EPA-600/2-75-029
September 1975
Environmental Protection Technology Series
MEASUREMENTS OF
ACTIVE BIOMASS CONCENTRATIONS IN
BIOLOGICAL WASTE TREATMENT PROCESSES
Municipal Environmental Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development,
U.S. Environmental Protection Agency, have been grouped into
five series. These five broad categories vere established to
facilitate further development and application of environmental
technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in
related fields. The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
A. Environmental Monitoring
5. Socioeconomic Environmental Studies
This report has been assigned to the ENVIRONMENTAL PROTECTION
TECHNOLOGY STUDIES series. This series describes research
performed to develop and demonstrate instrumentation, equipment
and methodology to repair or prevent environmental degradation from
point and non-point sources of pollution. This work provides the
new or improved technology required for the control and treatment
of pollution sources to meet environmental quality standards.
This document is available to the public through the National
Technical Information Service, Springfield, Virginia 22151.
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EPA-600/2-75-029
September 1975
MEASUREMENTS OF ACTIVE BIOMASS CONCENTRATIONS IN
BIOLOGICAL WASTE TREATMENT PROCESSES
By
F. 6. Pohland
S. J. Kang
School of Civil Engineering
Georgia Institute of Technology
Atlanta, Georgia 30332
Grant No. R-800354 (17050 GAI)
Program Element No. 1BB043
Project Officer
Ronald F. Lewis
Wastewater Research Division
Municipal Environmental Research Laboratory
Cincinnati, Ohio 45268
MUNICIPAL ENVIRONMENTAL RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Municipal Environmental Research
Laboratory, U.S. Environmental Protection Agency, and approved for publica-
tion. Approval does not signify that the contents necessarily reflect the
views and policies of the U.S. Environmental Protection Agency, nor does
mention of trade names or commercial products constitute endorsement or
recommendation for use.
ii
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FOREWORD
Man and his environment must be protected from the adverse effects of
pesticides, radiation, noise, and other forms of pollution, and the unwise
management of solid waste. Efforts to protect the environment requires focus
that recognizes the interplay between the components of our physical environ-
ment—air, water, and land. The Municipal Environmental Research Laboratory
contributes to this multidisciplinary focus through programs engaged in
t studies on the effects of environmental contaminants on the
biosphere, and
0 a search for ways to prevent contamination and to recycle valuable
resources
As part of these activities, the study described herein presents an
evaluation of the use of the determination of dehydrogenase enzyme activity
for the accurate measurement of active biomass in both batch and continuous
cultures grown on a variety of substrates in the biological wastewater
treatment processes and may be a useful tool for the determination and close
control of the biomass in the processes.
A. W. Breidenbach, Ph.D
Director
Municipal Environmental
Research Laboratory
iii
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ABSTRACT
This research was initiated to determine the applicability and limitations
of the dehydrogenase test for the measurement of active biomass in biological
wastewater treatment processes. Pure culture with E. coli and/or heterogeneous
culture batch studies were conducted on a variety of substrates including
glucose, galactose, sucrose, alanine, acetic acid, and selected industrial
wastewaters. Also conducted were continuous aerobic or anaerobic culture
studies with and without solids recycle. Dehydrogenase activity was monitored
along with other parameters including plate count, Coulter Counter enumeration,
adenosine triphosphate (ATP), and suspended solids to provide comparative and
complementary information on the biomass concentration.
Dehydrogenase activity was a very sensitive and accurate measure of active
biomass throughout the growth phases especially during endogenous growth but
showed limitations with the nutrient deficient cultures. The correlation
between dehydrogenase activity and suspend solids was constant at varying
retention times, or at all growth rates with or without solids recycle.
Consequently, a standard curve could be developed for given wastewaters by
operating the measurement of active biomass and thereby effectively controlling
the biological process.
The measurement of ATP was also a reliable new technique for measurement
of active biomass except more study on the extraction method is required as
well as investigations on the change of the correlation with suspended solids
with the change of growth, rate.
The technique for dehydrogenase activity measurement is simple, less costly
and gives more reliable and interpretable results.
This report was submitted in fulfillment of Grant No. R800354 (17050 GAI)
by the School of Civil Engineering, Georgia Institute of Technology under partial
sponsorship of the U. S. Environmental Protection Agency.
iv
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CONTENTS
SECTION PAGE
I CONCLUSIONS 1
II RECOMMENDATIONS 2
III REVIEW OF THE LITERATURE 3
IV INTRODUCTION 25
a) Experimental Apparatus for
Pure Culture Studies 25
Heterogeneous Culture Studies 28
b) Culture Preparation 30
c) Analytical Techniques 30
Coliform Analysis 31
Solids Determination 31
Substrate Concentration 32
Coulter Counter Analysis 34
Adenosine Tri-Phosphate Analysis 34
Dehydrogenase Analysis 39
V PRESENTATION AND DISCUSSION OF RESULTS 46
BATCH STUDIES WITH E^ coli 46
BATCH STUDIES WITH HETEROGENEOUS CULTURES . 70
CONTINUOUS CULTURE STUDIES 93
CONTINUOUS CULTURE STUDIES WITH SOLIDS RECYCLE 94
NUTRITIONAL DEFICIENCY STUDY 116
VI REFERENCES 127
VII APPENDICES 135
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TABLES
No.. Mi
1 Minimal Substrate for Batch Cultures 26
2 Comparison of ATP Contents Extracted by DMSO
and by Nitrogen Bombing 37
3 Effects of Freezing and Nitrogen Bombing on ATP
Contents Extracted 40
4 Comparison of Sample Pretreatraent Method for
Dehydrogenase Analysis 45
5 Pure Culture Batch No. 1 with Glucose 48
6 Pure Culture Batch No. 2 with Glucose 49
7 Pure Culture Batch No. 3 with Glucose 50
8 Pure Culture Batch No. 4 with Galactose 51
9 Pure Culture Batch No. 5 with Galactose 52
10 Pure Culture Batch No. 6 with Galactose 53
11 Pure Culture Batch No. 7 with Sucrose 54
12 Pure Culture Batch No. 8 with Acetic Acid 55
13 Pure Culture Batch No. 9 with Acetic Acid 56
14 Pure Culture Batch No. 10 with L-Alanine 57
15 Pure Culture Batch No. 11 with Benzoic Acid 58
16 Heterogeneous Culture Batch No. 1 with Glucose 72
17 Heterogeneous Culture Batch No. 2 with Glucose 73
18 Heterogeneous Culture Batch No. 3 with Galactose 74
19 Heterogeneous Culture Batch No. 4 with Galactose ..... 75
20 Heterogeneous Culture Batch No. 5 with Sucrose 76
21 Heterogeneous Culture Batch No. 6 with Acetic Acid ... 77
22 Heterogeneous Culture Batch No. 7 with L-Alanine 78
23 Heterogeneous Anaerobic Culture Batch No. 1 with
Leachate 86
24 Heterogeneous Anaerobic Culture Batch No. 2 with
Leachate 87
25 Heterogeneous Anaerobic Culture Batch No. 3 with
Leachate 88
vi
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TABLES (Continued)
No. Page
26 Ratio between Biomass Parameters during Log Growth
Phase of Batch Cultures 92
27 Continuous Culture Study with Glucose Substrate 97
28 Continuous Culture Study with Galactose Substrate .... 98
29 Continuous Culture Study with Shellfish Processing
Wastes 99
30 Continuous Culture Study the Chicken Processing
Wastes 100
31 Continuous Culture Study with Leachate 101
32 Anaerobic Digester with Heterogeneous Cultures in
Continuous Flow System 102
33 Summary of Correlations between Biomass Parameters
in Continuous Culture Studies 109
34 Kinetic Growth Constants 110
35 Continuous Culture Study with Galactose Substrate and
with Solids Recycle Ill
36 Summary of Growth Constants and Ratios between Para-
meters in Soilids Recycle Study with Galatose Sub-
strate 112
37 Nutrient Deficient Culture Batch No. 1 (C/N - 10) H7
38 Nutrient Deficient Culture Batch No, 2 (C/N » 20) H8
39 Nutrient Deficient Culture Batch No. 3 (C/N - 30) 119
40 Nutrient Deficient Culture Batch No. 4 (C/P - 150) ... 120
41 Comparison of Biomass from Nutrient Deficient Culture
Studies 121
vii
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FIGURES
No. Page
1 Biological Monitoring Tests 4
2 DNA Extraction-Analysis Procedure 8
3 Transfer Mechanisms of Intermediate Substrate
Metabo lism 22
4 Reactor Assembly for Pure Culture Studies 27
5 Reactor Assembly for Heterogeneous Culture Studies
with Solids Recycle 29
6 Comparison of ATP Contents Extracted by DMSO and by
Nitrogen Bombing 33
7 Apparatus for the Dehydrogenase Test AT
8 Correlation between Dehydrogenase Activity and Bio-
mass Concentrations ^
9 Pure Culture Batch No. 1 with £_._ coli and Glucose
Substrate 50
10 Pure Culture Batch No. 2 with E. coli and Glucose
Substrate g0
11 Pure Culture Batch No. 3 with E. coli and Glucose
Substrate 61
12 Pure Culture Batch No. 4 with E. coli and Galactose
Substrate 62
13 Pure Culture Batch No. 5 with E. coli and Galactose
Substrate &3
14 Pure Culture Batch No. 6 with E. coli and Galactose
Substrate 64
15 Pure Culture Batch No. 7 with E. coli and Sucrose
Substrate • 55
16 Pure Culture Batch No. 8 with E^_ coli and Acetic Acid
Substrate 66
17 Pure Culture Batch No. 9 with E^ coli and Acetic Acid
Substrate 67
lg Pure Culture Batch No. 10 with E. coli and L-Alanine
Substrate 68
viii
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FIGURES (Continued)
Np_._ Page
19 Pure Culture Batch No. 11 with E. coll and Benzole Acid
Substrate 69
20 Heterogeneous Culture Batch No. 1 with Glucose Sub-
strate 79
21 Heterogeneous Culture Batch No. 2 with Glucose Sub-
strate 80
22 Heterogeneous Culture Batch No. 3 with Galactose Sub-
strate 81
23 Heterogeneous Culture Batch No. 4 with Galactose Sub-
strate 82
24 Heterogeneous Culture Batch No. 5 with Sucrose Sub-
strate 83
25 Heterogeneous Culture Batch No. 6 with Acetic Acid
Substrate 84
26 Heterogeneous Culture Batch No. 7 with L-Alanine
Substrate 85
27 Heterogeneous Anaerobic Culture Batch No. 1 with
Leachate 89
28 Heterogeneous Anaerobic Culture Batch No. 2 with
Leachate 90
29 Heterogeneous Anaerobic Culture Batch No. 3 with
Leachate 91
30 Continuous Culture Study with Glucose Substrate 103
31 Continuous Culture Study with Galactose Substrate .... 104
32 Continuous Culture Study with Shellfish Processing
Wastes 105
33 Continuous Culture Study with Chicken Processing
Wastes 106
34 Continuous Culture Study with Leachate 107
35 Anaerobic Digester with Heterogeneous Cultures in
Continuous Flow System 108
ix
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FIGURES (Continued)
No. Page
36 Continuous Culture Study with Galactose Substrate
and with Solids Recycle 113
37 Active Biomass Measurements in Continuous Culture
Study with Solids Recycle 114
38 Effect of Specific Growth Rate on Correlations between
Biomass Measurements in Continuous Culture Study with
Solids Recycle 115
39 Nutrient Deficient Culture Batch No. 1 (C/N - 10) 122
40 Nutrient Deficient Culture Batch No. 2 (C/N =20) 123
41 Nutrient Deficient Culture Batch No. 3 (C/N =30) 124
42 Nutrient Deficient Culture Batch No. 4 (C/P = 150) ... 125
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ACKNOWLEDGEMENTS
The research reported herein was performed in the Sanitary Engineering
Laboratory, Georgia Institute of Technology, Atlanta, Georgia. The research
team which directed the project and prepared the report consisted of
Dr. Frederick G. Pohland, Project Director and Shin Joh Kang, Research
Associate.
This project was sponsored by the Environmental Protection Agency,
with Dr. Ronald F. Lewis serving as Project Officer.
xi
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SECTION I
CONCLUSIONS
The use of dehydrogenase activity for the measurement of active biomass
in the biological wastewater treatment process has proven very sensitive
and effective in both batch and continuous cultures grown on a variety of
substrates.
Definite relationships between plate count, Coulter Counter enumeration,
solids and dehydrogenase activity were established during the log growth
phase of batch cultures with E_._ coll.
Dehydrogenase activity is a more sensitive measure for active biomass
under substrate limited endogenous growth.
ATP and dehydrogenase activity have shown a very similar pattern of
measurements throughout the log and stationary phases of heterogeneous
batch cultures except for lag observed during the anaerobic culture studies.
The correlation between dehydrogenase activity and suspended solids
in continuous culture remained constant at all growth rates.
Except for nutrient deficient conditions where nitrogen and phosphorus
are limiting, dehydrogenase activity was an acceptable measure of active
biomass.
The correlation between dehydrogenase activity and suspended solids
was constant but lower in continuous culture with solids recycle than
without recycle. The existance of partial endogenous growth in the
cultures did not affect this relationship.
When measured with wastes of different organic characteristics, the
dehydrogenase activity per unit biomass changed in both batch and continuous
culture.
The technique for dehydrogenase measurement is less costly and tedious
than other biomass parameters such as ATP and yields more reliable and
interpretable results.
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SECTION II
RECOMMENDATIONS
Based on batch and continuous studies with pure and heterogeneous cultures
under aerobic and anaerobic environments, it is recommended that pilot plant
or field investigations at selected wastewater treatment plants be conducted
to establish the validity and applicability of the correlations determined
during these studies and to develop a standard techniques for the use of
dehydrogenase activity measurements in the design and control of biological
treatment processes.
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SECTION III
REVIEW OF THE LITERATURE
Introduction;
The adequacies of available parameters for design and operation of
water pollution control systems have become particurly apparent during
recent years as requirements for increased treatment efficiency have
intensified. Use of inappropriate analytical measurements has often
resulted in poor performance which must be elminiated in order to
meet current and projected demands for quality control. Biological
monitoring procedures also suffer from such deficiencies and of those
methods available (Figure 1), the determination of active biomass has
become one of the most controversial.
One approach to the measurement of active biomass has been the adapta-
tion of enzyme activity analysis to heterogeneous biological populations.
In order to be successful, certain criteria must be satisified including:
1. a definite relationship between the gross enzyme concentration
measured and the active (viable) biological population present
in the system;
2. a similar enzyme concentration level for all bacterial species
found in the system;
3. a resistance to variations in gross enzyme concentration with
changes in environment or exposure to stress;
4. a methodology providing quantitative information not influenced
by variations in the dissolved and suspended chemical constituents
or physical characteristics of the system; and
5. a sufficient quantity of measurable indicator of enzyme activity
to permit reasonable accuracy and precision.
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Total Mixed Liquor
Suspended Solids
MLSS
I
TSS
VSS
£
Suspended
Solids
Organic
Inert
Solids
Suspended
Solids
Inorganic
Cells
trate
DNA Content
Glucose Uptake Method
Substrate
Measurement
Inactive or
Dead Cells
1
Active, Viable
Cells
Warburg
* 02 Uptake
fc NAT) fc NAT)
Dehydrogenase Activity
(TTC—
Figure 1. Biological Monitoring Tests
(1)
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Three parameters which have received consideration for adaption to
biological treatment processes include the deoxyribonucleic acid (DNA)
content of bacteria, the dehydrogenase activity of bacterial cultures
and bacterial adenosine triphosphate (ATP) content. These methods are
based upon several fundamental premises, viz., DNA is a nuclear con-
stituent indigenous to all living cells; the dehydrogenases are vital
to all biologically mediated reactions; and, bacterial ATP is associated
only with living cells as a source of energy for biosynthesis and metabolism.
Since the thrust of the research reported herein has been to further develop
an adequate biochemical parameter to indicate active biomass content in
biological waste treatment systems, a critical review of the available
literature was considered in order. Similar reviews have been prepared
by Patterson, et al. and Weddle and Jenkins in separate investi-
gations. The former authors considered the measurement of cellular
ATP superior to dehydrogenase activity determinations because of inter-
ferences of test reagents (TTC) with normal cell metabolism and difficulties
in interpretation of results with the latter technique as compared with
rapidity, simplicity and sensitivity of the ATP analysis. Conversely,
the latter authors considered both parameters valid with ATP content
remaining constant over a wide range of growth conditions. Similarly,
the dehydrogenase enzyme activity as well as the oxygen uptake rate
remained constant over a net growth rate range of 0.03-6.0 day for
the systems investigated.
DNA Methodology and Application:
General Perspective - Numerous papers have been published over the
past twenty years on the quantitative determination of deoxyribonucleic
acid (DNA) in various biological systems. Almost all cellular DNA
(2 95%) is found in the nucleus and distributed throughout the nucleoplasm
as chromatin while the cell is in the resting state (i.e., engaged in
maintenance and growth between cell division). In anticipation of
division and during its course, the chromatin becomes highly organized
into distinct linear structures called chromosomes. The number of chromo-
somes per somatic cell is constant and this constant complement is passed
-------
on to a daughter cell as a result of mitotic divisionv . The microbial
concentration of DNA has been shown to be invariant with physiological
state and fairly constant among various bacterial species with an average
of 2 x 103 ug/mg of cell material.(5)6)
DNA is a macromolecule which consists of a sequence of nucleotides
linked together by phosphoric acid in a diester linkage; the nucleotides
in DNA molecule consist of a 2-deoxy-D-ribose (carbohydrate) moiety with
one of four bases (i.e., purines and pyriraidines) attached to the
hydroxyl group on C-l' carbon atom. These four bases are thymine,
(4 7)
cytosine, adenine, and guanine. '
Development of Analytical Methods - DNA determinations were originally
made indirectly by gently heating an assay sample in dilute alkali so
that the ribonucleic acid (RNA) would be degraded to acid soluble
nucleotides allowing the DNA fraction to remain unhydrolyzed and to
precipitate in dilute acid. By determining the carbohydrate and phos-
phate content of the acid-insoluble fraction, the initial DNA content
could be calculated . The ultraviolet absorption of the purine or
pyrimidine moieties in the region of 270-357 mu although specific
requires the selection of suitable DNA standards since the proportion
/Q\
of purines to pyrimidines varies according to the source . Webb
and Levy developed a colorimetric test for the direct measurement
of the deoxyribose moiety of DNA with p-nitro-phenylhydrazine to yield
a blue-colored product. DNA hydroloyzed in TCA (trichloroacetic acid)
reacted quantitatively with p-nitrophenylhydrazine. When the product
was separated from interfering substances, it was determinable colori-
raetrically in alkaline solution. The blue color developed in accordance
with Beer's law over a range of 10 to 300 ug of DNA producing five times
the color intensities and was more specific than the early diphenylamine
reagent used in tissue and microorganism studies. It was postulated
that the deoxypentose resulting from the hydroloysis forms a hydrazone
when heated with the phenylhydrazine derivative in the presence of TCA.
However, while the chromogenic compound was more intensely colored,
the color started to fade almost immediately so that optical densities
of all samples had to be recorded at approximately the same time. DNA
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values on biological materials determined by reaction with p-nitrodipheny-
Ihydrazine have been found to be generally lower than those values noted
by reaction with diphenylaraine reagent. These lower results may be
explained by assuming that p-nitrodiphenylamine does not give color
with protein or protein breakdown products resulting from TCA hydroly-
sis. It was observed, however, that in the presence of proteins and
protein degradation products, the diphenylaznlne reagent did give high
DNA values. Other carbohydrates present which might be capable of
reacting with p-nitrodiphenylhydrazine either do not form colored
products in alkaline medium or are extracted along with excess reagents
by butyl acetate when in TCA solution thereby eliminating the need
for any prior tissue or microorganism preparation. The DNA extracted
from pure culture organisms yielded an average DNA content of bacterial
cells of 3.8 percent on a dry weight basis.
(9-11)
Agardy and co-workers studied a method of determining the DNA
content of anaerobic fermentation but detected a green color developed
in the reaction with diphenylamine which tended to obscure the charac-
teristic blue color. Even with the purification of diphenylamine and
numerous washings of the cellular material, the green color could not
be controlled in anaerobic digesting sludges . The extraction and
quantification of bacterial DNA was a three-stage process consisting
of rupture of the bacterial cell, extraction of protein-bound DNA,
hydrolysis, and evaluation of DNA content. A schematic diagram of
the procedure has been included in Figure 2.
Agardy and co-workers also used DNA content determinations as a
digester loading parameter to relate the daily organic load applied
to a biological system to some measure of the system's microorganism
content. The values of DNA found in field digesters were higher
than expected when compared with values reported in the literature
for pure cultures of bacteria (3.1 to 4.8% of the volatile solids).
It was concluded that either the organism concentration during sludge
digestion at detention times in excess of 20 days was higher than
anticipated or interfering substances such as sugars and aldehydes
were present in relatively high concentrations so as to affect the
DNA determination.
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Bacterial Cell Suspension
Ultrasonic
Vibration
Broken Cells
Cold TCA
(centrifuge extraction)
Acid Soluble
Fraction
Residue
Ethanol
Extraction
Phospholipids
Lipids
Residue
Hot TCA
Extraction
Acid Soluble
Fraction
Residue
Hot Diphenylamine
Reaction
Blue Color
Figure 2. DNA Extraction-Analysis Procedure
(9)
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U-2)
Fisher used a modified method in analyzing digester sludge.
When the DNA in the sludge samples was allowed to react with hot con-
centrated sulfuric acid, a yellow color resulted during hydrolysis
and extraction; however, when concentrated sodium hydroxide was sub-
stituted in the first extraction-hydrolysis step, no interfering yellow
color resulted. Recovery studies proved the modified DNA extraction
technique to be quantitative.
Hattingh and Siebert ' increased the sensitivity of the DNA method
by adding an aldehyde to the diphenylamine reagent. The alcohol-ether
extraction proposed by Agardy was considered unnecessary. Moreover,
the acid-soluble compounds of the cell debris were considered responsible
for the interfering green color. DNA was liberated from the sludge
with sodium lauryl sulfate by ultrasonic vibration. The supernatant
liquor was collected after centrifugation and the DNA was precipitated
with perchloric acid at 70°C instead of the usual boiling for 15 minutes.
This method resulted in 99 and 100 percent recovery of DNA from anaerobic
sludge with the addition of aldehyde to the diphenylamine reagent serving
/g\
to stabilize and intensify the blue color .
Application and Results - Genetelli developed and evaluated organic
loading parameters for an activated sludge system based upon DNA and the
organic nitrogen used as measures of the system's organism concentration.
It was concluded that a loading parameter for activated sludge may be
based on pounds of BOD per day per pound of DNA in the aeration basin.
A parameter based upon DNA was more universally applicable with different
feed substrates than one based upon nitrogen. DNA content was considered
much more sensitive to changes in sludge volume index (SVI) than solids
or nitrogen concentration because it reflected the variations in
bacterial concentration and the shift in type of microorganism contributing
to the active biomass. The DNA content of sludge was applied as a process
control parameter indicative of the type of microorganisms present in
the sludge (i.e., zoogleal floes or a more filamentous masses). The DNA
content decreased prior to an increase in SVI and an abrupt change in the
DNA content indicated an approaching change in the settleability of the
sludge.
9
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(14)
Holm-Hansen applied DNA content to marine samples as a biomass
parameter and to calculate growth rates at various oceanographic depths.
A micromethod for DNA was developed based upon the measurement of the
fluorescence of the complex formed by samples containing DNA incubated
with diaminobenzoic acid dihydrochloride (DABA). The sensitivity of
the method was in the 0.2 ug to 40 yg DNA range. To use DNA as a biomass
indicator, it was considered necessary to relate DNA content of the
sample to a cellular entity such as total organic carbon (TOG). DNA
to TOC ratios were higher than those observed by methods such as plate
count or microscopic enumeration. The data indicated that there was
a considerable quantity of living material that was high in DNA and/or
that the DNA was associated with particulate non-living material.
Irgens investigated the use of DNA concentrations of activated
sludge as an estimate of the viable population based on the premise
that the DNA released from dead cells was readily degraded by activated
sludge. DNA constituted about four percent of the volatile matter of
_9
cells assuming that weight of one cell was 1 x 10 milligram. DNA
could then be expressed as a percent of volatile solids in the sludge
samples. The bacterial population of sewage, based upon DNA content, was
determined to be 4.1 x 10~ cells/ml of sewage.
ATP Methodology andApplication:
General Perspective - Adenosine triphosphate (ATP) , a high-energy
compound found in every living cell, is constantly re-formed in
metabolism. It functions in a catalytic capacity acting as a link
between reactions that serve as a source of energy for the organisms
and those that lead to biosynthesis and growth. Therefore, as a
catalyst it is conserved throughout the entire metabolic process and
is a relatively constant constituent of the cell . Fermentation,
respiration and photosynthesis are the three major processes used by
cells to extract energy from the environment and make ATP. The
netabolically available energy of ATP lies in the chemical hydrol-
ysis of the a, 3, and y_phosphate groups. The cell uses ATP to make
otherwise endergonic reactions exergonic
10
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NH,
9* 90 9r
HO— P~O— P~O-P-O— CH
o-
O"
OH OH
Adenosine Triphosphate
There are also some specialized energy liberating reactions, such as
"firefly luminescence" that require ATP. Many comprehensive accounts
on the various fundamental aspects and reaction mechanisms of bio-
(18—26)
luminescence and chemiluminescence have been published.
Development of Analytical Methods - This literature survey will con-
centrate on the application of the luciferin-luciferase bioluminescence
system in adenosine triphosphate measurement.
(27 28)
McElroy and Green * pioneered the discovery of the absolute and
integral requirement of ATP for Photinus pyralis (firefly) bioluminescence
and used purified luciferin and crystalline firefly luciferase to characterize
the light emission reaction as being associated with the utilization of both
luciferin and ATP.
11
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Lucifer-Luciferase Reaction
MR++
LH2 + ATP + E . * *. (E - LH2 - AMP) + PP
(E - LH2 - AMP) + 02—» (E - L - 0 ) + Products
(E - L - 0*)-» (E - L - 0~) + hv
E = enzyme luciferase
LH2 = reduced luciferin
ATP = adenosine triphosphate
11
Mg = magnesium ion
E - LH_ - AMP = luciferyl-adenylate complex
PP. = phosphate inorganic
0» = molecular oxygen
hv - photon or quantum of light
E - L - 0 = oxyluciferyl-adenylate complex, excited state
The number of light quanta emitted was determined to be directly pro-
portional to the initial ATP concentration of the reaction system. The
reaction of ATP with luciferin lead to the formation of pyrophosphate
and active luciferin. The latter compound could react with either
oxygen for light production or be hydrolyzed to luciferin and adenylie
acid (AMP) under anaerobic conditions. Moreover, the luminescent
reaction was inhibited by the end product of the reaction. Pyro-
phosphate liberated the enzyme from this inhibitory complex but at
(29)
the same time counteracted the activation step . It was noted that
the light producing step was not the rate-limiting step; luciferin
had to react with ATP before it could be oxidized with light production;
LH--AMP was shown to be the active intermediate and ATP did not act
(20 30)
as an energy donor in the luciferin-luciferase system. '
Considerable research has been performed on developing adenosine tri-
phosphate analysis based upon the firefly luminescence system. Attention
has been focused upon improving the methodology of this very complex bio-
chemical system in the varied biological systems studied.
12
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The basic methodology is initially concerned with complete extraction
of the ATP in the sample and this primary extraction has been performed
both on the reaction mixture as a whole and upon cells washed and filtered
from the growth media. Extraction has been achieved with dilute perchloric
acid , boiling HO, tris (hydro xymethyl) aminomethane (Tris) or N-tris
(hydroxymethyl) Methyl-2-aminoethane sulfonic acid (TES) ^ 5~37) trichloro-
acetic acid (TCAK , or dilute sulfuric acid ». Most recent studies
have shown that in aqueous systems, the ATP of bacterial cells was
quantitatively extracted with boiling (100°C) Tris buffer at pH 7.75^35' .
(31)
Cole found chilling and anaerobiosis of growing cells of E. coli
before extraction with perchloric acid caused a reduction in the ATP
(41)
pool. However, Knowles refuted this in a study of Azobacter vinelandii
where harvested, washed, and starved cells had aerobic ATP levels similar
to cells taken directly from the culture medium. In general, the efficiency
of ATP production was low in disrupted cells (lysis, ultrasonic rupture,
or Parr bomb) compared to ATP production measurements or unbroken cell
(31,32,41)
suspensions .
Preparation of the lucifer-Lucif erase system also has differed greatly.
Lypholyzed and dessicated firefly lantern extracts should be reconstituted
in a magnesium arsenate buffer which will both control the ionic strength
( 38)
of the medium and provide the magnesium ions essential as a catalyst
in the initiating step of the luciferin-luciferase bioluminescence
(18)
reaction . In addition, when ATP and firefly lantern extract (FLE)
are combined in the presence of an arsenate buffer, an intermediate level
of light emission occurs which decays steadily and exponentailly with
(19)
time . Some authors have preferred to use purified luciferin when
analyzing submicro quantities of ATP, since in the crude, commercially
available firefly extracts residual ATP and other high energy phosphate
I whi(
(28)
(42)
compounds are found which interfere with and obscure the analysis
of submicro quantities.
(28)
McElroy and Green observed that the light emission was modified
(38)
by the ionic strength of the reaction medium, and Aledortv evaluated
the optional ionic requirements for firefly bioluminescence. It was
concluded that light emission mediated by firefly luciferin-luciferase
and ATP was inhibited by increasing the ionic strength of the reaction
13
-------
medium. The inhibition of firefly luciferase by ions was shown to follow
+
certain trends, i.e., cationic inhibition occurred when Ca
occurr*
- (38)
Na
Rb > Li > Choline and anionic inhibition occurred when I
Br > C103 > Cl > Fl
HC0
COOCH
HO
/ \
COOH
Firefly D(-) Luclferin
(Active Form)
(23)
Some researchers have preferred to reconstitute the crude firefly
lantern extract (FLE) and incubate it at 0-4°C from 6 to 72 hours to
deplete the endogeneous luminescence associated with the residual
(31 35-38 44)
adenosine triphosphate found in FLEV ' ' . Others have used
a method of luciferin-luciferase enzyme dilution to reduce the enzyme
(29)
blank due to endogeneous ATP and ATP-AMP phosphotransferaseN
Incubating the enzyme with apyrase to eliminate endogeneous ATP and
(42)
lower the blank has also been used and numerous authors have
observed that the enzymatic activity and luciferin content of crude
firefly lantern extract varied from batch to batch thereby requiring
(27)
determination of background emission levels » or variation of
the quantity of enzyme preparation used in the reaction mixture to
(44)
yield a constant for a known amount of ATP.
Instrumental analysis of the photon emission by the firefly assay
also has been subject to a great variety of investigations. Historically,
14
-------
the recording D.C.1P121 photoraultiplier tube was first used to quantify
the light which was followed by a Farrand photomultiplier and the liquid
nitrogen quantum counter; the ultimate sensitivity reached with these
-9 (19 40 45)
instruments was in the order of 10 g ATP/ml ' ' . However, the
liquid scintillation spectrometer was quickly adopted to provide sensiti-
vities to 10 mole ATP/ml^ '. Many difficulties have been
encountered in standardizing the methodology using this instrument
due to the variabilities noted between models and type of liquid
scintillation counters (LSC). Therefore, each investigator has had
(29 31
to adopt his particular LSC to the particular research application ' '
' . Such problems as whether or not to use the "in-coincidence"
or "out-coincidence" mode are still a matter of controversy as is the
voltage to be applied ' ' . When using either a photomultiplier
tube system or a liquid scintillation counter method, a standardized
procedure for constant sample injection or timed reaction counting
has become vital since the kinetics of the light reaction are rapid
(49)
and subject to logarithmic decay. Many researchers have developed
unique apparatus to accomodate these variables ' ' and to achieve
(33)
quick sampling and injection into the reaction mixtures.
Instrument companies such as Du Pont and American Instrument Com-
pany have already recently developed luminescence "biometers" based
upon the luciferin-luciferase system to quantify ATP as a biomass para-
meter . Defresne and Gitelman developed another system
employing a standard Technicon Auto-Analyzer with a Packard flow
detector which in turn was connected to a Pachard Tri Garb 314F to
circumvent the problems of uniform sampling and constant timing,
App1ication and Regult s - Because the amount of endogenous ATP and
the ability of the organisms to further synthesize ATP were important
to bacterial survival, Strange investigated the effects of
starvation on Aerobacter aerogenes. When washed endogenous phase
organisms (10 ) were starved, the viability remained high (100-98%)
for at least 40 hours. Although ATP was required and formed during
this period of metabolism of reserves, the rate of formation of ATP
decreased as the reserves declined during prolonged starvation. The
content of freshly gathered and washed bacteria varied with oxygen
15
-------
tension and solute concentration of the suspending solution; both
anaerobic conditions and high solute concentration markedly reduced
the amount of ATP extracted. The synthesis of ATP which occurred when
bacteria were transferred from anaerobic to aerobic conditions or
from a solution of high to one of low solute concentration was
extremely rapid. Although no direct relationship seemed to exist
between ATP concentration and the viability of survival propects of
this bacterial population, evidence suggested that the ability of
bacteria to synthesize ATP in the absence of exogenous nutrient was
related to survival. Starvation progressively reduced the magnitude
of the increase in ATP which occurred when bacteria were transferred
(34)
from anaerobic to aerobic conditions.
(39 40)
Forrest ' studied the ATP pool in Streptoccoccus faecalis, an
organism with a comparatively simple anaerobic metabolism. It appeared
that a critical concentration of ATP was necessary for exponential ("log")
growth to occur and at levels lower than this critical concentration,
only linear growth occurred. The pool level of ATP of an organism
was defined as the balance between the demands of the organism for the
energy and the supply of energy derived from the catabolism of the
(39)
substrate. '
Holm-Hansen and Hamilton ' used ATP content data of the endo-
genous levels of ATP in laboratory cultured marine species to estimate
the active biomass in ocean samples. The ATP levels found at various
depths of the ocean indicated an oceanographic bacterial population
of 50-2000 times greater than estimated by standard plate culturing
techniques. Studying seven marine bacterial strains grown in a
chemostat, the ATP content per viable cell count (standard plate
count) was always high during a period of log growth and began to
decline during the early endogenous phase of growth. After a short
endogenous phase, all cultures studied showed a marked period during
which the viable cell count decreased drastically while the ATP
(36)
content in the cells decreased to a plateau level.
16
-------
Cole examined the ATP pool in batch cultures of Escherichia
coli since this organism could grow on complex or minimal media both
anaerobically and aerobically with a variety of energy sources. The
measurement of the ATP pool level throughout growth and starvation
would indicate the extent of ATP control by the organism. The rate
of ATP production was in balance with the rate of growth both aero-
-9
bically and anaerobically. The ATP pool (10 moles ATP/mg dry
weight of cells) remained fairly constant during "log" growth.
ATP metabolism in a strict anaerobe, Methanobacterium strain M.O.H.
was investigated and the results of growth yield studies indicated
that the ATP conservation was very inefficient (0.06 moles ATP/mole
(33)
hydrogen) under conditions used to grow the bacterium in a fermentor .
In whole cell studies of this organism, ATP formation was decreased
and AMP formation increased in the presence of air, chloroform, 2,
4-dinitrophenol, carbonylcyanide-m-chlorophenylhydrazone and penta-
chlorophenol. It was suggested that these substrates were either
inhibitors of electron transport or uncouplers of an energy-linked
process. The compunds also inhibited methane formation in cell-free
extracts, an ATP-requiring process.
(41)
Using obllgately aerobic Azobacter vinelandii which possessed a
very active respiratory chain system and no fermentation ATP synthesis,
aerobic ATP pool levels were always high and about the same level with
endogenous substrates and on anaerobiosis the ATP level fell to a quarter
of the aerobic ATP pool level. D'Eustachio and Levin reported the
level of endogeneous ATP in bacteria to be relatively constant for several
species and all phases of growth. Examining thirteen species of aerobic
gram-positive and gram-negative bacteria with the aid of a "Biometer",
a mean ATP content 4.7 x 10 yg/cell was determined. Microbial growth
was related to the amount of ATP present and/or produced by metabolism of
nutrient energy sources. A study of three taxonomically different organ-
isms (Escherichia coli, Pseudomonas fluorescens, and Bacillus subtilis)
during all growth phases yielded a relatively constant level of based
ATP at 1.45 x 10~ yg/cell which may have been low because of sample
pretreatment by sonic cell rupture . This steady-state level of
endogenous ATP suggested that a relatively constant level of cellular ATP
17
-------
was maintained to provide sufficient energy for maintenance for vital
enzyme systems during all growth phases. A linear correlation between
plate count and ATP content was obtained.
(53)
Kao , et al. reported that the rate of ATP increase was closely
related to the rate of growth through lag and log phases. However, con-
trary to other reports, oscillatory variations were followed as the
stationary phase began in pure culture studies with E. coli and
P. aeruginosa. A concentration of 2 yg ATP per ml of MLSS was found most
effective for the waste water having 240 mg BOD,./! in a field study by
f C J \
Biospherics. Excessive ATP was removed from the aeration basins by
lowering sludge return rates and still maintaining the same efficiency
of treatment.
(37)
Patterson and co-workers developed a method for ATP analysis in
activated sludge samples with relative standard deviations for replicate
samples of less than two percent and ATP recovery in "spiked" samples
of 98-100 percent. Results indicated the use of the ATP pool (endogeneous
level of ATP in the system) as an indicator in activated sludge toxicity
studies. The maximum ATP pool measured, approximately 2 pg ATP/ing of mixed
liquor suspended solids (MLVSS) was typical of endogenous ATP concentration
in activated sludge batch units. The maximum ATP pool occurred at pH
7.5 to 8.0 which represented normal operating conditions and was greatly
reduced at low (3.0) or high (11.0) pH conditions. The effect of various
concentrations of mercuric chloride on ATP levels in activated sludge
after one hour incubation indicated a rapid drop in ATP pool at low
I [
Hg levels, a more gradual decrease at intermediate concentrations
I j
and increased rate of pool reduction above 10 mg Hg /liter of sludge.
In studying the firefly bioluminescence analysis of ATP for toxic effects,
it was concluded that any substance which inhibited luciferase activity
would reduce light emission and yield false ATP results. The presence
of mercuric ions reduced the luciferase activity and consequently also
the light emission which effect had to be compensated for by filtration
or dilution of the sample containing heavy metals. Using an endogen-
ous ATP pool value of 2 ug/ml cell material, the pattern of the ATP
pool response to changes in metabolic activity of an activated sludge
18
-------
culture was determined. The results indicated a slow drop in ATP
pool during a 24-hour period prior to feeding with a subsequent constant
level being attained in the unfed sample during the next 24 hours as
(34)
also observed by Strange and co-workers . The immediate response in
the fed sample was a slight drop in ATP, followed by a rapid and significant
increase. The ATP pool was affected by the metabolic activity of an
activated sludge culture and responded rapidly to an increase in substrate
loading while being only gradually reduced as the organisms entered an endogen-
ous phase. The study confirmed previously published reports of a relatively
constant ATP pool under endogenous conditions although the pool in activated
sludge was significantly lower (0.8 ug ATP/mg of volatile suspended solids)
than reported for pure culture experiments (2yg per mg dry cell material).
This lower ATP pool in MLVSS indicated that only a fraction of the total
activated sludge solids was viable cell material. ATP was a specific
indicator of cell viability since ATP content completely disappeared
within 2 hours of cell death. '
Many applications of the luciferin-luciferase bioluminescence system
have been developed. For example, the differential determination of ATP
and ADP in the presence of each other is possible through the use of
myokinase, phosphocreatine may be determined by the use of transphosphori-
fase and AMP, and glucose can be determined by the use of hexokinase
(19)
and ATP by measuring the depression in luminescence . Therefore, any
component in a system which can be made to influence the level of ATP can
be studied by this method.
A method for the estimation of ADP in ethanolic extracts of plasma
was developed based upon the conversion of ATP with a pyruvate-kinase
(43)
system and subsequent assay of the ATP with FLE method . Studies
have been conducted on membrane adenosine triphosphotase and active
transport , the production of adenosine triphosphate in normal cells
(45)
and sporulation mutants of Bacillus subtills and using ATP levels
in food to indicate bacterial contamination .
While the FLE bioluminescence method has an absolute requirement
(21)
for ATP to initiate the reaction, many other bacterial luciferases
catalyze the bioluminescent oxidations of reduced flavin mononucleotides
in the presence of long chained aldehydes. A bioluminescent method,
19
-------
NADH + FMN + H+ dehydrogenase^ _>
z
,__.,. , f, luciferase ,
FMNH- +0- > hv
long chained aldehyde
therefore, has been developed to directly measure the NADH oxidation
in the reductive amination of a-ketoglutaric acid with NH_ by means
of a liquid scintillation counter and method of Stanley in cell-
free extracts of Nitrosomonas europaea.
Brolin has further developed a photokinetic micro assay based
on bacterial luciferases to include many compounds which are either
convertible in dehydrogenase reactions or compounds which are involved
in reactions leading to a dehydrogenase step because they can be
followed by quantitating the bioluminescence produced. The bacterial
luciferase may be coupled to enzyme utilizing or producing FMN or NADH
to produce dynamic measurements in situ or the system may be used for
measuring compounds which are conjugate in their action with NADH .
Dehydrogenase Activity Methodology and Application;
General Perspective - Attempts have been made to improve the method-
ology for analysing dehydrogenase enzyme activity so that it can be
applied as a parameter indicative of active biomass. Various dehydro-
genase enzymes or oxidoreductases are the key enzymes which catalyze
(4)
the redox reactions of biological metabolism . These enzymes catalyze
the oxidation of organic substrate by the removal of hydrogen atoms.
Every dehydrogenation must be coupled with a hydrogenation; the hydrogen
atoms removed from the substrate must be added to some other compound.
Many of the dehydrogenases have associated coenzymes which serve as
temporary acceptors of the substrate hydrogen in the electron transport
system. Two compounds which frequently serve in this capacity are the
pyridine nucleotides, nicotinamide-adenine dinucleotide (DPN) and
nicotinamide-adenine dinucleotide phosphate (TPN )
In the simplest type of enzyme catalyzed oxidation reaction, only one
electron carrier is interposed between the substrate molecule and molecular
20
-------
oxygen. In these dehydrogenase reactions the electron carrier is coupled
to the oxygen and no other substances. However, the dehydrogenase enzyme
can pass electrons to certain reducible dye-stuffs such as tetrazolium
salts(1).
Development of Analytical Methods - Lenhard and Nourse ' '
developed a test to measure dehydrogenase activity and applied this
method to soils, benthal deposits, activated sludge systems, and anaerobic
waste treatment systems. During the aerobic biological treament of organic
wastes, organic carbon is oxidized to CO with a concomitant reduction
of 02 to H20. A tetrazolium salt such as 2,3,5-triphenyltetrazolium
chloride (TTC) can be employed as the hydrogen acceptor during this
dehydrogenase-catalyzed oxidation reaction. The hydrogen released during
the reaction is attached to the salt yielding the highly red-colored
redcued compound, triphenyltetraformazan (TF) which is easily extracted
into alcohol and measured spectrophotometrically according to Beer's
Law. (See Figure 3)
Lenhard and Nourse ' also investigated the effects of pH, tempera-
ture and reaction time during analysis. The optimum pH was 8.4 for samples
obtained from anaerobic and aerobic systems; strongly alkaline pH values
resulted in possible tetraformazan production. The optimum temperature
for the incubation of the tetrazolium chloride with activated sludge and
anerobic digestion samples was 37°C. Batch studies indicated an increase
in dehydrogenase activity within one hour after substrate addition and
continuous culture (extended aeration) studies indicated that the dehydro-
genase test offered the possibility of measuring the availability of
endogenous substrates by comparing the reduction of TTC with and with-
out added glucose. In the absence of glucose, low values of dehydrogenase
activity would indicate a low concentration of endogenous metabolites
available for oxidation. Without siginficant modification of the analysis,
the dehydrogenase activity in anaerobic samples was tested both in the
presence of air and in vacuo; the in yacuo test results were slightly
higher but the difference was not considered significant enough to warrant
the further complication of the method. The rapidity of the dehydrogenase
test (1 hour), allowed for the detection of impending process difficul-
ties before any change in the quality of effluent could be measured.
21
-------
Organic
Material
— 2H
Oxidized Organic
Material
ADP P. ATP
NAD H2 FADH
-2H|f+2H -2H
NAD FAD
2,3,5 Triphenyl
Tetroformozan
( RED)
iCyeto ] crom
l/2 0,
Electron Transport
System
+• Microorgonismus
4-Dehydrogenaseenzymes
4-2,3,5, Triphenyltetroiolium Chloride
( Colorless )
2CH3— CH — C — OH
OH
Dehydrogenose ^ 2 CH,-C — C—OH -I- 2H
• *
Substrate
Enzyme
Oxidized Substrate
2H
)— C
\
—N-(O/
CL
,N NH
HCL + (0)-c
\
2,3,5 Triphenyltetrazolium
Chloride (Colorless)
2,3,5 Triphenyltefraformozan (Red)
Figure 3. Transfer Mechanisms of Intermediate Substrate Metabolism
22
-------
Bucksteeg expanded Lenhard^ work to include the evaluation of a
prototype activated sludge plant and improved the methodology to include
the exclusion of light during the incubation period which he extended
to one hour to eliminate the photochemical effect. These investigations
indicated that the overall activity of dehydrogenases in a wastewater
and activated sludge mixture increased with increasing concentrations of
the dry mass of sludge, whereas the biochemical efficiency of the sludge
relative to unit weight of dry mass decreased.
Ford further investigated the dehydrogenase activity reaction
time for samples obtained from a contact stabilization process and
determined that the production of tetrafortnazan declined rapidly after
60 minutes and was a direct function fo sludge age. The TTC-measured
dehydrogenase activity responded to significant changes in plant loading.
Jones examined the effect of extended aeration on the dehydrogenase
activity. Oxygen was demonstrated to be a competitive inhibitor in
TTC—»TF reduction. The reaction reached the same level of reduction
aerobically as it did anaerobically but it took longer to attain that
level (characteristic of competitive inhibition). It was concluded
that the dehydrogenase test more accurately measured the total biological
activity of an anerobic system since molecular oxygen acted as an inhibitor
and competed with TTC as a terminal hydrogen receptor.
(ft? "i
Shih and Stack used the dehydrogenase test to explore the
temperature effects on energy-oxygen requirements in an aerobic biological
oxidation process. Energy-oxygen was defined as the net consumption of
oxygen in support of synthesis reactions. The methodology was refined
during the studies to include the extraction and concentration of TF
with 1-butanol because all the TF produced (including that associated
with the solids) was concentrated in the limited butanol system. To
preclude a possible TF production interference by dissolved oxygen, it
was removed with sodium sulfite prior to the addition of TTC. The energy-
oxygen coefficient developed during the studies varied with substrate
oxidized and temperature conditions.
23
-------
(f T \
Marlar noted the inhibitory effect of oxygen on the dehydrogenase
test and by scrubbing the samples with nitrogen, increased the color
intensities and the reproducibility of the reduced and extracted TF.
Ghosh included initial cell disruption and a one-hour sample incubation
at 37°C with TTC under a nitrogen atmosphere to eliminate the effects of
dissolved oxygen and microbial growth during the test period. The dehydro-
genase activity was proportional to the solids concentration and the con-
stant of proportionality was independent of growth rates for a given micro-
bial population and substrate composition.
Dean and Rodgers investigated the steady-state levels of dehydro-
genases of Aerobacter aerogenes in a variety of nutrient-limiting chemostats
at various dilution rates between 0.1 and 1.0 hr . These dehydrogenase
activities were determined by the TTC reduction method and it was shown
that the dehydrogenase activities were generally higher when sugars provided
the carbon for growth than in other nutrient-limiting conditions. Randall^ '
investigated the use of dehydrogenase activity as a predictive parameter
for activated sludge drainability. Results indicated that a dehydrogenase
activity to solids ratio of less than 0.6 ymoles per gram of solids was
assurance of good sludge drainability.
24
-------
SECTION IV
INTRODUCTION
This research was initiated to define the applicability and limitations
of the dehydrogenase test for the measurement of active biomass content of
biological sludges used in the treatment of domestic and industrial waste-
waters. During the studies reported herein, experimental apparatus were
constructed, analytical techniques established, and pure and heterogeneous
batch culture and continuous culture studies with and without sludge re-
cycling were conducted.
Experimental Apparatus;
Pure Culture Studies - In this phase of the experimental studies a
pure culture of £_._ coli was grown in a minimal substrate (Table 1). The
ractor system was designed to permit aseptic techniques and to eliminate
possible external contamination. To accomodate this requirement, three
double sidearm, water-jacketed one-liter Spinner flasks were arranged
as shown in Figure 4. Both sidearms of each flask were sealed with
serum stoppers and hypodermic needles were passed through them to allow
for both gaseous interchange and for feeding and sampling. The compressed
air used for aeration was filtered through a Gelman filter holder contain-
ing a 0.45u, 25 mm diameter filter paper prior to its introduction and
diffusion into the culture medium.
Inside the flask reservoir, a glass air diffuser was attached with
tygon tubing to the needle extending through the serum stopper to provide
for aeration below the substrate level in the reactor. An air outlet
port was provided by attaching a cotton-plugged syringe barrel to a
hypodermic needle inserted through the stopper next to the air inlet.
The sampling port consisted of a similar arrangement including a length
of tygon tubing attached to the needle and extending well below the
surface of the substrate in the reactor. The cotton filled syringe
could be removed aseptically from the needle and either a filled or an
empty sterile syringe could be attached for feeding or sampling res-
pectively.
25
-------
TABLE 1
Minimal Substrate* for Batch Cultures
Nutrients
(NH4)2S04
Substrate
MgCl2'H2O
CaCl3'2H2O
FeCl3«6H2O
KH2P04
Na2HPO4
Concentration of
Stock Solutions
18.857 g/1
As selected
32 g/1
400 mg/1
400 mg/1
5.4436 g/1 (0.04M)
5.6784 g/1 (0.04M)
Volume of Stock
Solution Used
in the Reactor
As selected
As selected
5 ml
5 ml
5 ml
Final Concentration
in the Reactor
to maintain
C:N of 20:1
160 mg/1
2 mg/1
2 mg/1
Use equimolar vol-
umes of both and
bring final volume
up 1 liter, which
yields :
pH=6.88 and 0.08M
POj concentration
ro
* Minimal Substrate: a simple synthetic medium consisting of ammonium salts,
phosphates, sulfates, and other mineral salts with the addition of organic
compound as a source of carbon and energy.
-------
AIR LINE
COTTON FILTER
AIR OUTLET
'•;".;- •."•:• "M
;;
f
\
WATER BATH
RESERVOIR
PUMP
Figure h. Reactor Assembly for Pure Culture Studies
-------
A magnetic stirrer was placed beneath each reactor to provide internal
mixing by rotation of the magnetic impeller inside the flasks. A piece
of asbestos was placed between each reactor and the stirring apparatus
to control heat transfer. The temperature in the reactors was maintained
at 37°C by circulating water from a water bath through the water Jackets
surrounding each culture flask.
Prior to each experiement, one liter of substrate was measured into
the reactor flask and sealed with a dome-shaped top containing an inner
sleeve to align the impeller assembly in the center of the substrate
reservoir. The top was secured by a heavy wire flange clamp arid the
complete assembly was then autoclaved at 15 psi and 120°C for 15 minutes.
Heterogeneous Culture Studies - The 10-liter volume reactor unit is
schematically shown in Figure 5. The essential elements are the reactor
in which biological growth occurs, a mixing motor and propeller, an
air diffuser located in the bottom of the reactor, a variable speed
influent pump, and an electronic level controller. Compressed air was
supplied throughout the aerobic batch and continuous culture studies.
The air supply was regulated by Air Flowmeter (SHO-Rate, Model 1355,
Brooks Instrument Div., Emerson Electric Co.).
During the batch culture studies, nutrient media similar to that
used during the pure culture studies were seeded and pumped into the
reactor followed by a predetermined amount of selected substrate.
The mixing units consisted of a B & B Motor (1725 rpm, 115 V, 1/8 HP,
NSH 54 type) and controller (B & B Motor and Control Corp., N.Y., N.Y.)
and provided complete mixing along with air diffusion. The reactor
temperature was maintained at 20°C during these studies.
In the continuous culture study, both nutrient media and substrate
were stored in a reservoir and pumped into the reactor at a selected
flow rate to give a desired retention time. During the anaerobic
digestion study, however, the air supply line was sealed off and the
level controller was employed to regulate the reactor volume. An
effluent pump (Model 7015, Master Flex Pump, 500 rpm, 115 V. Cole-
28
-------
MOTOR CONTROLLER
VO
LEVEL
CONTROLLER
TEMPERATURE CONTROL
CULTURE
MEM
Figure 5- Reactor Assembly for Heterogeneous Culture Studies With
Solids Recycle
-------
Farmer Instrument Co., Chicago, 111.) was connected to the effluent
line and either activated or deactivated by the level controller as
soon as the reactor contents went above or fell below the 10-liter
mark, respectively. A temperature of 37°C was maintained by exterior
heating tape and a heating element inside the reactor controlled by
a temperature controller. During the sludge recycle studies, a
2.7-liter clarifier and recycle pump (115 V, 7.5 rpm. Gorman-Rupp
Corp., Bellville, Ohio) were connected to the reactor system as shown
on Figure 5. The biological seed was obtained from the activated sludge
process and anaerobic digester of the South River Water Pollution Control
Plant in Atlanta, Georgia for the aerobic and anaerobic studies res-
pectively.
Pure Culture Preparation;
The test organism for the pure culture studies, E. coli was cultured
,for 18 hours on a nutrient agar slant and then washed from the slant with
three separate applications of distilled water, concentrating the cells
by centrifugation after washing. The washed cells were resuspended in
sterile water and a known volume of E. coli suspension was used to
innoculate each reactor. Nutrient agar plates were also streaked at
this time to determine the absence of contaimlnation.
In some of the batches, growth and substrate were monitored from
the time of initial innoculation whereas in other cases, the population
was allowed to acclimate to the substrate over night (about 16 hours)
after which time additional substrate was added. The analyses were
then followed from the beginning of substrate removal thereby avoiding
the lag period observed when the population was first acclimating to
the culture medium. In either case, log growth was easily observed.
Analytical Techniques;
Each batch conducted during this phase was monitored by standard
plate counts, Coulter Counter enumeration, substrate removal, total
suspended solids determination, dehydrogenase activity and, on occa-
30
-------
sion, adenosine triphosphate (ATP) content. The analytical deter-
minations required a minimum sample volume of 30 ml and at least 30
minutes between samplings. The various tests could be performed con-
currently and some samples could be stored and frozen for later
analysis. When substrate volumes were reduced below 400 ml by samp-
ling, the culture medium was diluted back to one liter by addition
of sterile phosphate buffer solution. Usually one day was required
to prepare and sterilize the reactors, innoculate and grown the cul-
tures, and to prepare the necessary reagents and sampling vials
before a batch study could be initiated.
Coliform Analysis - The Millipore Filter plate count technique
for the analysis of coliform as described in Standard Methods for the
Examination of Water and Wastewater (Standard Methods) was employed
in these investigations for E. coli. A 1.0 ml sample of the proper
dilution was filtered and rinsed through a stainless steel Hydrosol
filter holder and the bacteria were retained on a 0.45y pore size, 47 mm
diameter, sterile Millipore filter. The filter was then placed onto a
sterile pad soaked in 2 ml of MF Endo Broth in a sterile petri-dish,
inverted and incubated at 37°C for 18 to 24 hours. The coliform analysis
was initiated as soon after the sample was drawn in order to prevent
growth or culture attenuation. The filter holder was maintained in
a sterile condition by exposure to ultraviolet light for at least one
/go\
minute before each filtration . Results were generally available
within 24 hours of sampling.
Solids Determination - Total suspended solids were determined gravi-
metrically on 0.45y, white grid, 47 mm Millipore filters. The filters
were Individually washed with distilled water under vacum for approxi-
mately two minutes in order to remove the glycerine and wetting agent
and to insure a constant tare weight. Each filter was numbered, dried
for 30 minutes at 103°C and desiccated for at least 30 minutes before
weighing. Filters were also stored in the desiccator prior to use.
An exact volume of sample was vacum filtered through the Millipore
filter and the filtered solids (cells) were dried with the filter at
103°C for 30 minutes, desiccated for at least 30 minutes, and then
weighed. The tare weight was subtracted from the final weight obtained
-------
and the difference was mutiplied by the volume factor of that sample
thereby giving the results in mg/1. The samples were analyzed for solids
shortly after removal from the reactors to prevent settling, clumping a*nd
possible changes in bacterial populations. The very low populations
(approximately 10 organisms/ml) and the limited sample volume (10-20 ml)
characteristic of the pure culture batch studies required considerable
care in analysis since samples often yielded less than a 10 mg weight
change. Later solids analyses were improved (Batches 7-10) by using a
double filter-tare method (Appendix A).
Determination of Substrate Concentrations - Various methods of
substrate determination were employed depending upon substrate and
technique available. Glucose and galactose concentrations were
monitored by "Glucostat and "Galactostat assay techniques
which measured the substrate concentration spectrophotometrically.
Standard glucose or galactose solutions of known concentrations were
determined with each set of samples analyzed in order to circumvent
the problem that slight changes in the incubation times of the sample
with the test reagent produced variations in color intensities even
with identical glucose or galactose concentrations. In addition,
all samples were filtered through a glass fiber filter to rid them
of bacterial solids which would otherwise assimilate glucose or
galactose during the test period and also contribute turbidity inter-
ferences during absorbance measurements. The glucose samples were
incubated with the test reagents for 30 minutes and the color developed
was stable for at least 12 hours thereby permitting delayed Beckman
DU analysis. The galactose samples required much more care in analysis
since the color development during the incubation period frequently
was poor and unstable so that samples could not be stored more than
3 hours.
Chemical oxygen demand (COD) determinations provided a measure of
the oxygen equivalent of that portion of organic matter in the sample
subject to oxidation. In monitoring sucrose in pure culture studies
with E^ coli, the COD assay was employed according to Standard Methods.
All samples were filtered through glass fiber filters to rid them of
bacterial solids which were also susceptible to strong oxidation.
32
-------
The samples were refluxed with known, amounts of potassium dichromate
and sulfuric acid, and the excess dichromate was titrated with ferrous
ammonium sulfate. The amount of oxidizable organic matter measured
as oxygen equivalent was proportional to the potassium dichromate
consumed.
The Beckman Total Carbon Analyzer was used to monitor the acetic
acid and L-alanine substrates in pure culture studies (Batches 8 and 10).
All samples were filtered through Millipore filters (0.45 u) and preserved
with HC1 to prevent further growth before analysis. When the sample was
injected into the Total Carbon Analyzer, it was completely combusted to CXL
and the CO was analyzed in the instrument by infrared spectroscopy.
Appropriate standards were analyzed and treated in the same manner for
calibration purposes.
Gas-liquid chromatography was also employed to follow the acetic
acid uptake (pure culture Batches 8 and 9). A model 700 F&M gas
chromatograph with hydrogen flame ionization detector and six feet,
1/8 inch-diameter stainless steel columns packed with 20% carbowax
4000 and TPA on 60 to 80 mesh WAWDMCA (high performance chromosorb
W, acid washed, silanized) was used. All samples were filtered
through Millipre filters (0.45 y) and preserved with HC1 to prevent
further growth. Retention time was five minutes using this column
packing with excellent resolution.
L-alanine in Batch 10 was monitored on both the Beckman Total Carbon
Analyzer and the Technicon Auto Analyzer for ammonia and total nitrogen.
Based upon the calculated value of the total nitrogen available in the
standard solution of pure L-alanine (1200 mg L-alanine or 192 mg nitrogen),
the results obtained from the Auto Analyzer indicated that apporximately
one-half (100 mg) of the total nitrogen content of L-alanine was available
as ammonia. The possible explanations for this inconsistency were (1)
the amino group of L-alanine was hydroloyzed upon standing in aqueous
solution, (2) the pure L-alanine crystals were contaminated with ammonia
salts, or (3) the amino group was cleaved from L-alanine when coming
into contact with the strong digestion mixture (H^SO,, HC10,, and catalyst)
used in the auto-analysis procedure.
33
-------
Coulter Counter Analysis - A methodology for employing the Coulter
Counter for correlation of bacterial populations with plate counts was
developed in accordance with the experiences of Swanton . By diluting
a known volume of sample in Isoton (particle-free saline), the bacterial
population at the time of sampling was fixed thereby allowing delayed
analysis. A 1.0 ml sample was most frequently used to provide an adequate
counting range; however, on occasion, the sample required further dilution
when the organism concentration exceeded 10 organisms per ml.
To eliminate the interference of very high background noise, a metal
wire mesh "cage" was positioned around the sampling namometer and aperture
tube stand of the counter. The instrument was calibrated with 3.49 u
diameter latex spheres and windowed to count all particles between 3-5 u
diameter. A 50 u aperture tube was employed and a 0.05 ml sample aliquot
was counted. To obtain a recordable population count, each sample was
counted five times, the total count averaged, corrected for background
and coincidence, and multiplied by the appropriate dilution factor. In
later batches (7-10), a multiplication factor of 100 was used for data
interpretation.
Adenosine Triphosphate (ATP) Analysis - The first method employed for
(37)
ATP analysis was patterned after the work of Patterson e_t al_. This
method entailed the preparation of a luciferin-luciferase enzyme reagent
and ATP standard solutions, ATP extraction from the samples, and determina-
tion of ATP content in the samples by counting the light emissions with a
Packard Tri Carb Liquid Scintillation Counter (Model 3320).
The luciferin-luciferase enzyme preparation required dissolving one
vial of Sigma desiccated firefly tails in 37.5 ml of deionized distilled
water and allowing it to stand at room temperature for one hour. The
solution was then filtered through a Watman #3 filter and then allowed
to incubate in an ice bath for 24 hours. The ATP standards were pre-
pared in 0.025 M tris buffer to desired concentrations.
For sample analysis, 2.0 ml of sample were transferred to 50 ml
NPN tubes containing approximately 40 ml of boiling tris buffer (0.025 M,
pH 7.75) and held in boiling water for 10 minutes with occasional shaking
34
-------
to kill the bacterial population and extract the ATP quantitatively.
The tubes were then rapidly colled and brought to volume with additional
tris buffer. For an immediate assay, the tube was then placed in an ice
bath; for later analysis, the samples were frozen at -20°C.
The scintillation counter was set with a gain at 100 percent amplifi-
cation, a window setting of 50-1000 on the "Red Channel", and the coin-
cidence mode was switched to off. Counts were taken for 0.1 min (6.0
seconds) and background from the luciferin-luciferase preparation was
measured prior to each analysis. Samples were inserted into the counter
by transferring 1.5 ml of enzyme preparation into a scintillation vial
followed by 0.5 ml of ATP sample (or standard) and mixing. Since the
luminescence decayed exponentially with time, the interval between addi-
tion of the ATP to the enzyme preparation and the initiation of the
counting sequence was carefully controlled. The data was analyzed
graphically due to the random variability of 6-second counts, extrapolating
the line of best fit back to one minute since exponential decay commonly
began one minute after the reaction was initiated. The graphical tech-
nique provided linear standard curves for a wide range of ATP concentrations,
Attempts to apply this methodology to pure culture batch studies during
the investigations have proven inconclusive and have further emphasized
the sensitive nature of the analytical procedures particularly where
relatively low biomass concentrations were developed. This sensitivity
can be ascribed to the necessity of control of physical parameters during
analysis. For example, the temperature at which ATP extraction from the
cells takes place has been found to be very critical and a few degrees of
temperature fluctuation will greatly change the ATP yield ' . More-
over, the pH and the volume of both the sample being extracted and the
sample aliquot being measured in the liquid scintillation counter are
very critical . Exact and consistent timing of all steps in the pro-
(29)
cedure is essential
Considerable difficulty has been experienced in standardizing these
variables and generating reliable and reproducible data. Adaptation of
the liquid scintillation counter became questionable primarily because of
the relatively low organism populations and the necessity for switching
35
-------
the counter out of coincidence mode of scaling due to the danger of
"stripping" the photomultiplier tubes when high intensity light is emitted.
The possibility of reducing the light intensity through both enzyme
dilution and aging, and decreasing the volume of sample aliquot being
counted were investigated without success. As a consequence, this
method was abandoned at this point.
The second method employed for ATP analysis was as proposed by
McElroy, e_t al^. ( ' This method entailed the preparation of a luciferin-
luciferase enzyme reagent and ATP content photometrically through con-
version of the light intensity and its proportional transfer to a digital
readout unit. The instrument was calibrated for each reaction mixture so
that the ATP concentration was read directly. The luciferin-luciferase
enzyme was supplied with a buffer salt in tablet form. After dissolving
one tablet of buffer salt in 3.0 ml of ATP-free Low Response Water (acid-
ified, boiled, neutralized to pH 7 with NaOH and autoclaved distilled
water), one vial of enzyme substrate was added and 0.1 ml transferred
into each reaction cuvette with an automatic pipettor.
In preparing the ATP standard, 100 ml of fresh 0.01 M morpholinopropane
sulfonic acid (MOPS) buffer and 100 mg of crystalline adenosine-51 -
triphosphate-disodium salt were mixed to make a stock solution. From this
solution, serial 1:100 or 10:100 dulutions with 0.01 M MOPS were made until
o
the final ATP concentration was 0.1 ug ATP/ml or 1 x 10 fg (femtogram)
ATP/ml. This final solution was dispensed in about 0.5 ml aliquots into
clean cuvettes, capped, frozen and stored. The frozen standard was thawed,
brought to room temperature and injected into the reaction mixture when
the samples were ready for analysis.
The ATP extraction method for the samples by Dimethysulfoxide (DMSO)
preceded by freezing and thawing was chosen as recommended by DuPont.
The extraction procedure is included in Appendix B. A comparison of
the ATP extraction method by DMSO and by nitrogen bombing was also
accomplished in an attempt to shorten and simplify sample preparation
as indicated in Table 2 and Figure 6 for data obtained from selected
batch and continuous culture studies. In the nitrogen bombing method,
the samples were treated in a Parr Bomb by exposing at 30 atmospheric
36
-------
TABLE 2
Comparison of ATP Contents Extracted by DMSO and by Nitrogen Bombing
unit: fg/ml
DMSO
6.78 x 107
8.56 x 107
,1.46 x 108
8.23 x 107
1.03 x Id7
1.9. x 107
1.31 x 107
3.46 x 107
1.56 x 107
1.27 x 107
9.60 x 106
8.46 x 106
8.52 x 106
9.48 x 106
7.26 x 106
7.56 x 106
Bombing
1.81 x 107
2.10 x 107
3.09 x 107
1.47 x 107
3.84 x 106
5.67 x 106
4.47 x 106
1.50 x 106
1.03 x 106
7.10 x 105 ,
1.22 x 106 i
1.64 x 106
5.03 x 105 :
1.13 x 106 1
6.29 x 105
8.56 x 106
Sources
Aerobic/
continuous
Cultures grown on
i
glucose.
n n
n n
tf it
M ii
Anaerobic batch
cultures grown on
leachate.
n n
n n
ii n
ii n
n n
n ii
DMSO
5.60 x 107
3.60 x 107
8.82 x 106
5.22 x 107
3.80 x 107
9.20 x 106
3.60 x 107
2.60 x 107
4.80 x 107
1.70 x 107
1.55 x 107
1.93 x 107
2.33 x 108
1.36 x 108
8.94 x 107
2.94 x 107
Bombing
1.25 x 107
3.75 x 106
2.14 x 105
2.78 x 106
5.41 x 106
8.20 x 106
1.00 x 107
4.10 x 106
7.40 x 106
5.13 x 106
2.02 x 106
2.80 x 106
7.17 x 107
3.63 x 107
2.96 x 107
4.61 x 106
Sources
Aerobic continuous cultures
Grown on chicken processing
wastes.
n n
n ii
Aerobic continuous cultures
grown on fish processing
wastes.
ii n
n ii
Aerobic batch cultures
grown on acetic acid.
n ii
n n
n n
n n
-------
*'
10'
E
a>
108
w
JO
i
a 7
510
10
6
Itttii ;
• Aerobic continyoui "itk (lucose
Q Unatrobit bitch ititk Itackate
A Aerobic continuous witk ckickti wastes
a (erobic continwis witk fisk waste
O Aerobic batch *ith acttic acid
/
/
£
0
i? i?"
ATP by DMSO.fg/ml
Figure 6. Comparison of ATP Contents Extracted by DMSO and by
Nitrogen Bombing
38
-------
pressure under nitrogen and stored in the freezer until ready for
Biometer analysis.
As shown in Figure 6, there was no definite numerical relationship
observed between the amount of ATP extracted by DMSO and by nitrogen
bombing, even though general similarity was shown over the indicated
ranges of concentrations. The nitrogen bombing technique did not
show as much reliability or reproducibility as the DMSO extraction
method. The amounts of ATP extracted by DMSO preceded by freezing
(Table 3) yielded the highest values, while those by nitrogen bombing
with freezing resulted in increased concentrations but much lower than
those observed by DMSO extraction. As a consequence, the DMSO extrac-
tion method was employed for the ATP analysis throughout present studies,
However, the continuous culture studies indicated definite relation-
ships between the two methods in certain retention time ranges; i.e.,
ATP concentration by bombing vs. by DMSO was close to 90 percent at
20 hours and 30 percent near 12 hours or shorter retention times.
Under consistent operation schemes as those used during activated
sludge treatment or other biological processes in the field, this
relationship would possibly be constant and the bombing method could
therefore give satisfactory results. This consistency is demonstrated
on Figure 6 where the higher magnitude correlations were obtained at
longer retention times" for the continuous culture study with glucose.
Additional effort must be directed toward definition and/or develop-
ment of a possible correlation between the two methods of ATP extrac-
tion of samples from aerobic and anaerobic processes during the sub-
sequent field studies.
Dehydrogenase Analysis - Numerous investigators have attempted
to develop a standardized method for the measurement of dehydrogenase
as an indication of active biomass. Most work has been performed
with heterogeneous continuous culture studies or pilot plant studies
of activated sludge systems. However, these studies were limited
to systems with high microbial populations as stimulated by non-
limiting substrate levels. The study reported herein emphasized
application with pure and heterogeneous cultures with various selected
substrates.
39
-------
TABLE 3
Effects of Freezing and Nitrogen Bombing on ATP Contents Extracted
Before Freezing
After Freezing
After Refreezing
Nitrogen
Bombing
fg/ml
1.18 x 106
1.07 x 106
1.82 x 107
2.03 x 107
DMSO
Extraction
fg/ml
1.06 x 108
1.14 x 108
1.23 x 108
8.90 x 107
-------
The method adopted for these investigations was proposed by Ghosh
and included initial cell disruption and incubation of the sample with
2,3,5-triphenyltetrazolium chloride under a nitrogen atmosphere to
eliminate the effects of dissolved oxygen and microbial growth during
the test period. The inhibitory effect of oxygen on this method was
tf o\
first observed in these laboratories by Marlar , when nitrogen was
bubbled through the test samples, the color intensities of the
tetraformazan became highly reproducible. The apparatus used in these
investigations is illustrated in Figure 7 and the analytical method
is detailed in Appendix C.
To properly interpret the results with respect to dehydrogenase
activity, it was necessary to be able to express a relationship between
dehydrogenase activity and biomass concentration which would permit
direct conversion of the analyses for dehydrogenase activity obtained
during these studies to an expression of active biomass. Since the
pure culture studies of this phase of the investigation utilized sub-
((."}}
strates similar to those reported by Ghosh , the empirical equation
developed by this author was also employed in these studies in accor-
dance with the following:
X = 4.4 + 536 A (1)
where:
X = biomass concentration, mg/1
A = dehydrogenase activity
Equation 1 was developed by multiple regression analysis of data from
a series of soluble substrate studies utilizing glucose and galactose in
which the biomass concentration was plotted against the dehydrogenase
activity as absorbance at 1.0 cm light path and 483 my wavelength. These
data and the resulting equation of the curve of best fit are shown on
Figure 8 together with the data obtained for substrates used during
these studies.
41
-------
SCREW COMPRESSOR MANIFOLD
CLAMP
FLOWMETER
NITROGEN
NPti DIGESTION TUBE
37°C WATER BATH
Figure 7- Apparatus for the Dehydrogenase Test
-------
0
x = 4.4 4- 536 A
Ltgtnd: A Goloeto« ( Ghosh)
O Glucose (Ghosh)
Q Glucose (This Study)
Galoctose (This Study )
Acelic Acid (This Study)
400 600 1200 1600 2000 2400
D«hydrofltnost Activity (Abserbonc* CP ' *"• Light Path , A )« I04
Figure 8. Correlation Between Dehydrogenase Activity and Biomass
Concentrations
43
-------
One of the difficulties experienced with the dehydrogenase measure-
ment was that when the samples were homogenized in Waring Blender, little
color was developed and large analystical variations existed between
duplicate sampels. Moreover, since the untreated samples had more
color than the homogenized samples, the use of the blender was dis-
(72)
continued and a cell disruption technique was
this variance and insure consistency of analysis.
(72)
continued and a cell disruption technique was developed to eliminate
In this method, the samples were treated in a Parr Bomb by exposure
at 30 atmosphers of nitrogen. Better color development was experienced
than with the untreated or homogenized samples and little significant
deviation was noted for duplicate samples, Moreover, as indicated
in Table 4, there was good correlation between the dehydrogenase
activity of samples analyzed immediately after distintegration in
the Parr Bomb and those frozen after treatment in the Bomb and
analyzed after thawing. This latter observations permitted storage
of samples for subsequent analysis in number.
44
-------
TABLE 4
Comparison of Sample Pretreatment Method for Dehydrogenase Analysis
Sample
Number
1A
IB
1C
2A
2B
2C
2D
2E
2F
3
4
5
6
7
Untreated
Sample
A W
0.1805
0.2381
0.1221
0.0883
0.1261
0.0841
0.1013
0.1255
0.0783
X (mg/1)
99. 9 w
135.2
66.3
51.2
72.5
48.9
57.5
71.9
44.6
Homogenized
Sample
A
0.0339
0.0283
0.0232
X
20.9
17.7
14.9
Disintegrated
Sample
A
0.2050
0.1760
0.1690
0.1840
0.1800
0.1640
0.0306
0.0381
0.2299
0.0391
0.1319
X
116.7
100.5
96.6
104.9
102.5
94.9
18.9
23.2
130.4
23.8
75.2
(c)
Disintegrated &
Frozen Sample
A
0.0320
0.0357
0.2182
0.0320
0.1249
X
19.8
21.9
124.1
19.8
71.9
(a) Homogenized in Waring Blender at 15,000 rpm
(b) Cell disruption by Parr Bomb
(c) Analyzed after thawing
(d) Optical density
(e) Dehydrogenase activity calculated from the empirical equation.
-------
SECTION V
PRESENTATION AND DISCUSSION OF RESULTS
Batch Studies with E. coll:
A representative sample of the data obtained during the various
batches utilizing glucose and galactose innoculated with E. coli are
included in Table 5 through Table 15 and on Figure 9 through Figure 19.
Each of these tables and figures indicate changes in substrate con-
centration, Coulter Counter enumeration, plate counts, suspended solids
concentration and dehydrogenase activity as expressed by the empirical
relationship or Equation 1 and Figure 8.
Various trends were established as the culture progressed through lag,
log growth and eventually endogenous phase after the substrate was nearly
depleted. Dehydrogenase activity was first observed at a limiting popula-
tion of approximately 10 organisms/ml and increased during log growth.
A similar behavior was observed for the solids analysis except, whereas
the solids concentration reached a limiting value for any one batch and
then leveled off for an extended period of time, dehydrogenase activity
declined immediately after the substrate had been essentiallt depleted.
The plate count and Coulter Counter data also followed dehydrogenase
activity during log growth but plate counts decreased during the endogen-
ous phase whereas the Coulter Counter enumeration remained more constant.
Not until the batch studies had been extended for a considerable period
beyond the time of depletion of the substrate did the counts and suspended
solids concentrations begin to decrease. The refined analytical technique
showed these trends more clearly in the later batches. (Appendix A)
The data in the tables and figures indicated that dehydrogenase
activity as defined was a more sensitive indicator of the activity of
the biomass with respect to its response to the growth limiting sub-
strate during exponential growth as well as when the substrate had
-------
reached a limiting concentration. During exponential growth, the
plate count, Coulter Counter, suspended solids and dehydrogenase
activity indicated similar trends. This similarity between parameters
is illustrated on Table 26 which also suggested a change in ratio be-
tween suspended solids and dehydrogenase activity as the carbon source
changed.
47
-------
TABLE 5
Pure Culture Batch No. 1 With Glucose
Time,
hour
0
1.75
2.16
2.50
2.75
67.00
117.0
157.00
Substrate,
mg/1
230.0
154.0
127.0
124.0
113.0
-
_
-
Biomass Concentrations By
Dehydrogenase
Activity, mq/1
-
38.0
-
54.0
61.0
21.0
18.3
18.7
Suspended
Solids, mq/1
-
130
-
140
180
-
-
220
Plate Counts
cells/ml
1.2 x 106
1.5 x 107
-
1.1 x 107
1.8 x 107
1.3 x 107
-
2.0 x 103
Coulter Counter
cells, ml
1.3 x 107
1.5 x 107
-
2.1 x 107
2.2 x 107
4.3 x 107
-
7.1 x 106
oo
-------
TABLE 6
Pure Culture Batch No. 2 With Glucose
Time,
hour
0
1.75
2.16
2.50
2.75
67.00
117.00
157.00
Substrate,
mg/1
420.0
324.0
—
222.0
214.0
-
-
-
Biomass Concentrations By
Dehydrogenase
Activity, mg/1
_
38.0
85.0
123.0
121.0
31.0
21.6
21.0
Suspended
Solids, mg/1
_
160
180
190
_
-
—
220
Plate Counts
Cells/ml
1.2 x 106
1.5 x 107
1.6 x 107
1.6 x 107
2.3 x 107
1.28x 107
-
1.7 x 105
Coulter Counter
cells/ml
1.9 x 107
2.2 x 107
2.4 x 107
2.1 x 107
3.0 x 107
-
-
1.03 x 107
VO
-------
TABLE 7
Pure Culture Batch No. 3 With Glucose
Time,
hour
0
3.00
5.00
6.00
7.00
7.50
12.50
21.00
21.75
23.25
24.50
26.25
29.00
30.00
49.50
75.00
107.00
Substrate,
mg/1
1,475.0
1,125.0
1,150.0
1,092.0
948.0
782.0
312.0
253.0
188.0
145.0
143.0
110.5
64.4
33.5
_
_
-
Biomass Concentrations By
Dehydrogenase
Activity, mg/1
-
18.9
23.2
52.7
130.4
-
156.0
-
-
65.1
60.5
52.2
-
25.4
-
-
-
Suspended
Solids, mg/1
_
-
30
55
125
-
-
-
-
230
180
220
140
-
85
80
190
Plate Counts
cells/ml
1.42 x 107
1.20 x 107
8.0 x 106
2.0 x 107
4.0 x 107
-
-
-
-
2.2 x 108
2.4 x 108
1.8 x 108
4.0 x 108
6.6 x 107
3.0 x 107
3.0 x 107
3.4 x 105
Coulter Counter
cells/ml
1.9 x 106
2.0 x 106
2.3 x 106
7.8 x 106
4.4 x 107
-
-
-
-
1.2 x 107
1.1 x 107
8.4 x 106
1.4 x 107
5.2 x 106
6.4 x 106
4.3 x 106
8.7 x 106
50
-------
TABLE 8
Pure Culture Batch No. 4 With Galactose
Time,
hour
0
1.50
2.50
5.00
7.00
14.50
30.50
55.00
76.00
100.50
146.25
Substrate,
mg/1
583.0
335.0
-
238.0
231.0
116.0
-
-
-
—
-
Biomass Concentrations By
Dehydrogenase
Activity, mg/1
-
-
36.0
46.0
69.3
-
48.9
42.0
37.7
24.6
-
Suspended
Solids , mg/1
165
-
180
210
-
-
200
170
280
230
270
Plate Counts
cells/ml
1.76 x 108
2.18 x 108
2.71 x 108
2.64 x 108
2.83 x 108
-
3.60 x 108
-
7.4 x 108
1.5 x 108
-
Coulter Counter
cells/ml
3.4 x 106
3.2 x 106
3.8 x 106
5.7 x 106
6.9 x 106
-
9.4 x 106
8.2 x 106
1.7 x 107
2.9 x 106
3.8 x 107
-------
TABLE 9
Pure Culture Batch No. 5 With Galactose
Ul
to
Time,
hour
0.25
3.25
5.00
6.50
12.50
31.00
52.50
77.50
132.25
Substrate,
mg/1
924.0
752.0
731.0
565.0
94.8
52.7
-
-
-
Biomass Concentrations By
Dehydrogenase
Activity, mg/1
86.7
150.3
101.9
109.0
86.3
53.0
69.1
41.1
-
Suspended
Solids, rag/1
320
270
340
-
298
-
390
557
530
Plate Counts
cells/ml
5.0 x 107
3.3 x 108
5.2 x 108
6.9 x 108
-
2.1 x 109
6.9 x 108
5.3 x 108
2.3 x 105
Coulter Counter
cells/ml
7.4 x 106
1.0 x 107
1.23 x 107
1.20 x 107
-
1.60 x 108
3.40 x 107
3.2 x 107
-
-------
TABLE 10
Pure Culture Batch No. 6 With Galactose
Time,
hour
0.25
3.25
5.00
6.50
12.50
31.00
52.50
77.50
126.00
Substrate,
mg/1
1,240.0
803.0
348.0
93.2
42.6
-
-
-
-
Biomass Concentrations By
Dehydrogenase
Activity, mg/1
231.9
233.4
121.1
125.3
75.2
113.4
117.7
105.4
24.0
Suspended
Solids , mg/1
430
490
530
500
567
510
580
600
430
Plate Counts
cells/ml
5.7 x 108
9.2 x 108
6.0 x 108
1.0 x 109
-
1.7 x 109
5.5 x 108
7.1 x 108
5.6 x 105
Coulter Counter
cells/ml
2.6 x 107
3.2 x 107
2.6 x 107
4.1 x 107
-
6.7 x 107
7.4 x 107
9.2 x 107
-
LO
-------
TABLE 11
Pure Culture Batch No. 7 With Sucrose
Ui
Time,
hour
0
1.50
2.50
3.50
4.50
6.00
7.00
25.00
29.00
35.50
47.00
Sucrose,*
mg/1
2,170
2,170
2,210
1,990
1,935
1,692
1,620
1,325
-
884
847
Biomass Concentrations By
Dehydrogenase
Activity, mg/1
-
12.1
19.6
26.1
131.4
91.6
61.6
43.5
-
41.5
52.6
Suspended'
Solids, mg/1
-
—
—
—
150
130
130
-
400
-
660
Plate Counts
cells/ml
1.5 x 106
3.8 x 106
7.5 x 106
4.0 x 107
4.6 x 107
9.1 x 108
9.3 x 108
7.5 x 109
9.4 x 10°
-
-
Coulter Counter
cells/ml
4.6 x 107
1.1 x 107
3.4 x 107
-
3.5 x 109
6.1 x 109
7.1 x 109
1.5 x 1010
1.7 x 1010
-
2.0 x 1010
* measured by Chemical Oxygen Demand (Standard Methods)
-------
TABLE 12
Pure Culture Batch No. 8 With Acetic Acid
Time,
hour
0
0.50
1.50
3.00
4.25
6.00
7.00
24.50
29.00
Substrate , mg/1
Acetic Acid
1,254
_
1,134
-
—
826
774
—
60
Total Organic
Carbon
553
_
517.4
-
—
430
384.6
—
103.8
Biomass Concentrations
Dehydrogenase
Activity, mg/1
84.1
77.4
-
-
159.9
165.5
224.4
87.9
-
Suspended
Solids , mg/1
120
130
170
-
190
340
350
320
-
Plate Counts
cell/ml
4.2 x 108
6.9 x 108
7.6 x 108
9.7 x 108
9.6 x 108
1.0 x 108
9.8 x 108
Coulter Counter
cell/ml
2.6 x 108
4.4 x 108
8.4 x 108
1.2 x 109
1.7 x 109
2.3 x 109
4.7 x 109
(Jl
-------
TABLE 13
Pure Culture Batch No. 9 With Acetic Acid
Time,
hour
0
2
3
5
6
7.5
14.75
24.0
26.0
29.0
48.0
55.0
79.0
Acetic Acid,
mg/1
2,250
2,100
2,222
2,100
2,100
2,100
—
1,080
1,020
640
380
-
—
Biomass Concentrations
Dehydrogenase
Activity, mg/1
-
11.0
14.9
21.0
38.3
60.6
238.6
507.6
446.0
527.0
856.0
514.0
28.6
Suspended
Solids, mg/1
33.3
53.2
59.8
54.4
90.0
190.0
330.0
380.0
470.0
500.0
720.0
-
560.0
Plate Counts
cell/ml
6.7 x 107
1.1 x 108
1.3 x 108
1.2 x 108
3.4 x 108
6.2 x 10 8
-
1.5 x 109
2.6 x 109
2.2 x 109
1.5 x 109
-
—
Coulter Counter
cell /ml
4.9 x 107
8.1 x 107
9.5 x 107
1.8 x 108
4.6 x 108
7.1 x 108
-
6.2 x 109
5.4 x 109
4.6 x 109
5.3 x 109
-
—
-------
TABLE 14
Pure Culture Batch No. 10 With L-Alanine
Time,
hour
0
1.0
2.0
4.50
6.00
24.50
29.50
L-Alanine
Total Organic
Carbon , mg/1
441.6
420
378
330
288
129.6
100.5
Organic
Nitrogen, mg/1
82
76
70
51
-
-
-
Biomass Concentrations
Dehydrogenase
Activity, mg/1
177.6
225.4
179.0
172.8
260.9
149.5
-
Suspended
Solids , mg/1
230
340
360
290
370
390
-
Plate Count
cell/ml
6.7 X 108
7.8 X 108
1.3 X 109
2.1 X 109
3.9 X 109
2.2 X 109
3.5 X 109
Coulter Count
cell/ml
6.8 X 108
8.4 X 108
1.1 X 109
1.8 X 109
1.8 X 109
3.5 X 109
-------
TABLE 15
Pure Culture Batch No. 11 With Benzole Acid
Ln
OO
Time,
Hour
0
0.75
2.50
3.25
4.50
6.00
11.00
29.00
52.00
75.75
95.75
166.00
Dehydrogenase,
Activity, mg/1
81.4
70.8
138.6
151.9
130.7
100.6
125.6
154.4
182.0
111.4
98.1
7.8
Suspended
Solids, mg/1
280
260
320
400
320
360
580
— _
620
620
600
Plate Counts
Cell/ml
4.9 X 108
5.8 X 108
3.0 X 108
8.0 X 108
1.2 X 109
6 X 108
2.5 X 109
1.3 X 109
6.7 X 108
1.6 X 108
Coulter Counter
counts/ml
1.2 X 109
7.9 X 108
8.4 X 108
8.8 X 108
9.3 X 108
9.7 X 108
_ —
__-
-------
VO
•JO
no
CM
MC
|«0
^•00
^ IM
s
"5. **°
? "°
0
•8 too
=5
•o «o
4O
- 700
i
— toe
- soa
^
- r
3
. 0
- BDC
• B0(
•
• K»
p
r^" ^^^-\
"•x
\s
\
.-•••" ^
y
Legwd : A Plaf« Count \
« • \
• \
Q • Coulter Counter Enumeration \
k •
O Suspended SoHdt ^ ^
t
O Dehydrogenase Activity
S • Subttrate
/ %'% ° *^
i i i '»*fj i i i i i i i i > i i i i i i i i
t 4 • t 0 tt M • •tOttM'r4«nMaOM4WMlM
Time In hours
Figure 9- Pure Culture Batch No. 1 With E. Coli and Glucose
H*
O
c
2
•
K>T "
o
i
I0«
10*
Substrate
-------
ON
O
- K>5 3
Plate Count
Coulter Counter Enumeration
Suspended Solids
Dehydroqenose Activity
Substrate
Time in hours
Figure 10. Pure Culture Batch No. 2 With IS. Coli and Glucose
Substrate
-------
SOOT BOOl-
_ z«o
o>
E zeo
~ 200
31
o>
o
I
•o
o
ISO -
ao
(n
4O
to
-iio"
Time in hours
Figure 11. Pure Culture Batch No. 3 With E_. Coli and Glucose
Substrate
-------
300 -
no -
o
?
Legend
a.
ro
MO
TOO •
1
J5
A
Q
O
o
Plate Count
Coulter Counter Enumeration
Suspended Solids
Dehydrogenase Activity
Substrate
-1(0°
Time in hours
Figure 12, Pure Culture Batch No. ^ With IS. Coli and Galactose
Substrate
-------
4O r-
SB
500
r
- •»!
•»<>
100
so
- too
o
Legend
- MO
A Rote Count
• Coulter Counter Enumeration
O Suspended Solids
O Dehydrogenose Activity
• Substrate
10 •
M u 10
i A i | | f
M f~HnM ieo
Time in hours
Figure 13. Pure Culture Batch No. 5 With E_. Coli and Galactose
Substrate
-------
MOO -
0>
E
000-
400-
2
ft)
o>
o
•o
o
c
0>
a
en
100
Legend ;
too
too
'•••III
Lnsr.«rr.*ifv.viF&:m'
A Plate Count
a Coulter Counter Enumeration
^ Suspended Solids
O Dehydrogenose Activity
o Substrate
W
M W 18 20 22
Time in hours
a4
S>
Figure Ik. Pure Culture Batch No. 6 With
Substrate
Coli and Galactose
o
o
\
BO
-------
Ui
2400—
40C
E
o
>hydrogenase
w
o
•o
c
0
•V IOO
0
(A
ipended
w
•3
CO
2200
ZOOC
1 BOC
1600
QI400
O
O
JJI200
. -1000
9
i
u>
400
2OO
O
^- — •- -" "n A~~
\ ,Va'I> --*' "**" 0-
/V x- •*"*'" Legend: O
.' ••&& ..."" A
/ r\ ...•-" a
' I / % ,-•* o
/ ' X'"
— • — -o_
Oehydrogenase Activity
Plate Count
Coulter Counter Enumeration
Total Suspended Solids
Substrate
/A/A X COD"'""..,,.
" V / *. ,... -
"X /
f\
1 \
tMMf**! 1 1 1 | | 1 | | | | J | | 1
2 4 6 • IO 12 14 16 IB 2O 22 24 26 2S 3O
Time in hours
Id
Counts, cells/ml.
-o "o
.0'
,oT
•o6
O
""• '
i i i A i
32 34 36 V4i
Figure 15- Pure Culture Batch No. 7 With E. Coli and Sucrose
Substrate
-------
10
-i 10
• 300
500
100
•o
V
,o
,o
o
o
o>
'"I
01
JlO
14 16 IB
Time in hours
Figure 16. Pure Culture Batch Ifo. 8 With T&. Coli and Acetic
Acid Substrate
-------
IO
10
MOO
900
•>
i»
g
* 80C
««00 .
I4OO .
600
•o
^500
3
V)
ieoo .
E 1200
401 _ £ 800
"C_ 400
100
fl
«...
Legend •.
O Dehydrogenose Activity
A Plot* Count
Q Coulter Counter Enumeration
<^ Total Suspended Solids
O Substrate
14 16 18
Time in hours
20 22 24 26 28 SO 32
I A I I
2 V 4872
10
10
10
10
72 96 120
O
O
O
•
Figure 17- Pure Culture No. 9 With E. Coli and Acetic Acid
Substrate
-------
500,
500
o
<
0300 _
00
Jzoc
MIOO
400
-i SO
Legend: O Dehydrogenose Activity
A Plate Count
Q Coulter Counter Enumeration
:> Total Suspended Solids
Substrate
"A" Organic Nitrogen
16 IB 20 22 24 26 28 SO
lime in hours
10
10
10
o
• o
10
Figure 18. Pure Culture Batch No. 10 With E. Coli and L-Alanine
Substrate
-------
700r
VD
© Dehydrogenase
Rate Count
o Coulter Counter Enumeration
Total Suspended Solids
K)
10
10* O
.
10 5T
10
24 28 32 36
Time in Hours
76 92 96 106
Figure 19. Pure Culture Batch No. 11 With E_. Coli and Benzole
Acid Substrate
-------
Batch -rtj^jgs with Heterogeneous Cultures;
Seven batch studies with heterogeneous aerobic cultures were conducted
with glucose, galactose, sucrose, acetic acid, and L-alanine substrates.
The data in Table 16-22 and Figures 20-26 indicated that dehydrogenase
activity was a very sensitive indicator of the activity of the biomass.
With the addition of ATP analysis, Figures 25 and 26 indicated the same
pattern between dehydrogenase activity and ATP concentrations throughout
the growth phase. A comparison at ATP content extracted by dimethyl
sulfoxide (DMSO) solvent and nitrogen bombing was also included in
Figures 25 and 26. A parallel relationship between the extraction
technique was reflected by curves. During exponential growth, suspended
solids and dehydrogenase activity maintained a similar trend as observed
in the pure culture studies as shown in Table 26.
In addition to the aerobic studies, an anaerobic digester was
maintained to demonstrate the possible applicability of the dehydrogenase
test in such a process. The digester had been fed with leachate from
a solid waste disposal site once a day and with initial COD concentrations
of 350,550, and 800 mg/1.
The data from three batch studies are included in Figures 27,28 and
29 and Tables 23,24, and 25. Leachate concentrations, biomass by all
parameters and gas (carbon dioxide and methane) production were measured.
It was during these batch studies that the sensitivity of the dehydrogenase
test became more pronounced when compared with other parameters. The
total suspended solids with both non-volatile and volatile fractions
including biomass did not reflect changes interpretable in terms of
activities of organisms in the system. The magnitude of increase in
solids concentration after 8-10 hours was much less than observed in
aerobic systems.
ATP concentrations were monitored in two of these batch studies and
showed a rapid decrease instead of an increase in the first six hours
followed by a slight increase (Figures 24 and 25). Similar observations
on ATP were reported by Forrest(39) during the growth of the anaerobic,
70
-------
Streptococcus faecalis, on a pyruvate substrate. Apparently, during
the first several hours of the experiment after growth began, synthesis
reactions made heavy demands on the ATP pool causing a rapid decrease in
the pool level. It appeared then to fall below the critical level
necessary to sustain exponential growth. Consequently linear growth took
place, limited by the availability of ATP for synthesis.
Contrary to these observations on ATP content or solids concentrations,
the dehydrogenase activity in the studies reported herein was consistently
sensitive to the behavior of the active biomass during the growth cycle
under anaerobic conditions. As the substrate was depleted and the
corresponding gas production rate decreased, the dehydrogenase activity
also decreased to a minimum value.
71
-------
K3
TABLE 16
Heterogeneous Culture Batch No. 1 With Glucose
Time/
hour
0
1.50
2.75
5.50
6.25
7.75
24.00
31.00
Glucose/
mg/1
1540
1252
948
153
70.2
8.1
0
0
Biomass Concentrations
Dehydrogenase
Activity , mg/1
19
24.8
33.2
47.0
60.0
4.14
20.1
16.2
Total Suspended
Solids, mg/1
40
65
90
120
-
150
185
225
Volatile Suspended
Solids, mg/1
40
60
75
110
-
110
135
145
-------
TABLE 17
Heterogeneous Culture Batch No. 2 With Glucose
•vj
CO
Time,
hour
0
1.25
2.75
4.75
6.25
13.25
23.75
28.50
30.00
53.50
72.00
79.00
Glucose,
mg/1
824
739
671
563
429
189
7.2
0
0
0
0
0
Biomass Concentrations
Dehydrogenase
Activity, mg/1
47.2
89.3
134.4
95.1
96.3
97.4
101.8
102.3
74.3
74.3
31.8
20.4
Total Suspended
Solids, mg/1
65
120
190
215
220
250
-
-
250
220
195
160
Volatile Suspended
Solids, mg/1
50
90
140
160
130
175
-
-
170
-
180
145
-------
TABLE 18
Heterogeneous Culture Batch No. 3 With Galactose
Time,
hour
0
1.0
2.50
4.50
6.25
7.25
8.50
11.50
13.50
24.00
26.75
29.50
32.50
48.00
52.25
Galactose,
mg/1
740
737
751
742
691
534
162
24.8
12.2
7.3
-
-
-
:
Biomass Concentrations
Dehydrogenase
Activity, mg/1
-
-
-
19.0
33.0
42.4
91.0
116.2
166.4
85.9
69.8
70.4
-
15.7
12.0
Total Suspended
Solids, mg/1
-
30
25
130
145
230
250
280
290
250
330
190
230
110
110
Volatile Suspended
Solids , mg/1
-
25
15
80
100
190
190
190
225
230
300
180
210
90
90 1
-------
TABLE 19
Heterogeneous Culture Batch No. 4 With Galactose
Time,
hour
0
1.25
2.75
4.75
6.00
7.00
8.50
12.25
14.50
23.50
26.00
29.50
30.50
48.50
53.50
73.00
77.00
Galactose.
mg/1
942
928
1010
950
842
-
788
763
595
161
-
6.4
-
-
-
-
-
Biomass Concentrations
Dehydrogenase
Activity, mg/1
-
-
-
11.6
15.2
32.1
-
130.6
184.4
148.2
131.6
88.8
-
72.6
-
20.8
-
Total Suspended
Solids , mg/1
-
-
15
-
15
-
85
140
155
320
-
320
315
285
265
-
-
Volatile Suspended
Solids, mg/1
-
-
5
-
-
-
50
140
185
280
-
305
285
260
-
170
155
75
-------
TABLE 20
Heterogeneous Culture Batch No. 5 With Sucrose
Time,
hour
0
1.0
2.0
3.5
5.0
7.0
26.25
29.5
Sucrose fiy
COD, mg/1
2440
2400
2360
2360
—
2280
2020
1940
Biomass Concentrations By
Dehydrogenase
Activity, mg/1
19.3
21.0
23.5
28.1
27.7
112.6
14.4
15.8
Total Suspended
Solids, mg/1
120
115
135
160
170
205
175
230
Volatile Suspended
Solids, mg/1
120
100
125
135
165
175
170
200
-------
TABLE 21
Heterogeneous Culture Batch No. 6 With Acetic Acid
Time,
hour
0
1.75
4.50
6.25
7.50
24.00
25.75
27.75
48.00
Acetic Acid by
Total Organic
Carbon, mg/1
940
-
-
325
308
30
19
18
-
Biomass Concentration By
Dehydrogenase
Activity,
mg/1
_
_
18.0
—
24.8
65.7
34.9
26.1
0
Total
Suspended
Solids, mg/1
_
250
188.5
192.5
225
225
250
300
265
Volatile
Suspended
Solids, mg/1
_
100
115
140
190
205
210
205
145
ATP , f g/ml
DMSO
1.55 X 107
1.93 X 107
1.36 X 107
1.51 X 107
1.92 X 107
2.32 X 108
1.36 X 108
8.86 X 107
2.89 X 107
Bombing
2.02 X 106
2.77 X 106
3.00 X 106
1.39 X 106
3.00 X 106
7.17 X 107
3.63 X 107
2.96 X 107
4.61 X 106
-------
TABLE 22
Heterogeneous Culture Batch No. 7 With L-Alanine
Time,
hour
0
1.0
3.0
4.5
6.0
7.5
9.0
15.0
25.0
29
L-Alanine by
Total Organic
Carbon, mg/1
420
396
_
288
184
126
_
56
_
27.5
Biomass Concentrations By
Deny dr ogenas e
Activity, mg/1
0
9.8
20.8
66.3
87.3
122.6
130.0
113.6
47.2
19.3
Total Suspended
Solids , mg/1
100
90
_
165
180
245
510
500
215
Volatile Suspended
Solids, mg/1
_
60
_
_
140
130
265
405
170
110
ATP, fg/ml, by
DMSO
_
2.00 x 108
2.12 x 108
1.56 x 108
2.12 x 108
2.83 x 108
3.35 x 108
6.06 x 108
_
2.09 x 108
Bombing
^
1.26 x 107
1.63 x 107
2.07 x 107
3.79 x 107
3.88 x 107
5.6 x 107
9.92 x 107
2.10 x 107
3.83 x 107
00
-------
240_
220
200
^.
» l«0
^ 160
>
o 140
«
o (20
220O
2000 _
1800 -
01
o
c
0
o
V)
100
GO
«O _
5. 20 _
la
3
V)
_ syi uw .
O Dehydrogenose Activity
Total Suspended Solids
Volatile Suspended Solids
Substrate
12 14 la
Time in hours
Figure 20. Heterogenous Culture Batch No. 1 With Glucose
Substrate
-------
Z6O
Z4O
220
200
1000
00
o
180
WO
I4C
TOO .
V
Oi
o
£ .00
•o
o
o
V)
c Z
•
Q.
«
3
CO
% • s
O Dehydroq«nase Activity
Total Suspended Solids
• Volatile Suspended Solids
O Substrate
10 12 14 16 16 20
Time in hours
22
24 26 26 3O 32
72 06
Figure 21. Heterogeneous Culture Batch Wo. 2 With Glucose
Substrate
-------
400 1000
9OO _
£ 300
>»
200
oo
o>
o
•o
o
j» 100
"5
TJ
c
I
CO
O Substrate
Legend: O Dehydrogenose Activity
A Plate Count
<3> Total Suspended Solids
• Volatile Suspended Solids
800 .
700 _
600
500 .
10
o
o
&
400 .
300
200 .
100 .
10 12 14 |6 |B 20 22 24 26 26 90 32 « 48 72
Time in hours
Figure 22. Heterogeneous Culture Batch No. 3 Galactose Substrate
-------
400
~ 300
o
(A
O
00
to
7*
£
•
O
c
o
•o
o
•a
«
•o
200
IOO
I 200
I 100
1000 .
Legend :
O Dehydrogenose Activity
O Total Suspended Solids
• Volatile Suspended Solids
Substrate
10 12 14 16 IB
120
Time in hours
Figure 23. Heterogeneous Culture Batch No. U With Galactose
Substrate
-------
2500r
oo
O
8
2000-
15001
O Dehydrogenase Activity
• Total Suspended Solids
•Volatile Suspended Solids
a Substrate
10 12 14 16 18 2O 22 24 26 28 30 32
Time in Hours
400
300
CD
O
01
8
- 200^:
•100
Figure 2k. Heterogeneous Culture Batch No. 5 With Sucrose
Substrate
-------
Legend
40O 10OOr
oo
o Oehydrogenase Activity
A Total Suspended Solids
• Volatile Suspended Solids
a ATP-OMSO
• ATP - Bombed
o Substrate
'*o—
-O-..
""" ^ **•
O "*--.
•'..
.•**••
to9
10
10'
10 12 14 16 18 20 22 24 26 28 30 32 " 46 48 50
Time in Hours
Figure 25- Heterogeneous Culture Batch No. 6 With Acetic Acid
Substrate
-------
oo
Legend: o Dehydrogenase Activity
A Total Suspended Solids
• Volatile Suspended Solids
° ATP-DMSO
• ATP-Bombed
Substrate
\l
2 4 6 8 10 12 14 16 18 20 22 24 26 28 30
Time in Hours
32 34
Figure 26. Heterogeneous Culture Batch No. 7 With L-Alanine
Substrate
50
10
•o
«
«*
-------
TABLE 23
Heterogeneous Anaerobic Culture Batch No. 1 With Leachate
Time,
hour
0
0.25
2.50
4.25
6.00
8.00
14.25
25.00
Leachate
by COD,
mg/1
330
307
279
250
213
160
133
Biomass Concentrations By
Dehydrogenase
Activity , mg/1
27.9
84.8
91.6
64.1
61.3
-
-
62.0
Total Suspended
Solids, mg/1
990
810
730
720
735
725
805
740
Volatile Suspended
Solids, mg/1
490
430
395
355
370
360
420
375
Total Gas
Production, ml
0
0
70
160
270
410
680
870
00
-------
TABLE 24
Heterogeneous Anaerobic Culture Batch No. 2 With Leachate
Time,
hour
0
1.00
2.50
4.00
5.25
9.50
19.50
23.50
27.50
Leachate by
COD, mg/1
538
-
518
489
-
446
363
326
Biomass Concentrations By
Dehydrogenase
Activity, mg/1
45.5
51.7
44.4
34.4
-
-
25.5
25.5
Total
Suspended
Solids, mg/1
525
490
470
460
455
550
490
525
515
Volatile
Suspended
Solids , mg/1
320
275
270
275
310
330
310
335
295
ATP, fg/ml
DMSO
2.55 x 107
1.80 x 107
1.35 x 107
1.24 x 107
8.76 x 106
8.64 x 106
9.18 x 106
7.47 x 106
6.51 x 106
Bombing
9.40 x 106
6.22 x 105
5.32 x 105
6.33 x 105
4.58 x 105
3.71 x 105
5.2 x 105
6.57 x 105
5.34 x 105
Total Gas
Production ,
ml
0
30
90
160
200
420
800
900
980
00
-------
TABLE 25
Heterogeneous Anaerobic Culture Batch No. 3 With Leachate
Time,
hour
0
1.5
3.0
4.5
6.0
7.5
9.0
13.5
24.0
54.0
Leachate by
COD, mg/1
774
738
-
760
741
723
701
647
621
Biomass Concentrations By
Dehydrogenase
Activity, mg/1
9.4
25.8
42.9
47.6
32.1
43.3
-
8.1
7.9
Total
Suspended
Solids, mg/1
3,190
2,990
3,405
3,380
3,220
3,350
3,170
3,100
2,930
2,775
Volatile
Suspended
Solids, mg/1
555
550
590
575
555
565
580
615
585
515
ATP By
DMSO
3.45 x 107
1.5 x 107
1.27 x 107
9.60 x 106
8.46 x 106
8.52 x 106
9.48 x 106
7.26 x 106
7.56 x 106
5.04 x 106
Bombing
1.48 x 106
1.07 x 106
7.11 x 105
1.24 x 106
1.64 x 106
5.03 x 105
1.13 x 106
6.33 x 105
8.63 x 105
3.62 X 105
Total Gas
Production ,
ml
0
40
180
280
360
490
570
780
1,020
1,210
00
oo
-------
00
Legend: o Dehydrogenase Activity
A Total Suspended Solids
® Substrate
• Volatile Suspended Solids
Gas
24 6 8 10 12 14 16 18 20 22 24 26 28 30
Time in Hours
2 X102
10
-9
8
ff
«3 -\
.
Figure 21. Heterogeneous AnaeroMc Culture Batch No. 1 With
Leachate
-------
VO
o
X «TP-Bomk . .
024 6 8 10 12 14 16 18 20 22 24 26 28
Time in Hours
x10
- 7.5 -fl
x10
-8'
(B
4!
1
0 -1
2
0)
~^
2.5 -
10
105
Figure 28. Heterogeneous Anaerobic Culture Batch No. 2 With
Leachate
-------
10
8
o Dehydrogenase Activity
• Volatile Suspended Solids
° ATP-DMSO
o Substrate
xlOO
10
10
o
**•
s»_
O
•o
O
>
•o
-------
TABLE 26
Ratios Between Biomass Parameters during Log Growth
Phase of Batch Cultures
Substrate
Glucose
Galactose
Sucrose
Acetic Acid
Alanine
Glucose
Galactose
Sucrose
Acetic Acid
Alanine
Dehydrogenase
VSS
0.60 - 0.70
0.45 - 0.6
0.66
0.60
0.70
0.66
0.90
0.60
0.40
0.60
Plate Count
Coulter Counter
0.82
0.35 - 0.40
0.42
0.41
0.78
-
-
-
-
-
Culture
Pure culture with
E. coli
ii
ii
ii
ii
Heterogeneous
culture
n
n
n
n
-------
Continuous Culture Studies;
The series of continuous culture experiments were continued to
study the application of dehydrogenase activity and ATP measurements
under steady state conditions. Steady state was established by operating
the reactors for periods of 3 to 4 retention times prior to sampling and
analysis for each of the substrates. The results are included in Tables
27-32 and Figures 30-35. The substrates used during the studies included
not only simple sugars like glucose (in aerobic and anaerobic) and
galactose but also industrial wastes from shellfish and chicken processing
plants and leachate from a solid waste disposal site. Parameters monitored
in these studies were similar to those used during the batch culture
studies. COD, 5-day biochemical oxygen demand (BOD-) and/or total organic
cabon (TOG) were used to measure the substrate concentrations for these
latter indistrial wastes.
The data on biomass measurement indicated similarity between parameter:
during the steady state observations. The observed ratio of the dehydro-
genase activity to suspended solids remained fairly constant and close to
unity with the glucose and galactose substrate in the aerobic cultures
and also constant but lower (0.70) in the anaerobic cultures except
when very long retention times were investigated. It was further observed
that this ratio was 0.35 on chicken processing wastes and 0.60 on shell-
fish processing wastes as shown in Table 33. Since the dehydrogenase
activity was reported decreasing in the endogenous growth phase during
batch culture, it become self-explanatory that the preceding ratio would
decrease at very long retention times where partial endogenous growth
existed. It became more evident when this ratio decreased to 0.30 with
the same galactose substrate in aerobic cultures and only the sludges
were being recycled as presented in the succeeding section of this
report. Unlike the correlations between ATP, organic nitrogen, or
other parameters and VSS varing with the specific growth rates, it is
very important to observe the consistent correlation between the
dehydrogenase activity and VSS with a given substrate in continuous
cultures.
93
-------
The different organic character of the substrate resulted in
changing ratios of dehydrogenase activity to weight of solids as
Indicated for the selected industrial wastewaters. It is noted that
not all the volatile solids reported represented biological mass. The
correlation established from the simple sugar substrate studies should
be different from those for the industrial wastes investigated here.
By plotting data from studies on chicken and shellfish processing wastes
the following emperical equation was obtained.
X = 930 A + 10 (2)
where: X = active biomass, mg/1
A = dehydrogenase activity measured as absorbance at 483 my
and 1 cm light path
From these data it follows that once the correlating ratio between the
parameters have been established in an actual waste treatment process
the active biomass could be monitored more accurately and more rapid
corrective measures taken as problems develop.
Kinetic constants for all studies were calculated and compared in
Table 34. The maximum specific growth rate was higher on glucose-grown
cultures than on the other sugars, amino acids or industrial wastes.
Continuous Culture Studies with Solids Recycle; A continuous culture
study with solids recycle was conducted with a galactose substrate as
shown in Table 35 and Figures 36 and 37. These results indicated that
the steady state galactose concentrations were much lower than those
observed in the same system without recycle. The biomass concentrations
as measured by VSS, dehydrogenase activity and ATP at shorter retention
times were observed almost doubled as the recycle factor increased from
1.6 to 5.8 and inversely porportional to the settling time in the clarifler,
Increased rates of substrate utilization with recycle were attributed to
opportunitites for more rapid growth and the magnitude of biomass con-
centration by recyle particularly at shorter retention times.
94
-------
To determine changes in active biomass with changes in specific
growth rate, dehydrogenase and ATP vs. VSS were plotted as shown on
Figure 38. These data are included in Table 36 and indicated that the
ATP content in the solids rapidly increased and then decreased gradually
with the increase of growth rate. At low specific growth rates (up to
1.5 day' ), ATP content increased to 0.45 mg ATP per gram VSS while at
high specific growth rates, ATP content decreased to a limiting range
of 0.25 - 0.27 mg ATP per gram VSS and finally decreased when organism
washout occurred. The cause of this rapid increase followed by a
decrease has not been well established. However, the same behavoir
(3)
was illustrated also by the data of Weddle and Jenkins on activated
sludge although this early increase was essentially ignored in their
analysis. The ATP content of pure cultures have been reported varying
(35 39)
from 0.02 to 1.2 percent on a dry weight bases * , while those
for activated sludge of 0.2 and 0.3 mg ATP/g dry weight by Patterson,
££. fLL- and UP to 2>0 mS ATP/8 ss by Biospherics are in good agree-
ment with the data obtained from the study with solids recycle. The
initital rapid increase of ATP content per weight of VSS could reflect
more growth due to the sufficiently high availability of substrates which
may not have been possible at longer retention times. The decrease of
ATP appeared to have resulted from either washout of certain organisms
of high ATP content or a smaller capacity for ATP storage inside the
cells when organisms grow faster by utilizing more energy at higher
growth rates. Therefore, the correlation between ATP and VSS could
be meaningfully applied for control of continuous culture type systems
within the growth ranges where essentially not much change occurred
(above 2.0 day ).
The dehydrogenase activity increased steadily with growth rate
( to a specific growth rate of 3 day ) and then remained essentially
constant until washout occurred. However, the ratio between the
dehydrogenase and VSS indicated virtually no sifnificant change through-
out the range of specific growth rates covered, even though it was
recognized that the overall 0.30 level was only one third of that from
the study without solids recycle (Table 28). The existence of a partial
95
-------
endogenous growth phase by solids recycle was considered the major cause
of a decreased ratio. Generally constant nature of this correlation
between dehydrogenase activity per weight of biomass permits the
determination of active biomass concentrations in cultures operating
at any practical growth rate by establishing a standard curve correlating
these parameters at exponential growth.
96
-------
VO
TABLE 27
Continuous Culture Study with Glucose Substrate
Re tens ion
Time, hr.
24.0
18.5
15.5
12.3
6.0
4.0
Glucose
mg/1
0.3
0.4
_
6.0
14.7
70.7
Biomass Concentra
Dehydrogenase
Activity, mg/1
89.9
_
121.2
140.0
111.6
135.5
71.5
33.0
Total Suspended
Solids , mg/1
115
125.0
127.5
120
150
97.5
105
65
32
tions
Volatile Suspended
Solids, mg/1
100
110
115
110
115
80
65
32
-------
TABLE 28
Continuous Culture Study with Galactose Substrate
oo
Retention
Timef Hrs.
5.92
5.83
3.81
2.96
2.84
2.06
1.65
Galactose,
mg/1
4.3
4.0
1.9
87.7
26.8
24.0
133.9
Biomass Concentrations
Dehydrogenase
Activity/ mg/1
83.5
63.9
92.5
40.0
98.8
53.9
54.5
Total Suspended
Solids, mg/1
90.0
66.0
102.0
60.0
100.0
63.0
53.1
-------
TABLE 29
Continuous Culture Study with Shellfish Processing Wastes
v£>
/
Retention
Time / Hrs .
12
10
8
6
4
2
Substrate
by BOD5,
mg/1
50.0
33.3
-
47.6
95.1
166.1
Biomass Concentrations By
Dehydrogenase
Activity
mg/1
-
41.7
58.0
-
66.7
8.8
Total
Suspended
Solids,
mg/1
88
84
83
-
102
94
ATP, fg/ml
DMSO X 106
9.1
26.0
36.0
-
49.2
17.5
Bombing
X 106
8.2
4.1
10.0
-
7.4
5.1
-------
TABLE 30
Continuous Culture Study with Chicken Processing Wastes
Retention
Time, Hrs.
20
16
11
8
6
2
Substrate
BOD5,
mg/1
116
118
130
125
160
COD,
mg/1
157
165
185
260
Biomass Concentrations By
Dehydrogenase
Activity ,
mg/1
54.6
30.7
69.9
45 .6
Total
Suspended
Solids ,
mg/1
193
220
116
150
176
75
ATP, fg/ml
DMSO
5.60 x 107
3.59 x 107
9.18 x 106
5.21 x 107
3.79 x 107
Bombing
1.26 x 107
3.75 x 106
2.14 x 105
2.78 x 106
5.42 x 106
o
o
-------
TABLE 31
Continuous Culture Study with Leachate
Retention
Time , Hrs .
15
10
5
2
Leachate by
TOC,
mg/1
273
307
385
596
COD,
mg/1
700
674
850
1860
Biomass Concentrations by
Dehydrogenase
Activity,
mg/1
495
672
1064
Total
Suspended
Solids,
mg/1
3116
3450
5470
3150
Volatile
Suspended
Solids ,
mg/1
1993
2040
2940
1235
ATP,
fg/ml
7.6 x 107
1.53 x 108
1.99 x 107
-------
TABLE 32
Anaerobic Digester with Heterogeneous Cultures
in Continuous Flow System
Retention
Time, Hr.
44.7
28.7
26.5
20.0
16.6
8.8
6.0
4,0
Glucose
mg/1
1.0
2.5
5.0
14.0
15.3
5.3
10.6
194.0
Biomass Concentrations by
Dehydrogenase
Activity, mg/1
154.9
219.4
250.0
300.0
284.0
347.0
116.0
93.6
Total
Suspended
Solids,
mg/1
384
458
384
355
365
307
279
235
Volatile
Suspended
Solids/
mg/1
110
270
180
173
177
166
132
95
-------
JSS
Dehydrogenase
10 12 14 16 18
Time in Hours
24 26 28 30
Figure 30. Continuous Culture Study With Glucose Substrate
-------
120-
100
t
.9
a
1
60
O
40
20
" Gilictost
234
Retention Time in Hours
6
Figure 31. Continuous Culture Study With Galactose
104
-------
200
TP-OMSO
6 7 8 9 10 11
Retention Time in Hour*
10
10
H
7 -o
-------
B 10 « f4 Y6 IB 20 22 24
-108
.6
Figure 33. Continuous Culture Study With Chicken Processing Waste
106
-------
xlO
6
O
O
o
25
-f10
OH
5*
3
3 -
10
10
11 i i i i i i i i
0 1 2 3 4 S 6 7 • I 10
Rvtontlon Tim* in Hours
tl 12 M 14 15
10 J
Figure 3^. Continuous Culture Study With Leachatp
-------
o
00
1O 12 14 16 18 2O 22 24 26 28 30 32 34 36
Retention Time in Hours
024
Figure 35- Anaerobic Digester With Heterogeneous Populations in
Continuous Flow System
-------
TABLE 33
Summary of Correlations between Biomass Parameters
in Continuous Culture Studies
Substrates
glucose
galactose
chicken waste
shellfish waste
galactose with Recycle
glucose
Dehydrogenase
VSS
0.8 ~ 1.0
0.9 ~ 1.0
0.3 ~ 0.4
0.5 " 0.6
0.2 ~ 0.3
0.6 ~ 0.8
Ma ATP
mq VSS
-
-
0.27 ~ 0.29
0.30 ~ 0.63
0.26 ~ 0.46
-
Remarks
aerobic cultures
ii
it
»
ii
anaerobic cultures
-------
TABLE 34
Kinetic Growth Constants
Experiment
Substrate
Maximum Specific
Growth Rate,
hour"1
Saturation
Constant,
mg/1
Pure Culture No. 1
Pure Culture No. 2
Pure Culture No. 3
Pure Culture No. 5
Pure Culture No. 6
Pure Culture No. 8
Pure Culture No. 9
Heterogeneous Culture No. 1
Heterogeneous Culture No. 2
Heterogeneous Culture No. 3
Heterogeneous Culture No. 4
Continuous Culture
Continuous Culture
Continuous Culture
Continuous Culture
Anaerobic Digester
Glucose
Glucose
Glucose
Galactose
Galactose
Acetic Acid
Acetic Acid
Glucose
Glucose
Galactose
Galactose
Glucose
Shellfish Waste
Leachate
Galactose
Glucose
0.040
0.079
0.056
0.037
0.025
0.021
0.017
0.457
0.328
0.141
0.098
0.625
0.40
1.0
0.46
0.435
118.2
416.0
538.0
96.9
181.0
1,165.0
4,230.0
995.0
2,460.0
150.5
146.0
4.1
45.0*
1,460 **
5.7
19.60
* BOD5 basis
** COD basis
-------
TABLE 35
Continuous Culture Study with Galactose Substrate and Solids Recycle
Retention
Time,
hour
11.9
6.0
2.5
1.25
0.80
Galactose,
mg/1
2.6
3.4
1.2
10.4
405.0
Biomass Concentrations By
Dehydrogenase Activity, mg/1
Reactor
114.5
141.0
212.0
445.0
249.0
Clarifier
Effluent
43.7
23.8
11.2
32.8
19.0
Volatile Suspended Solids,
mg/1
Reactor
655
570
770
1,270
840
Clarifier
Effluent
400
195
210
220
200
ATP, x 10 8, fg/ml
Reactor
1.91
2.62
2.29
3.58
2.16
Clarifier
Effluent
1.25
1.84
1.30
1.34
0.70
-------
TABLE 36
Summary of Growth Constants and Ratios
Between Parameters in Solids Recycle Study
with Galactose Substrate
Retention
Time
(hour)
11.90
6.00
2.50
1.25
0.80
Recycle
Factor*
1.64
2.92
3.67
5.77
4.20
Specific**
Growth ,
Rate , day"
1.22
1.37
2.46
3.23
7.22
ATP vs .
VSS,
mg ATP
g VSS
0.292
0.457
0.377
0.282
0.257
Dehydrogenase
Activity
vs. VSS
0.18
0.24
0.28
0.35
0.30
* Ratio of VSS in reactor to the VSS in clarifier effluent,
**
u - fl • 1 ,
M V (R.F.)
where p = specific growth rate, day
Q = influent, I/day
V = volume, 1
R.F. = Recycle Factor
112
-------
5 6 7 8 9
R*ttntion Time in Hours
10
11
12 13
Figure 36. Continuous Culture Study With Galactose Substrate and
With Solids Recycle
113
-------
2000 r
1800
1200
1000-
g 800-
10
o
0 600-
400
200-
Hydraulic Retention Time in Hours
2J5 1.25
0.8
-i500
/
' TSS, Effluent
Dehydrogenase, Reactor
7.2
Specific Growth Rate, day
Figure 37. Active Biomass Measurements in Continuous Culture Study
tftth. Solids Recycle
114
-------
LOT
0.6
M 0.6
(0
e
o»
2
•o
0-4
0.
Legend:
ATP/VSS
D*hydrog«nasa/VSS
0.*
0.4
0.3 g
I
0.2 j;
I
0.1
5- _-• -•
.4 3.6 4.8
Specific Growth Rate, day'1
8.0
Figure 38, Effect of Specific Growth. Rate on Correlations Betveen
Biomass Measurements in Continuous Culture Study With
Recycle
115
-------
Nutrient Deficiency Studies - Heterogeneous batch cultures on media
deficient in nitrogen or phosphorus were grown on glucose as the sole
carbon and energy source in order to investigate the effect on the
dehydrogenase actibity. In the nitrogen deficiency study, the ratios
of carbon to nitrogen were selected at 10, 20 and 30 to successive
batches (nos. 1, 2 & 3) and the results are shown in Tables 37,38 and 39
and Figures 39, 40 and 41. Carbon to phosphorus ratios of 150 and 200
were selected in phosphorus deficiency studies. No measurable growth
was observed in the latter and the results, of the former (Batch No. 4)
are shown in Table 40 and Figure 42.
It was observed that the dehydrogenase activity was again a very
sensitive indicator of the biomass and the same correlation was established
with VSS, except with cultures grown on extreme nitrogen and phosphorus
deficiencies, i.e., when C/N was 30 and C/P was 150. However, the ATP
data indicated good agreement with VSS even during extreme deficiencies.
For instance, about 75 percent of the maximum ATP and VSS in Batch No. 1
(Table 41) was observed in Batch No. 3 while only a third of the dehydro-
genase activities was indicated. Therefore, it could be deduced that
the dehydrogenase activity was limited in application under extreme
nutrient deficiency, while the ATP measurement was acceptable even under
this condidion. Since the carbon to nitrogen ratio in domestic sewage
normally does not indicate such a deficiency, the application of the
dehydrogenase test remains viable.
116
-------
TABLE 37
Nutrient Deficient Culture Batch No. 1 (C/N=10)
Time,
hour
0
1.0
4.5
6.0
7.0
8.0
13.0
24.0
28.5
30.5
Glucose,
mg/1
1,000
1,000
-
541
102
15
0
0
0
0
Biomass By
Dehydrogenase
Activity, mg/1
0
122.9
211.4
227
-
223
10
0
0
Volatile Suspended
Solids, mg/1
40
-
250
220
280
290
105
-
50
ATP
x 107, fg/ral
6.76
8.37
12.10
13.10
11.10
15.30
8.30
3.99
1.70
-------
00
TABLE 38
Nutrient Deficient Culture Batch No. 2, (c/N = 20)
Time,
hours
0
1.0
2.5
4.5
6.3
7.5
14.5
24.0
29.3
53.3
Glucose,
mg/1
1025
972
863
580
488
399
0
0
0
0
Biomass bv
Dehydrogenase
Activity, mg/1
-
8.5
104.0
210.0
185.0
222.0
205.0
71.0
84.8
-
Volatile Suspended
Solids, rag/1
52
68
92
200
190
185
280
280
255
185
ATP
x 10, fg/ml
-
6.72
-
12.30
15.10
26.70
-
13.40
8.88
5.43
-------
TABLE 39
Nutrient Deficient Culture Batch No. 3. (C/S = 30)
\o
Time,
hours
1.0
2.0
3.3
Glucose,
mg/1
975
988
940
5.3 1000
i
6.5
7.5
8.3
12.3
22.5
25.0
28.8
52.3
700
525
412
84
0
0
0
0
Biomass by
Dehydrogenase
Activity, mg/1
—
-
9.1
19.4
39.5
57.8
-
66.7
35.5
36.9
40.8
16.0
Volatile Suspended
Solids, mg/1
20
30
26
36
83.5
116.0
180.0
200.0
180.0
185.0
185.0
225
ATP
x 10 , fg/ml
1.80
2.27
2.43
6.12
8.84
5.98
5.98
11.30
9.32
7.93
7.28
8.02
-------
TABLE 40
Nutrient Deficient Culture Batch No. 4 (c/P = 150)
NJ
o
Time,
hours
0
2.0
4.0
6.0
7.5
11.5
24.5
29.0
Glucose,
mg/1
826
806
813
780
767
737
687
670
— • r
Rtoroass By
Dehyd r oge na s e
Activity, mg/1
-
6.8
7.9
18.8
13.9
-
-
-
Volatile Suspended
Solids, mg/1
-
25
50
60
55
65
75
65
ATP
x 107, fg/ml
-
1.08
1.26
1.87
2.36
3.08
2.81
2.64
-------
TABLE 41
Comparison of Biomass from Nutritional Deficiency Culture Studies
Biomass By
Max. VSS, mg/1
Max. Dehydrogenase , mg/1
Max. ATP, mg/1
Batch No.
i
C/N=10
290
227
150
2
C/N=20
280
200
134
3
C/N=30
200
70
114
4
C/P=150
75
19
31
-------
xlOO
NJ
to
4OO 10
300
200
o
100
10 12 14 16 18 20 22 24 26 28 3O
2
32
ixifl
>
TJ
(Q
3
IX It
1 xlO
Figure 39- Nutrient Deficient Culture Batch No. 1 (C/N=10)
-------
10
U)
30?°
25
20
15
8
10
vss
14 16 Y9 10 22 24 26 28 30 32
50
10
J5
Figure UO. Nutrient Deficient Culture Batch No. 2 (C/N=20)
-------
300-
280-
260
240
220-
200-
iao.
160-
140-
j? 120-
o
m
40-
20-
10-
xlO
10 12 14 16 18 20 22 24 26 28 30 32
468
Figure 1*1. Nutrient Deficient Culture Batch No. 3 (C/N=30)
-------
NJ
Ul
(O
CO
-------
Cost Analysis;
The results obtained to date are encouraging with respect to the
use of dehydrogenase activity as a measure of active biomass con-
centration in biological systems. Moreover, this analysis may prove
far less tedious and costly than other methods of analysis and
appears to be more reliable and interpretable than other techniques.
Some comparison of the cost of analytical techniques has been includ-
ed in Appendix D. It is anticipated that these studies will provide
the basis for an analytical method for the determination and applica-
tion of dehydrogenase activity to design and control of biological
waste treatment processes.
126
-------
SECTION VI
REFERENCES
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127
-------
A ardy F. •!•» Cole, R. E., and E. A. Pearsons, "Kinetic and Activity
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11. Agardy, F. J., and W. C. Shepherd, "DNA - A Rational Basis for Digester
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12. Fisher, W. L. "Determination of DNA in Digester Sludge", M.S. Thesis.
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15. Irgens, R. L. "DNA Concentration as an Estimate of Sludge Biomass",
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World, 2nd Edition, Prentice-Hall, Inc., Englewood Cliffs, N. J.,
p. 240 (1963).
17. Goldsby, R. A., Cells and Energy, MacMillan Co., New York, N.Y., p. 23
(1967).
18. Seliger, H. H., and W. D. McElroy, "Spectral Emission and Quantum
Yield of Firefly Biolutninescence", Archives of Biochemistry and
Biophysics,,88, pp. 136-141 (1960).
128
-------
19. Strehler, B. L., and J. R. Trotter, "Firefly Luminescence in the
Study of Energy Transfer Mechnistns. I. Substrate and Enzyme Deter-
minations", Archives of Biochemistry and Biophysics, 40, pp. 28-41,
(1952).
20. Rhodes, W. C., and W. D. McElroy, "The Synthesis and Function of
Luciferly-adenylate (LH--AMP) and Oxyluciferyl-adenylate (1-AMP)",
Journal of Biological Chemistry, 233 (2), pp. 1528-1537 (1958).
21. Mitchell, G., and J. W. Hastings, "Flavin Isomers and Color of
Bacterial Bioluminescence", Journal of Biological Chemistry, 244
(1), pp. 2572-2578 (1969).
22. Hastings, J. W. Riley, W. H., and J. Massa, "The Purification,
Properties, and Chemiluminescent Quantum Yield of Bacterial
Luciferase", Journal of Biological Chemistry. 240. pp. 1473-1479
(1965).
23. Hastings, J. W., "Bioluminescence", Annual Review of Biochemistry
J7_, pp. 597-627 (1968).
24. Henry, J. P., Isambert, M. F., and A. M. Michelson, "Studies in
Bioluminescence in the Pholas dactylus System", Biochimica Biophysica
Acta. 205. pp. 437-450 (1970).
25. Mitchell, G., "Light-induced Bioluminescence: Isolation and Character-
ization of a Specific Protein Involved in the Adsorption and Delayed
Emission of Light", Biochemistry, 9. (13), pp. 2699-2707 (1970).
26. Hastings, J. W., and L. Weber, "Structurally Distinct Bacterial
Lucif erases", Biochemistry, 8_, pp. 4681 (1969).
27. McElroy, W. D., "The Energy Source of Bioluminescence in an Isolated
System", National Academy of Science, 33, pp. 342-348 (1947).
129
-------
28 McElroy, W. D., and A. Green, "Function of Adenosine Trlphosphate
in the Activation of Luciferin", Archives of Biochemistry and Bio-
physics . 64_, pp. 257-271 (1956).
29. Schram, E., "Use of Scintillation Counters for Bioluminescence Assay
of Adenosine Triphosphate", The Current Status of Liquid Scintilla-
tion Counting, E. D. Brandsome, Jr., editor; Grune & Stratton,
New York, N. Y., pp. 129-133 (1970).
30. McElroy, W. D., Seliger, H. H. and E. H. White, "Mechanisms of
Bioluminescence, Chemiluminescence, and Enzyme Function in the
Oxidation of Firefly Luciferase", Photochemistry and Photobiology.
10_, pp. 153-170 (1969).
31. Cole, H. A., Wimpenny, J. W. T., and D. E. Hughes, "The ATP pool
in Escherichia coli. I. Measurement Using a Modified Luciferase
Assay", Biochemica et Bjophysica Acta, 143, pp. 445-453, (1967).
32. Welsch, F., and L. Smith, "Kinetics of Synthesis and Utilization of
Adenosine Triphosphate in Rhodo s p irjL 1 lum rub rum", Biochemistry, 8_,
pp. 3403-3408 (1969).
33. Roberton, A. M., and R. S. Wolfe, "Adenosine Triphosphate Pools
in Methanobacterium", Journal of Bacteriology, 102, pp. 43-51
(1970).
34. Strange, R. E., Wade, H. E., and F. A. Dark, "Effect of Staravation
on ATP Concentration in Aerobacter aerogenes", Nature,199, pp. 55-
67 (1963).
35. Holm-Hansen, 0., and C. R. Booth, "The Measurement of Adenosine Tri-
phosphate in the Ocean and Ecological Singificance", Limnology an
Oceanography. 11, pp. 510-519 (1966).
36. Hamilton, R. D., and Holm-Hansen 0., "Adenosine Triphosphate Content
of Marine Bacteria", Limnology and Oceanography. 12_, pp. 319-324 (.1967)
130
-------
37, Patterson, J. W. Brezonik, P. L., and H. D. Putnam, "Measurement
and Singificance of Adenosine Triphosphate in Activated Sludge",
Environmental Science and Technology. 4^ (7)> pp. 569-575 (1970).
38. Aledort, L,, Weed, R. I. and S. B. Troup. "Ionic Effect on Firefly
Bioluuminescence Assay of Red Blood Cells (RBC) ATP", Analytical
Biochemistry. 17» PP- 268-277 (1966).
39. Forrest, W. W. "ATP Pool during Growth Cycle in Streptococcus
faecalis", Journal of Bacteriology, 90, pp. 1013-1018 (1965).
40. Forrest, W. W., and D. J. Walker, "Synthesis of Reserve Materials
for Endogenous Metabolism in Streptococcus faecalia", Journal
of Bacteriology. 89_, pp. 1448-1452 (1965).
41. Knowles, C. J., and L. Smith, "Measurement of ATP Levels of Intact
Azobacter vinelandii", Biochimica et Biophysica Acta, 197, pp. 152-
160 (1970).
42. Lyman, G., and J. DeVincenzo, "Determination of Picogram Amounts
of ATP Using the Luciferin-Luciferase Enzyme System", Analytical
Biochemistry. 21, pp. 435 (1967).
43. Holmsen, H., Holmsen, I., A. Barnhardeas, "Microdetermination
of ADP and ATP in Plasma with Firefly Luciferase System", Analytical
Biochemistry. 17., pp. 456-473 (1966).
44. Lin, S., and H. P. Cohen, "Measurement of Adenosine Triphosphate'
Content of Crayfish Stretch Receptor Cell Preparations", Analytical
Biochemistry. 24_, pp. 531-540 (1968).
45. Klofat, W., Picciolo, G., Chappell, E. W., and E. Freese, "Production
of ATP in Normal Cells and Sporulation Mutants of Bacillus subtilis",
Journal of Biological Chemistry. 244. pp. 3270 (1969).
131
-------
46. Addanki, S., Sotos, J. F., and P. D. Rearick, "Rapid Determination
of Picomole Quantities of ATP with Liquid Scintillation Counter",
Analytical Biochemistry. 14, pp. 261-264 (1966).
47. Stanley, P. E., "Determination of Subpicomole Levels of NADH and
FMN Using Bacterial Luciferase and the Liquid Scintillation
Spectrometer", Analytical Biochemistry. 39. pp. 441-453 (1971).
48. Stanley, P. E. and S. G. Williams, "Use of Liquid Scintillation
Spectrometers for Determining Adenosine Triphosphate by the
Luciferase Enzyme", Analytical Biochemistry, 29, pp. 381-392
(1969).
49. St. John J. B., "Determination of ATP in Chlorella with Luciferin-
Luciferase Enzyme System", Analytical Biochemistry. 37, pp. 402-408
(1970).
50. D'Eustachio, A. J. and D. R. Johnson, "Adenosine Triphosphate Content
of Bacteria", Abstract #3062 from Federation Proceedings, p. 761
(1968).
51. Sharpe, A. N., Woodward, M. N. and A. K, Jackson, "ATP Levels
in Foods Contaiminated by Bacteria", The Journal of Applied Bac-
teriology. 33_, pp. 758-767 (1970).
52. Defresne, L., and H. J. Gitelman, "A Semiautomated Procedure
for Determination of Adenosine Triphosphate", Analytical Biochemistry.
37., pp. 402-408 (1970).
53. Kao, I. C., Chiu, S. Y., Fan, L. T., and Erickson, L. E., "ATP pools
in Pure and Mixed Cultures", Journal Water Pollution Control Federation,
45_, pp. 926-931 (1973).
54. Biospherics, Inc., "Biomass Determination -A New Technique for Acti-
vated Sludge Control", Project Report, No. 17050, EOY, U.S. Environ-
mental Protection Agency (1972).
132
-------
55. Post, R. L.f Merritt, C. R., Kinsolving, C. R. and C. D. Albright,
"Membrane ATP-ase and Active Transport", Journal of Biological
Chemistry, 235, pp. 1796-1802 (1960).
56. Nicholas, D. J. D., and C. R. Clark", Bioluminescent Method for
Determining Micro Quantities of Ammonia in a Liquid Scintillation
Spectrometer", Analytical Biochemistry, 42, pp. 560-561 (1971).
57. Brolin, S. E., "Photokinetic Micro Assay on Dehydrogenase Reactions
and Bacterial Luciferase", Analytical Biochemistry, 42, pp. 124-135
(1971).
58. Lenhard, G., Nourse, L. D., and H. M. Schwarts, "Dehydrogenase
Activity of Activated Sludges", Advances in Water Pollution Research
£, pp. 105-127 (1965).
59. Lenhard, G., "A Standardized Procedure for the Determination of
Dehydrogenase Activity in Samples of Anaerobic Treatment System",
Water Research, 2_, pp. 161-167 (1968).
60. Bucksteeg, W., "Determination of Sludge Activity - A Possibility
of Controlling Sludge Plants", Advances in Water Pollution Research
2., pp. 83-102 (1966).
61. Jones, P. H. and D. Prasad, "The Use of Tetrazolium Salts as a Measure
of Sludge Activity", Journal of Water Pollution Control Federation, 4^,
(11), pt. 2, R441-449 (1969).
62. Shih, C. S., and V. T. Stack, Jr., "Temperature Effects on Energy
Oxygen Requirements in Biological Oxidations", Journal Water Pollution
Control Federation. 4JL, (11), pt. 2, R461-473 (1969).
63. Marlar, J., "The Effect of Turbulence on Bacterial Substrate Utilization",
M.S. Thesis, Georgia Institute of Technology, Atlanta, Georgia (Dec-
ember, 1968).
133
-------
64. Ghosh, S., "Kinetics of Aerobic Utilization of Mixed Sugars by
Heterogeneous Microbial Populations", Ph.D. Thesis, Georgia
Institute of Technology, Atlanta, Georgia (Nov., 1969).
65. Dean, A. C. R., and P. J. Rodgers, "Steady State Levels of Dehydro-
genases and a-and $-Glucosidases in Klebsiella aerogenes", Journal of
General Microbiology, 5]_, pp. 102-122 (1971).
66. Randall, C. W., Turpin, J. K., and P. H. King," Activated Sludge
Dewatering: Factors Affecting Drainability", Journal Water Pollution
Control Federation. 43, (1), pp. 102-122 (1971).
67. Standard Methods for the Examination of Water and Wastewater, 13th
edition, APHA (1971).
68. Rhines, C. E., "Decontamination of Membrane Filter Holders by
Ultraviolet Light", Journal American Water Works Association. 57,
p. 500 (1965).
69. "Glucostat for the Enzymatic Determination of Glucose", Worthing-
ton Biochemical Corporation, Freehold, New Jersey (1965).
70. "Galactostat: A Coupled Enzyme System for the Determination of Galactose",
Worthing Biochemical Corporation, Freehold, New Jersey (1966).
71. Swanton, E. M. Curby, W. A., and H. E. Lind, "Experiences with the
Coulter Counter in Bacteriology", Applied Microbiology 10, pp. 480-
485 (1962).
72. Fraser, D., "Bursting Bacteria by Release of Gas Pressure", Nature,
167, pp. 33-34 (1951).
134
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SECTION VI
APPENDICES
Page
A. Total Suspended Solids Determination 136
B. ATP Extraction Procedure 138
C. Procedure for Dehydrogenaae Test 139
D. Time and Cost Summary of Various Analytical Techniques 140
135
-------
APPENDIX A
TOTAL SUSPENDED SOLIDS DETERMINATION
Procedure
1. Wash 0.45 y white grid 47 mm Millipore filters with distilled water
under vacuum for approximately two minutes in order to remove the
glycerine and wetting agent and to insure a constant tare weight.
Lable filters and dry filters for 30 minutes at 103°C and desiccate
for 30 minutes prior to weighing.
2. Weigh and record tare weight of each filter. Store filters in desi-
ccator prior to use.
3. Each sample is filtered through a pair of tared filters. The top
filter in the pair is the test filter which will retain the solids,
while the bottom filter in the pair is the control filter.
4. Using forceps, place the pair of filters on a fritted base of the
filter holder so that the test filter is above the control filter.
Place the funnel on top of the fritted based and clamp securely.
5. Pipet the selected volume of sample into funnel, apply vacuum to the
filter, wash inside of funnel free of any attached solids, then filter
to dryness.
6. Release vacuum, remove clamp, and funnel from the holder, and with
forceps carefully remove and separate the pair of filters. (Use
care not to disturb the surface of the test filter.) Place each
filter on a teflon pad (wet filter tend to stick to glass).
7. Dry filters at 103°C for 30 minutes. Desiccate for 30 minutes
after drying. Reweigh each filter and record its final weight.
136
-------
8. Subtract tare w«ight from the final weight of each filter. The
results obtained for the test filter gives the uncorrected weight of
the sample solids while that for the control filter gives the gain
or the loss in weight.
9. Apply control filter weight change as a correction factor to each
test filter result, substracting this factor when the control filter
shows a weight increase or adding the factor when the control filter
shows a weight decrease.
10. Compute the dry bacterial solids concentration from the determined
weight of solids and the known volume of the sample filtered.
137
-------
APPENDIX B
ATP EXTRACTION PROCEDURES
1. Add 0.5 ml sample to a test tube containing 1.0 ml of 90% Dimethyl
Sulfoxide (DMSO).
2. Mix 10 seconds by vortex mixer.
3. Allow to stand at room temperature for 2 minutes, the optimum
recovery time.
A. Add 5.0 ml of 0.01 M morpholinopropane sulfonic acid (MOPS) buffer.
5. Mix the solution thoroughly.
6. Place tube contianing test material into ice bucket until assayed.
7. Assay the solution directly using the following formula to convert
Biometer readings to units per milliliter of sample.
Units/ml - Biometer Reading x 13
138
-------
APPENDIX C
PROCEDURE FOR DEHYDROGENASE TEST
1. Set up 3 NPN tubes: one for blank and two for samples in duplicate.
Pipette 8 ml of distilled water into blank tube.
2. Pipette 1 ml Tris buffer into each tube.
3. Disintegrate the sample at 30 atmospheres of nitrogen (Parr Bomb)
or at 15,000 rpm in a blender for 2 minutes. Pipette 8 ml of sample
into two sample tubes.
4. Incubate at 37°C and bubble nitrogen at slow rate.
5. After 10 minutes, add 1 ml TTC-glucose to all tubes and return the
TTC-glucose to refrigerator.
6. Incubate for 60 minutes and continue nitrogen flow. Cover tubes with
black plastic sheet.
7. After 60 minutes add 1 ml of formaldehyde. Also add 1 ml of 4 N
HCI to each tube. Stop nitrogen flow.
8. Wash nitrogen purging tubes with 95% ethyl alcohol.
9. Dilute samples and blank to 50 ml mark • Mix.
10, Keep samples in darkness for 30 minutes.
11. Filter through cotton In a funnel and Into cuvette (10 or 1 cm
ligh path).
12. Read and note X transmittance at 483 my and 0.05 mm slit width.
139
-------
APPENDIX D
TIME AND COST SUMMARY OP SELECTED ANALYTICAL TECHNIQUES
Analytical Technique
Time to Perform Analysis
Cost of Specialized Equipment and Reagents
Dehydrogenase Test
Total Suspended Solids
ATP bytBio»eter
Glucostat Test
Galactostat
Coulter Counter
Enumeration
Millipore Filter Technique
2 1/2 hrs. (1 hr. incubation
and 30 min. color
development)
1 1/2 hrs. (30 min. drying
period & 30 min.
in desiccator)
10 min. (30 min. for enzyme
substrate and buffer
preparation prior to
analysis)
1 1/2 hrs. (30 min incuba-
tion)
3 1/2 hrs. (1-1 1/2 hrs.
incubation)
3-4 hrs.
20 min. (16 hr. incuba-
tion)
Parr Bomb - S678.00
2r3,5 Triphenyltetrazolium chloride -
$3.83/5g. (allows for 2500 tests)
No special equipment.
Reagents (substrate and buffer)
(allows for 500 tests) $160
Biometer $5,000
Glucostat reagents - $4.90/box of
5 sets of reagents (allows for 50 tests)
Galactostat reagents - $13.75/box of 5
sets of reagents (allows for 85 tests)
Isoton (particle-free electrolyte)
$17.00/5 gal. (allows for 190 tests)
MF sterile filter paper - $15.00/box
of 100 MF sterile plastic petri dishes
S6.00/box of 100
-------
TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
7" PEPORT NO.
EPA-600/2-75-029
3. RECIPIENT'S ACCESSION-NO.
ANDSUBTITLE
MEASUREMENT OF ACTIVE BIOMASS CONCENTRATIONS IN
BIOLOGICAL WASTE TREATMENT PROCESSES
5. REPORT DATE
September 1975 (Issuing Date)
6. PERFORMING ORGANIZATION CODE
8. PERFORMING ORGANIZATION REPORT NO.
F. G. Pohland and S. J. Kang
ORGANIZATION NAME AND ADDRESS
School of Civil Engineering
Georgia Institute of Technology
Atlanta, Georgia 30332
10. PROGRAM ELEMENT NO,
1BB043 (ROAP 21-ASR, Task 008)
11. CONTRACT/GRANT NO.
R800354
. SPONSORING AGENCY NAME AND ADDRESS
Municipal Environmental Research Laboratory
Office of Research and Development-
U.S. Environmental Protection Agency
Cincinnati. Ohio 45268 _
13. TYPE OF REPORT AND PERIOD COVERED
Final, 1970-1974
14. SPONSORING AGENCY CODE
EPA-ORD
^^SUPPLEMENTARY NOTES
ABSTRACT
This research was initiated to determine the applicability and limitations of the dehydrogenase
test for the measurement of active biomass in biological wastewater treatment processes. Pure
culture with E^ coli and/or heterogeneous culture batch studies were conducted on a variety of
substrates including glucose, galactose, sucrose, alanine, acetic acid, and selected industrial
wastewaters. Also conducted were continuous aerobic or anaerobic culture studies with and with-
out solids recycle. Dehydrogenase activity was monitored along with other parameters including
plate count. Coulter Counter enumeration, adenosine triphosphate (ATP), and suspended solids to
provide comparative and complementary information on the biomass concentration. Dehydrogenase
activity was a very sensitive and accurate measure of active biomass throughout the growth
phases especially during endogenous growth but showed limitations with the nutrient deficient
cultures. The correlation between dehydrogenase activity and suspended solids was constant at
varying retention times, or at all growth rates with or without solids recycle. Consequently,
a standard curve could be developed for given wastewaters by operating the measurement of active
biomass and thereby effectively controlling the biological process. The measurement of ATP was
also a reliable new technique for measurement of active biomass except more study on the extrac-
tion method is required as well as investigations on the change of the correlation with suspended
solids with the change of growth rate. The technique for dehydrogenase activity measurement is
simple, less costly and gives more reliable and interpretable results. This report was submitted
in fulfillment of Grant No. R8003S4 (17050 GAI) by the School of Civil Engineering, Georgia
Institute of Technology, under partial sponsorship of the U.S. Environmental Protection Agency.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
*Enzymes
*Assaying
Activated sludge
*Biomass
Process control
Waste treatment
Quantitative analysis
13B
18. DISTRIBUTION STATEMEN1
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)
UNCLASSIFIED
!0. SECURITY CLASS (This page)
UNCLASSIFIED
21. NO. OF PAGES
153
22. PRICE
gpA Form 2220-1 (9-73)
141
ftUSGPO: 1975 - 657-695/5304 Region 5-11
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