EPA-R2-73-147
JUNE 1973 Environmental Protection Technology Series
Biological Removal of
Colloidal Matter from Wastewater
Office of Research and Monitoring
U.S. Environmental Protection Agency
Washington, D.C. 20460
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and
Monitoring, Environmental Protection Agency, have
been grouped into five series. These five broad
categories were established to facilitate further
development and application of environmental
technology. Elimination of traditional grouping
was consciously planned to foster technology
transfer and a maximum interface in related
fields. The five series are:
1, Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental studies
This report has been assigned to the ENVIRONMENTAL
PROTECTION TECHNOLOGY series. This series
describes research performed to develop and
demonstrate instrumentation, equipment and
methodology to repair or prevent environmental
degradation from point and non-point sources of
pollution. This work provides the new or improved
technology required for the control and'treatment
of pollution sources to meet environmental quality
standards.
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EPA-R2-73-147
June 1973
BIODOGICAL REMOVAL OF COLLOIDAL MATTER
FROM
WASTEWATER
Walter J. Maier
University of Minnesota
Minneapolis, ffitBaabota 55455
Grant #17030 DGQ
Program Element 1B2043
Project Officer
Robert L. Bunch
U.S. Environmental Protection Agency
National Environmental Research Center
Cincinnati, Ohio ^5268
for the
OFFICE OF RESEARCH AND MONITORING
U.S. ENVIRONMENTAL PROTECTION AGENCY
WASHINGTON, D.C. 20k60
For sale by the Superintondenfolflipijilli'ff.&^JJoyermnent Printing Office, Washington, D.O. 20402
! OPO Bookstore
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EPA Review Notice
This report has been reviewed by the
Office of Research and Monitoring, EPA,
and approved for publication. Approval
does not signify that the contents
necessarily reflect the views and
policies of the Environmental Protection
Agency, nor does mention of trade names
or commercial products constitute endorse-
ment or recommendation for use.
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ABSTRACT
This research program was designed to provide a more basic under-
standing of the mechanism and rates of removal of organic materials
from waste waters by biological processes. Pure compound feed
materials were used with the exception of a series of tests on
sewage solids obtained from the Minneapolis-St. Paul Sewage Treat-
ment Plant by centrifugation of'primary effluent. Two model reactor
systems were used to measure rates of substrate removal. The film
flow reactor, characterized by a stationary biological slime layer,
was used to study removal kinetics under conditions where mass trans-
fer may be a limiting factor. A well mixed batch reactor was used
to study rates of removal under conditions where biological processes
are rate controlling. Rates of substrate degradation and carbon
removal are reported for each of the pure - compounds using acclimated
innoculum. Rate data from batch reactors are expressed in terms of
microbial growth rate coefficients. Rate data from the film flow
reactor is reported as the quantity of substrate removed per unit
of slime surface per unit of time.
This report was submitted by the University of Minnesota, Minneapolis,
Minnesota 55455, in fulfillment of Project No. 17030 DGQ under the
sponsorship of the Environmental Protection Agency.
iii
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CONTENTS
\
SUMMARY AND CONCLUSIONS
INTRODUCTION
Mechanisms and Controls of Biological Degradation 5
Colloidal Substrates - Physical Characteristics 6
Metabolism of Colloidal Substrates 7
Kinetics - Growth and Enzymes 8
Mathematical Analysis of Film Plow Reactor 11
Waste Water Composition 16
APPARATUS AND EXPERIMENTAL PROCEDURE 18
Batch Reactor 18
Film Plow Reactor 18
Continuous Propagator 21
Peed Solutions 21
Analytical Procedures 22
BATCH REACTOR TEST RESULTS 23
Chemistry of Starch 23
Characterization of Starch 24
Molecular Weights of Starches 25
Sephadex Gel Separation of Starches 25
Hydrolytic Degradation of Starch 29
Comparison of Biological Degradation of Different Starches 32
Soluble Exoenzyme Activity 37
Degradation of Starch-Glucose Mixtures 39
Amlno Acid Metabolism 42
Glutamic Acid Utilization 45
Glycene Utilization 48
Protein Chemistry 48
Protein Degradation 51
Milk Solids 54
Lipid Chemistry 58
Fatty Acid Degradation 60
Rate Studies Using Treatment Plant Biomass 68
Measurement of the Rate of Oxygen Uptake of Sewage Solids 74
iv
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FILM BLOW REACTOR 1EST RESULTS ?8
Mass Transfer Considerations in Biological Treatment 78
Biological Treatment 78
Biological Slime Layer 79
Film Flow Reactor-Operating;Procedure 81
Starch Degradation 81
Glucose-Starcli Mixtures 85
Amino Acids and Proteins 86
Laurie Acid 89
Mixed Feed Acclimated Slime Surface 91
Discussion - Summary of Film Flow Reactor Results 9^
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FIGURES
No.
INTRODUCTION
1 Schematic Diagram of Control Volume 12
2 Normalized Solution Equation 20 15
PART A - APPARATUS AND EXPERIMENTAL
PROCEDURE
Film Flow Reactor 20
PART B - BATCH REACTOR TEST RESULTS
4 Sephadex Gel Separation of Soluble Starch 27
5 Sephadex Gel Separation of Starch SD-5 28
6 Hydrolytic Degradation-Effect of Starch
Concentration 30
7 Rate of Degradation of Starch 31
8 Biological Degradation of Starches 33
9 Biological Degradation of Starches 34
10 Biological Incorporation of Starches-Removal
of Carbon 36
11 Soluble Exoenzyme Activity 38
12 Degradation of Starch-Glucose Mixtures 40
13 Degradation of Glucose from Starch Glucose
Mixture 111
14 Biological Degradation of Glutamic Acid-Effect
of Concentration 46
15 Biological Degradation of Glutamic Acid-Rate
of Carbon Removal-Effect of Concentration 47
16 Glycine Removal in Acclimated Innoculum 49
17 Amino Acid Degradation with Glycine Acclimated
Innoculum 50
18 Protein Carbon Utilization Using Mixed Feeds 53
19 Biological Degradation of Milk Solids 56
20 Milk Solids Carbon Utilization Rates 57
21 Fatty Acid Degradation-Carbon Removal 62
22 Degradation of Mixed Feed Solution by Laurate
Acclimated Innoculum 63
23 Removal of Glutamate and Glucose by Laurate
Acclimated Innoculum 64
vi
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No.
24 Mixed Peed Innoculum-Carbon Removal 66
25 Mixed Feed Innoculum-Glucose and Glutamate
Removal 67
26 Glucose Removal by Activated Sludge Biomass 70
27 Amino Acid Removal by Activated Sludge Biomass 71
28 Starch Removal by Activated Sludge Biomass 72
29 Protein Removal by Activated Sludge Biomass 73
30 Op Uptake Rate of Activated Sludge Using
Sewage Solids 76
31 Carbon Concentration of Sewage Solids 77
PART C - FILM FLOW REACTOR TEST RESULTS
32 Starch Concentration Versus Flow Rate 83
33 Starch Degradation Versus Feed Concentration 84
34 Glutamic Acid-Carbon Removal 87
35 Laurie Acid-Carbon Removal 90
36 Skim Milk Solids Removal 93
vii
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LIST OP APPENDIX TABLES
Title
Experiment No.
Page
Effect of Starch Concentration on 4-2-68
fetes of Biological Decomposition
Part I - Comparison of Biological 4-30-68
Decomposition fetes of Different
Starches.
Part II - Comparison of Biological 5-7-68
Decomposition Rates of Different
Starches.
Test for "Soluble" Exo-enzyme
Activity - Part I
Test for "Soluble" Exo-enzyme
Activity - Part II
Biological Decomposition of Starch
Glucose Mixtures
Effect of Glutamic Acid Concentra-
tion on Rate of Biological Decomp-
osition
Effect of Glutamic Acid Concentra- 2-5-69
tion on Rate of Biological Decomp-
osition
Glycene Degradation by Glutamic . 2-20-69'
Acid Acclimated Innoculum
Protein and Glutamic Acid Degrada- 3-12-69
tion on Glutamate Acclimated In-
noculum
Effect of Concentration on Biological 3-18-69
Degradation of Peptone and Glutamic ,•
Acid using a Peptone Acclimated Culture
95
98
100
2-9-68
2-10-68
8-29-68
12-20-68
101
102
104
107
109
111
113
116
viii
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List of Appendix Tables Cont.
Title Experiment No. Pag
Biological Degradation of Glu- 3-20-69 119
tamate, Glycine and Peptone using
Glutamate Acclimated Innoculum
Determination of Carbon Residues 6-19-69 122
from Glutamic Acid Metabolism
Effect of Milk Solids Concentra- 8-12-69 125
tion on Rate of Decomposition
Biological Degradation of Milk 8-18-69 127
Solids with Different Amounts of
Biologically Active Solids
Effect of Substrate Concentration 9-22-69 ,129
for Glycene Acclimated Innoculum
Effect of Substrate Concentration 10-7-69 132
in Degradation of Laurie Acid
Effect of Substrate Concentration 12-18-69 133
in Degradation of Laurie and Pal-
mitic Acid
Effect of Substrate Concentration 12-23-69 136
on Degradation of Palmitic and
Laurie Acid
Effect of Substrate Concentration 12-30-69 139
on Rate of Degradation of Mixed
Peed Solutions
Effect of Substrate Concentrations • 1-29-70 141
on Rate of Degradation of Mixed
Peed Solutions
Effect of Substrate Concentration on 2-23-70 ' 144
Rate of Degradation of Mixed Feed
Solutions
Ix
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List of "Cables Cont.
Title
Effect of Substrate Concentration on
Rate of Degradation of Mixed Peed
Effect of Substrate Concentration on
Rate of Degradation of Mixed Peed
Effect of Substrate Concentration on
Rate of Degradation Using Mixed Peed
and Excess Ammonia Nitrogen
Effect of Substrate Concentration on
Rate of Degradation Using Mixed Feed
and Excess Ammonia Nitrogen
Effect of Substrate Type on Rate
of Degradation Using Mixed Liquor
Activated Sludge from Minneapolis-
St. Paul Sewage Treatment Plant
Effect of Substrate Type on Rate
of Degradation using Mixed Liquor
Activated Sludge from Minneapolis-
St. Paul Sewage Treatment Plant
Effect of Milk Solids Concentration
on Rate of Biological Degradation
in a Film Plow Reactor
Effect of Milk Solids Concentration
on Rate of Biological Degradation
in a Film Flow Reactor
Effect of Laurie Acid Concentration
and Mixed Feed Solutions on Rate of
Biological Degradation in a Film
Flow Reactor
Experiment No,
4-30-70
5-21-70
6-19-70
7-15-70
7-23-70
8-11-70
8-20-69
9-4-69
3-17-70
Page
147
149
151
153
155
157
160
161
162
X
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List of Tables cont.
Title Experiment No. Pag
Effect of Flow Rate on Biological 4-9-70 164
Degradation of Mixed Feeds in a
Film Flow Reactor
Evaluation of Mixed Feed Solutions at 8-20-70 165
various Flow Rates in a Film Reactor
Evaluation of Starch, Protein and 8-29-70 166
Laurie Acid Feed Mixtures in a
Film Reactor
XI
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SUMMARY
This research program was designed to provide a more basic understand-
ing of the mechanism and rates of removal of organic materials from
waste waters by biological processes. Such information is needed to
develop fundamentally sound process variables correlations and to
stimulate development of new design concepts or process improvements
of existing biological treatment devices.
Typical municipal waste waters contain a wide variety of organic
materials; the major categories of biological compounds are represented;
colloidal materials include carbohydrates, proteins, and lipids; soluble
constituents include sugars, amino acids, and fatty acids. Soluble
constituents typically account for less than 25 wt.$ of the organic
fraction. It follows that degradation of colloidal and larger partic-
ulates is a crucial, if not controlling factor, in biological treatment.
Pure compound feed materials were used with the exception of a series
of tests on sewage solids obtained from the Minneapolis-St. Paul Sewage
Treatment Plant by centrifugation of primary effluent. Starch and
glucose were used as representative carbohydrates; peptone, glutamate,
and glycine were used as representative protein related materials and
palmitate and laurate were used as representative lipids. Each sub-
strate was tested individually and in admixture with other substrates
to test for Interactions. Two model reactor systems were used to
measure rates of substrate removal. The film flow reactor, character-
ized by a stationary biological slime layer, was used to study removal
kinetics under conditions where mass transfer may be a limiting factor.
A well mixed batch reactor was used to study rates of removal under
conditions where biological processes are rate controlling; the well
mixed batch reactor approximates process conditions encountered in
completely mixed activated sludge type treatment devices. Biological-
ly active sludge (innoculum) was obtained from continuous propagators,
starting with sludge obtained from the Mlnneapolis-St. Paul Sewage
Treatment Plant. In the film flow reactor, liquid medium flows over
a stationary slime layer in a thin film. Flow is parallel to the
slime surface so that direct contact between feed solution and cell
mass is limited to the interface. Mass transfer of the substrate to
the biologically active slime surface is slow, primarily by molecular
diffusion; the film flow reactor simulates some conditions encountered
in trickling filter processes.
Rates of substrate degradation and carbon removal are reported for
each of the pure compounds using acclimated innoculum. Rate data
from batch reactors are expressed in terms of microbial growth rate
coefficients; pure compound substrates show growth rates in the range
0.005-0.010 per minute on acclimated innoculum. The rates of degrada-
tion of colloidal, substrates cover a broader range, presumably because
hydrolytic cleavage, which is mediated by exoenzymes, precedes the
final utilization of the substrate, and may be a limiting factor if
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the microbial population is not acclimated to the colloidal substrate.
Data, usdng starch substrates, show that enzyme activity decreases as
the reaction proceeds. It appears that the hydrolytic exoenzymes are
inhibited by the products of hydrolysis; products of hydrolysis act as
competitive inhibitors as evidenced by the increase in the calculated
values of Michealis-Menton adsorption coefficients.
Substrate uptake of slime surfaces acclimated to single substrates
seem to be mass transfer limited at concentrations below 100 mg/1 and
reaction limited at higher concentrations. Rate data from the film flow
reactor are reported as the quantity of substrate removed per unit of
slime surface per unit of time, (mg/cm -min}. Maximum uptake with sin-
gle substrates is in the range 50-250 x 10~ mg/cm -min. Highest rates
were observed with lauric acid, glucose, and when mixed substrates
were used. Lowest uptake rates,-were observed with starch feed; starch-
carbon uptake was only 50 x 10 mg/cm -min. even though the rate of
starch degradation was 2-4 fold greater. By contrast, protein removal
and carbon disappearance are more nearly equivalent. Both starch and
protein molecules are far too large to be absorbed directly into
bacterial cells, leading to the conclusion that ezoenzymes, either
free or bound to the cell surface, are responsible for the hydrolytic
breakdown of colloids, followed by ingestion of soluble fragments.
The starch-carbon data suggest that the sites for hydrolysis and in-
gestion are independent and physically separated, as evidenced by the
low carbon uptake rate.
Acclimation of the slime surface is necessary when new substrates are
introduced; glucose is an exception. Acclimated surfaces may loose
their activity for metabolizing a given substrate if the substrate is
removed for any appreciable time, (a matter of hours). Acclimation of
the surface to an entirely new substrate may take several days. It
appears that an entirely different surface coating (different bacterial
species) is formed.
Use of mixed substrates generally results in higher rates of carbon
uptake. This is particularly pronounced when carbohydrates are added.
Preferential utilization of glucose In pure compound mixtures and lac-
tose from skim milk substrated have been observed. Nevertheless, there
is concurrent utilization of other substrated e.g. protein and starch
degradation as well as some uptake of carbon from noncarbohydrate
sources.
The results of this study provide- new insight on the behavior of bio-.
logically active slime layers and provide quantitative information on
rates of degradation of the major biochemical constituents of waste
water. The results have been used to guide pilot plant test programs
sponsored by the University of Minnesota for the development of more
fundamentally based engineering design correlations. A three vessel
prototype activated sludge unit with sludge recycle was used to study
the effects of residence time and substrate concentration using the
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same types of feed substrate mixtures as used in the model reactor test
program. The results will be published in the near future.
Data from the film flow reactor indicate that mass transfer may be the
rate limiting factor at low substrate concentrations. This suggests
that some commonly used operating modes for trickling filters may not
be sound and should be reconsidered. For example, the use of effluent
recirculation would appear to be undesirable at low BOD concentrations
because removal may well be mass transfer limited. The use of packing
which minimizes liquid hold up seems undesirable in a mass transfer
limited reactor. It is therefore recommended that trickling filter
plant tests be carried out to determine the effects of these process
variables under closely controlled conditions. In view of the high
potential rates of biological activity that are associated with the fix-
ed slime layer, further work is recommended to delineate optimal condi-
tions leading to new design of packing as well as the use of movable
surfaces.
INTRODUCTION
Biological degradation of waste matter is a naturally occurring pro-
cess, albeit a slow one, which functions in all receiving waters such
as lakes and rivers. However, the capacity of this natural process of
purification is limited. Overloading of receiving waters results in
gross pollution and makes waters undesirable for reuse. To avoid this
situation, treatment of wastewaters prior to discharge becomes increas-
ingly important. In fact, progressively more treatment will be requir-
ed in the future in order to keep pace with the rapidly increasing
quantities of wastes being disposed.
Man-made treatment devices such as activated sludge units and trickling
filters essentially are modifications of the naturally occurring process-
es. The same biological phenomenon of microbial metabolism underlies
the removal of waste materials. Man-made devices differ from the nat-
urally occurring purification process in that they are designed for
relatively short detention times in order to make them economically
feasible. However, increasing the. degree of treatment as measured by
the percentage removal of waste matter requires longer detention times
and increases the costs markedly. The cost increases exponentially
with percent removal. It follows that the increasing need for more
complete treatment of wastewaters which is projected for the future
will stimulate a search for cost reduction in the design of treatment
facilities.
Design of conventional treatment facilities is based on an impressive
accumulation of many years of practical experience. Within the context
of these correlations, many process design improvements have been devel-
oped and cost reductions achieved. However, it seems unlikely that
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any major design Improvements or cost reduction for convential treat-
ment facilities will be forthcoming from this approach in the future.
An alternate approach is to develop a more fundamental understanding
of the underlying principles of biological treatment in the hope of
arriving at new design concepts or uncovering new ideas.
To this end, there is need for descriptions of the mechanism of biologi-
cal purification at the microscopic and/or molecular level. Description
at the microscopic level must consider such factors as:
Size and composition of the waste material, e.g., sol-
utes and colloids, carbohydrates, proteins, and lipids.
Characterization of the microorganism population and
its physical distribution, e.g., predominant bacterial specie:;,
mobility relative to the solution, dispersion of cells.'
A measure of the rates of metabolism.
A measure of the effectiveness of mass transfer of war;te
matter to the cells.
It obviously is impossible to study the effects of all variables in one
set of experiments. Meaningful results can be obtained only by varying
a limited number of variables at one time while holding other conditions
constant.
With this in mind, a broad-based model study has been carried out using
a variety of soluble and collodial pure compound organic materials.
The materials where chosen with a view to approximating the types of
organic material that are found in municipal and industrial wastes.
The use of model reactors and pure compound substrates allows studying
complex systems under controlled conditions. The models do not dup-
licate all aspects of the reaction system, but are designed to isolate
process variable effects. This provides a more theoretical basis for
correlating process results, and leads to a better understanding of
the mechanisms of biological treatment.
The broad objective of this study was to Investigate the biologically
mediated removal of organic matter from a mechanistic standpoint in
order to provide a better understanding of the overall process. Such
information is needed.to develop fundamentally sound process variable
correlations and rate equations.
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MECHANISMS AMD CONTROLS OF BIOLOGICAL DEGRADATION
Biological treatment for the removal of organics from water comprises
a sequence of interrelated steps including mass transfer, adsorption
and a host of biochemical reactions. Considering the microbial cell as
the center of this activity, the sequence starts when dissolved, or
colloidal organics, salts, and oxygen from the bulk liquid phase are
transported to the surface of the bacterial cell. Mass transfer is by
molecular diffusion and by eddy current transport. At the cell surface,
utilization proceeds with adsorption and.ingestion of the nutrients
necessary for growth, followed by step-wise oxidation within the cell
to provide energy and building blocks for growth. By-products and oxi-
dation products are released into the bulk liquid phase.
Two limiting situations suggest themselves—the case where mass trans-
fer limits the rate of removal, and the case where cell metabolism limits
the rate of utilization. Process variable, effects will be different for
these two situations because mass transfer and cell metabolism are known
to respond differently to changes of such process variables as mixing,
nutrient concentration, and temperature. By studlng these limiting
situations it is possible to learn a great deal about the mechanisms
which control the overall process of removal of waste matter by bio-
logical means,
Two model reaction systems were used to simulate the above situations.
A well mixed batch reactor was used to simulate conditions of high rates
of mass transfer. In this situation both the fluid and the bacterial
cells are free to move in all directions and relative velocities are
high. This well-mixed reactor is used frequently as an idealized model
of the activate sludge process.
The second model is a film flow reactor which is characterized by a
stationary slime layer simulating trickling filter conditions. In this
model, liquid medium flows over the slime layer in a thin film. Flow
is parallel to the slime layer so that the degree of contact between
liquid and cell mass is limited to the Interface between the slime layer
and the liquid phase. This makes for relatively low rates of mass trans-
fer, especially if the liquid film is in laminar flow in which case mass
transfer is limited to molecular diffusion. The film flow reactor thus
provides a simulation of conditions where mass transfer could be the
rate limiting factor..
These reactor models were used to study the removal of soluble and
colloidal substrates. Glucose was used as a typical soluble carbohy-
drate and starch as the corresponding colloidal carbohydrate; glutamic
acid and glyclne were used as representative soluble amino acids and
peptone as a typical colloidal protein; lauric acid was used ^s a typi-
cal lipid; it is soluble at very low concentrations but behaves like a
colloid at higher concentrations. The results obtained with glucose
as the only carbon source have been published by the author (San. Eng.
Divt> ASCE, SA4 93, 91, 1967), and are briefly summarized as background
Information.
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Glucose Removal in Model Reactors
Process variable studies were carried out using glucose as the only
source of carbon In a liquid medium containing all other growth require-
ments in excess. Results indicate that glucose removal can be described
analytically in terms of growth kinetics 'of the microorganisms for the
well-mixed reactor and mass transfer of nutrients for the film flow
model.
In the well mixed reactor system, the rate of removal of glucose was
independent of glucose concentration (above ten mg/1) but directly
proportional to the amount of active cell mass. Ihis is consistent with
the fact that high rates of mass transfer are obtained in a well-mixed
reactor. Thus rate of glucose removal is determined by the rate of
metabolism of the microorganism.
On the other hand the film flow reactor, in which the bacterial mass
(slimelayer) remains stationary at the retaining wall while medium
flows' over it in a thin film, showed a marked reduction in the rate of
removal as glucose feed concentration was reduced below 100 mg/1. In
this situation the quantity of bacterial mass as measured by slime
thickness had no effect on the rate of removal. These results are con-
sistent with the fact that- the film flow reactor is a mass transfer
limited system because mass transport of nutrients from the liquid
film to the slime layer is by molecular diffusion, which is slow.
Colloidal Substrates - Physical Characteristics
Removal of colloidal substrates could be expected to follow a similar
pattern as soluble substrates except where particle size becomes a
factor. Pertinent physical characteristics of water, glucose, starch,
and bacterial cells are shown below.
TypicalHjDGlucoseStarchBacterium
Dimensions
Molecular weight, /- „
g/mole 18 180 10° 101
p ii
dam., angstrom 4 10 10 10
Diffusion
coefficient, 0.6 x lO"0 0.08 x 10 ') —
cm /sec.
Weight of 6.0 x 1023 cells.
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Molecular diinension have a direct effect on mass transfer rates because
larger molecules diffuse more slowly as shown by the lower diffusion
coefficient for starch vs. glucose. Mass transfer by molecular diffu-
sion is proportional directly to the diffusion coefficient and the con-
centration gradient. It follows that mass transfer of starch is approx-
imately eight times less than glucose under the same concentration grad-
ient .
Molecular size also is an important factor when considering permeabil-
ity of cellular membranes. Glucose molecules are sufficiently small
to pass through the cell wall and semipermeable membrane (cytoplasmic
membrane) which surrounds bacterial cells. However, the diameter of
starch molecules is some 100 times larger and approaches cellular
dimensions. As a result it is unlikely that starch molecules can be
absorbed directly.
Metabolism of Colloidal Substrates
The mechansim by which microorganisms metabolize large particles or
colloids centers on two possibilities. The microorganism either can
evolve a mechanism for engulfing and secreting the appropriate catabo-
lic enzymes within the cell confines, or they can excrete the necessary
enzymes into the surroundings to cause the reaction sequence to proceed
outside of the confines of the permeable membrane of the cell proper.
According to Pollock (The Bacteria, Vol. 4, Acad. Press, 1962), the
engulfing processes or direct passage of large molecules into cells
(referred to as phagocytosis and pinocytosis) generally are associated
with large cells and/or higher forms of life than the Eubacteriales.
The rigid cell wall of Eubacteriales would make it difficult to dev-
elop a mechanism for passage of large molecules through the cell wall,
whereas higher forms (e.g., amoeba) are surrounded by an elastic mem-
brane, free to fold and form vacuoles. Pollocks' review of some 280
references leads him to conclude that bacterial metabolism of large
molecules is mediated by exoenzymes which are available outside of
the semipermeable membrane (other'authors perfer the term extracellular
enzymes).
The mechanisms and controls by which exoenzymes are produced and/or
liberated outside of the semipermeable membrane is not understood. As
a result, it is not possible to predict the influence of process varia-
bles on the kinetics of colloid degradation. . Control mechanisms for
exoenzyme production have been deduced for a few pure culture systems
using simple substrates. The adaptive response mechanism is the control
mechanism most widely mentioned. Fukumoto (Proc. Intl. Symp. Enz. Chem,,
Academic Press, 1958) has shown that the formation of a-amylase in a
medium free of starch substrate is negligible but can be stimulated by
the addition of substrate even in the absence of nitrogen. The work
by Rogers (Bioch.•Jdurn. 39, 435, 1945) on synthesis of hyaluronldase
into the growth medium gives support to the concept of adaptive response
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In Streptococci but suggest that other influences can become predomin-
ant as evidenced by the need for a peptone factor to stimulate forma-
tion of hyaluronidase. If adaptive response is the controlling mech-
anism, then concentration of substrate should be an important parameter
in the description of the kinetics of colloid degradation.
Nomura (Bloch. Journ. 43, 84l, 1956) has explained the control mech-
anism in terms of the concept of competitive synthesis. The cell pro-
tein in synthesized preferentially and, in the absence of adequate
nutrients, synthesis of cell protein stops and amylase formation pre-
ceeds. If competitive synthesis is the controlling factor, then the
effects of colloid concentration would be secondary and the concentra-
tion of competing substrate solutes would be the most important para-
meter .
The work of Reynolds (Jour. Gen. Microbio., Britl, 11_, 150,- 195*0 on
chitinase activity is interesting particularly because the results show
that agitation of the medium and culture resulted in increased chitinase
activity. This suggests that mass transfer is a limiting factor in
certain colloidal systems and must be considered.
The present state of knowledge of metabolism of colloids can be sum-
marized by explaining that there-are various hypotheses which describe
specific observations but there is no generally applicable theory.
The available Information does not permit making apriori predictions
about the kinetics of colloid removal in the systems of interest in
waste water treatment.
The first phase of the experimental work therefore was designed to
study the effects of colloid concentration. Measurements were design-
ed to establish the relative rates of colloid degradation and utiliza-
tion of the degradation products using starch as a representative
colloid. Subsequent tests were carried out in the presence of other
substrates to establish whether there is a competitive effect. Five
different starches were evaluated. Protein and fatty acid degradation
was also studied.
Kinetics - Growth and Enzymes
The underlying process in biological treatment of waste waters is the
metabolic activity of microorganisms which utilize the organic materials
as food for growth. Oxidizable substrates such as carbohydrates, lipids
and proteins serve+as a source of carbon and energy. Inorganic sub-
strates such as NH|. and ELS can also serve as sources of energy for a
few species.
Microbial growth also requires a source of nitrogen, oxygen (aerobes),
sulfur, phosphorous and a broad range of inorganic salts in low concen-
trations. It may be assumed that all the required salts are present in
excess in municipal waste waters. However, nitrogen and phosphorous may
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be deficient in certain industrial wastes.
Given a system where all nutritional requirements are available in
excess, microbial growth will generally proceed rapidly until eventual-
ly one of the required nutrients is depleted. For this type of growth
situation the rate of growth can be described by:
(1) dN _
dt
Where N = cell mass concentration
y = growth rate coefficient
t = time
The growth rate coefficient has a unique maximum value for each species;
it is temperature dependent, affected by pH, and substrate concentration.
The effect of substrate concentration has been described by Monod and
can be represented by the equation:
g
(2) y = y max v , „
Where S = substrate concentration
K = adsorption coefficient
C
y max = maximum growth rate coefficient for the environ-
mental conditions.
The adsorption coefficient has the units of substrate concentration and
takes the numerical value of the concentration where the rate of .growth
is one half its maximum value. Substitution of equation (2) into (1)
leads to the generalized growth rate equation which now has two depend-
ent variables, S and N both of which are time dependent.
(3) dN S_ N
dt y mx K + S
c
One dependent variable can be eliminated by using the relationship be-
tween yield of cell mass and amount of substrate consumed.
The relationship between cell mass synthesis and substrate utilization
has been the subject of extensive investigations with pure cultures as
well as with mixed cultures. In theory, there is a unique relationship
between synthesis of cell mass and the substrate consumption which is
defined analytically by equation (4).
(4) dN _ v dS
dt " "Y dt
Where Y = yield coefficient, weight of dry cell mass produced
1! per unit weight of substrate consumed.
-------
The yield concept follows from the hypothesis that synthesis is the
net result of a series of biochemical reactions (metabolic pathways)
wlrLch produce protoplasm and release energy from the oxidation of
substrate. As long as the same metabolic pathways are involved, the
yield of cell mass per unit weight of substrate has a constant value.
In practice, this condition is at best an approximation, which is
acceptable as long as the changes in environmental conditions are not
extreme. Assuming the yield coefficient to be constant, equation (4)
can be integrated between limits to give:
(5) N - No = -Y(S - SQ)
Where N and S are the initial concentrations of cell mass
ans substrate respective2y. Using the yield relationships to eliminate
the dependent variable N from equation (3) gives:
(6) - dS = max S (SQ - S + NQ)
dt KC + S -y
A similar equation can be obtained in terms of cell mass, N. Equation
(6) is readily integrated for K .and Y constants, and gives:
(7) In S + B + K lnJs(S + B)
S + B c —/o f~ , p\ ( = y max t
o / ^Q^ T B;
Where In = natural logarithm
B= -VSo
Y
Equation (7) describes substrate concentration as a function of time in
terms of the maximum growth rate coefficient, the yield coefficient and
the adsorption coefficient K as parameters. For the special case where
K is negligibly small,, i.e., at high substrate concentrations, equation
(7) reduces to the form:
(8) In S + B max
S + B
o
Either of the above equations can be used to predict the course of sub-
strate depletion in a well-mixed batch reactor. As a rule, values of
N and S can be obtained by direct measurement. The yield coefficient,
Y, can be measured directly by harvesting the cell mass and simultaneous
measurement of substrate depletion.
The maximum growth rate coefficient y max may be evaluated from data
obtained at relatively high substrate concentrations using equation
(8); alternatively both K and y max can be evaluated by trial and
error from two independent sets of observations of substrate depletion
as a function of time.
10
-------
Data on the enzymatic degradation of colloids was analyzed in terms
of a simple model where the rate of hydrolysis is defined by equation
(9):
dt
Where C = concentration of starch
t = time, min.
K-, = rate constant
E = enzyme concentration.
Concentration of enzyme is assumed to be directly proportional to cell
mass concentration, E ^ N. Substituting into Equation (9):
Where k Incorporates a proportionality constant which relates
enzyme concentration and cell mass concentration.
The concentration of cell mass, N, can be replaced by the growth rate
equation which relates cell mass at any time, t, to the initial con-
centration N and the growth rate coefficient \i max as shown in
equation (11;:
(11) N = N [eymaxt]
Equation (11) applies as long as y max remains constant (logarithmic
growth phase)
(12) -dC=kN ^y^t-j
crc o
Integrating between limits,
(13) C -C = kN r P t m
v o o [e max - 1J
Hnax
Mathematical Analysis of Film Flow Reactor
Prom a theoretical viewpoint, the film flow reactor has three advantages.
(i) It holds the biologically active mass stationary and thus permits
the making of rapid changes in environmental conditions such as composi-
tion of the growth medium and concentration of substrate, (ii) It
allows for instantaneous control of the flow of nutrients, (iii) It
allows regulating contact time between the bacterial mass and the med-
ium, independent of flow rates.
11
-------
The disadvantages stem from the relatively low rates of mass transfer
which limit removal of nutrients at low concentrations of nutrient.
It has been shown from considerations of Reynolds number that the flow
on a flat, incline plane is in the laminar flow regime. Laminar flow
can also be demonstrated by the use of dye injections which show that
the flow lines are parallel to the surface of the slime layer. It
follows that mass transport of nutrients from the liquid film to the
slime layer is by molecular diffusion, which is relatively slow. How-
ever, the flat plate reactor has the advantage that the rate of mass
transfer by molecular diffusion can be described mathematically, so
that it becomes possible to distinguish between the situation where
mass transfer is the rate-limiting factor. A general mathematical
model which describes mass transfer and reaction at the solid surface of
the inclined plane has been described by the auther (San. Eng. Div.,
ASCE, 93, SA4, 91, Aug. 196?) and is briefly summarized below. The
mathematical model is obtained from a material balance over an infinite-
simal control volume as illustrated in Figure 1.
-Liquid
Solid w
Support / ^
FIGURE 1
SCHEMATIC DIAGRAM OF CONTROL VOLUME
At low energy gradients the flow regime is laminar and velocity is
described by equation (14):
(14) V = pgsing (62 -X2) = G(S2-X2)
2y
in which V = velocity, in grams per centimeter,
p = density, in grams per centimeter,
g = gravitational constant, in centimeters per second squared,
y = viscosity, in grams per centimeter-second,
g = angle in inclination,
5 = thickness of the liquid film in centimeters, and
X = distance from surface of film, in centimeters.
12
-------
Let CA represent the concentration of the limiting or potentially
limiting substrate (.Component A).
The material balance assumes that there is no production nor use of
component A in the control volume. In effect, this means that there
is no growth in the liquid film per se and that all metabolic activity
takes place in the slime layer. However., the possibility of growth and
use of nutrients in the liquid film was checked experimentally and
found to be below detectable limits. Flux of component A in the Z dir-
ection is approximated by convective transport (equation 14). Flux in the
X direction is limited to molecular diffusion. The resulting
differential equation for the steady state is:
2
(!5), -D 9 °A + G (<52 -X2) 3CA = 0.
3X2 3Z
Where D. i| the molecular diffusion coefficient of component A in
water, cm /sec. Proceeding on the assumption that the surface of
the slime layer is the major site for assimilation and metabolic de-
gradation of component A, the solution of equation (15) is subject to
three boundary conditions:
(16) Dfl 9CA = 0 at X = 0
H 3X
3CA
(17) -DA T~ -R" = 0 at X = 6
(18) CA = CAQ at Z = 0 for 0 < X < 6
The first boundary condition states that there is no mass transfer
across the interface between the liquid film and air at X = 0. The
second boundary condition states that the rate of transfer of component
A across the interface between the liquid and slime layers at X = 6, is
equal to the rate of removal of component A by assimilation at the slime
surface. The term R" is a function of the metabolic activity of the
microorganisms in the surface of the slime layer. Boundary condition
No. 3 states that the concentration of A in the liquid film at the inlet
to the plane (at Z = 0) is uniform and is represented as CAQ
Boundary condition No. 2 was approximated by assuming that for active
biological slime layers R" >»DA (3CA/3X) which leads to the approxima-
tion that the concentration of component A at the interface is zero.
The corresponding boundary condition-No. 2 becomes
(19) CA ~ 0 at X = 6.
13
-------
Integration of equation (15) and transforming the variables into
dimensionless form gives the following equation for c, the vertical
average concentration from the film flow reactor, at any point Z down-
stream from the inlet.
2 I
(20) c* = 3/2 I e ~AnZ* An ' (1-xJ) ^(X%,An)dX*
An 0
in which c% = dimensionless average concentration of nutrient at any
point Zx on the plane,
X = eigenvalue obtained as a result of boundary condition
no. 2, „
A = coefficient which is a function of A and Xx,
¥„ = power series in X^
and Xx = dimensionless distance from the surface of the liquid film,
and Zjj = dimensionless distance from the inlet. (The variables have
been expresses in dimensionless form to simplify the solution).
Equation (2) has been evaluated and the solutions compared with experi-
mental results. Solutions for equation (2} are graphed in Figure 2,
which relates the average concentration c (dimensionless) to the dimen-
sionless variable Z defined as follows:
— C X
c* = _A ^* = ~$
C AO
Where C. = average concentration any point Z, mg/1
C.Q = inlet concentration, mg/1
Z = distance from inlet, cm.
DA = molecular diffusion coefficient, cm /sec
G = p g sin 3/2y as defined by equation (14)
5 = thickness of the liquid, film, cm.
-------
FIGURE 2
SOLUTION EQUATION 20
C.CCCI
( DJrtAENStONLESS VARIABLE)
-------
Waste Water Composition £
A thorough, understanding of the composition of waste waters is essent-
ial in evaluating the effectiveness of various types of treatment and
pollution effects on the environment. Several investigators have ana-
lyzed composites of municipal sewage with a view to identifying and
measuring the organic constituents.
Composite samples were obtained from municipal sewage systems and
subjected to physical separation methods to recover solids. The solid
fractions were then analyzed for the major classes of organic materials.
Large particulate matter was removed by sedimentation for 1 hour. The
supernatant from sedimentation was then subjected to centrifugation to
remove a second fraction of solids. The centrifuged liquid was then
passed through a candle filter to remove large colloids. (Candle filters
have been used for removing bacteria from suspension. However, it is
most likely that colloids such as proteins and polysacherides can pass
through the candle filter). The final fraction of solids was determined
by evaporation and includes both solutes and small colloids. Results
are shown below.
*
Treatment Amount of Solids Removed as mg/1
of Carbon
1. Settled 1 hour 110
2. Centrifuged' 130
3. Candle filtered 54
4. Filtrate 118
(evaporated)
*Water polution Research, Report of the Water Pollution Research Board
Laboratory, Stevenage, England 1959-61.
A chemical analysis of the solids from fractions 1, 2, and 3 is shown
under the heading "suspended Matter". Composition of the solids from
the candle filtrate is shown as "filtrate".
Concentration as mg/1 of carbon
Constituent Total Suspended Solids Candle Filtrate
Carbohydrate 77-1 21.1 56.0
Amides 2.4 2.4
Amino Acids - Free 6 = 3 6.3
-Bound 70.7 63.3 7-4
Acids - soluble 17.3 6.7 10.6
- insoluble 71.6 71.6
Esters 46.9 46.9
Anionic Surfactants 9.3 2.0 7.3
Great inine 3.0 3.0
16
-------
Total Organic carbon
- by analysis 412 294 118
- by addition 304.6 214 90.6
Inspection of the results shows that the carbohydrates are primarily
solutes and small colloids. However, the bound amino acid group is
primarily large colloidal or particulate matter which can be removed
physically. The most noticeable group of constituents are the lipid
related compounds (acids and esters) which account for more that 45$
of the total organic carbon. The major portion of the lipid material
is in the large colloidal and particulate fraction with only small
quantities showning up in the candle filtrate.
The results obtained by Hunter (J. Water Poll. Cont. fled., 37, 1142
Aug. 1965) show similar trends. Composite samples of sewage were sub-
jected to successive physical separation steps, sedimentation, centri-
fugation and membrane filtration which are referred to as settleable,
colloidal and supra-colloidal particulates respectively. Total part-
iculate solids are approximately 80 percent organic matter, while the
soluble-fraction solids are approximately 30 percent organic matter.
.Approximately 64 percent of the total wastewater solids is contributed
by the soluble fraction, but only 40 percent of the total organic mat-
ter is contributed by this fraction.
The particulate fractions were composed largely of grease (17 percent),
amino acids (19 percent), and carbohydrates (24 precent). The grease
was found to be principally esterified fatty acids and unsaponifiable
matter. Free fatty acids were present only in small amounts. The
amino acid nitrogen content of the particulate fractions averaged only
about 50 percent of the particulate organic nitrogen.
The organic compositions of the three particulate fractions were some-
what similar. The main difference was found in the considerably high-
er amino acid and hemicellulose contents of the supracolloidal fraction
and the considerably higher cellulose content of the settleable solids.
The "soluble organic matter was found to be composed largely of ethyl-
ether-extractable matter, of which the organic acids were the primary
constituent (56 percent). The other organic constituents present in
significant quantities were the amino acids and sugars. ABS, volatile
acids, phenols, cholesterol, uric acid, and creatine-creatlnine were
found to be minor constituents.
On the basis of these analyses, it is clear that typical sewage is
a composite of a wide range of organics. All the major classes of bio-
chemical conpounds are represented, carbohydrates, amino acids - pro-
teins and lipids. For the purpose of this study substrates were chosen
to give one or more soluble and colloidal compounds from each of the
major classes of biochemical compounds.
17
-------
Soluble .Colloidal
s?f
Carbohydrates glucose starch
Amino Acid-protein glycine, peptone
glutamate
Lipids laurate palmitate, stearate
Preparation and analytical procedures are discussed in the following
section.
APPARATUS AND EXPERIMENTAL PROCEDURE.
BATCH REACTOR
Two model reactor systems were used to study the rate of biological
growth and the kinetics of removal of substrate. Batch reactors were
used to simulate conditions of high rates of mass transfer. " The vessels
were agitated by introducing air through sparging devices. Sparging
provides for both rapid mixing and an excess supply of oxygen for the
biological system. The sparging air was pre-saturated with water by
passing it through a wet packed column. Batch reactors in a size range
of 250 milliliters to 2 liters were used depending on the length of the
experiment and the number of samples to be obtained. Batch reactors
were used to study the effects of process variables such as temperature,
concentration of substrate, type of substrate, and concentration of
microbial cell mass. In any given series, all reactors were started
at about the same time using aliquots of the same feed materials and
innoculum. Samples were withdrawn at predetermined time intervals and
immediately filtered through a 0.^5 micron multipore filter to remove
active cell material. Samples were then stored under refrigeration
for anaylsis at a subsequent time.
FILM FLOW REACTOR
This reactor is characterized by a stationary slime layer. Peed sol-
ution is allowed to flow over the slime layer in a thin film. Plow is
parallel to the slime layer so that the degree of contact between li-
quid and biological film is limited to the interface between the slime
layer and the liquid phase. At the conditions used in this experiment,
namely relatively low flow rates, and very low hydraulic gradients, the
liquid film is considered to be in the laminar flow regime. It has
been shown by the.author (Journ. San Eng. Div., ASCE, 93, 91, 196?)
that under such conditions mass transfer is very slow because it is
limited to molecular diffusion from the liquid film to the interface
of the slime layer. The film flow reactor thus provides a simulation
18
-------
of conditions where mass transfer is, or could be, the rate limiting
factor in the consumption and removal of substrate and the growth of
the microbial cells.
The film flow reactor consists of a machined flat surface made of clear
plastic with the dimensions shown in Figure 3. The plastic is bolted
to a metal frame to prevent warping. Peed solution is introduced into
a distribution or stilling basin whence it passes over a precisely mach-
ined overflow weir. Channelling and short circuit ing through the still-
ing basin was observed initially, but was overcome by the use of a dis-
tribution grid. The grid consisted of wire mesh designed to provide
pressure drop and thereby randomize the flow pattern through the still-
ing section. The outlet section was separated into three parts. For
most analyses, samples were taken only from the center section which
collected about 2/3 of the flow. The end sections were generally dis-
carded because of wall effects, associated with the more rapid flow In
the vicinity of the side walls. The slope of the plane was adjusted
with a nut and bolt attachment at the upper end. The lower end was
supported by a two point swivel support. This method of attachment
facilitates leveling from side to side and allows changing the slope
very easily. :
The surface of the plate was covered with a fiberglass fly screen which
serves as a structural framework for building up the slime layer.
Growth of the slime was initiated by seeding the feed solution with
effluent from a continuous propagator. After a few hours of seeding,
the plane was allowed to stagnate and allow the biomass to settle into
the screen. Thereafter, feed solution was continually passed over the
plane and in general, growth was established within a matter of a few
days. Measurements of the screen volume showed that the void spaces
account for about 80$ of the volume defined by the edgeview volume of
the screen. After the biological materials had filled the voids,
excess growth was periodically removed by scraping the surface with
a knife edge. The thickness of the slime layer was thus controlled
to the approximate thickness of the screen, approximately 0.05 cm.
thick. Thicker slime layers were obtained by superimposing several
layers of screens; 0.08 cm. for 2 screens, 0.11 cm. for 3 screens and
0.14 cm. for 4 screens.
19
-------
F1GUHK 3
Fiberglass
screen
Overflow weir.
Feed
Inlet
FILM FLOW REACTOR
20
-------
CONTINUOUS PROPAGATOR
A small continuous flow, constant volume, agitated reactor was used as
a source of actively growing and acclimatized microorganisms (Chemostat
type system). A sparger was used to introduce air to insure high oxy-
gen concentrations and to provide good mixing. Peed was introduced by
gravity flow through a capillary tube to control flow rate. Excess was
allowed to overflow to waste or collected as needed. In this type of
system, liquid feed rate determines residence time and to some extent
the amount of growth. It also controls the rate at which microorganisms
are flushed out of the propagator and hence offers a simple method of
regulating the concentration of microorganisms. The continuous pro-
pagator was originally seeded with a small sample of sewage obtained
from the Minneapolis-St. Paul Sewage Treatment Plant and contained a
large variety of microorganisms. The propagator was operated for sever-
al weeks, using the feed solution to be tested, in order to acclimate
the microorganism population. Periodic inspection showed that a mor-
phologically similar culture was produced within a matter of days.
This is not to say that this was a pure culture, however, for all prac-
tical purposes the predominance of the acclimitised .species made it be-
have as though it were a pure culture. The continuous propagator efflu-
ent was used as innoculum for all batch tests as well as for seeding
the film flow reactor. A separate propagator was used for each new
feed stock or feed solution that was tested.
WED SOLUTIONS
Feed solutions were prepared using distilled water in a buffering salt
solution. The composition of the salt solution is listed below:
Chemical Formula Milligrams/liter
KH2P04 42
106
164
NH^Cl 259
106
CaCl2 12
—6H20 1
21
-------
Reagent grade chemicals were used throughout. Soluble organic substrat-
es consisted of glucose, glutamic acid, .dycine, and lauric acid. Col-
loidal substrates consisted of various, starches, and proteins. Fatty
acids, such as lauric, palmitic, and stearic, probably behave as col-
loids because of their low solubility . ' There was some evidence of
removal of fatty acids during millipore filtration, suggesting that some
of the fatty acids tend to agglomerate and form miscelles which are of
colloidal dimensions.
ANALYTICAL PROCEDURES
Glucose was measured using a commercially available enzyme system sold
under the name of Glucostat (Worthington Chemical Co., Freehold, N.J.).
This method of analysis has been used successfully by a number of in-
vestigators. Glucostat reagent contains glucose oxydase, which oxydizes
glucose to gluconic acid with the release of hydrogen peroxide. The
hydrogen peroxide is in turn oxidized by a chromogen in the presence of
the enzyme peroxidase. The oxydized chromogen has a distinctive yellow
straw color which has a strong absorption band in the 400-500 millimicron
range. Color intensity was measured in a Coleman Spectrophotometer and
glucose contents calculated by reference to known standards which were
tested with each batch of 10-15 samples. Samples containing high con-
centrations of cell materials were filtered before measuring transmit-
tance to eliminate absorption by cell material.
Carbon concentration was measured in a Beckman Total Carbon Analyzer,
as described by Van Hall (Anal. Chem. 35, 315, 1963). Samples were
filtered through a 0.45 micron millipore filter to remove cell material
prior to carbon analysis. The carbon analyzer consists of a. combustion
tube followed by an infrared cell to measure the concentration of carbon
dioxide produced in the combustion system. The combustion train operates
at 940°C in the presence of a cobalt molybdate catalyst, and air at at-
mospheric pressure. The combustion gases pass through a condenser to
remove water, and are then analysed for carbon dioxide in a balanced
infrared cell which gives a direct readout in terms of milligrams of
carbon. Samples containing more than 100nig/l of carbon were tested by
injecting smaller aliquots (ten microliters), or the samples were diluted
to the range of 1 to 100 milligrams per liter of carbon. The readout
system covers a range from 0 to 100 milligrams per liter of carbon.
-f
;
Amino acid concentration vras measured using ninhydrin reagent. The pro-
cedure was patterned after that described by Yemm and Cocking (Analyst,
Volume 80, March 1965, pp. 209-213). The method gives stoichiometric
reactions with ninhydrin reagent for most amino acids and is both easy
to use and reproducible. Ammonia does react and" has to be corrected for,
or eliminated from the sample prior to testing. The test procedure was
calibrated using both glutamic acid and glycine. Ammonia corrections
were made using direct Nesslerization to establish ammonia concentration,
22
-------
and then using a calibration chart of absorbance versus ammonia concen-
tration, to incorporate corrections for specific samples. The hydrol-
ysis products of proteins react like amino acids and give a positive
reaction.
Cell mass was measured by filtering and weighing the dried solids
through a 0.45 micron millipore filter. The technique is patterned
after that used by Engelbrecht and McKinney (Sewage and Industrial
Wastes, 28, 11, 1321, November 1956). This test provides a direct
measure of the biomass and suspended solids but does not differentiate
between viable cell mass and debris. The millipore filters were pre-
washed and predried prior to use and again weighed after drying at
103°C.
Proteins were measured using Polin-Phenol reagent. The method is pat-
terned after that described by Lowry et al. (Journal of Biological Chem.
193., 265, 1951). The Polin reaction involves the peptide bonds of ty-
ros ine and tryptophane in the protein. The final color is the result
of two reactions, the reaction of copper with the protein in an alka-
line solution, and the reduction of the phosphomolybdic—phosphotungs-
tic reagent contained in the Polin-phenol reagent. The Polin reaction
requires only 1 milliliter of sample. There are no apparent interfer-
ing substances and the determination is quite sensitive and easy to
perform.
Starch contents were determined in a modified iodine test. The iodine
test is described in section B of this report (Iodine Reaction and
Carbon Determination of Starches).
BATCH REACTOR TEST RESULTS
Chemistry of Starch
Starch is a naturally occurring polysaccharide material consisting of
glucose units jointed by a, 1, 4 linkages and to a lesser degree by a,
1, 6 linkages. Starches fall into two classes, amylase, which is be-
leived to be a long unbranched chain, and amylopectin which has been
shown to be a branched chain structure with one terminal glucose unit
for every 20 to 30 glucose residues.
Hydrolytic cleavage of starches is mediated by a variety of enzymes;
amylolitic enzymes are the most common; phosphorylases and amylcglucon-
sidases occur in special situations. The amylases fall into two cate-
gories; a amylases (a, 1, 4 glucan 4 glucanohydrolase) catalyse the
scission of a, 1, 4 glucosidic bonds at random resulting in- a rapid re-
duction in molecular weight, viscosity, and light scattering; B amylas-
es (a, 1, 4 glucose maltose) catalyse the scission of maltose units from
the non-reducing end of the chain; producing high yields of maltose but •
23
-------
a more gradual reduction In the physical characteristics of the starch
substrate. B amylase activity ceases when a branch point (a, 1, 6 bond)
is reached. The resulting polysaccharide fragments that remain after
B amylase hydrolysis are called dextrins or limit dextrins.
Enzymes that catalyse cleavage of the a, 1, 6 bond have been
found in both intestinal bacteria and associated with certain other
bacteria. However, relatively little information is available.
Characterization of Starches
Six different starches were tested. Soluble potato -starch (Reagent
grade) was used as a reference. The other starches were obtained from
A.E. Staley IManufacturing Company (P.O. Box 151, Decatur, Illinois
62525).
PPPS = Unmodified corn starch
ES-A = Eclipse A. lightly acid converted corn starch
ES-G = Eclipse G, highly acid converted corn starch
SD-5 = Stadex #5j lightly dextrinized corn starch
SD-80 = Stadex #80, highly dextrinized com starch
Iodine Reaction and Carbon Determination
The starch iodine test is a widely used and simple test. However, as
a quantitative test it is subject to a number of variables, specifical-
ly the molecular weight and bonding structure of different starches.
Iodine reacts with amylase to yield a deep blue color complex with „,
approximately one iodine molecule per 7-8 glucose units. (Physical •
Characteristics of Polysaccharide, Florkin and Stotz, p. 189).Iodine
is believed to be complexed in the core of the helically arranged glue-
cose chain. At least 30-35 glucose units are needed for full color
development. Shorter chains give a red color with maximum absorption
peaks in the 520 millimicron region. Chains of less than six glucose
units give no color reaction.
Iodine reagent used for starch determination was made up periodically
using 0.2 wt. % iodine in 2.0 wt. % solution of potassium iodide.
Equal volumes of iodine reagent and sample were mixed and allowed to
stand for 15 minutes. The color was measured in a Coleman Stectrophoto-
meter at 520 and 680 millimicrons. Absorbance at 680 millimicrons is
related to the concentration of amylase units with a long chain length
of the order of 35 and higher. Absorbance at 520 millimicrons is relat-
ed to the concentration of the shorter chain lengths, of the order of
8-12.
Carbon contents of starch solutions were measured in the Beckman Carbon
Analyzer. All starches gave essentially equivalent results, and approach
the theoretical value of 44.5 wt %, carbon. However, there are substant-
ial differences between starches in the iodine reaction as indicated
below.
-------
Starch Concentration for 10$ Transmittance, mg/1
520 my 680 m y
Soluble starch 96.5 123
PFPS 160 148
ES-A 109 112
ES-G 102 114
SD-5 116 143
SD-80 93* 84**
* 50%
**
Molecular Weights of Starches
Molecular weights were estimated from right angle light scattering
measurements of concentrated solutions as described by Debye (Journ.
Physical and Colloidal Chemistry'51, 18, 19^7). Molecular weights are
in the range 30 x 10 for the soluble starch to 220 x 10 for the ES-A
sample after 5 minute autoclaving in distilled water. These values are
substantially higher than the molecular weight determinations reported
in the literature using chemical methods. Stacy and Poster (Journ.
Polymer Science, 20, 57, 1956) have pointed out that physical methods
of molecular weight measurement generally give higher values than the
chemical methods; literature values-based on chemical determinations
are in the range of 160,000 to 360,000 grams per mole for corn amylo/-
pectin whereas light scattering gave values in the range 40-100 x 10
grams per mole. One likely explanation for these differences is that
starch is a mixture of different molecular weight fractions depending
on the source as well as prior treatment. Thus it appears likely that
processed starches would have a different mixture of high and low mole-
cular weight constituents. The work of Stacy and Poster provides sup-
port for this explanation; they report a molecular weight of approxi-
mately 30 x 10 for limit dextrins preparedgby B amylase degradation
of corn amylo pectin as compared to 80 x 10 for the untreated amylo
pectin. (It is assumed that B amylase does not catalyse internal
hydrolytic scissions).
Jeanes. (Encyclopedia of_ Polymer Science and Technologyg Vol. 4., )
reports weight average molecular weights of 40-50 x 10 for dextrans
produced in growing cultures of L. Mesehterloides. One dextran pre-
paration showed a molecular weight of 97 x 10 (weight avarage) usgng
light scattering and a number average molecular weight of 1.7 x 10 and
365,000 as determined by chlorous acid and copper reagent respectively.
Sephadex Gel'Separation of Starches
Sephadex gel columns were used to evaluate the molecular size distribu-
tion of various starches. The objective was to determine the quantity
25
-------
of low molecular weight polysaccharides associated with each type of
starch.
Three sizes of gel material were obtained from Pharmiacia Pine
Chemicals Inc. (800 Centennial Avenue, Piscataway, N.J.).
i
Sephadex Type Molecular Weight
Fractionation Range (Dextrans)
/
C-15 0-1500 '
G-75 1000-50,000
G-150 1000-150,000
The gels and the columns were prepared in accordance with the descrip-,
tive literature provided by the manufacturer. Columns 2.5 cm in dia-
meter and gel beds of 60 cm. were used. The starch was layered into
the column surface and eluted with distilled water. Eluent was tested
for carbon (carbon analyzer) and starch using the iodine test. The
; results from the starch determination appear to be more sensitive.
Typical eluent curves are shown in Figures 4 and 5. Blue dextran 2000
which has an approximate weight average molecular weight of 2 x 10 and
is believed to have essentially no small polysaccharide fragments was
used as a reference curve. The quantity of small diameter materials
was calculated from the extension of the eluent curve for each starch.
Gel Column 0-15 G-75 G-150
Percent of Starch Retarded by Gel %
Soluble Starch 0 0 0-19
PEPS 4 / 0-14 9
ES-A 0 58
ES-G 5 6 14
SD-5 2 18 32
SD-80 3 60 61
The results show that with the exception of SD-80 and SD-5 the starches
have relatively small amounts of polysaccharide below 150,000 molecular
weight, in the range of 0 - 14%. There is almost no material in the
0-1500 molecular weight range. The Stadex starches show substantial
quantities of the lower molecular weight fragments; SD-5 has 32$ below
150,000, molecular weight and 18% below 50,000 while the more dextriniz-
ed SD-80 sample has some 60% in the 1000 to 50,000 molecular weight
range. Those observations are in line with the poor iodine color re-
sponse of SD-5 and especially of the SD-80 sample. The SD series of
starches appear to have been treated (dextrinized) to such a degree
that molecular size does not allow for formation of the iodine complex.
26
-------
ro
FIGURE 4
SEPHADEX GEL SEPARATION OF SOLUBLE- STARCH
10
100
-------
FIGURE 5
GEL SEPARATION Of STAfcCM SO-5
FFFFI
34
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Hydrolytic Degradation of Starch
Enzymatic hydrolysis of starch was measured in batch reactors using
acclimated innoculum from a continuous propagator. Reactors were
sparged with air to provide excess oxygen and to keep the system well
mixed. The tests were designed to measure initial rates of hydrolysis
at various substrate concentrations and to see how the rates of hydroly-
sis change with tame.
Innoculum was collected overnight from a continuous propagator (des-
cribed in section on Experimental Procedure). Soluble starch concen-
itrate was prepared by autoclaving for 5 minutes. Small amounts of
1 starch concentrate were added to aliquots of innoculum and starch con-
centrations were measured every 10 minutes using a modified iodine
test as described earlier. After approximately one hour, another por-
tion of starch concentrate was added and the starch concentration was
again monitored.
Typical results are summarized in Figure 6 (complete data are listed
in the appendix). Starch concentration drops off rapidly at the start
of each test but slows down appreciably as the reaction proceeds. The
rate of starch removal is highest in the test reactors with the high-
est initial concentration of starch. Rates of removal after the second
addition of starch concentrate are not quite as high as the Initial
rates. Changes in rates are shown more explicitly in Figure J. Rates
of starch removal were calculated for each ten minute time increment,
and compared to illustrate the effect of starch concentration and the
effect of time. Figure 7 shows that the initial rate of removal is
Independent of starch concentrations above 100mg/l but drops off at
lower concentrations.
The initial rate data appears to follow. ?Hchaelis-Menton enzyme kinetics
which predict zero order reaction (in substrate) at high concentrations
of substrate and first order reaction in substrate at low concentration.
The calculated adsorption coefficient (Km) has a value of 35mg/l and
the calculated (extrapolated) maximum rate of reaction (Vm) has a value
of 66 mg/1 per minute for the given enzyme concentration. Tne exact
concentration of active enzyme is not known, however, suspended solids
concentration and hence the biomass was measured by millipore filtration,
(40.2, 38.9, 38.2, 37.7 mg/1 for reactors A,B,C,D respectively). The
calculated rate coefficient is therefore approximately 1.7 per minute
(mg/1 starch per mg/1 biomass per minute).
The open points in Figure 7 represent measured rates of removal at sub-
sequent time intervals. It is interesting to note the gradual decrease
in rate of degradation with time, despite the continuing growth of new
biomass which accumulates in the reactor, and is equivalent to an in-
crease of 30 to 50% In biomass from the beginning to the end of the test.
Furthermore, there was a noticeable increase In rate after the second
addition of fresh starch substrate. One possible explanation for these
29
-------
FIGURE 6
DEGRADATION - EFFECT OP STA&CM
18
-68
*•
.to
30
-------
'c >:
O \-
" 70
cn
? .
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OP
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CONCENTRATION
wg/l
-------
observations is that hydrolyzed polysaccharide fragments act as competi-
tive inhibitors of the hydrolytic enzyme; there should be a correspond-
ing increase in the adsorption coefficient, (Kin). The dashed lines in
Figure 7 are calculated using progressively higher values of (Km) at
otherwise constant conditions. Increasing (Km) by a factor of four
accounts for most of the observed drop in rates.
Comparison of Biological Degradation of Different Starches
Rates of biological decomposition for each of the 5 starches were det-
ermined in batch tests. Innoculum was taken from a continuous propaga-
tor operating on soluble starch solution (lOOmg/1 starch in salt sol-
ution). Starch concentrates (200mg/l) were prepared by autoclaving for
5 minutes to dissolve the starch in distilled water. (Details of pro-
cedure and data are listed in the Appendix under Exp. #4-30-68 and 5- "
7-68). Starch concentrate was added at the beginning of each test; air
was sparged into each batch reactor to provide oxygen and to keep the
system well mixed.
Starch concentrations are shown in Figures 8 and 9. More than 50% of
the starch is degraded in the first 30 minutes; thereafter the rate of
degradation slows dovn. The initial rates of degradation (first 30
minutes) are summarized and compared below.
Initial Rates of Starch Degradation
Starch Exp. # mg/1 (first 30 minutes) Relative Rate
Soluble
starch 4-30-68 119 I'.O
ES-A 4-30-68 100 .84
SD-5 , 4-30-68 103 .87
SD-80 4-30-68 78 .66
Soluble
starch 5-7-68 134 1.0
PFPS 5-7-68 151 1.13
ES-6 5-7-68 115 .86
Unmodified corn starch (PFPS) shows the highest rate whereas the dex-
trinized starches show the lowest rates. The most highly dextrlnized
starch (SD-80) shows a tendency to level off after 2/3 of the starch
has been degraded, thus leaving a substantial fraction, about 1/3 that
is apparently not degradable by the existing enzymes in the first few
hours of the test.
A smaller number of mlllipore filtered samples were also tested for
carbon content to determine the rate of removal of carbohydrates.
32
-------
6IOLOG»CAL DEGRADATION OF STARCHES
**
STANCH
10
$t> 40 -»0 «0
TIME, MINUTES
toe
33
-------
230
FIGURE
2 00
!&0
I&O
120
£
2
U
0
§ loo
V
fe4» 80
TIME, MINUTES
«00
-------
Disappearance of carbon was taken to represent biological- growth because
millipore filtration removes bacterial solids but does not remove the
starch polysaccharide products of hydrolysis. Figure 10 summarizes the
data and shows that carbon removal pattern is essentially similar for
all starches. Ihe sigmoidal curve is typical for this type of batch
test. The rate of carbon removal increases for the first few hours
while the biomass undergoes exponential growth; as growth proceeds,
substrate becomes limiting thereby reducing growth rate; the rate of
carbon removal also slows down and approaches zero (constant concentra-
tion) . The residual carbon concentration of 10-20 mg/1 is quite typical
for batch tests and probably represents non-biodegradable carbon for
biomass that is available. The implications of this residual carbon
are discussed elsewhere in this report. Close inspection of the data
on carbon removal shows that the rate of carbon removal for starch SD-80
is somewhat slower than that for the other starches. The effect is most
likely associated with the slower rate of hydrolysis of the SD-80 starch
which in turn slows down bacterial incorporation of hydrolyzed carbohy-
drates. The precise magnitude of the difference in rate of carbon re-
moval has not been established but could be as much as 50% in the course
of the test.
35
-------
FIGURE: 10
.BIOLOGICAL
! ; ; n , . . . : ; .T.
:-h::! . I
I N C OR POR AT IO N _Q,F__
REMOVAL OF
6 f
TIME, HOURS
.36
-------
Soluble Exoenzyme Activity
Ihe location and control mechanisms of the hydrolytic enzymes that cat-
alyze starch degradation are not well defined. There is considerable
support for the concept that in bacterial systems exo-enzymes are close-
ly associated with cell walls. Some enzymes are also released to the
medium in the form of small colloids which pass through 0.45 micron
filters and will be referred to as "soluble" enzymes. The following
experiments were designed to test for the presence of soluble enzymes
in bacterial decomposition of starches.
Innoculum was obtained from a continuous propagator using soluble starch
feed solution (lOOmg/1). Fresh feed solution was added at the beginning
of each test; one reactor received the innoculum as collected, the other
reactor received millipore filtered innoculum (0.45 micron). Starch and
carbon concentrations were measured for a period of several hours; re-
sults are summarized in Figure 11 detailed data are listed in the Append-
ix as Experiments #2-9-68 and 2-10-68. There is evidence of exoenzyme
activity, however, the observed rate is only 3% of innoculum. Similar
results have been reported by other investigators. Banerji (University
of Illinois, Sanitary Engineering Report, No. 29) has demonstrated the
presence of hydrolytic enzymes in activated sludge liquors. Millipore
filtration of mixed liquor, (0.45 micron filter) showed substantial am-
ylase activity; cell free filtrates from acclimated sludges in the en-
dogenous phase showed higher activity than filtrates from sludges in or
near the log phase of growth, 150mg/l/hr versus 20mg/l/hr respectively.
Thus, it appears that prior history and age of the biomass is an impor-
tant variable controlling production and release of exoenzymes.
This idea was confirmed in a test of effluent collected form the inclined
plane test reactor. Filtered effluent showed about 18$ of the hydrolytic
activity observed on unfiltered effluent. The slime layer consists of
considerably older biomass and some of which is undergoing endogenous
metabolism and releasing "soluble" enzymes to the feed solution passing
over the slime layer.
37
-------
FIGURE II
MILM PORE FILTERED
40 60 90
TIME , MINUTES
•38
-------
Degradation of Starch-Glucose Mixtures.
A series of batch, tests were carried out to measure the effect of add-
ing glucose and starch substrate to an innoculum that was previously
acclimated to starch as the only source of carbon. Concentrated sol-
utions of glucose and starch were added at the start of each test;
Reactor Nominal Initial Concentrations, mg/1
Glucose Starch
A 0 100
B 100 100
C 100 200
D ICO 0
See appendix Table Experiment No. 8-29-68 and 9-4-68 for detailed
description. Samples were removed periodically and tested for glucose,
carbon, and starch concentration.
Introduction of glucose with the feed solution has no measurable effect
on the hydrolytic degradation of starch as illustrated in Figure 12.
The decay curve for starch is typical and shows a high initial rate
which then levels off at lower starch concentrations. Comparisons of
the rates of glucose degradation are shown in Figure 13- The presence
of starch appears to slow down the initial rate of glucose utilization.
However, the effect is small at equal concentrations (lOOmg/1 each)
and some 20$ greater when 200mg/l of starch was added along with lOOmg/
1 of glucose. Tne rate of disappearance of carbon is essentially the
same in all tests, indicating that growth of biomass is not affected
by the relative concentration of glucose and starch when the innoculum
is acclimated to starch feed. However, the accumulation of biomass is
greater at the end of the tests which received the largest amount of
degradable carbon.
39
-------
FIGURE 12
-------
FIGURE 13
L.D.E 6 RAO ATI O N OF
ISO 140
TIME , MINUTES
420
4*0
-------
Amino Acid Metabolism
Mlcrobial utilization of amino acids has received considerable attention
in the Microbiology, Biochemistry and Sanitary Engineering Literature.
Rates and mechanisms of catabolism, and rates of direct incorporation
into protein have been described. .Amino acid transport has been studied
for a number of microorganisms. Kay and Gronlund (Journ. Bact., 97,
p. 273; 1968) have presented an interesting comparison of the rate of
transport for Pseudomonas aeruginosa. The results show large differen-
ces between amino acids, 40 fold differences from a low of 1.92 x 1^
micro-moles per minute per milligram by weight of cells to 80 x 10 y
moles/min. - mg. (dry weight) as shown below.
Amino Acid Rate of Transport*
Hydroxyprollne 0
Cysteine 1.92 x 10
Threonine 3-54 x 10
Aspartate 4.11 x 10
Serine 4.20 x 10
Glutamic 4.67 x 10
Proline 5.75 x 10~4
Glycine 5.90 x IQ~^
T^rosine] 1.40 x 10~3
Methionine 1.99 x 10~3
Valine 2.36 x 10~3
Histidine ^ 2.74 x 10~3
Alanine 2.76 x 10~3
Phenylalanine 3.23 x 10~3
Lysine 3.36 x 10" 3
Isoleucine 3.92 x 10~3
Tryptophane 5.20 x 10~3
Arginine 6.75 x 10~3
Leucine 8.00 x 10~3
*Micromoles/minute - mg. of dry weight of cells using an external con-
centration of 10~" moles/liter.
These values are particularly interesting because they also provide some
insight on the relative rates of removal of amino acid from the surround-
ing medium; removal rates are in the range of .02 to 0.1 wt. % on cell
42
-------
mass per minute; thus indicating that these ceils are highly effective
for the removal of amlno acids from dilute solutions. It has also been
shown that incorporation of amino acids is energy dependent, and is
probably mediated by transferases, therefore energy metabolism is a
prerequisite for incorporation of amino acids. The transfer mechanism
appears to be extremely effective; during exponential growth observed
amlno acid concentrations in the cell interior are 100 to 300 fold great-
er than in the medium.
Cells which had been in the stationary growth phase for 3 hours, showed
essentially complete removal of amino acids, except for methionine, from
the surrounding medium and from the cell interior.
Based on their results and extensive review of the literature, Kay and
Gronlund conclude that:
"The enzymatic nature of metabolite incorporation into
cells, first demonstrated with carbohydrates, has been
extended in recent years to encompass the amino acids,
as well as other compounds. The data presented here
revealed that the amino acid transport systems of P_.
aeruginosa have all the properties ascribed to active
transport systems; the transport process is seemingly
energy dependent, temperature sensitive, and saturates
at high substrate concentrations.
Amino Acids which are transported into P_. aeruginosa
equilibrate with amino acids synthesized de novo which
reside in the intracellular pool, and also with amino
acid pools which have been transiently preestablished
from an exogenous source. This demonstrates that, in
this organism, as in E_. coli and in Neurospora crass a,
incorporated amino acids equilibrate with, and are
subject to, the same metablic fates as amino acids
synthesized de novo.
The total amino acid pool levels for P_. aeruginosa
during exponential growth are of the same order of
magnitude as the levels reported for E. coli."
It has also been demonstrated that P_. aeruginosa can utilize the maj-
ority of commonly occurring amino acids for growth as either the sole
carbon source or as the sole source of nitrogen (Journ. Bact. 100,276-
282, 1969).
Utilization of amino acids by activated sludge has been studies by sev-
eral investigators. Carlson and Polkowski (Journ. Water Pol. Control
Fed., 34, 8l6, 1962) showed that activated sludges can be maintained on
single amino substrates as the nitrogen source. Acclimation of the
sludge before exposure to a single amino acid increased rates of met-
abolism, most noticeable with glycine and to a lesser extent with
-------
L-cystlne and L-arginine. Rate of glyclne depletion was reported to be
48 mg glycine per hour per gram of sludge solids. This is equivalent to
0.08 wt. % on cell mass per minute and is in reasonably close agreement
with the values reported in the microbiology literature for incorpora-
tion rates. Malaney and Gerhold (Journ. WaterPol. Cont. Fed., 41, Rl8,
1969) list a number of studies evaluating the rates of utilization of
amino acids under aerobic conditions. The individual amino acids show-
ed substantial variations in oxidizability as measured by oxygen uptake.
However, their conclusion is that amino acids can be considered as a
family of similar compounds readily susceptible to biological oxidation.
The reported oxygen uptake is reproduced below:
P , Percentage of Theoretical Oxygen Demand
ComPQund & 6hr. 12hr. 24hr.
Aminoethanoic acid (Glycine) 4.1 8.1 16.9
2-aminopropanoic acid (a-DL-alanine) 11.7 27.0 43.0
3-aminopropanoic acid (3-alanine) 1.7 6.9 16.0
2-amino-3-methylbutanoic acid (DL-valine) 2.1 4.3 9-4
2-amino-3-hydroxypropanoic acid (L-serine) 8.6 21.0 29.0
2-amino-3-mercaptopropanoic acid (cysteine) 7,5 8.6 11.2
Dicysteine (Lr-cystine) 1.5 2.4 4.7
2-amino-3-hydroxybutanoic acid (DL-threonine) 3.9 8.2 16.2
2-amino-4-hydroxybutanoic acid (DL-homoserine) l.l 2.3 4.4
2-amino-4-mothylthlo'butanoic acid (DL-methionine) 1.5 2.4 2.6
2-aminohexanoic acid (DL-norleucine) 2.3 5.2 12.9
2-amino-3-methylpentanoic acid (L-isoleucine) 2.4 5.3 14.8
2-amino-4-methylpentanoic acid (L-leucine) 1.4 3.6 9.9
2-amino'outanedioic acid (DL-aspartic acid) 8.9 16.2 28.8
2-aminopentanedioic acid (L-glutamic acid) 14.0 25.1 35.8
2-aminobutanedioic amide (L-asparagine) 10.3 19.5 24.7
2-amihopentanedioic amide (L-glutamine) 9.2 19.2 30.7
2,6-diaminohexanoic acid (L-lyslne) 1.8 4.5 14.1
2-amino-5-quanidopentanoic acid (L-arginine) 2.1 7.7 16.5
2-amino-3-phenylpropanoic acid (DL-phenylalanine) 2.2 5.6 16.4
2-amino-(4-hydroyphenyl) propanoic acid (DL-tyrosine) 3-4 9-7 22.6
1-pyrrolylmethanoic acid (L-proline) 4.8 13.1 25.5
l-(2-hydroxy) pyrrolylmethanoic acid (L-hydroxyproline)l.O 2.9 18.2
2-amino-3-imidazolypropanoic acid (L-histidine) 2.8 6.3 16.5
44
-------
2-amino-3-lndolypropanoic acid (DL-tryptophane) 0.6 1.4 4.6
N-acetyl-2-amlnoethanoic acid (acetylglycine) 9.3 10.0 18.5
Glutamylcysteinylglycine (glutathione) 5.1 8.2 22.0
The theoretical oxygen demand represents a calculated value based on
complete combustion to COp and ELO. The low oxygen demand for glycine
as opposed to glutamate is consistent with the notion that amino acids
are utilized for energy, as a source of nitrogen and also incorporated
into cellular protein directly. The small carbon chain of glycine is
obviously not as readily incorporated into the metabolic pathways as
the larger glutamic acid carbon chain.
Glutamic Acid Utilization
A series of batch tests were carried out to measure rates of biological
degradation of glutamic acid at various feed concentrations. Innoculum
was obtained from a continuous propagator using 100mg/l glutamic acid
feed solution without any other sources of either carbon or nitrogen.
Concentrated glutamic acid solution was added to each reactor at the
beginning of each test and samples were withdrawn periodically. The
ninhydrin test was used to measure glutamic acid concentrations and
carbon concentrations were measured independently with the Beckman Car-
bon Analyzer. Typical decay curves of glutamic acid as measured by nin-
hydrin are shown in Figure 14. The results show an increasing rate of
removal initially, followed by a decreasing rate as substrate concentra-
tion drops off. The initial rates are essentially independent of sub-
strate concentration. Data for carbon concentration follow a similar
trend. Analysis of the data in terms of the simplified rate equation
for batch systems as described earlier confirms the conclusion that
utilization of glutamate is independent of concentration except at very
low concentrations. Figure 15 shows that the rate data for carbon re-
moval for all 3 reactors fall on a single straight line regardless of
initial substrate concentration. The Monod adsorption coefficient (Kc)
has been estimated to be approximately l6mg/l. This estimate is pro-
bably on the high side because it is based on residual organic carbon
concentration which includes the concentration of nonbiodegradable car-
bon residues. The corresponding growth rate coefficient is 0.0073 per '
minute and the yield coefficient was measured to be 0.33 grams cell mass
per gram of glutamic acid utilized. The yield coefficient is consistent
with values reported in the literature.
-------
FIGURE 14
BIOLOGICAL DEGRADATION OF; GLUTAMICJ
CONCENTRATION
ooo a
-------
FIGURE 15
I ..... BIOLOGICAL DEGRADATION OF GLUTAMIC ACI0 '
RATE OF
REMOVAL - EFFECT OF COMCENT(?ATIOM
SLOTAMIC AC50 SOSST»?ATE i
CONCENTRATION OF
I..:(.7rX- of ".C"
I1O 163 100 240
TIME , MINOTES
280
-------
Utilization
Glycine was evaluated in a series of tests to determine the effect of
substrate concentration and the effect of acclimation of the innoculum.
Experiment No. 2-20-69 was carried out using innoculum from a continuous
propagator, using glutamic acid as the only source of carbon. The re-
sults show essentially no removal for the first 6-12 hours . It is par-
ticularly interesting to note that longer incubation periods were need-
ed when higher initial concentrations of glycine were used. Carbon re-
moval showed similar trends. Residual carbon at the end of the test
(after 24 hours) accounted for 26-32$ of the original feed carbon from
glycine. Rate of glycine utilization was determined using an acclimat-
ed innoculum obtained from 3 continuous propagators using glycine feeds
of different concentrations, 100, 500 and 1000 mg/1 respectively. (Ex-
periment No. 7-11-69). Half the effluent from each propagator was milli-
pore filtered to remove cell mass and the filtrate added back. This
allowed making comparisons of the effect of cell mass concentration as
well as substrate concentration. The results show that the growth
rate coefficient ( y max) is in the range of 0.0020-0.0049 and the Monod
adsorption coefficient is approximately 80mg/l. The growth rate coef-
ficient for the acclimated innoculum is comparable to the values observ-
ed for glutamic acid. However, the high value of the adsorption coeffi-
cient is unusual. The most likely explanation for this phenomenon is
the presence of protozoa which act as predators and retard bacterial
growth accumulation. In effect, substrate removal kinetics become lin-
ear after a short initial period of exponential growth. The subsequent
prey -predator balance results in a uniform rate of glycine removal as
shown in Figure 16 .
As a further check on glycine utilization a -new series of batch tests was
carried out using glycine acclimated innoculum. Glycine and glutamic
acid concentrates were added in different proportions as shown in Appen-
dix Table 15 (Exp. No. 9-22-69). The carbon data are summarized in
Figure 17- Glycine carbon is not utilized as rapidly as glutamic acid
as shown by the slower rate of carbon removal in Reactors A and B. The
highest rate of removal is observed in reactor C and E where glutamic
acid concentration is 50mg/l. These data confirm earlier indications
that glycine is not as readily utilized as the larger molecular weight
amino acids .
Protein Chemistry
Proteins are polymers of amino acids which are linked by peptide bonds
between the carboxyl and amino groups of adjacent amino acids. Ele-
mental composition tells relatively little of the structure or function
but approximates 45-55 wt. % carbon, 6-8 wt. % hydrogen, 19-25 wt. %
oxygen and 14-20 wt. % nitrogen. Sulfur and phosphorous are,~also present
in small amounts. Molecular weights range from 10,000 to 10 , which
still places proteins in the collodial size range which passes., through
0.45 micron millipore filters. "" ,
48
-------
\ 6
vo
SOp mg/l..;
• i :: :i.. ....'.
(OOO mq/|
... •!. .;.• I ' ..
fOOO mq/l
: I i w
a co 400
TIME , MINUTES
-------
FIGURE 17
H T i ,
„ i
} - i >
i ' !
i '
AMI
1 |
NO
A
!
t—
., — _
CID
'!
DIE
ACID DEGRADATION! WITH! GtyciP^E ; ACCLI MATED
-------
The protein used In this work was obtained from Difco Laboratories
(Detroit, Michigan) under the trade name Bacto Peptone. Typical com-
position is listed below:
Wt. %
Total Nitrogen 16.16
Peptone Nitrogen 15-38
Ammonia Nitrogen 0.04
Organic Sulfur 0.33
Inorganic Sulfur 0.29
Phosphorous 0.22
Ash 3.53
\
Hydrolysis of proteins yields amino acids and polypeptide intermediates.
Hydrolysis is mediated by acids or alkalies in boiling water or by cer-
tain proteolytic enzymes. Enzymatic hydrolysis yields free amino acids.
An excellent review of the proteolitic enzymes and hydrolysis of peptide
bonds is presented by Boyer (The Enzymes Vol. Ill, Academic Press 1971,
vn-d Vo1- IV» I960, p. 193). There 'has been a considerable accumulation
of information on the mechanism, rates and products of protein hydrolysis.
Matsubara (as above, p. 721) points out that microbial proteases are pre-
dominently extracellular and can be isolated from filtrates of bacteria,
molds and yeasts. Most of the common proteolytic enzymes of animal ori-
gin are present in the filtrate from bacterial and mold or yeast cultures,
including amino- and carboxypeptidases and a variety of endopetidases.
Lake animal enzymes, bacterial and mold proteases include enzymes which
are active in acidic, neutral and alkaline pH. The majority do not re-
quire activation, but+s_ome
-------
Innoculum was prepared in a continuous propagator using lOOmg/1 br.cto-
peptone feed solution. The innoculum was collected overnight and aerat-
ed continuously. Concentrates of bacto-peptone and glutamic acid were
added at the beginning of each test to give concentrations in the range
of 120-200 mg/1 protein and 0- 90 glutamic acid.
The results of this test are summarized below:
Initial concentration, mg/1 Growth Rate coefficient, min
Reactor* Protein Glutamic acid Carbon data protein measure
ments.
A
B
C
D
E
120
195
260
185
175
0
0
0
47
90
0.0077
0.0087
0.0087
0.0095
0.0101
0.0087
0.003^
0.0034
_ _ _ _
_ _ _ _
* Data from Exp. No. 3-18-69 In Appendix)
Growth rate coefficients were calculated using the simplified form of
the batch reactor equation (zero order substrate kinetics). This ap-
proach is quite justified because the analysis was limited to the init-
ial part of the test when the substrate concentrations were high. Com-
parison of the growth rates illustrate that the addition of glutamic
acid increased growth rate somewhat, 10-20%. The increase in rate ap-
pears to be the result of incorporating glutamic acid into the cell
growth metabolism. This is illustrated in Figure 18 which compares
protein removal and carbon removal for batch tests which had received
proteins only and mixtures of protein and glutamate. Batch tests with
glutamate feed show a decidedly higher carbon uptake for a given removal
of protein, indicating that glutamate is being removed from solution.
Glutamate acclimated innoculum was used to evaluate it capacity for
degrading protein (peptone). Six tests were carried out using 50> 90
and 120 mg/1 of protein and glutamate solution. The results show es-
sentially no activity for utilization of protein, thus indicating that
this innoculum had lost its hydrolytic activity through acclimation to
glutamic acid as the only carbon and nitrogen source (Exp. No. 3-12-69).
52
-------
(8
*•
lew
tec
T
i
£»N CARSON UTILISATION USING
FEEDS
|
O
Z
•*
u
o
or
6.
160
„
120
80
to
40
10
_j.
i •
:NT
3-'8-
REACTORS iA, 5, c ( NO
li REACTOR pj...i_
I : , : . • I • • : . i
J4_ .REACTOR e L_
8—REACTOR _F_
i
- T_.
-t—
:—1_.
11
-*>••—i-
.._!...
zia
-11.4 ._4
_ ^ ,
. i
i.
..:j: .
:il
i
t • t
•M-
_L--.
_:!__.
.^
.:.L
_.:ON
T
_l_J_
_(_
.,.[..,. L_>_
, , . . 1
^1-11-
_Lc H e mi C*HL_ co N\ Pp s iTi ON .
:.• l:-•;••:••"••""!"•: r "—T"-i--7t~'|—~"f
^-N-^-*-lr-f^-:-^-f
-y
-L2L.
_4_v
,"~t~
[._
10
20
30
CO
..A:
-f-rf-r-
-i
^•^•4U
7*
60
REMOVED
m9/l
53
-------
A similar experiment using an innoculum that had not been operating as
long on glutamic acid showed similar results, except that both glycine
and peptone, were eventually utilized though at a slow rate.
Growth rate , Adsorption coeffici-
Substrate coefficient, rain." ent mg/1
Reactor A Glutamic acid 0.0043 20
Reactor B Glycine 0.002 34
Reactor C Peptone 0.0001
Glycine
Glutamic acid
Observed value for peptone indicates that at the existing conditions,
hydrolysis of proteins was repressed either by preventing the formation
of hydrolytic enzymes or by inhibiting their funtioning.
Milk Solids
A number of investigators have used solutions of milk solids for pro-
cess variable studies. This is one step closer to simulating munici-
pal waste constituent as compared to the use of pure organic compounds
and mixtures. Milk solids contain a large variety of organic materials
such as proteins, carbohydrates and minor quantities of lipids as shown
below:
Analysis of Milk Solids - wt. %
Butterfat (lipids) 0.9
Protein 36.9
Lactos e (carbohydrate) 50,5
Total solids 96.7
Organic solids 88.6
BOD (5 day) 75(mg/100 mg)
Organic Carbon 39.2
Milk solids acclimated innoculum was obtained from a continuous propag-
ator using 100 mg/1 feed solution. Concentrates of milk solids were
added to each of 3 batch reactors and utilization of carbon, protein
and carbohydrate were measured.
The results are summarized in Figure 19. Initial carbon removal is
Independent of the concentration of substrate but the rate slows down
as substrate is depleted. Protein degradation proceeds rapidly and is
-------
essentially complete after 400 minutes. The residual concentration is
probably the result of small peptides and interferences in' the Folin
test. It is interesting to note that carbohydrate removal is slower
than protein degradation. Figure 20 shows the carbon data anaylzed
in terms of the simplified batch rate equation; the results show that
rates of removal are independent of concentration. Growth rate coef-
ficient (y) is 0.0016-0.0019 per minute and the adsorption coefficient
(Kc) is in the range 18-31 mg/1. based on total carbon. If the non-
biodegradable carbon is taken into consideration, the value of Kc is
in the range 6-13 mg/1.
55
-------
PK30RE 19
BIOLOGICAL DEGRADATION OF MILK SOUOS
REACTORS
56
-------
FIGURE 2O
ui
—3
1,00
*»r
Coo
•coo t2e»
TIME, MINOTC?
-------
Lipid Chemistry
The term lipid refers to a large group of organic compounds including
fatty acids and their alcohol and sterol esters. The most prevalent
lipids in municipal waste waters are the glyceride esters and the alkali
salts of fatty acids.
Tne most significant chemical characteristic of lipids is their high car-
bon and hydrogen content and relatively low oxygen content. Where car-
bohydrates have 40-^5 wt. % carbon, fatty acids have upwards of J0% car-
bon and very little combined oxygen. The result is that lipids have a
much higher free energy content (available energy) than any other or-
ganic material. The yields of cell mass per unit weight of substrate
are correspondingly high and the relative amounts of .oxygen needed for
complete oxidation (ultimate BOD) are much higher than those for carbo-
hydrates and amlno acids - proteins. Lipids are generally deficient
in nitrogen, phosphorous and sulfur and thus resemble carbohydrates.
The role of microbial enzymes in transformation of lipids has been the
subject of extensive research and publication (Microbial Transforma-
tions of Steroids, A handbook by Charney and Herzog, Academic Press
1967; Fatty Acids, Edited by Markley, Interscience Publishers, 196?).
The action of microbial systems includes a wide variety of enzyme cat-
alyzed reactions from oxidations and reductions to isomerization and
hydrolysis.
"As far as we know, transformations of steroids, carried
out with intact microbial cells, occur within the cell
and not in the medium surrounding the cell. To enter the
cell the steroid being transformed must dissolve to some
extent in the medium so that it can diffuse through the
cell wall and into the enzyme-rich interior. The prac-
tical implication of this requirement is that solubility
and rate of diffusion may become the rate-limiting factors
for transformation. Most steroid substrates ordinarily
employed have modest, though measurable, solubilities in
water and in the aqueous media used for microbial culture.
To ensure saturation of the medium and to minimize this
rate-limiting effect, steroids are often introduced into
reactions in micronized form or, more conveniently, in
solution in a water-miscible solvent from which precipi-
tation in very/fine particles occurs upon dilution with
the aqueous medium containing the microorganisms.
The experimental findings may be interpreted reasonably to
show that microbial enzymes are not highly substrate speci-
fic. The alternate explanation for the diversity of sub-
strates which a given species can transform is that the
organism has a different enzyme for each new substrate.
The latter explanation is much less satisfying, and no
evidence has been adduced in its support."'
58
-------
One possible explanation for the nonspecificity of microbial enzymes
is that microbial organisms are also producers of lipids internally and
therefore requires the capability to catabolize lipid materials in the
natural growth cycle.
The hydrocarbon chain and the carboxyl group are the most prominent
characteristics of lipids. They impart special physical characteristics
which make for.low solubility and surface activity in water solutions.
Solubility in water is a strong function of molecular size, pH and salt
concentrations.
Solubilities of Fatty Acids in Water
Grams acid per 100
Acid
Caproic
Heptanoic
Capryllc
Nonanoic
Capric
Hendecanoic
Laurie
Tridecanoic
Myristic
Pentadecanoic
Palmitic
Heptadecahoic
Stearic
0°C
0
0
0
0
0
0
0
0
0
0
0
0
0
.864
.190
.044
.014
.0095
.0063
.0037
.0021
.0013
.00076
.00046
.00028
.00018
20
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
°c
968
244
0068
026
015
0093
0055
0033
0020
0012
00072
00042
00029
g. water
30
1.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
0.
°c
019
271
079
032
018
Oil
0063
0038
0024
0014
00083
00055
00034
45°C
1
0
0
0
0
0
0
0
0
0
0
0
0
.095
.311
.095
.041
.023
.013
.0075
.0044
.0029
.0017
.0010
.00069
.00042
60°C
1
0
0
0
0
0
0
0
0
0
0
0
0
.171
.353
.113
.051
.027
.015
.0087
.0054
.0034
.0020
.0012
.00081
.00050
GJ. Org. Chem., 7, 546, 1942)
Substantially higher solubilities
are obtained in alkaline solution
Lithium Salts
of the long carbon chain fatty acids
by salt formation, (saponification).
gm/100 gm H90 (25°C)
Laurie
]Y(yristic
Palmitic
Stearic
0.187
0.036
0.015
0.01
59
-------
It has been observed that a number of the higher molecular weight fatty
acids can be dispersed in water in excess of their reported solubility
and maintained in suspension. One mechanism for such dispersion is
the formation of a thin film at the air-water interface, (frequently
assumed to be a monomolecular film). This in effect makes the air-
water interface a rich source of organic material and oxygen for those
microorganisms that metabolize fatty acids. Another phenomenon which
enhances the "apparent" solubility of fatty acids is the fcarnation of
micelles. Micelles are aggregations of molecules'to form colloids.
According to Jirgenson, micelles are formed by molecules with large
hydrophobia parts and a strongly polar group in the same molecule.
(Organic Colloids, Jirgenson, p.50, Elseier Press 1958). Arrangement
of molecules is believed to be such that the long carbon skeletons are
parallel to each other with the carboxyl group extending into the sol-
ution in order to minimize water contact. This type of behavior is
evidenced by decreasing osmotic activity at increasing concentrations
of the hydrophobic material. Micelle formation is dependent on several
variables, including concentration of the molecular species. Critical
concentration which leads to micelle formation have been reported.
Potassium laurate 0.02 moles/1. (26°C)
Potassium Linoleate 0.0002 moles/1. (26°C)
Fatty Acid Degradation
Three fatty acids were tested, lauric, palmitic, and stearic. Feed
solutions were prepared by solubilizing the fatty acid in approximately
200ml. of 1.0, normal sodium hydroxide solution. This concentrate was
then dispersed rapidly into feed solution (5 gallon batch) which con-
tained all the necessary salts. The pH was adjusted by adding hydroch-
loric acid. The resulting solutions frequently developed a cloudy
appearance, Indicating that there was some formation of colloidal
dispersions (micelles). •
In order to test for colloid formation, the feed solutions were occas-
sionally filtered through millipore filters. It became apparent that
at the desired concentrations, up to lOOmg/1, only lauric acid solu-
tion remained in reasonable dispersion. As a result, most of the bio-
degradation studies were carried out with lauric acid.
/
/
Considerable effort was expended on developing or adapting analytical
procedures for the precise determination of fatty acids at the concen-
trations in question. (0 to 100 mg/1). Unfortunately, the available
methods are not sufficiently sensitive at low concentrations. Further-
more, extractions'with fat specific solvents were not quantitative nor
sufficiently reproducible to allow measuring the small changes in fatty
acid content that are obtained in kinetic studies of this type. As a
result, it was,necessary to calculate or estimate fatty acid degradation
from carbon removal data and specific analysis for the other substrates
60
-------
in order to back calculate removal of fatty acids.
Biological uptake of lauric acid was measured in batch tests using
innoculum from a continuous propagator using 50 mg/1 lauric acid feed
as the only source of carbon. The innoculum was thus well acclimated
to metabolizing the lauric acid substrate. Different amounts of fresh
feed solution (50 mg/1) were added at the start of each batch test, and
samples removed periodically for carbon analysis (Experiment No. 10-7-69)
and 10-18-69). The results show that acclimated innoculum is an effec-
tive biological system for removing fatty acids as illustrated in Pig.
21 which shows the removal of carbon as a function of time. The results
were correlated using the simplified batch reaction equation (assumes
adsorption coefficient K is negligible). The calculated growth rate
coefficient is'0.0037 per minute (25°C), carbon residue was approximately
6 mg/1 and corresponds to 15 wt. % of the initial feed carbon. Higher
growth rates were obtained in batch tests which started with higher
dilution ratios of feed to innoculum, up to 0.006 per minute, (Exp. No.
12-18-69 and 12-23-69) reason for this is not known. It is unlikely
that substrate concentration is responsible because the calculated
asdorption coefficient (Kc) from these batch tests on fatty acids is
quite low, 7-16 mg/1. One possible explanation is that the fatty acids
are removed from solution-suspension by adsorption on the biomass sur-
face and are therefore not as readily available for ingestion into the
microbial cells.
A similar series of batch tests (Exp. No. 12-23-69) using palmitate
acclimated innoculum shows similar results. The calculated growth rate
for these tests in the range of 0.0057 - 0.0064 and the adsorption co-
efficient K is 7-16 mg/1. The average value of the calculated yield
of cell mass is 95 wt. % based on carbon removed. The carbon residues
were very low, indicating that substrate was almost completely removed
from solution. Initial rate data using palmitic acid feed solution may
not be reliable because there is some evidence that palmitate was re-
moved by the millipore filters prior to analysis. Innoculum which was
acclimated to lauric acid feed (50 mg/1) was contacted with feed concen-
trates of glucose and glutamic acid in a series of batch tests (Exp.
No. 12-30-69 and 1-29-70). The results show that the microbial pop-
ulation is capable of metabolizing these substrates without any dif-
ficulty and without any lag. Figure 22 shows the removal of carbon;
the initial rate is independent of total carbon concentration and
substrate added. The initial growth rate coefficient is 0.0065 per
minute. , However, it is interesting to note that the -calculated values
of Kc are higher for the batch tests which had received glucose and
glutamic acid, 6 mg/1 for Reactor A using only laurate and 40-50 mg/1
for the mixed feeds. This suggests that the lauric acid acclimated
microorganism population does not proliferate as well where lauric acid
is no longer available. Further evidence for this is shown in Figure
23 which summarizes the data for removal of glucose and glutamic acid
measured independently. It is interesting to note that the removal of
glutamate begins 200 minutes after the start of the test and glucose
61
-------
FIGURE" 21
FATTY ACID DEGRADATION - CARSON
a RE/HCTOR *
• REALTOR R
* RETACTOR C
+ REACTOR D
O REACTOR E
X REACTOR F
t , \ B t I . I t ( . n r . i . . . i -i r FT T - . . L . | f r n . . t r i [r r I - [r [r r j r I I I f - I ' t *)-. - -i I rlilrri) ••i^.j^X . . i H L I [ I n > -4 • • •
''
cct
TIME ,
62
-------
FIGURE 22
DEGRADATION OF MIKED FEED SOLUTION BY
LAURATE ACCLimATED INNOCULC/A/t
REACTOR
A
B
C
D
LAUl$!C ACIP
-I-
4- 6LUTAMATE
t 6LUCOSE 4
TIMS, MINUTES
63
-------
FIGURE 23
R£MO\/Al. OF ©LUTAMATS" AMD GLUCOSE
LAURATE ACCUMAT£f>
jcc Stic iccc n<>o I2oc lice
106 260 VCO 4CO ?CC
o
TIME ,
-------
removal is delayed for 300-400 minutes. Thus lauric acid was removed
preferentially and the other substrates were utilized only after most
of the lauric acid substrate had been consumed. The utilization of
glutamate before glucose is especially noteworthy. The batch tests
were fed with a nitrogen limited solution so that this may account for
some preferential uptake of glutamate, however, it is also evident
that glucose was not used until most of the glutamic acid was degraded
so that the nitrogen deficiency is not the whole answer. ,
Additional tests using innoculum from a propagator using mixed feed
solutions were carried out (Exp. No. 2-23-70, 4-30-70, 6-19-70, and
7-15-70). Concentrates of lauric, glutamate and glucose were added
at the start of each batch test. To overcome the nitrogen deficienc-
ies observed in some tests, Exp. No. 6-19-70 and 7-15-70 were mod-
ified to include a source of ammonium ions when glucose or laurate
concentrates were added. Figure 24 shows the removal of carbon when
mixed feed innoculum is exposed to lauric acid, and glutamic acid
alone and in admixture. Initial rates of removal are essentially
equivalent; carbon concentration drops off to approximately 30 mg/1
in less than 600 minutes except for the mixed substrate. It is
also noteworthy that the residual carbon level remains high (30 mg/1)
even after 100 hours. Comparison of the rates of glucose, and glu-
tamic acid removal show that the latter is used preferentially over
glucose, Figure 25. This holds for the pure substrate tests as well
as the mixed feed test. Furthurmore, the removal of lauric acid is
at least as fast as the other substrates. The more gradual removal
of carbon from the mixed feed test finds a parallel in the more
gradual removal of glucose shown in Figure 25.
65
-------
FIGURE 24
M
-------
FIGURE 25
FEED INN0CULUM
GLUCOSE AND 6LUTAJAATE
• REACTOR 6
* REACTOR C
4 REACTOR 0
i«c
TilV»e , MINUTES
tec icO
TIMC, MIMUTES
67
-------
Rate Studies Using Treatment Plant Biomass
A series of batch tests were carried out using activated sludge from
the Minneapolis-St. Paul Sewage Treatment Plant.
The tests were designed to evaluate the activity of activated sludge
biomass and to measure the relative rates of removal of the three major
categories of substrate, namely carbohydrates, proteins and lipids, (Exp.
Nok 7-23-70 and 8-11-70). Activated sludge was taken at the plant just
prior to the tests. Concentrates of various substrates were added at
the start of each test. Samples were withdrawn for analysis as pre-
viously described. Experiment No. 8-11-70 consisted of 7 batch tests
each starting with 200. ml of dilute activated sludge (492 mg/1 sus-
pended solids). Concentrates of pure substrates were added as shown
below.
Volumes of Concentrate Added*, ml.
Glucose Starch Glutamate Peptone Laurate Reactor
i|0 A
40 B
^ _ _ 1|0 C
i. - - ' 40 D
_-_ 160 E
20 20 40 F
u A* _ 20 20 40 G
*1QOO mg/1 except for laurate which was 100 mg/1 as lauric acid.
Comparison of the concentration versus time profiles of each substrate
and carbon gives some insight on the relative rates of removal and the
ability of the biomass to handle single substrates in large doses. A
second batch of concentrate was added after 400 minutes and substrate
removal measured for an additional 1000 minutes.
Analyses of the carbon data shows that glucose was removed more
rapidly than any other substrate. Glutamate was removed rapidly after
the second addition of concentrate but showed a lag during the first
part of the test. The most rapid removal of carbon was noted with the
mixture of glucose-glutamate-laurate. By contrast, degradation rates
of the colloidal substrate, starch and protein, were slower in both
parts of the test. Data for glucose removal are shown in Figure 26.
Glucose is removed completely and at a constant rate from the beginning
of the test. Figure 27 shows the corresponding data for amino acids.
Removal rates are more variable. The gradual increase in amino acid
concentration in Reactor G is probably the result of hydrolytic degra-
dation of proteins to form amino acids which are subsequently removed
68
-------
by the biomass. Removals of starch and protein are summarized in Fig.
28 and 29 respectively. The rates of removal after the second addition
of concentrate are higher in each case, suggesting that the sludge as
received is not sufficiently acclimated to hydrolyze colloidal substrates
at high rates. The slow rate of colloid degradation seems surprising
because municipal sewage has some 50% suspended material of which half
is settleable and half is in the colloidal size range. One possible
explanation for the hydrolytic activity may be the prior history of the
recycled sludge biomass. Free exoenzyme being relatively small colloids,
molecular weights of 100,000 or less, are more likely to be carried out
with the effluent from the final sedimentation tank. Ihe hydrolytic
enzymes associated with the cell surface are frequently subjected to a
period of endogenous metabolism in the final sedimentation tank and in
the latter part of the aerator itself where the detention time is long.
69
-------
26
GLUCOSE REMOVAL £y ACTIVATED SLUDGE
• REACTOR A
X REACTOR F
|OC
TIME ,
70
-------
FIGURE 27
AMINQ ACID REMOVAL
ACTIVATED SLUDS£ BlOMASS
REACTOR C
REACTOR P
REACTOR F
G
71
-------
FIGURE: 28
STARCH REMOVAL BY
ACTUATED SLUO6E
8
REACTOR (5
ICO
TIME, MINUTES
72
-------
FIGURE 29
ACTIVATED 5UID6£
73
-------
Measurement of the. Rate of Oxygen Uptake of'.Sewage Solids
This experiment was designed to measure the rate of oxygen uptake re-
sulting from the addition of typical raw sewage solids to a typical
mixed liquor activated sludge innoculum. Parallel experiments were
carried out to measure oxygen uptake using mixtures of soluble sub-
strates (glucose and glutamic acid) and colloidal substrates (peptone
and starch) and aliquots of the same innoculum. Tests were carried
out in a 1.5 liter enclosed vessel. Oxygen determinations were made
using a dissolved oxygen electrode probe; samples were analyzed for
organic carbon.
The sewage solids were obtained by centrifuglng primary effluent from
the Mlnneapolis-St. Paul Treatment Plant. The solids were collected and
autoclaved for 10 minutes and stored under refrigeration until use.
Prior to the test the solids were treated in a blender for about 5 min-
utes to disperse large flocculated material and to reduce particle size
somewhat. Feed concentrates of glucose, glutamlc acid, peptone, and
starch were also autoclaved prior to use.
At the beginning of each test, sewage solids or fresh feed solutions
were added as shown below:
Reactor A - 2.0 liters mixed liquor; no feed added, mixed liquor
suspended solids concentration was 1,330 mg/1.
Reactor B - 1.5 liters mixed liquor, added 0.25 liters each of
1600 mg/1 concentrate of glucose and glutamic acid,
mixed liquor suspended solids concentration was 890
mg/1.
Reactor C - 1.5 liters mixed liquor, added 0.25 liters each of
1600 mg/1 concentrate of peptone and starch, mixed
liquor suspended solids concentration was 875 mg/1.
Reactor D - 1.5 liters mixed liquor, added 0.5 liters of sewage
solids obtained from centrifugation of raw sewage,
mixed liquor suspended concentration was not determined
after solids addition but was calculated to be 860 mg/1
based on the innoculum alone. The solids alone had
a suspended solids concentration of 2,715 mg/1.
Reactor E - 2.7 liters of mixed liquor from composite of reactors
B, C, and D, added 0.1 liters each of 900 mg/1 glucose
and glutamic acid solution.
Reactor F - 2.7 liters of mixed liquor from composite of reactors,
B, C, and D, added 87.5 ml. of sewage solids.
The mixtures were aerated and agitated continuously except for the time
during which oxygen measurements were carried out. The mixture was
transferred into the special reactor and oxygen determinations were made
-------
continuously usjjig a strip chart recorder. The rate of oxygen consump-
tion was then determined from the strip chart recordings. Sarrples were
also removed and total organic carbon measured for each oxygen consump-
tion test.
Oxygen uptake rate expressed as milligrams of oxygen consumed per hour
per gram of suspended solids as a function of time in minutes are shown
in Figure 30, the corresponding organic carbon concentrations are shown
in Fig. 31. Reactor A represents the base case mixed liquor activated
sludge without any extraneous addition of fresh substrates. The oxygen
consumption rate remains fairly constant at about 10 milligrams per hour
per gram of suspended solids. By constrast both reactors B and C show
a marked peak after the addition of fresh substrate. Oxygen uptake in
Reactor C using collodial substrates (Peptone and Starch) are approximat-
ely the same as for Reactor B using soluble substrates (glucose and
glutamic acid). This suggests that mixed liquor contains sufficient
concentrations of hydrolytic enzymes to convert colloidal materials
into assimilable soluble substrates. Reactor D which received the sew-
age solids additionally showed the highest oxygen uptake rate. This is
particularly surprising in view of the fact that the dissolved carbon
of Reactor D was if anything somewhat lower than Reactor C. One possible
explanation for this observation is that the sewage solids contain a broad
range of substrates which favor growth of a large variety of microorgan-
isms, such as are found in sewage. By contrast it may be that speciali-
zed substrates, such as pure compounds, may be restrictive in the spec-
ies that can thrive immediately on the limited substrate variety.
Test results using mixed liquor from an activated sludge plant as in-
noculum show that there is sufficient enzyme activity to degrade col-
loidal materials (as measured by oxygen uptake). Tnis conclusion is
based on the observation that the addition of colloidal substrate gave
approximately the same rate of increase in oxygen uptake as when soluble
substrates were added. Tne initial dosing of food materials is consum-
ed during the first few hours but then levels off leaving a large resi-
due of organic carbon in solution. Oxygen uptake rate also levels off
but continuous for more than 20 hours without any substantial changes
in dissolved organic carbon. The results indicate that continuous
aeration leads to endogenous respiration of the cell mass but does not
reduce the dissolved organic carbon concentration of the liquor to any
appreciable extent during the first 20 hours or more.
75
-------
CT\
RAT£ OF i ACTIVATEo
SEWAGE ! SOUOS
3.COC
-------
Me
FIGURE 31
2s:
2w
FOR
MEM
: . ^^ ^^ ^^ —^^^ '^ ^MB - ^k ^B • -i ............. ; - - - - I • -t
CARBON CONCENTRATION
SOUOS
REACTOR
REACTOR
REACTOR
+ RCACTOR
o
X
-------
FILM FLOW REACTOR TEST'RESULTS
There Is. growing interest in the quantitative, description of biologically
mediated reactions In connection with, waste, water treatment and natural
purification of waters. IVbst of the published data in this field concerns
well mixed reaction systems in which, the active cell mass Is suspended
in the growth medium by agitation. Relatively little Information is
available on the kinetics of stationary, biologically active surfaces
(slime layers). Stationary slime layers are important in at least two
areas; namely, in the trickling filter process for sewage treatment and
in the attached biomass of stream beds.
This section describes results of studies using fixed biologically ac-
tive slime layers to determine the kinetics of removal of organic sub-
strates. The substrates were chosen with a view to simulating the maj-
or constitutents of domestic waste water; namely, carbohydrates, amino
acids-proteins, and fats. The immediate objective is to provide a better
understanding of the trickling filter process in sewage treatment. How-
ever, the approach and results may be of general interest in biological
processing.
MASS TRANSFER CONSIDERATIONS IN BIOLOGICAL TREATMENT
It is generally recognized that there are many similarities between
enzyme kinetics and heterogenous chemical reaction kinetics. Reactants,
intermediate adsorbed species and product species can be defined and
measured. By contrast, the biological activity of whole microorganisms
is much more complex. Mcrobial metabolism is the sum of thousands of
enzyme catalyzed reactions, simultaneous and sequential, dependent and
Independent of each other. Nevertheless, it has been found useful to.
apply some of the concepts of heterogenous catalysis to evaluate biolo-
gical kinetics; namely, mass transport of substrate to the active sites
and adsorption and reaction at the surface. In heterogenous catalysis,
mass transfer limitations are likely to occur when, (a) concentration of
reactants is low, (b) catalytic sites are inaccessible to bulk flow, and
(c) potential reaction rate is high. Liquid phase reaction systems are
frequently limited by mass transfer because diffusional transfer through
the boundary layer is very slow. Some of the same conditions prevail
in biological,waste water treatment systems such as trickling filters
and to a lesser extent in the floe particles in activated sludge treat-
ment.
A number of investigators have shown that biological reaction rates
are limited by the availability of either substrate, oxygen, or salts.
Rashevsky (Mathematical Biophysics; volume I; page 36, Dover Press) has
described an analytical model for a spherically shaped microorganism
Immersed in a bulk fluid. The model describes concentration gradients
78
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in the exterior boundary layer of the cell assuming that the layer is
stagnant and mass transfer is by molecular diffusion. Mueller, Boyle,
and Lightfoot, (21st Purdue Industrial Waste Conference, May 1966)
studied the effects of floe size in a wen mixed agitated reaction vessel
to simulate the conditions obtained in activated sludge aeration tanks.
For dispersed cells of Zooglea ramigera, minimum oxygen concentration
for uninhibited growth was found to be 0.1 mg/1. For floe particles,
minimum 02 concentration was 0.6-2.5 mg/. depending on floe size. Re-
sistance to oxygen mass transfer in the liquid film surrounding floe
particles was negligible compared to the resistance within the clump
of cells.
Studies using fixed slime layers to simulate some aspects of trickling
filter operations have been reported by several investigators. Gulevich
(Johns Hopkins diversity Report, Jan. 1967) used rotating disks covered
with biological slime and immersed in feed medium. Glucose uptake rate
was found to be a function of the velocity field. Hartmant (J. Wat.
Poll. Control Fed., 39, 958, June 196?) used a tubular reactor coated
with biological slime and measured the effects of flow rates on oxygen
consumption. The results show that increasing the velocity increased
oxygen consumption, except at very high flow rates. He ascribes this
to increased mass transfer from the bulk fluid to the slime layer.
Swilley (Riee University Report, Feb. 1965) and Maier (San. Eng. Div.,
ASCE, SA4, p.91, Aug. 1967) used a flat surface slime layer to study
biological uptake of glucose from medium percolating over the slime in
a thin film. These studies have demonstrated that mass transfer limits
uptake at low concentrations. The slime layer-film flow reactor data
have been considered in the light of analytical models patterned after
the film flow reactor models used in heterogenous catalysis. A der-
ivation and solution for the flat plate reactor has been published by
the author (Thesis, Cornell University, Sept. 1966) and will not be
repeated here. The model allows making predictive statements about the
maximum amount of substrate available at the slime-liquid interface
as a function of flow rate, slope of surface, substrate concentration
in the bulk liquid phase and substrate type. This model has also been
used as a reference point for the studies to be described below.
BIOLOGICAL SLIME LAYER
Most of the literature discussion of slime layers is with reference to
trickling filters. Holtje (Sew. Works Journ., 15, 14, Jan. 19^3) has
described the development and species composition of slime layers in
trickling filters. Tygically, the slime consists of large numbers of
microorganisms (3 x 10 cells/cm^); a large variety of microorganisms,
namely bacteria, fungi, protozoa, and higher forms of life have been
identified. Popluation distribution and density reflects environmental
conditions such as composition and concentration of wastes, temperature,
and hydraulic loading. Surface growth starts with the attachment of
79
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zoogleal bacterial at the solid surface. Wattle (Sew. Works Journ.,
15, 476, 1943) has described the cultural characteristics of pure cul-
tures isolated from trickling filter slimes; they, are gram negative
short rods, enmeshed in a gelatinous mass. Their characteristics.are
similar to Zooglea ramigera as described in Sergey's Manual of Deter-
minative Bacteriology" Hawkes (The Ecol. of Waste Water "Treatment,
McMillan Co.,,1963) has published the most comprehensive- description
of the ecology of slime layers. It has been suggested that bacterial
slimes form a gelatinous matrix which acts as a binder and holds the
bacteria together. The slime layer thus provides a habitat for other-
species. Filamentous forms such as Sphaerotilus and Beggiatqa are
frequently present. Nitrite and nitrate producing bacteria build up,
given sufficiently long residence time. The occurrence of fungi depends
on operating conditions. .Since bacteria and fungi are. in competition /-
for soluble substrates, the bacteria generally predominate except at
unusual pH. It is believed that the development of protozoa is closely
tied to the bacterial population • on which they feed. However, Brink
(Intl. Rev. Gas. Hydr. 52, 1, 1967) questions the importance of bacteria
as food sources for protozoa and suggests that ciliates feed mainly on
dead organic matter. Higher forms of life, such as worms and insects
are considered to be. secondary feeders in that they derive their nutri-
ents from the degradation of other microorganisms. The thickness of the
biological slime layer varies from a few microns to 0,5 cm; the steady
state thickness depends on hydraulic load and availability of nutrients.
Bacteria predominate at the liquid interface where there is direct ac-
cess to soluble and colloidal nutrients.. Fungi and filamentous forms
predominate in the inner layers next to the solid support.
During the last few years a number of investigators have studies the
kinetics of biological surfaces under more controlled conditions -in .
order to obtain more detailed descriptions of,the effects, of operational
variables and to allow .making more precise measurements of the effective
slime thickness. Sanders (Air and Water Poll., Int. Jt, 1(D,,235) stud-
ied the attachment and formation of slime layers on submerged, surfaces
in a flow system using nutrient broth medium. He reports a value,of
10 microns (10 cm) for the average thickness of the first ^ attaching
layer of microorganisms. Thickness increases with time, but a steady
state rate of oxygen utilization was observed after the slime^ thickness
reached a depth of approximately 20 microns. The limiting thickness
(just prior to complete break away) varied from 80 to 400 microns. It
is clear that the slime layer is not a smooth and uniform- surface.
Sanders reports topographical variations of the same order of magnitude
as the average thickness of the slime layer. The slime is periodically
torn away from its attachment (after about two weeks) and the build up
process starts anew. Bulk flow velocity has the expected effects; low
flow rates (O.lft/sec.) ultimately result in thicker slime layers where-
as higher flow rates (1.Oft/sec_.>increase the maximum uptake rate, of
oxygen from 38 xlO to.68 x 10 milligrams per minute per centimeter
squared of surface.
80
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The Stevenage Laboratory of the Institute of Sewage Purification
(1956 Report) has run tests using a rotating cylindrical surface to
measure, the effect of slime accumulations in sewage treatment. Iheir
data show that the rate of purification increases, with increasing slims
mass until an amount equivalent to a thickness of 0.012 cm. has built
up. Further accumulations had no effect on the rate of uptake of nut-
rients. Komegay and Andrews (J. 'Wat. Poll. Control Fed., ijp_, R400,
Nov. 1968) report that the maximum effective thickness of slime in an
immersed film reactor is approximately 70 microns. In a study using
a flat surface slime layer, the author showed that increasing, slime
thickness In the range of 0.05 to 0.2 centimeters had no effect on glucose
uptake rate. The measured rate of glucose uptake has been compared with
uptake rates measured in well mixed reaction vessels to allow estimating
the effective thickness of the slime layer. A cell size of 0.5 to 1.0
microns was assumed. The results indicate that an active layer of one
to two cells could account for the observed glucose uptake rate.
Studies of a number of investigators confirm the conclusion that the
biological activity responsible for substrate uptake is localized in a
thin film at the interface between the slime layer and liquid film. This
lends support to the idea of modeling the behavior of the slime layer in
terms of a catalytically active surface akin to heterogenous catalysis
in a film flow reactor.
FILM FLOW REDACTOR—OPERATING PROCEDURE
A flat inclined plane, illustrated in Figure A-l (Section A) was used
as a support for the slime surface. The plate, 60 cm. long and 11 cm.
wide, was covered with a wire mesh as a framework to' hold the slime layer
in place. Growth medium was passed over the surface in a thin film.
The surface was scraped periodically with a knife edge to remove cell
rass and ensure uniform slime thickness. Scraping did not affect the
rate of uptake of substrate, Feed and effluent samples were tested to
determine substrate removal. Samples were collected over a period of
1 - 10 minutes and filtered through a 0.^5 micron millipore filter to
remove active 'cell material. The surface was allowed to equilibrate for
at least 30 minutes after each change in operating conditions. Previous
experience' has shown that this was more than adequate to achieve steady
state provided that there is no need for acclimatization of the cells
to a new substrate. The data are listed in Appendix Tables 29-34-
STARCH DEGRADATION
A'''slime layer was grown using a feed solution containing 100mg/l._ starch
as the only source of carbon, and salts. The original microorganism
seed was obtained from the Mlnneapolis-St. Paul Sewage Treatment Plant.
81
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Four levels of starch concentration were tested at various flow rates
ranging from 1 to 75 milliliters per nimute per centimeter of plane
width. Starch concentration was determined colorimetrlcally using
iodine reagent and the Beckman Carbon Analyzer was used to determine
organic carbon as described in Section A.
Typical results using soluble starch are shown in Figure 32, where
effluent concentration is correlated with reciprocal flow rate. Efflu-
ent concentration was found to vary inversely with flow rate. This is
in accord with earlier observations using glucose which showed that the
quantity of substrate removed is essentially independent of flow rate.
It follows that the slope of the correlation lines is a measure of the
rate of substrate uptake. Increasing feed concentration from 20 mg/1
starch to 50 mg/1 more than doubles the slope and the rate of starch
degradation. A smaller increase was observed for the 100 mg/1 liter
feed, while above 100 mg/1, there was essentially no change in slope.
These data are summarized in Figure 33, the rate of substrate degrada-
tion is expressed as milligrams per square centimeter of slime surface
per minute and is correlated with feed substrate concentration. The
measured rates of starch removal are compared with the rates of starch
mass transfer calculated from the diffusion control model. A value of
0.08 cm /sec. was used for the molecular diffusion coefficient of starch
is water at 20°C. There is close agreement up to 50 mg/1 feed concen-
tration. At higher feed concentrations, the measured rates of degrad-
ation- level off and become independent of feed concentration. Starch
degradation in excess of that calculated by the mass transfer model was
observed occasionally at low concentrations. This is believed to be
due to the counter diffusion of hydrolytic enzymes and cells from the
slime surface into liquid film. Spot tests show that there was contin-
uing degradation of starch in the receiving vessel at rates of 0.1 -
0.2 milligrams per liter per minute.
Removal of organic carbon proceeds at a slower rate than starch de-
gradation. Nevertheless, the effect of feed concentration follows the
same pattern, carbon removal increases with feed carbon concentration
then levels off at high feed concentrations.
The observed pattern of starch degradation and organic carbon consump-
tion lend support to the explanation that starch degradation is mass
transfer limited at low concentration and reaction rate limited at
higher concentrations. The slime layer behaves like a catalytically
active surface which catalized hydrolysis of starch colloids into
soluble fragments; part of the solubilized carbon is consumed while the
remainder diffuses back into the liquid film.
Measured carbon uptake accounts for less than 50% of the degraded starch
at feed concentrations above 50 mg/1 of starch. The difference between
rate of carbon uptake and starch degradation indicates that these pro-
cesses occur independently and possibly at different locations on the
surface. The presence of hydrolytic activity in the liquid film (with-
out any measurable carbon removal gives further support to this explana-
tion.
82
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Figure 32
Starch Concentration Versus Plow Rate
160
140 I
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1/FLOW RATE
83
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Figure 33
Starch Degradation Versus Feed Concentration
20 40 60 80 100 120 140
SUBSTRATE CONC. mg/l
-------
GLUCOSE-STARCH. MIXTURES
Mixed feed solutions of starch and glucose were tested to determine
the behavior of the slime layer In the presence of both colloidal and
soluble substrate. A slime surface, which had been acclimated to feed
solutions containing starch as the only source of carbon, was used.
Feed and effluent samples were millipore filtered then analyzed for
starch, organic carbon, and glucose. Glucose was measured colorimetri-
cally using an enzyme reagent available from Worthlngton Biochemical
Corporation under the trade name Gluostat. The test sequence was start-
ed with a lOOmg/1 starch feed followed by mixtures of starch and glucose,
glucose alone and finally a mixture. Each feed was evaluated over a ran-
ge of flow rates. However, the quantity of substrate removal was essent-
ially independent of flow rate and uptake rate was expressed in terms of
milligrams per centimeter squared per minute. Measured rates of removal
of starch, glucose, and organic carbon are summarized below.
Peed Feed Concentra- Carbon Concentra- Rate of Removal*-
tipnmg/1 tion mg/1 mg/cm -min.xlO Test
Starch Glucose
Starch
Starch
Glucose
Starch
Glucose
Glucose
^W-MMMMMIIBwi-^VIMMMHM^V
Glucose
Starch
107
105
53
100
99
100
98
48
44
64
82
45
— — —
63
309
330
267
132
_ _ _
190
362
380
320
Carbon
52
129
154
170
~~161
1
2
3
4
—
Addition of glucose had no immediate effect on the rate of starch de-
gradation Tests #2 and #3 were carried out over a period of 4-5 hours.
However, addition of glucose did increase the rate of organic carbon
uptake by a factor of 2-3. Glucose uptake was nearly doubled by using
a feed concentration of 100 mg/1 compared to 50 mg/1 in Test #2. How-r
ever, the rate of glucose and carbon uptake remained essentially con -
stant when starch was deleted during Test #4. At the end of Test #4
('about 3 hours), starch was reintroduced at 50 mg/1 concentration.
Starch degradation was observed, but the rate was 1/3 of that observed
earlier. The reduction is larger than expected for this change in feed
concentration and it seems likely that some of the enzyme activity was
lost during Test #4 when growth was fueled by glucose alone.
Comparison of the rates of carbon uptake versus glucose uptake shows
that glucose is almost completely consumed. By contrast, only a fraction
of the degraded starch is consumed, less than 40$. These observations
85
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are consistent with the concept of a surface. which, has catalytic
activity for both hydrolysis and carbon uptake and where the active
sites are independent and physically separated. The. marked increase
in carbon uptake observed when glucose was added along with starch
clearly shows that the carbon consigning activity was not fully ut-
ilized when starch was used. It seems plausible that carbon uptake
is limited by the transport of the products of starch hydrolysis to
the active carbon consuming sites. Such transport would have to be by
molecular diffusion, which would account for the. low rate of carbon
uptake as well as the "loss" of hydrolysis products into the main liquid
film.
AMINO ACID AND PFDIEINS.
Experimental work using glutamic acid, glycine, peptone, and mixtures *
was designed to establish the difference in rates of uptake of soluble
and colloidal substrates. Bacto peptone and bacto agar (Difco) were
used as a source of peptones. Amino acids were measured with ninhydrin
as described in Section A. This test is both sensitive and reproducible
but interpretation of the results is difficult because ammonium ions
also react with the reagent. Protein was measured using Folin Phenol
Reagent. The reagent reacts with reducing groups in proteins to give
misleading results with partially hydrolized proteins. Organic carbon
was measured to determine complete uptake.
In the first test, a glutamic acid feed solution (100 mg/1) plus salts
and buffer was used to grow an acclimated slime layer using seed from
the Minneapolis-St. Paul Sewage Treatment Plant. Pour feed concentra-
tion levels of glutamic acid were tested at various flow rates. As
with the starch tests, the quantity of glutamic acid removed was found
to be independent of flow rates and the rates have been expressed in
terms of mg/cm -rain. Carbon rate data are shown in Figure 34 as a fun-
ction of feed carbon concentration. Calculated rates of carbon trans-
fer' using the diffusion control model are shown for comparison. A
diffusion coefficient of 0.63 x 10 cm /sec. was used for glutamic
acid in water at 20°C and a carbon content of 40.8 wt.% was assumed for
glutamic acid. Measured rates of carbon uptake increase with increasing
feed concentration but level off at concentrations about 40 mg/1. Up-
take of carbon is many fold less than that calculated from mass trans-
fer considerations. Nevertheless, the initial effect of feed concentra-
tion, suggests that mass transfer is a limiting factor at low concentra-
tions .
The corresponding rates of disappearance of glutamic acid (measured with
ninhydrin) are nearly independent of feed concentration. It appears that
this is due to the interference of ammonium ions in the test and does
not necessarily reflect the kinetics of the reactions.
86
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Figure 34
Glutajiic Acid-Carbon Removal
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320 i
280
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MASS TRANSFER MODEL
CARBON
200
0
20 40 60 80 100
CARBON CONC. mg/l
87
-------
When a glutamic acid acclimated slime layer was exposed to. a solution
containing glyclne as the only carbon source, there was essentially
no uptake of glyclne and no uptake of carbon. Furthermore, when mix-
tures of glycine and glutamate were used, the uptake rate was essent-
ially that of glutamic acid. It appears that the microorganism pop-
ulation was unable to metabolize glycine. Mien peptone was substituted
for glutamic acid, degradation of peptone and consumption of carbon was
observed as shown 'below. •
? —6
p , Concentration, mg/1 Substrate Removal,mg/cm -min x 10
feed carbon carbon "Protein
Glutamic
acid 110 46 213
Peptone 200 91 120 466
Mixture* - - 69 213 80
*50~50 mixture
Carbon removal for the peptone feed was less than that observed with.
glutamic acid. Mixed feeds (glutamic + peptone) showed higher carbon,
uptake than peptone alone. However, the slime layer had surprisingly
high activity for removal of peptone as measured- with Polin Phenol re-
agent. The measured rate of protein disappearance (460 mg/cm -min,.) is
if anything somewhat larger than that predicted from mass transfer'con-
siderations (340 mg/cm min.), using a diffusion.coefficient of 0.056 x
10~DcmVsec for proteins in water at 24°C. This'high proteolitic activity
was not observed in all cases. It appears that under certain (as yet
undefined conditions) a glutamic acid acclimated* slime layer may have
very little initial proteolitic activity.
is--'
Slime surface acclimated to protein substrate as its only carbon.1;'source.
is capable of shifting to the use of glutamic acid or mixtures of glut-
amic and protein without any reduction in organic carbon uptake.. If
anything, there appears to be a slight rate enhancement with glutamic
acid as shown in the tabulation below:
2 -6
Concentration, mg/1 Substrate Removal, mg/cm -min x 10 •••.
feed carbon carbon protein
Protein 128 53 1?4 . • 373-**
Glutamic ' ....
acid 97 49 200
Mixture - - 47 160 173
!
The observed disappearance of protein is in excess of that calculated,
from mass tmasfer considerations using a diffusion coefficient of 0.056 x
10 cm /sec. Carbon removal is essentially equivalent to protein removal
-------
and indicates that mostly protein hydrolisates are consumed as they are
formed, and only small amounts diffuse back into the liquid film When
peptone,- feed concentration was cut in half, the rate of disappearance
was reduced proportionately. This effect of ..feed concentration is In-
dicative of a mass transfer limitation.
In comparing the kinetics of removal of glutamic acid and protein, it
is interesting to note that glutamic acid shows evidence of mass trans-
fer limitation even though the diffusion coefficient is some 10 fold
larger than that of proteins. Glutamic acid removal is less than 30$
of the maximum calculated from mass transfer. Whereas, observed protein
degradation rates are of the same magnitude as calculated from mass
transfer considerations. This suggests that uptake of glutamic acid is
limited by an additional resistance, either in terms of mass transfer,
adsorption, or reaction.
LAURIC ACID
High molecular weight fatty acids are very sparingly soluble in water.
The use of alkali salts (soaps) allows forming suspensions at higher
concentrations; however, these suspensions are unstable. Plocculation
and settling ,as well as adsorption on filters makes it difficult to
work .with such suspensions. In order to minimize these problems and
yet work with a reasonably high molecular weight fatty acid, lauric acid
was used to study the kinetics of fats. As an example, lauric acid solu-
tions could be filtered through millipore filters without substantial
loss of fatty acid. However, palmitic and stearic acid could not be
handled''in this manner. :
Biological uptake of lauric acid (no other carbon source) was tested
at three concentration' levels,. 25, 50, and 100 mg/1. All other nutritional
requirements were available in excess. A slime layer was acclimated to
lauric acid" feed over a period of several weeks. Carbon removal was used
as a measure of biological -uptake because chemical analyses for fatty acids
are not sufficiently precise to measure small differences in concentration.
Each feed*was,tested over a range of flow rates. The results show that
uptake is essentially independent of flow rate. The rate data are sum-
marized in Figure 35. Lauric acid shows the same trends as reported for
other substrates, namely increasing rates of uptake as feed concentration
is increased. It was not feasible to evaluate higher concentration levels-
due to the low solubility of lauric acid. However, i| appears that max-
imum uptake rate is in the range 200-250 x 10" mg/cm -min. The observ-
ed rate data are indicative of mass transfer limitation in the same manner
observed with glutamic acid substrate. The rate of mass transfer calcul-
ated from fche9diffusion control model using a diffusion coefficient of
0.43 x 10~5cm /sec at 20°C is shown In Figure 35, the calculated values
are substantially higher than the measured rates of uptake .
-------
Figure 35
Laurie Acid-Carbon Removal
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240
200
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160
120
20 40 60 80 100
FEED CARBON CONC. mg/1
90
-------
The lauric acid acclijnated slime layer was also tested with a mixed feed
consisting of approximately IQOmg/l each of lauric acid and glutamic
acid and glucose. The initial rate of organic carbon removal and glucose
disappearance are summarized below.
Substrate Concentration* Substrate Removal,-
mg/1 mg/cm -nrin. x 10
Total carbon 139 238
Glucose (100) 48
*Mixed feed, glucose, glutamic acid and lauric acid, nominally lOOmg/1
of each.
Total carbon removal is essentially the same as observed with lauric
acid alone. However, some glucose removal was observed and accounts for
about 20% of the carbon uptake.
MDED FEED ACCLIMATED SLIME SURFACE
A series of tests were made to determine the kinetics of substrate re-
moval after slime layers had been acclimated to mixed substrates. The
mixed substrate consisted of 100 mg/1 each of glutamic acid and lauric
acid and glucose. Rates of removals were measured at two levels of feed
concentrations (nominally 50 and 100 mg/1 each) and over a range of flow
rates. Peed and effluent samples were analyzed for carbon, glucose, and
amino acid concentration.
Data from the 100 mg/1 feed mixture show substantially higher rates of
substrate uptake then had been observed on slime layers acclimated to
a single substrate. Data for carbon and glucose removal are shown below:
Mixed feed concentration
(glucose, glutamic acid, 100 50
lauric acid, mg/1 of each )
Substrate Removal, mg/cm -rain, x 10
Total Carbon ^10 160
Glucose 760 ^53
Glucose disappearance accounts for the major part of the carbon uptake.
Measurements of amino acid removal are inconsistent but the data clearly
point to the conclusion that rates of removal of glutamic acid are much
smaller than the rates of glucose removal.
91
-------
As a further test of the behavior of mixed substrates, a slime surface
was acclimated to feed solutions containing skim milk solids. Skim
milk solids are known to consist of high concentrations of lactose (50-5
wt.JO and protein (36.9 wt.J?) plus traces, of fats (0.9 wt.JB) and in-
organics. Growth of the slime layer was noticeably more rapid than that
observed with single substrates. Rates of uptake were measured from
carbon and carbohydrate disappearance using four concentration levels and
a range of flow rates. Protein degradation was not determined quant-
itatively because the analytical techniques were Inadequate. Average
rates of carbon uptake and carbohydrate degradation are shown in Fig.
36 as a function of substrate concentration in the feed solution. Car-
bon uptake increased with increasing feed concentrations in the same
manner as observed with single substrates. It appears that higher
rates of removal could have been obtained by further increasing the
milk solids concentration (range covered was 50-200 mg/1 of dry milk
solids). Carbohydrate removal accounts for most of the observed carbon
uptake at all concentration levels. Nevertheless, protein degradation
was noted in all tests. At low feed concentrations, protein degrada-
tion was nearly complete.
92
-------
Figure 36
Skim Milk Solids Removal
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SUBSTRATE CONG mg/l
-------
DISCUSSION-^UMARY
Substrate uptake of slime surfaces acclimated to single substrates appear
to be mass transfer limited at concentrations below 100 mg/1, and
reaction limited at higher concentrations. This conclusion is based on
the observed effects of feed substrate concentration on uptake rate±g
Faximum uptake with single .substrates is in the range 50 - 250 x 10
mg/cm -min. Highest rates were observed with lauric acid, glucose, and
when mixed substrates were used. Lowest uptake, rates were-observed with
starch feed; starch-carbon uptake was only 50 x 10 mg/cm -min. even
though the rate of starch degradation was 2-4 fold greater. By contrast,
protein removal and carbon disappearance are more nearly equivalent.
Both starch and protein molecules are far too large to be absorbed dir-
ectly into bacterial.cells. Based on an extensive literature review,
Pollock (The:Bacteria, Vol. 4, Academic Press, 1962) has concluded that
exoenzymes, either free or bound to the cell surface, are responsible
for the hydrolytic breakdown of colloids, followed by ingestion of sol-
uble-fragments. The starch-carbon data'suggest that the sites for hyd-
rolysis and ingestion are independent and physically separated, as
evidenced by the low carbon uptake rate.
Acclimation of the slime surface is necessary when new substrates
are introduced; glucose is an exception. Acclimated surfaces may loose
their activity for metabolizing a given substrate if the substrate is
removed for any appreciable time. Some loss in activity may occur in
a few hours as evidenced by the loss of hydrolytic activity for starch
when glucose was substituted for approximately four hours. Acclimation
of the surface to an entirely new substrate may take several days.
Acclimatization to fatty acids and to glycine are examples. It is quite
likely that an entirely different surface coating (different bacterial
species) is formed.
Use of mixed substrates generally results in higher rates of carbon up-
take. This is particularly pronounced when carbohydrates are added.
Preferential utilization of glucose in pure compound mixtures and lac-
tose from skim milk substrates have been observed. Nevertheless, there
is concurrent utilization of other substrates e.g., protein and starch
degradation as well as some uptake of carbon from noncarbohydrate
sources.
The results of this study provide some new insights on the behavior of
biologically active slime layers in the presence of various' substrates.
The practical 'significance of this work has not been evaluated although
the results do raise some questions about the practical value of certain
operating practices commonly used in trickling filters. For example,
the use of effluent recirculation would appear to be undesirable if BOD
removal is mass transfer limited. Also the use of packing which nlnimizea
liquid hold up seems undesirable in a mass transfer limited reactor.
Further work is needed to evaluate the potential value of slime layers in
biological processings and to delineate optimum operating conditions.
94
-------
TABLE NO. 1
Effect of Starch Concentration on Rates of Biological Decomposition
Experiment No. 4-2-68, Batch Reactor, Well Mixed.
Preparation of Innoculum
Collected 1300 ml overflow from continuous propagator (overnight)
using standard soluble starch feed solution (100 mg/1)
Preparation of Concentrates
0.8 grams of starch in 399 ml distilled water was autoclaved for 5
minutes to dissolve starch. Predetermined amounts of concentrate
were added at the beginning of each test.
Sample Time Starch Concentration, mg/1 Carbon Concentration
No A=520 X=680 mg/1
Reactor A - 300 ml innoculum + 10 ml starch concentrate
A-0 10:00 63 65
A-l 10:10 38 27,5 112.5
A-2 10:20 27 15-5
A-3 10:30 18.5 10
A-4 10:40 15 8.5
added 30 ml of starch concentrate
A-0 11:23 162 179
A-l 11:33 157=5 151
A-2 11:43 144.5 116
A-3 11:53 127 87
A-4 12:03 H2 65
A-5 12:23 88.5 39
A-6 12:43 76 27
A-7 1:03 59-5 21
A-8 1:23 ^8.5 17
A-9 1:56 38 14.5 95
Reactor B - 300 ml innoculum + 20 ml of starch concentrate
B-0 10:02 126 128.5
B-l 10:12 90 75.5
B-2 10:22 68.5 44
B-3 10:32 56 28
B-4 10:42 46 20
B-5 10:52 35 15-5
B-6 11:02 31 13
95
-------
Table No. 1 con't
Sample
No
added
B-0
B-l
B-2
B-3
B-4
B-5
B-6
B-7
B-8
B-9
Reactor
C-0
C-l
C-2
c-3
c-4
C-5
C-6
C-7
added
C-0
C-l
C-2
c-3
C-4
C-5
C-6
C-7
C-8
C-9
Time
25 ml of starch
11:25
11:35
11:45
11:55
12:05
12:25
12:45
1:05
1:25
1:58
Starch Concentration, mg/1 Carbon
X=520
concentrate
182
149
126
109
95.5
75.5
61
-51
43
34
C - 300 ml innoculum + 25 ml
10:04
10:14
10:24
10:34
10:44
10:54
11:04
11:14
20 ml of starch
11:27
11:37
11:47
11:57
12:07
12:27
12:47
1:07
1:27
2:00
153
121.5
99
80
68.5
59.5
51
45
concentrate
170
146
125
109.5
98.5
81
68
58
51
55
A=680 i
171
125
86
61
44
28.5
21
17
15.5
14 !
of starch concentrate
160
107.5 :
71
48
34
26.5
21
17.5
150
114
82
60
46
31
24
18.5
18
16
mg/i
97
124
94
96
-------
Table No. 1 con't
Sarrple Time St ^Concentration, mg/1 Carbon Concentrate
^u A=520 X=680
Reactor D - 300 ml of innoculum + 30 ml of starch concentrate
D-0 10:06 180 183
D-l 10:16 144.5 lofi
D-2 10:26 117 88
D-3 10:36 96,5 57-5
D-4 10:46 83 41
D-5 10:56 69.5 30.5
D-6 11:06 61 24
D-7 11:16 53 18.5
added 10 ml of concentrate
D-0 .11:29 111 82.5
D-l 11:39 8? 47.5
D-2 11:49 76 ' 32.5
D-3 11:59 67 25
D-4 12:09 59 21
D-5 12:29 48.5 17
D-6 12:49 40 14
D-7 1:09 35 13-5;
D-8
D-9 / : 75
Footnotes: Temperature, 23 centigrade
Innoculum was filtered (0.45 micron filter) to determine
suspended solids and the "dissolved" carbon concentration.
Innoculum before addition of starch concentrate suspended
solids, mg/1 35.2, 47.6
dissolved carbon, mg/1 21.0, 26.0
Yield of suspended solids in continuous propagator was
determined from meansurement of dry mass and carbon
concentration in filtrate and feed.
cell mass yield 'g nlmM 0-51,0.795
97
-------
TABLE NO. 2
Part I - Comparison of Biological Decomposition Rates of Different
• Starches
Experiment No. 4-30-68, Batch Reactor, Well Mixed
Preparation of Innoculum
Collected 1250 ml overflow from continuous propagator overnight (18
hours), using standard 100 mg/1 soluble starch feed. Added 550 ml fresh
feed (100 mg/1) at 10:00 AM and continued aeration.
Preparation of Starch Concentrate
0.8g starch added to 399 ml distilled water and autoclaved for 5 minutes
to dissolve starch. Concentrate was added to innoculum to bring starch
concentration to 200 mg/1.
Sample No Time Starch Concentration, mg/1 Carbon Concentrate
X=680 X=520 mg/1
Reactor A - Evaluation of SD-5 Starch
A-0 Innoculum 4.5 5.0 16 50
A-l 11:00 198 213 88'
A-2 11:10 161 155 82
A-3 11:20 122 130 71
A-4 11:30 93 108 63
A-5 11:40 72 97 50
A-6 11:50 57 87 10
A-7 12:00 43 76
A-8 12:20 29 59
A-9 12:40 21 45
Reactor B - Evaluation of SD-80 Starch
B-0 Innoculum 4.5 6.0 16.50
B-l 11:02 192 220 96
B-2 11:12 155 192 87
B-3 11:22 128 168 83
B-4 11:32 114 152 71
B-5 11:42 99 136 62
B-6 11:52 92 127 ' 23
B-7 12:02 78 113
B-8 12:22 71 100
B-9 12:42 65 80
98
-------
Table No. 2 can't.
Sample. No Time Starch Concentration, mg/1 Carbon Concentrate
X=680 x=520 . rng/1
Reactor C - Evaluation of ES-A
c-o
C-l
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
Reactor
D-0
D-l
D-2
D-3
D-4
D-5
D-6
D-7
D-8
D-9
Innoculuni
11:15
11:15
11:25
11:35
11:45
11:55
12:15
12:25
12:45
D - Evaluation
Innoculum
11:07
11:17
11:27
11:37
11:47
11:57
12:07
12:27
12:47
4.5
194
164
129
93
72
55
41
27
18
of Standard
4.5
183
134
93
64
45
35
27
18
13
5.5
220
167
142
118
103
91
79'
62
47
Soluble Starch
5-0
212
154
126
107
93-5
81
71
54.5
42
16.50
85
86
70,5
60
49.5
12.5
16.50
82
79-5
68.5
52
41.5
21
Footnotes: Dry weight of biomass measured oh selected samples by
filtering through 0.45 micron Millipore filters
Biomass, mg/1
Innoculum 32»3> 34-5
Reactor A 105>8, 104.0
Reactor B 104.0
Reactor C 94.6
99
-------
TABLE NO. 3
Part II - Comparison of Biological Decomposition Rates of Different
Starches
Experiment No. 5-7J-68
Preparation of innoculum and concentrates same as experiment 4-30-68.
Sample , Time Starch Concentration, mg/1
No X=680 A=520
Reactor A - Evaulation of PPPS Starch
A-0
A-l
A-2
A-3
A-4
A-5
A-6
A-7
Reactor B -
B-0
B-2
B-3
B-4
B-5
B-6
Reactor C
C-0
C^l
C-2
C-3
C-4
C-5
C-6
C-7
.10.29
10:39
10:49
10:59
11:09
11:19
11:29
11:49
Evaluation
10.31
10:41
10:51
11:01
11:11
11:21
11:31
11:51
Evaluation
10.35
10:45
10:55
11:05
11:15
11:25
11:35
11:55
230
161
112
80
58
44
34
23
of ES-G Starch
175
125
84
60
44
33
26
17
of standard soluble
172
96
56
38
26
20
16
11
220
174
145
126
107
91
75
53
164
129
105
90
79
67
56
39
starch
162
117
95
79
66
55
46
32
100
-------
TABLE NO.4
Test for "Soluble" Exor-enzyme Activity
Part I - Experiment No. 2-9-68, Batch Reactor, Well Mixed
Innoculum collected from continuous propagator using standard soluble
starch feed solution of 100 mg/1. Fresh feed solution was added at
the beginning of the test. Part of innoculum was filtered through
0.45 micron Millipore filter to remove cell mass.
Sample No Time Starch Concentration, mg/1
X=520 X=680
Reactor A - 100 ml innoculum + 200 ml feed solution
A-l 2:40 61.5 62.5
A-2 2:55 36.0 28.5
A-3 3:10 23.0 13.0
A-4 3:25 10.5 7.0
Reactor B - 50 ml millipore filtered innoculum + 100 ml feed solution
B-l 2:43 66.0 64,0
B-2 2:58 66.0 63..0
B-3 3:13 65.0 62.5
B-4 3:28 64.5 61.5
B-5 3:50 64.0 59.5
B-6 4:40 63-5 58.5
Footnotes: Temperature, 20 C. „,,«*,«„
Innoculum starch concentration, non detectable • carbon
concentration, 25 mg/1
101
-------
TABLE NO,5
Part II - Experiment No, 2-10-68, Batch Reactor, Well Mixed
(InnQculum collection and test procedure same as Part I test
No, 2-9-68)
Sample
No
Reactor
E-l
E-2
E-3
E-4
E-5
E-6
E-7
E-8
E-9
E-10
E-ll
E-12
E-13
Reactor
P-l
F-2
P*3
F-4
P-6
Time
Starch Concentration mg/1 Carbon Concent
X-520
E - 100 mg innoculujn + 300 ml
9:43
9:58
10:13
10:28
10:43
10:58
11:13
11:28
11:43
11:58
12:28
12:58
1:28
P - 50 ml
9:00
10:05
10:20
10:35
11:05
80.0
57.0
42.0
31.5
24.5
19-0
15.5
13,5
11.5
8.5
4.0
2.0
2.0
millipore filtered
80
80
80
80
79
X=680
feed solution
49-5
31.5
20.0
14.0
10.0
8.0
7.0
6.0
5.0
3-5
2.0
2.5
innoculum + 150 ml
73
74
74
74
74
mg/i
38.5
37.0
35.0
34.0
33-0
32.0
30.0
31.5
fresh feed
Footnotes: Temperatures 20 centigrade
102
-------
Table No.5 con't
Sample
No
Tame
Starch Concentration rng/1
X=520 X=680
Reactor G - 100 ml Innoculum + 300 ml fresh feed
G-l
G-2
G-3
G-4
G-5
G-6
G-7
G-8
G-9
G-10
G-ll
1:
1:
1:
1;
2:
2:
2:
3:
3:
4:
10
25
40
55
19
35
50
05
35
05
4:55
79.0
61.0
52.0
46.0
40.0
35,
34,
32.0
27 = 5
22.0
14.5
,5
-5
75.0
57-5
44.0
35.0.
26.5
20.0
18.5
17.0
12.0
10.0
6.0
Reactor H - 50 ml filtered innoculum + 150 ml fresh feed
H-l
H-3
H-5
H-6
H-7
H-8
H-9
H-10
44
21
55
3:10
3:50
4:15
5:00
75.5
76.0
74,
75,
after 17 hours
74.0
73-5
68.0
56.5
Footnotes: Temperature 21 centigrade
Innoculum starch concentration, 3 mg/1
103
-------
TABLE NO. 6
Biological Decomposition of Starch-Glucose Mixtures
Experiment No. 8-29-68, Batch Reactor, Well Mixed
Preparation of Innoculum
Collected 1500 ml of overflow from continuous propagator using standard
100 mg/1 soluble starch feed solution. Innoculum was collected over-
night in an aerated vessel and diluted with salt solution-of equal
volume. Each reactor was started with 600 ml of inncoeulum.
Preparation of Concentrate
One gram of starch and glucose were dissolved in 500 ml and 250 ml
respectively of distilled water and autoclaved for 5 minutes. :Concentrates
were added to each of four reactors as shown.
Reactor
A
B
C
D
Concentrate added, ml
Starch Glucose
30
30
60
0
0
15
15
15
Sample No.
Reactor A
A-0
A-l
A-2
A-3
A-4
A-5
A-6
A-7
A-8
A-9
A-10
A-ll
A-12
A-13
A-16
A-17
A-18
A-19
Time
10:36
10:40
10:55
11:10
11:25
11:40
11:55
12:10
12:25
12:40
1:11
' 1:40
2:
2:
5:
5:
6:
4o
40
30
50
Starch Concentration, mg/1
X=680 X=520
2.0
9.8
82.5
62
54
48
43
39-
36.
30.
25,
21.
7-5
1.5
2.5
104
92
82
76
71
67
65
62
72
53
46
38
Carbon
mg/1
Glucose
mg/1
7:56
53 (11:03)
591(12:05)
49 (12:58)
33 ( 3.04)
28 ( 3-58)
25
25
25
5
1
1
1
1
3
104
-------
Table No. 6 con't
Sample No. Time Starch Concentration, mg/1 Carbon Glucose
mg/1 mg/1
Reactor B
B-0 10:37 2.5 3- 0
B-l 10:42 94.5 99 - 103
B-2 10:57 79,5 85 122
B-3 11:12 66.5 77 86 (11:11) 101.5
B-4 11:27 56.5 71 102
B-5 11:^2 49. 66 98
B-6 11:57 43.5 63 108
B-7 12:12 39.5 61 97
B-8 12:27 35 57 81
B-9 12:42 31.5 55 92
B-10 1:12 27 86 (1:08) 88.5
B-ll 1:42 23 45 78
B-12 2:12 19,5 35 66
B-13 2:42 67 (2:30) 53
B-14 3:22 57 (3:10) 28.5
B-15 3:51 41 (4:09) 14.5
B-17 6:48 18 32 (5:30) 15-
B-18 25 (6:50)
B-19 24 (7:56)
Reactor C
C-0 10:38 2.5
56 53
49
105
3.5
r iR 7-ns> "R1 ^ 56
C-18 7.02 31.5 49 (8;05)
113
C-3 11: 16 149 l8l 97
C-4 11:31 138-5 175 97
C-5 11:47 126 l6l 94
C-8 : 12:31 100 137 .
^ %$ M li S7 (!:») 3.5
73(2:»2) 5
C-13 2=53 52-5 9 ,
C-ii) 3:23 48 88 90
?•§ S3'5 ° 68(^:2^ 18-
(600) 25
-------
Table No. 6 can't
Sample No. Time Starch Concentrate, mg/1 Carbon Glucose
A=680 A=520 rag/1 mg/1
Reactor D
D-0 10:39 24 0
D-l 10:48 2 3 109
D-2 11:03 2 3 109
D~3 11:18 57 (11:22) 109.5
D-4 11:33 105
D-5 11:48 - 105
D-6 12:03 ' 103
D-7 12:18 55 99
D-8 12:33 97
D-9 1:18 47 (1:30) 87
D-ll 1:48 76
D-12 2:18 68
D-13 2:56 40 58
D-14 3:22 34 (3:28) 49
D-15 3:56 36
D-17 4:25 25 (4:40) 4
D-18 7:02 16 (6:00) 11
D-19 14 (7:18)
D-20 11 (8:05)
Reactors A B C
Innoculum Composition. Before Test
blonass .(milipore filtered), mg/1 11 9 10
carbon content of filtrate, mg/1 12 13 ll
After Test
blomass, mg/1 36 66 94.
carbon content of filtrate, mg/1 11.5 16 4
106
-------
TABI£ NO. 7
Effect of Glutamic Acid Concentration on Rate of Biological Decomposition
Experiment No. 12-20-68, Batch Reactor, Well Mixed.
Innoculum Preparation
Collected overflow from continuous propagator (110 ml/19 hours) at an
average of 58 ml/hr using 100 mg/1 glutamic acid feed solution. Prior
to the test the Innoculum was diluted with an equal volume of fresh
feed solution.
Preparation of Concentrate
0.25 grams of glutamic acid was dissolved in 1000 ml of distilled
water and sterilized for 5 minutes.
Sample No Time Concentration, mg/1
Carbon Amine Nitrogen
Reactor A - 200 ml of innoculum
A-0 11:30 26 .62
A-2 12:00 • 26 54
A-3 12:30 60
A-4 1:00 27 60
A-5 1:30 53
A-6 2:00 25 52
A-7 2:30 51
A-8 3:00 25 58
A-9 3:30 52
A-10 4:00 22 54
A-ll 6:00 21 30
A-12 7:00 20 32
A-13 10:00 A.M. 10 35
Reactor B - ,
B-0 11=32 26 24
added 40 ml concentrate .,
B-l 11:33 37 33
B-2 12:02 37 18
B-3 12:32 26
B-ll 1:02 36 2f
E-5 1:32 32
B-6 2:02 34
B-7 2:32 g
B-8 3:02 32 26
B-9 3:32 7
B-10 4:02 30 37
B-ll 6:01 25 3b
B-12 7:01 27 &
ill3 10:02 A.M. 14 37
107
-------
Table No.7 con't
Sample No Time
Concentration, mg/1
Carbon Amine Nitrogen
Reactor C -
C-0
added 80 ml
C-l
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-ll
C-12
C-13
Reactor D -
D-0
added 120 ml
D-l
D-2
D-3
D-4
D-5
D-6
D-7
D-8
D-9
D-10
D-ll
D-12
D-13
11:34
concentrate
11:34
12:04
12:34
1:04
1:34
2:04
2:34
3:04
3:34
4:04
6:02
7:02
10:04 A.M.
11:36
concentrate
11:37
12:06
12:36
1:06
1:36
2:06
2:36
3:06
3:36
4:06
6:03
7:03
10:06 A.M.
26
47
47
48
47
46
41
36
36
15
26
53
55
54
53
52
50
43
36
16
112
114
112
110
110
108
108
53
40
50
38
33
43
45
40
42
47
44
46
45
45
46
40
43
43
Footnotes: Suspended solids concentrations were exceedingly low, 1-2
mg/1 which is difficult to measure precisely.
108
-------
TABLE NO. 8
Effect of Glutamic Acid Concentration on Rate of Biological Deconposition
Experiment No. 2-5-69, Batch Reactor, Well Mixed
Preparation of Innoculum
Collected overflow from 250 ml continuous propagator (overnight) using
100 mg/1 glutamic acid feed at flow rate of 110 ml/hr. Each reactor was
started with 245 ml of innoculum.
Preparation of Concentrate
0.200 grams of gultamic acid and 200 ml of distilled water were sterilized
for 5 minutes. Concentrate lost some water by evaporation, final
concentration was 1.03 mg/ml. Concentrate was added to each of four
batch reactors at the beginning of the test.
Sample No Time
Reactor B
B-l
B-2
B-3
B-4
B-5
B-6
B-7
B-8
B-9
B-10
B-ll
B-12
B-13
B-14
B-15
Reactor C
C-l
C-2
C-3
C-4
C-5
C-6
: 31
10:
10;
10:31
11:01
11:31
12:01
12:31
1:01
2:00
3:01
4:01
5
5
8
9
9:30
10
10
10
11
11
12
49
15
40
:20
:33
:33
:02
:32
;02
Concentration, mg/1 •
Carbon Amine Nitrogen
6.0 2.0
added 25 ml concentrate
49-0 8.4
38.0 9.3
28.0 8.0
22.0 3.4
13.0 2.2
8.0 1.8
8.0 1.6
12:32
9-0
8.0
8.0
6.0
added 40 ml
71.0
67.0
57-0
51.0
43.0
1.6
0.8
1.6
1.5
1.8
1.9
2.0
concentrate
13-2
13-7
12.7
8.5
7-4
109
-------
Table No. 8 can't
Sample No Time
Reactor C con't
C-7
C-8
C-9
C-10
C-ll
C-12
C-13
C-14
C-15
Reactor D
D-l
D-2
D-3
D-4
D-5
D-6
D-7
D-8
D-9
D-10
D-ll
D-12
D-13
D-14
D-15
1:
2:
3:
4:
02
00
02
02
5:13
5:52
8:15
9:40
9:30
:23
:36
:36
:03
:33
:03
:33
:03
:00
:03
:03
:16
:55
:15
10:
10:
10:
11:
11:
12:
12:
1:
2:
3:
4:
5:
5:
8:
9:40
9:30
Concentration, mg/1
Carbon Amine Nitrogen
24.0
9.0
9-0
10.0
7.0
11.0
11.0
4-7
2.4
1.0
1.0
1.1
1.2
1.7
2.2
2.7
6.0 2.1
added 50 ml concentrate
85.0
80.0
71.0
61.0
52.0
34.0
12.0
13.0
14.0
13.0
13-0
16.0
16.0
16.0
.4
.3
.7
16.
17.
15-
11.0
11.1
6.7
0.3
1,
0.
1.
1,
2.1
2.6
3.1
Footnotes: a) The quantity of suspended material (cell mass) was
determined by filtering through 0.45 micron filters and
drying and weighing.
Sample filtered Solids collected Concentration of filtrate
mg/1 Carbon, mg/1 Amine Nitrogen mg/1
30.0 10.0 0.6
51.0 9-0
82.5 12.0
79.0 16.0
Innoculum
Reactor B, end of test
Reactor C, end of test
Reactor D, end of test
1
— j - - - •-- -Q
b)Temperature was maintained at approximately 21 centigrade
8
2.2
1.3
110
-------
TABLE NO. 9
Glycine Degradation by Glutamic Acid Acclimated Innoculum
Experiment No. 2-20-69. Batch Reactor, Well Mixed.
Innoculum Preparation
Collected overflow from continuous propagator, 2,200 ml in 15 hours,
using 100 rng/1 glutamic acid feed.
Preparation of Concentrate
0.404 grams of glycine was dissolved in 200 ml distilled water and
sterilized for 5 minutes. Concentrate was added to innoculum at the
start of each test.
Sample No
Time
Concentration, mg/1
Carbon Glycine
Reactor A
A-l
A-2
A-3
A-4
A-5
A-6
A-7
A-8
A-9
A-10
A-12
11:
11:
11:
12:
12:
1:
1:
2:
3:
3:
5:
11:
35
40
40
10
40
10
38
10
11
55
10
44
7 7-5
added 5.0 ml concentrate to 250 ml of innoculum
18.
23.5
23
20.5
27
25
23-5
20
23
19
47
50
52
52
50
48
37
32.
20
21
Reactor B
B-l
B-2
B-3
B-4
B-5
B-6
B-7
B-8
B-9
B-10
B-12
11:35
11:41
11:41
12:10
12:41
38
7 7.5
added 15 ml concentrate to 250 ml innoculum
143 112
149 118
1
1
2:10
3:12
3:55
5:11
11:44
49-
47.
.46
48
56
51.
49
40
125
125
125
126
125
127
122
96
111
-------
Table No. 9 con't
Sample No
Reactor C
C-l
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-12
Reactor D
D-l
D-2
D-3
D-4
D-5
D-6
D-7
Time
D-9
D-10
D-12
11:
11:
11:
12:
12:
1:
1:
2:
3:
3:
5:
11:
35
42
42
11
42
12
39
11
12
56
11
44
11:
11:
11:
12:
12:
1:
1:
2:
3:
3 =
5:
11:
37
43
^3
12
43
12
39
11
13
56
12
44
Concent rat ion 3 mg/1
Carbon Glycine
7 9.5
added 25 ml concentrate to 250 ml. innoculum
66 165
79
75
74
75
83
100
83
100
,5
,5
.5
.5
187
187
187
187
187
187
245
176
5 7.5
added 35 ml concentrate to 250 ml innoculum
93
98
99
97
100
95-100+
74
102
82
102
235
235
235
235
235
235
187
245
187
245
Footnotes: Suspended solids measured by millipore filtration
Innoculum 2.03 8.2, mg/1
Reactor A 20.0 mg/1
Reactor c 33-7 mg/1
112
-------
TABLE NO. 10
Protein and. Glutamic Acid Degradation on Glutamate Acclimated Innoculum
Experiment No. 3-12-69, Batch Reactor, Well Mixed.
Innoculum Preparation
Collected overflow from continuous propagator overnight (150 ml/hr)
using 100 mg/1 glutamic acid feed solution. Microscopic examination
showed predominance of rod shaped bacteria (length to width ratio of
3-4). Mostly single cells except for a few small clumps.
Preparation of Concentrates
0.200 grams glutamic acid + 200 ml distilled water.
0.200 grams peptone + 200 ml distilled water.
Concentrates were autoclaved for 5 minutes.
Sample No Time Concentration, mg/1
Carbon Amino Acid Protein
Reactor A -
A-0 10:56 10 24 0
added 30 ml protein concentrate + 245 ml innoculum
A-l 10:59 54 51 115
A-2 11:30 55 52 125
A-3 12:01 52 45 125
A-4 12:37 52 10 110
A-5 1:39 48 28 115
A-6 2:43 •. 48 25 110
A-7 3:55 45 25 HO
A-8 4:58 45 33
A-9 6:23 ^5 25
A-10 9:10 52 27
A-ll 10:25 44 25
A-12 9:07 45 43
Reactor B - ft n
B-0 11:01 10 18 0
added 60 ml protein concentrate + 245 ml innoculum
R_l 11:03 90 92
-------
Table No. 10 can't
Sample No Time Concentration, mg/1
Carbon Amino Acid Protein
Reactor C -
C-0
added 90
C-l
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-ll
C-12
Reactor D -
D-0
added 60
11:05
ml protein
11:07
11:33
12:05
12:39
1:41
2:45
2:57
5:00
6:24
9:11
10:26
9:09
11:08
ml protein
10
concentrate +
118
120
123
116
116
109
105
105
105
100
99
89
10
concentrate +
34
0
245 ml innoculum
137
129
152-170
17
43
60
60
75
72
75
55
75
29
15 ml glutamate
295
290
310
255
275
250
230
concentrate
+ 245 ml innoculum
D-l
D-2
D-3
D-4
D-5
D-6
D-7
D-8
D-9
D-10
D-ll
D-12
Reactor E
E-0
added 60
+245 ml
E-l
E-2
E-3
E-4
E-5
E-6
E-7
Er8
E-9
E-10
E-ll
E-12
11:11
11:34
12:06
12:40
1:42
2:48
3:58
5:01
6:24
9:11
10:26
8:09
11:12
ml protein
innoculum
11:15
11:34
12:06
12:43
1:43
2:47
3:59
5:02
6:25
9:12
10:26
9:11
108
105
108
100
94
86
80
81
77
74
74
60
12
concentrate +
115
105
108
118
110
105
91
82
80
80
80
76
160
157
174
25
50
43
86
99
90
67
49
94
15
30 ml gltamate
157
174
25
118
105
121
129
105
75
. 89
200
220
210
190
195
185
180
concentrate
200
220
210
210
185
180
185
114
-------
Table No. 10 can't
Sanple No Time Concentration, mg/1
Carbon Amino Acid Protein
Reactor F -
F-0 11:16 11 26
added 60 ml each of protein and glutamate concentrate + 245 ml
inncoulum
F-l 11:19 144 224 175
F-2 11:37 148 24? 190
F-3 12:08 156 236 195
F-4 12:43 156 26 165
F-5 1:44 140 163 175
F-6 2:47 132 115 180
F-7 4:00 124 192 175
P-8 5 = 03 111 217
F-9 6:25 88 207
F-10 9:12 78 181
F-ll 10:28 76 149
F-12 9:11 70 115
Reactor G - (No concentrate added)
G-0 (22°c) 11:17 6 25
G-2 (23°c) 11:38 11
G-3 (24°c) 12:11 11 25
G-4 " 12:45 16
G-5 " 1:47 14
G-6 " 2:47 12
G-8 5:04 20
G-9 " 6:26 19
G-10 " 9:13 16
Footnotes: Temperature of reactors is indicated by numbers in paren-
thesis in Reactor G data columns
Suspended solids measured by millipore filtration and drying
and weighing
Innoculum 26.5 mg/1
Reactor D
115
-------
TABLE NO. 11
Effect of Concentration on Biological Degradation of Peptone and
Glutamlc Acid using a Peptone Acclimated Culture
Experiment No. 3-18-69, Batch Reactor, Well Mixed.
Preparation of Innoculum
Collected effluent from continuous propagator overnight using 100
rng/1 Bacto-Peptone feed at a flow rate of 150 ml/hr. Each reactor was
started with 245 ml of innoculum.
Preparation of Concentrates
a) Dissolved 0.400 grams of Bacto-Peptone in 400 ml distilled water
b) Dissolved 0.200 grams of glutamic acid in 200 ml. distilled water
Both concentrates were autoclaved for 5 minutes
Sample No
Time
Reactor A
A-0
added
A-l
A-2
A-3
A-4
A-5
A-6
A-7
A-8
A-9
A-10
A-ll
Reactor B -
B-0 '
added
B-l
B-2
B-3
B-4
B-5
B-6
B-7
B-8
B-9
B-10
B-ll
10:07
30 ml of peptone
10:32
11:01
11:29
11:59
12:59
1:58
2:55
3:58
4:56
8:18
10:25
10:07
60 ml of peptone
10:33
11:02
11:29
11:59
12:59
1:39
2:55
3:55
4:57
8:18
10:28
11
concentrate
51
50
48
48
40
28
24
16
23
16
15
11
concentrate
83
82
81
78
70
57
49
41
38
26
15
Concentration, mg/1
Carbon Amine Nitrogen
17.8
16.5
17 = 5
17 = 5
18.0
13.9
16.4
14.8
14.8
16.2
17.6
17.5
15-1
17.5
16.9
16
17.2
17.8
17.8
15-2
14.0
16.8
15.5
17 = 5
Peptone
30
120
120
120
110
100
90
60
45
35
25
20
25
195
190
185
150
170
140
110
90
70
40
20
116
-------
Table No. 11 con't
Sample No Time Concentration, mg/1
Carbon Amine Nitrogen Peptone
Reactor C -
C-0
added 90 ml
C-l
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-ll
Reactor D -
D-0
added 60 ml
D-l
I>-2
I>3
D-4
D-5
I>6
D-7
D-8
D-9
D-10
D-ll
Reactor E -
E-0
added 60 ml
E-l
E-2
E-3
*»rf ^
E-4
E-5
E-6
E-7
L-^ f
E-8
E-9
E-10
E-ll
10:08
of peptone
10:35
11:02
11:30
11:59
12:59
1:59
2:55
3:58
4:57
8:18
10:28
10:10
of peptone
10:37
11:09
11:35
12:03
1:06
2:02
3:01
4:13
5:09
8:22
10:33
10:10
of peptone
10-: 38
11:09
11:36
12:03
1:06
2:03
3:02
4:14
5:09
8:30
10:40 ,
11
concentrate
115
115
110
106
102
89
74
62
60
37
32
9
concentrate
100
100
92
92
83
68
57
46
32
30
22
9
concentrate
115
115
106
104
96
79
65
53
40
28
13
15.3
16.2
14.8
13.6
14.1
19.7
15.0
16.2
15-1
12.8
13-6
14.3
17.8
+ 15 ml glutamate
20.2
18.7
24.0
27.0
22.8
23-9
20.4
18.3
18.0
13.9
14.5
14.7
+ 30 ml glutamate
21.7
25.1
23.9
27-7
26.0
27-0
22.3
18.6
17.6
18.8
19-3
25
*«••> ^
260
265
245
245
240
205
180
185
125
55
40
25
concentrate
185
200
175
170
155
140
115
185
85
45
25
25
concentrate
17.5
175
165
165
150
135
110
95
75
40
35
117
-------
Table No. 11 con't
Sample No Tame Concentration, mg/1
Carbon Amine Nitrogen Peptone
Reactor P - l '
F-0 10:10 9 19.5 25
added 60 ml each of peptone and glutamate concentrate
F-l 10:39 129 30.3 165 * '
F-2 11:04 ' 124 34.4 160 .
F-3 11:36 127 33.0 150
F-4 12:03 125 37-5 150
F-5 1:06 117 30.6 140
F-6 2:03 104 28.3 130
F-7 3:02 88 21.0 115
F-8 4:14 76 21.2 100
F-9 5:10 65 20.6 85
F-10 8:35 35 18.7 50
F-ll 10:45 31 13-7 40
Footnotes: Temperature - 25°
Innoculum suspended solids concentration, 22.7, 35.2 mg/1
118
-------
TABLE NO. 12
Biological Degradation of Glutamate, glycine and Peptone using
Glutamate Acclimated Innoculum.
Experiment No. 3-20-69, Batch Reactor, Well Mixed.
Preparation of Innoculum
Collected effluent overnight from continuous propagator using 100 mg/1
glutamic acid feed solution at approximately 130 ml/hr. Each batch
test was started with 245 ml of innoculum.
Concentrate Preparation
a) 0.400 grams peptone + 400 ml distilled water
b) 0.200 grams glycine + 200 ml distilled water
c) 0.200 grains glutamic acid + 200 ml distilled water
Each concentrate was autoclaved for 5 minutes
Sample No Time Concentration, mg/1
Carbon Amine Nitrogen
Reactor A -
A-0 11:00 7 1-9
added 30 ml glutamic acid concentrate
A-l 11:07 ^9 12-5
A-2 11:40 46 12.5
A-3 12:11 41 11.0
A-4 12:46 31 . 6.5
A-5 1:48 20 6.0
A-6 2:47 15 3.4
A-7 3:45 11 2.0
A-8 4:55 13 2.5
A-9 7:37 12 2.0
A-10 9:05 13 2.0
A-ll 11:07 11 2.5
Reactor B - ,
B-0 11:10 ? 0.6
added 15 ml each of glycine and glutamic acid concentrate
B-l 11:08 45 7.0
B_2 11:40 43 6.6
B-3 12:11 37 6.0
B-4 12:46 32 6.2
1:49 28 6.0
** 3 2-0
- o i "
119
-------
Table No. 12 con't
Sample No Time
Reactor C. -
C-0
added
C-l
C-2
C-3
C-4
C-5
C-6
C-7
C-8
C-9
C-10
C-ll
D-ll
Concentration, mg/1
Carbon Amine Nitrogen
Reactor D -
D-0
added
D-l
D-2
D-3
D-4
D-5
D-6
D-7
D-8
D-9
D-10
11:00
30 ml
11:15
11:43
12:15
12:48
1:54
2:50
3:50
5:00
7:40
8:08
Reactor E -
E-0
added
E-l
E-2
E-3
E-4
E-5'
E~6
E-7
E-8
E-9
E-10
11:00
30 ml
11:16
11:44
12:15
12:48
1:55
2:51
3:50
5:00
7:41
9:08
E-ll
11:00
30 ml
11:10
11:41
12:12
12:46
1:49
2:4?
3 = 45
4:55
7:38
9:05
11:07
11:00
30 ml
11:15
11:43
12:15
12:48
1:54
2:50
3:50
5:00
7:40
8:08
11:10
11:00
30 ml
11:16
11:44
12:15
12:48
1:55
2:51
3:50
5:00
7:41
9:08
7:30
7
0.6
glycine concentrate
41
43
47
44
50
43
42
44
43
40
38
7
glutamate and 30
72
72
69
66
45
38
32
29
23
22
21
7
each of glycine,
102
99
95
91
74
70
61
58
49
49
46
9.6
9.5
9.5
11.6
9.3
10.0
9.1
8.9
8.3
8.7
0.6
ml glycine
9.4
9.5
9-3
10.5
8.0
7.2
5.5
3.3
2.0
2.5
1.7
0.4
glutamate and peptone
9.3
7.3
7.5
9.0
8.0
6.5
6.0
4.7
2.5
2.8
2.0
120
-------
Table No . 12 can't
Sample No
Reactor F
F-l
F-2
F-3
F-4
F-5
F-6
F-?
F-8
F-9
F-10
F-ll
added
Time
11:00
Concentration, mg/1
Carbon Amine Nitrogen
12
0.4
30 ml each of glycine and glutamate and 90 ml peptone
11:18
11:44
12:16
12:48
1:55
2:51
3:51
5:00
7:42
9:08
11:10
156
156
156
152
143
132
121
106
97
98
93
9.5
8.7
9.7
8.7
10.4
5.0
8.8
6.3
4.7
3.0
2.5
Footnotes: Imoculiin suspended solids concentration, 23-6, 23.2 mg/1
•> Suspended solids at end of batch test
Reactor B 12.5 mg/1
/; Reactor C 14.2 ng/1
r Reactor & &%**%
' Reactor E J3.0 mg/1
. Reactor F I1-
121
-------
TABLE NO. 13
Determination of Carbon Residues from Glutamic Acid Metabolism
Experiment No 6-19-69, Batch Reactor, Well Mixed
Preparation of Innoculum
Collected effluent from continuous propagator overnight (125 ml/hr).
Propagator feed solution contained 100 mg/1 glutamic acid. About one
half of the innoculum material was filtered through a 0.45 micron
Millipore filter.
Concentrate Preparation
0.400 grams of glutamic acid was dissolved in 400 ml of distilled
water and autoclaved for 5 minutes.
Sample No Time Concentrate, mg/1
Carbon Glutamic Acid
Reactor A - 200 ml of innoculum, no concentrate added
A-l 11:35 4 13
A-3 12:53 9 13
A-4 2:26 4 8
A-5 4:40 7 7
A-6 7:35 5 7
A-7 11:17 6 8
A-8 8:07 8 10
Reactor B - 200 ml of innoculum + 20 ml of concentrate
B-l 11:35 4 11
B-2 11:47 added concentrate 104
B-3 12:53 38 100
B-4 2:26 8 25
B-5 4:40 7 13
B-6 7:35 5 10
B-7 11:17 7 8
B-8 8:20 5 10
Reactor C - 200 ml of innoculum + 40 ml concentrate
C-l 11:35 5 4
11:48 added concentrate
C-2 11:52 68 158
C-3 12:54 73 200
C-4 2:27 43 116
C-5 4:40 12 38
C-6 7:30 8 30
C-7 11:17 10 25
C-8 8:10 12 8
122
-------
Table No. 13 con't
Sample No Tame Concentrate, mg/1
Carbon Glutamlc Acid
Reactor D - 50 ml each of raw innoculum and filtered innoculum
D-l 11:35 4 3
D-3 12:55 4 5
D-4
D-5
D-6
D-8
Reactor E
E-l
E-2
E-3
E-4
E-5
E-6
E-7
E-8
Reactor F
P-l
F-2
F-3
F-4
F-5
F-6
F-7
F-8
3:00
4:40
7:28
11:17
8:12
2
4
3
5
4
50 ml each of raw and filtered innoculum +
11:40
11:50 added
11:54
12:59
2:31
4:45
7:26
11:17
8:14
3
concentrate
40
33
20
6
5
5
5
2
1
2
15
5
10 ml of concentrate
4
67
108
60
13
a5
4
3
20 ml raw innoculum + 80 ml filtered innoculum + 10 ml
of concentrate
11:40
11:52 added
11:56
1:00
2:36
4:45
7:25
11:17
ft-1R
2
concentrate
39
36
33
16
5
5 '
4
8
78
108
90
43
15
7
3
123
-------
Table No. 13 con't
Sample No Time Concentrate, mg/1
Carbon ' ' Glutamic Acid
G 29 ml raw innoculum + 80 ml filtered^innoculum
G-l 11:40 '3
G-3 1:00 14
G-4 2:32 2 2
G-5 4:45 4 1
G-6 7:24 3 2
G-7 11:17 3 2
G-8 8:17 2 0
Footnotes: All reactors were filtered at the end of the experiment
Reactor Concentrate, mg/1
carbon suspended solids
A 6 21
B 7 37-4
C 11 40
D 68
E 6 24
F 6 26
G 4 N.D.
124
-------
TABLE NO. 14
Effect of Milk Solids Concentration on Rate of Deconposition
Experiment No. 8-12-69, Batch Reactor, Well Mixed
Preparation of Innoculum
Continuous propagator was operated on 100 mg/1 milk solids feed
solution at 148 ml/hr. Innoculum was collected overnight.
Concentrate Preparation
0.400 grams of milk solids were dissolved in 400 ml distilled water
and autoclaved.
Batch Tests
Four batch tests were set up using different amounts of concentrate.
Samples were withdrawn periodically and analyzed for total carbon,
carbohydrate, and protein. Effluent was collected overnight.
Batch Reactor Innoculum Concentrate
A
B
C
D
Sample No
A-l
B-l
C-l
D-l
A-2
B-2
C-2
D-2
A-3
B-3
C-3
D-3
A-4
B-4
C-4
D-4
A-5
B-5
C-5
D-5
A-6
B-6
C-6
D-6
Time
9:20
9:20
9:20
9:20
9:
9:
9:
10
10
10
10
11
11
11
11
12
12
12
12
1
1
1
27
29
30
.30
31
32
:33
:25
:25
;24
:26
:28
:29
:25
:29
:34
:37
:33
>300 ml
300 ml
300 ml
300 ml
At
Minutes
1:33
7
9
10
70
71
72
73
125
125
124
126
188
189
185
189
254
257
253
253
0 ml
30 ml
60 ml
-100 ml
Concentrate, mg/1
Carbon Carbohydrate
14
12
13
13
33
49
73
13
40
61
77
12
38
59
75
16
33
55
71
13
29
52
62
6
5
5
46
65
78
7
48
65
78
1
43
63
88
8
41
62
77
5
41
63
75
Protein
13
8
5
8
18 -
30
65
10
45
66
72
15
25
66
62
8
32
61
32
8
25
48
40
125
-------
Table No.l4 con't
Sample No Time At Concentrate, mg/1
Minutes Carbon Carbohydrate Protein
A-7 3:21 361 13 4 10
B-7 3:27 367 18 8 20
C-7 3:25 365 38 45 32
D-7 3:26 366 55 70 35
A-8 9:20 720 14 6 8
B-8 9:28 728 13 8 18
C-8 9:44 744 18 6 25
D-8 9:31 731 14 38 20 ,
A-9 8:10 1370 14 10 10
B-9 8:19 1379 17 6 8
C-9 8:14 1374 18 13 18
D-9 8:16 1376 14 12 13
126
-------
TABLE NO. 15
Biological Degradation of Milk Solids with Different Amounts of
Biologically Active Solids
Experiment No. 8-18-69, Test #17
Three continuous propagators using milk solids feed solutions were
set up. CP #1 ran on a 100 mg/1 milk solid solution, CP #2 on a
300 mg/1 solution and CP #2 on a 500 mg/1 solution. They were
allowed to run overnight then 100 ml aliquoto from each was filtered
on 0.45 millipore filter pads to remove bacteria.
Two batch reactors were set ~up with innoculum taken from each
continuous propagator
Batch Reactor Innoculum
A
B
C
D
E
F
Sample No
A-l
B-l
C-l
D-l
E-l
P-l
A-2
B-2
C-2
D-2
E-2
F-2
A-3
B-3
C-3
D-3
E-3
P-3
Time
:11
;20
:11
:21
10:
10;
10:
10:
10:24
10:24
12:10
12:11
12:13
12:12
12:16
2:10
2:11
2:15
2:13
2:18
2:19
Raw
200 ml CP#1
20 ml CP#1
200 ml CP#2
20 ml CP#2
200 ml CP#3
20 ml CP#3
At
Minutes
0
9
0
10
13
13
119
120
122
121
125
239
240
244
242
24?
248
Filtered
0 ml
100 ml CP#1
0 ml
100 ml CP#2
0 ml
100 ml CP#3
Concentration, mg/1
Carbon Carbohydrate
28
23
72
72
96
90
28
17
72
72
96
85
21
17
64
70
92
84
27
11
81
80
86
87
21
5
78
80
86
86
13
4
76
79
81
86
127
-------
Table No .igcon't
Sample No Time At Concentration, rng/1
Minutes Carbon Carbohydrate
A-4 4:09 358 16 6
B~4 4:11 360 16 5
C-4 4:15 364 63
DH» 4:12 36l 6? 77
E-4 4:25 374 73 70
F-4 4:17 366 74 86
A-5 9:07 1376 16 4
B-5 9t09 1378 17 7
C-5 9:11 1380 33 36
D-5 9:13 1382 46 56
E-5 9:24 1393 27 18
P-5 9:25 1394 53 67
A-6 10:01 2870 16 5
B-6 9:55 2864 17 7
C-6 9:43 2852 21 13
D-6 9:41 2850 29 33
B-6 9:33 2842 24 22
P-6 9:35 2845 28 27
128
-------
TABLE NO. 16
Effect of Substrate Concentration for Glycine Acclimated Innoculum
Experiment No. 9-22-69, Test #19, Barch Reactor, Well Mixed
Innoculum and Concentrate Preparation
Effluent from a continuous propagator
solution was used. This material was
tests. Concentrate of glycine (0.200
and glutamic acid (0.200 grams in 200
pared and autoclaved
Batch Reactor Innoculum
A 200 ml
B 200 ml
C 200 ml
D 200 ml
E 200 ml
using 100 mg/1 glycene feed
used as innoculum for 5 batch
grams in 200 ml distilled water)
ml distilled water) were pre-
Concentrate Additions
25 ml of glycine
50 ml of glycine
50 ml of glutamic acid
25 ml of each
50 ml of each
Sample No
A-l
B-l
C-l
D-l
E-l
(Concentrate
A-2
B-2
C-2
D-2
E-2
A-3
B-3
C-3
D-3
B-3
A-4
B-4
C-4
D-4
E-4
A-5
E-5
C-5
D-5
E-5
Time
10:10
10:10
10:12
10:14
10:1?
was added
10:21
10:23
10:23
10:27
10:28
11:30
11:31
11:33
11:35
11:36
12:31
12:32
12:36
12:37
:39
:42
:45
St Concentration, mg/1
Minutes Carbon Ammonia Amino Acid
Nitrogen (as
72
93
76
75
77
12:
1;
1:
1:46
1:49
1:49
0
0
2
4
7
prior to
11
13
13
17
18
80
81
83
85
86
141
142
146
147
149
212
215
216
219
219
27
27
27
28
27
second sample)
58 7.6
82 5.8
92 12.7
89 4.7
134 12.0
58
84
95
88
132
59
84
92
89
132
62 11.0
85 6.8
92 12.7
89 12.9
132
169
152
177
167
148
176
178
153
176
168
154
183
129
-------
Table No.l6 con't
Sample No
A-6
B-6
C-6
D-6
E-6
A-7
B-7
C-7
D-7
E-7
A-8
B-8
C-8
D-8
E-8
A-9
B-9
C-9
D-9
E-9
A-10
B-10
C-10
D-10
B-10
A-ll
B-ll
C-ll
D-ll
E-ll
A-12
B-12
C-12
D-12
E-12
A-13
B-13
C-13
D-13
E-13
Time
At
Minutes
2:35
2:37
2:39
2:41
2:42
,4:24
4:24
4:26
4:27
4:29
6:01
6:02
6:03
6:06
6:07
10:57
10:57
11:00
11:00
11:02
12:28
12:30
12:31
12:32
12:37
7:23
7:23
7:27
7:30
7:35
8:58
8:59
9:08
9:08
9:10
11:15
11:16
11:17
11:18
11:18
265
267
269
271
272
374
374
376
377
379
471
472
473
476
477
767
767
770
770
772
858
860
861
862
867
1273
1277
1280
1285
1368
1369
1374
1378
1380
1505
1506
1507
1508
1508
Carbon
59
87
93
89
136
59
81
88
80
130
54
79
85
79
124
51
73
63
50
110
48
59
64
49
106
37
51
17
20
71
30
50
18
19
63
22
41
20
18
46
Concentration, mg/1
Ainmonla Amlno Acid
Nitrogen (as glycine)
164
147
180
,4
• 7
6.3
12.1
6.6
8.2
6.3
12.9
10.2
9.8
17.0
11.
14.
11.0
9.3
17.0
12.9
16.
18.
17.4
24.8
12.9
23.0
20.0
21.6
27-6
20.8
29.1
23-
27-
24.
19-
,5
.3
.7
,1
,2
,5
35.2
164
148
177
163
147
177
162
129
162
159
128
163
105
179
55
74
185
83
167
48
52
173
66
142
38
40
151
130
-------
Table No. 16 con't
Sample No Time At
Minutes Carbon Ammonia Amlno Acid
Nitrogen (as glycine)
A-14 1:50 1660 19 24.0 48
B-14 1:50 1660 28 31.3 77
C-14 1:55 1665 20 27.0 48
D-14 1:58 1668 17 17-7 '" 39
E-14 2:02 1672 30 37.3 102
A-15 12:47 4477 12 11.1 23
B-15 12:49 4479 16 15.2 31
C-15 12:52 4482 16 20.3 36
D-15 12:54 4484 28
E-15 1:03 4493 20 18.9 27
131
-------
TABLE NO. 1?
Effect of Substrate Concentration in Degradation of Laurie Acid
Experiment No. 1O-7-69, Test #20, Batch Reactor, Well Mixed
Effluent from a continuous propagator using 50 mg/1 lauric acid feed
was collected and used as innoculum in a series of batch tests.
Different amounts of fresh feed (50 mg/1 lauric acid) were added at
the beginning of each batch test. The contents of the lauric acid
propagator were used as innoculum.
in lieu of concentrate.
Batch Reactor Innoculum, ml
A 400
The aluric acid feed was used
B
C
D
Sample No
Time
200
200
40
At
Minutes
Fresh Feed, ml
0
200
200
360
Mixing Time
9:44 A.M.
9:44 A.M.
9:49 A.M.
9:4? A.M.
Carbon Concentration, mg/1
A-"l
B-l
CXL
D-l
A-2
B«i2
C-i2
D^-2
A"-3
A-4
C^-3
1X3
A-4
B-4
C^4
D*>4
A-5
B-5
C-5
D-^S
A-6
B-6
C-6
D-6
A-7
B-7
C-7
D-7
A-8
B^8
C-8
D-8
Footnote :
10:05
10:00
10:09
10:03
11:37
11:26
11:35
11:29
2:20
2:22
2:30
2:26
4:32
4:36
4:43
4:43
7:46
7:50
7:58
8:00
9:55
10:00
10:09
10:10
11:20
11:28
11:25
11:26
11:12
11:28
11:17
11:32
Fa'tty acid
5
1 0
9
3
97
86
95
89
260
262
270
266
392
396
403
403
586
590
598
600
715
720
729
732
1320
1528
1525
1526
2952
2968
2957
2972
analyses were erratic
19
21
31
6
14
15
25
7
7
6
19
6
5
6
15
6
6
6
6
7
5
7
7
7
5
5
5
8
10
8
4
132
-------
TABLE NO. 18
Effect of Substrate Concentration in Degradation of Laurie and
Palmitic Acid
Experiment No. 12-18-69, Test #21, Batch Reactor, Well Mixed
Effluent from a continuous propagator using 50 mg/1 laurlc acid feed
was collected and used as innoculum in a series of batch tests.
At the beginning of each batch test, fresh feed solution of lauric
or palmitic acid were added.
Batch Reactor Innoculum Fresh Peed Added at Start of Tests
50 mg/1 100 mg/1 50 mg/1
Lauric Laurie' Palmitic
A 190 0 0 0
B 190 200 0 0
C 190 0 200 0
D 50 0 350 0
E 50 350 0 0
F 100 0 0 300
Sample No Time At Carbon Concentration, mg/1
A-l 10:02 11
B-l 10:05 11
C-l 10:05 11
Fresh feed added before taking sample #2
B-2 10:28 3 21
C-2 10:35 10 *»1
D-2 10:36 11 5«
E-2 10:37 12 36
p-2 10:38 13 1°
A-3 11:00 35 13
B-3 11:02 37 24
C-3 11:04 39 35
D-3 11:06 41 58
E-3 11=12 *7 ?2
p_3 11:14 49 1'
A-4 12:00 95 H
B-4 12:02 97 23
C-4 12:04 99 34
D-4 12:06 101 56
E-4 12:13 108 • 32
F-4 12:15 HO ^
A-5 I'M 159 oo
B-5 1 = 06 161 22
C-5 1:08 163 30
D-5 1=10 165 53
1 = 15 170 26
1:17 172 17
133
-------
Table No. 18 con't
Sample No
A-6
B-6
C-6
D-6
E-6
F-6
A-7
B-7
C-7
D-7
E-7
F-7
A-8
B-8
C-8
D-8
E-8
F-8
A-9
B-9
C-9
D-9
E-9
F-9
A-10
B-10
C-10
D-10
E-10
F-10
A-ll
B-ll
C-ll
D-ll
E-ll
F-ll
A-12
B-12
C-12
D-12
E-12
F-12
Time
At
3:00
3:02
3:04
3:06
3:12
3:14
5:00
5:02
5:04
5:08
5:16
5:18
7:45
7:45
7:50
7:50
8:00
8:00
10:20
10:20
10:30
10:30
10:35
10:35
9:13
9:15
9:17
9:19
9:24
9:28
12:00
12:02
12:04
12:06
12:14
12:16
3:30
3:32
3:34
3:36
3:50
3:54
275
277
279
281
287
289
395
397
399
403
411
413
560
560
565
565
575
575
715
715
725
725
730
730
136
1370
1372
1374
1379
1383
1535
1537
1539
1541
1549
1551
1745
1747
1749
1751
1765
1769
Carbon Concentration, mg/1
19
25
48
26
16
13
17
42
21
12
1
2
3
33
14
6
1
0
0
14
4
4
0
0
0
4
2
2
1
1
1
3
2
2
1
1
2
1
3
134
-------
Table No. 18 can't
Sample No Tome At Carbon Concentration, mg/1
B-13 11:45 2960 5
C-13 11:58 2973 3
D-13 12:10 2985 2
B-13 11:52 2967 5
F-13 11:50 2965 5
Analysis of feed solutions (F = filtered, UP = unfiltered)
50mg/l Laurie UP 10:15 35
50mg/l Laurie P 10:15 35
lOOmg/1 Laurie UP 10:15 65
100mg/l Laurie F 10:20 65
50mg/l Palmitic UP 10:20 33
50mg/l Palmitic F 10:18 15
Footnotes: Temperature was approximately 24°c
Cell mass determination by millipore filtration
Reactor Sample Suspended Solids
Concentration-, mg/1
A 4
B 18
C 22
D 48
E 32
p 16
135
-------
TABLE NO. 19
Effect of Substrate Concentration on Degradation of Palmitic and
Laurie Acid.
Experiment No. 12-23-69, Test #22, Batch Reactor, Well Mixed
Effluent from a continuous propagator using 50 mg/1 palmitic acid
feed solution was collected and used as innoculum in a series of
batch tests. At the beginning of each batch test, fresh feed
solutions of lauric or plamitic acid were added.
Fresh feed added at start of test
Reactor Innoculum Lauric Acid Feed (m/1) Palmitic Peed
A
B
C
D
E
F
Sample No
A-l
B-l
C-l
D-l
E-l
F-l
A-2
B-2
C-2
D-2
E-2
F-2
A-3
B-3
C-3
D-3
E-3
F-3
A-4
B-4
C-4
D-4
E-4
F-4
200
200
200
50
50
100
lime
10;
10;
10;
10:
10;
25
24
26
32
44
10:55
11
11
11
11
11
12
12
12
12
12
12
1
1
1
1
1
29
30
31
39
45
20
21
22
23
29
51
30
21
22
23
35
1:49
50 mg/1
0
200
0
0
350
0
At
1
0
2
8
20
31
65
66
67
75
81
116
117
118
119
125
147
206
177
178
179
191
205
100 mg/1
0
0
200
350
0
0
50 mg/1
0
0 •
0
0
0
300 ml
Carbon Concentration, mg/1
5
20
34
54
34
15
14
28 '• :
41 •••-•
21
8
O -"•
7
20
48
23
8
6
4
12
45
21
6
136
-------
Table No-19 cpn't
Sanple No Time At Carbon Concentration
A-5 3:26 302 3
B-5 3:14 290 3
C-5 3:28 304 4
D-5 3:11 287 34
E-5 3:39 315 14
F-5 3:39 315 5
A-6 4:45 381 3
B-6. 4:42 378 3
C-6 4:57 393 4
D-6 4:52 388 23
E-6 5:06 402 4
P-6 5:1^ 410 3
A-7 8:10 586 0
B-7 8:15 591 0
C-7 8:20 596 2
I>7 8:20 596 5
E-7 8:30 606 2
F-7 8:30 606 2
A-8 9:50 686 2
B-8 9:50 686 2
C-8 9 = 55 691 2
D^8 9:55 691 5
E-8 10:10 706 2
F-8 10:10 706 2
A~9 9:24 1380 3
B-9 9:25 1381 3
C-9 9:26 1382 4
M 9:27 1383 3
E-g 9:48 1404 2
p-9 9:50 1406 3
A-10 12:00 1536 ^
B-10 12:01 1537 2
C-10 12:02 1538 3
D-10 12:03 1539 2
E-10 12:24 1560 3
jui 12:25 1561 3
137
-------
Table No. ig con't
Sample No Time
B-ll
C-ll
D-ll
E-ll
F-ll
At
:58
;59
;QQ
;02
3:18
1715
1716
1718
1734
Carbon Concentration, nig/1
3
3
0
3
Footnotes: Temperature was approximately 25° c
Analysis of feed solution (F = filtered, UP = unfiltered)
50 rag/1 Laurie Feed UF 37
50 mg/1 Laurie Feed F 38
100 mg/1 Laurie Feed UF 64
100 mg/1 Laurie Feed F 62
50 mg/1 Palmitic Feed UF 28
50 mg/1 Palmitic Feed F 15
Cell Mass Determination by millipore filtration
138
-------
TABLE NO • 20
Effect of Substrate Concentration on Rate of Degradation of Mixed
Peed Solutions
Experiment No. 12-30-69, Test No. 23, Batch Reactor, Well Mixed
Effluent from a continuous propagator using 50 mg/1 lauric acid
feed solution was collected and used as innoculum in a series of
batch tests. At the beginning of each test, fresh feed solutions
were added as shown:
Concentration
Feed Solutions
Lauric Acid
Glutamic
Glucose
Reactor
A
B
C
D
E
Sample
A-l
B-l
0-1
D-l
E-l
A-2
B-2
0-2
D-2
E-2
A-3
B-3
0-3
D-3
E-3
A-4
B-4
C-4
D-4
E-4
A-5
B-5
0-5
E-5
Acid
Innoculum
ml
200
200
200
200
200
Time
9:00
9:00
9:01
9:01
9:02
9:19
9:20
9:20
9:21
10:00
10:01
10:02
10:03
10:08
11:00
11:01
11:02
11:03
11:11
12:00
12:01
12:02
12:03
12:15
At
0
0
1
1
2
19
20
20
21
60
61
62
63
68
120
121
122
123
131
180
181
182
183
195
100 mg/1
1000 mg/1
1000 mg/1
Fresh Feed Solutions Added,.ml
Lauric Feed Glucose Feed Glutamic Feed
200 0 0
200 50 0
200 0 50
200 50 50
200 0 0
Glutamic
Concentration
mg/1
108
81
Carbon
Concentration
mg/1
3
3
3
4
6
23
74
61
95
26
75
63
97
3
16
68
57
87
1
6
55
44
77
1
Glucose
Concentration
mg/1
0
0
72
70
74
71
73
61
71
67
107
99
106
102
92
97
139
-------
Table No. 20 con't
Sample Time At Carbon Glucose Glutamlc
Concentrate Concentrate Concentrate
mg/1 mg/1 mg/1
4
47 66
37 98
67 32 71
3 32 71 t
4
39 ^5
23 87
47 13 83
4 0
13 1
7 66
10 1 68
4
4
6 2
8 71
8 9 80
4
5
5 2
7 88
6 1 65
4
4 1
6 72
7 1 74
5
3
2 0
5 85
3 8 75
2
7
10 8
10 80
10 8 83
Footnotes: a) Temperature was approximately 28°c, b) Suspended solids
(cell mass) was measured by filtering through a membrane filter and
weighing the dry mass. Reactor Suspended Solids, mg/1
B 34
C 34
c) High amino acid concentration is due to presence of ammonia nitrogen
Sample C-l C-6 C-ll D-l D-5 D-10
Arrmonia Nitrogen, mg/1 106.5 80.5 95-0 115.5 85.0 78.5
140
A-6
B-6
C-6
D-6
E-6
A-7
B-7
C-7
D-7
E-7
A-8
B-8
C-8
D-8
E-8
A-9
B-9
C-9
D-9
E-9
A-10
B-10
C-10
D-10
E-10
A-ll
B-ll
C-ll
D-ll
B-ll
A-12
B-12
C-12
D-12
E-12
A-13
B-13
C-13
D-13
1:00
1:01
1:01
1:02
1:24
1:59
2:00
2:01
2:02
2:28
4:30
4:41
4:36
4:57
4:41
6:40
6:41
6:42
6:43
7 = 13
9:15
9:16
9 = 17
9:18
9:48
9:04
9:05
9:26
9:07
9:32
2:05
2:17
2:15
2:35
2:09
2:29
2:34
2:58
3:20
240
241
241
242
264
299
300
301
302
328
450
461
456
477
461
580
581
582
583
613
735
736
737
738
768
1444
1445
1446
1447
1472
1745
1757
1755
1777
1749
4649
4654
4678
4700
-------
TABLE NO. 21
Effect of Substrate Concentrations on Rate of Degradation of Mixed
Feed Solutions.
Expertaent No. 1-29-70, Test No. 24, Batch Reactor, Well Mixed
Effluent from a continuous propagator using 50 mg/1 lauric acid feed
solution was collected and used on innoculum in a series of batch
tests. At the beginning of each test, fresh feed solutions were
added as shown.
Feed Solution 'Concentration, mg/1
Lauric Acid 100
Glutamic Acid 1000
Glucose 1000
Reactor Innoculum (ml) Fresh Feed Solutions Added
Lauric Feed Glucose Feed Glutamic Feed
A 200 200 ml 0 ml 0 ml
B 200 200 ml 50 ml 0 ml
C 200 200 ml 0 ml 50 ml
D 200 200 ml 50 ml 50 ml
E 200 0 ml 0 ml 0 ml
Sample Time -At Carbon Glucose Glutamic Acid
Concentrate Concentrate Concentrate
(mg/1) (mg/1) (mg/1)
A-0 10:48 0 12
B-0 10:48 0 16 2
C-0 10:49 1 14 .12
D-0 10:49 1 14 8 20
E-0 10:54 6 14
A-l 11:00 12 35
B-l 11:01 13 79 78
0-1 11:02 14 63 72
D-l 11:04 16 96 73 87
E-l
A-2 11:55 67 37
B-2 11:56 68 77 80
0-2 11:57 69 59 90
D-2 11:58 70 97 79 »?
E-2 12:01 73 12
A-3 1=06 138 29
B-3 1:07 139 70 81
0-3 1=08 140 53 89
D-3 1 = 09 1^1 90 77 88
E-3 1=15 1^7 9
A-4 2:00 192 25
B-4 2:01 193
-------
Table No. 21 con't
Sample
A-5
B-5
C-5
D-5
E-5
A-6
B-6
C-6
D-6
E-6
A-7
B-7
C-7
D-7
E-7
A-8
^8
C-8
D-8
E-8
A-9
B-9
C-9
D-9
E-9
A-10
B-10
C-10
D-10
E-10
overnight
A-ll
B-ll
C-ll
D-ll
E-ll
A-12
B-12
C-12
D-12
E-12
Ttaie
3:04
3:05
3:06
3:07
3:12
4:03
4:05
4:07
4:09
4:12
6:25
6:25
6:30
6:30
6:35
8:02
8:03
8:04
8:05
8:16
9 = 57
9 = 58
9:59
10:00
10:08
11:59
12:00
12:03
12:05
12:11
1/29 to
10:27
10:32
10:39
10:54
10:31
12:48
12:52
1:00
1:07
12:50
At
256
257
258
259
264
315
317
319
321
324
457
457
462
462
467
554
555
556
557
568
669
670
671
672
680
791
792
795
797
803
1/30
1419
1424
1431
1446
1423
1560
1564
1572
1579
1562
Carbon
Concentrate
(jflg/D
16
56
44
79
5
14
55
40
76
5
11
54
24
57
4
9
51
10
40
4
6
49
9
23
4
H
45
9
18
6
5
39
10
14
8
5
33
10
13
9
Qlucose Qlutamlc Acid
Concentrate Concentrate
, (mg/D
80
74
81 55
79
70
79 69
80
44
73 45
77
20
43 28
78
21
9 26
70
21
2 12
26
25
3 18
21
25
5 11
142
-------
Table No. 21 con't
Sample Time At Carbon Giucose Qlutamic Acid
Concentrate Concentrate Concentrate
A-13 9:45 2757 g ) CinS/1) (rng/1)
B-13 9:46 2758 5 ?
C-13 9:47 2759 9 ^
D-13 9:48 2760 u n ft
E-13 10;05 2777 8
Footnotes: a) Temperature was maintained at approximately 27°c
b) Suspended solids were measured by membrane filtration
and dry weighing
Reactor Suspended Solids, mg/1
__ g
B 34
C 26
D 58
E 4
c) Greenish tint developed in Reactor C and D after 12 hours
d) Glucose analysis by phenol sulfuric acid test
143
-------
TABLE NO. 22
Effect of Substrate Concentration on Rate of Degradation of Mixed
Peed Solutions
Experiment No. 2-23-70, Test No. 25, Batch Reactor, Well Mixed
Effluent from a continuous propagator using a mixed feed consisting
of 100 mg/1 lauric acid, 100 mg/1 glutamic acid and 100 mg/1 of
glucose was collected and used as innoculum in a series of batch
tests. At the beginning of each test, fresh feed solutions were
added as shown:
'Feed Solutions Concentration, mg/1
Lauric Acid
Glutamic Acid
Glucose
Inncoulum (ml)
Reactor
A
B
C
D
E
Sample
A-0
B-0
C-0
D-0
E-0
A-l
B-l
C-l
D-l
E-l
A-2
B-2
C-2
D-2
E-2
A-3
B-3
C-3
D-3
E-3
200
200
200
200
200
Time
10:48
10:49
10:50
At
0
1
2
10:51
11:49
11:49
11:50
11:50
11:51
1:02
1:09
1:21
2:26
2:28
2:30
2:34
3:31
3:43
3:49
3
61
61
62
62
63
134
141
153
218
220
222
226
283
295
301
1000
1000
Freeh Feed Solutions Added
Lauric Feed Glucose Feed Glutamic Feed
(ml) (ml) (ml)
0 25 0
0 0 25
200 0 0
200 25 25
000
Carbon
Concentrate
(mg/1)
44
42
42
41
76
58
42
34
72
45
35
35
22
63
20
27
27
18
Glucose
Concentrate
(mg/1)
34
Glutamic Acid
Concentrate
(mg/1)
89
67
74
30
68
9
70
45
13
5
144
-------
Table No. 22 con't
Sample
i
A-4
B-4
C-4
D-4
E-4
A-5
B-5
C-5
D-5
E-5
A-6
B-6
C-6
D-6
E-6
A-7
B-7
c-7
D-7
E-7
A-8
B-8
c-8
D-8
B-8
A-9
B-9
C-9
D-9
E-9
A-10
B-10
C-10
D-10
E-10
Time
;io
4:22
4:53
5:14
5:18
7:24
7:25
7:26
7:27
8:06
9:38
*9:39
9:40
9:41
10:40
9 = 30
9:30
9:31
9:32
10:15
12:05
12:06
12:07
12:08
12:46
2:28
2:29
2:30
2:31
3:18
9:52
9:53
9:54
9:55
10:24
At
322
334
365
386'
390
516
517
518
519
558
650
651
652
653
712
1362
1362
1363
1364
1407
1517
1518
1519
1520
1558
1660
1661
1662
1663
1710
2824
2825
2826
2827
2856
Carbon
Concentrate
Crag/1)
58
16
24
12
13
45
15
17
11
13
' 40
15
11
7
13
20
21
6
11
16
13
21
7
8
15
11
20
7
10
15
12
21
7
11
15
Glucose Glutamlc Acid
Concentrate Concentrate
Org/l) (mg/1)
43
7
12
0 4
21
14
12 6
25
22
80 4
23
21
34 6
11
26
12 12
14
16
8 19
145
-------
Table No. 22 con't
Sample Time At , Carbon Qyucgs.e Qlutaraic Acid
Concentrate Concentrate Concentrate
Crag/1) (jng/1)
A-ll 10:17 4289 15 19
B-ll 10:18 4290 20 11
C-ll 10:19 4291 7
D-ll 10:20 4292 11 3 24
E-ll 10:58 4330 16
Footnotes: a) Temperature was approximately 2? c
b) Suspended solids were measured at the end of each test by
filtering through a membrane filter and dry weight
Reactor Suspended Solids Concentrate3 rng/1
A 112
B 102
C 50
D 92
E 96
c) Some protozoa were detected in batch tests B, C3 D, and E
146
-------
TABLE NO. 23
Effect of Substrate Concentration on Rate of Degradation of Mixed
Peed
Experiment No. 4-30-70, Test No. 26, Batch Reactor, Well Mixed
Effluent was collected from a continuous propagator using a mixed
feed solution of 100 mg/1 each of glucose, glutamic acid and lauric
acid and was used as innoculum in a series of batch tests. Fresh
feed solutions were added at the beginning of each batch test as
shown:
Fresh Feed Solution
Lauric Acid
Glutamic Acid
Glucose
Reactor
A
B
C
D
E
Sample
A-0
B-0
C-0
D-0
E-0
A-l
B-l
C-l
.D-l
E-l
A-2
B-2
C-2
D-2
E-2
A-3
B-3
C-3
D-3
E-3
Innoculum
(ml)
200
200
200
200
200
Time
At
1:08
1:04
1:06
1:08
1:15
1:43
1:44
1:45
1:44
1:44
4:39
4:37
4:45
4:45
5:00
6:08
6:06
6:12
6:16
6:39
4
0
2
4
11
39
40
41
40
40
175
173
181
181
196
304
302
308
312
335
Concentration, mg/1
100
1000
1000
Fresh Feed Solution Added
Lauric Glucose Glutamic
Concentrate Concentrate Concentrate
(ml) (ml) (ml)
200 0 0
200 50 0
200 0 50
200 50 50
000
Carbon
Concentrate
(mg/1)
39
37
37
37
39
^5
72
66
. 93
47
45
71
43
76
33
33
65
27
59
27
Glucose
Concentrate
(mg/1)
33-8
25.0
70.0
.80.0
95-0
80.0
70.0
77.0
Glutamic Acid
Concentrate
(mg/1)
5
7
73
45
40
10
8
147
-------
Table No. 23 con't
Sample Time At Carbon Qlucose Glutami.c Acid
Concentrate Concentrate Concentrate
(jng/1). (jng/1) (mg/1)
A-4 7:38 394 30
B-4 7:39 395 56 47.3
C-4 7:40 396 14 8
D-4 7:41 397 38 56.3 10
E-4 7:42 398 24
A-5 10:30 566 22
B-5 10:30 566 51 81,3
C-5 10:32 568 10 12
D-5 10:32 568 22 25.6 10
E-5 10:34 570 14
A-6 9:10 1206
B-6 9:11 1207 34 48.8
C-6 9:11 1207 10 13
D-6 9=23 1219 10 15.0 5
E-6 9:23 1219 10
A-7 4:22 1638 15
B-7 4:23 1639 28 42.3
C-7 4:23 1639 11 11
D-7 4:32 1648 13 22.0 8
E-7 4:32 1648 10
A-8 1:06 2882 12
B-8 1:20 2896 16 19.0
C-8 12:58 2874 12 12
D-8 1:20 2896 13 20.0 13
E-8 1:11 2887 12
148
-------
TABLE NO. 24
Effect of Substrate Concentration on Rate of Degradation of Mixed
Feed
Experiment No. 5-21-70, Test #273 Batch Reactor, Well Mixed
Effluent was collected from a continuous propagator using a mixed
feed of 1QO mg/1 each of glucose, glutamic acid and lauric acid and
was used as innoculum in a series of batch tests. Fresh solutions
were added to at the beginning of each batch test.
Feed Solution Concentration, mg/1
Lauric Acid 100
Glutamic Acid 1000
Glucose 1000
Reactor Innoculum Feed Solutions Added
(ml) Lauric Feed Glucose Feed Glutamic Feed
(ml) (ml) (ml)
A 200 0 0 25
B 200 0 25 0
C 200 200 0 0
D 200 200 25 25
E 200 0 0 0
Sample Time At Carbon Glucose Glutamic Acid
Concentrate Concentrate Concentrate
(mg/1) (mg/1) (mg/1)
A-0 11:59 12 11 92
B-0 11:52 5 10 6
C-0 11:48 1 9
D-0 11:50 3 11 **
E-0 11:47 0 11
A-l 12:27 40 39 141
B-l 12:30 43 30 45
C-l 12:23 36 27
D-l 12:22 35 ^8 23
E-l 12:27 40 9
A-2 2:44 177 H 117
B-2 2:53 186 7 12
C-2 2:38 171 12
D-2 2:41 174 14 3
£-2 2:43 176 7
A-3 6:50 423 8 160
B-3 6:55 428 7 6
0-3 6:40 413 6
D_3 6:42 415 8 2
E-3 6:44 417 6
149
-------
Table No. 24 can't
Sample
A-4
B-4
C-4
D-4
E-4
A-5
B-5
C-5
D-5
E-5
A-6
B-6
C-6
D-6
E-6
A-7
B-7
C-7
D-l
E-7
Time '
8:53
8:57
8:43
8:49
8:47
11:07
11:08
10:50 '
10:50
10:51
1:33
1:36
1:25
1:28
1:28
2:50
2:56
3:43
3:49
2:48
At
546
550
536
542
540
680
681
663
663
664
826
829
818
821
821
1623
1629
1616
1622
1621
Carbon
Concentrate
(ms/ll
11
10
7
8
7
8
10
7
8
7
10
6
6
6
8
7
6
7
8
5
Qlucose
Concentrate
(mg/1)
Glutamic Acid
Concentrate
(rag/1)
135
140
8
2
0
2
0
0
127
103
Footnotes: a) Sample D-l foamed when glutamic acid was added and some
foam was lost
150
-------
TABLE NO . 25
Effect of Substrate Concentration on Rate of Degradation Using
Mixed Peed and Excess Ammonia Nitrogen
Experiment No. 6-19-70, Test #29, Batch Reactor, Well Mixed
Effluent was collected from a continuous propagator using a mixed
feed of 100 mg/1 each of glucose, glutamic acid and lauric acid
and was used as innoculum in a series of batch tests. Fresh solutions
were added to at the beginning of each batch test.
Peed Solutions Concentrations, mg/1
Glutamic Acid 200
Lauric Acid 200
Glucose 200
NHjjCl 200
Reactor Innoculum Lauric Glucose Glutamic Acid NK^Cl
ml Concentrate Concentrate Concentrate Concentrate
ml ml ml ml
A 100 200 0 0 100
B 100 0 200 0 100
C 200 0 0 200 0
Sample Time At Carbon Glucose Glutamic Acid
Concentrate Concentrate Concentrate
(mg/1) (mg/1) (mg/1)
A-0 9:55 0 10 16 165
B-0 9:58 0 12 16 112
C-0 10:04 0 13 16 Ho
A-l 10:12 2 128
B-l 10:10 0 64 51
C-l 10:10 0 56
A-2 11:18 68 123
B-2 11:20 70 60 53
C-2 11:18 68 53
12:39
B-3 12:42 152 57 37
0-3 12:59 169 *»? 153
A-4 1:48 218 94
B-4 1:55 225 ^ ^
C-i, 1:59 229 O 72
US *
C-5 2:49 279 31
A-6 3:52 342 g
B-6 4:00 350 27 (
C-6 4:00 350 24
151
-------
Table No. 25 can't
Sample Time At Carbon Gfluco.se Glutamlc Acid
Concentrate Concentrate Concentrate
Ong/1) (ing/1) (mg/1)
56
27 8
. 20 50
41
20 6
15 •• '71
38
19 -9 , •::•:,.;•.;
12 59
33
11 7
9 • 77
29
15 8
9 75
30 :
10 ' 8
7 92
32
9 7
9 62
A-7
B-7
C-7
A-8
B-8
C-8
A-9
B-9
C-9
A-10
B-10
C-10
A-ll
B-ll
C-ll
A-12
B-12
C-12
A-13
B-13
C-13
4:48
4:52
4:52
8:31
8:32
8:33
10:33
10:30
10:37
12:22
12:21
12:23
9:47
9:45
9:45
8:15
8:13
8:13
6:43
6:43
6:43
398
402
402
621
622
623
743
740
747
852
851
853
1417
1415
1415
2045
2043
2043
3397
3393
3393
152
-------
TABLE NO. 26
Effect of Substrate Concentration on Rate of Degradation Using lyiixed
Feed and Excess Attimonia Nitrogen
Experiment No. 7-15-70, Test No. 30, Batch Reactor, Well Mixed
Effluent was collected from a continuous propagator using a mixed
feed of 100 mg/1 each, of glucose, glutamic acid and lauric acid
and was used as innoculum in a series of batch tests. Fresh solutions
were added to at the beginning of each batch test.
Feed Solutions Concentration,
Lauric Acid 1000
Glutamic Acid 1000
Glucose 1000
Ammonium Chloride 1000
Reactor Innoculum Lauric Glucose Glutamic Acid NtLCl
Volume Concentrate Concentrate Concentrate Volume
(ml) Volume(ml) Volume(ml) Volume(ml) (ml)
A 340 40 0 0 20
B 340 0' 40 0 20
C 360 0 0 40 0
D 280 40 40 40 0
Sample Time At Carbon Glucose Glutamic Acid
Concentrate Concentrate Concentrate
(mg/1) (mg/1) (mg/1)
A-0 11:30 0 79 30 4
B-0 11:30 0 78 55 20
C-0 11:30 0 76 33 22
D-0 11:30 0 80 75 2B
A-l 12:05 35 129
B-l 12:05 35 H7 I40 R
IP-OR 35 98 |°
lillO 40 160 135 87
A-2 1*09 99 120
B-2 It 10 100 105 128
C-2 1:11 101 94 68
D-2 1:12 102 155 139 87
A-3 2:19 169 H° ,
2:20 170 99 125
2-20 170 87 51
2:17 167 15J 133 *
A-4 3:17 227 13J
B-4 3:18 228 94 116
C-4 3:19 229 77
D-4 3:45 245 167
153
-------
Table No. 26 con't
Sample Time
At
A-5
B-5
C-5
D-5
A-6
B-6
C-6
D-6
A-7
B-7
C-7
D-7
A-8
B-8
C-8
D-8
A-9
B-9
C-9
D-9
A-10
B-10
C-10
D-10
A-ll
B-ll
C-ll
D-ll
A-12
B-12
C-12
D-12
A-13
B-13
C-13
D-13
4:11
4:13
4:14
4:18
5:45
5:46
5:47
5:50
6:05
6:08
6:15
6:18
8:25
8:27
8:32
8:30
9:35
9:36
9:37
9:31
10:25
10:26
10:26
IP: 27
9:46
9:47
9:47
9 = 50
4:23
4:23
4:21
4:27
9:38
9:35
9:37
9:50
281
283
284
288
375
376
377
380
395
398
405
408
535
537
542
540
605
606
607
601
655
656
656
657
1336
1337
1337
1340
1733
1733
1731
1737
2768
2765
2767
2780
Carbon
Concentrate
(rag/1)
98
83
63
149
80
63
49
133
126
114
95
187
42
45
30
109
32
39
31
115
31
28
32
107
24
20
31
62
24
20
34
47
21
17
31
Qlucose
Concentrate
Crag/1)
109
125
88
108
87
87
47
93
22
68
10
48
2
5
2
7
4
2
Qlutaraic Acid
Concentrate
(rag/D
17
22
6
7
16
9
5
6
5
7
5
5
8
6
16
6
18
6
Footnotes: Temperature was approximately 30°c-at the end of each test
the material was filtered and dried to measure suspended-solids (cell
mass)
Reactor Suspended Solids,, mg/1
A H8
B 84
C 50
D 152
154
-------
TABLE NO
27
Effect of Substrate Type on Rate of Degradation Using Mixed Liquor
Activated Sludge from Minneapolis-St. Paul Sewage Treatment Plant
Experiment No. 7-23-70, Test #31, Batch Reactor, Well Mixed
Mixed liquor was obtained from the treatment plant at about 9:00 A.M.
Aliquots were used as innoculum of six batch reactors. Feed solutions
were added as shown.
Feed Solutions Concentration, mg/1
Glucose 1000
Starch 1000
Glutamic Acid 1000
Peptone 1000
Laurie Acid 100
Reactor
A
B
C
D
E
F
Innoculum
Used
200
200
200
200
200
200
Glucose
(ml)
50
0
0
0
0
25
Feed Solutions Added
Starch Glutamic Acid Peptone
(ml)
0
50
0
0
0
0
(ml)
0
0
50
0
0
25
(ml)
0
0
0
50
0
0
Laurie Acid
(ml)
0
0
0
0
200
50
Sample Time At Carbon Inorganic Protein Glucose Glutamic Carbohydrate Ammonia Starch
No
AI
BI
CI
DI
El
AO
BO
CO
DO
EO
FO
Al
Bl
Cl
Dl
El
Fl
Min
1:25 0
1:25 0
1:25 0
1:25 0
1:43 18
1:40 15
2:45 80
2:45 80
2:45 80
2:45 80
2:57 92
2:53 88
Total
mg/1
89
93
96
93
93
132
135
132
159
89
129
111
119
114
119
34
94 -
Carbon
mg/1
26
27
27
27
27
21
26
21
26
30
27
21
19
24
23
31
25
mg/1 mg/1
250
128
54
95
190
Acid
mg/1
5
7
150
59
74
125
28
mg/1
7
7
152
18
111
146
mg/1 mg/1
7.0
6.6
4.9
9.0
9.5
9
11
2.3
155
-------
Table No. 27 con't
At Carbon Inorganic Protein Glucose Glutamic Carbohydrate Ammonia Starch
mg/1 mg/1 Acid mg/1 mg/1 mg/1
mg/1
78 98
130 0
101
270 30
4 3
57 87
132 0
58 14.3
255 34 12.9
7 3 2.7
21 47 0
120
22
235 25
7 20
122
17 11.8
225 20 10.5
6 3 3.0
4 21
112
18
215 16
6 2
5 15
124
15
205 16
6 7
5 19 . " -
118
11
199 18
Footnotes: Temperature was approximately 28°c
Volatile suspended solids of inncoulum was 670 mg/1
Total suspended solids of innoculum was 837 mg/1
156
Sample Time At
No
A2
B2
C2
D2
E2
P2
A3
B3
C3
D3
E3
P3
A4
B4
C4
D4
E4
F4
A5
B5
C5
D5
E5
F5
A6
B6
C6
D6
E6
F6
A7
B7
C7
D7
E7
F7
A8
B8
C8
D8
E8
F8
Min
3:45 140
3:45 140
3:45 140
3:45 140
3:55 150
3:57 152
4:55 210
4:53 208
4:56 211
4:57 212
5:03 218
5:02 217
7:15 350
7:15 350
7:20 355
7:23 358
7:25 360
7:25 360
9:04 459
9:04 459
9:06 461
9:06 461
9:07 462
9:07 462
10:45 560
10:46 561
10:45 560
10:54 569
10:50 565
10:49 564
9:23 1198
9=23 1198
9:26 1201
9:31 1206
9:31 1206
9:32 1207
4:03 1598
4:03 1598
4:03 1598
4:10 1605
4:12 1607
4:12 1607
Carbon
Total
mg/1
108
89
105
111
94
81
94
108
86
103
94
81
81
111
81
100
100
78
75
105
81
97
105
81
75
100
75
86
103
73
75
105
78
86
94
75
75
100
81
86
84
Inorgan:
Carbon
mg/1
20
20
25
25
30
25
20
19
22
22
32
25
19
20
23
23
32
24
19
20
21
21
32
23
19
20
22
21
32
24
21
21
23
23
34
25
20
21
22
21
26
34
-------
TABLE NO. 28
Effect of Substrate Type on Rate of Degradation Using Mixed Liquor
Activated Sludge from Minneapolis-St. Paul Sewage Treatment Plant
Experiment No. 8-11-70, Test #32, Batch Reactor, Well Mixed
Mixed liquor was obtained from the treatment plant at 10:00 A.M.
just prior to start of the test. Aliquots were used as innoculum
for each batch reactor. Peed solutions were added at the beginning
of each test as shown. A second addition of fresh feed solution
was also made after sample number five (after about 7 hours)
Feed Solutions Concentration, mg/1
Glucose 1000
Starch 1000
Glutamic Acid 1000
Peptone 1000
Laurie Acid 100
Anmonium Chloride 1600
Reactor Innoculum Feed Solutions Added, ml
ml NHnCl. Glucose Starch Glutamic Peptone Laurie
Acid Acid
A
B
C
D
E
P
G
200
200
200
200
200
200
200
5
5
0
0
5
5
5
0
0
0
0
20
0
0
40
0
0
0
0
20
0
0
40
0
0
20
0
0
0
0
40
0
0
20
0
0
0
0
160
40
40
Sample Time At Inorganic Total Glucose Protein
No Carbon Carbon mg/1 mg/1
mg/1 mg/1
A-I 12:40 0 27 90 0
B-I 12:40 0 26 88
C-I 12:40 0 27 91
D-I 12:45 5 26 91 73
E-I 12:45 5 26 90
P-I 12:45 5 27 91 20
G-l 12:45 5 26 88 110
A-0 1:25 45 22 137 164
B-0 1:25 45 22 140
C-0
D-0
E-0
P-0
0-0
A-l
B-l
C-l
D-l
1:27 47
1:30 50
1:30 50
1:32 52
1:32 52
2:30 110
2:32 112
2:35 115
2:36 116
16
22
17
19
20
19
16
20
120
144 167
120 62
130 140
116 110
118
122
123 153
Glutamic
Acid
mg/1
0
2
3
7
Oo
82
26
43
20
41
36
Starch Ammonia
A=520 X=680 mg/1
2 0
0 0
64 24
21 7
25
32 10 16
12
12
157
-------
Table No. 28. con'.t
Sample
No '
B-l
F-l
G-l
A-2
B-2
C-2
D-2
E-2
F-2
0-2
A-3
B-3
.03 ,
D-3
E-3
F-3
G-3
A-4
B-4
C-4
D-4
E-4
F-4
G-4
A-5
B-5
05
D-5
E-5
F-5
G-5
Time At
2:38 118
2:40 120
2:42 122
3:35 175
3=37 177
3=39 179
3:40 180
3:42 182
3:42 182
3; 44 184
4:30 230
4:33 233
4:35 235
4:38 238
4:40 240
4:40 240
4:42 242
5:23 383
5:25 385
5:26 386
5:28 388
5:30 390
5:30 390
5:32 392
6:15 435
6:16 436
6*17 437
6:19 439
6:20 440
6:22 442
6:24 444
Inorganic
Carbon
mg/1
15
17
18
19
19
17
22
16
17
. 17
18
18
35
22
16
17
17
17
18
21
22
16
18
17
18
18
20
19
15
17
13
Total
Carbon
mg/1
54
93
104
99
114
134
120
54
72
93
73
109
111
118
55
62
90
64
109
103
114
55
63
86
64
85
72
87
50
59
62
Glucose Protein
mg/1
38
68
0
21
0
0
0
16
0
mg/1
123
153
110
130
95
135
88
115
73
Glutamic
Acid
mg/1
11
20
23
33
6
61
26
6
45
31
7
22
13
28
7
Starch
A ==520
5
18
1
0
0
7
0
6
0
X=680
1
4
0
3
0
3
0
2
0
Ammonia
mg/1
12
19
21.6
11
14
17
14
10
13
20
Additional Feed Solutions added to each reactor
A-7
B-7
07
D-7
B-7
F-7
G-7
6:42 462
6:44 464
6:45 465
6:47 467
6:50 470
6:52 472
6:53 473
15
14
11
20
14
16
14
134
165
131
166
51
93
124
165
56
205
100
106
66
27
22
165
57
132
25
158
-------
Table No.
Sample Time At
No
A-8 8:58 598
B-8 8:59 599
C-8 9:00 600
D-8 9:01 601
E-8 9:02 602
G-8 9:04 604
A-g 9:45 645
B-9 9:45 645
C-9 9:46 646
D-9 9:46 646
E-9 9:47 647
G-9 9:47 647
8-12-70
A-10 10:151395
B-10 10:151395
C-10 10:153395
D-10 10:151395
E-10 10:151395
F-10 10:151395
G-10 10:15395
28 con't
Inorganic
Carbon
mg/1
12
12
18
19
16
13
11
10
19
19
13
13
14
13
14
17
18
12
16
Total Glucose
Carbon mg/1
mg/1
62 27
136
90
120
145
84
51 0
129
75
116
43
77
75
51
47
78
69
134
64
Protein
mg/1
160
81
155
75
105
75
Glutamic Starch Ammonia
Acid A=520 X=680 mg/1
mg/1
42 5
85
71
37 00
19 5
56
59
solids were determined on the innoculum by
'and drying and showed 484 and 512 mg/1 respectively
on repeat tests.
159
-------
TABLE NO- 29
Effect of Milk Solids Concentration on Rate of Biological Degradation
in a Film Plow Reactor
Experiment No. 8-20-69, Plane Test #7, Film Flow Reactor Using Fixed
Slime Layer on Inclined Surface
Film flow reactor was acclimated for 3 weeks on dry milk solids feed
solution. Growth was spotty, yellowish-beige in color and had a
gelatinous surface. Slope of the surface was maintained at 0.0777
inches/24 inches. Samples were collected from the mid section (4.4 in.
wide) for analysis.
Sample Flow Rate Carbohydrate Organic Carbon Protein
ml/min Concentration Concentration Concentration
mg/1 mg/1 mg/1
100 mg/1 feed solution 38 40 81
P-l 30 3 25 24
P-2 21 0 29 40
P-3 8.3 0 24 30
P-4 1.5 0 16 3
100 mg/1 feed solution 47 40 65
PL-1 17 34 27 5
PL-2 8.2 21 24 1
50 mg/1 feed solution 18 24 19
PL-3 43 13 18 5
PL-4 25 11 17 5
PL-5 8,1 4 16 5
PL-6 3.5 0 10 2
200 mg/1 feed solution 89 139
Pl>7 42 60 73
PL-8 26 75 68 132
PL-9 5.6 62 117
PIXLO 2.4 51 io4
500 mg/1 feed solution 162 220 308
PL-11 51 156 195 292
PL-12 18 132 180 243
PL-13 7.3 87 180 266
PL-14 2.4 56 120 139
160
-------
TABLE NO. 30
Effect of Milk Solids Concentration on Rate of Biological Degradation
in a Film Plow Reactor ;
Experiment No. 9-4-69, Plane Test #8, Film Flow Reactor Using Fixed
Slime Layer on Inclined Surface
This is an extension of Experiment #8-20-69, Test #7
Sample No Flow Rate Organic Carbon Carbohydrate Protein
mg/1 Concentration Concentration
mg/1 mg/1
Feed Solution (50mg/l) 28 11 40
P-l T.Oml/lOmin 9 4 11
P-2 4.8ml/min 10 5 " 5
.P-3 15ml/min , 14 12 •• 10
P-4 , 28ml/min 15 14 I8
200 mg/1 feed 78 76 •' 133
P-5 26ml/min 63 41 102
P-6 12ml/min 54 10 65
P-7 3.2ml/min 39 5 • 87
P-8 3.5ml/10min 15 2 35
125 rag/1 feed 50 33 ' 88
p-9 27ml/min 44 1 70
125 mg/1 feed 51 H . 88
p-10 7.6ml/10min 12 3 10
p-11 6.3ml/min 25 10 23
P-12 13ml/min 31 8 32
P-13 23ml/min 40 8 5«
161
-------
TABLE NO . 31
Effect of Laurie Acid Concentration and Mixed Feed Solutions on Rate
of Biological Degradation in a Film Flow Reactor
Experiment No. 3-17-70, Plane Test #9, Film Flow Reactor using Fixed
Slime Layer on Inclined Surface
Film flow reactor was acclimated to a 50 mg/1 lauric acid feed solution
for a period of several days. The slope of the plane was maintained at
0.0782 inches per 24 inches. Samples were collected from the center
portion 4.4 in. width and analyzed for organic carbon. Initial part of
test used only lauric acid feed. Mixed feed solutions were used at the
end of the test as shown.
Sample Feed Rate Organic Carbon Glucose Glutamic Acid
ml/min mg/1 mg/1 mg/1
100 mg/1 lauric acid 69
P-l 60ml/5min 52
P-2 12.1ml/min 67
P-3 25ml/min 64
100 mg/1 lauric acid 67
P-l 22.3ml/min 63
P-2 12ml/min 6l
P-3 12.Jjnl/5min 46
P-4 57ml/min 66
P-5 4l.0ml/min 65
P-6 21.0ml/5min 51
P-7 22.1ml/5min 54
50 mg/1 lauric acid 42
P-l 29ml/min 38
P-2 l8.1M/min 38
P-3 11.9ml/min 35
P-4 llml/min 33 •
P-5 10.1ml/5min 28
P-6 15.3ml/2min 33
P-7 11.0ml/5min 28
P-8 lOml/lOmin 21
25 mg/1 feed 21
P-l 47ml/min 18
P-2 34.2ml/min 17
P-3 44ml/2min 18
P-4 25.0ml/2min 17
P-5 37.0ml/6min 16
P-6 l6.4ml/5min 10
P-7 17.0ml/10min 7
100 mg/1 mixed feed (glucose, 71 130
glutamic acid, Lauric acid)
162
-------
Table No. 31 con't
Sample
P-l
P-2
P-3
P-4
P-5
P-6
P-7
100 mg/1
P-l
P-2
P-3
P-4
P-5
P-6
P-7
P-8
P-9
Feed Rate Organic Carbon Glucose
ml/mln
6lml/min
47.0ml/min
38.0ml/mln
25.5ml/min
17.3ml/mln
20.0ml/2min
30.5ml/4min
mixed feed •
51.0ml/mln
43 . 8ml/mln
33.0ml/min
27 . Oml/mln
20.4ml/mln
I6.8ml/mln
17 . 2ml/2mln
19 . 6ml/4mln
5.8ml/20mln
mg/1
140
138
134
134
138
132
128
144
70
69
71
71
72
70
69
67
48
mg/1
72
72
71
72
76
72
75
75
75
74
75
76
75
73
72
69
45
Glutamlc Acid
mg/1
126
132
127
131
140
114
123
109
114
113
130
126
120
..121
117
97
24
163
-------
TABLE NO i 32
Effect.of Plow Rate on Biological Degradation of Mixed Feeds in a
Film Flow Reactor
Experiment No. 4-9-70, Plane,Test #10, Film Flow Reactor Using a
Fixed Slime Layer on an Inclined Surface
Film flow reactor was acclimated using mixed feed (glucose, glutamic
acid and lauric acid) for a period of 5 days. Flow was interrupted
for one day on 4/5/70. The slope of the plane was maintained at 0.0782
inches per 24 inches. Samples were collected from the center section
(4.j4 in< wide) for analysis.
Glucose
mg/1
71
71
70
71
71
73
73
68
61
Continuation 4^10-70, Plane Test #11, 4/16/70
Feed Solution 124 69
P*a 49ml/min 120 89
P-2 35.9ml/min n4 84
P-3 21.0ml/min 116 82
P-4 24;ML/min 108 79
P-5 36,9ml/2min 102 70
P-6 24.8ml/2min 98 64
P-7 l6.ML/3min 95 72
Sample Feed Rate Organic Carbon Glutamic Acid
Feed
P-l
P-2
P-3
P^4
P^5
P-6
P-7
P^-8
Solution
36ml/min
29.8ml/min
25.4ml/min
33.0ml/2min
l8.0ml/2min
I6ml/2.5min
9.0ml/3min
6-3nLL/7min
mg/1
136
134
132
132
130
128
120
106
96
mg/1
68
89
85
79
83
81
74
75
81
164
-------
TABLE NO. 33
Evaluation of Mixed Feed Solutions at Various Plow Rates in a
Film Reactor
Experiment No. 8-20-70, Plane Test #13, Film Flow Reactor Using A
Fixed Slime Layer on a Inclined Surface
The film flow reactor was acclimated to a mixed feed of glucose, glutamic
acid and lauric acid. Ammonia addition was limited in order to'
avoid interference with amino acid test. The slope of the plane was
maintained at 0.0782 inches per 24 inches, Samples were collected from
the center reaction (4.4 in. wide) for analysis.
Sample Feed Rate Organic Carbon Glucose Glutamic Acid
ml/min mg/1 mg/1 mg/1
Feed Solution 140 138 102
p-1 11.4 124 111 102
p-2 7-3 120 92 95
P-3 3.7 98 62 , 98
p-4 8.8 120 75 90
p-5 22. 130 116 93
p-6 28.6 130 77 96
P_7 40. 128 118 63
Continuation - Plane Test #14
Feed Solution 78 59 50
P-1 100 71 58 45
p-2 78 73 55 f
P-3 60 76 54 87.5
P-4 36 .74 54 47.5
111 H 74 50 55
P-7 20 76 54 56
S3 ll n %
as u 11 i %
165
-------
TABLE NO. 34
Evaluation of Starch, Protein and Laurie Acid Peed Mixtures in a
Film Reactor
Experiment No. 8-29-70, Plane Test #15, Film Flow Reactor Using a
Fixed Slime Layer on an Inclined Surface
The film flow reactor was, acclimated to a mixed feed of 100 mg/1
starch and .protein and 50 mg/1 of lauric acid feeds. The slope of
the plane was maintained at 0.0782 inches/24inches. Samples were
collected from the center reaction (4.4 in. wide) for analysis.
Sample Feed Rate Starch, mg/1 Organic Carbon Peptone
ml/min X =520 X =680 mg/1 mg/1
Feed Solution 104 99 54 118
P-l 51 104 93 55 143
P-2 33 76 72 53 118 •••
P-3- 20.3 76 66 54 118
P-4 13. 60 48 53 108
P-5 8. 58 38 53 115
P-6 5. 33 15 52 118
»U.S. GOVERNMENT PRINTING OFFICE: 1973 546-308/8 1-3
166
-------
SELECTED WA TER
RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
w
Biological Removal of Colloidal Matter From Wastewater
5. i
6.
8. f rformti.g 0rga,. '•• ation
Au-thor(s)
Maier, W. J.
9. Organization
University of Minnesota
Minneapolis, Minnesota 55455
12. Sr i,tsor;> «: Organ; »
i'j. Project No.
7 . CeinractlGtantNc.
17030 DGQ
$ Type fJRepo, t and
Period Coveie ~^~
Environmental Protection Agency report
number, EPA-R2-7 3-147, June 1973.
If
This research program was designed to provide a more basic understanding of the
mechanism and rates of removal of organic materials from waste waters by
biological processes. Pure compound feed materials were used with the exception
of a series of tests on sewage solids obtained from the Minneapolis- St. Paul
Sewage Treatment Plant by cent rifugat ion of primary effluent. Two model reactor
systems were used to measure rates of substrate removal. The film flow reactor,
characterized by a stationary biological slime layer, was used to study removal
kinetics under conditions where mass transfer may be a limiting factor. A well
mixed batch reactor was used to study rates of removal under conditions where
biological processes are rate controlling. Rates of substrate degradation and
carbon removal are reported for each of the pure compounds using acclimated
innoculum. Rate data from batch reactors are expressed in terms of microbial
growth rate coefficients. Rate data from the film flow reactor is reported as
the quantity of substrate removed per unit of slime surface per unit of time.
J7a. .Descriptors
*Biological Treatment, *Biodegradation-Rates, *Growth Rates-Microbial Population,
*Mass Transfer-Membrane Processes-film flow reactor, Growth Rates, Enzymatic
Degradation, Colloid Removal Colloid Degradation, Exoenzyme Activity
17b. Identifiers
Biological Removal, Biological Treatment, Organic Colloid Removal
; 7c C 0 WR H Field & Group
.' ty
"'/. $<••. v/if-y
Hi
21, No. of
Send To:
WATER RESOURCES SCIENTIFIC INFORMATION CENTER
US DEPARTMENT OF THE INTERIOR
WASHINGTON. D. C. 2O24O
\ institution University of Minnesota
VVRS1
(HEX JUMP 1871,
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