UTILIZATION OF FREE AND COMBINED
AMINO ACIDS BY ACTIVATED SLUDGE
Prepared by
DALE A. CARLSON
Associate Professor of Civil Engineering
UNIVERSITY OF WASHINGTON
COLLEGE OF ENGINEERING
Department of Civil Engineering
Funded by a Public Health Service Research Grant
WP-00247
Division of Water Supply and Pollution Control
June 1965
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UTILIZATION OF FREE AND COMBINED AMINO ACIDS
BY ACTIVATED SLUDGE
Prepared by
Dale A. Carlson
Associate Professor of Civil Engineering
University of Washington
College of Engineering
Department of Civil Engineering
Funded by a Public Health Service Research
Grant WP 00247 "*
Division of Water Supply and Pollution Control
June 1965
-------
TABLE OF CONTENTS
Title Page
INTRODUCTION 1
THE UTILIZATION OF FREE AMINO ACIDS 2
Isomeric Effects on Amino Acid Utilization 4
Laboratory Procedures 7
THE UTILIZATION OF PEPTIDES 10
Effect of Temperature on Oxidation with Glycine Substrates 11
Isomeric Effects on Peptides 13
STUDIES ON ALANYL PEPTIDES 13
Comparison of Alanyl Glycine and Glycyl Alanine 15
Temperature Effects on Peptide Utilization 28
APPLICATION OF A DNA TEST FOR DETERMINING CELL GROWTH ON AMINO ACID
SUBSTRATES 32
Deoxyribonucleic Acid and its Determination 33
Deoxyribonucleic Acid in Microorganisms 34
Activated Sludge Growth 36
Extraction and Determination of DNA from Activated Sludge Cultures 36
Preliminary Tests of DNA Procedure 38
Results of DNA Studies with Amino Acid Substrates 43
Field Studies on DNA Concentrations of Activated Sludge 62
Effects of Toxic Compounds on DNA Production 62
Discussion of DNA Test 66
Summary on Use of the DNA Test 72
UTILIZATION OF THE KERATIN HAIR - PROTEIN RESISTANT TO BIOLOGICAL
DEGRADATION 73
Formation of Keratin 75
Morphological Aspects of Hair 77
Structural Features of Hair 78
Dissolution of the Hydrogen Bond 79
Theoretical Considerations 82
Experimental Tests on Dissolution of Hair 84
Analytical Methods 85
ii
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Title Page
Results of Test Procedures 86
Discussion 87
SUMMARY AND CONCLUSIONS 105
RECOMMENDATIONS FOR FUTURE WORK 108
ACKNOWLEDGEMENTS 109
LIST OF REFERENCES 110
APPENDICES
I - GRAPHICAL PRESENTATION OF DATA BY DIGITAL COMPUTER 115
Data Processing and Data Display Systems 115
Program Format and Presentation 116
Results of the Warburg Respirometer Studies 117
Summary 118
II - KWIC LITERATURE RETRIEVAL SYSTEM 119
Input Deck Preparation 120
III - EFFECTS OF CHLORIDES ON ACTIVATED SLUDGE CULTURES 123
Chloride Study Results 123
IV - CHEMICAL DATA ON PEPTIDES AND AMINO ACIDS 128
ill
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LIST OF TABLES
Number Title Page
1 The Effect of Acclimation on Utilization of Glycine
Peptides 11
2A Free Energy and Oxygen Uptake Rates for Amino Acids 15
2B Free Energy and Oxygen Uptake Rates for Peptides 21
3 Nucleic Acid Content of Microorganisms 35
4 Extraction of DNA from Bacteria 39
5 Extraction of DNA from Activated Sludge Cultures Us-
ing 0.5N HC104 for 10 Minutes at 70°C 40
6 DNA Content of Various Activated Sludges 62
7 Partial Composition of Some Keratins 74
8 Thermodynamic Data on Formation of Glycyl glycine 76
9 Chloride Effect on Microorganisms 124
10 Entropies, Heats of Formation and Free Energies of For-
mation @ 298.1°C 128
iv
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LIST OF FIGURES
Number Title Page
1 Effect of Molecular Size on Utilization of D-isomers
of Amino Acids 5
2 Effect of Molecular Size on Utilization Rate of D-
isomers of Amino Acids at 20°C 6
3 Supernatant Concentrations of Tryptophan Isomers
After Batch Feeding 8
4 Supernatant Concentrations of Phenylalanine Isomers
After Batch Feeding 9
5 Effect of Temperature on Oxygen Uptake With Glycine
Substrates 12
6 Effect of Stereoisomerism and Amino Acid Sequence on
Leucyl Peptide Utilization Rates at 20°C 14
7 Oxygen Uptake for Culture Acclimated to Glycyl DL
Alanine Test Temperature 10°C 16
8 Oxygen Uptake for Culture Acclimated to Glycyl DL
Alanine - 20°C 17
9 Oxygen Uptake for Culture Acclimated to Glycyl DL
Alanine - 30°C 18
10 Oxygen Uptake for Culture Acclimated to Glycyl DL
Alanine - 35°C 19
11 Peptide Depletion with Time 20
12 Free Energy vs. Oxygen Uptake Rate 22
13 Change in Volatile Suspended Solids with Alanyl Pep-
tide Substrates 23
14 Ammonia-Nitrogen and Nitrate-Nitrogen vs. Time 24
15 Alkalinity and Carbon Dioxide vs. Time 25
16 Oxygen Uptake Rates vs. Temperature - Substrate:
Glycyl DL Alanine 29
17 Total Oxygen Uptake Rates vs. Temperature - Substrate:
DL Alanyl Glycine 30
18 Total Oxygen Uptake Rates vs. Temperature - Substrate:
DL Alanyl DL Phenylalanine 31
19 DNA Absorption Spectra -<. 41
20 DNA Standard Curve 42
21 Oxygen Uptake with Arginine - HC1 Substrate 44
22 Oxygen Uptake with DL Alanine Substrate 45
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Number Title Page
23 Oxygen Uptake with Phenylalanine Substrate 46
24 Oxygen Uptake with Na-Glutamic Acid and Glucose Sub-
strates 47
25 Oxygen Uptake with Glutamic Acid and Glucose 48
26 Maximum Oxygen Uptake with Amino Acid Substrates 49
27 Growth Study - Arginine HC1 Substrate 50
28 Growth Study - DL Alanine Substrate 51
29 Growth Study - Phenylalanine Substrate 52
30 Growth Study - Na-Glutamic Acid Substrate 53
31 Growth Study - Arginine and Glucose Combined Substrate 54
32 Growth Study - DL Alanine and Glucose Combined Substrate 55
33 Growth Study - Phenylalanine and Glucose Combined Sub-
strate 56
34 Growth Study - Na Glutamic Acid and Glucose Combined Sub-
strate 57
35 DNA Production vs. Amino Acid Substrate Concentrations 59
36 DNA Production vs. Nitrogen Content of Substrate 60
37 Volatile Solids Production vs. DNA Production 61
38 Volatile Solids Production vs. Substrate Concentration 61
39 Oxygen Uptake Toxicity Test with CuSO, DL Alanine Sub-
strate - 618 mg/1 64
40 Oxygen Uptake Toxicity Test with NaCN Arginine HC1 Sub-
strate - 1000 mg/1 65
41 Growth Study - Toxicity Test with CuSO, DL Alanine Sub-
strate - 618 mg/1 67
42 Growth Study - Toxicity Test with NaCN - Arginine HC1
Substrate - 1000 mg/1 68
43 Absorptive Spectra of DNA Standard and Activated Sludge
Extract 71
44 Cross Section of Hair 77
45 Hair Follicle 77a
46 The Disulfide Bond 79
47 Hydrophobic Bond Between Two Side'tlhains Showing Exclu-
sion of Water 80
48 Urea Breaking a Hydrogen Bond 81
49 Relative Effect of Various Solvents on Dissolution of
Hair 89
vi
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Number Title Page
50 Effect of Different Concentrations of Sodium Hydroxide
Upon Dissolution of Hair 90
51 Titration of a Solution of Hair 91
52 Absorption Spectra of Keratin 92
53 Net DNA Content of Activated Sludge Solids 93
54 Protein Depletion 94
55 Variation in Suspended Solids 95
56 Oxygen Uptake of a Culture Fed Undissolved Hair 96
57 Effect of Increasing Concentrations of Substrate on
Oxygen Uptake 100
58 Affect of Supplementation with Glucose on Oxygen Uptake
of a Culture Fed Dissolved Hair 101
59 Effect of Supplementation of Keratins with Nutrient Broth
on Oxygen Uptake 102
60 Acclimation Effect on Oxygen Uptake 103
61 Change in Volatile Suspended Solids with Various NaCl
Concentrations 125
62 Oxygen Uptake with Variation in Chloride Concentration 126
vii
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UTILIZATION OF FREE AND COMBINED AMINO ACIDS
BY ACTIVATED SLUDGE
INTRODUCTION
The removal of nitrogen from waste waters is of primary concern in waste
treatment especially where receiving waters are slow moving and warm. In such
situations where algal populations can build to sufficient concentration to cause
taste and odor problems, the concentration of nitrogen delivered by waste streams
can be the growth triggering or growth limiting element for these nuisance algal
blooms. Inhabitants of urban and resort areas adjacent to lakes have become in-
creasingly aware of the effect of waste streams in such quiescent waters and ex-
pect that control agencies will require treatment removal methods that will alle-
viate the eutrophication problems brought about by nitrogen and phosphorus enter-
ing into lakes from waste water and treatment plant effluent streams. These
problems need attention on all phases of the nitrogen cycle from degradation of
protein material to reincorporation into living matter. Research has been under-
way on the various aspects of utilization or disposal of ammonia, nitrites, and
nitrates as well as on the algal metabolic pathways and on means of limiting
growth.
A major problem with conventional waste treatment plants is that, although
nitrogen is converted from one form to another in passage through the plant, much
of the incoming nitrogen remains in the effluent of the plant in soluble forms
such as nitrates or in escape of volatile suspended solid material.
Desirable means of removal of nitrogen are (a) by incorporation into vola-
tile suspended solids materials and complete elimination of these solids from
the liquid fraction, or (b) by exhaust of the nitrogen into the atmosphere as
nitrogen gas.
The rate of incorporation of nitrogen into volatile solids is of concern to
the engineer who must design treatment plants so that adequate contact time is
available for satisfactory nitrogen removal. This rate of incorporation is de-
pendent upon the type of compound in which the nitrogen is bound at the start of
the process and upon the metabolic pathways utilized in converting the nitrogen
-------
to a form which can be separated from the liquid stream. Municipal sewage streams
have been recognized as containing a large variety of compounds ranging from the
very simple to the complex and varying as much as degradability.
Studies on variation in degradability have been made for many of the sac-
charide compounds and have shown the influence of chemical bonding on the ease
of biological utilization. For example, the monomers and dimers of glucose have
been noted for their rapid uptake into biological systems while such long chain
polymers of glucose as cellulose have been cause for concern because of the slow
rate of utilization in aerobic treatment systems.
The bonding of nitrogen into monomers, dimers, and longer units is important
as well in the bioutilization of waste streams. The research reported herein is
concerned with utilization of nitrogen in the form of amino acids both singly
and bound together in peptides and in proteins. Such proteinaceous materials
comprise the major nitrogen fraction in domestic sewage and, hence, studies on
optimum conditions required for high rate biological utilization of proteins can
be useful in the design of more efficient biological treatment facilities for the
removal of nitrogen from waste waters.
This report is divided into several sections covering the various aspects
of the research effort. The study was concerned first with the utilization of
individual amino acids as separate entities, then as components of di- and tri-
peptides, and finally as components of keratins—a protein type very resistant
to degradation.
Complementary sections of the report describe several phases of the overall
study which were inaugerated as auxiliary investigations for obtaining and pre-
senting necessary information for the project. These auxiliary topics covered
(a) modification of Burton's DNA test for use with the mixed cultures of acti-
vated sludge. (DNA was used as a measure of new cell production) (b) develop-
ment of a graphical digital computer program for evaluating and presenting War-
burg respirometer data and (c) development of a KWIC program for retrieval and
storage of literature pertinent to the project.
THE UTILIZATION OF FREE AMINO ACIDS
The smallest integral molecular units of the simple proteins are the amino
-2-
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acids. Thus, the initial studies on this project were conducted on the utiliza-
tion of these primary units of protein structure. These studies were an expan-
sion of earlier research at the University of Wisconsin (1) and covered several
of the aspects of amino acid utilization for such compounds as glycine, glutamic
acid, tryptophan, arginine, leucine, cystine, methionine, alanine, and phenyla-
lanine. These amino acids are available to microorganisms as sources of energy
(The free energies of the amino acids are listed in Appendix IV) , and as compo-
nents in the synthesis of new cellular material. The pathways of amino acid
disappearance in activated sludge cultures were traced by following amino acid
depletion, changes in supernatant conductivity, nitrate, nitrite, and ammonia
concentration, pH, solids, oxygen uptake, C0_ production and alkalinity.
Thus, the pathway of amino acid oxidation:
and further:
n°2 - *" WC02
*» *• . .
NH3 + 02
+
could be traced by following amino acid depletion (removal of C H 0 N ), oxygen
w x y z
uptake (a measure of oxygen utilization in the several equations), and then trac-
ing NH-, N0~, N0~ and pH changes. Conductivity was used to measure variations
in ion concentration. Carbon dioxide evolution was indicated by following gas
volume changes on the Warburg respirometer without the absorption of C0_ in potas-
sium hydroxide and by following alkalinity and free CO^ changes. As is noted
later in the appendix of this report the problem of calculating and plotting up
the large quantity of Warburg respirometer data was alleviated by using a plotting
program on the IBM 709 digital computer (2).
The purpose of this portion of the study was to show that:
1. The amino acids serve as a source of energy and of constituent material
for synthesis of new cellular material.
2. The enzymes and permease sites used for amino acid oxidation and incor-
poration into the cell mass are Inducible in an activated sludge culture
and hence the utilization of amino acids is accelerated by acclimation
of the culture to an amino acid substrate.
-3-
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3. The enzyme and permease systems for amino acid utilization are stereo-
specific and unnatural isomer utilization can be limited by racemase
activity converting the D to the L form of the amino acids prior to
further utilization.
4. The incorporation of combined amino acids is related to the number of
peptide linkages and the restriction to usage increases with increase
in the number of peptide linkages in the test compound.
5. The utilization of compounds is related to the energy available in the
compound.
The research on the availability of the amino acids as sources of energy
and of constituent material for synthesis has been reported previously in the
literature (2) and is developed in the section on use of the DNA test for measur-
ing growth. The advantages of acclimation for single amino acids also was cover-
ed previously but in this report research on the effects of acclimation has been
expanded for the incorporation of peptides and is developed later.
Of interest is the effect of isomerism on the utilization of amino acids
and peptides. These studies—oxygen uptake, amino acid depletion, nitrate pro-
duction—all have shown that isomerisra is very important in the utilization of
both amino acids and peptides.
Isomeric Effects on Amino Acid Utilization
The D-isomers of the amino acids are the unnatural form and are converted
to the L form by the bacteria before they are utilized in the cell's machinery.
The utilization of the amino acids, as measured by oxygen uptake, is usually
limited by the racemase activity; this limiting effect is progressively more pro-
nounced as the size of the amino acid increases. As shown in the bar graphs in
Figure 1 the racemase does not limit oxygen uptake for alanirie, the smallest iso-
meric amino acid. The metabolic use of both D and L alanine is in agreement with
this. Alanine (3) appears in the D form in the cell walls of both gram positive
and gram negative bacteria. Also D alanine appears to have a special role in
biological metabolism (4). Note that for tryptophan, however, the oxygen uptake
for the D isomer is only 38% of that of the L form. These data have been plotted
in Figure 2 to give a relationship between the number of carbons in the molecule
and the restriction placed on oxygen uptake by the necessary racemase activity.
These data were obtained from cultures acclimated to DL amino- acids for a period
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(OXYGEN UPTAKE RATE-D-ISOMER )
( OXYGEN UPTAKE RATE—L-ISOMER )
FI6.2 EFFECT OF MOLECULAR SIZE ON
UTILIZATION RATE OF D-ISOMERS
OF AMINO ACIDS AT 20*C
-6-
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of several weeks so that acclimation was accomplished for both the D and L iso-
mers.
From Figure 2 an equation can be established for D isomer utilization by
activated sludge. If X = (100) (oxygen uptake rate - D-isomer)
(oxygen uptake rate - L-isomer)
and C = number of carbon atoms in the amino acid molecule.
Then: log X = 2.362 - 0.76 (log C)
or: X - 231
C0.76
Thus the effect of isomerism on oxygen uptake at 20°C can be predicted for an
araino acid by using the number of carbons in the molecule to give the relative
uptake rate for the D isomer. As will be developed later this same equation
applies as well to the utilization of D isomers in peptides.
The depletion studies on amino acid substrates correlate with the oxygen up-
take results obtained with the Warburg respirometers. The area of the spot appear-
ing with ninhydrin reagent on paper chromatograms is proportional to the concen-
tration of amino acid remaining in the supernatant of the activated sludge test
culture;thus these spot areas can be used as a measure of the depletion of amino
acid substrates with time (5).
As shown in typical depletion studies in Figure 3 and A, the D isomer is
depleted at a much slower rate than the L isomer for both tryptophan and phenyla-
lanine. Note that in both cases a considerable fraction of the D isomer remains
even after twenty-four hours aeration time after the batch feeding.
Respirometer studies have been executed for many amino acids at several dif-
ferent temperatures. The graphical display of these data is available at the
University of Washington. The D isomer effect noted above appears to be temperature
dependent as well for many of the amino acids studied. The data obtained indi-
cate that the racemase activity is sufficiently temperature sensitive so that
the ratio of D-isomer uptake to L isomer uptake increases as the temperature is
raised from 10eC to 40°C.
Laboratory Procedures
The test procedures used in this study were taken insofar as practicable
from Standard Methods (3). Paper chromatography, Warburg respirometer tests,
free amino acid depletion, activated sludge culture techniques were as reported
-7-
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O D-TRYPTOPHAN
D L-TRYPTOPHAN
A DL-TRYPTOPHAN
DL-TRYPTOPHAN
AND L-TRYPTOPHAN
UNDETECTABLE AT 24 HOURS
AFTER ADDITION AS SUBSTRATE
HOURS
FI6.3 SUPERNATATANT CONCENTRATIONS
OF TRYPTOPHAN ISOMERS
AFTER BATCH FEEDING
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UNDETECTABLE AT 8
HOURS AFTER ADDITION
AS SUBSTRATE
L-PHENYLALANINE
UNDETECTABLE AT 3
HOURS AFTER ADDITION
AS SUBSTRATE
HOURS
FIG.4 SUPERNATATANT CONCENTRATIONS
OF PHENYLALANINE ISOMERS
AFTER BATCH FEEDING
-------
in previous publications (1,4). Other special tests used are incorporated below
in the body of the report.
THE UTILIZATION OF PEPTIDES
Following the studies on the free amino acids the next logical research area
was the effect of the peptide bond on the utilization of amino acids. General
evaluation of oxygen uptake rates for a large group of peptides was carried out
but special emphasis was given to glycyl, leucyl, and alanyl peptides. These
studies are related below.
The literature contains little information on the metabolism of peptides.
Peptides are formed by the condensation of the carboxyl group of one amino acid
with the amino group of another to form a peptide bond. Simple proteins are long
chains of amino acids linked together in peptide bonds. Protein synthesis occurs
on messenger and transfer RNA molecules associated with ribosome particles (6).
In a review of Jacob and Monod's theory on genetic regulatory mechanisms (7),
Stent (8) presents some of the current theories on the process of protein synthe-
sis. Howe (9) discusses the peptide*s importance in synthesis as an intermediate
between the free amino acids and proteins. The overall dehydration reaction for
peptide and protein formation is
n(RCHNH2COOH) >• RCHOT^CO — (NHCHRCO) (n-2) — NHCHRCOOH -I- (n-1) ^0
where R varies with each combining amino acid.
Evidently, the primary atom of a peptide bond is the oxygen atom. Combina-
tions of electron acceptors with the oxygen weaken the carbon-oxygen bond and
strengthen the carbon-nitrogen bond. The hydrogen bonding between peptide bonds
produces an appreciable degree of stiffening of the bond itself (10).
The catabolic utilization or incorporation of peptides by microorganisms
such as the bacteria may follow several pathways. Like the single amino acids,
the smaller peptides may be incorporated directly into the interior of the bac-
terial cell. Following entry into the interior of the cell the peptide may be
utilized in the synthesis of new protein material, catabolized for energy pro-
duction and, sometimes, stored in pools within the cell (5).
Other peptides may not be able to pass directly into the cell's interior
and, hence, must await biochemical modification before being utilized inside the
-10-
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cell. In such cases, processes such as hydrolysis of the peptide can limit the
rate of utilization of the peptide.
The effects of peptide bonding on oxygen utilization rates were studied for
a variety of compounds. Studies with peptides of glycine provide some interest-
ing information on the controlling oxidation rates for monomers and peptides of
glycine. For activated sludge cultures acclimated to the glycine monomer, the
peptide bond controlled the oxidation rate of the glycine as evidenced in the
table below.
TABLE 1: THE EFFECT OF ACCLIMATION ON UTILIZATION OF GLYCINE PEPTIDES
Test
No.
6-1
7-1
7-3
19-0
19-3
Culture
Acclimated
To
Glycine
Glycine
Glycine
Glycyl-Glycine
Glycyl-Glycine
Test Run
On
Glycyl-Glycine
Gly cy 1-Glycy 1-Glyc ine
Glycyl-Glycyl-Glycyl-Glycine
Glycine
Glycyl-Glycyl-Glycine
02 Uptake
% of Uptake Rate
on Ace. Substrate
64
66
51
100
100
Temp.
°C
20
20
20
20
20
However, the table shows also that after the culture has been acclimated to gly-
cyl peptides, the oxygen utilization rates of glycyl peptides are no longer re-
tarded by the peptide bond.
Effect of Temperature on Oxidation with Glycine Substrates
Figure 5 indicates the effect of temperature on oxygen uptakes with glycine
substrates. The apparent energy of activation, p, lowers slightly as the pep-
tide bonding linkages are increased. Whereas, v is about 8600 cal/mole with gly-
cine, tetra-glycine showed an apparent energy of activation of about 7000 cal/
mole. These values were determined from the modified Arrhenius equation:
log
K,
2.303R
where: R is the universal gas constant (1.987 cal/degree) and the temperatures
T.are in degrees K. This gradual diminution in energy of activation corresponds
with the free energy in the glycine substrates since glycine substrate added had
-11-
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LEGEND
GLYCINE |J= 8600 CAL./MOLE
GLYCYL-GLYCINE /
TRI-GLYCINE
TETRA-GLYCINE fJ = 7000 CAL./MOLE
10
20 30
TEMPERATURE —°C
40
FIG. 5 EFFECT OF TEMPERATURE ON OXYGEN
UPTAKE WITH GLYCINE SUBSTRATES
-12-
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503 cal/liter, glycyl-glycine 442.6 cal/liter, glycyl-glycyl-glycine 397 and
glycyl-glycyl-glycyl-glycine 369.2 cal/liter. Thus the energy of activation can
be correlated approximately with the energy available in the glycine peptide for
this particular series of tests.
Isomeric Effects on Peptides
As noted previously with the single amino acids, the D isomer restricts the
utilization of peptides at 20°C. This depression of oxygen uptake was noted for
all D isomers tested except alanine. The effect is vividly evident in the dis-
play of peptides of leucine and glycine in Figure 6. Note that uptake rates in
this case are independent of molecular arrangement (i.e. whether the peptide is
leucyl glycine or glycyl-leucine) but are significantly different for the D iso-
mer of leucine.
Especially important is the fact that the utilization of the D isomer pep-
tide follows the same equation as that developed for the utilization of D isomers
of the free amino acids.
where: C is, in this case, the number of carbon atoms in the D isomer of the
dipeptide, D-leucine.
Now this equation holds for D leucyl glycine but it holds as well for D
leucyl glycyl glycine. Thus, the ratio of utilization for
D leucyl glycine ^ D leucyl glycyl glycine _ ,
L leucyl glycine L leucyl glycyl glycine
since: C0.76 = 60.76 in both cases.
STUDIES ON ALANYL PEPTIDES
For this portion of the study three dipeptides, glycyl DL-alanine, DL alanyl
glycine, and DL alanyl DL phenylalanine, were chosen and experiments were carried
out with these peptides as nutrient sources for activated sludge cultures.
The purpose of this peptide study was to investigate: (1) the significance
of the location of the peptide bond for these peptides, (2) possible differences
in utilization due to the presence of a peptide bond as compared with previous
findings made with free amino acids, and (3) effects of temperature variations
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FI6.6 EFFECT OF STEREOISOMERISM AND AMINO
ACID SEQUENCE ON LEUCYL PEPTIDE
UTILIZATION RATES AT 20°C
-14-
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on the peptide utilization. Also, the effects of varying the concentrations of
an inorganic salt, sodium chloride,was studied.
In these studies, as before, aliquots of activated sludge were grown on a
batch fill and draw basis with a single peptide (or Bacto Nutrient Broth for con-
trol cultures) as the nitrogen source supplied in the nutrient substrate. The
cultures, after at least one week of acclimation to the substrate, were subjected
to the following studies: (1) measurement of oxygen uptake using the Warburg
respirometer, (2) measurements of suspended solids to indicate cell mass changes
over a given period, (3) concurrent chromatographic studies to show depletion
of the peptide in the liquid supernatant of the activated sludge, (4) measure-
ments of pH, alkalinity, nitrates, and ammonia concentrations to give an indica-
tion of breakdown or synthesis of the peptide.
Comparison of Alanyl Glycine and Glycyl Alanine
The oxygen uptake for glycyl DL alanine was almost double that for DL alanyl
glycine in cultures acclimated to glycyl DL alanine, DL alanyl glycine, or DL
alanyl DL phenylalanine (64). This confirms results obtained earlier where oxy-
gen uptake rates for glycyl DL alanine were double those for DL alanyl glycine
for cultures acclimated to either glycine or alanine. Figures 7 to 10 show the
curves for oxygen uptake plotted against time.
As shown on Figures 8 and 9, when the endogenous oxygen uptake is subtracted
from total uptake the net uptake curve becomes horizontal as the peptide supply
becomes depleted. This peptide depletion is confirmed by chromatographic studies
which are plotted on Figure 11.
Oxygen uptake rates are plotted against free energy on Figure 12 (See Table
2).
TABLE 2A: FREE ENERGY AND OXYGEN UPTAKE RATES FOR AMINO ACIDS
Substrate
glycine
DL alanine
DL phenylalanine
DL glutamic acid
Oxygen Uptake Rate
at 20eC
yl/hr/me Solids
8.5
10
21
6.9
Free Energy*
Calories/Liter
593
502.31
161.74
594.32
*See Appendix IV
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• GLYCYL DL-ALANINE
• DL-ALANYL GLYCINE
A DL-ALANYL DL ALANINE
0 ENDOGENOUS
SUBSTRATE - 500 MG/L
TEST TEMPERATURE- IO°C
0
10 15
HOURS
20
25
FIG. 7 OXYGEN UPTAKE FOR CULTURE ACCLIMATED TO GLYCYL DL-ALANINE
-------
O GLYCYL DL-ALANINE
a DL-ALANYL GLYCINE
A DL-ALANYL ALANINE
SUBSTRATE-500 MG./L.
TEST TEMPERATURE-20°C
TOTAL UPTAKE
NET UPTAKE
HOURS
FIG. 8 OXYGEN UPTAKE FOR CULTURE ACCLIMATED TO GLYCYL DL-ALANINE
25
-------
900
800
LEGEND
700
SUBSTRATE-
CD GLYCYL DL-ALANINE 500 MG./L
D DL-ALANYL GLYCINE 500 MG./L.
A DL-ALANYL DL- ALANINE 500MG./
TEST TEMPERATURE-30°C
600
HOURS
FIG. 9 OXYGEN UPTAKE FOR CULTURE ACCLIMATED TO
GLYCYL DL-ALANINE —30°C
15
-18-
-------
500
400
C/5
tr
UJ
o
a:
o
UJ
UJ
X
o
300
200
100
LEGEND
O GLYCYL DL-ALANINE
D DL-ALANYL GLYCINE
A DL-ALANYL DL ALANINE
O ENDOGENOUS
TEST TEMPERATURE-35°C
SUBSTRATE- 500 MG/L
FIG.IO OXYGEN UPTAKE FOR CULTURE ACCLIMATED TO GLYCYL DL-ALANINE
35°C
-------
400
300
i
NJ
o
I
Ul
o
I-
o.
LEGEND
O 6LYCYL DL-ALANINE—ACCLIMATED
• GLYCYL DL-ALANINE—UNACCLIMATED
D DL-ALANYL GLYCINE-ACCLIMATED
• DL-ALANYL GLYCINE-UNACCLIMATED
TEST TEMPERATURE-20°C
200
100
FIG.II PEPTIDE DEPLETION WITH TIME
-------
TABLE 2B: FREE ENERGY AND OXYGEN UPTAKE RATES FOR PEPTIDES
Substrate
glycyl DL alanine
DL alanyl glycine
DL alanyl DL phenylalanine
Oxygen Uptake Rate
at 20°C
yl/hr/mg solids
13.02-13.1
7.09-9.12
10.79-20.6
Free Energy*
Calories/liter
399.63
399.63
175.96
*See Appendix IV
The oxygen uptake rates for free amino acid substrate cultures, when plotted
against the free energy of the amino acid used, fall along a straight line such
that the approximate oxygen uptake could be determined from the free energy by
the equation:
Y - 26.5 - 0.0325 F
where: F is the free energy in the substrate in calories per liter
and: Y is the oxygen uptake rate in microliters per hour per mg sludge solids.
For sludges not acclimated to the peptide, DL alanyl DL alanine and DL alanyl
glycine, a second line can be approximated. The spread of data points is not suf-
ficient to write an equation for the line of best fit. The line CD as drawn on
Figure 12 has the equation:
Y = 15.4 - 0.018 F
which indicates a curve with about half the slope and initial value of the accli-
mated cultures. When the culture has been acclimated to DL alanyl Dl alanine,
the oxygen uptake rate fits the "acclimated" curve. Acclimation, however, did
not have this effect on the utilization of DL alanyl glycine. Note also that
glycyl DL alanine was utilized as well as the constituent amino acids even prior
to acclimation. This evidence indicates that some reaction, such as hydrolysis
to constituent amino acids, is evidently limiting utilization of the peptide
sequence DL alanyl glycine but is not a limiting condition when the constituent
acids are in the sequence glycyl DL alanine.
It is quite interesting that sequential arrangement could influence oxygen
uptake even with the smallest possible length of peptide chain.
Figures 8 to 11 give further evidence of differences in the utilization of
glycyl DL alanine and DL alanyl glycine. After eight hours an acclimated culture
of glycyl DL alanine had essentially depleted its supply of peptide while a DL
-21-
-------
to
NJ
o
\0
LEGEND I
O DL-ALANINE —ACCLIMATED
• GLYCINE—ACCLIMATED
D DL-GLUTAMIC ACID—ACCLIMATED
• DL-PHENYLALANINE — ACCLIMATED
A DL-ALANYL GLYCINE—ACCLIMATED
A DL-ALANYL GLYCINE—UNACCLIMATED
O DL-ALANYL GLYCINE—UNACCLIMATED
* DL-ALANYL DL~4> ALANINE-ACCLIMATED
O DL-ALANYL DL-<|> ALANINE—UNACCLIMATED
• DL-ALANYL DL-0 ALANINE—UNACCLIMATED
9 GLYCYL DL-ALANINE—ACCLIMATED
9 GLYCYL DL-ALANINE —UNACCLIMATED
GLYCYL DL-ALANINE—UNACCLIMATED
TEST TEMPERATURE-20aC
200
300 400 500 600
FREE ENERGY— CALORIES/LITER
700
800
900
FIG.I2 FREE ENERGY VS. OXYGEN UPTAKE RATE
-------
I
r-o
LEGEND
O GLYCYL DL-ALANINE ACCLIMATED CULTURE
A DL-ALANYL GLYCINE ACCLIMATED CULTURE
D DL-ALANYL DL-PHENYLALANINE ACCLIMATED CULTURE
TEST TEMPERATURE— 20° C
125
GLYCYL
DL-ALANINE
SUBSTRATE
4 8
HOURS
751
J
\,
o
DL-ALANYL
GLYCINE
SUBSTRATE
4 8
HOURS
100
J
"N
o
75
o
CO
- 50
ui
o
X
o
25
DL-ALANYL
DL-PHENYL-
ALANINE
SUBSTRATE
4 8
HOURS
12
FIG.I3 CHANGE IN VOLATILE SUSPENDED SOLIDS
WITH ALANYL PEPTIDE SUBSTRATES
-------
50
LEGEND
GLYCYL DL-ALANINE SUBSTRATE
O NITRATE-N
D AMMONJA-N
DL-ALANYL GLYCINE SUBSTRATE
• NITRATE-N
• AMMONIA-N
TEST TEMPERATURE- 20°C
8
16 24
HOURS
32
FIG.I4 AMMONIA-NITROGEN AND
NITRATE-NITROGEN VS. TIME
-24-
-------
100
e
L E G E N D
GLYCYL DL-ALANINE — 500 MG/L
O ALKALINITY
n C02
DL-ALANYL GLYCINE—500 MG/L
• ALKALINITY
• C02
TEST TEMPERATURE-20°C
16 24
HOURS
32
40
48
FIG. 15 ALKALINITY AND CARBON DIOXIDE VS. TIME
-25-
-------
alanyl glycine culture had not fully utilized its supply in 24 hours as shown in
Figure 8.
From Figure 9 it is apparent that the cell synthesis is twice as great for
glycyl DL alanine as for DL alanyl glycine. At the same time, Figure 10 and 11
respectively show a significant production of ammonia and carbon dioxide for the
glycyl DL alanine culture while the DL alanyl glycine culture shows quantities
which were hardly measurable.
The following equations can be derived by comparing peptide depletion with
ammonia and nitrate production and oxygen uptake rates over a given period of
time.
Glycyl DL Alanine
A. Initial reaction rates (until the 500 mg/1 of peptide is depleted in the
supernatant - 6.9 hours).
Energy
72.4 mg/l/hr
peptide depletion"
in the supernatant
27.8 mg/l/hr of peptide to ammonia (net)
14.2 mg/l/hr of peptide to nitrate
14.9 mg/l/hr of peptide to cell storage
and unidentified products
13.4 to 25.3 mg/l/hr of peptide to C02
and HO (from 0 uptake
measurements)
Synthesis
15.5 mg/l/hr
(21.4% of peptide)
B. Reactions 6.9 to 25 hours after feeding.
Cell storage and unidentified products + NH,
77.4 mg/1
as peptide
cell material
43.6 mg/1
as peptide
-26-
NO,
240 mg/1
as peptide
-------
PL Alanvl Glycine
A. Initial reaction rates (first 10 hours).
Energy
32.5 mg/l/hr
peptide depletion in the
supernatant
1.04 mg/l/hr of peptide to NH (net)
0.32 mg/l/hr of peptide to nitrate
26.14 mg/l/hr of peptide to unidentified
products and cell storage
7.75 to 13.7 mg/l/hr of peptide to CO
and H.O
Synthesis
5 mg/l/hr
(15.4% of peptide)
Reactions 10 to 24 hours after feeding.
175 mg/1 of peptide —>• 66 mg/1 of peptide
+ 2 mg/1 of peptide
to NH3 (net)
+ 2.5 mg/1 of peptide
and cell storage
+ 104.5 mg/1 of peptide to unidentified products
From the preceding equations it is seen that glycyl DL alanine is depleted
from the supernatant in 6.9 hours with ammonia and carbon dioxide as the initial
products. The pH increased from 6.5 to 7.9 during this same period. After the
peptide was depleted in the supernatant, the ammonia and carbon dioxide concen-
trations were reduced and nitrates continued to increase. The increase in ni-
trates was greater than the decrease in ammonia and it is therefore assumed that
the cell storage proposed above, plus material from volatile solids utilization,
were sources of the resulting nitrate production after the peptide was depleted.
The pH decreased to 5.9 by 24 hours.
The DL alanyl glycine was not utilized as completely as was glycyl DL alanine,
Sixty-five percent of the peptide was removed from the supernatant in 10 hours
and 87% after 24 hours. Trace amounts of ammonia and nitrate were measured in
the supernatant of the culture. Although oxygen uptake measurements indicated
possible CO production, the alkalinity measurements showed a reduction in CO .
The pH increased from 6.7 at the time of feeding to 7.7 after 6 hours and de-
-27-
-------
creased to 5.9 after 24 hours. A possible explaination of the results obtained
for the DL alanyl glycine culture might be that the culture is tying up the pep-
tide as unidentified products and cell storage, but it is not able to degrade
the peptide to NH, and CCL readily. Therefore, the ammonia production is low
and alkalinity is being removed from the supernatant by the culture as a carbon
source. Further verification of these hypotheses awaits additional studies in
this area. Cell synthesis was indicated by solid studies results. It is possi-
ble that the culture was producing a product from the peptide which was poison-
ous or inhibitory to further metabolism by the culture and thus resulted in a
reduction in peptide depletion rates and a minimum of deamination.
Temperature Effects on Peptide Utilization
A ten degree centigrade increase in temperature should approximately double
the oxygen uptake rate. Plotting temperature on an arithmetic scale vs. oxygen
uptake rate on a log scale should give a straight line variation in the
range of 10° to 30°C. In some cases there was a reduction in oxygen uptake rates
at 35°C, probably due to the population of the particular culture.
Temperatures in the range of 30° C are approaching the thermal death point of
protozoa and if the protozoan concentration of the culture were high the curve
would tend to break over as shown on Figures 16 to 18. On the other hand, bac-
teria have a thermal death point which is much higher, above 42°C, and if the
protozoan population is low a curve similar to the left hand curves on Figure 16
would be more likely where the straight line relationship holds up to a tempera-
ture of 35°C. K-/K1 values for a 10°C increase in temperature were equal to
2+0.2 for oxygen uptake rates obtained in this investigation. Taking the values
from a typical curve the following value for y was obtained.
For glycyl DL alanine:
^ = 26.2
K2 - 52.4 TX = 10°C
KZ/KI - 2 T2 = 20°c
U - 11,400 calories/mole
This is within the values suggested in the literature (10,000 to 20,000 calories/
mole). However, this value may change from day to day with variations in the
population of the culture within the above range.
-28-
-------
300
15
10
LEGEND
O GLYCYL DL-ALANINE CULTURE
A DL-ALANYL GLYCINE CULTURE
D DL-ALANYL DL-PHENYLALANINE CULTURE
SUBSTRATE— GLYCYL DL-ALANINE-
500 MG./L
TOTAL UPTAKE
20 30
TEMPERATURE—°C
FI6.I6 OXYGEN UPTAKE RATES
VS. TEMPERATURE FOR GLYCYL DL-ALANINE
-29-
-------
L E G E N D
O DL-ALANYL GLYCINE CULTURE
A GLYCYL DL-ALANINE CULTURE
D DL-ALANYL DL-PHENYLALANINE CULTURE
SUBSTRATE—DL-ALANYL GLYCINE-
500 MG. L
o 100
x
w
oc
UJ
O
o:
o
70
50
UJ
tu
30
z
ui
o
>•
X
o
eo
15
10
20 30
TEMPERATURE— *
40
FIG.I7 TOTAL OXYGEN UPTAKE RATES
VS. TEMPERATURE FOR DL-ALANYL GLYCINE
-30-
-------
LEGEND
O GLYCYL DL-ALANINE CULTURE
A DL-ALANYL GLYCINE CULTURE
D DL—ALANYL DL-PHENYLALANINE CULTURE
SUBSTRATE— DL-ALANYL DL-PHENYLALANINE-
500 MG./L.
20 30
TEMPERATURE —
40
FIG. 18 TOTAL OXYGEN UPTAKE RATES
VS. TEMPERATURE
FOR DL-ALANYL DL-PHENYLALANINE
-31-
-------
APPLICATION OF A DNA TEST FOR DETERMINING CELL GROWTH
ON AMINO ACID SUBSTRATES
Commonly used methods for direct measurement of growth in activated sludge
cultures are based on the determination of the dry weight of cell mass or the
assay of one of their elementary constituents such as carbon or nitrogen. Other
methods are based on the determination of the rate of respiration or, in the case
of unicellular pure cultures, viable or plate counts can be used. These conven-
tional methods present difficulty in interpretation as applied to mixed cultures
because:
a) An increase in dry weight or in carbon or nitrogen content does not
necessarily reflect an increase in population size of microorganisms. For in-
stance, the formation of extracellular capsular material or intracellular food
reserves may cause a considerable increase in dry weight without an increase in
the number of living organisms.
b) An increase in respiration rates may correlate closely with the rate of
substrate utilization by a culture, but, like methods based on determination of
the dry weight, does not give information on the increase in population size.
c) The viable count methods, although tedious to perform, is a direct method
of enumeration of viable population size. However, the tremendous dilution ratios
involved render its accuracy doubtful. In addition, the application of this
method to mixed cultures is questionable.
In view of the limitations and disadvantages inherent in conventional methods,
it is of importance in the field of biological waste treatment to develop a method
capable of reflecting the population size and activity of a mixed culture.
A brief survey in the structure and composition of living cells indicates
that all cells share a common general chemical composition, the most fundamental
feature of which is the invariable presence of three types of complex organic
macromolecules: proteins, deoxyribonucleic acid and the ribonucleic acid. De-
oxyribonucleic acids (DNA) are the genetic material of the cell (11); that is,
the hereditary information needed to specify and direct the growth and repro-
duction of the cell is contained in the structure of DNA molecules and is passed
from generation to generation in this form. Aside from its genetic role in liv-
ing systems, bacteria and other microorganisms are characterized by a high DNA
-32-
-------
content in comparison with higher organisms. In addition, studies on the DNA
content of microorganisms show that the DNA in the cells is constant, with the
exception of the period just prior to cell division (12,13). These striking
facts, as Vendrely (14) stressed, are of great interest for the biologist from
the theoretical as well as the practical point of view. The discovery of the
constancy of the amount of DNA per nucleus in all tissue of the same animal or
in cells of the same microorganism, and the fact that the sperm contains half
of the DNA content of somatic cells is confirmation of the theory that DNA is
an important component of the gene. From the practical point of view, the con-
stant amount of DNA per nucleus can serve as a measure of cell multiplication
and as a standard of reference in the expression of biological changes in liv-
ing systems.
The purpose of this research phase was to develop a suitable method of DNA
determination in activated sludge cultures and to evaluate growth and activity
of the culture by virtue of DNA determinations.
Deoxyribonucleic Acid and its Determination
The nucleic acids are polymeric molecules of great size with molecular weights
reaching several million. They are complex polymers, made up of repeating non-
identical units known as nucleotides. Each nucleotide is composed of a ring
shaped nitrogenous base, a sugar and a phosphate group, all linked together. The
individual nucleotides are joined through their sugar and phosphate group in the
following manner (15):
base base base
i i i
- sugar - phosphate - sugar - phosphate - sugar - phosphate
There are two types of nucleic acids, the ribonucleic acids (RNA) and de-
oxyribonucleic acids (DNA), which differ from each other in the nature of the
base and sugar components of their nucleotide sub-units. In RNA, the bases are
adenine, guanine, cytosine and uracil; the sugar in ribose. DNA also contains
adenine, guanine and cytosine but has thymine instead of uracil as the fourth base
constituent. In addition, the DNA nucleotides differ from those of RNA in that,
instead of the sugar ribose, they contain its reduced derivative, deoxyribose.
DNA itself has a unique macromolecular structure. Watson and Crick (16)
proposed that two long chain molecules are coiled around one another to form a
double helix which is held together by very weak hydrogen bonds formed between
-33-
-------
their bases. The configuration of the bases is thought to be such that the base
adenine can bind only with the base thymine in the opposite chain, and cytosine
can bind only with guanine. Thus, a DNA macromolecule is thought to be composed
of two complementary DNA chains (16,17).
The determination of nucleic acids requires, first, isolation of the nucleic
acids from cells and then the actual determination. Nucleic acids are quite easy
to detect cytochemically because all three of their essential components, phos-
phoric acid, pentose, and the purine or pyrimidine bases lend themselves well to
that purpose (18). Quantitative determinations of the nucleic acids are based
usually on (a) their phosphorus content, after separation of RNA and DNA or (b)
the ribose of RNA and the deoxyribose of DNA or (c) the ultraviolet absorption
of the purine and pyrimidine components in the region of 270 to 350 my (14). The
last two methods are more specific than the first; the method using the color
reaction of the sugars of nucleic acids is perhaps the most widely used and the
better tested method. Of the color reactions available for DNA, the most commonly
used method is the reaction with diphenylamine in a mixture of acetic and sulfuric
acids at 100°C (19). However, this method, prior to the modification by Burton
in 1956, involved substantial error which could arise from incomplete separation
of DNA from interfering cellular constituents such as glucose, glycogen, mono-
saccharides, proteins, or amino acids (20).
Convenient and reliable methods for determining the nucleic acid content of
cells were not available until Schmidt and Thannhauser (21) and Schneider (22)
published their procedure of nucleic acids isolation in 1945.
Deoxyribonucleic Acid in Microorganisms
Microorganisms present a special problem in the isolation and determination
of their constituent DNA because microorganisms, in mixed culture, vary greatly
in the ease with which- their cell walls can be ruptured, in their content of cap-
sular polysaccharides which are difficult to separate from DNA, and in the assoc-
iation of DNA to protein which influences the ease of DNA purification (23).
Several procedures have been used for DNA isolation from microorganisms.
Vendrely (24) and Burton (20,25) have successfully applied Schneider's method
(22) to microorganisms. Bacteria are particularly rich in nucleic acids. The
nucleic acids may account for as much as 15% of the dry weight (26). Table 3
below gives nucleic acid concentrations isolated from several cultures by Ven-
-34-
-------
drely (24). The ratio of RNA to DNA is usually in the range of 2:1 to 3:1.
Little information is available on the DNA content of higher protista; but the
concentration is believed to be lower than for the bacteria. Scherbaum,(27)
using the Schmidt - Thannhauser procedure (21) and ultra-violet spectrophoto-
metry, concluded that a normal culture of Tetrahymena pyriforrois contained 6%
RNA and 0.33% DNA on a dry weight basis.
Aside from the high content of nucleic acids in microorganisms, a study on
the dynamics of the nucleic acid content in microorganisms showed that the total
nucleic acid content undergoes considerable variation and is closely connected
with the physiological state of the culture, i.e., with its age, activity and
growth. Investigators (12,13) have noted that young cultures are always charac-
terized by higher nucleic acid contents than old cultures and, in the process of
aging, there is always a regular decrease in the nucleic acid content. However,
for DNA alone, all results have shown that the DNA content in cells of microor-
ganisms is constant, with the exception of the period just prior to cell divi-
sion. Thus, the DNA concentration can be used as a measure of cell concentra-
tion in a volatile solids mass. The DNA content of the nucleus, therefore, is
a useful tool in measuring activated sludge cell concentrations.
TABLE 3: NUCLEIC ACID CONTENT OF MICROORGANISMS (24)
Microorganisms
Stapholococcus albus
Escherichia coli
Various strains
Bacillus aertryaclce
Yeast (baker's)
Total
11.57
13.90
13.12
14.67
12.37
13.98
8.40
10.64
4.26
Percent Dry: Weight
RNA
8.75
9.73
8.72
10.43
8.59
10.09
5.40
7.00
3.95
DNA
2.83
4.17
4.40
4.24
3.78
3.89
3.00
3.64
0.31
-35-
-------
Activated Sludge Growth
The use of the term "growth" without clear definition has been a source of
confusion in studies on activated sludge cultures. Growth in activated sludge
cultures means synthesis of protoplasm and hence cell multiplication of microor-
ganisms due to the utilization of organic substrate. However, because there has
been no suitable and efficient growth parameter to show actual increase in the
population of microorganisms, growth has been conventionally defined as an in-
crease in biological mass and the solids concentration has been used widely as
a growth parameter. Many workers have realized the limitation of this assumption.
Gaudy and Engelbrecht (28) used volatile suspended solids, protein, carbohydrate
content and substrate utilization as parameters of activated sludge growth. They
concluded that, although synthesis is measured usually as sludge production (mass)
and interpreted as an increase in microbial population, such assumptions should
be used with caution. Heukelekian and Manganelli (29) stated that the increase
in the quantity of sludge in the activated sludge process is a result of a com-
bination of the following influences: (a) incorporation in the floe of resist-
ant and unavailable organic materials (b) residual available and oxidizable or-
ganic materials removed from the sewage and which, because of the shortness of
sludge age, have not been fully oxidized but which could be oxidized further if
longer periods of aeration were available, and (c) the increase in the bacterial
protoplasm resulting from the oxidation of the portion of available food mater-
ials removed from the sewage.
McCabe (30) noted that a crude estimate of the active biological solids can
be obtained from the volatile suspended solids content, but gradual buildup of
inert organic materials frequently interferes with this estimate.
Thus, growth parameters in use cannot accurately determine the concentra-
tion of active viable cells in activated sludge nor can they indicate the genesis
of new cells. The studies herein presented on DNA were predicated on the idea
that DNA concentration determinations could provide a measure of the cell con-
centration as well as follow the generation of new cells or the gradual deple-
tion of existing activated sludge populations.
Extraction and Determination of DNA From Activated Sludge Cultures
Schneider and other investigators (23,31,32) have described the isolation
of DNA from selected groups of microorganisms. The procedures involved are not
-36-
-------
complicated, no special equipment is required and successive extractions are
possible from the same sample. Schneider's procedure, with some modification,
was used in this study. Detailed procedures of the DNA determination are pre-
sented below:
I. Materials: (a) Standard Deoxyribonucleic Acid (DNA) Na salt, A grade,
extracted from salmon sperm as prepared by the California
Corporation for Biochemical Research.
(b) Diphenylamine (Fisher certified reagent grade)
(c) Acetic Acid (glacial)
(d) Aqueous acetaldehyde
(e) Perchloric acid, 0.5 and l.ON
(f) Concentrated sulfuric acid (reagent grade)
II. Materials: (a) Safety centrifuge (Fisher 1725 RPM)
(b) 12 ml graduated conical centrifuge tubes
(c) 70°C water bath
(d) Ice bath
(e) 20 ml test tubes
(f) Spectrophotometer (Spectronic 20)
III. Diphenylamine Reagent — prepared by dissolving 1.5 grams of Fisher certi-
fied reagent grade diphenylamine in 100 ml of glacial acetic acid and adding
1.5 ml of concentrated sulfuric acid. This preparation is stored in the dark
•
in a brown bottle. On the day it is to be used, 0.1 ml of aqueous acetalde-
hyde (15 mg/ml) is added for each 20 ml of reagent required. Acetic acid
must be glacial or redistilled since low grade acetic acid may cause the
development of a blue color in the reagent which interferes with the colori-
raetric estimation of DNA. The stored reagent should be transparent.
IV. Preparation of DNA Standard Solution — DNA used was a highly polymerized
sodium salt, A grade, extracted from salmon sperm. The stock solution was
0.1 mg DNA standard/ml of distilled water. From the stock solution, desired
concentrations of working solutions were prepared by mixing a measured volume
of the stock solution, which was adjusted to 1.25 ml with distilled water,
with 1.25 ml of 1 N perchloric acid to give a final 0.5 N perchloric acid
solution. After heating for 10 minutes at 70°C two volumes (5 ml) of dipheny-
lamine reagent were added. The total volume (7.5 ml) of solution was then
incubated at room temperature (25 to 35°C) for twenty hours. The incubation
-37-
-------
temperature is not critical provided the constancy of temperature can be
maintained. The intensity of the developed blue color was measured with a
spectrophotometer at 600 my.
V. DNA Determination Procedure —
(1) Centrifuge a 10 ml aliquot of sample for 10 minutes. Carefully waste
clear supernatant and save centrifuged solids in the bottom.
(2) Add 2.5 ml cold 0.25 N perchloric acid. Place in an ice bath for 20
minutes.
(3) Centrifuge for 10 minutes. Waste supernatant, add 2.5 ml of 0.5 N per-
chloric acid and start extraction. Let stand for 10 minutes in 70°C
water bath with occasional stirring of solids.
(4) Centrifuge for 10 minutes and save the supernatant (first extraction),
re-extract solids with another 2.5 ml of 0.5 N perchloric acid.
(5) Centrifuge for 10 minutes and save the supernatant (second extraction).
(6) A series of extractions can be obtained by the same procedure to find
the total DNA in the sample. In the case of activated sludge cultures,
three successive extractions gave nearly 100% of the extractable DNA.
(7) The supernatants from each extraction are retained for the DNA deter-
mination. To conserve reagents, smaller aliquots of the total extrac-
tion mixture may be used.
(8) Add two volumes of diphenylamine reagent to one volume of the extrac-
tion mixture aliquot. Incubate at room temperature for 17 to 20 hours.
(9) The characteristic reaction of diphenylamine to DNA is the development
of a blue color. The optical density or percentage transmission is
measured against a blank (2.5 ml of 0.5 N perchloric acid plus 5 ml of
the diphenylamine reagent) using a spectrophotometer at a wavelength of
600 millimicrons. The value obtained is compared with the prepared DNA
standard curve, A known concentration of DNA standard is tested as a
reference for each sample set run.
(10) Calculations:
(me of DNA read from standard curve) (x3)
DNA mg/1 = (ml of sample used)
Preliminary Tests of DNA Procedure
Because DNA determinations on heterogeneous biological systems such as acti-
-38-
-------
vated sludge are a relatively new procedure, preliminary tests were carried out
to determine:
(1) The applicability of Schneider's and Burton's methods to activated
sludge cultures.
(2) The required number of extractions to obtain complete extraction of
detectable DNA from activated sludge.
(3) The range of DNA standard concentrations that obey Beer's Law.
(4) The suitable sample sizes and concentrations.
Burton (20) applied Schneider's DNA extraction method to various strains of
Escherichia coli and obtained the results shown in Table 4.
TABLE 4: EXTRACTION OF DNA FROM BACTERIA (19)
Conditions
Experiment I
0.5 N HC10,
@ 70°C ^
1 N HC10
@ 70°CT
.5% Trichlor-
acetic
Experiment II
0.5 N HC10
@ 70°C 4
Extraction
Time
10
20
30
10
20
5
10
10
15
Vol.
ml
10
10
10
10
10
10
10
10
10
% of DNA Extracted
1st
Extract
63
88
88
48
52
68
84
45
71
2nd
Extract
27
8
8
38
33
23
11
46
25
3rd
Extract
6.4
1.7
1.7
10
10
5.5
2.7
7.2
1.4
Total
96.4
97.7
97.7
97.0
95.0
96.5
97.7
97.2
97.4
1st Two
Extracts
90
96
96
86
85
91
95
91
96
Table 5 shows the results obtained with varying solids concentrations of
activated sludge. From the results shown in the table it was concluded that for
a suspended solids concentration of 1000 mg/1, three successive extractions with
0.5 N perchloric acid would obtain nearly 100% of the extractable DNA.
The DNA standard curve was prepared from the standard stock solution. The
working standards were adjusted to 2.5 ml with distilled water and 1 N perchloric
acid to a final concentration of 0.5 N perchloric acid and heated in a water bath
-39-
-------
TABLE 5: EXTRACTION OF DNA FROM ACTIVATED SLUDGE CULTURES USING 0.5 N
HC104 FOR 10 MINUTES AT 70°C
Cultures
I Alanine Substrate
S.S. 200 rag/1
500
750
1000
2000
II Glycine Substrate
S.S. 200
500
1000
2000
III Nutrient Broth
S.S. 200
500
750
1000
2000
% of DNA Extracted
Supernatant
0
0
0
0
0
7.1
0
0
1.9
0
0
0
0
0.3
1st
Extract
96.2
93.5
94.3
68.5
69.7
21.2
13.3
49.0
19.2
64.0
52.0
54.3
20.5
34.7
2nd
Extract
3.8
6.5
5.7
30.2
25.3
71.7
86.7
49.0
67.3
36.0
41.0
42.3
67.3
56.8
3rd
Extract
0
0
0
1.3
5.0
0
0
2.0
11.1
0
7.0
3.4
12.2
7.9
1st Two
Extracts
100
100
100
98.7
95.0
92.9
100
98.0
86.5
100
93.0
96.6
87.8
91.5
for 10 minutes at 70°C. After adding two volumes (5 ml) of diphenylamine reagent,
the total volume of 7.5 ml was then stored at room temperature for 20 hours.
The absorption spectra of the DNA standards in the visible light range (X =
350 to 750 millimicrons) were scanned by a Perkin-Elmer Automatic Recording Spec-
trophotometer. The peak of absorbance, as shown in Figure 19, occurred at a wave
length of 600 millimicrons. The intensities of the blue color, developed in the
reaction of diphenylamine with DNA were then measured on a Spectronic 20 at the
600 millimicron wave length. The results indicated that, in the DNA concentra-
tion range of 0.01 mg/7.5 ml to 0.10 mg/7.5 ml (1.3 to 13.3 mg/1), the relation-
ship of percentage transmission (% T) to DNA concentration obeyed Beer's Law as
shown in Figure 20.
-40-
-------
SAMPLE- DNA
ORIGIN-DNA STD. SOLUTION
SOLVENT- HCI04+ REAGENT
SPECTROPHOTOMETER-PERKIN-ELMER
CONCENTRATION-O.OI 0.125 MG. DNA
CELL PATH- 10 MM.
REFERENCE-BLANK, HCI04+REAGENT
SCAN SPEED-RAPID
SLIT-25
OPERATOR-F.L.
WAVELENGTH-VISIBLE RANGE
0.0 i—
0.6
350
400
450
500 550 600
WAVELENGTH M(J
650
700
750
FIG.I9 DNA ABSORPTIVE SPECTRA
-------
z
o
CO
z
cr
UJ
o
a:
Ul
0.
90
80
70
60
40
30
20
10
SPECTRONIC- 20, X = 600
SAMPLE- 2.5 ML+ 5.0 ML. REAGENT
BLANK- 25 ML- 0.5 N HCI04+5.0 ML REAGENT
0.00
Q02
0.04
0.06 0.08
ON A- MG.
0,10
FIG.20 DNA STANDARD CURVE
0>i 2
-------
To utilize the most accurate portion of the standard curve (% T from 20 to
80%) the sample should contain 0.02 to 0.16 mg of DNA. Therefore, sample sizes
were increased to 10 ml to better fit this range for the standard curve and for
activated sludge cultures of approximately 1000 mg/1 suspended solids concentra-
tions .
Toxicity tests, using copper sulfate and sodium cyanide, were carried out to
evaluate the activity of activated sludge cultures and the inhibitory effects of
these bacteriocides on sludge growth. In toxicity tests, the experimental pro-
cedure followed that used in growth studies, except for the addition of the bac-
teriocidal agent. Solids, amino acid depletion, oxygen uptake and DNA tests were
performed on the activated sludge cultures slugged with bacteriocides. The
changes in these measured parameters were compared with results of cultures with
normal feedings.
Results of the DNA Studies With Amino Acid Substrates
Data obtained in the study appears in work by Li (33). To facilitate dis-
cussion some of the data are shown graphically. Amino acid substrates were de-
veloped on a C:N ratio of 5:1. All amino acids used were the L isomers except
for DL alanine. These studies have shown that DL alanine is used equally as well
as the L isomers alone.
Oxygen uptake studies were used for evaluating the biochemical activity of
activated sludge cultures with given substrates. Oxygen uptake tests were run
on the Warburg respirometer with four amino acids. To correlate results with
growth studies, samples were adjusted to approximately 1000 mg/1 suspended solids.
Figures 21 through 25 show the uptake curves for L - arginine, DL alanine, L -
phenylalanine, and L - glutamic acid.
Because the cultures were acclimated to the desired amino acid and glucose
and then subjected to the amino acid as the sole carbon source, differences in
oxygen uptake rates were anticipated. However, as shown in Figure 25 no differ-
ence was obtained with L - glutamic acid vs. L - glutamic acid and glucose. Fig-
ure 26 summarizes the maximum oxygen uptake rates of the four amino acids.
The results obtained in the growth studies appear graphically in Figures 27
through 34. In Figures 27 to 30, the results are shown for a single amino acid
as sole carbon source. In Figures 31 through 34 the results are shown for the
respective amino acid and glucose as the combined substrate. In both cases, the
-43-
-------
DOSAGE
• 1470 MG/L.
O 1175 MG/L
D 735 MG/L.
A 294 MG/L
• ENDOGENOUS
LEGEND
TEMPERATURE- 20°C
SUSPENDED SOLIDS-
ACCLIMATED SLUDGE 1000 MG/L
500
2 3
HOURS
FIG.2I OXYGEN UPTAKE
WITH ARGININE-HCI SUBSTRATE
-44-
-------
LEGEND
DOSAGE
• 1235 MG/L
O 865 MG/L
D 618 MG/L.
A 247 MG/L.
• ENDOGENOUS
500
400
i
ui
0.
ID
z
LU
X
O
300
200
TEMPERATURE- 20°C
SUSPENDED SOLIDS-
ACCLIMATED SLUDGE 1000 MG/L.
100
HOURS
FIG.22 OXYGEN UPTAKE
WITH DL-ALANINE SUBSTRATE
-45-
-------
LEGEND
DOSAGE
• 765 MG./L
O 535 MG/L
D 383 MG/L
• ENDOGENOUS
TEMPERATURE- 20°C
SUSPENDED SOLIDS-AC CLIMATED
SLUDGE 1000 MG/L
500
HOURS
FIG.33 OXYGEN UPTAKE
WITH PHENYLALANINE SUBSTRATE
-46-
-------
LEGEND
DOSAGE TEMPERATURE- 20°C
D No-GLUTAMIC ACID 987 MG/L SUSPENDED SOLIDS- ACCLIMATED
O GLUCOSE 625 MG/L. SLUDGE 1000 MG/L.
• ENDOGENOUS
500
400
I
LU
Q.
ID
LU
CD
>-
X
O
300
200
IOO
H OU RS
FIG.24 OXYGEN UPTAKE WITH Na-GLUTAMIC ACID
AND GLUCOSE SUBSTRATES
-47-
-------
LEGEND
O No-GLUTAMIC ACID
D Na-GLUTAMIC ACID
AND GLUCOSE
• ENDOGENOUS
SUSPENDED SOLIDS- 1000 MG/L
ACCLIMATED TO Na-GLUTAMIC
ACID AND GLUCOSE
500
400
300
Ul
UJ
x
O
200
100
FIG.25 OXYGEN UPTAKE WITH GLUTAMIC
ACID AND GLUCOSE
-48-
-------
500
FIG.26THE MAXIMUM OXYGEN UPTAKE RATE
WITH AMINO ACID SUBSTRATES
-49-
-------
LEGEND
• SUBSTRATE RESIDUE- M6./L.
A VOLATILE SOLIDS-MQ./L.
• ON A CHANGE-MG./L.
OT
O
O
O
O
O
in
v>
o
o.
10
o
o.
2
o
o-
«o
DOSAGE (BOD 200 MG./L.)
ARGININE-HCI 238 M6./L.
o
O-
o
o
o-
O
O-
10
O
o.
o
o
o-
OJ O-l O
DOSAGE (BOD 700 MG./L.)
ARGININE-HCI 1090 MG./L.
§.
m
:>
o
o
£
o
o.
2
O
o-
10
DOSAGE (BOD SOP MQ./L. )
ARGININE-HCI 735 MG./L.
2 4
HOURS
o
o-
K)
g
^
o-
IO
OJ OJ O
IO
DOSAGE (BOD 1000 MQ./L.)
ARGININE-HCI 1470 MG./L.
2 4
HOURS
FIG.27 GROWTH STUDY—ARGININE-HCI SUBSTRATE
-50-
-------
LEGEND
• SUBSTRATE RESIDUE-MO./L.
A VOLATILE SOLIDS-MG./L.
• DNA CHANGE-MO./L.
o
o.
m
o
o
o
o
o-
in
in
O
O
in
O-1 0-"
DOSAGE (BOD 200 MO./L.)
DL-ALANINE 247 M6./L.
O
o-
en
o
81
O
ta-
in
OJ OJ
DOSAGE (BOD 700 M6./L. )
DL-ALANINE 865 MG./L.
DOSAGE (BOD 500 MG./L]
DL-ALANINE 618 MO./L.
O-
O
O
O
in
O
o-
o
o.
o
o-
m
o
o-
o
o
o-
(O
o
o-
m
o
o
9
o
o-
m
DOSAGE (BOD 1000 MG./L.)
DL-ALANINE 1235 MG./L.
FIG.28 GROWTH STUDY-DL-ALANINE SUBSTRATE
-51-
-------
LEGEND
• SUBSTRATE RESIDUE-MG./L.
A VOLATILE SOLIDS-MG./L.
• DNA CHANGE-MG./L.
K
to
o
o-
o
o
o
in
o
o
o
o.
o
o
o-
1ft
DOSAGE (BOD 200 MQ./L. )
PHENYLALANINE 193 MQ./L.
o
o-
2 4
HOURS
oj o
DOSA6E (BOD 700 MO./L.)
PHENYLALANINE 535 MG./L.
HOURS
CO
o
O-
O
O
O-
in
V)
o
o
IT)
O
O
O
o
o-
in
OJ Q-i
DOSAGE ( BOD 900 MG./L.)
PHENYLALANINE 383 MG./L.
O
o-
10
to
O
o-
O
OJ
o
OJ O-" O
DOSAGE (BOD 1000 MG./L.)
PHENYLALANINE 765MG/L
FIG.29 GROWTH STUDY-PHENYLALANINE SUBSTRATE
-52-
-------
LEGEND
• SUBSTRATE RESIDUE- MG./L.
A VOLATILE SOLIDS-MG./L.
• DNA CHANGE-MG./L.
O
O-
o
in
o
o-
o
o
o-
DOSAGE (BOD 200 MG. A.)
Na-GLUTAMIC ACID SUBSTRATE
282 MG. L.
tr
at
o
O-
O
I
O
o-
o
o
o-
O- 0J O
o.
DOSAGE (BOD 7 00 MG./L)
No-GLUTAMIC ACID SUBSTRATE
987 MG. L.
oc
vi
o
o-
o
o
o-
in
o
o-
o
o
o-
in
o- o-
DOSAGE (BOO 500 MG./L.)
No-GLUTAMIC ACID SUBSTRATE
701 MG. L.
o
o-
o
o
o-
m
§J
DOSAGE (BOD 1000 MG /L. )
No-GLUTAMIC ACID SUBSTRATE
1401 MG. L
HOURS
FIG.30 GROWTH STUDY—Na-GLUTAMIC ACID SUBSTRATE
-53-
-------
IEQEND
• SUBSTRATE RESIDUE-MG./L.
A VOLATILE SOLIDS - MG./L.
• ONA CHANGE-MG./L.
K
(ft
o
o
n
O
O-
O
o
o.
o
o
o-
m
j o
DOSAGE (BOD 200 MQ./L.)
AR6ININE-HCI 92 MG./L.
GLUCOSE 200 MG./L.
O
O-
2
I-
o
o-
o
o.
o
o
o-
in
oj oj o
o-
DOSA6E (BOD 700 M6./L.I
ARGININE-HCI 199 MG./L.
HOURS
O
O.
O
CO
o
o.
m
DOSAGE (BOD SOO MQ. A.)
ARGININE-HCI 143 MG./L.
GLUCOSE 900 MG./L.
O.
O
o.
o
o
o-
tt-
O
O-
O
O-
J O-1 O(
o
O-
o
o
o-
4
HOURS
o-i oj
DOSAGE (BOD 1000 MQ./L.)
ARGININE-HCI 285 MG./L.
GLUCOSE 1000 MG./L.
FIG.3I GROWTH STUDY—ARGININE-HCI AND
GLUCOSE COMBINED SUBSTRATE
-54-
-------
LEGEND
• SUBSTRATE RESIDUE - MG./L.
A VOLATILE SOLIDS-MG./L.
0 DNA CHANGE- MG./L.
at
CO
o
§'
8-
§.
o
O
O
o
o
10
o-l o
DOSAGE (BOD 200 MG./L.)
DL-ALANINE 127 MG./L.
GLUCOSE 122 MG./L.
O
o.
o
o
o
o
81
OJ OJ
DOSAGE (BOD 700 MG./L.)
DL-ALANINE 445 MG./L.
GLUCOSE 928 MG./L.
ol
CO
O
o.
o
z
o
O
8-
§
O"
DOSAGE (BOD 800 MG./L.)
DL-ALANINE 318 MG./L.
GLUCOSE 306 MG./L.
o
o
m
o-i
§•
o
o-
irt
2-
DOSAQE (BOD 1000 MG./L.)
DL-ALANINE 636 MG./L.
GLUCOSE 610 MG./L.
FIG.32 GROWTH STUDY—DL-ALANINE AND
GLUCOSE COMBINED SUBSTRATE
-55-
-------
LEGEND
• SUBSTRATE RESIDUE MO. /L.
A VOLATILE SOLIDS MG./L.
• DNA CHANGE MG./L.
at
v>
o
OJ
o"
o
o
o
o
o.
in
o
o.
OJ OJ O
o.
DOSAGE (BOD 400 MG./L.)
PHENYLALANINE 153 MG./L.
GLUCOSE 250 MG./L.
§
o
o.
n
DOSAGE (BOD
1400 MO./L.)
PHENYLALANINE 535 MG./L
GLUCOSE 875 MG./L.
o
o.
o
o
o
IO"
o
o
o
o
o.
ID
OJ O- Ol
DOSAGE (BOD 1000 MG./L.)
PHENYLALANINE 383 MG./L.
GLUCOSE 625 MG./L.
o
o.
6
OJ
m
O
o-
IO
DOSAGE (BOD 2000 MQ./L.)
PHENYLALANINE 765 MG./L.
GLUCOSE 1000 MG./L.
HOURS
FI6.33 GROWTH STUDY—PHENYLALANINE AND
GLUCOSE COMBINED SUBSTRATE
-56-
-------
LEGEND
• SUBSTRATE RESIDUE - MG./L.
A VOLATILE SOLIDS -MG./L.
• DNA CHANGE - MG./L.
<
O
DOSAGE (BOD 400 MG./L.)
No-GLUTAMIC ACID 282 MG./L.
GLUCOSE 250 MG./L.
o
o
o
o
o
in
O
O^
O :
0-1
in I
in
oc
OT
OJ
o
o-
10
DOSAGE (BOD 1400 MG/L.)
No-GLUTAMIC AC1D 987 MG./L.
GLUCOSE 875 MG./L.
(T
(O
8,
Q
o
o
m
OJ
DOSAGE (BOD 1000 MG./L.)
No-GLUTAMIC ACID 701 MG/L
GLUCOSE 625 MG./L.
K
(A
O
O
O
I
DOSAGE (BOD 2000 MG/L.)
HOURS
FIG. 34 GROWTH STUDY—Na-GLUTAMIC ACID AND
GLUCOSE COMBINED SUBSTRATE
-57-
-------
concentrations of substrate have been adjusted to carbon equivalents of 100, 250,
350 and 500 mg/1. For comparison, the three measured growth parameters, suspend-
ed volatile solids content, substrate residues, and change in DNA content are
plotted on the same figure. As can be noted on the figures, the three measured
parameters correlate with one another quite well. In general, increases in both
volatile solids and DNA content are seen after the feeding of the substrate. In
terms of DNA content, an increase is seen two hours after feeding for the four
cultures fed amino acid substrates. However, the ultimate increase and the pat-
tern of increase seem to be dictated by the concentration and type of substrate
applied.
Increases in DNA content at eight hours after feeding of single amino acid
substrates are shown in Figure 35. Note that there is a given concentration for
each substrate at which maximum increase of DNA is obtained and above which DNA
increase is depressed. For example, L - phenylalanine produces maximum DNA in-
crease at 400 mg/1 whereas L arginine - HC1, DL alanine and Na - L glutamic acid,
as a group show the maximum DNA increase at a substrate concentration nearly 1000
mg/1. It is quite interesting to point out that alanine, arginine, and glutamic
acid are aliphatic acids and phenylalanine. is an aromatic acid. When the optimum
concentration of 400 or 1000 mg/1 is exceeded, decreases in DNA production are
observed. This decrease is considered as due to the inhibitory effect of the
high concentration of substrate.
In Figure 36, DNA increases are plotted against the nitrogen content of the
substrate. This figure indicates that the DNA increase is related to the nitro-
gen content of the substrate; however, it is seen also that the addition of glu-
cose to a single amino acid substrate promotes DNA production.
Growth has frequently been defined as an increase in suspended volatile sol-
ids content. Experimental results in this study as plotted in Figures 37 and 38
show that the increase in volatile solids correlate with an increase in substrate
concentration rather than an increase in DNA content. The experimental evidence
showed that in an unsteady-state situation of organic substrate, increase in vola-
tile solids is mainly the result of the accumulation of organic compounds rather
than an increase in active solids. This fact suggests that in an unsteady-state
situation, volatile solids content is not an efficient growth parameter.
-58-
-------
15
10
z
o
o
ft
a.
z
o
LEGEND :
• ARGININE-HCI
A DL-ALANINE
• Na-GLUTAMIC ACID
• PHENYLALANINE
TIME- 8 HOURS AFTER FEEDING
200
400
600 800
SUBSTRATE CONCENTRATION
1000
MG./L.
1200
1400
1500
FIG. 35 ON A PRODUCTION VS. AMINO ACID SUBSTRATE CONCENTRATIONS
-------
18
16
14
6 12
I
z 10
O
»-
o 8
o
o 6
en
o.
< 4
z
Q
-2
SINGLE AMINO
ACID SUBSTRATE
0
100 200 300
NITROGEN CONTENT— MG/L
400
AMINO ACID
AND GLUCOSE
COMBINED
SUBSTRATE
0 100 200 300
NITROGEN CONTENT— MG./L
FIG.36 DNA PRODUCTION VS. NITROGEN CONTENT OF SUBSTRATE
-------
o
ID
Q
O
oc
a.
CO
Q
_l
O
(O
UJ
o
>
800
d>
O
0
•£
O
0
ro
o
O
4 HOURS AFTER
FEEDING
TILE SOUDS PRODUCTION- H
ro .^ en a
O O O C
o o o c
I
t
•
•
3 HOURS AFT
•EEDING
I
ER
i
0 2468
DNA PRODUCTION- MG/L
0 2468
DMA PRODUCTION-MG/L
FIG. 37
VOLATILE SOLIDS PRODUCTION
VS. DNA PRODUCTION
SOU
o
**SI y. f~\ S~\
VOLATILE SOLIDS PRODUCTION-
— — ro w -t
o o o o c
o o o o o c
•
i
•
• •*
*• X
•X Y
Xj
V"*
0.45X-
•
Z
- 184
•
X
V
•/
•
X
•
'i ArSn ' l?hO 16
bo
SUBSTRATE CONCENTRATION-MG/L.
FIG.38 VOLATILE SOLIDS PRODUCTION
VS. SUBSTRATE CONCENTRATION
-61-
-------
Field Studies on DNA Concentrations of Activated Sludge
The amount of DNA in activated sludge plants is of interest from the practi-
cal point of view because it represents the active portion of the sludge and could
function as an index indicating the activity and potential purification ability
of the sludge.
Activated sludge samples were collected in the Seattle Metropolitan Waste
Treatment Plant, the Safeway Bellevue Milk Waste Treatment Plant and the Holiday
Inn Waste Treatment Plant. The samples, after addition of perchloric acid to a
final concentration of 0.25 N were brought back to the laboratory and immediately
subjected to DNA and solids tests. The results of these field tests are compiled
in Table 6 below.
TABLE 6: DNA CONTENT OF VARIOUS ACTIVATED SLUDGES
Samples
DNA (Average)
mg/1000 mg Volatile Solids
Sludge Ty
Lake City Municipal Waste
Treatment Plant
Bellevue Milk Waste Treat-
ment Plant
Holiday Inn Waste Treat-
ment Plant
Arginine - HC1 Cultures
DL Alanine Cultures
L - Phenylalanine Cultures
Na - L - Glutamic Acid
Cultures
8
17
11
13 - 23
12 - 37
18 - 33
23 - 32
Biosorption
Extended Aeration
Extended Aeration
Laboratory Cultivated
Laboratory Cultivated
Laboratory Cultivated
Laboratory Cultivated
Laboratory sludge cultures grow in a more regulated environment than actual
field cultures. Thus, as would be expected, the average DNA content in labora-
tory cultures is higher than in cultures from treatment plant installations. The
laboratory saturation concentration of DNA appears to be about 37 mg per 1000 mg
of suspended volatile solids.
Effects of Toxic Compounds on DNA Production
Several methods have been used to evaluate the acitivity of activated sludge
-62-
-------
processes. Among them the use of activated sludge oxygen requirements is perhaps
the most widely used method. In general, the purpose of these methods is to de-
termine the rate of metabolism of the culture. Because the metabolism of any or-
ganism is directed primarily toward assuring its growth and reproduction, DNA
tests could be a direct method to evaluate the activity of a culture.
The validity of this concept was tested experimentally by noting the effect
of bacteriocides. Copper as copper sulfate and cyanide as sodium cyanide were
used as the bacteriocidal agents in this study.
An examination of the literature on the toxic effects of bacteriocides on
activated sludge provides rather wide and diverse information on the toxic effects
of various substances on waste treatment. From laboratory experience, Oetning
et al (34) reported that 0.5 mg/1 CuSO, as copper was toxic to all microorganisms
and 0.1 mg/1 had a toxic effect on most bacteria. In addition, they concluded
that waste water must not contain more than 0.1 mg/1 of copper if the water is to
be treated biologically. Working with activated sludge, Ridenour (35) observed
a decrease in nitrate formation and ammonia reduction as a result of 2 to 3 mg/1
of cyanide in the form of NaCN. He stated, however, that at no time was nitri-
fication completely disrupted even when an amount as high as 40 mg/1 was added.
Oeming (34) and Siebert (36) found that cyanides from metal plating wastes were
toxic to biological activity in concentrations as low as 1.0 mg/1. According to
Ridenour et al (35) sludge has a tolerance to 3 mg/1 of Na CN when first intro-
duced, but that a build-up in tolerance was noticed after a few days until a
large daily increment could be absorbed.
The critical limits of toxicity set by the different investigators cited
above clearly show the wide range of diversity in the results shown. These con-
flicting reports, according to Moulton et al (37) can be explained by the fact
that each investigator used different test parameters, experimental conditions,
and experimental units and methods.
In viewing these reported facts, oxygen uptake tests were carried out with
a Warburg Respirometer as screening tests to determine critical limits of two
bacteriocides. In Figures 39 and 40, the oxygen uptake curves for CuSO^ and
Na CN slugs are shown. In the case of CuSO^, the inhibitory effect became clear
at a concentration of 5 mg/1. Between 5 to 10 mg/1, the inhibitory effects re-
mained about the same. Exceeding this limit, an evident lowering in the maximum
oxygen uptake rate is observed. In the case of sodium cyanide, no clear effects
-63-
-------
LEGEND
DOSAGE
• 0 MG/L
A 5 MG/L
D 10 MG/L
O 50 MG/L
• ENDOGENOUS
500
FIG.39 OXYGEN UPTAKE TOXICITY TEST WITH CuSO,
DL-ALANINE SUBSTRATE- 618 MG/L
-64-
-------
500
400
i
Ld
i-
Q-
UJ
o
o
300
200
100
LEGEND
DOSAGE
• 0 MG/L
A I MG/L
D 5 MG/L
O 10 MG/L
• ENDOGENOUS
FIG.40 OXYGEN UPTAKE TOXICITY TEST WITH NaCN
ARGININE-HCI SUBSTRATE- 1000 MG/L
-------
are detectable in the dosage range of 1 to 5 mg/1. The critical limit in this
case is found at 10 mg/1.
With this background information on the toxic effects on activated sludge,
growth studies were initiated. Figure 41 shows the changes of DNA content at
different CuSO^ levels and in Figure 42 the results are shown using NaCN as the
bacteriocide. From these figures, two varied toxic effects on activated sludge
cultures can be shown. The shock dosage of CuSO seems to affect respiratory
activity of the culture, whereas NaCN appears to inhibit the reproduction acti-
vity directly.
Discussion
The purpose of this portion of the study was to develop a valid and effi-
cient test procedure for the determination of growth and activity of activated
sludge.
The composition of activated sludge suggests that it can be divided into
active and inactive portions. The absorptive power of activated sludge floe sur-
face and successive biochemical transformations of absorbed organic materials
through microbial activities are responsible stabilizing processes for the treat-
ment of wastes. The bacteria and protozoa of the floe are the responsible agents
for biological waste treatment and, thus, the overall efficiency or treatment
capabilities of an activated sludge process could be evaluated from the size of
the active microbial population responsible for treatment.
The content of active solids or microorganisms in activated sludge and their
pattern of growth are significant because they serve as an index to the potential
treatment capabilities of the sludge and, as well, as an index to the biodegra-
dability of a given waste.
Traditionally, the fraction of active solids in activated sludge has been
indicated by the volatile suspended solids content and growth by increases in
the volatile suspended solids. Volatile solids are actually a mixture of micro-
organisms and non-living organics. In a steady state condition, where the compo-
sition and concentration of organic waste do not fluctuate greatly, a constant
relationship could exist for a period of time between active and volatile solids.
However, in unsteady state, where composition and concentration of waste vary
greatly, the volatile solids fraction of the sludge will vary greatly so that
volatile solids cannot be assumed to hold any specific relationship to active
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LEGEND
DOSAGE
• 0 MG/L
A 10 MGA
D 50 MG/L
8
FIG.4I GROWTH STUDY—TOXICITY TEST WITH CuS04
DL-ALANINE SUBSTRATE— 618 MG/L
-67-
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_j
\
o
z
o
t-
o
o
o
cr
o.
<
z
o
LEGEND
DOSAGE
• 0 MG/L
A I MG/L
D 5 MG/L
O 10
HOURS
FIG.42 GROWTH STUDY—TOXICITY TEST WITH NaCN
ARGININE-HCI SUBSTRATE-1000 MG/L
-68-
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microorganisms. Many investigators have realized this problem in attempting to
relate volatile solids to microbial populations. For this reason a new parameter
was needed to indicate populations of microorganisms.
In the biological sense, growth is an orderly increase of all the components
of an organism. In all cellular organisms, cell multiplication is a consequence
of growth. The definition of growth thus suggests one of the direct measurements
of growth in a biological system could be based on the assay of one of the fun-
damental cellular materials shared by all cells. Recent studies in biology have
indicated that deoxyribonucleic acid is one of the fundamental and invariable
components of living cells. The characteristics of DNA, i.e., as the invariable
component of cells and the fact that the amount of DNA per nucleus is constant,
bear practical significance because the assay of DNA content in a biological
system directly reflects the population size and can be a growth parameter.
The determination of DNA on microbial cultures requires two procedures, i.e.,
isolation of DNA in the pure state and then the qualitative and quantitative de-
termination of DNA. Because microorganisms vary greatly in the ease of cell wall
lysis and in their content of capsular polysaccharides which interfere with DNA
determination, there was no procedure available for the isolation of DNA from a
diverse group of microorganisms. In this study, Schneider's procedure of DNA
isolation was adapted with a slight modification because the method has been
applied successfully to several typical activated sludge bacteria such as Es-
.cherichia coli and Staphylococcus albus. The simplicity of the procedure and
equipment involved, and the successive steps of extraction possible on the same
sample are advantages of this procedure.
As for the method of DNA determination, the diphenylamine reaction discover-
ed by Dische in 1933 and modified by Burton (19) in 1955 was adopted. Burton
stated his modified method is more sensitive and specific than the original meth-
od, and less susceptible to interferences by other compounds. He concluded that
approximately 700 mg/1 of each of the following substances gave no detectable
color development: sucrose, glucose, inositol, ascorbic acid, bovine plasma,
glutathione, cystine hydrochloride, tryptophan, glycine, histidine hydrochloride,
potassium gluconate, adenine sulfate, uric acid, adenosine-51 phosphate, creatine
hydrate and chloral hydrate. The low susceptivity of this method to diverse groups
of organic compounds presents advantages if it is to apply in activated sludge
cultures where varied groups of organics are present.
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Although voluminous studies are available in the fields of biochemistry and
microbiology pertaining to the DNA determination on pure cultures, no account is
available for the DNA determination on mixed aerobic cultures. The first phase
in this study was therefore devoted to the applicability of Schneider and Burton's
method to activated sludge cultures. As shown in Figure 43, the absorption spec-
tra of the DNA standard solution and the solution extracted from activated sludge
using Schneider's method shows identical absorptive characteristics.
The oxygen uptake tests using four amino acids as sole substrate for accli-
mated activated sludge cultures all resulted in increases in the maximum oxygen
uptake rate. These increases due to the feeding of substrate suggested that the
four substrates used in this study were all biodegradable. The growth resulting
from the biological oxidation of the substrate was then evaluated by the increase
In DNA content.
From the results several items could be cited. They are:
1) DNA increases were seen for all four amino acid substrates.
2) Tho extent and duration of DNA increase, or in other words, growth
phase, depends on the types of amino acids and concentration of the
feeding.
3) Although an increase in DNA content and hence growth was seen for a
sole amino acid substrate, the presence of glucose in addition to the
amino acid accelerated the growth.
Furthermore, from Figure 35, it is seen that there are two optimum dosages
for the varied amino acids to give the maximum DNA increase. On the basis of
chemical structure, the aliphatic amino acids, arginine-HCl, DL-alanine, Na-
glutamic acid, as a group show a peak at a certain dosage; and phenylalanine,
an aromatic acid, shows a different peak at another dosage. Mills and Stack (38)
and other investigators (39,40) found that biological oxidation of pure compounds
was related to the chain structure, molecular size, and the functional group pres-
ent in the molecule. Figure 35 also implied that the aliphatic group is more
amenable to biodegradation than the aromatic group.
The DNA test as an activity index of activated sludge was tested by means
of artificial slugging of bacteriocidal agents,CuSO, and NaCN.
Copper and cyanide are classified as enzyme inhibitors that damage the cell
by inhibiting the action of its enzymes. Copper and other heavy metals combine
with the sulfhydryl (SH) groups of important cell components. Since the SH groups
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0.0
0.6
SAMPLE-DNA, ACTIVATED SLUDGE EXTRACT
ORIGIN-DNA STD. SOLUTION AND ACTIVATED SLUDGE
SOLVENT- HCI04 + REAGENT
SPECTROPHOTOMETER -PERKIN-ELMER
CELL PATH-10 MM.
REFERENCE-BLANK , HCI04+REAGENT
SCAN SPEED-RAPID
SPLIT-25
OPERATOR-F.L.
WAVELENGTH-VISIBLE RANGE
ACTIVATED SLUDGE
EXTRACT
350
40X)
450
500 550 600
WAVELENGTH MJJ
650
700
750
FIG.43 ABSORPTIVE SPECTRA OF DNA STANDARD
AND ACTIVATED SLUDGE EXTRACT
-------
of many enzymes play an important role in catalysis, the effect of heavy metals
is to cause a general dearrangement of metabolism. The toxic effect of copper,
although it is very powerful, can, within limits, be reversed by the addition of
chelating agents such as proteins and amino acids. A more specific group of en-
zyme inhibitors are compounds such as cyanide, which owe their toxic effects to
their ability to combine with and inactivate the terminal cytochrome oxidase of
aerobic organisms (15).
Thus, according to their mechanisms of toxic action, copper was expected to
block the general metabolic activity and cyanide the respiratory activity of acti-
vated sludge cultures. Within the range of tested concentrations, the differences
in their theoretical toxic action were not evident in this study. In the case of
CuSO, , the critical limits of the toxic effect appeared at about 50 mg/1, which
was almost fifty times as much as that quoted in the literature. This high cri-
tical limit was believed to be caused by the chelating effect of amino acids which
were used as substrates in this study. Although the inhibitory effect on respira-
tory activity of activated cultures was not evident at the tested concentration
range of sodium cyanide, the inhibitory effect of DNA production or growth was
evident, as shown in Figure 42.
Summary on Use of the DNA Test
Having applied the Schneider and Burton methods of DNA isolation and deter-
mination to activated sludge cultures and having evaluated the DNA content of the
culture as a growth and activity parameter, the following conclusions were esta-
blished.
4
1) The required steps to isolate all the detectable DNA from an activated
sludge culture depend on the solids content of the sample. In a sample of 10 ml
with a suspended solids content of 1000 mg/1, three serial steps of DNA extrac-
tion by perchloric acid give nearly complete extraction of detectable DNA from
the sample (c.f. Table 5).
2) Because of the highly polymerized state of DNA solutions, the workable
concentration of DNA stock solution was found about 0.1 mg/ml. It is recommend-
ed that working standard solutions be prepared from this stock solution.
3) The absorption spectrum of the blue colored mixture of DNA and disphenyl-
amine reagent showed the maximum absorbance at a wavelength of 600 millimicrons.
The maximum sensitivity in spectrophotometry is, therefore, obtainable at this
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wavelength (Figure A).
4) The DNA standard solution obeys Beer's Law in the concentration range of
0.01 mg/7.5 ml to 0.10 mg/ml (1.3 to 13.3 mg/1). Experience showed that a sample
size of 10 ml with a suspended solids content of 1000 mg/1 gave a DNA content with-
^in this range (Figure 5).
5) With experience in running the DNA test procedures, the accuracy of the
test was found to be +0.002 mg/10 ml sample for a 95% confidence interval.
UTILIZATION OF THE KERATIN HAIR-PROTEIN RESISTANT TO BIOLOGICAL DEGRADATION
All proteins share one common structural feature, their amino acid content.
A special group of protective proteins, the keratins, have reached a degree of
differentiation which confers a number of characteristics not present in other
proteins. Among these special properties are their remarkable insolubility and
resistance to biodegradation. Thus, these keratins present an interesting remov-
al problem in waste streams from slaughter houses, tanneries, glue manufacturing
operations, and meat packing plants where hair, feathers and epidermal layers are
a significant part of the waste constituents. Keratins present a number of prob-
lems in waste treatment facilities ranging from the clogging of distribution de-
vices to flotation in activated sludge tanks and buildings and interference with
digester performance (41). These investigations have shown that with appropriate
treatment these insoluble keratins may be transformed into a soluble material
accessible to bacterial enzyme systems and, thus, to biological degradation.
This portion of the Amino Acid Study was concerned primarily with the degra-
dation of resistant proteins and, specifically, with the bioutilization of hair
especially as concerned with the pretreatment necessary for facilitating biologi-
cal attack and with the factors affecting the biodegradation of keratins such as
hair. Different methods for dissolving hair were investigated and microbiologi-
cal behavior was analyzed when dissolved keratin was the sole carbon and energy
.source. The results obtained indicated that the methods developed provide a good,
feasible means of utilizing keratins biologically.
The term, "keratin" is applicable to proteins which exhibit characteristics
°f insolubility in hot water, organic solvents, dilute bases and acids, and re-
sistance to digestion by pepsin and trypsin. It could also be defined as a pro-
tein stabilized by disulflde linkages. This definition will be developed more
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fully later.
Keratins are found in nature as an evolutionary development of epithelial
tissue for protective body functions including acting as a water barrier. They
assume, in mammals, the form of hair, wool, hoofs, horn and epidermis. In birds
they appear, as well, as feathers, and, in reptiles, as scales.
As shown in Table 7, the chemical composition of keratin varies widely. Al-
so, the molecular structure presents different arrangements in response to pecu-
liar physiological functions. Two types of keratin have been described. This
distinction between a and 6 keratin stems primarily from the diffraction pattern
obtained by x-ray analysis. This technique yields information on the arrangement
of atoms in the crystalline regions.
All mammalian tissues yield an a pattern. Feather keratin and reptile scales,
on the other hand, yield a very distinct 6 pattern. In addition, mammalian kera-
tin of the a type may assume the 3 configuration as a type of stereoisomer. In
some cases, such as in the hair cuticle, an amorphous highly cross-linked keratin
is found (42).
TABLE 7: PARTIAL COMPOSITION OF SOME KERATINS (42)
(grams of component from 100 grams of dry keratin)
Keratin Source
Type
Sulfur
Nitrogen, Total
Glycine
Alanine
Glutamic Acid
Arginine
Cystine
Methionine
Hair
Human
Hard
5.0 - 5.24
15.5 - 16.9
4.1 - 4.2
2.8
13.6 - 14.2
8.9 - 10.8
16.6 - 18.0
0.7 - 1.0
Horn
Cattle
Hard
3.7 - 3.9
14.8 - 16.9
9.6
2.5
13,8
6.8 - 10.7
10.5 - 15.7
0.5 - 2.2
Feather
Chicken
Hard
2.9
15.0 - 16.2
7.2
5.4
9.0 - 9.7
6.5 - 7.5
6.8 - 8.2
0.4 - 0.5
Epidermis
Human
Soft
1.9
14.2 - 15.5
6.0
9.1
5.9 - 11.7
2.3 - 3.8
1.0 - 2.5
"Keratinization may be considered as a specific form of cell differentiation
in which metabolically highly active epithelial cells pass through cytomorphic
and physiologic changes while they reach the terminal stage and become filled
with a resistant and considerably insoluble horny material". (43)
The horny cells resulting from the keratinization process may be of a single
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type or a complex type depending on the character of the germinative cells. Ger-
ntinative cells of some epithelia such as epidermis, nail, claw, hoof, horn, beak
or keratinized tooth, give rise only to a single type of specialized cell and e-
ventually to a single type of structure. Germinative cells from hair, wool, quills
and feathers give rise to different specialized cell types which eventually pro-
duce complex structures of horny cells.
Certain epithelial cells produce exclusively cytoplasmic granules of an amor-
phous material, others produce fibrils or both fibrils and granules. Cells pro-
ducing cytoplasmic granules are usually responsible for the formation of amorphous
keratin. A good example is the cell of the cuticular line of the hair. The cells
producing cytoplasmic fibrils are usually responsible for the formation of fibrous
keratin as found in the cortical line of the hair. A third type cell producing
both cytoplasmic fibrils and granules is characteristic of the epidermis (43).
Formation of Keratin
Biochemically, the production of the terminal keratin, either amorphous or
fibrous, proceeds in the same fashion as in the synthesis of other proteins.
The mitochondrion is the organ of respiration in the cell with the citric
acid cycle enzymes located in the matrix or soluble inner portion and the cyto-
chromes located in the membranes in the form of molecular assemblies (44); there-
fore, the mitochondrion is the site, as well, of the synthesis of the energy car-
rier, adenosine triphosphate (ATP).
It now appears clear that proteins are polypeptides containing peptide bonds
formed by the reaction:
R'CH(NH2)COOH + R"CH(NH2)COOH + Energy > R 'CH(NH2)CONHCH(R")COOH -I- H20
where the equilibrium would be favored in the direction of synthesis by the for-
mation of insoluble products as is the case with keratin.
From thermodynamic data on reactants yielding peptides the free energy neces-
sary for the synthesis of a peptide bond from unmodified amino acids has been
determined to be in the range of 2000 - 4000 cal/mole as shown in Table 8.
From the equilibrium constant it can be shown that, at equilibrium,. 99% of
the material is on the hydrolysis side of the equation. Obviously a continuous
supply of energy is necessary.
The incorporation of labelled amino acids into microsomes proceeds only in
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TABLE 8: THERMODYNAMIC DATA ON FORMATION OF GLYCYLGLYCINE (46)
N+H3CHRCOO + N^CHR'COO" = NH2CHRCONHCHRf COOH + HO
_ Glycylglycine _
N — ,„, , » 9 — U . UUJ.
(GlycineH
A F = -RT In K
where R = 1.98 Cal/mole/deg°K
T = 300°K
In K = 2.3 log K
A F = 4.1 K cal/mole of glycylglycine
the presence of ATP suggesting that an activated amino acid is probably an inter-
mediate in the synthesis process and is assembled in a stepwise addition to a
growing chain (45). The initial step is the formation of amino acyl AMP.
5' AMINOACYL PHOSPHORYL ADENOSINE DERIVATIVE
R-C-C-0-P-O-CH
Mg
ATP + AA + E ——*" E [Amino Acyl - AMP ] + PP
The same enzyme that catalyzes the above reaction catalyzes the transfer of
acid residues to the acceptor or soluble RNA.
E [Amino-Acyl - AMP] + S - RNA —>• Amino-Acyl - RNA + E -I- AMP
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where PP = pyrophosphate
AMP = adenosine monophosphate
E = Aminoacyl - RNA synthetases
The S - RNA transfers the amino-acyl residues to the ribosomes where the final
steps of protein synthesis take place. This final assembly of amino acids takes
place in two steps according to Haurowitz, a) Formation of definite polypeptide
sequences on a template and b) folding of a polypeptide to form a three dimension-
al molecule (46).
Morphological Aspects of Hair
Because hair was the primary keratin used in this study a few morphological
characteristics should be considered. The wool research workers, mainly from
Australia, have provided much of the information available.
In a cross section of hair, three zones can be distinguished: the cortex,
the cuticle, and the medulla. (See Figure 44)
FIGURE 44: CROSS SECTION OF HAIR
CUTICLE: Amorphous granules of
keratin, scale-like cells
CORTEX: Fibrous keratin and
amorphous cement
MEDULLA: trichohyalin, dif-
ferent composition than kera-
tin, sometimes absent
The association of sulfur rich and sulfur poor constituents in different
ratios appear to be an essential factor determining the nature of the final kera-
tin, its stability and its chemical properties (42).
Human hair is fully stabilized at a level about one-third of the total length
above the bulb up to the epidermal surface. This keratinization zone up from the
bulb can be divided into two zones: the lower zone B where synthesis and orienta-
tion is complete but structural stability is poor and the upper zone A which is
an area of structural stability (see Figure 45).
Perhaps the most important chemical feature of the keratinization process
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EPIDERMAL SURFACE
ZONE "A"- PROGRESSIVE
ADDITION OF DISULFIDE
BONDS
ZONE "8"-STABILIZED
THRU HYDROGEN BONDS
KERATINIZED ZONE-
NOT AFFECTED BY UREA.
FULLY STABILIZED BY
DISULFIDE BONDS.
SWELLS BUT DOES NOT
DISSOLVE IN UREA- ZONE "A"
1MH ^KERATINIZATION ZONE
•SWELLS AND DISSOLVES
IN UREA.
-------
is the fact that the keratinizing layers give a positive reaction for thiol (SH)
groups and that this reaction disappears as the tissues harden. The reaction in-
volved is the oxidation of SH groups to produce cystine bridges.
2 - SH + 0 ^ -S-S- + H20
In Figure 45, zone B is stabilized primarily through hydrogen bonding but
zone A is consolidated as well by the progressive introduction of cystine bridges.
Frazer (47) showed that wool keratin consists of a matrix (a component) of
very high sulfur content but with no particular orientation of the fibers, and
regularly oriented fiber embedded on it. The peptide chains of the matrix con-
tain approximately 24% cystine with an average molecular weight of 25,000 to
28,000 (42).
Structural Features of Hair
Keratin fibers of hair are extensible. Ashbury and Street (48) discovered
that stretched hair gave a 6 x-ray pattern, while unstretched hair gave an a pat-
tern. From these observations they inferred that the structure was developed by
successive foldings of the structure. They further assumed that the a form is
half the length of the 3 form (42).
Many attempts were made to find a structure that would account for the char-
acteristic x-ray pattern of the a form. Pauling (49) formulated the a helix based
on geometrical requirements deduced from the known structures of small peptides.
Later it was postulated that the a helices were tilted to form a super helix or
a coiled coil with a radius of 10 A°. Six helices would twist around a seventh
straight a helix (50).
The actual cross section of the elementary filaments of hair keratin is of
the order of 60 A°. What is visible in the light microscope of the order of 2000 -
10,000 A° is a fibril composed of a large number of filaments (42).
Experiments with radioactive labelled materials indicate that the contribu-
tion of sulfur to the hair comes from methionine and not cystine in the diet and
enters directly into the keratinization zone. No radioactivity is noted in the
bulb. This evidence is taken to mean that sulfur enters at the level of the kera-
tinization zone through the walls of the follicle (42).
The primary structure for structural bondings in the keratins are covalent
cross linkages with bond energies greater than 35 kilocalories per mole. The di-
sulfide bond between cysteine residues forms a dihedral angle of 90°; rotation
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is hindered around it. The disulfide bond possesses a bond energy of 60 kilo-
calories per mole (50). (See Figure 46)
FIGURE 46: THE DISULFIDE BOND
R
Secondary structural effects from regular and periodic foldings occur because
of the formation of hydrogen bonds. Hydrogen bonds possess a bond energy of the
order of 6 to 8 kilocalories per mole in vacuo and 1.5 kilocalories per mole in
aqueous solution (51). They are caused primarily by the electrostatic interactions
of two electronegative ions, such as of oxygen, nitrogen or sulfur, separated by
a hydrogen atom. An electronegative atctr. covalently bonded to a hydrogen local-
izes an electron pair between the hydrogen atom and the electronegative atom; this
allows a large portion of the electropositive hydrogen atom to interact with
neighboring electronegative atoms.
- C = 0 H - N^
Another type of bond, the hydrophobic bond, results when apolar side chains
from alanine, leucine, isoleucine, valine, phenylalanine, proline, tyrosine and
methionine come together. When this happens, water is excluded and a hydrophobic
region is created. The formation of hydrophobic bonds is an exergonic process of
about - 0.7 kilocalories per mole due to the combination of a slight endothermic
process with a high negative entropy. Because of its endothermicity, hydrophobic
bonds become more stable with temperature up to about 60°C. The strength of a
hydrophobic bond lies mainly in the reorganization of the water molecules around
it which become more hydrogen bonded (45) (See Figure 47). The importance of
hydrophobic bonds in protein stability has not been completely elucidated.
Dissolution of the Hydrogen Bond
The disulfide bonds react through three different mechanisms which have been
studied: oxidation, reduction and cleavage.
Alexander and Earland (52) obtained soluble derivatives from keratins by oxi-
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dizing the disulfide with peracetic acid. After oxidation, keratins are readily
soluble in dilute alkalis; the addition of ammonium sulfate or acid brings about
FIGURE 47: HYDROPHOBIC BOND BETWEEN TWO ISOLATED SIDE CHAINS SHOWING EXCLUSION
OF WATER (45)
CH
CH
CH3
: x x- CH, '0
CH3 CH3
CH3 CH3
CH
0 = water molecule
a partial precipitation. The precipitated material, a keratose, has a low sulfur
content and a molecular weight of 50,000. The portion remaining, the 7 keratose
has a higher sulfur content and a lower molecular weight of about 3000 (52). In
general, the oxidation of the disulfide bond gives two molecules of sulfonic acid.
RSSR + 6(0) >-2R - SH
One cleavage reaction occurs typically in keratins using sodium sulfite.
RSSR + NA S0_ RSNa + RSSO Na
been used (42). The reductive process produces two moles of thiol from the disul-
fide bond.
RSSR >• 2R - SH
The reduction of the disulfide bond has been carried out at high pH values
using thioglycollic or mercaptoacetic acid. Also sulfides at high pH values have
The sulfite cleaves the disulfide bond to produce one S - H group. This is a re-
versible reaction requiring a high concentration of sulfite and a low concentra-
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tion of RS~. The concentration of R - S can be kept low by keeping the reaction
at a low pH, thereby keeping the SH group unionized. Also the SH group can be re-
moved as a heavy metal mercaptide.
Urea is the most widely used reagent for rupturing the hydrogen bond. The
action of urea involves interference with the internal bonding of the molecule as
shown in Figure 48 below.
FIGURE 48: UREA BREAKING A HYDROGEN BOND
R-C-H-C-0-R'
i i
HN 0 HN - C - NH
|
Affected Bond' H New Bond
HN
I
-C-
The effect of urea is quite dramatic in structures where hydrogen bonding is
the primary stabilization factor.
The zone of fibrilar differentiation and growth and the lower zone of kera-
tinization are completely dissolved by an 8M solution of urea, but the dissolving
action of urea stops when the S - S bonds take over the primary stabilization role.
(Refer to Figure 45) The addition of urea to a protein unfolds the molecule ex-
posing the disulfide groups thereby facilitating any reaction involving them.
Enzymatic action on unmodified keratins is restricted to the cell membranes
and the intracellular cement; the keratin itself is invulnerable to proteolytic
enzymes.
Experiments with pronase indicate that enzymatic activity is definitely stop-
ped at the fully keratinized zones with less than 20% of the fiber solubilized.
The percentage is not any better than the results obtained with other proteases,
in spite of the fact that pronase has exo- and endo- peptidase activity together
with a very broad specificity spectrum (53).
The reagent used for dissolving the keratin should be able to reach and cleave
the disulfide bonds. At the same time, the entrance of water into the hydrogen
bonded material breaks a large number of hydrogen bonds disrupting the original
structure and favoring enzymatic action.
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Theoretical Considerations
The chemical properties of the hair cell membrane care are complimentary to the
keratinized protein. When keratin is dissolved through oxidation-, reduction or
cleavage, the insoluble residue is found to be cell membranes and cement (42).
Appropriate treatment for solubilization must take this relationship into consi-
deration. Bacteria require modification of the keratinaceous material because
this is the fraction resistant to protease activity.
As previously stated, the insolubility characteristics of the keratins de-
pend largely on the covalent disulfide cross linkages. The importance of the
covalent cross linkages in protein behavior can be anticipated by an analysis of
their influence in the denaturation process.
The standard free energy of denaturation, AF, is, primarily, the sum of the
free energy involved in unfolding the helical backbone in the absence of side
chain interactions, AFu, plus the free energy due to the presence of covalent
cross linkages in the crystalline phase, AF . Other interactions, for the sake
X
of simplicity, are not taken into consideration (45).
AF - AF + AF -1-
u x
AF - AH - TAS -2-
XXX
If covalent cross linkages are introduced the entropy, AS , decreases. Assuming
X
no enthalpy contribution to AF then AH » 0 so AF » -TAS where the value of
X X X X
the entropy change, AS , is given by Flory (45) as
AS - ^ (ln n. + 3)
where 4> • number of polypeptides in a cross sectional area
n'» number of statistical elements between cross links
Even when the maximum number of hydrogen bonds are formed in a polypeptide,
four CO and four a amino groups cannot form hydrogen bonds. These end effects
WH1 alter the value of AF by the quantity C
Then AF - nAF ., + C -4-
u residue
where n - number of peptide units
AF . , - free energy change per residue
1T6S1QU6
- AH° . . -TAS° ,, -5-
residue residue
C - -4AH° . , + TAS° . . -6-
residue residue
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Now combining (4), (5) and (6)
AF =• (n-4)AH° . . - T (n-l)AS° ., -7-
u residue residue
or AF_ = (n-4)AH° ., - T (n-l)AS° . , -TAS° -8-
D residue residue x
The transition temperature, T , is the temperature at which the peptide helix
melts or AF = 0.
"" -9-
'"^'residue +AS°x
Since AS0 is negative (entropy decreases with increased S-S bonds) , an in-
X
crease in the covalent cross links leads to an increased temperature of transi-
tion. That is, cross linkages, such as S-S bridges, stabilize the protein in its
native state.
The resultant product from the cleavage of the disulfide bonds is readily
hydrolyzed by proteolytic enzymes (54). The pathway for keratin utilization in
nature usually involves the cleavage of S - S bonds.
An outstanding example is represented by insects such as the cloth moth which
possess, in their digestive tract, some type of sulfhydryl compounds which effect
the reduction of the disulfide bond thereby allowing the utilization of wool as
food. It is quite well established that microbial solubilization of wool is ef-
fected through a joint action of disulfide reducing agents and proteolytic agents
(55). Streptomyces fradie solubilize 80 to 90% of wool and feather keratin after
4 days of incubation (56).
The successful utilization of a substrate by a microorganism depends on its
ability to convey the substrate through energy and synthesis pathways. Microbial
systems will provide their energy through the conveyance of the molecules to the
TCA or Krebs cycle for the production of ATP which will store the energy neces-
sary for protein synthesis and other endothermic reactions in the cell. Schemati-
cally, modified keratins can be hydrolyzed by proteolytic. enzymes to their compo-
nent amino acids. Pyruvate, acetyl Co-A, a -ketoglutaric and oxaloacetic acid
Provide the prime matter for the functioning of the TCA cycle. The main link be-
tween carbohydrate and amino acid metabolism is provided by the reduction amina-
tion of a -keloglutaric acid into glutamic acid where ammonia is the nitrogen
aource and NADPH is the reducing agent.
Once ammonia is converted to the amino N, the transamination process can
transfer it to other carbon skeletons. Obviously this reaction represents a most
important pathway for the formation and deactivation of amino acids.
For average values, protein can be considered to be 50% carbon and 16% nitro-
-83-
-------
gen (55). For an activated sludge culture, nutritionally balanced feed with a
C:N ratio of 5:1 is appropriate (1). Besides the conversion of amino acids in
the TCA cycle, intermediates provide N as NH_ which is thought to be 100% avail-
able for bacterial utilization (57). Since the proper amount and ratios of re-
quired elements to sustain microbial life are contained in keratin, activated
sludge cultures should be able to utilize keratinaceous material as a carbon and
energy source.
The basic problems considered in this research effort were the elucidation
of the behavior of organisms utilizing keratinaceous material and the extent of
biodegradation that could take place. Improvement of the rate and extent of mi-
crobial action were considered as areas of interest in practical applications and
in the overall evaluation of the problem of protein utilization. The initial
phase of this research was concerned with the problem of insolubility of the kera-
tins and with the search, then, for an appropriate keratin solvent.
Experimental Tests on Dissolution of Hair
The study of keratins in this research was concerned mainly with the kera-
tin of human hair. Preparation of the cut hair prior to use in the experiment
consisted of triple washing with distilled water in an erlenmeyer flask followed
by three vigorous rinses with 50% solution of benzene in 95% ethanol. The hair
was then air dried under a hood and stored in a closed container.
A solution of hair was prepared by placing 416 mg of cleaned hair in 20 ml
of 0.5 N NaOH and leaving in a covered beaker overnight. The mixture was then
placed in a 50°C water bath until complete dissolution was evident. After dis-
solution, the mixture was neutralized to pH7 with 1 N H-SO, and then was ready
to use as a feed for activated sludge cultures. Supplementation of the keratin
with glucose and nutrient broth was used when desired.
The above methods for dissolving hair were determined after a series of tests
indicated that these procedures were the best for the results desired. The sev-
eral determining tests are listed below.
The following series of tests were performed in 50 ml pyrex test tubes us-
ing reagents in the proportion of 20ml reagent to 0.5 gm of hair.
1. Storage for 10 days at room temperature
A. Acids
50% (18N) H2S04
-84-
-------
25% (9N) H2S04
ION HC1
Concentrated (12N) HC1
18 N H3?04
B. Bases
0.2 N, 0.5 N, 1 N, 2 N, 3 N NaOH
0.5 N, IN KOH
0.5 N Ca(OH)2
0.5 N Ba(OH)2
C. Combined Treatment
Reducing agents:
1 M Thioglycollic acid in 0.1 N NaOH
Na2S03(l gm/gm of hair) in 0.2 N NaOH
Hydrogen bond breakers:
0.5 N NaOH in a saturated solution of urea
Na2SO.(l gm/gra of hair) in 0.25 N NaOH in a saturated solution
of urea
2. At 35°C for 50 hours:
50% H2S04
0.5 N NaOH
1 M Thioglycollic acid in 0.1 N NaOH
Na2S03 (1 gm/gm of hair) in 0.2 N NaOH
Estimation of dissolution was made by pouring the tube contents into a 4 inch
Petri dish set over a white surface and comparing with 2 blanks; one containing
°nly distilled water, the other containing 0.5 gm of hair.
Analytical Methods
Protein determination followed the method given by Lowry (58). This method
consists of the reduction of Folin's phenol reagent (phosphomolybdic acid) by
alkaline copper treated protein. The intensity of the blue color developed is
Measured at 750 my and the protein concentration calculated from a previously
established standard curve. Experiments were done in triplicate with sample size
adjusted to approximately 80 yg of protein.
-85-
-------
Oxygen uptake, suspended solids, and DNA determinations were as previously
decribed, COD determinations followed Standard Methods (59).
Paper chromatography was used for determination of hydrolytic produsts us-
ing descending chromatography on 8" x 17" Whatman No. 1 paper and a solvent system
of butanol : acetic acid " water in a 4:1:1 ratio. A platinum loop was used to
apply the spot. Spot locations were obtained colorimetrically by spraying a solu-
tion of 0.2% ninhydrin in water saturated n-butanol and dried in a 100°C oven for
three minutes (60).
Results of Test Procedures
1. Of all the agents used, sodium hydroxide was the best solvent for human hair.
Optimal concentrations were between 0.5 N and 1 N.
2. As a general rule, the application of heat favors the dissolution process.
3. The ratio of 0.5 grams of hair to 20 ml of reagents was satisfactory.
4. The dissolution reaction consumes 5.4 milliequivalents of sodium hydroxide
per gram of hair.
5. Neutralization of the mixture can be accomplished without precipitation of
the proteins. A light smell of sulfur develops during neutralization. Below
pH5 proteins begin to precipitate, but a fraction remains in solution.
6. Treatment with 0.5 N NaOH partially hydrolyzes hair. Hydrolysis seems to be
produced in the less keratinized portion of the hair. When purified keratin
was used, the same treatment did not liberate hydrolysis products detectable
by paper chromatographlc techniques used.
7« Biological utilization of hair occurs at a slow rate as shown by Warburg
respirometer studies. The maximum initial oxygen uptake rate was 31 micro-
liters per hour followed by a rate of 11 microliters per hour. The material
oxidized in 44 hours represented 44.8% of the calculated COD. The oxidation
curve appears to follow a zero order rather than a first order curve.
8. The best efficiency obtained for removal of keratin derived protein from the
culture supernatant was 76%. The mechanism of removal appears to be one of
adsorption and metabolism of adsorbed products. Addition of supplements such
as glucose and nutrient broth did not improve the rate or efficiency of the
utilization process.
9« A rate increase in oxygen uptake of 45% occurred with acclimating of the cul-
ture to keratin.
-86-
-------
10. Net DNA production in the activated- sludge solids was negligible through the
first 30 hours of the experiment and thereafter increased slightly indicat-
ing some increase in number of cells.
11, Protein content of the supernatant generally followed a downward trend, ex-
cept at six hours when an increase was detected. This corresponded to a de-
crease in suspended solids at six hours.
12. Boiling the feed mixture for six hours under reflux prior to feeding did not
significantly improve the rate of oxidation and no appreciable change in the
U. V. spectra was obtained.
13. No net oxygen uptake was obtained from a culture fed undissolved hair.
14. The net solids yield at the end of 44 hours was approximately 57.6% of the
dry weight of substrate fed.
15. Powdered purified keratin was brought into solution at a concentration of 1.25
gm/L in a KH.PO, + NaOH buffer at pH 8.5 and was available as a substrate to
acclimated activated sludge cultures.
16. Activated sludge cultures acclimated to keratin retained floe formation and
settling properties and microscopic examination did not reveal appearance
changes in the culture. However, excessive foaming required addition to Dow
Corning Antifoam B and mineral oil to keep foam from overflowing the culture
containers.
Discussion
Several interesting points should be presented on the dissolution of hair.
For example, the outer membrane of hair seems to have selective permeability to
4" "f* I I ^ I
Na ions as against K , Ca and Ba ions. The selective permeability of bio-
logical membranes is a well established fact. Hendricksshowed that the outer mem-
brane of frog skin was impermeable to K and permeable to Na ions (61). Also it
is well established that the inner structure of hair is unavailable to molecules
larger than propanol (62). The experimental results suggest that the size and
nature of the anionic component of the base plays a significant role in reaching
the internal structure of the keratin.
The action of NaOH on hair may be a cleavage reaction of the disulfide bond
and formation of a sodium salt upon ionization of the free carboxyl and amide
groups in the keratin molecule.
-87-
-------
The reaction of the disulfide.bond with metal ions seems to proceed with a
direct attack of the metal on the disulfide (45).
2 RSSR + 2 M+ —--="• 2 RS - SR+
I
M
2 RSM + 2 RSOH
2 RSSR+
i
M
2 RSOH — *
RSH + M+ —
+ 2 OH —
i RS02H + RSH
-=*• RSM + H+
As the concentration of NaOH is increased, a point is reached where the con-
centration of Na is high enough to cause a salting out effect because of the com-
petition between the proteins and the salt ions for the molecules of water.
The absence of hydrolysis products from the solubilized pure keratin in con-
trast with the hydrolysis products of natural hair with dissolution by 0.5 N NaOH
indicate that possibly a less stabilized fraction of the hair was being hydrolyzed.
Figure 49 indicates the effect of several chemicals on dissolution of hair.
Long digestion periods with 50% H?SO, can accomplish dissolution, but at that con-
centration, a considerable amount of base is necessary for neutralization. Another
inconvenience of treatment with H-SO, is that, when the reaction is accelerated
with heat, a powdery humin precipitate appears.
Thio glycollic acid in alkaline solution at 35°C is a satisfactory treatment
for solubilizing hair but presents the problems of strong mercaptide odor produc-
tion plus the release of hydrogen sulfide at low pH.
Sulfitolysis at 35°C in 0.2 N NaOH is satisfactory. Since a smaller amount
of base is required this treatment might be considered if the incoming waste can
be or is heated.
Figure 50 shows that the best concentration of sodium hydroxide for dissolu-
tion lies between 0.5 N and 1 N. The existence of an optimum between these con-
centrations was not pursued.
In the biological degradation of complex molecules like the keratins, the
extreme specificity of the proteases must be considered. This specificity may
vary from a single absolute type in which there exists one and only one substrate
for the enzyme to a relative type in which a specific linkage is attacked by the
enzyme.
Several factors will affect substrate specificity such as the structure,
-88-
-------
TEMPERATURE-35°C AND ROOM TEMPERATURE
DIGESTION TIME-10 DAYS
PROPORTION-0.5 GM OF HAIR PER 20ML OF REAGENT
I
DO
•O
I
100
75
o
Ul
o
CO
CO
o
8? 50
a
Ul
w^f
2
^K
CO
Ul
25
O
.
-
_
o
CM
r^^^m
•MM
c?
CO
X*
b
IO
dT
CO —
N O
Q
^* z
£ o
M —
n n
f
O *>
^ X
z z
CM SB
^™"
nn
•MMM
x
O
o
z
X
o
o
z
IO
•_••«
X
O
0
z
•-«••
X
o
e
Z
10
o
MN^BB
X
O
e
Z
CM
O
X
o
o
m
IO
o
•M-W
X
0
o
o
z
IA
.
o
n
IB^B^H
m
0
CO
CM
0
ta^^H
Ul
!«
_i
0
5
o
o
X
•*••••
^
111
K
*
X
o
o
z
z
o
r
••••
Ul
3
+
m
0
CO
N
o
Z
•J
^
•^•M
o
CO
ev
o
* N
•••^H
(
Ul
h-
_l
•
•J
O
o
5
o
o
z
FIG.49 RELATIVE EFFECT OF VARIOUS SOLVENTS
ON DISSOLUTION OF HAIR
-------
LEGEND
O
I
4
K
Z
Z
ui 3
a.
(O
z
5l 2
•
z
UJ
i .
a.
u.
0
o
*
0
-
-
(
A 0.5 N NaOH TEMPERATURE- 2O°C
B I.ON NoOH TIME OF DIGESTION- 5 HOURS
C 2.0N NaOH WEIGHT OF HAIR- O.I GM
D 3.0N NaOH VOLUME- 10 ML
E 4.0N NaOH
F I.ON K OH
1 1 1 1
B C D
CONCENTRATIONS
FIG.50 EFFECT OF DIFFERENT CONCENTRATIONS OF SODIUM
HYDROXIDE UPON DISSOLUTION OF HAIR
-------
0.215 GM. OF HAIR DISSOLVED IN 75 ML. OF
1.0 N SODIUM HYDROXIDE.
TEMPERATURE 20°C
70 71 72 73 74
AVERAGE ML. OF 1.0N H2S04 ADDED
75
EQUIVALENCE POINT- 73.82 MEO OF SULFURIC
ACID ADDED.
1.18 MEQ OF NoOH CONSUMED IN THE REACTION.
FI6.5I TITRATION OF A SOLUTION OF HAIR
-91-
-------
AFTER
ULTRAVIOLET SPECTRA OF DISSOLVED KERATIN.
ULTRAVIOLET SPECTRA OF DISSOLVED KERATIN
BOILING THE MIXTURE FOR 6 HOURS.
DIFFERENTIAL SPECTRA OF ® AND (J) WITH 0 AS REFERENCE.
VISIBLE SPECTRA OF DISSOLVED KERATIN.
60
O
CO
m
70
50
40
30
O
O
to
CD
20
NOTE- ABSORPTION BETWEEN 250 AND 30O MU
— DUE TO AROMATIC RESIDUES IN THE
PROTEIN.
10
400 500 600 700 250
WAVELENGTH- MU
30O
350
FIG.52 ABSORPTION SPECTRA OF KERATIN
-------
LEGEN0
O CULTURE FED DISSOLVED HAIR
A CULTURE SUPPLEMENTED WITH
NUTRIENT BROTH
SUSPENDED SOLIDS- 2000 MG/L
ROOM TEMPERATURE.
FEED- 416 MG/L OF HAIR
TIME AMOURS
FIG. 53 NET DNA CONTENT OF ACTIVATED
SLUDGE SOLIDS
-------
LEGEND
AO SUSPENDED SOLIDS AT 2000 MG/L
D SUSPENDED SOLIDS AT 1000 MG/L
ROOM TEMPERATURE
600
Ul
g
NOTES -
p
^^
PROTEIN DEPLE
SUPERNATANT F
VARIOUS CONCEI^
SHARP DEPLETI
PROBABLY DUE
==^
TION MEASURED
ROM ACTIVATE!!
ITRATIONS OF H
ON IN THE FIRS
TO ADSORPTION
PRO
0 Q
I IN THE CENT
) SLUDGE BATC
AIR.
»T FEW MINUTE
TEIN/SUSPENDE
-
RIFUGED
H FED
S
D SOLIDS- 0.2
10
20 30
TIME—HOURS
40
50
FIG.54 PROTEIN DEPLETION
-------
600
i
vO
LEGEND
D HAIR FED AT 416 MG/L
O HAIR (416 MG/L) PLUS NUTRIENT
BROTH (52.5 MG/L AS BOD).
INITIAL SUSPENDED SOLIDS- 2000 MG/L
ROOM TEMPERATURE
20 3O
TIME—HOURS
20O
FIG. 55 VARIATION IN SUSPENDED SOLIDS
(CHANGE IN ACTIVATED SLUDGE SOLIDS AFTER BATCH FEEDING
OF HAIR AND HAIR WITH NUTRIENT BROTH.)
-------
LEGEND
EENDOGENOUS CULTURE
AUNDISSOLVED HAIR (416 MG/L )
O CULTURE FED ON POWDERED KERATIN (416 MG/L)
DISSOLVED IN KH2P04+NoOH BUFFER AT PH 8.5
SUSPENDED SOLIDS- 2000 MG/L
TEMPERATURE- 20*C
600
4 6
TIME- HOURS
8
10
FIG.56 OXYGEN UPTAKE OF A CULTURE FED
UNDISSOLVED HAIR
-96-
-------
spatial configuration and polar groups of the substrate. The nature of the enzyme
itself, its structure, the prosthetic group, the metals involved, all these are
of extreme importance on enzyme specificity. It follows, therefore, that a poly-
peptide or protein will be hydrolyzed at definite loci in the chain.
Chemical treatment disrupts a number of disulfide and hydrogen bonds and di-
minishes electrostatic and hydrophobic attractions. As well, the protein can be
assumed to change from the helical to a random coiled form.
Because the hydrolysis of the peptide bond is an exergonic process favored
by the ionization of the amino acids involved, bacteria are not thermodynamically
hindered from effecting the hydrolysis. Thus, the slow rate of oxidation of the
keratins cannot be explained by lack of energy. The keratin supplemented with
glycose and nutrient broth — both substrates readily available as energy sources
— did not change significantly in rate of utilization.
The highly complex keratin molecule must be reduced to simpler building blocks
before it is readily accessible as a bacterial food source. Thus either physical
or chemical transformations will be required as pretreatment or exoenzymes will
have to hydrolyze a sufficient number of bonds to form small peptides or free
amino acids that can travel to the interior of the bacterial cell and be incor-
porated in the cell's biochemical activities.
The highly specific enzymatic degradation process has been shown by this re-
search to be accelerated by acclimating the activated sludge culture to the kera-
tin substrate thus building up the microbiological populations and enzyme systems
utilized in the keratin degradation process. The parameters measured indicate
that the respiration and growth of the cells occurs at a low rate which can be
attributed to the difficulties incurred by the bacterial cell in hydrolyzing and
utilizing the substrate.
The proteolysis of the oxidized high sulfur component of wool (approximately
30% of the dry weight) is about 3.2 times faster than that of the low sulfur com-
ponent (approximately 60% of the dry weight) and this latter fraction reaches only
26% of the total hydrolizable material in six hours. This difference in proteoly-
8is has been explained as due to the structural difference of the two components
(53).
In analyzing the kinetics of the reaction of the biological degradation of
hair the rate expressions of Henri (50) are
-97-
-------
k k
[E] + [S] ^7^ [E-S] — =*> [P] +
k
where [E] ±s the concentration of enzyme
[E-S] is the concentration of enzyme-substrate complex
[P] is concentration of products
and the k's are the various reaction rates shown
-1-
Now
K » _2_ - [E][S] for the equilibrium between reactants and
68 k [E-S]
the enzyme intermediate. The total enzyme concentration is
[ET] - [E] + [E'S]
and - v -
ee
[ET] - l+-f [E-S]
M J
The initial rate of product formation is given by
v.dlZl „
-2-
-3-
or
dt
w
S + K
[E-S]
-5-
eg
A plot of V (velocity of reaction) versus S (substrate concentration) will yield
a curve
For large values of S, equation -5- for V indicates that the reaction rate
will be insensitive to substrate concentration. That is, when s>>Keg> equation
~5- yields V « k.E,,,
-6-
-98-
-------
Vm
Therefore, the maximum reaction, V , for a given total enzyme concentration,
m
E_, should, under these conditions, be dependent only on the equilibrium constant,
k_. This is why the rate of oxygen utilization of a culture fed a simple fully
utilizable substrate which is present in excess proceeds in a straight line fash-
ion. Under this condition, the rate of substrate depletion is expressed by —r-
" k , k being the rate constant.
The equilibrium constant, k_, and hence the rate constant, k , appear to be
a function of the substrate characteristics and will remain constant only when
those characteristics are invarient with time in the period where S»K
eg
In the case of keratin, however, the substrate is highly complex and is char-
acteristics are not apt to remain constant throughout the reaction. The curves
in Figure 57 indicate a time variant equilibrium coefficient as shown by the change
in slope of the oxygen uptake curve at 2 1/4 hours elapsed time in batch feeding
°f keratin. Such a slope change may indicate a change in vulnerability of the
keratin.
The oxygen uptake rates shown in Figure 58 show that with the long time ex-
periment, the condition of S»K prevailed probably because of the low concentra-
tion of E'S.
If the substrate concentration, S, is very small when compared with K , the
rate of reaction or enzyme activity is proportional to the substrate concentra-
tion.
Prom -5-
wMch plots as a straight line with slope -
-7-
K
-99-
-------
LEGEND
<3> ENDOGENOUS
A4I6 MG/L OF HAIR
D 830 MG/L OF HAIR
01660 MG/L OF HAIR
TEMPERATURE- 20°C
S. SOLIDS- 2000 MG/L
600
234
TIME ~ HOURS
FROM ZERO TO 2J4 HOURS CURVES CD AND g> FALL ON THE
SAME SLOPE, WHICH CURVE 0 KEPT UNTIL 3/4 HOURS.
AFTER 3# HOURS CURVES ® AND ® TAKE THE SAME SLOPE
AS (3>
FIG.57 EFFECT OF INCREASING CONCENTRATIONS
OF SUBSTRATE ON OXYGEN UPTAKE
-100-
-------
1400
1200 —
i
t-1
o
O ENDOGENOUS CULTURE
D CULTURE FED GLUCOSE (0.77 MG /ML )
O CULTURE FED DISSOLVED HAIR (416 MG/L )
A CULTURE FED HAIR (4I6MG/L) AND
GLUCOSE (0.77 MG/ML)
SUSPENDED SOLIDS-2000 MG/L
TEMPERATURE-20° C
30 40 50
TIME~ HOURS
FIG.58 OXYGEN UPTAKE OF A CULTURE FED DISSOLVED HAIR
AFFECT OF SUPPLEMENTATION WITH GLUCOSE
-------
2000
1600
1200
(9
i
u.
o
(O
(T
UJ
O
ce
o
800
400
DHAIR (416 MG/L) SUPPLEMENTED WITH NUTRIENT
BROTH, (52.5 MG/L AS BOD)
ANUTRIENT BROTH (52.5 MG/L AS BOD)
OENDOGENOUS
30 40
TIME~HOURS
FIG.59 EFFECT OF SUPPLEMENTATION OF KERATINS WITH
NUTRIENT BROTH ON OXYGEN UPTAKE
-102-
-------
LEGEND
©ENDOGENOUS (ACCLIMATED AND UNACCLIMATED)
AUNACCLIMATED CULTURE
Q ACCLIMATED CULTURE
OACCLIMATED CULTURE (FEED WAS BOILED 6 HOURS)
FEED- 416 MG/L OF KERATIN
TEMPERATURE- 20°C
SUSP. SOLIDS- 2000 MG/L ACTIVATED SLUDGE SOLIDS
600
468
TIME~ HOURS
FIG.60 ACCLIMATION EFFECT ON OXYGEN UPTAKE
-103-
-------
The final stage in the oxidation scheme of keratin is that of a rate of reac-
tion limited by the amount of substrate present. The oxygen uptake of this period
must be reflected by a first order reaction curve,
. -kt
02 = Ae -8-
where 0~ = oxygen remaining at time t
A = total oxygen available or remaining at time t = 0
The fact that no first order reaction curve is evident from the experiments
suggests that the reaction stops before substrate concentration was noticeable as
the limiting factor. Two factors may help to account for the incompleteness of
the reaction: toxic products might have accumulated to concentrations sufficient
to stop the reaction or the remaining fraction of substrate was fully unutilizable.
It may be also that this first order portion of the curve occurred in a time in-
terval not perceptible in the time units measured.
One other important aspect needs consideration. The dissolution of the hair
was done in a highly alkaline medium and amino acids racemize at high pH values,
the natural L form being converted to the D form. Bacterial systems are highly
sensitive to isometric configurations and it has been established that, excepting
alanine, the utilization of the D amino acid occurs at a slower rate than for the
L form (1). The organisms must change the racemized amino acid back to the L form
before it can be utilized. The production of the responsible enzymes, the race-
mases, could well limit the reaction although racemic mixtures have been shown to
be utilized at rates near that of the L form.
Even when the aeration solids yield was high, no significant increase in DNA
was obtained which indicates a slow rate of cell division. The net increase in
mass may be due to cell volume growth or to accumulation of cell storage material,
slime, and debris incorporation in the floe.
-104-
-------
SUMMARY AND CONCLUSIONS
The utilization of compounds by mixed biological cultures is influenced by
a variety of factors. In this study the central theme has been the consideration
of some of these factors that affect the utilization of amino acids by activated
sludge systems. Experimental data has been developed on the effects of tempera-
ture, stereo-isomerism, peptide linkage sequence and length. In the pursuance of
these studies it has been necessary to develop or adapt experimental techniques
to reflect or present the desired information. Thus, digital computer programs
were designed to present experimental data graphically as well as to store perti-
nent abstracts in a literature retrieval system. A DNA test was adapted for use
with activated sludge cultures and chemical procedures were developed for dis-
solving keratins prior to use as a substrate.
On the basis of these and other experimental determinations, a number of con-
clusions can be drawn on the utilization of free and combined amino acids by acti-
vated sludge cultures. The summary points outlined below indicate that enzyme
systems for incorporation and oxidation of amino acids are increasingly stereo
specific with molecular size, are temperature sensitive, increasingly hindered by
increasing lengths of peptide chains and, in some cases, are responsive to amino
acid sequence in the peptide chain. Further, new cell production, as indicated
by DNA, does not directly follow cell mass increases of activated sludge.
In keratins such as hair the complex nature of the incorporated mechanisms
thwarting biological breakdown of the compound offer a real challenge to those
interested in providing biological decomposition of these compounds in the rela-
tively short time interval used in contemporary waste treatment facilities.
As might be expected the biological utilization of a compound is dependent
on the available free energy stored in the compound and in the relative ease with
which this energy can be released from the compound.
Some of the other salient observations and conclusions from this research are
listed below.
1. D-isomers of amino acids and peptides are not utilized by activated sludge
cultures as well as is the natural isomer. The isomeric effect increases
with molecular size such that
-105-
-------
, f-,nn\ D isomer oxygen uptake
where x = (100) L isomer oxygen uptake
and C = no. of carbon atoms in the D isomer
2, Where acclimation is effective, oxygen uptake rates for amino acids and
peptides are related to the free energy in the substrate. The data in
this study seemed to fit the straight line:
Y = 26.5 - 0.0325F
where F = free energy in the substrate in calories per liter
Y = oxygen uptake rate in microliters per hour per milligram
sludge solids
3. Special peptide studies with glycyl DL alanine and DL alanyl glycine in-
dicated that, for these two peptides, the sequence of the amino acids in
the peptide is very important in their utilizability. While the glycyl
DL alanine was essentially removed from the supernatant in 8 hours, DL
alanyl glycine was still present after 24 hours. Uptake rates, CO. and
NH production, were all significantly higher for glycyl DL alanine than
for DL alanyl glycine.
4. For temperatures in the range of 10 to 30°C, with a 10°C change in tem-
perature, k-i/k, ratios were approximately 2+0.2 for oxygen uptake rates
for glycyl DL alanine, DL alanyl glycine and DL alanyl DL phenylalanine.
A typical experimental value for the apparent free energy, y, was 11,400
calories/mole for glycyl DL alanine.
For temperatures variations above 30°C there was some variation in
doubling of oxygen uptake with a 10°C temperature rise because of dif-
ferences in culture populations. If the activated sludge culture was
comprised largely of bacteria, the doubling effect could be expected up
to 35°C. However, if a large protozoan population were present tempera-
tures in the 30°C range would be approaching the thermal death point of
the protozoa and hence oxygen uptake rates would tend to be depressed
above 30°C.
5. The Schneider and Burton method of DNA isolation and determination has
been applied to activated sludge cultures. Recommended test conditions
are listed under the "Summary on Use of the DNA Test" page 36 .
6. DNA tests on acclimated activated sludge cultures using the amino acids,
arginine, alanine, glutamic acid, and phenylalanine, as substrates show-
-106-
-------
ed increases in DNA content after feeding. Thus, all four amino acids
are an adequate substrate for production of new cells if other environ-
mental conditions are not limiting.
a. The pattern of ultimate increase in DNA content depends on the
type and concentrations of amino acids.
b. In terms of DNA change at eight hours after feeding, the aliphat-
ic amino acids (arginine-HCl, DL alanine, and Na-glutamic acid)
showed maximum DNA increase at a substrate concentration of 1000
mg/1 as a group, whereas phenylalanine, an aromatic amino acid,
showed maximum DNA increase at the much lower concentration of
400 mg/1. This experimental evidence suggests that amino acid
structure could have a general effect on new cell production with
the aliphatic group in one category and the aromatic group in
another (See Figure 35).
c. Exceeding these optimum concentrations, a decrease in DNA pro-
duction indicates the inhibitory effect of overdosage. Also,
it should be noted that activated sludge cultures are more toler-
ant of higher dosages of aliphatic amino acids than of the aro-
matic acid.
d. Although activated sludge can utilize the four tested amino acids
as both carbon and nitrogen sources and can use them for new cell
production, the addition of glucose to the substrate increases
DNA production (Figure 36).
In sanitary engineering reports, growth frequently has been defined as an
increase in suspended volatile solids. For the unsteady state phase of
batch culture feeding the experimental results in this study showed that
the increase in volatile solids correlated with an increase in substrate
concentration rather than an increase in DNA content. This experimental
evidence from batch cultures indicates that initial unsteady state vola-
tile solids increases result from organic compound accumulation rather
than increase in active solids; thus, in this activity phase volatile
solids concentration increases would not be a reliable growth parameter.
The keratin studies showed that hair can be dissolved at room temperature
with a solution of 0.5N to IN sodium hydroxide. The dissolved keratin
is then available for biological degradation in an activated sludge system
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with approximately 75% of the material fed removed from solution. The
sludge yield is about 58% of the dry weight fed. However, the net DNA
content of the activated sludge solids were approximately constant
throughout the experiment.
9. Warburg respirometer studies with the dissolved hair as the activated
sludge substrate gave zero order reaction rate curves. At the end of
44 hours the material oxidized accounted for 45% of the COD fed. The
best efficiency obtained for removal of keratin derived protein was 76%.
The addition of substrate supplements such as glucose and nutrient broth
did not improve the rate or efficiency of the utilization process.
RECOMMENDATIONS FOR FUTURE WORK
Further research should be concerned with the behavior of other keratins
where the difference in composition and structure might affect dissolution and
biological utilization. Preliminary work on sea gull feathers show them to be
insoluble in 0.5 N NaOH at room temperature until the oily film, which renders
them impermeable, is removed with detergent or solvents such as benzene or carbon
tetrachloride.
Further study should be given to the possibility of dissolving keratin through
biological action because biological dissolution may provide a product more amen-
able to degradation than chemically dissolved keratin.
The tendency of keratin to foam under aeration may have a practical applica-
tion in foam fractionation procedures for the removal of dissolved organics in
advanced waste treatment. Recirculation of the foamed mixture may enhance the
utilization of refractory fractions from the waste or the keratin itself by in-
creasing the detention time in the system.
Further work is needed too on the relationships between cell production and
substrate oxidation versus the free energy and molecular configuration and com-
plexity of the substrate. From the indications obtained in this study some under-
lying biochemical mechanisms should soon become apparent that would be of value
in waste treatment system development.
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ACKNOWLEDGEMENTS
Information presented in several sections of the report are a contribution
from research assistants at the University of Washington. Mr. John Osborn's re-
search was on the utilization of alanyl-glycine and glycyl-alanine, George Capes-
tany developed the keratin studies and the section on the DNA test resulted from
the efforts of Feng Li. Tom Shen adapted the KWIC program to the literature of
this study. Charles Bowers was consultant and programmer on the graphical dis-
play of Warburg respirometer data and William Fung was laboratory assistant for
the initial Warburg studies. The author wishes to express his sincere apprecia-
tion to these co-authors and investigators.
Acknowledgements are extended to the Public Health Service for their support
of this research and to Prof. R. 0. Sylvester for his encouragement and coopera-
tion throughout the study period. Professors R. T. Oglesby and R. F. Christman
extended advice on biological and chemical aspects of the project. Secretaries
Karen Salvesen and Judy Deal patiently and competently typed the drafts and final
manuscript.
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17. Feughelman, M., and Langridge, R., "Molecular Structure of Deoxyribonucleic
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20. Davidson, J. D., "Tissue Nucleic Acids", Biochemistry Journal, 38. 39. (1944).
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22. Schneider, W. C., "Phosphorus Compounds in Animal Tissue", Journal of Biolo-
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23. Marmur, J., "A Procedure for the Isolation of Deoxyribonucleic Acid from Mi-
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24. Vendrely, R., "Un Symposium sur les Proteines", Masson et Cie (1964).
25. Burton, K., "The Relation Between the Synthesis of Deoxyribonucleic Acid and
the Synthesis of Protein in the Multiplication of Bacteriophage T-2", Bio-
chemistry Journal, 61., 473 (1955).
26. Davidson, J. N., The Biochemistry of the Nucleic Acids. John Wiley and
Sons, New York (1960).
27. Scherbaum, 0., "The Content and Composition of Nucleic Acids in Normal and
Synchronously Dividing Mass Culture of Tetrahymena pyriformis". Experimental
Cell Research, JL3., 30 (1957).
28. Gaudy, A. F., and Engelbrecht, R. S., "Basic Biochemical Considerations Dur-
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Treatment, Proc. of the 3rd Conference on Biological Waste Treatment, Perga-
mon Press, New York (1963).
29. Heukelekian, H., and Manganelli, R., "Factors Affecting the Quantity of Sludge
Production in the Activated Sludge Process", Sewage and Industrial Wastes Jour-
nal, 23, 945 (1951).
30. McCabe, B. J., "Mathematical Formulation of the Biological Oxidation Process",
Advances in Biological Waste Treatment, Proc. of the 3rd Conference on Bio-
logical Waste Treatment, Pergamon Press, New York (1963).
31. Hotchkiss, R. D., Methods in Enzymology. Vol. 3, Academic Press Inc. New York
(1957).
32. Zaroenhoff, S., Reiner, B., DeGiovanni, R., and Rich, K., "Introduction of Un-
natural Pyrimidine into Deoxyribonucleic Acid of Escherichia coli". Journal
of Biological Chemistry, 219, 165 (1956).
33. Li, Feng Tsang, The DNA Test - A New Method for Determining Growth and Acti-
vity of Activated SludRe Cultures. M. S. Thesis, University of Washington
(1964).
34. Oeming, L. F., "Industrial Wastes and Their Effects on Municipal Sewage Treat-
ment Plants", Sewage Works Journal, .9, March (1937).
35. Ridenour, G. M., and Backus, R. D., "Effect of Polysulfide Treated Cyanide
Case Hardening, Copper and Zinc Plating Wastes on Sludge Digestion", Sewage
Works Journal, 17., May (1945).
36. Siebert, C. L., "The Effect of Industrial Wastes on Sewage Treatment", Sewage
Works Journal, 9., January (1946).
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37. Moulton, E. Q., and Directo, L. S., "Standardized Procedure for Evaluation
of Toxic Effects on Activated Sludge", Engineering Experiment Station Bulle-
tin, Vol. XXXI, No. 3, Ohio State University, May (1962).
38. Mills, E. J., Jr., and Stack, V. T., Jr., "Biological Oxidation of Synthetic
Organic Chemicals", Proc. 8th Ind. Waste Conf., Purdue University (1953).
39. Gellman, I., and Heukelekian, H., "Studies of Biochemical Oxidation by Direct
Method", Sewage and Ind. Wastes, 27. July (1955).
40. McKinney, R. E., and Jeris, J., "Metabolism of Low Molecular Weight Alcohols
by Activated Sludge", Sewage and Ind. Wastes, 28, November (1956).
41. Nemerow, N. L., Theories and Practices of Industrial Waste Treatment, Addi-
son Wesley Publishing Co., Inc., Reading, Massachusetts (1963).
42. Mercer, E. H., Keratin and Keratinization. Pergamon Press, New York (1961).
43. Butcher, E. 0. and Sognnaes, R. E., "Fundamentals of Keratinization", Ameri-
can Association for the Advancement of Science, Washington, D. C. (1962).
44. Lehninger, A. L., "Energy Transformation in the Cell", Scientific American,
£9_, 1 (1960).
45. Neurath, H., The Proteins. Composition, Structure and Function. Second ed.,
Academic Press, New York-(1963).
46. Hawrowitz, F., The Chemistry and Function of Proteins. Academic Press, New
York (1963).
47. Fraser, R. D. B., Macrae, T. P., Rogers, G. E., "Molecular Organization in
Alpha Keratin", Nature, 193. 1052 (1962).
48. Astbury, W. T. and Street, A., "X-ray Studies of the Structure of Hair, Wool
and Related Fibres", Phil. Transactions, A230, 75 (1931).
49. Pauling, L., Corey, R. B., Branson, H. R., "The Structure of Proteins: Two
Hydrogen-bonded Helical Configurations of the Polypeptide Chain", Proceedings
of the National Academy of Sciences, 37. 205 (1951).
50. Martin, R. B., Introduction to Biophysical Chemistry. McGraw-Hill, New York,
(1964).
51. Bray, H. G., and White, K., Kinetics and Thermodynamics in Biochemistry, Aca-
demic Press Inc., New York (1957).
52. Alexander, P. and Earland, C., "Structure of Wool Fibres", Nature, 166, 396
(1950).
53. Springell, P. H., "Proteolysis of Wool and its S-Carbonxymethyl Derivatives
by Pronase and Other Proteases", Australian Journal of Biological Sciences,
16., 727 (1963).
54. White, A., Handler, P., Smith, E. L., Stetten, D. W. , Principles of Biochemis-
try. McGraw-Hill, Inc., New York (1959).
55. Fruton, J. S., and Simmonds, S., General Biochemistry. Second ed., John Wiley
and Sons, New York (1963).
56. Benesh, R., Benesh, R. E., Boyer, P. D., Klor, I. M., Middlebrook, W. R.,
Szent-Gyorgyi, A. G., Schwartz, D. R., Sulfur in Proteins, Academic Press,
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New York (1959).
57. McCabe, B. J., Eckenfelder, W. W., Biological Treatment of Sewage and Indus-
trial Wastes, Vol. I, Chapman and Hall, Ltd., London (1960).
58. Lowry, 0. H., Resenbrough, N. H., Farr, A. L., Randall, R. J., "Protein Meas-
urement with the Folin Phenol Reagent", Journal of Biological Chemistry, 193,
265 (1951).
59. Standard Methods for the Examination of Water and Wastewater, Eleventh ed.,
American Public Health Assoc., Inc., New York (1960).
60. Berry, H. K., Button, H. E., Cain, L., Berry, L. S., Biochemical Institute
Studies IV. II Development of Paper Chromatography for Use in the Study of
Metabolic Patterns, The University of Texas Publication 5109 (1951).
61. Hendricks, S. B., "Salt Transport Across Cell Membranes", American Scientist,
52., 306 (1964).
62. Harrison, D. and Speakman, J. B., "The Pore Size of Keratin", Textile Research
Journal, .28, 1005 (1958).
63. Capestany, G. J., Laboratory Studies on Keratin. Typewritten M. S. Thesis,
University of Washington (1964).
64. Osborn, J. E., The Utilization of Peptides and the Effect of Chlorides in
Activated Sludge Cultures, Typewritten M. S. Thesis, University of Washington
(1964).
65. Horwood, E. M., and Rogers, C. D., "Research and Development of Electronic
Mapping", The Trend in Engineering, 1_3., No. 4 (1961).
66. Sylvester, R. 0., Carlson, D. A., Bergerson, W. W., Benson, D. J. and Bacon,
V. W., "Computer Analysis of Water Quality Data", Journal of the Water Pollu-
tion Control Federation, 34. pp. 605-615 (1962).
67. International Business Machines Corporation, General Information Manual. Key-
word- In-Context (KWIC) Indexing, IBM Technical Publications Department, White
Plains, New York (1962).
68. Labron, C. M., and Myers, D. H., IBM 1401 KWIC Index Systems, 10.3.010. IBM
Corporation, Philadelphia (1962).
69. Wadding, R. V., 7090 KWIC I. IBM Corporation, Space Guidance Center, Owego,
New York (1963).
70. Wadding, R. V., 7090 KWIC II. IBM Corporation, Space Guidance Center, Owego,
New York (1963).
71. Strominger, D. H., NC 138 Modified PK KWIC Program Share Distribution 884.
North American Aviation, Inc., Columbus, Ohio (1960).
72. Shen, T. T., KWIC Literature Retrieval. An unpublished project report, Amino
Acid Study, University of Washington, February (1964).
73. Klein, L., Aspects of River Pollution. Academic Press Inc., New York (1957).
74. Klein, L., River Pollution II, Causes and Effects, Butterworths, London (1962).
75. Sawyer, C. N., Chemistry for Sanitary Engineers, McGraw Hill, New York (1960).
76. Sachiko, Y., "Effect of Sodium Chloride on the Sewage Microorganism", Biolo-
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gical Journal of Nara Women's University, 11. 22 (1961) C.A. 57. 2001b.
77. Stewart, M. J., Ludwig, H. F., and Kearns, W. H., "Effects of Varying Sali-
nity on the Extended Aeration Process", J.W.P.C.F., 34, 1161 (1962).
78. Parfentjev, I. A. and Calelli, A. V., "Tolerance of Staphylococcus aureus
to Sodium Chloride", Bacteriological Proceedings 1963, RT5 (1963).
79. landolo, J. J., and Ordal, F. J., "The Effects of NaCl, pH, and Temperature
on the Growth of Staphylococcus aureus MF31", Bacteriological Proceedings
1963, A27 (1963).
80. Greenstein, J. P. and Winitz, M., Chemistry of the Amino Acids. John Wiley
and Sons, New York (1961).
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APPENDIX I
GRAPHICAL PRESENTATION OF DATA BY DIGITAL COMPUTER
Data Processing and Data Display Systems
Electronic data processing has been incorporated as a beneficial tool in many
professional fields in the past decade. Most procedures utilized have been limit-
ed to computing, tabulating and summarizing data.
Developments in the past three or four years have shown the feasibility and
economy of programming data for automatic graphic display. The processes utilized
can make the necessary computations to convert raw to finished data and then pre-
sent the desired information graphically. This graphical data display can be ap-
plied to present scales or plot sizes or, as developed for this Warburg data pres-
entation, the computer program can automatically select the scale dimensions and
the size of drawing which most appropriately fit the computed data. Computer sys-
tems are especially valuable where large volumes of reiterative data are processed
and where many graphical displays are desired.
Applications of computer displays are found in many fields such as presenta-
tion of isobars by the U. S. Weather Bureau, population distributions by age groups
as prepared by the U. S. Census Bureau, isodose plots indicating the distribution
of radioactive iodine in radiotracer thyroid studies or displays of radiation in-
tensities in the earth's atmosphere as relayed by the Tiros satellite.
Several of the electronic data processing programs have been applied to areas
of civil engineering and related fields such as in regional planning, transporta-
tion and population studies (65). Data displays are available in mapping outputs
or in bar graph array outputs. These bar graph arrays may appear with different
symbols to show confidence or distribution limits. Machine mapping outputs appear
as a series of numbers or symbols. This background of data can be overlaid with
a location map transparency by means of check points printed on the machine out-
put and photographed to give a map with the pertinent data appearing in the appro-
priate map areas. Such processing systems have been used in urban planning studies
for plotting housing and population trends. Also, mapping can be photographed from
an image displayed on a cathode ray tube. This type of projection has been used to
indicate traffic patterns out of Chicago. The desired data is printed as short
vectors and the intensity of traffic is indicated by the concentration of vectors
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along the arterials emanating from the city center. Other slower processes in-
volve the transferring of data to an inking head that may be directed to any spot
on a plane surface by use of either punch card or taped information.
A formidable amount of computations for the Warburg respirometer studies in
this project made electronic data processing a realistic means of obtaining the
desired data and for graphical presentation of the data. Further, because the
various Warburg runs were interrelated it was desirable to be able to compare
graphically the results from different runs on a single graphical display.
Because none of the available programs were satisfactory for the processing
of data obtained from the Warburg respirometer, it was necessary to develop a
new program and display techniques that would provide the necessary flexibility
in presentation and the means of computing and comparing the raw input data.
The requirements imposed on the program display were that the computer system
present from the raw data the quantity of oxygen utilized as a function of time,
the quantity of oxygen utilized per unit of cell mass per unit of time, the rela-
tive rates of oxygen used for each substrate expressed in percent and the change
in the respiration quotient with elapsed time fater substrate addition to the cul-
ture. Also, it was desirable to compare data from several different Warburg runs
on a single plot.
Dimensioning demands were placed on the computer program because the tir.e and
oxygen uptake scales varied with each batch of data. Also, the areal size of the
graphical display was left as a variable with several print-out sizes available
to the selection of the programmer.
Program Format and Presentation
The Warburg Respirometer Program as developed for this study is actually a
system of subprograms with one main or controlling program. The base program
was written in FORTRAN (Formula Translation System) language with the subroutines
written in FAP (Formula Assembly Program). These programs have been described
previously in the literature as formats for the IBM 709 computer system (66). No
tapes, other than standard input and output units, are required. The present for-
mat for the computer program now determines from the respirometer manometer read-
ings, the quantity of oxygen utilized per unit of time, the quantity of oxygen
used per unit weight of solids per unit of time, and the respirometer quotient
for each test run. Also, the program will express oxygen uptake of up to ten
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substrates as percentages of any given substrate used as the control or 100 per-
cent value. The program will search the raw data, find the tests and test cases
required for any graphical display, perform the desired calculations and then
print and plot the results. The program can be expanded by adding subroutines
to furnish any other desired calculations or displays from the Warburg data.
The input for the program falls into three major classifications, standard
data, test data and plot requests.
Standard Data: The standard data consists of the K-factors for each flask
and the abbreviations for the cultures and substrates used on the test data cards.
Test Data: The test data contains two types of cards, test cards and case
cards. The test cards contain milli-grams of solids, culture name, number of ob-
servations and the time value for each observation. The case cards contain the
substrate name, flask number and a respirotneter reading fro each observation.
Also, the case cards indicate whether the case is a control case and whether the
Warburg flask contains potassium hydroxide.
Plot Requests: The plot requests define the type of calculation to be per-
formed, the size of the plot, and the cases to be used. A maximum of ten cases
can be plotted together.
The output is both printed in tabular form and plotted. The plot may be varied
in size from one to nine pages. Any cases not printed by the computer system are
listed in the output.
The actual program used is presented in the appendix. Also representative
samples of the output data and graphical display are presented below and in the
appendix. The total machine output is on file at the University of Washington,
Department of Civil Engineering.
Results of the Warburg Respirometer Studies
The computer system display program developed especially for Warburg respiro-
meter studies permits a convenient means of rapidly computing and displaying the
respirometer data. The display program accepts a wide range of values for time
and oxygen uptake. The computer automatically set the range of values on the or-
dinate and abscissa to best fit the data to any desired size of graph up to 99
inches in length. The data plots shown were printed on a preset printout 2 pages
long or 15 inches by 22 inches in size.
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Summary:
A survey of existing electronic graphical display techniques indicated the
necessity of developing a special program for presenting information from respi-
rometer tests. The program designed for the Warburg respirometer is capable of
calculating the desired data, presenting this calculated data in tabular form
and then displaying the data graphically. The computer system program is flexi-
ble in that dimensions of the areal display and of its coordinates can be varied
to fit the range of values fed to the program. Also, subroutines can be added
to the program which can present additional calculations or graphical presenta-
tions as desired by the programmer.
The tabulations and graphical displays in this Warburg respirometer program
present the total volume of oxygen used per increment of elapsed time, the volume
of oxygen used per unit weight of suspended solids per increment of elapsed time,
the respiration quotients and a percentage comparison basis of oxygen uptake rates
with any designated control oxygen uptake rate. Other determinations and displays
can be added to the output at the discretion of the programmer.
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APPENDIX II
KWIC LITERATURE RETRIEVAL SYSTEM
The KWIC (Keyword in Context) Literature Retrieval Program as herein present-
ed is based on formats presented in IBM general information manuals (67 through
71). This program may be applied to either the title, the abstract or the entire
text of an article. In this research project the program was restricted to KWIC
indexing of titles (72).
To produce a KWIC index of titles, bibliographical entries consisting of at
least title, author and literature source are recorded in machine readable form,
such as punched cards or paper tape, so that processing can produce a printed in-
dex with its related bibliographical data.
The KWIC deck produced contains on each card a portion of a title so chosen
that an imbedded keyword starts at column number 25. This deck, consisting of
one card for each keyword found in the titles, may be sorted with either a stand-
ard tape sort program for a large deck of cards or an EAM sorter for a small deck
of cards. The sorting can then produce the KWIC index with keywords aligned on
a particular column in printout in alphabetical sequence. Each such contains as
well an appropriate "eleven character index word" as a controlling function.
After standard tape or EAM sorting, the program will produce a bibliography
listing the articles alphabetically by author and by title. Each entry in the
author index and in the title index carries the "eleven character index word".
The "index word" is developed by the IBM computer from the input deck of
author, title and source cards. The first six characters of the "index word" are
the first four letters of the senior author's last name and his first two initials.
The next two characters are the last two digits of the year of publication. The
last three letters are first letters of the first three significant words in the
title. For example:
1. Author card: BROWN, J P JONES A B
2. Title card: FACTORS AFFECTING THE EFFICIENCY OF TREATMENT PROCESSES
3. Source card: JWPCF - 1960, 29, 12, 1341-55
The "eleven character index word" is then "BROWJP60FAE". This "index word"
is inserted in columns 61-71 of every output card.
Keywords to be used in the program indexing may be determined by either ex-
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elusion of inclusion in an internally stored word list. For this project, "ex-
clusion criteria" were used whereby a list of unimportant words was prepared.
These words such as prepositions, articles, and conjunctions, were then eliminated
from the sort and listing process for the key words in the output of the KWIC pro-
gram. All other words in article titles are listed as subject words in the KWIC
output.
Input Deck Preparation
1. Bibliography (Master Record Format)
The input for the KWIC program normally consists of a set of references each
of which is processed independently. The format of a single reference is describ-
ed below.
Each card punched for a given reference has an information field (columns 1-
60) which contains the information on the literature reference; a major control
column (Column 73) and a minor control column (Column 74) used for sequencing with-
in a particular type of cards. In addition, each card contains the literature
identification number (Column 77-80) which is used as an accession number and for
bookkeeping purposes. For example, the literature identification number provides
a quick means of re-establishing order if card mixups occur. Also, this number
can be used to check the completeness of a collection of cards.
The content of the control columns identifies the type of cards according to
the following code:
Major Control Minor Control
Card Type Column Contents Column Contents
Author Card 1 punch 1-9
Title Card 2 punch 1-9
Source Card 3 punch 1-9
Each reference listing as a separate item in itself should consist of at least
one of each of the legal type of card in the order author, title, and source.
2. Author Card (Major Control Code 1)
This card carries the author's name, then a space and then the author's ini-
tials. If there are several authors, either a separate card is made for each, or
as many as three authors are recorded on one card in column 1-18, 21-38, and 41-
58, two columns remaining blank between authors. Corporate names are treated as
authors if no author is given. When several cards are required to record one
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author or corporate entry, the information on the second card is indented four
spaces (to column 5) to indicate that it is a continuation of the previous infor-
mation.
1 21 41
JONES A B SMITH C D NEWTON E P
3. Title Card (Major Control Code 2)
The title of the reference item is punched in the information fields of as
many title cards as are necessary to completely contain the title. No words
should be omitted or abbreviated except in accordance with conventions or esta-
blished rules as set up in the write up of the KWIC program. Individual words
should not be split between information fields of consecutive cards as this would
cause them to be interpreted as separate words. When additional cards, beyond
the first card, are used, the title continuation is indented two spaces on each
card following the initial card.
Card 1:
1 60_
USE OF MICROORGANISMS FOR THE QUANTITATIVE DETERMINATION OF
Card 2:
1 3 60.
AMINO ACIDS IN NATURAL MATERIALS
A. Source Card (Major Control Code 3)
The source card contains the name and location of the publisher for books
or the journal title, volume, and pages, and the date of publication in accord-
ance with conventional library practice. The date field is the last two columns
of the information field (columns 90 and 60) and normally contains the last two
digits of the year of the title. The date field information is used in process-
ing the cards.
1 60.
JWPCF, 1962, 26, 152-162 6 2
5. Word List
The word list cards contain a single information field (Columns 1-72). The
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field begins with Column 1 on the first card of the set and ends with Column 72
on the last card so that no indented spaces are required if several cards are used,
The word list itself consists of a series of word entries separated by a comma
and a space. The word list is terminated by using a period after the last word.
A typical exclusion list, which was used for title words to be omitted from
the KWIC critical word listing, would be as follows:
1 72
ON, IN, BEFORE, AFTER, OF, UNDER, THAT, ITS, NOT, SOME,
1 72.
UPON, BY CERTAIN, A, AN, THE, WILL, DOES.
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APPENDIX III
EFFECTS OF CHLORIDES ON ACTIVATED SLUDGE CULTURES
Variation in salt concentrations in a nutrient may affect the rate of nutri-
ent utilization by activated sludge. Studies in which various concentrations of
chloride were added to cultures of microorganisms are shown in Table 9. The stud-
ies reported in Table 9 indicate that the investigators are not in agreement over
maximum permissible chloride concentrations. This may be due to differences in
laboratory method. landolo used a pure culture and controlled pH quite closely.
Sachiko used a mixed culture and found that fungi tended to take over and bacter-
ia were reduced at chloride concentrations above 5000 ppm.
Normally concentrations as high as 5000 ppm would not be of concern in the
treatment of domestic sewage. Klein (73) reports the following chloride ion con-
centrations for domestic sewage:
Weak Sewage 70 ppm Cl~
Medium Sewage 100 ppm Cl
Strong Sewage up to 500 ppm Cl~
The Public Health Service suggests a maximum of 250 ppm Cl~ for drinking water
(74), but some communities have been known to use water with chloride concentra-
tions as high as 2000 ppm (75). From this information it seem safe to assume that
a domestic sewage, even under the worst conditions, should not have a chloride con-
centration much greater than 2000 ppm. However, industrial wastes and sea water
infiltration might increase this value considerably. Sea water has a chloride con-
centration of about 20,000 mg/1 (73) and infiltration might give a sewage with chlo-
ride concentrations greater than 5000 mg/1. According to Sachiko, chloride concen-
trations as high as 5000 mg/1 would be harmful to the activated sludge culture.
Therefore, a study of chloride ion concentration effects on activated sludge might
be of value.
In this investigation volatile suspended solid growth and oxygen uptake were
studied at various chloride concentrations and an attempt was made to determine
maximum permissible chloride ion concentrations.
Chloride Study Results
Solid growth studies showed a stimulation in growth rates with chloride con-
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TABLE 9: CHLORIDE EFFECT ON MICROORGANISMS
Organism
Concentration
of Cl
Comment
Reference
Sewage
Bacteria
Sewage
Bacteria
Sewage
Bacteria
Staphylococcus
aureus
Staphylococcus
aureus
Staphylococcus
aureus
4000 ppm
5000 ppm
5800 ppm
5160 ppm
in distilled
water
< 24250 ppm
> 24250 ppm
microorganism
affected
reduction in
microorganisms.
Increase in fungi
(raw sewage fed as
nutrient source)
adequate treatment
to sewage if organic
loading is closely
controlled
80% Staph. died in
15 minutes
100% in one hour
(addition of small
amount of nutrient
kept Staph.alive at
least 24 hours at
37°C)
growth rates stimulated
at pH 7.0 to 7.5 at
37°C
rapid decrease in
population
Sachiko (76)
Sachiko (76)
Stewart (77)
Parfentjev
(78)
landolo (79)
landolo (79)
centrations up to 2250 mg/1 as shown on Figure 61. Good growth was obtained for
concentrations as high as 4500 mg/1. Higher concentrations, 5000 mg/1 and greater,
resulted in excessive foaming and bulking of the culture making solid determina-
tions unreliable. Values for these higher concentrations are given by Osborn (64)
but are not plotted.
Warburg studies showed oxygen uptake without inhibition for chloride concen-
trations of 2430 mg/1 and less, these values are plotted on Figure 62. A culture
containing 6075 mg/1 of chloride had some inhibition for the first ten hours but
seemed to acclimate and at 20 hours the oxygen uptake was as high as for a culture
containing no chloride. For the culture containing 12,150 mg/1 of chloride there
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400
300
I
9
o
CD
5200
o
3!
O
100
SODIUM CHLORIDE
O 0 MG /L AS
• 0 MG /L
25 MG /L
450 MG /L
500 MG /L
2250 MG /L
4500 MG /L
TEST TEMPERATURE-20°C
SUBSTRATE-NUTRIENT BROTH
AND SODIUM CHLORIDE
FIG.6I GROWTH IN VOLATILE SUSPENDED SOLIDS
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900
800
OMG / L
304 MG/L
I,2I5MG /L
2,430 MG/L
6,075 MG/L
12,150 MG/L
24,300 MG /L
48,600 MG /L
100
0 5 IOHOURS l5 20 25 30
FIG. 62
OXYGEN UPTAKE WITH VARIATION IN CHLORIDE CONCENTRATION
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was definite inhibition, although not excessive. Higher concentrations showed
increasing degrees of inhibition.
These results might be compared with those found in the literature. Accord-
ing to Sachiko (76) the culture is adversely affected at a chloride concentration
of 5000 mg/1. This is substantiated by the foaming and bulking experienced at
similar concentrations in this investigation. landolo, however, reports good
growth at chloride concentrations as high as 24,250 mg/1 if pH is maintained in
a range of 7 to 7.5. This might also be reasonable since oxygen uptake results
show good uptake for a culture containing 24,300 mg/1 of chloride even though
there is inhibition.
A review of the results of this investigation combined with data from the
literature might lead to the following conclusions:
Maximum chloride concentration for unbuffered cultures is 5000 mg/1. If
buffers and conditioners are used to reduce foaming and bulking chloride concen-
trations as high as 24,300 mg/1 might be allowed without harm to the culture.
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APPENDIX IV
CHEMICAL DATA ON PEPTIDES AND AMINO ACIDS
TABLE 10: ENTROPIES, HEATS OF FORMATION, AND FREE
ENERGIES OF FORMATION @ 298.1°C (80)
Cpd
L-Alanine
DL-Alanine
L-Asparagine
L-Asparagine-H-O
L-Aspartic Ada
L-Cysteine
L-Cystine
Glycine
L-Glutamic Acid
Hippuric Acid
L-Leucine
D-Leucine
DL-Leucine
DL-Leucylglycine
L-Tyrosine
Carbon Dioxide (Gas)
Water (Liquid)
Q
Entropy
31.6
31.6
41.7
51.0
41.5
40.6
68.5
26.1
45.7
57.1
49.5
49.5
49.5
67.2
53.0
Entropy0
Formation
-153.7
-153.7
-208.0
-254.4
-194.2
-152.3
-286.1
-126.6
-222.6
-192.1
-233.6
-233.6
-233.6
-312.8
-227.4
Heat of
Combustion
387,210
386,620
460,850
458,130
382,720
532,200
997,700
232,600
537,450
1,007,860
856,090
856,110
855,320
1,093,330
1,058,450
Heat offe
Formation
-134,600
-135,190
-189,360
-260,390
-233,330
-127,880
-251,920
-126,660
-271,160
-147,710
-153,390
-153,360
-154,160
-207,100
-165,430
-94,240
-68,310
Free Energy,
of Formation
-88,780
-89,380
-127,360
-184,560
-175,440
-82,480
-166,630
-88,920
-174,800
-90,440
-83,750
-83,720
-84,520
-113,850
97,640
-94,100
-56,720
aln entropy units, calories per degree per mole.
Calories per mole.
Peptide
DL-Alanyl gly-
cine
Glycyl glycine
Hippuric Acid
DL-Leucyl gly-
cine
Molecular
Weight
146.2
132.1
179.2
188.2
- AH
k cal/mole
625.9
471.4
1007.8
1093.4
' AHf a
k cal/mole
185.8
178.0
145.9
205.4
S
51.0
45.4
57.2
b.2
AS
-231.3
-204.4
-192.0
- 12.8
F °
i ;/ i b
k cal/mole
-116.85
-117.10
-88.63
-112.13
Free Energy
of Hydrolysis
-3730
-3230
-2260
-2960
aHeat of formation of water taken as -68,317 calories and of C02 from graphite as
-94.030 calories.
From classic relation: AF.
AHf - T A s
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Glycyl-L-Leucine bond = 2110+50
Glycyl-(J) Ala " = 2550+50
Tyrosine-glycine " «= 1550+100
"The energy functions of the peptide bond constitute a key to the solution
of many problems of protein synthesis. It is evident from the table above that
hydrolysis of these peptides proceeds spontaneously, with a net loss of approxi-
mately 3,000 calories per mole".
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