WATER POLLUTION CONTROL RESEARCH SERIES • 17O7ODHOO2/71 DMA CONCENTRATION AS AN ESTIMATE OF SLUDGE BIOMASS ENVIRONMENTAL PROTECTION AGENCY WATER QUALITY OFFICE ------- 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. ------- 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 ------- 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 ------- 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 - ------- 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 ------- 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 - ------- 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- ------- 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. ------- 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 - ------- (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 - ------- 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 - ------- 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 - ------- 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 ------- 400 - z Q 234-56789 SONICATION TIME—MINUTES FIGURE 2. THE EFFECT OF SONICATION ON THE RELEASE OF DNA FROM ACTIVATED SLUDGE - 6 - ------- 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 - ------- 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 ------- 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 - ------- 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 - ------- 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 - ------- 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 - ------- 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 - ------- 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 - ------- 250 LJ < CO 200 cr ui CL 150 — 100 — 0 I 234567 TIME- HOURS FIGURE 4. DEGRADATION -OF DMA BY ACTIVATED SLUDGE 24 - 15 - ------- 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 - ------- 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 - ------- 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 - ------- 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 - ------- 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 - ------- 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 - ------- 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 ------- |