WATER POLLUTION CONTROL RESEARCH SERIES • 17O7ODHOO2/71
DMA CONCENTRATION
AS AN ESTIMATE OF SLUDGE BIOMASS
ENVIRONMENTAL PROTECTION AGENCY WATER QUALITY OFFICE
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WATER POLLUTION CONTROL RESEARCH SERIES
The Water Pollution Control Research Series describes
the results and progress in the control and abatement
of pollution in our Nation's waters. They provide a
central source of information on the research, develop-
ment, and demonstration activities in the Water Quality
Office, Environmental Protection Agency, through inhouse
research and grants and contracts with Federal, State,
and local agencies, research institutions, and industrial
organizations.
Inquiries pertaining to Water Pollution Control Research
Reports should be directed to the Head, Project Reports
System, Office of Research and Development, Water Quality
Office, Environmental Protection Agency, Room 1108,
Washington, D.C. 20242.
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DNA CONCENTRATION AS AN ESTIMATE OF SLUDGE BIOMASS
by
Southwest Missouri State College
Springfield, Missouri 65802
for the
WATER QUALITY OFFICE
ENVIRONMENTAL PROTECTION AGENCY
Project #17070 DHO
February 1971
For sale by the Superintendent of Documents, U.S. Government Printing Office, Washington, D.C. 20402 - Price 40 cents
Stock Number 5501-0112
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EPA Review Notice
This report has been reviewed by the Water
Quality Office, EPA, and approved for publication.
Approval does not signify that the contents
necessarily reflect the views and policies of
the Environmental Protection Agency, nor does
mention of trade names or commercial products
constitute endorsement or recommendation for
use.
11
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ABSTRACT
The objective of this project was to determine the feasibility
of using DNA concentration as an estimate of sludge biomass. Such
an estimate would be valid provided the DNA present in the sample
represented only viable cells. This assumption was satisfied by
experimentation. Since DNA constitutes about four percent of the
organic matter of bacterial cells, DNA expressed as percent of
volatile solids was used to estimate the amount of organic matter
represented by viable cells in a sludge sample.
Sludge population in terms of cells per ml was estimated by
assuming the weight of one cell to be 1 x 10~" mg. The population
size as based on DNA analyses was then compared with that of a cell
count obtained from the most probable number (MPN) method. Popula-
tion estimates of this type were performed on activated sludge.
This report was submitted in fulfillment of Project Number
17070DHO, under the partial sponsorship of the Water Quality Office,
Environmental Protection Agency.
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TABLE OF CONTENTS
1. Introduction and Background Information 1
2. Materials and Methods „ 2
Preparation of Sample „ 2
Colorimetric Analysis of DNA 3
Extraction of Crude DNA From Activated Sludge 4
Media 4
3. Experimental Phase and Discussion » 7
Effect of Sonication on MPN Count <, 7
Effect of Sonication on Quantity of DNA Released 8
Effect of Ultra Violet Light „ 9
Degradation of DNA by Activated Sludge 11
Population of Activated Sludge « 16
Population of Sewage <,..„....<> 19
4. Literature Cited „ „ 0 21
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LIST OF FIGURES
Figure
1. DNA S tandard Curve „ 5
2. The Effect of Sonication on the Release of DNA from
Activated Sludge <,. .. 6
3. Degradation of DNA by Activated Sludge 13
4. Degradation of DNA by Activated Sludge 15
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LIST OF TABLES
Table
1. Composition of Medium Used in Estimating Sludge
Population. „<,.„ 7
2. Effect of Sonication Time on Most Probable Number
Count „ „ 7
3. Killing Effect of UV Exposure on Population Size
o f S ludge 10
4. The Effect of UV on the Rate of D.egradation of DNA 11
5. DNA Content of Various Sludges - October through
December, 1969. o 17
6. DNA Content of Various Sludges - January through
August, 1970 o 18
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CONCLUSIONS
This investigation has shown that DNA released from dead cells is
rapidly degraded by activated sludge. The DNA concentration of this
sludge may therefore be used as an estimate of the viable population,
or the amount of organic matter represented by viable cells.
It was found that viable cells may represent from 75 to 100
percent of the organic matter of activated sludge. The average of 20
representative analyses during the months of July and August, 1970,
were 98 percent.
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INTRODUCTION AND BACKGROUND INFORMATION
The purpose of this project was to determine whether the biomass
of activated sludge could be estimated by a DNA analysis of that sludge.
It is well known that a cell count of activated sludge cannot be
obtained by the conventional plate count as used for uniform suspensions
of cells. The main difficulties that prevent the use of the plate count
are the unknown nutritional requirements of the many species present in
the sludge and the incorporation of the organisms in a gelatinous matrix.
DNA is a unique constituent of living protoplasm, and since the DNA
content of bacterial cells is fairly constant, it was proposed that the
quantity of bacterial protoplasm present in the sludge could be esti-
mated from the DNA content of that sludge.
DNA constitutes about four percent of the volatile matter of
bacterial cells. In order to make use of this relationship, one must
assume that the DNA becomes degraded when the organism dies. This DNA
degradation could possibly be catalyzed by enzymes that normally func-
tion in the synthesis of DNA in the viable cell, or the DNA may be
degraded by other organisms present in the environment.
In order to use DNA as an estimate of viable cells, it must first
be demonstrated that DNA from dead cells does not contribute signifi-
cantly to the total content of sludge DNA. This was done by adding DNA
extracted from sludge to activated sludge. DNA degradation was deter-
mined by periodic sampling.
Once the DNA content of the sludge has been determined, the percent
DNA of volatile solids is easily obtained:
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(1) ug DNA x 100
ug volatile solids
The percent of sludge organic matter that is represented by viable
bacterial protoplasm may be calculated as shown below:
(2) % DNA x 100
4.0
The biomass may be obtained as follows:
(3) mg DNA/ml x 100 = mg biomass/mi
4.0
For example, if the sludge was found to contain 160 ug DNA/ml, the
biomass would be 4000 ug or 4.0 mg per ml.
An estimate of the number of cells per ml of sludge or per gram of
dry weight may be obtained by assuming the weight of one cell to be
approximately 1 x 10 mg. The number of cells/ml would be repre-
sented by the expression:
(4> mg biomass/ml = mg biomass/ml x 109 cells/mg =
1 x 10~y
mg DNA/ml x 100 x 1Q9 cells/mg = cells/ml
4.0
MATERIALS AND METHODS
Preparation of Sample.
The sludge used in this research was obtained from the Southwest
Springfield Waste Treatment Plant. The samples were stored in ice
during the transport from the plant to the laboratory.
The procedure for extraction of DNA is based on that of Agardy and
Shephard (1965).
1. Filter chilled sludge through cheese cloth.
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2. Pipette 1.0 ml of chilled and filtered sludge into 4.0 ml of
12.5% trichloroacetic acid, (TCA), or 2.0 ml of sludge into
3.0 ml of 17.0% TCA. In either case the final concentration
of TCA is 10%. Mix sludge and TCA by pipetting.
3. Centrifuge in the cold at 8.2 x 1000 G for 10 minutes.
4. Discard supernatant.
5. Add 5.0 ml of 95% ethanol to pellet. Mix well.
6. Centrifuge at 8.2 x 1000 G for 10 minutes. Discard supernatant.
Steps 5 and 6 may need to be repeated if the sample contains a
large amount of lipid material.
7. Add 4.0 ml of 0.5 N perchloric acid, (PCA), to pellet. Heat
at 90°C for 15 minutes. Mix 2 or 3 times during this period.
8. Centrifuge at 8.2 x 1000 G for 10 minutes. Save supernatant.
This is the first DNA extract.
9. Add 2.0 ml of 0.5 N PCA. Heat at 90°C for 20 minutes. Mix 2
or 3 times during this period.
10. Centrifuge at 8.2 x 1000 G for 10 minutes.
11. Combine supernatant with extract from step 8.
12. Repeat steps 9-11.
13. Perform DNA analysis of combined extracts.
The DNA analysis of supernatants was performed in the following
manne r:
1. Dilute supernatant with PCA to make the final concentration
0.5 N PCA, e.g. 9.0 ml of supernatant may be mixed with 1.0
ml of 5.0 N PCA, or 5.0 ml of supernatant may be mixed with
5.0 ml of 1.0 N PCA.
2. Heat the acidified supernatant for 15 minutes at 70°C.
3. Do DNA analysis.
Colorimetric Analysis of DNA
This analysis is based upon that of Burton (1965).
Reagents:
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1. Aqueous acetaldehyde, 1.6 g per 100 ml.
2. Diphenylamine reagent: dissolve 1.5 g of diphenylamine in
100 ml of acetic acid. Add 1.5 ml of concentrated H2S04-
Store in the dark. Just before use add 0.5 ml of aqueous
acetaldehyde for each 100 ml of reagent.
3. 1.0 N perchloric acid, (PGA).
4. 0.5 N PGA.
5. Stock standard DNA: prepare by dissolving 40 mg highly poly-
merized calf thymus DNA in 100 ml of 5 mM NaOH.
Standard DNA solutions are obtained by diluting the stock
solution with 5 mM NaOH. The standard solutions must be
heated at 70°C for 15 minutes with equal volumes of 1 N PGA.
The standard curve is prepared by mixing 2.0 ml of standard
DNA with 4.0 ml of diphenylamine reagent containing acetalde-
hyde. The tubes are incubated for 16-20 hours at 25-30°C.
The OD is read at 600 nonemeters.
The range of the analysis is from about 5 to 80 micrograms DNA/ml.
A typical DNA standard curve is shown in Figure 1.
Extraction of Crude DNA from Activated Sludge.
1. Strain sludge through cheese cloth and allow to settle. Pour
off a volume of supernatant equal to one half of the original
volume.
2. Resuspend the solids and sonicate to rupture cells and release
DNA. The time required for maximum release of DNA will depend
upon the size and concentration of the sample. See Figure 2.
3. Centrifuge the sonicated sludge at 12.8 x 1000 G for 10 min-
utes .
4. Heat the supernatant from step 3 for 15-30 minutes at 70°C in
order to pasteurize the sample and coagulate protein.
5. Centrifuge at 21.6 x 1000 G for 10 minutes. The supernatant
contains crude DNA.
Media.
The composition of the medium used in estimating the cell popula-
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E 0.8
0.7
8 0.6
to
£ 0.5
0.4
z
LJ
0.3
O 0.2
I-
Q.
0 O.I
1
1
1
0 10 20 30 40 50 60 70 80
DMA- jjg PER ML
FIGURE I. TYPICAL STANDARD DNA CURVE
_ c M
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400 -
z
Q
234-56789
SONICATION TIME—MINUTES
FIGURE 2. THE EFFECT OF SONICATION ON
THE RELEASE OF DNA FROM ACTIVATED SLUDGE
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tion of the sludge samples by the MPN method is given in Table 1.
Table 1. Composition of Medium Used in Estimating Sludge Population.
Component g/liter
Glucose 5.0
Yeast extract 10.0
Nutrient broth powder 4.0
K2HP04 1.0
EXPERIMENTAL PHASE AND DISCUSSION
Effect of Sonication on MPN Count.
In order to determine the sonication time that would give the max-
imum number of cells as determined by the most probable number (MPN)
method, 10 ml of 5 times concentrated sludge was sonicated for various
periods of time at 80 watts.
The results are shown in Table 2.
Table 2. Effect of Sonication Time on Most Probable Number Count.
Time MPN
minutes cells/ml
0 6.9 x 108
1 4.8 x 109
2 3.2 x 1010
4 3.4 x 109
8 1.6 x 109
10 1.8 x 108
The results indicate that the floe particles are broken apart and
the individual cells of the floe are released within the two first min-
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utes of sonication. Further sonication causes destruction of the cells.
It was found that in general, the largest population of cells was
obtained after two minutes of sonication. However, this was not always
true. The time required appears to be determined by the characteristics
of each individual sludge sample.
It must be made clear that even though the largest population was
obtained after two minutes of sonication, this number does not represent
the total population since many cells would have been destroyed by this
time. Other experiments, such as the one shown in Figure 2, indicate
that the number of cells killed during the two first minutes of sonica-
tion approximates 10 percent of the total population.
Effect of Sonication on Quantity of DNA Released.
In order to determine the effect of sonication time on the quantity
of DNA released from sludge floe, the following experiment was performed.
Activated sludge from the nitrification tank was strained through cheese
cloth and centrifuged. The solids were concentrated 2.5 times by resus-
pending in part of the supernatant. Ten ml samples were sonicated for
0, 1, 2, 3, 4, 5, 6, 7, 8, and 10 minutes. The sonicated sludge was
centrifuged and a DNA analysis was performed on the pellet and the super-
natant. The results are shown in Figure 2.
It is evident from the graph that the DNA is released very rapidly
from the floe during the first three minutes. After 10 minutes of soni-
cation there is a leveling off where about 24/360 x 100 = 67% of the
total DNA has been released, i.e. at this time about 677=, of the popula-
tion has been killed. Supernatant DNA obtained in this manner was
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partially purified by heating at 70°C for 30 minutes in order to coagu-
late proteins which were removed by centrifugation. The partially puri-
fied DNA was added to activated sludge to check for DNA degradation.
Effect of Ultra Violet Light.
In order to demonstrate the degradation of DNA of dead cells,
several experimental techniques were employed. Ultra violet light was
employed as the lethal agent in a series of experiments. The idea
behind these experiments was to destroy 99 percent of the population by
exposure to ultra violet light. The DNA of the non-viable cells would
either be destroyed by the enzymes released from the dead cells or by
the remaining viable cells. However, any time a microbial population is
exposed to a pasteurizing agent such as ultra violet light, heat, or
chemicals one must consider the possible growth of the remaining viable
cells provided the conditions following exposure are favorable for
growth.
In our experiments where we destroyed cells by exposure to ultra
violet light, heat, or chemicals we were unable to demonstrate a degra-
dation of DNA corresponding to the initial reduction in population. In
one experiment using ultra violet light as the pasteurizing agent, the
population was reduced initially from 4.4 x 10 cells per ml to less than
8.0 x 10^ cells per ml. During four hours of incubation following the
exposure, the population increased to 1 x 10 cells per ml. Consequently,
in experiments of this type, where there is a simultaneous degradation
and synthesis of DNA, one cannot demonstrate the degradation of the DNA
of the non-viable cells.
The experiments using ultra violet light as a pasteurizing agent
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were performed as described below.
1. Sonicate 10 ml of strained and concentrated sludge for one
minute at 80 watts.
2. Expose 5.0 ml of sonicated sludge to UV light for 7 minutes,
Use 60 mm petri dishes and keep the sludge stirring during
the exposure.
3. Incubate the UV treated sludge on rotary shaker. Sample
periodically for DNA analysis.
4. Determine viable population at zero time, and periodically
thereafter.
Table 3. Killing Effect of UV Exposure on Population Size of Sludge.
Exposure time Population Size
minutes cells/ml ug DNA/ml
0
5
7
6.4 x 109
6.8 x 106
2.8 x 106
192
194
195
This shows that the population may be reduced by 99 percent by
exposure to ultra violet light for five minutes. Further exposure has
little effect. It is evident that the DNA as determined in our analysis
is not effected by the ultra violet light, since the variation between
the zero time and seven minute determinations are insignificant.
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Table 4. The Effect of UV on the Rate of Degradation of DNA.
Control
No UV
UV
Treatment
7 Minutes
Time
hours
0
1
2
3
4
0
1
2
3
4
MPN DNA
cells/ml ug/ml
4.4 x 107 156
136
130
113
108
136
131
121
117
113
DNA Degraded
ug/ml
0
20
26
43
48
0
5
15
19
23
As may be seen from Table 4 there is a decrease in the DNA of the
control population as well as in the UV treated population. This sug-
gests that the DNA degraded is that which was released from the one
minute sonication of the sample in order to break apart the floe.
Since the control sample has a considerably larger population than
the UV treated sample, it is expected that the DNA is degraded more
rapidly in this population. Unfortunately, separate analyses of the DNA
of the supernatant and of the solids were not performed in this experi-
ment. Such analyses would have indicated whether the DNA broken down
was that released from the initial sonication.
A repeat of the above experiment gave very similar results.
Degradation of DNA by Activated Sludge.
The rapid degradation of DNA by activated sludge was finally demon-
strated in the experiments described below.
The DNA used in this experiment was prepared as described on page 4
in this report.
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The experimental procedure was performed as follows:
1. Filter activated sludge.
2. Allow to settle.
3. Pour off a volume of supernatant equal to half of the original
volume. Save.
4. Add DNA extract.
5. Add supernatant to obtain original concentration of sludge.
6. Incubate on rotary shaker and sample periodically. Do separate
analyses on supernatant and solids.
7. As control use DNA extract diluted with sterile water.
8. A second control consisting of the activated sludge without the
added DNA may also be included.
The results of the above experiment are shown in Figure 3.
The data for the two controls are not shown on the graph. DNA
extract diluted with water to a final concentration of 175 ug DNA per ml
was not effected by four hours of shaking. The concentration after four
hours of shaking was the same as that of zero time.
Activated sludge without the addition of DNA maintained about three
micrograms of DNA per ml of supernatant. The DNA of the pellet varied
between 105 and 110 micrograms per ml.
From Figure 3 it may be seen that there is a slight increase in the
DNA of the pellet at the end of the first half hour of incubation fol-
lowing the addition of DNA. This increase is probably due to absorption
of DNA. There is, however, no increase in the DNA of the pellet corre-
sponding to the DNA decrease of the supernatant. This indicates that
under the conditions of this experiment the DNA is degraded and used
mainly as a source of energy and cell material other than DNA. Otherwise
there would have been an increase in the DNA of the pellet.
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140
FLASK A. SUPERNATANT
FLASK B, PELLET
FLASK A, PELLET
UJ 90
FLASK B> SUPERNATANT
123
TIME — HOURS
FIGURE 3. DEGRADATION OF DNA BY
ACTIVATED SLUDGE
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From the data shown in Figure 3 it may be calculated that 27.5
percent of the DNA added to flask A was degraded in the first half hour
of contact, and 48.5 percent was degraded after four hours of contact.
In flask B, 47.0 percent of the DNA was degraded at the end of one
half hour of contact and 76.0 percent was degraded at the end of four
hours.
It is quite evident from the graph that the initial absorption and
degradation occurs very rapidly. Then, as the cells become saturated,
the rate at which the DNA is being degraded decreases from 1.57 ug per
minute to 0.14 ug per minute. That is, the final rate is less than one
tenth of the initial rate.
The above results do not take into account the amount of degradation
that must have taken place during the initial mixing before the first
sampling. In the case of flask B, the zero time concentration of DNA in
the supernatant should have been 117 ug per ml. The graph shows 100 ug
per ml. However, if the graph is extrapolated to take into account four
minutes of sample preparation, the initial concentration becomes 117 ug
DNA per ml of supernatant.
Figure 4 shows the results of an experiment very similar to that
mentioned above. In this case the DNA extract was clarified by centrifu-
gation, but was not heated. The results are similar to those of the
previous experiment. Twenty percent of the DNA added was degraded in
the first 15 minutes of the experiment, 28.0 percent in the first 30
minutes and 80.0 percent in the first four hours. At the end of 24 hours,
94.0 percent of the DNA had been degraded.
In this experiment the DNA of the pellet increased from 140 ug per
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250
LJ
<
CO
200
cr
ui
CL
150 —
100 —
0 I
234567
TIME- HOURS
FIGURE 4. DEGRADATION -OF DMA BY
ACTIVATED SLUDGE
24
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ml at zero time to 195 ug per ml at the end of four hours. This corre-
sponds to a population increase of 27 percent.
The results of these two experiments seem to indicate that the
manner in which the DNA is utilized by the cells depends upon the condi-
tion of the floe, that is, the phase of growth that the cells are in.
These experiments were repeated using calf thymus DNA rather than
sludge DNA. The results were very similar. In one experiment the DNA
concentration of the supernatant dropped from 58 ug per ml to 17 ug per
ml in three hours, that is, a reduction of 41 ug per ml or 71.0 percent.
At the same time, the DNA of the solids increased from 138 to 161 ug per
ml, an increase of 23 ug per ml. This corresponds to a population
increase of 17 percent.
From these experiments and from the fact that there is only a trace
of DNA present in the supernatant of activated sludge, it is apparent
that any DNA present in the sewage or released from dead cells is rapidly
degraded by the sludge floe. The DNA of the sludge therefore represents
viable cells.
Population of Activated Sludge.
If one assumes the weight of one bacterial cell to be 1 x 10~" mg,
the population size as based on DNA analyses can be compared with that
of a cell count obtained from the most probable number method.
Population estimates of this type were performed on activated sludge,
return sludge and nitrification tank sludge. The latter tank contains
return sludge mixed with supernatant from the anaerobic digester. The
results are shown in Table 5.
When comparing the population estimates obtained from the two
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methods mentioned above, it was found that the population based on the
DNA content of the sludge was greater in 70 percent of the cases. This
is as would be expected due to the flocculant characteristic of the
sludge inoculum and the inability of many bacteria to grow in the count-
ing medium.
From the data in Table 5 it may be shown that in many cases the
viable bacteria represent nearly 100 percent of the organic matter in
activated sludge.
Table 5. DNA Content of Various Sludges, October - December, 1969.
Cells/ml x IP"9
Date
Oct.
Nov.
Dec.
22
24
27
29
3
5
10
12
14
17
19
24
26
3
12
15
17
Type of
Sludge
A
A
A
A
A
A
N
A
N
N
N
N
N
A
R
N
N
A
R
N
N
N
ug DNA/ml
112
47
72
54
61
60
126
71
109
196
192
151
156
70
65
144
124
53
58
132
139
134
% DNA
2.6
1.7
3.1
2.1
2.4
2.5
2.1
2.5
2.3
2.9
2.4
2.4
2.5
2.5
2.4
2.4
2.1
2.1
2.3
1.8
1.9
2.1
MPN
0.4
0.7
2.6
32.0
4.8
2.6
3.7
5.6
4.4
6.4
2.2
2.2
0.1
0.3
0.5
1.0
0.2
0.3
007
1.0
2.2
2.2
Based on
DNA
2.8
1.2
1.8
1.4
1.5
1.5
3.2
1.8
2.7
4.9
4.8
3.8
3.9
1.7
1.6
3.6
3.1
1.3
1.5
3.3
3.5
3.4
A indicates aeration tank
N indicates nitrification tank
R indicates return sludge
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Table 6. DNA Content of Various Sludges, January - August, 1970.
Cells/ml x 10"9
Date
Jan.
April
May
23
Feb. 20
22
18
June 24
29
July 4
8
15
Aug.
20
27
17
24
Type of
Sludge
A
R
N
A
R
N
A
R
N
A
R
N
A
R
N
A
R
N
A
R
N
A
R
N
N
N
N
A
R
N
A
R
N
A
R
N
A
R
N
A
R
N
A
R
N
ug DNA/ml
66
52
142
63
56
142
86
183
212
55
57
65
58
42
156
34
37
132
60
44
175
79
208
170
242
243
300
108
200
240
88
175
200
93
250
250
88
295
280
100
185
255
100
185
255
% DNA
2.5
2.2
2.2
2.6
2.4
2.2
3.5
3.0
2.8
2.5
2.4
2.3
2.6
2.4
2.1
2.3
2.3
2.0
2.6
2.0
2.4
3.3
2.7
2.3
3.9
4.2
4.2
4.4
5.6
3.4
3.0
3.7
3.0
3.6
4.1
4.1
4.0
3.9
3.9
3.6
4.0
3.7
3.5
4.0
4.0
MPN
1.1
0.2
1.1
0.3
0.5
0.5
0.7
1.4
0.2
1.1
0.1
0.5
6.4
3.7
6.4
0.2
0.5
1.4
0.7
3.2
1.0
Based on
DNA
1.7
1.3
3.6
1.6
1.4
3.6
2.2
4.6
5.3
1.4
1.4
1.6
1.5
1.1
4.4
0.9
0.9
3-. 3
1.5
1.1
4.4
2.0
5.2
4.5
6.1
6.1
7.5
2.7
5.0
6.0
2.2
4.4
5.0
2.3
6.3
6.3
2.2
7.4
7.0
2.5
4.6
6.4
2.5
4.6
6.4
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Bacterial Population of Sewage as Based on DNA Analysis.
To obtain an estimate of the bacterial population of sewage, 40 ml
of strained sewage was centrifuged at 8.2 x 1000 G for 10 minutes. The
resulting pellet was analyzed for DNA. The sewage was found to contain
1.63 ug DNA per ml and 0.38 percent DNA. By the use of Equation (2) on
page 2 it may be calculated that viable bacteria represent only 9.5
percent of the organic matter in sewage. The population may be estimated
by use of Equation (4) on page 2.
1.63 ug/ml x IP"3 mg/ug = 4>1 x 1Q6 cells/ml
4.0 x 10~9 mg/cell
This value is in accordance with most estimates of bacterial popula-
tion of sewage.
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ACKNOWLEDGMENTS
The author, Roar L. Irgens, wishes to express his sincere apprecia-
tion to Dr. H. Orin Halvorson, who initiated the idea for this research.
Appreciation is also expressed to Mrs. Glenda Marshman, Research
Assistant, and to Mr. Paul J. Cameron, Laboratory Assistant, during
various phases of the project.
In addition, the author would like to thank the personnel at the
Springfield Waste Treatment Plant for their cooperation in obtaining
sludge samples.
The Project Officer for the Water Quality Office, Environmental
Protection Agency, was Dr. Robert L. Bunch.
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LITERATURE CITED
1. Agardy, F. J. and W. C. Shephard. 1965. A rational basis for
digester loadings. Jour. Water Pollution Control Federation,
J7: 1236-1242.
2. Burton, K. 1955. A study of the conditions and mechanisms of the
diphenylamine reaction for the colorimetric estimation of
deoxyribonucleic acid. Biochem. Jour. 62: 315-323.
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1
5
Access/on Number
2
Subject
Field & Group
05 D
SELECTED WATER RESOURCES ABSTRACTS
INPUT TRANSACTION FORM
Organization
Southwest Missouri State College. Sprinefield. Missouri (SS80?
Title
DNA CONCENTRATION AS AN ESTIMATE OF SLUDGE BIOMASS.
10
22
Authors)
ROAR L. IRGENS
11
Date
February 1971
16
12
Pages
21
Project Number
17070 DHO
21
1 c- Contract Number
Note
Citation
Descriptors (Starred First)
25 I Identifiers (Starred First)
27
Abstract
The objective of this project was to determine the feasibility of using DNA
concentration as an estimate of sludge biomass. Such an estimate would be valid
provided the DNA present in the sample represented only viable cells. This assump-
tion was satisfied by experimentation. Since DNA constitutes about four percent of
the organic matter of bacterial cells, DNA expressed as percent of volatile solids
was used to estimate the amount of organic matter represented by viable cells in a
sludge sample.
Sludge population in terms of cells per ml was estimated by assuming the
weight of one cell to be 1 x 10~9 mg0 The population size as based on DNA analyses
was then compared with that of a cell count obtained from the most probable number
(MPN) method. Population estimates of this type were performed on activated sludge,
This report was submitted in fulfillment of project 17070 DHO under the
sponsorship of the Water Quality Office,
Abstractor
Roar L. Irgens
Institution
Southwest Missouri State College
WRjlOZ (REV. OCT. 1B68)
WRSIC
SEND TO; WATER RESOURCES SCIENTIFIC INFORMATION CENTER
U S. DEPARTMENT OF THE INTERIOR
WASHINGTON, D.C. 20240
* GPO: 1969—324-444
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