dilute-out pattern which is the expected behavior
according to equations 37 and 38^  The_dotted curves labelled X-| and
S^ show the predicted levels of X and S calculated from equations 37
and 38 using values of the biological constants, ymax' KS' anc' ^
determined for the cells present at each dilution rate.  The ym:3V and


          "10     20     30     40     50     60     70
                      DAYS OF  OPERATION

Q 200
     Q 150
                  30     40     50
                DAYS  OF OPERATION
  Figure 14.  Operational  data for  a  laboratory  reactor
  operated according to the kinetic model  of  Herbert
  with cell recycle at a constant recycle  ratio,  c, of
  1.5.  S-j = 1060 mg/1 glucose COD, a =  0.25,  aerator
  detention time = 3 hr (27).

                                                                 CARBOHYDRATE (S
                               DILUTION RATE, D, HOURS"
      Figure 15.   Comparison of observed (solid lines) values of  X and S with values
      predicted (broken line curves)  by the kinetic model equations of Herbert using
      experimentally determined values of the biological constants (see text for

                                                   TABLE III

Rate, hr" Time, t, hr
1/7.5 6
1/5 4
1/3.75 3
1/2.5 2
3 . A ™
b. c^ =
c. S =
d. 3, =
e. S_ =
f. c
a =
c =
Si =
a bed
X cX S" 5i
mg/1 mg/1 mg/1 mg/1
837 1295 60 1040
797 1236 87 1060
753 1121 79 1067
500 861 277 1065
mean biological solids
mean biological solids in
mean effluent COD
mean influent COD
mean effluent carbohydrate
COD removal rate
1060 mg/1 glucose COD

mg COD/
mg SS/hr

Ib SS/day

COD Removal

the recycle

Ks values were obtained by taking a sample of sludge as seed material
at various times during the operational period and using these samples
for batch growth studies run as previously described.  The value for Y
was calculated directly from the pilot plant data.  The curves labelled
Xp and So were calculated using average values of ymax, Ks> and Y for
all the dilution rates examined.  In general, it can be concluded that
the model does describe (predict) fairly well the behavior of these
systems in which the microbial population was not a pure culture but a
heterogeneous bio-mass, i.e., an activated sludge.

After a large expenditure of research effort we are thus able to con-
clude that the model proposed by Herbert does describe the observed
biological behavior.  It is appropriate to examine the model, and to
defend and criticize it with regard to the reasonableness of the
approach before going further.  It is first and most importantly a
simple one unencumbered by a multitude of biological  constants which
need to be determined; only V-max, K§, and Y are required.  It may be
argued that in setting up the materials balance there was no term for
substrate consumption for maintenance of existing solids, i.e., sub-
strate was considered as being consumed only for respiration and growth
of the bio-mass.  The amount of exogenous carbon source used by a
microbial cell simply to maintain itself intact is very, very small
compared to the amount needed for growth.  It is so small that it is
not a significant factor and, indeed, is very difficult to measure
except for extremely high cell concentrations.  Also, in the mass bal-
ance for X, no term was inserted for a decrease in X  due to "endogen-
ous" degradation of the bio-mass.  This omission is also defensible.
In the presence of exogenous substrate (S,- coming into the reactor con-
tinually), the net effect is an increase in the sludge concentration.
If this were not so, there would be no excess sludge  to dispose of.
(The special situation in which endogenous metabolism, i.e., autodiges-
tion, is extremely important, the extended aeration process, will  be
discussed in Chapter VIII.)  At any rate, it is impossible to deter-
mine whether, or how much, endogenous metabolism occurs in the pres-
ence of exogenous substrate, and non-measurable quantities have no
practical value.  Since net increase in solids is measurable, endogen-
ous metabolism is automatically accounted for if it does occur, and
there is really no good reason for complicating general, working-
model  equations for activated sludge by insertion of  a term for endog-
enous degradation.   The question posed is:  Why overcomplicate for the
sake of not missing anything which could possibly affect X and S?
There seems to be no justifiable reason for doing so.  One can surely
be cognizant of the fact that these phenomena can occur, but if they
do not play a significant role (maintenance) or are not measurable as
separate reactions  (endogenous metabolism), they can  and should be
omitted from the general  model.

Thus, it can be seen that the model  presented could be used as an
approach to plant design,  an approach which is in accord with biolog-
ical  principles relating the major parameters which govern the function


(operation) of the plant.   Equalization tanks could help even out
fluctuations in D and Si;   a can be controlled fairly easily, but a
constant sludge concentration factor causes difficulty in operation.
It was observed in once-through systems (e.g., Figure 11) that while  S
attained a relatively steady level, a steady state with  respect to X  was
only roughly approached.   When sludge recycle is practiced (e.g.,
Figure 14), X is subject to great fluctuation.  If one operates in
accordance with the model, it is required that c remain  constant.
In order to accomplish this, one is required to change the recycle
sludge concentration, X^,  when he detects a change in X  in the
reactor.  This mode of operation requires rather close monitoring of
the plant (which should be done in any event).  A more serious defect
of this model results from solids variations due to heterogeneity of
the population, so that operation at a constant c actually militates
against steadiness in X.  The operator may take a sample of mixed
liquor and find that since the last sample (e.g., the previous day or
shift), the aeration solids concentration has increased.   To hold c
constant, he then must increase X^.  This will not help  to steady X,
but will tend to cause a further increase in X.   Thus, while the model
may work well with pure cultures and while it offers  an  excellent
approach to kinetic picturization of the behavior of  S and X for the
activated sludge process and could be employed as a design tool,
operation in accordance with this model  is difficult, and militates
against steadiness in X.  One could let X vary,  so long  as S remain-
ed steady, since the prime concern is naturally with  the effluent
quality and with reliable  delivery of as low an S as  is  economically
possible.  However, since  constancy of c is the most  difficult oper-
ational requirement of the model and militates against predictability
of X in activated sludge systems (heterogeneous  microbial  popula-
tions), we asked the following question:  What happens to the model
if, instead of holding c as an operational constant,  we  hold X^ con-
stant (an engineering expedient which we could facilitate in practice
much more easily than constant c)?

Cell Recycle with Constant Return Solids Concentration

A flow sheet for the system operated with XR constant is given in
Figure 16.  It is essentially the same as Figure 12,  but an aerated
sludge consistency and holding tank (aeration tank 2) is placed in
the line.  Exiting the process is the effluent substrate concentra-
tion S and excess sludge,  Xe.  The total flow leaving the system is
F (part of the flow is the supernatant and part is the flow of waste

Assuming, as in previous calculations, that the biological solids in
the incoming waste flow is negligible compared to solids in the
reactor, the balance equation for the rate of change  in  reactor sol-
ids can be written as follows:

             TANK  1
                        TANK 2
Figure 16.  Flow diagram for a continuous flow, completely mixed reactor
employing cell  recycle at a constant recycle solids concentration, XR.
Diagram represents the kinetic model proposed by Ramanathan and Gaudy

               (recycle)   (growth)    (outflow)

      VdX/dt =   aFXD    +   V MX   -  (1  + a)FX                (39)

Proceeding as before, and setting dX/dt =  0:

       y= D   [0  +a)  -  (aXR/X)]                            (40)

The symbol X has now been replaced by the  average steady state value
X, in accordance with attainment of steady state  in X;  i.e.,
dX/dt = 0.

The materials balance equation for the change in  S through the
reactor is:

               (inflow)   (consumption)   (outflow)
      VdS/dt =    FSi   -   yXV/Y       -  (1 + a)FS            (41)

from which

      X = (YD/u) [S1 - (1 + a)s]                              (42)

Substituting_y from equation 40 in equation 42, an expression for X
in terms of S is obtained, and one for S in terms of X  is_pbtained
by substituting the Monod relationship for u in terms of S in equa-
tion 40.  Solution of these simultaneous equations leads to the
quadratic form in which X and S are given  as follows (for more detail-
ed presentations of the intermediate steps in the derivation, see
reference 32) :
                       a)S"]  +aX
      X =         (1+a)   -                              (43)
      5   - b ± N b2 - 4ac                                     ....
      S = - - -                                     (44)


      »'  "max' (' +a'D                                     (45)
      b-D[S,- (l+.)ig  -         [s.  t o,xR/Y]            (46)

      c =  KsDSi                                                 (47)

These equations can be employed to depict the behavior of X and S
for various values of VLax, K$, Y, S., D, a, and XR.  It is readily
appreciated that the calculations can be facilitated by use of a
computer.  It is interesting to compare the values of X and S for
this  model with those for the recycle system shown in Figure 13.
Such  a comparison is presented in Figure 17.  The curves labelled B
are those representing Herbert's model and are replotted from Figure
13.   The curves labelled A were computed from equations 43 through
47.   The same values for all system parameters except recycle solids
were  used in calculating the two sets of plotting points.   The only
difference in the two systems is that c, i.e., XR/X, is held con-
stant at a value of 4.0 for curves B, while XR is held constant at
a value of 10,000 mg/1 for curves A.  The latter value for recycle
solids concentration was chosen as one which is readily attained by
quiescent settling.  Two things are particularly important to note
in this figure.  First, operation at constant XR flattens  the
dilute-out pattern of the system, i.e., provides more stability at
high  dilution rates; secondly, at dilution rates which would usually
be considered for use in activated sludge systems (certainly those
below 1.0), the values for S are the same for both models.

The model in which c is a system constant is more in keeping with
justifiable kinetic theory, because it contains no assumption which
prevents a system from diluting out.  In the model we are  recommend-
ing for activated sludge, total dilute-out is impossible (XR is a
constant), and X can reach only a lower level of XR (a /I  + a); also
the assumption of zero substrate concentration in the recycle flow
(see  substrate balance equation 41) is unrealistic at extremely high
values of D because S cannot rise to Sj but can only approach
S.j(l/l + a).  However, at realistic values of D, the consequences of
these assumptions are immaterial; thus the engineering expediency in
making XR a system (design) constant is entirely defensible and
advisable.  These points are developed more fully in reference 32.

The behavior of the kinetic model  in X and S which we are  herein
recommending has been examined computationally for various  values of
%ax> Ks, Y, a, XR, and S-j  at various values of D (.32).  All of these
factors are important kinetic parameters which affect both  X and S.
However, at a reasonably high value for S^  (e.g., 1000 mg/1), changes
in the biological  constants ymax» Ks> and Y do not appear  to exert as
much effect on X and 5 as do the physical  constants a and  XR (32).
In general, selection of a = 0.25, which is a value widely employed
traditionally, appears to be a rather good one for XR values which
can be reasonably attained in practice (e.g., 10,000 mg/1).  The
values for these parameters which are in one case (i.e., a) nomin-
ally attained in the field, and in the other case (i.e., XR) are
usually attained fortuitously, are rather satisfactory for dilution


  O8           1.2

    Figure 17.   Comparison of values for X and 5 at various  dilution
    rates predicted by the two kinetic models for cell  recycle.
    Curves A are those predicted by the model equations of Ramanathan
    and Gaudy (32).  Curves B are those predicted by  the model  equa-
    tions of Herbert (31).

rates of less than 1/2 hr"  and organic substrate loadings of 1000 mg/1
and below.

In general, the model we have presented tends to explain why acti-
vated sludge plants have, on the basis of experience, gained a good
reputation as substrate removers.  Also, the model shows that unless
one does control a, XR, S-j, and D, they can give variable results in
S (the most critical parameter).  It is rather fortunate that the
biological parameters, umay, KS, and Y, which are not really subject
to engineering control (unless predominance of species can be con-
trolled at some point in the future), can be expected to exert less
effect on performance than do the parameters which can be subject to
engineering control (a, XR, 85, and D).

It is emphasized again that the operating engineer stands little
chance of operating the system steadily and reliably unless the means
for exerting control over these selectable variables are provided for
by the design engineer.  Conversely, if they are provided in the de-
sign, they are of little avail unless used by the operating engineer.

In Figure 18, the computed steady state levels of S and X at various
dilution rates for S^ levels up to 5000 mg/1 are plotted in accordance
with our kinetic model (equations 43 through 47).  The values employed
for the biological constants as well as the engineering constants are
those used previously (e.g., for calculating the curves shown in Fig-
ure 17), since they appear to represent values that are reasonably
expectable on a biological basis and reasonably attainable from an
engineering viewpoint, respectively.  It can be seen that the effi-
ciency of substrate removal remains fairly high over a wide range of
feed substrate (S-j) concentrations, especially for dilution rates of
0.5 hr~l and below.  This dilution rate corresponds to a reactor
dilution rate, Dr, of 0.625 hr"1, i.e., a reactor detention time of
1.6 hours.  At higher dilution rates, as Sn- is increased, substrate
leakage increases significantly.  In Figure 19, the curves for D val-
ues of 0.1, 0.5, 1.0, and 2.0 hr'1 for Si up to 1000 mg/1 are plotted
in expanded scale.  Figure 18 was intended to show the overall  behavior
of the model equations, but the range of S-j up to 1000 mg/1 is prob-
ably the one of most interest, since for many types of waste water the
biologically available organic matter (measured as ACOD) is within
this range.

A graph such as this could be employed as a design "guide," but its
real  value is that it shows the potential capability of the process.
Estimates for design should be revised upward from this baseline guide.
A constant balancing of "theory" vs. "engineering judgment" or
"experience" is as necessary (or even more so) in this area as it is
in any other area of engineering. , For example, in Figure 19, the
"chart" says that at a feed COD of 1000 mg/1, just over 60 mg/1 COD
ought to appear in the effluent at a D of 1.0 (reactor detention time
of 0.8 hour)-   Thus well over 90% efficiency is predicted by the
model  at this low detention time.  In determining whether the


3600 .-
                          2000   Z500   3000

                            FEED COO (S, I . mg/l
    Figure 18.   Predicted  levels of aeration solids,  X,  and
    effluent COD, S, for  a  range of organic loadings,  S-j, at
    various dilution rates.   Curves were computed  according
    to the model equations  of Ramanathan and Gaudy (32)  using
    the following values  for system constants:  vimax,  0.5 hr-
    Ks, 75 mg/l; Y, 0.60; a, 0.25; XR, 10,000 mg/l?

 i of
                    DILUTION  RATE, D, hr".
     Figure 19.   Expanded scale plot  of the curves from  Figure 18
     for organic  loadings up to 1000  mg/1  COD.

prediction is reasonable, e.g., in checking performance in treatabil-
ity studies, one needs to consider first that the substrate COD of
the chart is just that:  not total COD,  but the portion of organic
matter in the waste which is capable of  being utilized as carbon
source by an acclimated heterogeneous microbial  population, i.e., the
parameter ACOD developed in Chapter II.   For example,  a waste con-
taining biologically resistant organic matter may exhibit a very large
residual  COD even after the biologically usable (i.e., biologically
oxygen-demanding) organic matter has been removed.   Thus one could
not expect to register COD values as low as shown,  even when these
values, on the basis of utilizable carbon source, were attained.  Use
of a test for residual biologically degradable material  in the efflu-
ent, as recommended in Chapter III would, however,  demonstrate the
high efficiency of the process.  It should also be  realized that CODs
run on potable tap water can sometimes be registered as high as 25
mg/1.   Also, a very important point to make concerning this model
(which we believe to be the most realistic one available) is that it
is an  engineered one, i.e., D,a, Sj, and XR, the engineering para-
meters which can be controlled or for which engineering control can
at least be approached have, in accordance with the model, been con-
trolled.   One cannot approach these results unless  one approaches
this degree of control.  Also, it should be re-emphasized that the S
refers to organic substrate in solution.  Any kinetic  biochemical
model  yet devised is addressed only to this aspect, i.e., the assump-
tion is made that the biological  solids  have been separated from the
mixed  liquor before the effluent is discharged.   If the separation
is not essentially complete, the COD of  the cells which remain in
suspension will add to the values for effluent COD  predicted by the
model.  Thus, although we can recommend  the graphs  of  Figure 19 as a
guide  in selection of reactor detention  time at rather steady loads
of S-j, the model cannot replace the judgment inputs of the engineer.
It can enable more enlightened and reliable judgment;  it provides a
way to balance rational kinetic theory and experimental  data (lab
and field experience).

Employing the model equations (43-47), one could construct his own
"design"  charts using biological  constants for the  specific waste
being  investigated.  These can be determined as previously described
(Chapter IV), using populations acclimated to the waste in question.
Also,  during the treatability study, the expected amount of biolog-
ically available carbon source (ACOD) is determined.   Then the
behavior of X and § for various dilution rates and  engineering con-
trol design parameters can be computed and charted.  The resulting
charts can be employed as the design guides.  One of the most impor-
tant things to be considered is that there needs to be some provision
in the design to attempt, or approach, control of the controllable
parameters, i.e., S-j, F, a, and XR.

Possible ways and means of gaining some  engineering control over
variation in S. and F will depend upon analysis of  the individual


waste water situation.  For some industrially produced wastes, con-
trol of S-j and F may be possible at the various individual sources
comprising the total waste.  This is not always feasible, and in the
case of municipal wastes, this type of control may be even more dif-
ficult to attain.  Control at the treatment plant would seem to
dictate use of some sort of surge basin.  For a waste containing
settleable material, it would be best to place the settling tank be-
fore the surge basin.  There seems little to gain in using a surge
basin as a settling basin.  Depending upon the size of the surge
basin required, the cost of providing for removal of sludge from the
basin could be prohibitive.  Also, depending upon the size of the
basin, relative seeding population and strength of the waste, etc.,
some slight degree of mixing and aeration in the surge basin may be
required.  Thus, it seems best to let the settling tank ride on the
flow in the usual manner and to even out F and S-j to the activated
sludge tank.  Microbial growth in the surge basin can be somewhat con-
trolled by providing a paucity of aeration, e.g., enough to keep it
from going anaerobic but not enough to allow the carbon source to
limit growth.  These are aspects which are naturally subject to engi-
neering investigation in the treatability study stage which precedes
the design stage.  Some wastes will be grossly deficient in essential
nutrients such as nitrogen and phosphorus, and this situation may
obviate the need for providing aeration in the surge basin.  If there
is much growth in the surge tank, the biological solids concentration,
X.,-, entering the activated sludge reactor might be included as a term
in the materials balance equation.  This would lead to some modifi-
cation in the resulting model equations (equations 39 through 47).
In most cases, however, the amount of biological solids which grew in
the surge tank would be small in relation to those in the aeration
tank (which contains the recycle solids) and the assumption of neg-
ligible X in the aeration tank influent would appear to be satisfactory.

When the use of a surge basin is contemplated, it is advisable to
determine ymax» Ks, Y, and ACOD, using the effluent after the needed
period of storage in the surge tank.  Metabolic activity in the surge
tank even under severe 62 limitation can lead to chemical alteration
of the carbon source in the waste.  Thus, although significant sub-
strate removal may not take place, the original organic carbon source
may, in some degree, be converted to metabolic products (e.g., some
fermentation products).  This may have the effect of reducing the
variability of the kinds of organic compounds in the waste.  This
slight amount of pre-treatment may be highly desirable (more research
is needed here) but the point emphasized is that one should determine
the characteristics of the system as they apply to the reaction ves-
sel, and operations on the raw waste preceding the reactor can affect
these characteristics; thus it is not necessarily the raw waste the
designer is concerned with when considering the design of the acti-
vated sludge process.

Surge or equalization basins may or may not be considered in design


depending upon the expected variation of S^  and waste flow rate in
any particular situation.  As we shall see in the next chapter,
completely mixed systems are inherently provided with some degree of
internal stability and do resist changes in  S in response to changes
in Si and F.  Control of XR can be facilitated by the incorporation
in the return sludge line of an aerated sludge consistency and
storage tank from which sludge at constant concentration XR is
pumped at flow rate aF.

It might be argued that prolonged holding of sludge in the consis-
tency tank might affect the biological constants.  Some aerobic
digestion could and would take place in this tank in which concen-
trated sludge is aerated.  In this case, portions of sludge itself
represent substrate, and preliminary studies indicated that even
aerated storage up to 12 days did not cause  larger variations in
^max (when the sludge was used as seeding material in growth studies)
than those normally obtained with freshly grown populations.  A
greater amount of experimental data on this  aspect would be desir-
able, but it does not appear that the placement of an aerated sludge
consistency tank in the return line will cause any drastic change in
the biological constants.

Summary and Conclusions

Equations have been developed in this chapter for three kinetic
models describing the steady state in continuous flow, completely
mixed, microbial growth reactors.  The first model considered was
that for the simple once-through reactor generally used in microbio-
logical research with pure cultures.  Experimental evidence for the
applicability of this model to "steady state" growth of heterogen-
eous populations was presented.  Two models  describing systems more
operationally similar to the activated sludge process, i.e., contin-
uous flow reactors with cell recycle, were then developed and com-
pared with each other and with the once-through model.

Herbert's model, in which the ratio of recycle solids to aerator
solids is maintained constant, was originally developed for use
with pure cultures.  Experimental testing of the model showed that
it provided a fairly accurate prediction of  the behavior of hetero-
geneous populations.  However, operational control, even in the
laboratory, was difficult and a steady state in solids concentration
could not be achieved.

The kinetic model which we have proposed in  equations 43 through 47
is more operationally feasible since the system constant employed
is the concentration of recycle solids.  While this model is not
theoretically correct in that prediction is  poor for conditions
approaching dilute-out, it is realistic and  is applicable over the
range of values of the various kinetic parameters which might be


encountered in practice in biological treatment.  This model is
recommended for use in the design of activated sludge processes,
with the caution that provision for control of the parameters S-j,
F,'a, and XR must be made in the design and must be utilized in
operation.  The flow sheet for this model includes a sludge consis-
tency tank required for maintaining constant recycle solids concen-
tration, and a surge tank is recommended for systems expected to
be subjected to significant variations in volume rate of flow or in
waste concentration.

Both the surge basin and the sludge return consistency tank repre-
sent departures from "standard" or "traditional" practice.  They
represent changes which are in accord with more sound conceptual
principles of microbial metabolism and accommodate the particular
needs for engineering control of the process for the heterogeneous
bio-mass, i.e., an activated sludge.

The kinetic model herein recommended and the suggested changes in
unit processes which are desirably included in the flow sheet in
order to best accommodate the model are of practical utility to engi-
neers and operators.  The model is by no means the "last word" con-
cerning activated sludge, nor do we claim that it comprises the
ultimate in kinetic theory.  It is recommended simply as a practical
advance in conceptual understanding and in design and operational
control of the biological process.



In the previous chapter we presented a kinetic model for activated
sludge of the completely mixed type in which the system is designed so
that it can approach a steady state condition.  The steadiness is
encouraged by engineering actions in the design phase which are intended
to smooth out fluctuations in S^ and F, and operationally the system is
encouraged toward steadiness by controlling a and holding XR, rather
than c, constant.  These are things engineers can do to make an acti-
vated sludge process operate more in keeping with the premise on which
it was designed.

Even with the incorporation of these steadiness-enhancing modifications
in the design of the system, there can be times when the activated
sludge may be subjected to changing environmental conditions which tend
to disrupt the steady state.  Any environmental change tending to do this
can be termed a shock load.  Those environmental changes which cannot be,
or have not been, smoothed by preventive engineering expedients must be
accommodated solely by successful biological response or by combined bio-
logical and engineering remedial responses and/or measures.  It is some-
what paradoxical that although the response of natural  (heterogeneous)
microbial populations to environmental changes has been one of the prime
research interests of the senior author for more than a decade, the
space which can be given to this subject in this particular document is
the short chapter which follows.  This chapter is intended to serve pri-
marily as an introduction to biological responses to environmental
changes, or shock loads.  One can begin to appreciate the vastness and
depth of this area by remembering that the biological world as we know
it today evolved in response to environmental change.  Often the envi-
ronmental change was one which was imposed externally on the biological
system and often the change in environmental conditions was produced by
species within the "bio-mass."  Adaptation and selection of species occur
in response to environmental change.  Also individual species can make
an internal (molecular level) adjustment and become acclimated to a
change in environmental conditions.  An activated sludge, or for that
matter any naturally selected microbial population, represents a micro-
cosm which in itself can be said to serve as a crude model of the
evolutionary process.  Thus it possesses many of the complexities which,
if ultimately resolved through continued investigation, could add in
great measure to attainment of the ultimate goal of the life sciences,
i.e., understanding of the biological world.

Whether or not one agrees with these statements there can be little
argument concerning the mechanistic complexity of these responses, and
oversimplification of the situation would be misleading and a dis-
service to the profession.  On the other hand, in-depth discussion at
the molecular and ecological level would require a voluminous document


and would probably necessitate greater amounts  of study and reference
reading in the area of biology than most practicing engineers would be
able to devote to it.  While one could surely give valid arguments for
such a course of study by pollution control  engineers,  it is not appro-
priate for the present document.  We shall,  however, attempt to intro-
duce this important subject and set forth some  rough and tentative
guidelines which engineers can use.  These are  based primarily upon
research in our laboratories, much of which  is  yet to be published.

Quantitative and Qualitative Changes in Substrate

A quantitative change in S^ (concentration of influent  carbon source)
is the type of perturbation usually and traditionally envisioned as a
shock loading.  In the usual jargon, a shock loading is envisioned as
an increase in "BOD loading," although it could also be a decrease.   A
decrease in organic loading, if not too severe, e.g., a sudden halving
of the loading, would precipitate some adjustment in X  (concentration
of biological solids) but would not be expected (in our experience)  to
cause a severe leakage in substrate (i.e., increase in  S) in the
effluent.  Such shocks cannot be discounted, but our experience indi-
cates that they are less deleterious than a  comparable  increase in

An increase in loading can come on the line  in  various  forms.   There
can be a rise in concentration of metabolizable carbon  source with no
change in F, i.e., a quantitative shock load.   There can be a rise in
organic substrate concentration which is accompanied by a change in F,
i.e., a combined quantitative and hydraulic  shock loading.   Also there
may be a rise in the total concentration of  organic substrate coming
into the system, and the additional organic  matter may  be of an entirely
different type than the steady state substrate.  Alternatively, the
entire organic loading may change from one type to another, probably
also involving a change in total organic concentration.  In brief, the
quantitative shock loading in S-j may often be accompanied by a quali-
tative shock loading in S-j.  Also the change in Sj  may  be more or less
gradual and of variable duration.  Or it may be applied as  a relatively
short-lived pulse or slug dose.  Slug doses  to  the sewer are commonly
experienced and, where the run to the treatment plant is short, the
activated sludge aeration tank may receive a relatively unattenuated
slug.  The use of completely mixed reactors  is  helpful  in this regard
because of the instantaneous dilution of the inflowing  material (see
Figure 10).

Thus it can be seen that even the "simple" quantitative shock loading
(e.g., an increase in BOD loading) is not really a simple concept for
which it would be easy to provide equations, "models,"  and/or rules of
thumb of unquestionable predictive value. However, some approaches to
conceptual  analysis can be initiated, by considering the system which
might be expected to be the most susceptible to deleterious changes in


S-j.  Some conceptual insight into the ability of such systems to respond
successfully to changes in S^ may form a basis for making conservative
estimates for a more shock-resistant system.  The least resistant system
is the once-through system with no cell recycle.  Activated sludge
processes are not designed to operate as once-through systems, but the
study of such systems provides conservative insight.  The recycle of bio-
logical solids would be expected to provide resistance to the leakage of
substrate in a shock-loaded process and, in our experience, it does.
Therefore, systems without recycle are more suitable for studies design-
ed to determine how each type of shock affects the steady state and
limits within which the effects are acceptable.

A quantitative shock load occurs in a once-through system operating under
steady state conditions of X and S CSf and D constant) when the inflowing
feed, S.j, is increased.  If the increment of increase in S-j were not
metabolized by the sludge, substrate concentration in the reactor (and
the effluent) would increase along a dilute-in curve (Figure 10) until
the new effluent_substrate level would be equal to S + AS-j, i.e., the
former level of S plus the new increment in substrate concentration due
to the increase in S-j.  The system would be said to have undergone a
decided functional failure, i.e., the effluent substrate concentration
would have increased to an unsatisfactory level.  For a successful
response to occur, S in the reactor would have to be held approximately
at the steady state level of S by increased growth (increased X) due to
consumption of the new amount of carbon source.  Thus X would increase
to a new steady state level, while S would remain the same or, in any
event, the same percent substrate removal efficiency would be maintained.
In accordance with the hyperbolic equation (Monod equation, 18) the
increase in substrate can be expected to cause an increase in the specific
growth rate, y, thus increasing X.  The increase in X uses up the new sub-
strate (Y remains "constant"), preventing S from rising.  The behavior of
X and S, i.e., rise in X to maintain S constant, forms the transient
stage of successful response as the system approaches the new steady
state in accordance with the new concentration of Sn-.

In an approximate way such a response can be shown to occur, but one
must use "conceptual caution" tempered with empiricism and experimental
observation in analyzing this important response.  The hyperbolic (Monod)
equation is in itself an approximation.  It will be noted that in equa-
tion 18 we have employed SQ as it refers to batch (transient system)
kinetics and have indicated that the substrate value, S, could be used
provided it was understood that it referred to S in continuous flow sys-
tems under steady state conditions (equation 31).  In a continuous flow
system when S-j  in the incoming flow is changed, steady state conditions
no longer exist (because S is changed) and it has been shown that y does
not change instantaneously in response to a change in S (20, 21, 33).
Therefore, equation 18 really cannot be expected to predict precisely
the concentration of S and X during the transient resulting from an
increase in S-j, although this relationship does provide rather good pre-
diction of the new steady state levels of S and X.


Figure 20 shows a response in X (upper portion)  and in S (as COD and
carbohydrate) when a once-through system,  which  was operating previously
in a "steady state" with S,  of 450 mg/1  COD,  was shock-loaded by chang-
ing S-j to 1450 mg/1 COD.  The dilution rate,  F/V, was  0.244 hr~l, i.e.,
a reactor detention time of  slightly over  four hours.   The response to
this rather severe shock loading was an  increase in X  during the tran-
sient phase as X approached  its new steady state level, but X did not
rise rapidly enough to prevent substrate leakage (see  bottom portion of
the figure).  Approximately  1.5 to two detention times were required
before  the system recovered, i.e., regained  the steady state level of S.
Obviously this shock was greater than the  system could successfully accom-
modate, unless one accepts a six-to-eight  hour functional  disruption.

This severe shock loading was purposely  applied  so that COD would leak
from the system in order that the Monod  equation could be  tested during
the transient state.  The computations involve numerical integration
techniques applied to the balance equations in X and S (equations 25
and 28) for once-through systems (i.e.,  dX/dt and dS/dt not equal to 0
in the transient state).  These calculations  are discussed in reference
33.  It is important here to note that the dotted curves for S and X
labeled "predicted by the Monod equation"  provide at best  a rough pre-
diction of the observed transient response.

An important consideration in response to  shock  loads  is illustrated in
the plot of filtrate carbohydrate COD (values designated on the figure
by triangles).  These values were obtained by analyzing filtered sam-
ples for carbohydrates (the  synthetic waste being fed  was  a carbohy-
drate), and it is seen that  during the transient rise  in COD a very sig-
nificant portion of the substrate which  leaked from the system was not
the original carbon source being fed but consisted of  organic compounds
produced by the organisms from the original carbon source.  Thus, during
the quantitative shock loading there was also a  qualitative change in S.
Even a "simple" quantitative shock can cause  complications which can
affect the mechanistic and kinetic response to the change  in the envi-

It could be argued that attempts to provide mathematical models for
depicting or predicting transient responses are, at this stage of the
knowledge, somewhat futile because to cover all  of the metabolic con-
tingencies, they would need  to be very complicated and unwieldly to em-
ploy.  Any relatively simple model would have to be such a gross over-
simplification of the system that it would not provide meaningful output.
Obviously, there is need for much data obtained  by study of defined and
controllable environmental changes before  satisfying mathematical models
for shock loadings can be produced.  Such  endeavors are an important
area for continued research.  However, from the  standpoint of the con-
sulting engineer interested  in the design  of  the process,  mathematical
models for the steady state  operation are  of  more immediate utility, and
what are vitally needed are  guidelines regarding the extent of change
in S. which can be accommodated without severe disruptions of the


              THE MONOD EQUATION
                               TIME, HOURS

lo 200
                                    o FILTRATE COD
                                    A FILTRATE CARBOHYDRATE  COD
                               TIME, HOURS
     Figure 20.  Response of a continuous flow, completely mixed
     reactor of the once-through type to a severe quantitative
     shock load consisting of a change in S-; from 450 mg/1 to
     1450 mg/1 COD.  The dilution rate was 6.244 hr-1 (33).

designed plant efficiency.   Obviously,  a 200% increase in S-,-  under oper-
ational conditions extant for the system of Figure 20 would not be
desirable.  The prime question which should be answered now is how much
change in Sj could be accommodated without a rise in S.  Even this infor-
mation, while more easily provided than a mathematical  model  depicting
the transient, is not easily given.  We know from our own experiments
that the change in S-j which can be accommodated is dependent  upon the
reactor dilution rate (or reactor detention time) and upon the biological
solids level in the system.  At reasonably selected reactor dilution
rates, i.e., those not close to Mmax> even a once-through system will
undergo little if any leakage of substrate during the transient state
when S-j is increased 50% and the reactor detention time is approximately
four hours.  At a detention time of eight hours, a 100% increase in Si
can be expected to cause only a minor and short-lived increase in
effluent S.  With sludge recycling (maintaining X higher than in once-
through systems), the range of increase in Sn- which can be expected to
be accommodated without a significant increase in S can be revised up-
ward.  We have obtained a large amount of experimental  data for quanti-
tative shock loadings at various detention times and recycle  sludge
levels and will be able, after more complete analysis of these data, to
refine the ranges of increase in S-j which can be reasonably expected to
be accommodated without system upset.  For the present it seems reason-
able, although somewhat conservative, to conclude that at detention times
of six to eight hours one can allow increases in S-j of approximately 100%
without expectation of significant loss of efficiency during  the tran-
sient stage.

In Figure 20 the immediate metabolic response to a shock load in S-j was
shown.  This is the portion of the response which may eventually be
depictable by some sort of kinetic model (for a recent attempt see the
article by  Young , et al.,  34).  There is, however, the possibility of
ecologically important aftereffects which are manifested, not during the
transient, but after the system has reached an apparent new steady state.
These were uncovered during a recent investigation by Thabaraj and Gaudy
(35), and Figure 21 is reproduced from that report.  The data of par-
ticular interest in this figure are those in the lower graph  showing
biological solids concentration and substrate.  This completely mixed
continuous flow system (once-through) was operated at a dilution rate of
0.125 hr-'  (detention time of eight hours) with S-j = 1000 mg/1.  It had
attained a relatively steady condition and S-j was then changed to 2000
mg/1.  A successful response to the change was registered, i.e., the
biological solids "rose to the occasion" and S remained steady.  The
immediate (primary) response was one of successful accommodation to the
100% increase in S^, and it appeared that a new steady state condition
was being approached.  However, between the thirtieth and fiftieth hours
after changing S-s, there was a severe disruption in the steadiness of X
and S, and the C6D in the effluent (filtrate) rose to nearly 800 mg/1.
The situation was not self-correcting until the seventieth hour after the
shock (i.e., nearly nine reactor detention times).  This secondary
response was accompanied by a noticeable shift in predominating species


              - 300
            LLJ c
            O E

            z a:
            z *:
            uj -

                                                                       T   TV-IT-
                                                                             —o-o—taWratrrn	|-
                                                            - FEED = 2000mg/C GLUCOSE	
                                            100   120
                                                      140    160   ISO   200   220   240   260

                                                         TIME, hrs
                                                                             280   300  322
               Figure 21.   Response  of a continuous flow,  completely mixed  system of  the once-
               through type to a quantitative  shock load consisting  of a two-fold increase in
               Si.   The dilution rate was 0.125  hr-1  (35).

of microorganisms.   The secondary response is  one for which  there may be
little hope of developing any sort of reasonably reliable mathematical
or "mechanistic" models, at least in the relatively near future.   The
possibility of this type of response does emphasize the need for  caution
and some conservatism with regard to engineering guidelines  for allow-
able limits of increase in S-j.

In many cases the approximate allowable limit  we have tentatively recom-
mended, i.e., up to 100% increase in S^, does  not seem unduly difficult
to attain by a combination of on-line procedures and installation of an
equalization basin at the plant site.

Qualitative Shock Loads

Over the years, pollution control engineers have become accustomed to
thinking of the incoming organic loading, i.e.,  BOD, as a specific enti-
ty.  When it changed, as under shock loading conditions, one had  more
(or less) of the same kind of organic matter.  Also, because of the diver-
sity of the microbial population, as well as the diversity in the kinds
and amounts of organic compounds in the wastes,  one could aptly envision
the ecosystem as one in which each species metabolized that  portion of
the incoming waste to which it was acclimated.   Thus all component sub-
strates in the waste were thought to be metabolized concurrently.

However, early studies employing mixed (heterogeneous) microbial  popu-
lations in experimental systems in which two compounds comprised  the
organic substrate indicated that the presence  of one substrate could
prevent or hinder the metabolism of the other  compound even  though the
population had previously been acclimated to it  (36).   In accord  with
the general beliefs previously outlineds this  would not have been
expected to occur with mixed populations.

It was known from basic research investigations  with pure cultures that
certain enzymes required to metabolize specific  substrates are not pres-
ent in the cell at all times (i.e., they are not constitutive enzymes)
but are produced only when needed (i.e., they  are inducible  enzymes).
The required inducible enzymes are thus produced by the cell in response
to the presence of the particular organic substrate for which they are
needed.  The time required to turn on the genetic synthesizing machin-
ery represents the acclimation period.  If the organism is not geneti-
cally coded for this particular machinery, it  cannot make the requisite
enzymes and it cannot use the substrate.  This is one of the ways in
which changes in predominance of species are brought about in natural
populations when changes in the available substrates occur.   Whether
an organism has the ability to acclimate biochemically determines its
fitness for survival in the adaptation process.   When one develops an
"acclimated sludge," both selection of species (adaptation of the mix-
ed population) and acclimation can occur.  One of these processes, bio-
chemical acclimation or synthesis of inducible enzymes, is an intra-
cellular response, whereas the other, adaptation, is an inter-cellular


or inter-species phenomenon.  Both are important processes which are set
in motion in response to qualitative shock loadings.  In response to the
qualitative change in S-j, the cells in the reactor can acclimate, or
there may be an adaptation of the population or a combination of both.
Both mechanisms of response may overlap or go on concurrently, with or
without a disruption in system efficiency, i.e., leakage of organic
substrates.  On the other hand, these responses may occur in sequence.

Waste streams usually do not completely "turn over" during shocks; some
of the former substrates are present with the new, and one would think
that the former carbon stream would continue to be removed while the sys-
tem attempted to accommodate to the new.  This is, of course, one possi-
bility, but experimental observations indicate that the reverse situation
can also occur.  That is, the new substrate may prevent (repress) con-
tinued synthesis of the enzymes required for metabolism of the former
carbon source(s).  The control mechanism by which the synthesis of one
group of enzymes is blocked and another triggered has become known as
metabolite repression, and a detailed discussion of this phenomenon is
beyond the scope of the present report.  However, at this point one may
wonder concerning the functioning of the enzymes which have already been
synthesized; cannot these continue to function?  There is, after all,
quite a large amount of acclimated sludge in the reactor when the change
in Si occurs.  Should not these organisms be expected to continue func-
tioning on the old substrates even though the new substrates may have
prevented the continued production of enzymes?  This was a question we
posed as investigators and, in the process of experimentation, we came
to the conclusion that there was, in addition to the blockage of the
synthesis of enzyme systems, i.e., metabolite repression, another more
immediately acting mechanism by which the functioning of the already
existing enzyme system may be inhibited (either partially or completely)
by the presence of new incoming substrate.

While the discovery of this type of control  mechanism for enzyme sys-
tems in oxidative metabolic reactions contributed to the knowledge in the
basic field, it also was of practical significance in the area of waste
water pollution control, and a considerable experimental program was
undertaken in our laboratory in efforts to elaborate both basic and
applied insight into the phenomenon and the factors which might tend to
attenuate or to exaggerate the magnitude of disruption ensuing because
of changes in composition of the incoming waste.  A considerable amount
of these results have been published (3, 8,  36-46).  However, much of
the work remains to be published, and even more research is needed on
this type of shock.

In view of the infinite variety of qualitative changes in inflowing sub-
strate which can occur, it is impossible to predict the effects of such
changes upon purification efficiency.  It is, however, possible to pro-
vide some guidelines regarding factors which seem to attenuate disruption
of system efficiency due to qualitative shock loadings.   In some cases,
the age or maturity of the sludge seems to provide an attenuating effect


on the degree of interference which one substrate may exert on the removal
of another or, in any event, on the manifestation of deleterious effects.
Thus freshly grown, highly active cells, while admittedly exhibiting in-
creased substrate removal  capability, may be more susceptible to a change
in the substrate composition than the older ecosystem which evolves upon
longer retention of cells  in the system.  The employment of completely
mixed reactors also helps  to attenuate qualitative shocks since the change
in incoming substrate is diluted into the system thus tending to permit
more time for the system to generate a response (metabolic and ecologi-
cal).  The equalization basin, which was recommended for smoothing out
variations in flow rate and concentration of substrate,  will  also be use-
ful in preventing abrupt changes in the composition of the waste.

Changes in Chemical Composition of the Inflowing Waste Other Than the
Organic Substrates

The incoming waste can undergo significant chemical  changes involving, not
the amount or kind of carbon source, but other chemical  constituents which
may affect metabolism of the carbon source.   Among changes of this type,
the effects of introducing toxic components, either inorganic or organic,
are readily appreciated, and it is also readily appreciated that the best
way of handling such situations is to require treatment of waste streams
containing toxic components at the source of the waste water production
prior to their entry to the waste stream influent to the biological  treat-
ment plant.  Various wastes which would in general be toxic may be sub-
jected to biological treatment if the toxfcant stream is steady or con-
trollable.  For example, several decades ago phenolic wastes  were consid-
ered toxic, but it is now  well known that such compounds are amenable to
biological treatment.  An  activated sludge can be developed for phenol
metabolism, but the population "natural" to this environment can be ex-
pected to be a rather restricted one and would be predominant only if
phenol was the predominant carbon source in the waste.   If phenol were
not the predominant steady carbon source, but were to come on and off the
waste stream as a shock loading, it could inhibit many of the species
developed in response to the dominant carbon source(s)  and it would not
be readily metabolized because the relatively fewer species which can
metabolize phenol would not likely be present in the sludge when the shock
occurred, i.e., the sludge would have to re-adapt to it each time.  The
same generality might be applied for considering inorganic toxicants, e.g.
cyanides, chromium, etc.  Sludges can be developed which exhibit increas-
ed tolerance to some of the inorganic toxicants, but again only when the
system is fed a rather steady diet of the toxicant.

Certain normally nontoxic  inorganic components, when applied to the sys-
tem in high concentration  as shock loads, can also cause severe disrup-
tion of substrate removal  efficiency before the system can adjust.  So-
dium chloride is an example of compounds in this category.  We have ob-
served that activated sludges developed on relatively fresh water are con-
siderably more resistant to shocks of high sodium chloride concentration


than are sludges developed at high sodium chloride concentration to
shocks with fresh water.  Here again, it might be expected that the
sludges developed in high salt concentration contain a narrower selec-
tion of species and, in general, a selection of species which may
require salt (rather than, or in addition to, being tolerant to it).
Thus, when the salt concentration is drastically reduced, e.g., to the
fresh water level, these species die and undergo lysis, and during the
interim transient period of adaptation, substrate leakage occurs.  Since
sodium chloride is not disruptive to biological metabolism at relatively
low concentrations, only a rather drastic change would cause a deleter-
ious transient response.  For example, in a once-through, completely
mixed system operating with an 8-hour aerator detention time, the effi-
ciency of substrate removal was disrupted for nearly two days when the
sodium chloride concentration in the reactor was increased to 30,000
mg/1 over a period of one day (see Figure 1, reference 47).  However,  an
increase to 8000 mg/1 sodium chloride in the same period caused no dis-
ruption in substrate removal efficiency (see Figure 5, reference 47).
It cannot be said that changes of the same magnitude,  or even smaller
changes, involving other inorganic constituents of the waste would not
have more deleterious effects.  In general, the effects of changes in
the concentration and types of inorganic constituents  of the carriage
water should be the subject of continued investigation as the neces-
sity for exertion of greater technological control  of  the aqueous
environment increases.

Changes in pH also constitute chemical alterations  of  the growth envi-
ronment which can cause severe transient responses.   It is rather well
known that systems can be adapted to pH levels considerably below or
above neutral (i.e., pH 7.0) when the pH is held relatively steady.
Here again it should be kept in mind that most biological forms function
best at or near a neutral pH.  When one develops an activated sludge at
either a high (e.g., 8.5-9.5) or low (e.g., 4-5) pH, he restricts or
narrows the diversity of species which can exist in the system and
thereby may be narrowing the diversity or range of  successful ecological
response to all  types of changes in environment, i.e., to al 1  types of
shock loadings,  which that system can accommodate.   If one can be sat-
isfied that the particular waste stream in question will  be subject to
only small  variations in chemical composition, pH,  etc.,  operation of
activated sludge processes at either high or low pH values may save
some costs  of neutralizing chemicals and devices for their addition,
etc.  However, pH control and the monitoring of pH  in  the process are
among the more readily facilitated engineering controls which can be
applied either manually or automatically, and the control of pH either
at or near  neutrality is certainly recommended from the standpoint of
optimum function of the plant in steady state and in response to shock
loading as  well  as from the standpoint of possible  deleterious inter-
action between the waste and the materials of construction.  A system-
atic study  of response of completely mixed systems  to  changes in pH has
been made in our laboratories and these results will be prepared for
publication.  The present aim is to provide guidelines for allowable


variation in pH which should not produce serious  disruption of system
efficiency.  In this regard, our studies indicate that for systems  oper-
ated in the "steady state" at neutral  pH (6.5-7.0) a  change in pH,  i.e.,
a shock load, consisting of either an  increase or decrease of one pH
unit can be accommodated with little or no  disruption of the metabolic
efficiency of the system.  Systems can recover from more severe pH
shocks but a transient leakage of substrate can be expected to occur as
well as retardation of the flocculation and sedimentation of the bio-
mass, especially in the case of acid shocks.   In  general, acid shocks
can be expected to be more deleterious than alkaline  shocks.

Changes in Environmental Conditions not Involving Chemical  Composition
of the Waste

Two major types of system shock loadings in this  category are thermal
shocks and hydraulic shocks.  These, like the  environmental  changes  dis-
cussed previously, engender biochemical  and ecological  responses within
the activated sludge ecosystem which,  if the shock is great enough,  can
lead to substrate leakage from the process. Concerning these types  of
shock, systematic studies of the transient  behavior of completely mixed
systems have been accomplished in our  laboratory  (more extensively with
regard to hydraulic shocks), and these results will be readied for  pub-
lication in the research literature.  In this  report  we will  attempt to
provide some approximate guidelines for allowable limits of change which
can be expected to cause minimal  or no leakage of substrates.   As with
all types of shock loading, it is rather difficult to provide simple
dependable rules of thumb because shock loadings may be imposed in combi-
nations and what one observes concerning any specific type  of shock  may
not hold when that type of shock comes on to a system in combination
with another.  Also, differences in the immediate past history of the
system can engender different responses to  the same shock.   For example,
the dilution rate (or detention time)  at which the system is  operating
prior to a certain change in temperature may certainly affect the re-
sponse.  Also the steady temperature at which  the system was  operated
prior to the change may affect the response.   Furthermore,  for a given
dilution rate the rapidity with which  the change  in the reactor temper-
ature occurs can affect the response.   Thus the response to environ-
mental changes is an area which cannot be overly  simplified,  and there
are many facets to construction of a reasonably reliable set of concep-
tual guidelines for design and/or operation.

Regarding changes in temperature, we have examined the responses of
completely mixed systems operated at detention times  of four and eight
hours at a steady state temperature of 25°C.   Increases and decreases
in temperature were applied.  At an 8-hour  detention  time,  the system
could accommodate, with no excessive leakage of substrate,  an increase
of 10° (25 to 35°C) and with some, but not  severe, substrate leakage,
a decrease from 25 to 18°C.  These were not immediate changes in tem-
perature.  On the downward side the drop in temperature was slightly in


excess of 1  per hour and on the upward side somewhat less than 1   per
hour.  At the lower detention time, a change from 25 to 35°C could be
accommodated without substrate leakage, but a change in temperature from
25 to 18°C caused a significant and long-lived leakage of substrate.
Thus the bio-mass can respond somewhat more successfully to shocks of
increased temperature than to those leading to temperature decreases.
This seems reasonably predictable since increases in temperature (within
reasonable biological limitation) increase the rate of metabolic reac-
tions, thereby engendering more rapid response.  Also, microorganisms
capable of growth between 25 and 40° (mesophiles) are much more common
than those capable of growth below 20°C (psychrophiles).  Shocks con-
sisting of increases to temperatures above 35°C could be assimilated by
the system but not without severe transient leakage in substrate.   As a
rough tentative guideline, it would seem reasonable to recommend for a
system operating normally at or around 25°C that the reaction liquor
should be protected as much as possible from daily variations (i.e.,
changes in temperature taking place over a 24-hour period) of ± 10°C,
with particular precautions being necessary to prevent sudden drastic
decreases in temperature.

Since the reactor volume V is fixed, a change in F causes a change in D
(or in detention time) and, as was seen previously, D is related to the
exponential growth rate, y.  Thus a change in the rate of flow of  incom-
ing waste imposes a change in y.  How the system adjusts itself to this
hydraulic change determines the response, i.e., the loss or maintenance
of substrate removal efficiency during the transient period between
initial and final steady states.  There is a limit to the change in F
from which the system can recover, since if D exceeds Uraax for a suf-
ficient period of time, the solids will be completely diluted out.   If
a system which was formerly operating at a given (steady) flow rate
with substrate concentration, S^, is now shocked by an increase in F
while S-j remains constant, it can be seen that not only has D been
changed, but the overall average (daily) organic loading has also  been
increased simply because the rate of inflow of organic matter has  been
increased.  The system has not undergone a quantitative shock loading
(change in substrate concentration) but the daily organic loading  has
definitely increased.  This is a rather severe type of "purely" hy-
draulic shock loading.  Often hydraulic shocks are accompanied by  a
concomitant decrease in S^, and at times the shock may be accompanied
by an increase in S^.  We have examined responses of completely mixed
systems to both general types of hydraulic shock under a variety of
conditions (with various synthetic wastes employing specific carbon
sources and mixtures of carbon sources, changes in dilution rate,  etc.).
In accordance with our purpose of providing tentative guidelines for
accommodation of shocks on the basis of our experimentation, it is
recommended that the process be protected from fluctuations in F (with
no change in substrate concentration) larger than 100% for design
reactor detention times of approximately eight hours.  That is, if a
system is being operated at a detention time of eight hours on the basis
of the design average daily flow, protection (by way of an equalization


basin or a bypass of flow to another unit) against a two-fold increase
in F (leading to a decrease in detention time from eight to four hours)
should be provided in the interest of ensuring reliable and steady
delivery of the design substrate removal level.   This precaution cannot
be expected to provide absolute protection against disruption of the
system but it can be expected that in this range of change in F (pro-
vided there is no change in S^, temperature, etc.) the transient leakage
of S will be rather short-lived and the system will rather rapidly
recover its former steady state level of S.   Again, as a rough guide for
design detention times less than eight hours, changes in F somewhat less
than 100% should be protected against, and for design detention times
greater than eight hours, changes in F somewhat higher than 100% may be


This chapter has served as an introduction to various types of shock load-
ings involving chemical changes, both quantitative and qualitative, of
the incoming carbon source, changes in the chemical composition of waste
water constituents other than the carbon source and physical  changes such
as temperature and volumetric rate of flow.   The behavioral response of
the bio-mass (activated sludge) in completely mixed continuous culture
reactors is a complex subject, and in-depth  understanding of transient
kinetics may rightfully be a subject of continuing basic environmental
engineering research.  Before any sort of useful  mathematical  models for
the transient response will be forthcoming,  the molecular and ecological
physiology of these systems will need to be  penetrated.   These problems
are of enormous future interest to the entire area of engineering manage-
ment of the aqueous environment.  However, the most immediate concern of
the design engineer is not the ways and means to predict transient re-
sponses, but ways and means to prevent deleterious transient responses
leading to substrate leakage and guides to the extent of environmental
change (shock loading) the process can accommodate without the need for
preventive procedures.  Thus we have attempted to provide rough but
quantitative (and conservative) guideline limits for changes which the
system can be expected to accommodate without serious metabolic upset.
From our experimental observations, it appears that at reasonable design
aerator detention times (six to eight hours), periodic increases in S^
of 100% of the average daily design concentration can be accommodated
without serious or long-lived leakage of substrate in the effluent.  Con-
cerning changes in F, one can expect that hydraulic overloads represent-
ing a change of 100% in the average daily design flow (with no change of
organic carbon concentration, S^) can be accommodated at detention times
of approximately eight hours.  Where variations  larger than these are
expected or cannot be controlled in-line or  at the waste source, the use
of an equalization tank ahead of the aeration tank seems advisable to
smooth out peaks above this range.  Equalization tanks or basins for
high volume plants (e.g., those in large metropolitan areas treating
combined municipal and industrial discharges) may not be practical,


depending upon the variation in flow.  However, the range of change in
flow rate for larger systems is considerably less than for smaller ones.

Effective control of recycle solids concentration, XR, can also provide
a useful tool for attenuating effects of shock loadings.  One relatively
reliable and easily understood concept is that, in general, the higher
the concentration of cells, the more organic matter can be removed in a
fixed time.  Activated sludge plants are usually operated with much
higher concentrations of biological solids than of substrate.  For
example, an organic concentration in the waste of 200 mg/1 "BOD" and a
concentration of biological solids in the aeration tank of approximately
2000 mg/1 are not uncommon.  Even allowing for the fact that we cannot
say that all of the biological solids represent substrate users, a sub-
strate to cell ratio of this magnitude indicates that activated sludges
are essentially run under starvation conditions.  This situation repre-
sents a potential reserve capability which can be drawn on during
increases in S-j.  The reserve capacity that is found in excess metabolic
capability might also be expected to be beneficial during increases in
F which decrease the reactor detention time.  However, it must be
remembered that the hydraulic shock loading which is applied to the
aeration tank is also passed along to the secondary clarifier, and the
decreased settling time may be more of a problem than the decreased
aeration time.  It may not only cause loss of solids in the effluent,
but may also decrease the solids concentration in the sludge recycle
flow.  Thus the combined use of equalization basins and facilities to
control XD (see Figure 16) can help to steady the system under both
normal and shock loading conditions.



Nitrogen Supplementation for Biological Treatment

In previous chapters, removal of organic material from waste waters and
growth of microorganisms have been treated as interdependent processes.
It was stated that it is the usual practice in biological treatment to
add nutrients such as nitrogen and phosphorus to wastes deficient in
these elements to ensure that the carbon source is the limiting nutrient.
This procedure has been recommended for maximum purification efficiency
and it is a logical procedure since removal of the oxygen-demanding
organic components of the waste has traditionally been, and still is,
the foremost goal of biological treatment.  The recommended levels of
supplementation are based upon the ratios of carbon, nitrogen, and
phosphorus required by microorganisms for "balanced" growth.  This means
simply that if one considers synthesis of microbial cells, as described
in Chapter III, as the essential mechanism by which organic material is
removed from the waste, the amounts of nitrogen and phosphorus required
are dependent upon the amount of organic carbon which is to be removed.
Based upon studies of optimum nutritional conditions for purification,
ratios of 20:1 for BOD:N and 100:1 for BOD:P are commonly accepted and
supplementation is provided to achieve these ratios if necessary.

The addition of nutrients represents a continuous operational cost which
may amount to a significant fraction of the total cost of operation.
Any means of reducing the supplementation required could therefore allow
a significant reduction in the operational cost.  An additional reason
for re-examination of the practice of adding excess nutrients is the
recent concern over stream enrichment, i.e., the concern that leakage of
nitrogen and phosphorus from treatment plants may contribute to exces-
sive algal growth in receiving waters.  While much of the nitrogen and
phosphorus in many waters is undoubtedly contributed by natural and
agricultural runoff and while no nitrogen is required for growth of
blue-green algae which are capable of using atmospheric nitrogen, it is
important that all possible sources of nutrient addition be curtailed.
For wastes which contain nitrogen in excess of the required BOD:N ratio,
e.g., municipal wastes, the problem is removal of the excess nitrogen.
However, wastes which are initially deficient in, or devoid of, nitrogen,
e.g., many industrial wastes, present a different problem.  For these
wastes it is necessary to avoid adding excess nitrogen which may leak
from the system while maintaining maximum purification efficiency with
regard to removal of organic material.  In a laboratory pilot plant or
an industrial fermentation process using a pure culture and medium of
known and constant composition, it would theoretically be possible to
exactly balance the amounts of carbon and nitrogen sources so that each
would be maximally consumed.  However, on a practical basis using whole
wastes which vary in composition both qualitatively and quantitatively,


this is an impossible achievement, and it is necessary to consider other
means of avoiding leakage of added nutrients.

Studies of the applicability of the Monod equation (equation 18)  to the
relationship between nitrogen concentration and growth rate of hetero-
geneous populations have shown that the relationship provides a good
approximation of the data (48).  Values for maximum growth rate,  ymav?
were comparable to those measured in studies of carbon source limitation
(see Chapter IV).  The values of Ks ranged from 1.5 to 4.0 mg/1.   Based
on comparison of amounts of carbon source and  nitrogen source required
for maximum specific growth rate determined in these and  other studies,
a ratio of BOD:N of 25:1 appeared to be optimum for maximum growth rate.
This indicates a nitrogen requirement only slightly less  than that found
in earlier studies using different methods (49).

The studies cited above were performed in batch systems.   Rather  exten-
sive studies of the effect of nitrogen level upon  the performance of
once-through, continuous flow, completely mixed systems have also been
made in our laboratory (50).  The laboratory pilot plant  was operated
at various dilution rates with a synthetic waste containing 1060  mg/1
glucose COD, and three levels of nitrogen supplementation were employed.
These studies showed that purification efficiency  and leakage of  nitrogen
in the effluent depended not only upon the level of nitrogen supplemen-
tation, but also upon the dilution rate.   The  nitrogen content of the
sludge was also determined in these studies since  this parameter  appears
to be related to the purification capability of the sludge under  some
conditions (49, 51).

Figure 22 shows the variation in effluent COD  at three levels of  nitro-
gen supplementation and various dilution rates, and Figure 23 shows
ammonia-nitrogen levels in the effluent under  the  same conditions.   It
is apparent from Figure 22 that at longer detention times there is much
less effect of the COD:N ratio on purification efficiency than there is
at shorter detention times.   At a detention time of twelve hours, fairly
good purification was achieved, even in a once-through system, at a
COD:N ratio as high as 70:1.  (In a system with solids recycle, one
might expect greater purification efficiency under otherwise equal  con-
ditions.)  Very little additional purification efficiency was gained by
decreasing the COD:N ratio from 40:1 to 25:1.   With increasing detention
time, nitrogen leakage increased sharply at a  COD:N ratio of 25:1, while
no leakage was observed at any of the dilution rates at a COD:N ratio
of 70:1.

Figure 24 shows the relationship between nitrogen  content of the  sludge
and purification efficiency at four COD:N ratios and various dilution
rates.  As these data show,  even with severe nitrogen limitation, i.e.,
at a COD:N ratio of 70:1, the nitrogen content of  the sludge can  vary
depending upon dilution rate.  It is also apparent from this figure
that the same purification efficiency may be achieved with sludges of




              15        20        25        30       35

                NH3-N  CONCENTRATION  IN  THE FEED,
       Figure 22.  Relation between effluent substrate concentration,  S, and NH3 -
       concentration in the feed at three COD:N ratios for dilution rates ranging
       from  1 to 1/12 hr'1.  Feed COD was 1060 mg/1  (50).













                              |COD/N = 40/I
                                                                  D=I/I2 HR
                                                                  D= I/8HR
               15      20       25       30       35      40        45

                   NH3-N  CONCENTRATION  IN  THE  FEED, mg/Ji
         Figure 23.   Relation between effluent and influent concentrations of NH3 - N at three

         COD:N ratios for dilution rates ranging from 1 to 1/12 hr~l (50).


                          NITROGEN   IN BIOLOGICAL SOLIDS,  %
      Figure 24.  Relation between purification efficiency,  nitrogen content of  the sludge,
      dilution rate, and  feed COD:N ratio  (50).

quite different nitrogen content when other design parameters  are var-
ied.  For example, 90% COD removal  might be chosen as  the desired
purification.  From the figure,  one can determine  that this  degree of
purification could be achieved with:   a 12-hour  detention time at a
COD:N ratio of between 70:1  and  40:1  and a  sludge  nitrogen content of
2.5%; an 8-hour detention time,  COD:Nratio of 40:1, and  sludge nitrogen
content of  slightly  above 4%; or a 4-hour  detention time, COD:N ratio
between 25:1 and 10:1 and sludge nitrogen content  of 9%.   However, con-
cern over stream enrichment dictates  that consideration also be given to
efficient utilization of nitrogen,  and the  data  shown  in  Figure 23 indi-
cate that the lower COD:N ratios which enhance sludge  nitrogen content
also enhance nitrogen leakage in the  effluent.   Therefore, it  should  be
emphasized that no single factor such as a  standard ratio of nitrogen to
BOD or COD or sludge nitrogen content can be chosen as the basis for
design.  Both COD and nitrogen utilization  must  be considered  in choos-
ing the design detention time and the level  of nitrogen supplementation
to be employed.  It should also  be  pointed  out that a  system in which
solids are recycled would be expected to require less  nitrogen than a
once-through system where cells  are growing more actively and  that
nitrogen leakage would probably  be  greater  in a  system with  recycle.
Treatability studies carried out in the design stage should  include
determination of effluent nitrogen  levels.   Especially for industrial
wastes which are not devoid of nitrogen but contain available  nitrogen
at a level below that usually considered optimum,  i.e.,  wastes for
which the BOD:N ratio is higher  than  20:1,  treatability studies should
be designed to minimize or possibly eliminate nitrogen supplementation.
For wastes which contain little  or  no nitrogen,  a  completely different
mode of treatment may be preferable.

Our studies of nitrogen requirements  for the activated sludge  process
suggested the possibility that substrate removal might be accomplished
by oxidative assimilation without the addition of  nitrogen.  Concurrent
studies on the kinetics of purification in  relation to initial  biolog-
ical solids level (52, 53) showed that the  COD:  solids ratio is impor-
tant in determining the mechanism of  substrate removal.   These findings
led us to suggest a possible means  of utilizing  oxidative assimilation
to effect a savings in nitrogen  supplementation  and avoid the  possi-
bility of nitrogen leakage while maintaining purification efficiency
(54).  The new modification of the  activated sludge process  which we
have proposed for nitrogen-deficient  wastes is described  below.  This
rather radical departure from traditional methods  of treatment of such
wastes has been described in a series of publications  (54-60)  and some
of the data obtained in laboratory  pilot plant studies will  be pre-
sented herein.  Before discussing the process itself,  it  is  desirable
to describe oxidative assimilation  and to discuss  some of the  basic
kinetic studies which led to the proposal of the new process.

Oxidative Assimilation

In a system containing metabolizable organic compounds and other required
inorganic nutrients, but devoid of an external supply of nitrogen, rep-
lication of microbial cells cannot occur because synthesis of essential
proteins, nucleic acids, and some lipids requires nitrogen.  However,
many bacteria are capable of converting a large fraction of the avail-
able organic material into internal storage products, while oxidizing
the remainder of the organic matter to provide energy for the synthesis
of the storage product and for other activities of the cell.  The spe-
cific product synthesized varies with the bacterial  species.  The most
common internal storage product is a polymer of glucose which is very
similar to glycogen, the storage product synthesized by the human liver.
Other organisms synthesize a lipid polymer, poly-3-hydroxybutyric acid.
These storage products may accumulate to a level of  50% of the dry
weight of the cells.  Some organisms, if inorganic phosphate is present
in excess, also form a polyphosphate which serves as a store of both
phosphate and energy rather than as a carbon source  store.  The storage
of polyphosphate is undoubtedly responsible for much of the biological
phosphate removal which can occ'jr on prolonged aeration of activated
sludge if the organisms which possess this ability are present in suf-
ficient numbers.  Other organisms may synthesize external  polysaccharide
which is deposited as a capsule or slime layer outside the cell  wall.
Upon addition of nitrogen to the system, internal storage products are
utilized for growth.

Gaudy and Engelbrecht (61) studied removal of COD by activated sludge
in the presence and absence of an external nitrogen  source and showed
that oxidative assimilation could occur in such systems, i.e., COD was
removed in the absence of a nitrogen source, and the organic material
was stored primarily as carbohydrate.  These studies were carried out in
batch at a solids:COD ratio of approximately one, using sludge harvested
from the effluent of a continuous flow system.  Interestingly, analyses
of protein and carbohydrate content of the sludge throughout the period
of aeration in batch showed that both systems,,with  and without nitrogen
added, initially synthesized carbohydrate.  In the system containing
nitrogen, the carbohydrate content of the sludge later decreased while
the protein content increased.

In later studies of the effect of initial biological solids concentra-
tion upon the kinetics of COD removal and solids synthesis, it was
found that as initial solids level was increased in  batch systems, the
autocatalytic curves for both COD and solids typical of low initial
solids systems (see Figure 1) became more nearly linear.  That is, re-
actions which exhibited first-order increasing kinetics at low initial
solids concentrations approached zero-order kinetics or, in some cases,
first-order decreasing kinetics, when high initial solids were used
(52, 53).  Since replication of microbial cells is an autocatalytic proc-
ess, simply because replication occurs by division of one cell to form
two cells, the kinetics observed in these studies were atypical for


replicating systems.   This suggested that replication (cell  division)
actually does not occur to an appreciable extent during  the  period of  sub-
strate removal  in a batch system when the ratio of initial  solids to COD
is high.  Krishnan and Gaudy (53) showed that,  at an initial  COD:biolog-
ical solids ratio of approximately 0.5,  COD was efficiently  removed in
the presence of an inhibitor which completely prevents replication of
cells by preventing synthesis of proteins.   Figure 25 (taken from this
publication) shows the course of COD removal  and synthesis  of biological
solids, both of which approximate zero-order kinetics during most of the
substrate removal period.  These data demonstrate that replication of
cells is not necessary for COD removal.   Sludge synthesis  in this case
does not represent synthesis of new cells but rather an  increase in the
weight of the cells due to synthesis of  storage products,  primarily car-
bohydrate as shown by the analysis of the sludge.  More  detailed studies
of rates of COD removal and synthetic reactions during the  period of sub-
strate removal  in relation to the initial COD:solids ratio,  provided more
complete evidence that as the initial COD:solids ratio is decreased, COD
removal and sludge synthesis follow more similar patterns during the
period of COD removal in systems with and without nitrogen  (62).  In
short, the mechanism of COD removal in batch systems containing  high
initial levels  of biological solids is primarily oxidative  assimilation,
not cell growth.  Subsequently, if nitrogen is  present,  the  stored car-
bohydrate disappears and protein is synthesized, i.e., the  storage prod-
ucts are converted to new cellular components required for  replication
(see Figures 3  and 4 of reference 53, and Figure 3 of reference  62).

This behavior of high solids batch systems  may  be explained  as follows:
When microbial  cells are placed in fresh medium, replication is  not
initiated immediately unless the cells are  from a population actively
growing in the  same medium.  In most cases, e.g., when a culture is
grown to maximum numbers and a portion is transferred to fresh medium,
a period of adjustment is required before replication can  begin.  Dur-
ing this period, the lag period, the cell synthesizes the  proteins and
other cellular  components required for growth under conditions which
differ from those in which the cells had previously existed.   The longer
the period between cessation of growth and  transfer to fresh medium,
the greater is  the amount of adjustment  required, in part  because
enzymes no longer used once the food supply has been exhausted may be
degraded by endogenous metabolic reactions.  The cells used  in the
batch experiments described above were taken from batch  activated sludge
systems maintained by feeding once in twenty-four hours  with one-third
of the sludge and two-thirds of the supernatant  wasted  each day before
feeding (52, 53).  The wasted sludge was used for experimentation.
This sludge was certainly not composed of young, actively  growing cells.
Thus, when the  sludge was contacted with fresh  waste, it was not capable
of immediate replication.  However, it was  capable of removing COD, as
our studies showed, and indeed it is necessary  that COD  be  removed to
supply energy for the formation of cellular components required  for rep-
lication.  As our studies have also shown,  the  sludge is,  in addition,
capable of forming storage products which it may later use  as a  carbon


                     BIOLOGICAL SOLIDS
           TOTAL COD
              GLUCOSE  COD
                      SLUDGE PROTEIN
                                            5000  ?\


                  TIME, HOURS
      Figure 25. Substrate removal, sludge accumulation,
      and sludge composition in the absence of protein

      synthesis in a batch reactor  (53).





source for synthetic reactions.   Thus,  sludge synthesis  (increase in
weight of biological solids, X)  is  not  impeded but replication of cells
is delayed.   If only small  numbers  of cells  are present  (low initial
biological solids), a significant amount of  COD cannot be removed dur-
ing the lag  phase and COD removal  is  dependent upon formation of new
cells, giving rise to the autocatalytic curves for substrate removal
typical of a replicating system.   However,  if a sufficient number of
cells is present in relation to  the available carbon source, all  of the
carbon source can be removed even before replication begins.  If large
numbers of cells are present, the carbon source can be exhausted during
the oxidative assimilation phase which  precedes replication, and there-
fore replication may not occur,  and is  not  required, for  COD removal at
low COD:solids ratios.   However,  for  this process  to be repeated, it is
necessary that a nitrogen source be available so that the storage prod-
ucts may be  utilized for synthesis  of normal  cell  components.   In batch
systems containing very high solids,  this conversion can  take place
after the purification  phase is  complete, i.e., after the carbon source
(COD) has been taken up by the cells.

Since the purification  of the waste,  i.e.,  removal  of COD,  and the syn-
thesis of nitrogen-containing cellular  components  can occur sequentially
even in the  presence of nitrogen,  it  should  be possible to  separate these
phases physically.  It  seemed that  several  advantages might be realized
by such a treatment scheme for nitrogen-deficient  wastes.   Accordingly,
studies were undertaken to determine  the feasibility of the process
described in the following section.

Proposed New Process for Treatment  of Nitrogen-deficient  Wastes

The flow sheet for the  proposed  method  of treatment is shown in Figure
26.  After primary settling or other  required pre-treatment, the
nitrogen-deficient waste flows into the feeding aerator,  or activated
sludge tank, where it is contacted  with a high concentration of recycled
sludge in a  completely  mixed reactor.   In contrast to the usual  mode of
operation, no nitrogen  source is added, although phosphorus should be
added if necessary.  The waste is purified  by oxidative assimilation;
that is, the organic material is removed from the  waste and converted
to intra-cellular storage products.  The mixed liquor exiting the
aerator is settled in the usual  manner  (clarifier) and the supernatant
is discharged as plant  effluent.  At  this point, the settled sludge
differs  from the usual  activated sludge in  that it contains a higher
percentage of carbohydrate and possibly lipid, and a lower percentage of
protein.  The effluent  should be free of nitrogen, since  the nitrogen-
starved cells would remove any trace  of nitrogen present  in the waste.
The waste has been purified without nitrogen having been  added.

Before the sludge can be recycled to  the aerator,  its ability to con-
vert external organic carbon to  internal storage products must be regen-
erated by allowing the  cells to  utilize the  material already stored for


                                                   EXCESS SLUDGE
                                     NITROGEN  SUPPLY
      Figure 26.  Proposed flow sheet for  the treatment of nitrogen-deficient industrial
      wastes by the continuous oxidative assimilation modification of the activated sludge
      process (54).

synthesis of the nitrogen-containing compounds  (proteins, nucleic acids,
and some lipids) which are required for continued metabolic activity and
for replication.  However, only that portion of the sludge which is to
be recycled requires regeneration,  and the amount of nitrogen required
is therefore less than that usually added to allow unrestricted synthe-
sis of nitrogenous compounds by all of the sludge in the aerator.  The
portion of sludge to be recycled is pumped to a second  aerator, called
the endogenous aerator because no external carbon source is present.
Nitrogen is added, as NH/1", and the stored carbon source is used by the
cells for synthesis of nitrogenous  compounds, largely protein, and for
replication.  This regenerated sludge, now depleted of  storage products
and high in protein content, is recycled to the feeding aerator, where
it again removes COD by oxidative assimilation.

Initial tests of the feasibility of this mode of treatment were carried
out in batch systems using a synthetic waste containing glucose as the
sole organic carbon source (54).  Parallel systems were operated in an
identical manner except that nitrogen (as NH4+)  was added to the feed-
ing aerator in one system and to the endogenous  aerator in the other.
The necessity for regeneration was  also tested  by recycling sludge from
two endogenous aerators, to only one of which nitrogen  was added.  All
sludges were analyzed during each aeration period (feeding or endog-
enous) for carbohydrate and protein content.   These studies showed that
the physical separation of waste purification and replication of cells
was possible in practice.  COD was  removed with  approximately equal
efficiency in the presence and absence of nitrogen in the feeding
aerators, and was stored primarily  as carbohydrate by the sludge in the
aerator to which nitrogen had not been added.   In the endogenous aeration
period, the stored carbohydrate was converted to protein in the presence
of ammonia-nitrogen, and the recycled regenerated sludge removed COD as
efficiently as the recycled sludge  which had been treated in the usual
manner by addition of nitrogen to the waste before treatment.   Without
the regeneration step, the sludge rapidly lost  its ability to remove COD.

Since glucose is rather easily polymerized to form the  glycogen-like
carbohydrate storage product, it was important  to test  the feasibility
of the process for other carbon sources.  Batch  studies (56) were carried
out using synthetic wastes containing a non-carbohydrate, acetic acid,
and lactose, a compound which is metabolized by  inducible enzymes.  The
use of lactose was designed to determine whether inducible enzymes neces-
sary for oxidative assimilation would be degraded during endogenous aer-
ation in the absence of the carbon  source.  The  process operated suc-
cessfully with both synthetic wastes.

The feasibility of the process for  continuous flow operation was studied
in a laboratory-scale pilot plant (Figure 27) using two synthetic wastes
and a whole waste.  In the synthetic wastes, glucose (57) and acetic
acid (58) were used as carbon sources, and the  whole waste was a sugar
refinery plant effluent obtained from the Imperial Sugar Company refin-
ery at Sugarland, Texas (55).  The  nitrogen supplement  for the endogenous


Waste Holding Tank
Recycle Pump'
             Feeding Phase
        Sludge Holding
                                                   Waste Sludge
                                                                 Diluent for sludge
Figure 27.  Laboratory-scale pilot plant employed in operational feasibility
studies for the continuous oxidative assimilation process (55).

aerator was calculated as a ratio of the COD added to the feeding
aerator, and various ratios were used to determine the minimum require-
ment for the process.  These data have been published in detail  in the
references cited above.  With nitrogen-free glucose synthetic waste
(1060 mg/1 COD), 95% removal of COD was accomplished with a 4-hour
detention time in the feeding aerator at a COD:N ratio of 70:1 (approx-
imately equivalent to BOD:N = 50:1).  Sludge wasted was 17%, and no
NH4+ could be detected in the effluent.  With acetic acid (1060 mg/1  COD)
at the same COD:N ratio and feeding aeration time, COD removal was 90%.
Under the same conditions, 94% of the COD was removed when the COD:N
ratio was 50:1.  The sugar refinery waste varied considerably in COD
content, and no attempt was made to adjust its concentration since this
variation allowed assessment of the ability of the process to withstand
quantitative shock loads.  The COD of the waste varied from 380 to 1375
mg/1, and BOD from 271 to 841 mg/1.  Approximately 35% of the BOD of  the
waste was carbohydrate.  At an aerator detention time of five hours,  a
COD:N ratio of 60:1  and a COD:P ratio of 140:1, good purification effi-
ciency was maintained.  No serious disruption resulted from the quanti-
tative shock loads applied to the system, and no MM/*" was detected in
the effluent.   Sludge wastage was approximately 32%.

In all continuous flow operations, as shown in Figure 27, two aerators
were used for the regeneration and recycling of sludge.   These were
operated as batch systems.  Each twelve hours, sludge was withdrawn from
the clarifier underflow, diluted with effluent to a constant concentra-
tion (to maintain XR constant), and a portion was wasted.  The remainder
was aerated for twelve hours in the presence of NH^+ at a concentration
based on the amount of COD fed to the feeding aerator during the pre-
ceding twelve hours.  The regenerated sludge was then pumped to the aer-
ated holding tank, from which it was recycled at a constant rate during
the succeeding twelve hours while a new batch of sludge was being regen-
erated.  Preliminary studies of required regeneration time indicated  that
the detention time in the endogenous aerator could be reduced.

Figure 28 shows typical data for effluent analyses during continuous  flow
operation of the oxidative assimilation process with synthetic waste  con-
taining acetic acid as the sole carbon source (58).  The hatched area
represents the synthesis of storage products.  The synthetic waste con-
tained no nitrogen,  and the ratio of COD:N used as a basis for nitrogen
supplementation in the endogenous aerator was 70:1.  The efficiency of
solids removal in the clarifier was approximately 96%.  These data dem-
onstrate the potential of the proposed process for treatment of nitrogen-
deficient industrial wastes.

There is some obvious similarity in the flow sheets for the oxidative
assimilation process and the biosorption, or contact stabilization
process, and it might be possible in some cases to convert biosorption
plants to operation as oxidative assimilation processes.  Both types  of
treatment involve dual aeration tanks; however, relative detention times
for the first and second aeration periods may be quite different.  The


                                                       FEED FLOW = 400 mi/hour
                                                       RECYCLE FLOW = 200 mi/hour
                                                       MIXED LIQUOR DETENTION TIME = 4hrs
                                                        BIOLOGICAL SOLIDS
                                       CELL CARBOHYDRATE
                     CELL  PROTEIN
                EFFLUENT  COD (FILTRATE)
                   i    i    i    i    i    i
                                           8       10
                                            TIME, DAYS
        Figure 28.  Operational data for treatment of nitrogen-free acetic acid waste by continuous
        oxidative assimilation process with nitrogen supplementation in the endogenous aerator at a
        COD:N ratio of 70:1 (58).

fundamental difference between the oxidative assimilation process and
biosorption lies in the point of addition of nitrogen.   In the bio-
sorption process, nitrogen is added to the waste before the carbon
source is removed, and in oxidative assimilation, the carbon source is
removed before nitrogen is added.   In the latter process, nitrogen is
added only to the sludge, not to the waste, and only to that portion
of the sludge which is to be recycled.  In the biosorption process,
nitrogen cannot be used efficiently and much of the added nitrogen is
likely to be lost in the effluent.  This occurs because,  as our studies
have shown, at very high solids:COD ratios, such as those used for bio-
sorption, the carbon source is removed and stored in the  cells before
synthesis of nitrogen-containing compounds occurs,  even if nitrogen is
present.  Thus, it is likely that most biosorption  plants actually
remove carbon source largely by oxidative assimilationand that much of
the added nitrogen is wasted because it is made available at a time
when the cells cannot utilize it efficiently.   The  oxidative assimila-
tion process eliminates leakage of nitrogen in the  effluent and allows
a considerable reduction in the cost of nitrogen supplementation.

Summary and Conclusions

Studies of the kinetics of substrate removal  showed that  at high ini-
tial ratios of biological solids to COD, nitrogen utilization and cell
replication occur primarily after the COD has  been  removed.  In these
systems COD removal is largely accomplished,  even in the  presence of
nitrogen, by oxidative assimilation.  The carbon source is removed and
converted to internal storage products, which  are then  used for syn-
thesis of the nitrogen-containing compounds necessary for formation of
new cells.  Since carbon source assimilation and nitrogen utilization
occur sequentially in the cell, it is possible to separate these
processes physically in a new flow sheet proposed for treatment of
nitrogen-deficient industrial wastes.

The proposed flow sheet, shown in Figure 26,  utilizes two aerators.  In
the first, or feeding, aerator, nitrogen-free  waste is  treated by oxi-
dative assimilation.  A nitrogen-free effluent is produced since no
nitrogen is added to the waste.  The sludge to be recycled is regener-
ated by addition of ammonia-nitrogen in a second (endogenous) aerator.
A considerable savings in the cost of nitrogen supplementation is
realized, since a COD:N ratio much higher than that used  in traditional
methods of treatment can be employed.  Laboratory-scale pilot plant
studies using several synthetic wastes and a  sugar  refinery waste have
demonstrated the feasibility of the proposed continuous oxidative
assimilation process for treatment of nitrogen-deficient  industrial


                      EXTENDED AERATION PROCESS

The present document is concerned with conceptual principles relevant to
the•purification of wastes, i.e., with metabolic occurrences taking
place largely in the aeration chamber.  It is not our intention to cover
the topic of sludge disposal, although ultimate disposal of organic or
biological sludges is a vital concern in the water pollution control
field.  Most engineers are, however, aware of the "extended aeration"
process or the "total oxidation" process, in which biological treatment
of the incoming waste is effected via an activated sludge and, concur-
rently with this function, 'the activated sludge is subjected to aerobic
digestion—the end result supposedly being net conversion to C02 and  HoO
of all of the organic matter originally in the waste stream or at least
of that portion of it which is biologically treatable.  Concurrent waste
water purification and aerobic digestion are accomplished by returning
all sludge to the aeration chamber.  The organic matter in the sludge is
subject to wet biochemical combustion, and is dissipated as C02 and F^O
just as when it is ultimately disposed of by chemical combustion or
anaerobic digestion and burning of methane.  Thus, if the approach to
ultimate disposal is recycle of the waste organic carbon by converting it
to innocuous inorganic carbon, C02> combustion, i.e., consumption of
oxygen, is inevitable.  Since in an activated sludge process the oxygen-
ation energy which is supplied to the system is much in excess1 of the
metabolic requirement, the attractiveness of accomplishing both treat-
ment of the waste and wet combustion of the sludge developed during
treatment can be readily appreciated.

This unique modification of the activated sludge process was suggested
by Forges and his co-workers as a consequence of their research on the
biological treatment of dairy wastes (63).  It was suggested that if  the
biological solids were continually returned to the aeration chamber (no
wasting of excess solids), endogenous respiration would tend to decrease
the solids concentration by about the sa'me amount that the new incoming
carbon source in the waste would tend to increase it, and eventually  a
state of equilibrium might be approached at which there would be no net
solids accumulation.  Thus, the dual function of biological waste water
purification and sludge digestion (aerobic) could be accomplished con-
currently and without recourse to disposal of any biologically produced

At the time the process was suggested, the term "endogenous respiration"
was subject to various interpretations; it was at that time a rather  new
term to the field of biological waste treatment.  Interpreted solely  as
the intra-cellular oxidation of organic carbon, one could argue against
the theoretical  possibility of total oxidation, since it is obvious that
a given microbial cell cannot oxidize itself totally to inorganic car-
bon.  Also, it could be reasoned that if one cell died, not all of its


organic carbon might serve as available sources of carbon for another
species; thus the validity of the concept was suspected.  In any event,
the suggested process modification precipitated much interest and
engendered much research activity by other investigators (e.g., 64, 65).
Two general conclusions emanated from these investigations.  First, it
was concluded that certain component parts of the biological cells syn-
thesized in the purification phase were nonmetabolizable.  Thus there
was a fraction of organic matter which was inert from two points of
view:  it was dead organic matter, therefore incapable of removing sub-
strate, and it could not serve as food material for live cells.  As time
went on and this material was retained in the system, the bio-mass would
thus consist of an ever-increasing inert fraction, and eventually the
system must fail.  The other conclusion, which was somewhat related to
the first, was that a condition of biological  solids equilibrium could
not be attained, i.e., the biological solids concentration would con-
tinually build up.  It was generally felt that biological solids had to
be wasted, or the system could not function.  The concept of total  oxi-
dation was generally concluded to be theoretically untenable, and bio-
logically unsound.  Regardless of these conclusions, many extended
aeration plants have been built, and operational  experience has varied
regarding their worth and the need to waste biological  sludge period-

Recent Investigations of Extended Aeration

A few years ago, based upon some of the experimental observations made
during investigations pertinent to our research on "kinetics and mech-
anism of activated sludge processes" there was, we believed, some cause
to doubt the finality of the previous conclusions concerning the possi-
bility of total  oxidation.  Also, the paradoxical situation in the
field wherein extended aeration plants were being installed in opposi-
tion, as it were, to the conclusions of some engineering researchers,
certainly seemed to require some reconciliation.   Also,  there were no
definitive data on how long an extended aeration  activated  sludge might
continue to function until it experienced failure, i.e., until  it no
longer could function as an effective means of removing  soluble organic
matter from waste waters.  It was reasoned that even if, after a year or
so, some sludge had to be wasted and new bio-mass developed, the process
would still be advantageous since continuous operation of a sludge treat-
ing and disposal  facility would not be necessary.  Thus  it  might have
considerable utility and applicability to treatment of various  industrial
wastes.  All of these considerations contributed  to a decision to enter
upon long-term investigations of the process.

In order to test the theoretical possibility of attaining total  oxida-
tion, i.e., to determine whether continual buildup of biological solids
containing an ever-increasing fraction of biologically  inert solids
occurred, it was  necessary to devise a laboratory pilot  plant scheme
which could guarantee that all  biological  solids  were retained in the


 system.   The  problem of  inadvertent  loss of biological solids  in the
 effluent  had  been  experienced  by  other workers,  and was one of the fac-
 tors which  originally  caused us to doubt the validity of some of the
 conclusions which  had  been drawn  from their results.  The pilot plant
 and operational  techniques we  employed are described in detail in the
 research  literature  (66-68).   Briefly, positive  control over retention
 of biological  solids in  the pilot plant system was maintained by centri-
 fuging all  effluent from the clarifier and returning the solids to the
 aeration  tank.   Thus,  even at  times when solids  could have exited a more
 usual pilot plant, they  did not exit the one which we operated.  A
 small amount  of  solids was removed for analysis.  Such removal amounted
 to no more  than  0.2 per  cent of the bio-mass.

 The organic feed to the  unit consisted of a "synthetic waste" in which
 the carbon  source  was  a  soluble carbohydrate (glucose).  It is impor-
 tant to consider why a simple  soluble compound such as glucose was
 employed, since  it has been the experience of researchers that some in
 the pollution control  field have  a tendency to discount work with "syn-
 thetic wastes."  Some  of the pros and cons of this and other approaches
 to research in the environmental  field have recently been presented by
 Gaudy (69).   In  the present instance, it is important to point out that
 the crux  of the  question concerning the theoretical unsoundness of the
 total oxidation  process  was the supposed non-biodegradability of various
 fractions of  the microbial cells  of which the activated sludge is com-
 posed, e.g.,  the extra-cellular polysaccharide slime layer (64).   It is
 essential,  then, to make sure  that the activated sludge one is examining
 does consist  wholly of microbial  cells.  Obviously, entrapped organics
 such as coffee grounds,  and inorganics such as silt, etc., are relatively
 or completely inert to aerobic biological  treatment.  It is emphasized
 that the  previous  conclusion that the principle of total  oxidation was
 unsound was based  upon experimentations employing simple soluble carbon
 sources,  e.g., acetic  acid (64),  and glucose (65).  While we are in dis-
 agreement with the conclusions of these workers, we agree that their use
 of simple soluble  carbon  sources was wholly justified.

 If one retained all of the biological solids in the system, and if there
 were a permanently inactive organic fraction, the biological  solids con-
 centration should continue to  increase; i.e., one would expect continual
 accumulation  of biological solids.  Also,  if bacterial  polysaccharide in
 the cell capsule or slime layer were biologically inert,  the carbohydrate
 content of the sludge would gradually increase.  The ability of the acti-
 vated sludge  to remove the carbon source in the waste would gradually
 decrease.  Another indication of an increasing inactive fraction of the
 sludge might  be a decrease in basal  rate of respiration (02 uptake rate
 per unit of sludge under  endogenous conditions, i.e., in the absence of
 an extra-cellular source  of carbon).   These and other aspects were inves-
 tigated during a three-year period of continuous pilot plant operation
with centrifugation and  total  solids retention.

 It was found  that the biological solids concentration did not reach an


equilibrium, or steady, value in which "endogenous respiration" balanced
new synthesis.  However, neither did the biological  solids concentration
continually increase.  There were periods of solids  increase and periods
of decreasing biological solids concentration.   During a decreasing
cycle in biological  solids concentration, the organic matter (COD) in
the effluent did not rise, as would have been the case if some cells had
undergone lysis and  the organic matter thus released were not metabo-
lized by the remaining intact cells.  One of the most dramatic downward
cycles occurred during the latter part of the first  year of operation
(67).  Figure 29 shows the operational behavior of the system during this
cycle.  From day 285 to 307, the biological solids concentration decreased
from approximately 8500 to 2400 mg/1.   Despite this  rather sharp drop,
the filtrate COD (effluent) did not rise appreciably.  In fact, there was
only a slightly perceptible rise with  a rapid return to the previous
level.  The COD of the synthetic waste was approximately 530 mg/1 (500
mg/1 glucose), and the filtrate COD was for the most part less than 40
mg/1 except for the  short-lived transient as the solids began to de-
crease, when it rose to approximately  100 mg/1.

Since solids were not wasted (purposely or accidentally), and since they
decreased without any concomitant rise in COD,  and since no external
operational changes  were imposed upon  the system, this downward cycle in
biological solids was a natural one brought about by the system itself.
During such a decreasing cycle in biological solids  concentration with
no rise in effluent  COD, the most apparent explanation is that a por-
tion of the sludge (microbial population) is serving as a source of car-
bon (substrate) for  another portion of the microbial population.  In
short, total oxidation of the organic  material  in the waste must occur
by a combination of  the reactions shown in equations 3 and 13, with
recycling of the carbon through the latter.  It cannot be expected that
the amount of new growth on the incoming substrate will always be bal-
anced by cannibalism of the past growth so as to produce a steady or
equilibrium level of solids.  The predominance of species is constantly
shifting, and it is  necessary that the ecology of the system be such
that some of the cells extant in the system act as food material for
other cells which play the role of feeders.  Neither the periodicity nor
the extent of cyclic accumulation and  de-accumulation of biological sol-
ids can be predicted, but the results  shown in Figure 29 surely attest
to the occurrence of such periods.

It should also be pointed out that during more than  1000 days of oper-
ation there was no evidence for the buildup of carbohydrate content in
the sludge.  Samples taken for measurement of protein and carbohydrate
content of the sludge close to the end of the pilot  plant operation
[e.g., see Figure 5, reference (68)3 snow that the carbohydrate content
was approximately 20%, which represents a normal value for microbial

Many batch feeding tests to determine  the substrate  removal capability
of the sludge were periodically performed (67,  68).   Figure 30 shows


300   305   310
Figure  29.  Performance data  for a continuous flow  extended aeration activated sludge in a
laboratory pilot  plant operated with  total  solids recycle, showing a period of accelerated
autodigestion  (67).




722 MAR 21,1969 |




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                       15     30    45    60    75
                          TIME, MINUTES




                                                                  DAY 987: DEC. II, I969~|
                B.IOLOGICAL SOLIDS
                       A     I
                                                                       FILTRATE  COD
12 g
          15    30     45     60    75
              TIME,  MINUTES
                 Figure  30.   COD removal  capability of  extended aeration activated  sludge
                 after the  number of days of continuous operation with  total solids
                 recycle indicated on each graph (68).

the results of four such experiments.  Even after 987 days of oper-
ation with no sludge being wasted, the system removed the daily feed,
added as a slug dose, within thirty minutes.  Considering the fact that
the retention time in the aeration chamber was sixteen hours under con-
tinuous flow conditions, the capacity for substrate removal was far in
excess of that required even after nearly three years of aging.  This
would not have been possible had the sludge not possessed the ecological
and biochemical capacity for self-renewal, i.e., internal feeding.  It
is important to point out that the basal respiration rate (endogenous 0?
uptake rate; mg Oz/hr/gm sludge) was considerably lower than for a
younger microbial population.  This is an indication that the sludge was
somewhat less active than young cells on the basis of unit weight.  For
example, the endogenous uptake rate for this sludge appeared to level  off
between 1 and 2 mg/hr/gm, as is shown in Figure 31.  Young populations
freshly grown from a small inoculum of cells from the aeration chamber
manifested basal respiration rates approximately ten times higher.  The
rising trend in the values between days 300 and 350 occurred immediately
after the period of accelerated autodigestion (decline in solids)  shown
in Figure 29.  During and after such a period of declining biological
solids concentration, there is a larger fraction of younger cells  in the
sludge because of the higher substrate concentration (i.e., the canni-
balized cells), and as a consequence of this physiological  condition,
higher endogenous 0^ uptake rates were manifested.

The facts that biological solids did not continually accumulate, that
the carbohydrate content did not attain abnormal  proportions, and  that
the system continued to deliver excellent substrate removal efficiency
after three years of continuous operation with total cell recycle, bring
us to the conclusion that the conceptual principle of the total  oxidation
or extended aeration process is not theoretically unsound, and work
designed to gain a more definitive insight into the metabolism of  cell-
ular components is continuing.

In addition to the long-term studies just described, we have run other
experiments in closed batch systems which demonstrate that the total
oxidation of the bio-mass is possible.  Results of one such experiment
are shown in Figure 32 (62).  In this experiment, a relatively low ini-
tial  concentration of acclimated sludge was fed 2280 mg/1 glycerol COD
and various parameters were monitored during the ensuing growth and
"endogenous" periods.  The top portion of the figure shows changes in
protein and carbohydrate content of the sludge as well as ammonia
nitrogen, which was the only source of nitrogen in the system.  The
lower portion of the figure is particularly important for present  con-
siderations, because it can be seen that the concentration of biologi-
cal solids, for all practical purposes, returned in the prolonged  endog-
enous phase to the initial concentration; thus there was no "permanent"
synthesis product.  Essentially total oxidation of the assimilated car-
bon was effected in this closed batch system.

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" A

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250 300 400 500 600 700 800 900 1000 110
             Figure 31.  Effect of aging on endogenous oxygen uptake by an extended aeration acti-
             vated sludge during long-term pilot plant studies employing total solids recycle (68).

20   60    100

   TIME, hrs
400  600  800
   Figure 32.  Total oxidation, during  the  endogenous
   phase, of the biological solids  synthesized  during
   the substrate removal phase in a batch system  using
   a heterogeneous population developed from  a  sewage
   seed (62).

Engineering Control of Total Oxidation

Because the total oxidation process has been shown to be conceptually
sound does not automatically confer upon it practicability or engineer-
ing feasibility, but removal of the onus of conceptual  unsoundness from
the process allows engineering considerations to bring  theory to prac-
tical fruition.  No one can predict how long a total  cell  recycle sys-
tem will continue to accumulate biological  solids before the ecological
(predator-prey) relationship is such as to  initiate a period of accel-
erated autodigestion.  No one can predict the extent of the ensuing
decrease in biological solids concentration.  All that  can be said is
that if the cells can be retained in the system, periods of de-
accumulation will occur.  At times, solids  may accumulate to very high
concentrations, thus making retention difficult with currently employed
field procedures.  It seemed essential, therefore, to explore engineer-
ing possibilities for attaining control of  biological solids concen-
tration, especially ways to initiate the autodigestive  process.

When cells are totally oxidized, all portions of the cell  must be con-
verted to simple inorganic compounds which  include C02» H20, NH3, and
small amounts of inorganic salts.  Even for readily metabolized com-
pounds, approximately five passes through the food chain would be
required for reduction of the original amount of carbon source to the
desired level of purification.  This is based on an assumption of 50%
average yield for metabolism, i.e., the assumption that approximately
50% of a substrate is oxidized and 50% converted to new cell material.
When the newly formed cell is oxidized by other cells,  if  the yield is
again 50%, the amount of original carbon source still present as cell
material is 25%, and each succeeding pass reduces this  by  a factor of
one-half.  For components of the cell which are metabolized with diffi-
culty, long periods of time may be required for complete oxidation.
Initiation of oxidation of cellular components may depend  upon access to
them by other cells which are capable of utilizing them.  Some of the
carbon in a microbial cell is in soluble form, but a large part of it is
incorporated in larger insoluble macromolecules in the  cell membrane, cell
wall, and extra-cellular capsular material, e.g., polysaccharide,-and in
the proteins and nucleic acids within the cell.  Some of the predatory
activity which occurs in the sludge involves the ingestion of particulate
organic matter, e.g., ingestion of some species of bacteria by some spe-
cies of protozoa and other predatory microorganisms,  and other activity
involves the use of organic carbon of some  species of bacteria by other
species of bacteria.  This organic substrate must be transported into the
cell if it is to be metabolized.  Therefore, the first  and probably the
most difficult metabolic task is the breaking down of the  organic macro-
molecules, i.e., their hydrolysis to smaller fragments  which can be taken
into the cell.  It is indeed a very complex ecology which  is needed for
all of these steps to occur.  Our results have demonstrated that they do
occur; i.e., these biological occurrences are not theoretically impos-
sible.  However, one cannot "engineer" the  ecology of the system, at


least not at present.  We are continuing studies to gain insight into
understanding this useful natural occurrence (a prerequisite to attempts
to engineer the natural ecosystem).

There are ways to approach control of the system other than learning to
exert environmental control of the ecosystem.  One such channel, which
we felt warranted investigation, was to combine aspects of chemical and
biological digestion of the sludge.  One of the mildest digestive chemi-
cal operations and the easiest to perform is hydrolysis, the preparatory
step to biological digestion.  If one provided such an engineering assist
to the system, the autodigestive process might be subjected to direct
engineering control.  With the hydrolysis portion of the digestion proc-
ess subject to engineering manipulation, the heterogeneous microbial pop-
ulation might be better able to metabolize that portion of the sludge
which had been hydrolyzed and returned to the aeration compartment.
This process, like the one discussed in the previous chapter,  would rep-
resent a rather radical departure from current practice or concept, but
it seemed a plausible one in view of our previous findings and was con-
sistent with the general aim and need for innovation in the pollution
control field.  Experiments were initiated to test this concept.

Figure 33 shows the course of COD removal when hydrolyzed sludge was
fed to the extended aeration pilot plant in a batch experiment similar
to those shown in Figure 30 in which the synthetic waste (glucose) was
fed.  It is seen that the hydrolyzed sludge was metabolized rather rap-
idly (compare with Figure 30) by the extended aeration activated sludge.
These hydrolyzed cells had been rendered essentially soluble by acid
hydrolysis (pH = 1) for five hours at 15 psi (1.05 kg/sq m) and 121°C.
The hydrolyzed sludge was neutralized to pH 7 and fed as a slug dose to
the pilot plant.

On the basis of such results, we have proposed the modification of the
extended aeration process embodying the concept of the hydrolytic assist
shown in Figure 34 (68).  The type of extended aeration process shown is
the compartmentalized aeration chamber-clarifier with internal  sludge
recycle (dotted arrow).  The lower portion of the flow diagram shows the
hydrolysis process.  This portion of the plant can be considered as an
aid to aerobic sludge digestion in which a portion of the total return
sludge is solubilized before return.  In the extended aeration process,
the return sludge itself is really a waste stream being fed to the sys-
tem like the incoming waste.  The modification proposed in Figure 34
simply prepares a portion of the sludge so that it becomes a more read-
ily available substrate, thus allowing some engineering control over the
biological process.  With the chemical hydrolysis modification, one is
provided with a sludge disposal  aid which need be used only when requir-
ed, and for which there is no long startup time.  The  hydrolysate could
be stored without neutralization, thus preventing odors, etc., and
neutralized as it was channeled back to the aeration chamber at a rate
controlled and selected by the operator.  Such a system uses both chem-
ical  and biological methods of sludge disposal  to their best advantage.


                                               BIOLOGICAL  SOLIDS
  40      50

                Figure 33.  Metabolism of hydrolyzed activated sludge by an extended aeration
                activated sludge  (68).

                                                            EXTENDED AERATION
                                                             HYDROLYSIS PROCESS
           Figure 34.  Proposed extended aeration activated sludge process  incor-
           porating chemical hydrolysis for control of sludge concentration (68).

It performs chemically a function which is biologically difficult, and
performs biologically a function which is difficult and costly to perform
chemically.  Additional  investigational work is warranted and is in
progress; to date we have successfully operated the bench scale pilot
plant with periodic sludge hydrolysis, and shortly a report on these
operational experiences will  be prepared for publication in the techno-
logical literature.

It may be noted in Figure 34  that "raw waste" flows into the aeration
tank.  The figure was drawn in this way because it is generally the
case that where extended aeration is employed, an attempt is made to
require the process to perform the function of primary treatment as well
as secondary treatment and sludge disposal, i.e., there is no primary
settling tank.  In the "traditional" flow sheet for treatment of muni-
cipal wastes, secondary and primary sludges are combined and channeled
to anaerobic digestion, so it is probably natural that with the extended
aeration process, both types  of sludge would be expected to be combined
for aerobic digestion in the  aerator; i.e., it is natural  to look upon
the non-soluble organic material in primary sludge as a substrate just
as the return sludge, the microorganisms, are considered as a particu-
late substrate in the extended aeration process.   However, one does not
expect such panaceas with other activated sludge processes, and there
is no real justification for  expecting an extended aeration activated
sludge to totally oxidize primary sludge.  The probability is that it
will not.  The extended aeration process is best considered as a sec-
ondary biological treatment process.  Also, it is ideal for waste con-
taining primarily dissolved organic matter.  In this regard, it is bet-
ter suited to treatment of some industrial wastes than to municipal
sewage (where it has been largely employed).  However, with the hydro-
lytic assist process we are recommending, it may be possible that some
particulate organic matter in the raw waste which becomes entrapped in
the microbial sludge (and, indeed, is partially metabolizable) can be
hydrolyzed under the same conditions which solubilize the cells.  In
this case, some of the organic matter which normally would be removed by
primary settling is treatable "chemo-biologically."  Some organic com-
pounds in "primary" municipal sludge can undoubtedly serve as excellent
microbial substrates with or  without chemical hydrolysis.  However, con-
stituents such as coffee grounds, for example, would be difficult to
hydrolyze chemically or to metabolize aerobically; thus for municipal
sewage some organic residue should be expected, i.e., total oxidation of
such sludges may not be possible.  However, the residual organic sludge
which would have to be disposed of should be low in amount and, by
virtue of its resistance to both chemical hydrolysis and biological
attack, should be relatively  non-putrescible.  Thus, addition of the
hydrolytic assist should allow the extended aeration process to be very
usefully employed even for municipal wastes.  The non-hydrolyzed sludge
would have to be separated from the hydrolysate prior to channeling the
hydrolysate to the aeration chamber.  Removal of finely divided suspend-
ed organic matter in the hydrolysate could be accomplished with chemical
addition (coagulation, flocculation, and settling) following the hydrolysis


unit.  This would also remove finely divided inorganic matter which may
pass the grit chamber and be entrapped in the sludge.  It should be
apparent that suspended matter which is initially inert, i.e., non-
biodegradable, should remain so after passage through the aeration cham-
ber, after possible entrapment in the bio-mass and after hydrolysis.
Indeed, the hydrolysis modification including chemical precipitation of
permanently inert material originally in the waste stream provides a
useful  way of separating the pollutional (treatable) from the non-
pollutional material in the waste.


In summary, based upon long-term and thorough studies, we are able to
conclude that the extended aeration or total oxidation process is not
conceptually unsound.  The proposed modification of the process embody-
ing the hydrolytic assist offers an effective way to combine ideal
features of chemical and biological treatment for concurrent treatment
of the waste water and essentially total aerobic digestion of sludge
developed in the process.



At the outset it was emphasized that this document was not intended to
serve as a manual for design and operation of activated sludge process-
es, but as a conceptual guide for updating and unifying mechanistic and
kinetic principles which bear strongly on design and operation.  The
real aim was to help close the so-called "gap" between research and
practice, and this we have attempted to do by relating, largely in
readily understood engineering terms, many of the findings of our funda-
mental research effort and the concepts which have been developed over
the past decade.  While our research has been, and in large measure will
always be, designed to gain better basic understanding of biological
processes, our ultimate aim is the useful application of the informa-
tion.  Thus, throughout this document, presentation and discussion of
conceptual principles have been accompanied by embodiment of these prin-
ciples in recommended procedures, models, process innovations, etc., for
the consideration of the profession.

Beginning with generalized concepts of exertion of biochemical oxygen
demand and use of ACOD as the measure of biologically treatable organic
material, through discussion of the stoichiometry of the treatment proc-
ess and development of growth kinetics, incorporation of the kinetic
relationships into a relatively simple model  for description of com-
pletely mixed growth reactors and, finally development of a modified
kinetic model specifically for the activated sludge process, the approach
has been to foster better understanding and to provide recommendations
leading to engineering control over the biological treatment process.
The model finally evolved in Chapter V, equations 43 through 47, embodies
the concepts developed in the previous chapters, and represents an engi-
neering modification of basic kinetic theory.  The modification, i.e.,
holding XR rather than c as a system constant, complicates the kinetic
equations somewhat but allows for practical attainment of greater stead-
iness in X and S.  The goal in biological treatment is attainment of
steady, reliable delivery of the needed degree of process efficiency,
and the recommended model for design and operation should foster this
aim.  The graphs of Figure 18 or 19 can be used as a rough guide to de-
sign, although the engineer should ideally determine values of the bio-
logical constants ^^v, KS» anc' Y for the particular waste in question.
In computing the graphs shown in Figures 18 and 19, values of the phys-
ical constants chosen were XD = 10,000 mg/1, and a = 0.25, which are
reasonably selected values; however, the design engineer can, with the
model, select values of his own choice and can test the effect of his
selection on X and S.

It has been emphasized that if one desires a reasonable biological engi-
neering model for a process, he must provide as best he can in his de-
sign the facilities for control of the parameters included in the model.


Thus, means of controlling XR nearly constant at the selected value
should be provided.  This requires the incorporation of a  sludge con-
sistency tank, i.e., a revision of the "traditional" flow  sheet.  The
sludge consistency tank is somewhat analogous to the make-up tank for
chemical dosage used in chemical  treatment.   The sludge consistency
tank is one in which the biochemical  "dosage" to the activated sludge
aeration tank is prepared.  Also, some attempt to even  out fluctuations
in F and S^ may be required either on the line or at the treatment
plant.  The use of an equalization basin to  accomplish  this is recom-
mended where practicable.  While  equalization basins have  been used in
some installations, their use is  a departure from the general  practice.
Thus, the newer concepts we have  introduced  for the  "normal" activated
sludge process, involve some changes  in the  usual  or traditional  flow
diagram.  These changes are intended  to help the system approach  the
"steady state" assumption of the  kinetic model.

One should realize that regardless of which  processes one  employs in
the treatment of waste waters—chemical, physical, or biological, or any
combination thereof—an effort must be made  to hold  the system steady or
to protect against drastic changes in the operational environment.
After all, chemical-physical treatment plants are just  as  likely  to
undergo stress leading to fluctuations in efficiency, due  to changes in
S-j, F, or the amount of coagulant or  adsorbent chemicals added,  as  are
biological treatment plants.  For example, one would not think of chem-
ical treatment without control of chemical dosage.   In  like manner, one
should also provide control of the biological  dosage, i.e., XR,  or, in
any event, should not let XR fall below a selected level.   Thus,  build-
ing in the engineering control to make the process function in accord
with the preconceived notion (some sort of design model) is not incon-
gruous with accepted engineering  practice.   It is probably more true of
biological treatment systems than of  chemical-physical  ones, that the
system possesses an internal resistance and  responsiveness to external
change.  For example, the agents  for  removal  of organic matter (the
microorganisms) are self-producing, and do essentially  "rise to the
occasion" of high organic loadings, i.e., there is a self-correcting
feature in biological systems which,  if not  overloaded  by  abusive exter-
nal  change, serves to protect the system and to provide some internal
regulation and restoration of efficiency when disruption does occur.

While the material in Chapters II through VI can be  said to pertain to
general understanding of activated sludge processes, and in these chap-
ters some engineering innovations which depart from  traditional  practice
were introduced, in Chapters VII  and  VIII more radical  departures from
the norm were covered.  In Chapter VII, the  effect of nitrogen as limit-
ing nutrient and the effect of biological solids concentration on the
predominant biological mechanism  of substrate removal were delineated.
The new process modification which we have termed "continuous oxidative
assimilation," has been demonstrated  to be feasible, at least in labor-
atory scale pilot plant studies.   The process has unique possibilities
for nitrogen-deficient industrial wastes and, in addition, should serve


to demonstrate that some of the older concepts and nutritional guide-
lines are by no means inviolable.

It should perhaps be pointed out that the finding that COD may be
removed primarily by oxidative assimilation in batch or continuous flow
systems containing high concentrations of biological solids does not
negate the use of kinetic models for nitrogen-containing wastes, based
on equations for microbial growth, such as those presented in Chapter V.
The continuous oxidative assimilation system is one in which substrate
removal and cell replication are separated artificially.  The cells are
forced to carry out oxidative assimilation and protein synthesis in a
completely sequential manner by addition of carbon source and nitrogen
source to separate aerators.  The sequential synthesis of storage prod-
ucts and nitrogen-containing cellular components observed in batch sys-
tems, even in the presence of nitrogen, would undoubtedly be observed in
activated sludge systems closely resembling plug flow reactors.   It is
not possible to determine whether such sequential synthesis occurs to an
appreciable degree in completely mixed reactors with high solids, i.e.,
in reactors employing cell recycle, because one cannot examine synthesis
in individual cells and it is the newly recycled cells which would be
most likely to carry out oxidative assimilation.  We consider it likely
that oxidative assimilation does occur to some degree in these reactors.
Activated sludge is not completely comparable to the usual  growing sys-
tems of the microbiologist or fermentation engineer.  In the first
place, as was brought out in Chapter V, the recycling of cells forces a
very slow rate of replication, a rate which is only a fraction of the
system dilution rate.  Secondly, the recycled cells have an immediate
past history of existence in an environment essentially devoid of car-
bon source (the clarifier).  Because of these two reasons,  as well  as
the general starvation condition of the system, these cells cannot be
classified as physiologically young, actively growing microorganisms.
Therefore, when sludge is returned to the aerator, the cells may remove
carbon source initially by oxidative assimilation, possibly to an extent
depending primarily upon the retention time in the secondary clarifier.
However, in systems in which the sol ids:COD ratio is high,  i.e., sys-
tems in which 1 + a - ac is small, the doubling time for the population
may extend over several  aeration periods.  Therefore, in high solids
systems, a very long period of time is available to the cell to  prepare
for replication.  For maintenance of a steady state in X in systems with
recycle, it is only necessary that cells  replicate at a very slow rate
or, in other words, that a relatively small percentage of the cells in
the aerator replicate during one detention period.  Since batch  studies
have shown that in high  solids systems the rate of increase in X (meas-
ured in units of mass, not cell  numbers)  is not very different for cells
which are utilizing carbon source in the  presence of nitrogen or in its
absence, the occurrence  of oxidative assimilation during part of the
cell  division cycle of individual cells should have no noticeable effect
on the kinetics of the system.

Chapter VIII deals with  the extended aeration process, which incorpor-
ates the important aspects of sludge disposal  and secondary treatment of


the waste.  It was shown that this process is  not biologically unsound
and a new modification of the process (the hydrolytic assist), which
allows engineering control  of the process, was proposed.   This modifi-
cation is a union of chemical and biological  treatment which can pro-
vide a useful alternative or replacement for  anaerobic digestioir-end/or
incineration of waste organic matter from treatment plants.   It accom-
plishes the same thing as these processes, i.e.,  conversion  of organic
carbon to inorganic carbon.   In the long range, any method of disposal
which provides for recycle  of carbon via conversion of waste organic
carbon to CO? for ultimate  reconversion via the photosynthetic process,
may become obsolete, since  this process is a  slow recycle and the
organic carbon in the waste may need to be recycled for man's use by
more direct means; e.g., it may eventually be  a usable byproduct of water
rejuvenation or a unique raw material  for production of useful  commodi-^
ties.  However, in the near future, the conversion of these  waste organic
sludges to C02 by whatever  means, and the dissipation of  C02 into the
atmosphere represents, if not the ultimate, the "penultimate" sludge
disposal  method.  This is by any means a combustion process, and the
chemically-assisted wet aerobic biological combustion we  have herein
recommended for consideration of the profession should be accomplishable
without the remedial and often costly expedients  to abate air pollution
problems usually required with other means of  disposal.

Figure 35 shows a possible  flow diagram for an activated  sludge plant
which embodies many of the  conceptual  principles  discussed in this
report.  Since there are really no standard wastes, there is no standard
way of treating all wastes.   Accordingly, all  activated sludge process
schemes should not be expected to look the same,  but each should be
tailored to the unique requirements of the particular situation.  Unique-
ness and engineering innovation rather than standardization  should be the
rule, provided that the engineer can defend the reasonableness of the
concepts and has performed  pre-design investigations to justify omission
or addition of any particular unit operation  or process in his overall
plant design.  While we feel that the flow diagram shown  in  Figure 35
may offer considerable improvement over the more  or less  standardized
flow diagrams which have traditionally become  associated  with activated
sludge processes, we do not offer it as a new  standard flow  sheet but
as the general embodiment of many of the conceptual principles we have
developed in the previous chapters.  It is in  a design sense, a tenta-
tive "diagrammatic" conclusion which seems a meaningful result of the
transference of conceptual  principles and research findings  to function-
al design.  It is readily appreciated that this flow diagram differs
from the traditional representation of an activated sludge plant in a
number of ways.

The essential units are shown in solid lines  and  those which may or may
not be needed, depending upon the particular  situation, are  shown in
broken lines; the surge basin falls into the  latter category.  If the
waste flow and strength can be regulated within the guideline limits
previously given without a  surge basin, and if drastic changes in the


  i—i   i     i
  GRIT   L-I-J

                                                            N, P, ETC.
                                  | PRECIPITATION


     Figure 35.   Possible flow diagram  for  activated sludge
     process incorporating suggested modifications for oper-
     ational control of purification efficiency and including
     sludge disposal by aerobic autodigestion aided by chem-
     ical  hydrolysis.  This diagram is  based  on the conceptual
     principles  and research findings presented in this report,

types of organic compounds in the waste are not expected to occur, the
surge basin is not needed.  The main aerobic reactor (aeration tank 1),
the clarifier, and the sludge consistency tank (aerator 2)  are in accord
with Figure 16, and permit design (and operation)  in accordance with the
model equations (43 through 47) presented in Chapter V, holding XR as a
system constant.  Also, the flow sheet accommodates  the principle of
total (wet biological) oxidation and employs the hydrolytic assist dis-
cussed in Chapter VIII.  Thus, disposal  of biological  solids is effected
via the concept of extended aeration, but the substrate removal and the
autodigestive phases are separated by design in order that  biological
solids concentration in aerator 1 and subsequently in the clarifier, can
be controlled within operational limits selected by  the design.  Also,
separation of the purification (aerator 1) and "endogenous" phases
(aeration tank 3) should enhance control  of the latter (i.e.,  the auto-
digestive) phase.

Into aeration tank 1 flow the major portion of the influent waste, the
recycle sludge XR from aeration tank 2,  and a significant portion of the
hyrolysate, and all mixed liquor exits to the clarifier.  Into aerator
2, the sludge consistency tank, flow  a portion of the clarifier under-
flow, and three additional inflows.   One consists  of biological solids
from aerator 3; these may be bled in continuously, or as  needed to help
maintain constant XR.  Both of the remaining inflows consist essentially
of substrates (some of the waste inflowing to aerator 1 and some of the
hydrolysate).  These bleeder lines of inflowing carbon source  function
essentially as sludge "fresheners,"  and help pre-condition  the biological
solids for reentry to aeration tank 1.  The sludge has been existing
outside of the growth environment of aeration tank 1 for some  time, and
addition of some of the substrates with which it will  shortly  be con-
tacted should help to reduce the readjustment or lag period in aerator 1.
A lag period at high solids concentration can force  oxidative  assimi-
lation and, for wastes which contain ample amounts of nitrogen, it is
best to curtail oxidative assimilation because this  process conserves
nitrogen and could possibly increase nitrogen leakage in the effluent.

Aeration tank 3 receives the excess  sludge from the  clarifier.  This
tank can be looked upon as a modified aerobic digester which receives,
in addition to biological solids, some of the incoming waste and some
hydrolysate.  The dual function of the aeration tank of an  extended
aeration plant is here accomplished  in two separate  tanks,  substrate
removal in aerator 1 and autodigestion of the excess solids in aeration
tank 3.  The major portion of the outflow from aeration tank 3 is cycled
through the hydrolysis unit, and a small  portion may be recycled through
aeration tank 2 to aeration tank 1.

The hydrolysis unit receives mixed liquor from aerator 3.  Also, pro-
vision can be made to pump some clarifier underflow  directly to the
hydrolysis unit.  For wastes containing settleable organic  solids, the
primary sludge can be channeled to the hydrolysis  unit, where the
hydrolyzable portion can be liquified and recycled to the treatment


plant, i.e., to aeration tank 1, 2, or 3.  For waste containing non-
hydrolyzable participate organic matter or suspended inorganic matter,
effluent from the hydrolysis unit can be subjected to chemical treat-
ment (shown in the lower portion of Figure 35) to precipitate this
material prior to recycling hydrolysate to the treatment plant.  These
precipitated solids, i.e., non-hydrolyzable particulate organic matter
and suspended inorganic solids are labeled "inert solids."  They are not
inert solids produced in the treatment process, i.e., they are not pro-
duced from the biochemically Op-demanding organic matter in the waste
(ACOD), but are materials which were initially present as biologically
inert organic or inorganic matter.  It can be expected that municipal
sewage and some industrial wastes would contain some such materials and
the chemical treatment unit could prove useful in effecting separation
of these "inerts" while the chemical hydrolysis-assisted aerobic digestion
is used to provide "ultimate" disposal of the biological solids.  The
type of flow diagram shown in Figure 35 is, to be sure, a radical  de-
parture from current picturizations of activated sludge plants.  This
flow sheet permits incorporation of new principles and there are various
modifications of the flow sheet which could easily be devised.

Figure 36 shows one such modification which uses essentially the same
core unit processes in a somewhat different mode of operation.  In the
flow diagram of Figure 35, the hydrolysis unit was set up for continuous
operation.  Alternatively (Figure 36), aeration tank 3 and the hydrolysis
unit could be operated essentially as a separate extended aeration proc-
ess which receives a portion of the waste flow, diverted from the inflow
to aeration tank 1, and all of the excess sludge from the clarifier.   The
primary difference between the two flow sheets is the inclusion of a
settling compartment ir. aerator 3 in Figure 36.  In this case, the hydro-
lytic unit could be used intermittently or continuously for control  of
solids level in aerator 3, as discussed in Chapter VIII (see Figure 34).
In addition, aerator 3 could be used to accept increases in flow,  thus
helping to buffer aerator 1 against hydraulic shocks (changes in F).
In both flow diagrams (Figures 35 and 36), the portion of the plant
which includes aerator 1, the clarifier, and aerator 2 is designed to
operate according to the kinetic model developed in Chapter V (equa-
tions 43-47).  It should also be pointed out that recycling of hydro-
lysate to aerator 1 can be used to conserve nitrogen for wastes which
are low in nitrogen.

While these flow schemes may seem innovative or imaginative, the concep-
tual scheme does not require a "far stretch" of the engineering imagi-
nation.  All new conceptual principles require scrutiny and question by
the field and considerable developmental work before being put into gen-
eral practice.  The extrapolation of research findings represented by
the flow diagrams in Figures 35 and 36 is presented in order that its
pros and cons can be considered by the whole of the profession.

In closing, let us re-emphasize what was said at the beginning:  This
report is presented as an advancement of conceptual principles, surely


Figure 36.  One of several  possible modifications
of the flow diagram shown in  Figure 35.  See text
for details.

not an attempt to write finis to principles applicable to biological
treatment.  The essence of engineering is an ordered and justifiably
cautious change based upon definitive scientific fact.  We have docu-
mented the concepts with investigational  findings, and extrapolated
the concepts to possible process modifications for further scrutiny by
the field.  Our aim has been to improve and, if we can hope to write
finis to anything at all, it is to the "bio-stasis" and dogmatized
concepts with which engineering of the biological  process has  been
traditionally approached.


We are grateful to the Environmental Protection Agency for its sup-
port of this project (17090 FQJ).  Moreover, we gratefully acknow-
ledge its support and that of the federal agencies preceding it
(FWPCA and USPHS-NIH) for support of three major research projects
(WP-00075, WP-00325, and WP-00786) which in considerable measure
made possible the present research undertaking.  Also, some of the
experimental work which contributed to the concepts herein developed
was supported under project A-017, WRRI-USDI, and this support is
gratefully acknowledged.  Our research was accomplished in associa-
tion with many of our MS and PhD graduate students.  Some students
were employed as research assistants on the projects cited above,
and others received financial support as graduate trainees (WP-19)
or were institutionally supported.  In addition to the technological
information and principles represented by this report, the graduates,
who have helped accomplish and have been helped by accomplishment of
this research, are themselves no mean by-product of this effort.   We
gratefully acknowledge their help as students and as colleagues in
the work, and they join us in acknowledging the supporting agencies.
We are also grateful to Mrs. Grayce Wynd for her invaluable assist-
ance in preparing the final manuscript and for the contributions  she
has made to the research team over the past decade as our research


 1.  Jennelle, E. M., and Gaudy, A.  F.  Jr.,  "Studies  on  the  Kinetics
     and Mechanism of BOD Exertion in Dilute Systems."   Biotech.
     Bioeng., 12, 519-539 (1970).

 2.  Gaudy, A. F. Jr., "Biochemical  Oxygen Demand."   In  Water Pollu-
     tion Microbiology, John Wiley (in  press)(1971).

 3.  Bhatla, M.  N., and Gaudy,  A.  F.  Jr.,  "Sequential Substrate Removal
     in a Dilute System by Heterogeneous  Populations."   Appl. Microbiol.,
     13, 345-347 (1965).

 4.  Busch, A. W., "BOD Progression  in  Soluble  Substrates."  Sewage Ind.
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 5.  Wilson, I.  S., and Harrison,  M.  E.,  "The Biochemical Treatment of
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 6.  Gaudy-, A. F. Jr., Bhatla,  M.  N., Follett,  R.  H., and Abu-Niaaj, F.,
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     of BOD."  J. Water Pollution  Control  Fed.,  37, 444-459  (1965).

 7.  Gaudy, A. F. Jr., Bhatla,  M.  N., and  Abu-Niaaj, F., "Studies on the
     Occurrence of the Plateau  in  BOD Exertion."   Proceedings 18th
     Industrial  Waste Conf., Purdue  Univ., 183-193  (1963).

 8.  Bhatla, M.  N., and Gaudy,  A.  F.  Jr.,  "Studies on the Causation of
     Phasic Oxygen Uptake in High  Energy  Systems."  J. Water Pollution
     Control Fed., 38, 1441-1451 (1966).

 9.  Bhatla, M.  N., and Gaudy,  A.  F.  Jr.,  "Studies on the Plateau in
     Oxygen Uptake During Exertion of Biochemical  Oxygen Demand by Pure
     Cultures of Bacteria."  Biotech. Bioeng.,  7,  387-404 (1965).

10.  Bhatla, M.  N., and Gaudy,  A.  F.  Jr.,  "Role  of Protozoa in the
     Diphasic Exertion of BOD."  J.  San.  Eng. Div., Proc. ASCE, 91,
     SA3, 63-87 (1965).

11.  Gaudy, A. F. Jr., and Gaudy,  E.  T.,  "Microbiology of Waste Water
     Purification."  Ann. Rev.  Microbiol., 20,  319-336 (1966).

12.  Symons, J.  M., McKinney, R. E.,  and  Hassis, H. H.,  "A Procedure
     for Determination of the Biological Treatability of Industrial
     Wastes."  J. Water Pollution  Control  Fed.,  32, 841-852  (1960).

13.  Hiser, L. L., and Busch, A. W.,  "An  8-Hour  Biological Oxygen Demand
     Test Using Mass Culture Aeration and  COD."  J. Water Pollution
     Control Fed., 36, 505-516  (1964).

14.  Isaacs, W.  P=, and Gaudy,  A.  F.  Jr.,  "Comparison  of  BOD  Exertion
     in a Simulated Stream and  in  Standard BOD  Bottles."   Proceedings
     22nd Industrial  Waste Conf.,  Purdue Univ.,  165-182  (1967).

15.  Isaacs, W.  P., and Gaudy,  A.  F.  Jr.,  "Atmospheric Oxygenation  in
     a Simulated Stream."   J. San.  Eng. Div.  Proc. ASCE,  94,  SA2, 319-
     344 (1968).

16.  Gaudy, A.  F.  Jr.,  Bhatla,  M.  N.,  and  Gaudy, E. T., "Use  of Chem-
     ical Oxygen Demand Values  of  Bacterial Cells in Waste Water
     Purification."  Appl. Microbiol.,  12,  254-260 (1964).

17.  Gaudy, A.  F.  Jr.,  Turner,  B.  G.,  and  Pusztaszeri, S., "Biological
     Treatment  of Volatile Waste Components."  J. Water Pollution Con-
     trol Fed.,  35, 75-93  (1963).

18.  Goswami,  S. R.,  and Gaudy, A.  F.  Jr.,  "Removal of Aldehydes and
     Ketones by  Stripping  and by Combined  Stripping and Microbial
     Metabolism."   Proceedings, 4th Mid-Atlantic Industrial Waste Con-
     ference,  University of Delaware  (in press)(1970).

19.  Herbert,  D.,  Elsworth, R., and Telling,  R. C., "The  Continuous
     Culture of  Bacteria;  a Theoretical and Experimental  Study."
     J. Gen. Microbiol., 14, 601-622  0956).

20.  Gaudy, A.  F.  Jr.,  Yang, P. Y., Bustamante, R., and Gaudy, E. T.,
     "'Slippage1 in the Monod Equation."   (Submitted for  publication.)

21.  Gaudy, A.  F.  Jr.,  Obayashi, A.,  and Gaudy, E. T., "Control of
     Growth Rate by Initial  Substrate  Concentration at Values Below
     Maximum Rate."  (Submitted for publication.)

22.  Gaudy, A.  F.  Jr.,  Ramanathan, M.,  and  Rao, B. S., "Kinetic Behavior
     of Heterogeneous Populations  in Completely Mixed Reactors."
     Biotech.  Bioeng.,  9,  387-411  (1967).

23.  Monod, J.,  "Recherches sur la Croissance des Cultures Bacteriennes."
     Hermann et  Cie,  Paris (1942).

24.  Schaefer, W., "Recherches  sur  la  Croissance du Mycobacterium
     tuberculosis  en  Culture Homogene.  Ann.  Inst. Pasteur, 74, 458-
     463 (1948).

25.  Monod, J.,  "The  Growth of  Bacterial Cultures."  Ann. Rev. Microbiol
     3, 371-394  (1949).

26.  Peil,  K. M.,  and Gaudy, A. F. Jr., "Kinetic Constants for Aerobic
     Growth of Microbial Populations  Selected with Various Single Com-
     pounds and  with  Municipal  Wastes  as Substrates."  Appl  Microbiol
     21, 253-256 (1971).

27.  Ramanathan, M., and Gaudy, A.  F.  Jr., "Effect of High Substrate
     Concentration and Cell Feedback on Kinetic Behavior of Heter-
     ogeneous Populations in Completely Mixed Systems."   Biotech.
     Bioeng., 11, 207-237 (1969).

28.  Ramanathan, M., and Gaudy, A.  F.  Jr., "Studies on Sludge  Yield
     in Aerobic Systems."  Proceedings 26th Industrial Waste Conf.,
     Purdue Univ. (in press)(1971).

29.  Gaudy, A. F. Jr., and Ramanathan, M., "Variability  in Cell  Yield
     for Heterogeneous Microbial Populations  of Sewage Origin  Grown
     on Glucose."  Biotech. Bioeng., 13, 113-123 (1971).

30.  Monod, J., "La Technique de Culture Continue.   Theorie et Appli-
     cations."  Ann. Inst. Pasteur,  79, 390-410 (1950).

31.  Herbert, D., "A Theoretical Analysis of  Continuous  Culture  Sys-
     tems."  In Continuous Culture  of  Micro-organisms.    Soc.  Chem.
     Ind. Monograph No. 12, 21-53 (196TT

32.  Ramanathan, M., and Gaudy, A.  F.  Jr., "Steady-State  Model for
     Activated Sludge with Constant Recycle Sludge  Concentration."
     Biotech. Bioeng., 13, 125-145  (1971).

33.  Storer, F. F., and Gaudy, A. F. Jr., "Computational  Analysis of
     Transient Response to Quantitative Shock Loadings of Heterogen-
     eous Populations in Continuous  Culture."  Environ.  Science  Technol.
     3, 143-149 (1969).

34.  Young, T. B., Bruley, D.  F., and  Bungay, H.  R.  Ill,  "A Dynamic
     Mathematical Model of the Chemostat." Biotech.  Bioeng.,  12, 747-
     769 (1970).

35.  Thabaraj, G. J., and Gaudy, A.  F. Jr., "Effect of Dissolved Oxygen
     Concentration on the Metabolic  Response  of Completely Mixed
     Activated Sludge."  J. Water Pollution Control  Fed., 41,  R322-
     R335 (1969).

36.  Gaudy, A. F. Jr., "Studies on  Induction  and Repression in Acti-
     vated Sludge Systems."  Appl.  Microbiol., 10,  264-271  (1962).

37.  Gaudy, A. F. Jr., Gaudy, E. T., and Komolrit,  K., "Multicomponent
     Substrate Utilization by Natural  Populations and a  Pure Culture
     of Escherichia coli."  Appl. Microbiol., 11, 157-162 (1963).

38.  Gaudy, A. F. Jr., Komolrit, K., and Bhatla, M.  N.,  "Sequential
     Substrate Removal in Heterogeneous Populations."  J. Water
     Pollution Control Fed., 35, 905-922 (1963).

39.  Gaudy, A. F. Jr., Komolrit,  K.,  and  Gaudy,  E.  T.,  "Sequential
     Substrate Removal in Response to Qualitative  Shock Loading  of
     Activated Sludge Systems."   Appl.  Microbiol.,  12,  280-286  (1964).

40.  Komolrit, K., and Gaudy,  A.  F. Jr.,  "Substrate Interaction
     During Shock Loadings to  Biological  Treatment  Processes."   J.
     Water Pollution Control Fed., 38,  1259-1272 (1966).

41.  Komolrit, K., and Gaudy,  A.  F. Jr.,  "Biochemical Response of
     Continuous-flow Activated Sludge Processes  to  Qualitative Shock
     Loadings."  J.  Water Pollution Control  Fed.,  38, 85-101  (1966).

42.  Krishnan, P., and Gaudy,  A.  F. Jr.,  "Studies on  the Response of
     Activated Sludge to Shock-loadings."  Biotech.  Bioeng.,  7,  455-
     470 (1965).

43.  Kincannon, D. F., Gaudy,  A.  F. Jr.,  and Gaudy,  E.  T.,  "Sequential
     Substrate Removal by Activated Sludge After a  Change in  Salt Con-
     centration."  Biotech.  Bioeng.,  8, 371-378  (1966).

44.  Grady, C. P. L.  Jr., Gaudy,  A. F.  Jr.,  and  Gaudy,  E. T., "Control
     Mechanisms Operative in a Natural  Microbial Population Selected
     for Its Ability to Degrade L-Lysine.  I.  Effect of Glucose in
     Batch Systems."  Appl. Microbiol., 18,  776-784  (1969).

45.  Grady, C. P. L.  Jr., and  Gaudy,  A. F. Jr.,  "Control Mechanisms
     Operative in a  Natural Microbial  Population Selected for Its
     Ability to Degrade L-Lysine.   II.  Effects  of  Fructose and
     Ribose in Batch Systems." Appl.  Microbiol., 18, 785-789 (1969).

46.  Grady, C. P. L.  Jr., and  Gaudy,  A. F. Jr.,  "Control Mechanisms
     Operative in a  Natural Microbial  Population Selected for Its
     Ability to Degrade L-Lysine.   III.   Effects of Carbohydrates in
     Continuous-flow Systems Under Shock  Load Conditions."  Appl.
     Microbiol.,  18, 790-797 (1969).

47.  Kincannon, D. F., and Gaudy,  A.  F. Jr., "Response  of Biological
     Waste Treatment Systems to Changes in Salt  Concentration."
     Biotech. Bioeng., 10, 483-496 (1968).

48.  Goel, K. C., and Gaudy, A. F. Jr., "Studies on  the Relationship
     Between Specific Growth Rate and  Concentration  of  Nitrogen  Source
     for Heterogeneous Microbial  Populations of  Sewage  Origin."
     Biotech. Bioeng., 11, 67-78  (1969).

49.  Sawyer, C. N.,  "Bacterial Nutrition  and Synthesis."  In  Biolog-
     ical  Treatment  of_ Sewage  and  Industrial Wastes,  edited by J. McCabe
     and W. W. Eckenfelder, Jr.,  3-17.  Reinhold Publishing Corp.,
     New York (1956).

 50.   Goel,  K.  C.,  and  Gaudy, A.  F.  Jr.,  "Regulation of Nitrogen Levels
      for Heterogeneous Populations  of  Sewage Origin Grown 'in Completely
      Mixed  Reactors."   Biotech.  Bioeng.,  11, 79-98  (1969).

 51.   Helmers,  E. N., Frame, J. D.,  Greenberg, A. E., and Sawyer, C. N.,
      "Nutritional  Requirements in the  Biological Stabilization of
      Industrial  Wastes.   III.  Treatment  with Supplementary Nutri-
      ents."  Sew.  Ind.  Wastes, 24,  496-507  (1952).

 52.   Rao, B. S., and Gaudy, A. F. Jr., "Effect of Sludge Concentration
      on  Various  Aspects  of Biological Activity in Activated Sludge."
      J.  Water  Pollution  Control  Fed.,  38, 794-812 (1966).

 53.   Krishnan,  P., and Gaudy, A. F. Jr.,  "Substrate Utilization at
      High Biological Solids Concentrations."  J. Water Pollution Con-
      trol Fed.,  40, R54-R66 (1968).

 54.   Komolrit,  K., Goel,  K. C.,  and Gaudy, A. F. Jr., "Regulation of
      Exogenous  Nitrogen  Supply and  Its Possible Applications to the
      Activated  Sludge  Process."  J. Water Pollution Control Fed., 39,
      251-266  (1967).

 55.   Gaudy, A.  F.  Jr.,  Goel, K.  C., and Freedman, A. J., "Activated
      Sludge Process Modification for Nitrogen-deficient Wastes."
      In  Advances in Water Pollution Research, Pergamon Press, New York,
      613-623  (1969T.

 56.   Goel,  K.  C.,  and  Gaudy, A.  F.  Jr., "Regeneration of Oxidative
      Assimilation  Capacity by Intracellular Conversion of Storage
      Products  to Protein."  Appl. Microbiol., 16, 1352-1357 (1968).
                                                ,          *,
 57.   Gaudy, A.  F.  Jr., Goel, K.  C., and Gaudy, E. T., "Application of
      Continuous  Oxidative Assimilation and Endogenous Protein Synthe-
      sis to the  Treatment of Carbohydrate Wastes Deficient in Nitro-
      gen."  Biotech. Bioeng., 11, 53-65  (1969).

,58.   Gaudy, A.  F-  Jr.,  Goel, K.  C., and Gaudy, E. T., "Continuous
      Oxidative Assimilation of Acetic Acid and Endogenous Protein
      Synthesis Applicable to Treatment of Nitrogen-deficient Waste
      Waters."  Appl. Microbiol., 16, 1358-1363 (1968).

 59.   Goel,  K.  C.,  and  Gaudy, A.  F.  Jr.,  "Development of a New Method
      for Treatment of  Nitrogen-deficient  Wastes."  Proceedings 2nd
      Mid-Atlantic  Industrial Waste  Conf., Drexel Institute of Tech-
      nology, Philadelphia, 194-216  (1968).

 60.   Gaudy, A.  F.  Jr., and Goel, K. C.,  "Oxidative Assimilation of
      Nitrogen^deficient Industrial  Waste."  Proceedings 16th Ontario
      Industrial  Waste  Conf., 111-123  (1969).

61.  Gaudy, A.  F.  Jr., and Engelbrecht,  R.  S.,  "Basic  Biochemical  Con-
     siderations During Metabolism in Growing versus Respiring  Sys-
     tems ."  Advances in Biological  Waste Treatment. Vol.  II, Pergamon
     Press, New York, 11-26 (1963).

62.  Thabaraj,  G.  J., and Gaudy,  A.  F. Jr., "Effect of Initial  Biolog-
     ical Solids Concentration and Nitrogen Supply on  Metabolic Pat-
     terns  During Substrate Removal  and  Endogenous Metabolism."
     J. Water Pollution Control  Fed., 43, 318-334  (1971).

63.  Porges, N., Jasewicz, L., and Hoover,  S. R.,  "Aerobic Treatment
     of Dairy Wastes."  Appl.  Microbiol.  1, 262-270 (1953).

64.  Symons, J. M., and McKinney, R.  E.,  "The Biochemistry of Nitrogen
     in the Synthesis of Activated Sludge." Sew.  Ind.  Wastes,  30,
     874-890 (1958).

65.  Busch, A.  W., and Myrick, H. N., "Food-Population Equilibria  in
     Bench-scale Bio-oxidation Units."   J.  Water Pollution Control Fed.,
     32, 949-959 (1960).

66.  Ramanathan, M.,  Gaudy, A. F. Jr., and  Ragthaidee,  W., "Responses
     of Extended Aeration Activated  Sludge  to Quantitative Shock
     Loads."  Proceedings 19th Oklahoma  Industrial Wastes and Pollution
     Control Conf., Oklahoma  State Univ.  (1968).

67.  Gaudy, A.  F.  Jr., Ramanathan, M., Yang, P- Y., and DeGeare, T. V.,
     "Studies on the Operational  Stability  of the Extended Aeration
     Process."   J. Water Pollution Control  Fed., 42, 165-179 (1970).

68.  Gaudy, A.  F.  Jr., Yang,  P.  Y.,  and  Obayashi, A. W., "Studies  on
     the Total  Oxidation of Activated Sludge With and  Without Hydro-
     lytic  Pretreatment."  J.  Water  Pollution Control  Fed., 43, 40-54

69.  Gaudy, A.  F.  Jr., "Comparative  Utility of  Research Employing Model
     Synthetic  Systems and Natural  Field  Systems."  Biotech. Bioeng.
     (in press)(1971).

                      PATENTS AND PUBLICATIONS
Although several of the process modifications described herein would
seem to constitute patentable art, no patents have been applied for.

Publications completed during the tenure of this research project and
supported in part by it include numbers 20, 21, 28, and 32 of the
preceding list of references.


 Definition of  Terms                                         introduced

 Autotroph  - an organism capable  of  utilizing  C0?  as  the
             sole  carbon source for  growth                          8

 Carbon  source  - a compound  which furnishes  carbon for syn-
             thesis of  cellular components                          5

 Carbonaceous BOD  - biochemical oxygen demand  due  to
             organic  carbon  compounds                               8

 Endogenous  phase  - metabolism  in the absence  of exogenous
             carbon compounds.  In a strict  sense, for a
             pure  culture this  metabolism involves oxida-
             tion  of  its storage  materials or  other cellu-
             lar components  by  an individual cell.  In a
            mixed population,  cells may feed  on other
             cells                                                 9

 Organotroph -  an  organism which  requires organic  compounds
            as  carbon  source                                       8

 Oxidative assimilation  - utilization of carbon source only
            for respiration and  synthesis of  storage
            products,  i.e., without cell replication             100

 Photosynthesis  -  synthesis of  organic compounds from C02
            using  light as a source of energy                     15

 Substrate - any compound which is acted upon  by an enzyme;
            therefore,  a compound which is used by the
            cell  for energy and/or synthesis                       1

ACOD - a measure  of the amount of biologically available
            organic matter in a waste sample                       6

Abbreviations and  Symbols

A           A collective constant replacing the term
            (1  + a - ac)                                          58

BOD         Biochemical oxygen demand                              5

            Biochemical oxygen demand which has been express-
            ed  in five days, i.e., y5                             11


Abbreviations and Symbols                                  introduced

c           Sludge recycle concentration factor, equal
            to the ratio between the recycle solids
            concentration, XR, and the biological solids
            concentration in the reactor, X                       56

C           Initial concentration                                 47
C.          Concentration at any time, t                          47

COD         Chemical oxygen demand; measure of total  chem-
            ically oxidizable organic matter                       6

COD         Effluent COD exiting the biological  reactor
   e        system, or COD remaining at the end  of a  batch
            experiment                                            10

CODf        The COD of biological solids at the  end of  an
            experiment                                            19

COD.        COD at the beginning of a batch experiment, or
   1        influent COD to aerator                               10

COD.        COD of the biological solids at the  beginning
   1        of an experiment                                      19

COD         COD of the waste, not including biological
            solids if present                                     19

D           Dilution rate.  Ratio of the rate of inflow, F,
            and the volume of liquor in the aeration  tank,
            V.  It is equal to the reciprocal of the  mean
            residence time in a completely mixed reactor          46

D           The dilution rate for the reactor alone in  sys-
            tems employing sludge recycle.  It is always
            larger than the dilution rate for the entire
            system, i.e., Dr = D(l + a), a being the  ratio
            between the hydraulic rate of recycle flow  and
            the flow of incoming waste, F                         56

DO          Dissolved oxygen concentration                         5

e           Base of Naperian logarithms                           26

F           Rate of flow of incoming substrate or waste
            water                                                 45

Abbreviations and Symbols                                 introduced

K           A biological "constant" used in the hyper-
            bolic expression relating specific growth
            rate to substrate concentration.  It is known
            as the saturation constant.  It is numeri-
            cally equal to the substrate concentration at
            which the specific growth rate is equal to %
            the maximum specific growth rate for the system       29

LQ          Ultimate BOD                                           8

S           Substrate concentration                                6

S           Steady state concentration of substrate in a
            completely mixed continuous flow reactor              52

S           Initial concentration of substrate in batch
            systems                                               29

S.          Concentration of substrate in the inflowing feed
            in continuous flow operation                          45

t           Time                                                  47

t           Mean residence time in a completely mixed contin-
            uous flow reactor, V/F                                46

t.          Doubling time, time required for one doubling in
            weight of the bio-mass growing in an exponential
            phase                                                 28

V           Volume of reaction fluid under aeration               45

X           Biological solids concentration, weight per volume     6

X           Steady state biological solids concentration in a
            completely mixed continuous flow reactor              52

X           Initial biological solids concentration in a
 0          batch reactor                                         26

X           Excess biological solids produced in a process
 e          (sludge wasted)                                       56

XR          Biological solids concentration in the recycle
            solids flow to an aerobic reactor in a continuous
            flow system                                           56

Abbreviations and Symbols                                 introduced

X           Biological solids concentration at time t             26

y           BOD expressed, i.e., accumulated oxygen uptake         6

Y           Sludge or cell yield; mg biological solids
            produced per mg organic substrate used                21

a           Recycle flow ratio in a continuous flow bio-
            logical reactor system employing cell  recycle.
            It is the ratio between the rate of flow of
            recycle solids and the rate of flow of incoming
            waste water                                           56

A           Change in value of a parameter, or difference
            between initial and final  values                       6

y           Specific growth rate in an exponential phase
            of growth                                             26

u           The maximum specific growth rate for a system
            in exponential growth                                 29
                                                       .NG Off ICS:

i 1 Accession Number
f. Organization

n Subject Field & Group
Oklahoma State University. Cpnt.pr for Ulatpy Rpcpavrh in Fnn-i napvi nn
               Biological  Principles for Design and Operation of the Activated
               Sludge Process
•J Q Authors)
Anthony h. baudy. Jr.
Elizabeth T. Gaudy

Project Designation
17090 FQJ
21 I Note
Descriptors (Starred First)
   Activated Sludge*, Biological Treatment*, Aerobic Treatment*, Ultimate Disposal*
   Purification*, Oxidation, Oxygen Demand                                          '
Identifiers (Starred First)
   Kinetic Models*,  Shock Loads*, Purification Mechanisms*, Nitrogen Requirements*,
   Oxidative Assimilation*, Endogenous Phase*, Purification Efficiency, Metabolism
       Generalized  concepts  of  BOD exertion,  the use of ACOD as a design and operational
       tool, the stoichiometry  and mass  balance concepts of treatment, and kinetic
       equations for microbial  growth  are presented.  Design models are discussed, and
       a model for  completely mixed reactors  holding recycle solids, X^, constant is
       recommended.  Some guidelines for accommodation of various types of shock load-
       ings are included.  Concepts of oxidative assimilation and the multiple effects
       of solids concentration,  nitrogen concentration, and detention time are related;
       a new activated sludge process  (continuous oxidative assimilation) for nitrogen-
       deficient wastes  is presented.  Data supporting the concept of total oxidation
       are presented, and a  modification of the extended aeration process incorporating
       chemical hydrolysis of portions of the sludge is recommended.  In the final
       chapter, some possible flow diagrams for complete aerobic treatment (purifica-
       tion and sludge disposal) of metabolizable organic wastes are presented.
        Anthony F. Gaudy,  Jr.
                                  Oklahoma State University
 WR;!02 (REV. JUUY 19*9)
                                                  U.S. DEPARTMENT OF THE INTERIOR
                                                  WASHINGTON, D. C. 20240
                                                                          GPO: 1970 - 407 -891