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.
                                 - ill -

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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
                                - V

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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
                                - v i -

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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
                                 -vii-

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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:
                                 - 1 -

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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.
                                 - 2 -

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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:
                                 — 3 -

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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-
                                 - 4 -

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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
                       - 6 -

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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-
                                 - 7 -

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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
                                 - 9 -

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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.
                                 -  10  -

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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.
                                 - 11 -

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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.
                                 - 12 -

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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
                  - 13 -

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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
                                 - 14 -

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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
                       - 15 -

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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
                                 - 16 -

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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
                                  -  17  -

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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
                                 -  18  -

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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.
                                 - 19 -

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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.
                            - 20 -

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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.
                                 - 21 -

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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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