APPLIED MICROBIoLoGY, Feb. 1967, p.

358-361

Copyright @ 1968 American Society for Microbiology

Vol. 16, No. 2 Printed in U.S.A.

Microbiology of Anaerobic Sludge Fermentation'

I.

Enumeration of the Nonmethanogenic Anaerobic Bacteria

of Environmental

Department

ROBERT A. MAH AND CAROL SUSSMAN Sciences and Engineering, University of North Carolina, Chiapel Hill, North Carolina 27514 Received for publication 8 November 1967

An anaerobic medium containing sludge supernatant fluid and glucose was used for enumeration of bacteria from the sludge fermentation. Comparison of viable counts from several separate samples consistently showed 10 to 100 times more anaerobic than aerobic bacteria. However, viable counts of the various samples differed by as much as 10 times; this variation probably reflects a change in the natural environment or sampling errors, or a combination of the two. Direct microscopic counts yielded values of about 10'0/ml. The discrepancy between viable (108 to 109/ml) and direct counts may be due to large numbers of dead cells. Random isolates of representative colonies from high dilutions exhibited the ability to ferment sugars and are not likely to be methane bacteria.

Previous reports pertaining to the enumeration value reported was 8.2 x 107/ml for "Clostridium of bacteria in the anaerobic sludge fermentation carnofoetidum." Viable counts of cellulolytic can be divided into two categories, those dealing sludge bacteria yielded values as great as 1.7 x with methanogenic bacteria and those dealing 105/ml (11), but this figure was unusually high; with nonmethanogenic bacteria. The former other counts of cellulolytic bacteria were always group of organisms, all strict anaerobes, was in the range of 103/ml or lower (8, 11). Recent reportedly present at concentrations as high as studies by Torien (15) did not include quantita7 x 109/g of sludge solids and 1.6 x 109/ml of tive data. sludge liquid (6). Smith (12) reported figures as The methanogenic bacteria are restricted to high as 108/ml. All of these values are higher the dissimilation of certain organic acids and than those given for nonmethanogenic bacteria. alcohols (1). The recent findings of Bryant et al. Hotchkiss (7) found a total count of 8.5 x 107/ml (Bacteriol. Proc., p. 19, 1967) of an organism for nonmethanogenic bacteria in sludge from an growing in close association with and accounting Imhoff tank. These results were obtained by for the ability of the ethyl alcohol-nonoxidizing inoculation into various complex organic media, Methanobacillus omelianskii to oxidize ethyl alincluding nutrient agar, and incubation under cohol may lead to the disclosure of other bacaerobic conditions. It was not surprising that terial relationships which further reduce the such counts of aerobic organisms were lower than range of methanogenic substrates. Thus, the role the anaerobic methane producers, since the sludge of nonmethanogenic sludge anaerobes becomes environment has a measured E, as low as -530 of increasing importance because of their greater mv (5). The presence of higher numbers of physiological diversity. The initial substrates obligately anaerobic bacteria rather than aerobic present in sludge are complex organic compounds or euryoxic bacteria might, in fact, be expected. not known to be metabolized by methane bacHowever, it was not known whether nonmethano- teria. There is apparently a dependency of these genic anaerobes were also present in higher latter organisms on the fermentation products numbers than aerobes. produced by the dissimilation of the initial subInformation concerning the nonmethanogenic strates by the nonmethanogenic organisms. It anaerobes has been scarce. The numbers reported seems unlikely that the methane organisms with by Cookson and Burbank (4) were based on their presumed narrow substrate specificity would direct counts of normal sludge after acid-fast be the only predominant group of anaerobes. staining. Identification of several morphologically MATERI4LS AND METHODS nondistinctive bacteria was apparently made Sludge. A laboratory digester was inoculated with by direct microscopic observation; the highest sludge from the primary digester of the Third Fork I Department Publication No. 175. sewage plant at Durham, N.C.; this plant treats
358

VOL. 16, 1968

ANAEROBIC SLUDGE FERMENTATION

359

sewage primarily of domestic origin. An 8-liter bottle with gas, sample, and feed ports served as the receptacle; it was fitted with an airtight seal through

which a stainless-steel shaft and propeller were inserted. The shaft was connected to a 116 hp motor, and the sludge was stirred at hourly intervals for 5 min at a time. Gas was produced at the rate of approximately 3.5 liters per day and contained about 65% methane. All other conditions were the same as previously described by Smith and Mah (14). Media. Rumen fluid was tested because it contains fatty acids and unidentified growth factors necessary for culturing fastidious anaerobes (10). The inorganic salts solution was that reported by Smith (13). It was used in the preparation of the following media: rumen supematant fraction, sludge supernatant fraction, and the anaerobic synthetic medium (SM) of Caldwell and Bryant (3). A 1.5% agar medium containing 33% (v/v) clarified rumen fluid in inorganic salts solution was later supplemented with 0.01% glucose (2). Prior to use, the rumen fluid was centrifuged for 20 min at 36,400 X g. The gas atmosphere was 70% N2-30% CO2, and the medium was buffered with 0.4% NaHCO3 at about pH 6.8 to 7.0; 3.0 X 10-5% resazurin served as oxidation-reduction indicator and 0.05% cysteine and 0.025% Na2S as reducing agents. Cysteine was added during the preparation of the medium; Na2S was added just before inoculation. In the habitat-simulating medium, sludge supernatant fluid was substituted for the rumen fluid; other constituents were the same. Sludge supernatant broth was similar except for omission of agar. The synthetic medium of Caldwell and Bryant (3) was modified by the substitution of the inorganic salts used in the previous two media. Nutrient agar and E M B (Difco) were also examined under anaerobic conditions. Culture method. The anaerobic procedure used was the roll-tube method described by Hungate (8). Prior to inoculation, the sludge was mixed vigorously for 1 min under anaerobic conciitions by use of a

blendor. A 1-ml inoculum was introduced into 9 ml of sludge supernatant broth and a decimal dilution was made. Media to be tested were inoculated with 0.5 ml of broth from the same tube at the appropriate dilutions. Because of the slow growth rates exhibited by the sludge organisms, it was necessary to incubate for extended time periods (up to 6 weeks). For aerobic counts, plates were incubated in a humid chamber; the temperature of incubation was 35 C. Direct counts. Total counts were made by use of a Petroff-Hauser counting chamber and a Zeiss phasecontrast microscope at 600 and 1,200 X. The samples were diluted in physiological saline and blended for 1 min prior to counting.
RESULTS

Numbers of anaerobic sludge bacteria were estimated by decimal dilution in broth followed by inoculation into various agar media. Colony counts were made on all anaerobic cultures after incubation for 5 to 7 days and again after 4 to 5 weeks. Duplicate tubes containing between 30 and 300 colonies were counted. The difference in counts between successive tubes in the same decimal dilution series was always 10-fold at the higher dilutions. Table 1 shows the results of counts obtained in rumen fluid, the SM medium, and varying concentrations of sludge supernatant fluid. All media in each group received the same inoculum. In group 1, the media were tested in the presence and absence of low concentrations of glucose. Differences between media with and without glucose were not greater than 10-fold. However, since glucose supported a more rapid development of colonies, it was subsequently employed in other media. A comparison of 15, 33, and 50% sludge supernatant concentrations in the media yielded similar counts. The differNo. of cells/ml

TABLE 1. Anaerobic viable counts from laboratory digester inoculum

Medium
No glucose

5-7 days

4-5 weeks
No glucose

0.01% Glucose

0.01% Glucose

Group 1 15% SSa....................

33% SS .................... 50% SS .................... 33% rumen fluid ...........

Group 2 SMb 33% SS Group 3 SM 33% SS
a
.............

1.5 1.3 5 4 1.4

X X X X

108 108 107 108

2.0 X 108 1.9 X 108 1.8 X 108 1.5 X 108

1.2 X 108 9.9 X 107

1.9 1.6 1.6 1.4

X X X X

109 109 109 109

2.0 1.6 1.5 1.0

X 109 X 109 X 109 x 109

2.3 X 108 1.8 X 108 1.0 x 109 4.6 X 108

....................

1.6 X 108 1.3 X 108

SS = sludge supernatant medium.

SM

synthetic medium.

360

MAH AND SUSSMAN

APPL. MICROBIOL.

ences observed were small enough that data from larger numbers of samples would have to be statistically analyzed to determine whether they were actually significant. The counts obtained with 33 % rumen fluid were also in the same range. Ignoring these small differences, the 33% sludge supernatant concentration was selected for comparison with other media. In two other experiments (Table 1), counts on 33% sludge supernatant fluid were compared to those obtained on SM medium. The results indicated no gross differences between these two media. Table 2 shows the results of experiments in which the same inoculum was introduced into anaerobic as well as aerobic media. In group 1, sludge medium was compared with nutrient agar and E M B under anaerobic conditions. The sludge medium was also tested under aerobic conditions. These same media were compared with a second inoculum (group 2); in addition, aerobic counts were made on both sludge supernatant medium and nutrient agar. In both groups, the anaerobic counts on nutrient agar were surprisingly higher than those on sludge media after 5 days of incubation. However, the terminal counts after 5 weeks of incubation showed equivalent or better growth on sludge media. The anaerobic E M B counts were about 10 times lower. All counts obtained under aerobic condi-

TABLE 3. Viable counts of anaerobic and aerobic organisms from treatment plant inoculum
No. of cells/ml

Media

Anaerobic
1 week
6 weeks

Aerobic
6 weeks

Group 1

33% SSGa. 33% rumen fluid ........ SMb .......... Nutrient agar.

3.3 X 107 1.2 X 108

4.0 X 107 1.3 X 108 5.2 X 107 1.4 X 108 2.3 X 106 4.0 X 107 9.2 X 107 1.2 X 107

a SSG = sludge supernatant glucose. b SM = synthetic medium.

tions were considerably less than those observed under anaerobic conditions. A comparison of aerobic counts was made on E M B and sludge medium in the group 3 inoculum, and on nutrient agar and E M B in the group 4 inoculum. Sludge medium inoculated under anaerobic conditions served as the control in both samples. The results showed that E M B supported better growth than sludge under these conditions, but that nutrient agar and E M B were about the same. Anaerobic counts were again 10 to 100 times higher than aerobic counts. Table 3 shows the results of a direct comparison of anaerobic counts from a single inoculum taken TABLE 2. Comparison of anaerobic anid aerobic from the primary digester of the Third Fork viable counts of laboratory digester inoculum sewage plant at Durham, N.C. The following No. of cells/ml media were inoculated: sludge supernatant fraction, rumen fluid, nutrient agar, and SM medium. Media Aerobic Anaerobic Aerobic plate counts were also made on nutrient agar and SM medium. Anaerobic counts on all 4-5 weeks 5-7 days 5-11 days four media were nearly the same, except for nutrient agar which may be slightly lower. The Group 1 aerobic counts on nutrient agar were 10 times 8.3 X 107 1.3 X 109 33% SSGa. lower than the anaerobic counts. The SM medium Nutrient agar. 1.2 X 108 3.8 X 108 did not support aerobic growth equivalent to E M B ........ 2.1 X 107 8.0 X 107 2.0 X 105 nutrient agar. 33% SSG. Group 2 DISCUSSION 9.6 X 107 6.5 X 108 33% SSG. The selection of E M B and nutrient agar for Nutrient agar. 2.0 X 108 6.5 X 108 viable counts was based on past reports of the EM B ....... 9.0 X 107 9.3 X 107 1.2 X 105 presence of coliform and aerobic or euryoxic 33%0 SSG..... 3.8 X 106 organisms. On the basis of the present findings Nutrient agar. Group 3 on E M B, it is concluded that coliforms are 5.5 X 107 7.8 X 108 33% SSG. not the most numerous organisms in this habitat, 33% SSG. 26 X 107 although they are certainly easily demonstrated. E M B ........ 9.2 X 10~ Of all the media tested under aerobic conditions, Group 4 nutrient agar yielded the highest number of 1.4 X 108I 1.0 X 109 33% SSG.. colonies. Counts of nutrient agar pour plates 2.4 X 106 Nutrient agar. 2.1 X 106 showed that the aerobic bacteria were not the E M B ........ most numerous organisms present in the sludge a SSG environment. sludge supernatant glucose.
=

VOL. 16, 1968

ANAEROBIC SLUDGE FERMENTATION

361

The use of habitat-simulating media for enumeration of organisms has the disadvantage of variability from one batch to another. However, until a more precise comparison with synthetic media can be made, such media are chosen because of the probable presence of unknown growth requirements. The differences in anaerobic viable counts on sludge supernatant fluid, rumen fluid, and SM media are not decimal differences, and the counts obtained probably reflect a fairly accurate estimate of the actual numbers. Application of the most-probablenumber method of using sludge supernatant broth to estimate numbers of anaerobic organisms yielded results in the same range (109/rnl) as the colony counts. None of these media was purposely selective of specific physiological types. Accurate, direct microscopic counts are difficult to make in a heterogeneous sample such as sludge. Estimates from several samples yielded values at the level of 1010/ml. The discrepancy between the direct and culture (108 to 109/ml) counts may be due to the presence of large numbers of dead cells. Yet, the possibility of inadequate culture media or techniques cannot be excluded. Such a discrepancy also exists for the better-studied rumen habitat (9). Decimal differences in viable counts exist among the various samples regardless of the inoculum source, although counts from samples of the laboratory digester might be significantly higher than those from the treatment plant. These differences probably reflect changes due to actual differences in cell numbers in the natural environment or to sampling errors. Such fluctuations are not unexpected in a heterogeneous system of this type. More important, the results consistently showed an anaerobic viable count 10 to 100 times greater than the aerobic count, regardless of samples. This finding supports the hypothesis that anaerobic organisms are present at higher numbers. No attempt was made to discriminate between methanogenic and nonmethanogenic colonies. However, random isolates of representative colonies from high dilutions exhibited the ability to ferment sugars; this property is not characteristic of methane organisms. Characterization of these isolates is in progress.
ACKNOWLEDGMENTS

This investigation was supported by Public Health Service grant WP-00921-01. LITERATURE CITED 1. BARKER, H. A. 1956. Bacterial fermentations. John Wiley & Sons, Inc., New York.
2. BRYANT, M. P., AND I. M. ROBINSON. 1961. An improved nonselective culture medium for ruminal bacteria and its use in determining

diurnal variation in numbers of bacteria in the rumen. J. Dairy Sci. 44:1446-1456.

3. CALDWELL, D. R., AND M. P. BRYANT. 1966.

Medium without rumen fluid for nonselective enumeration and isolation of rumen bacteria. Appl. Microbiol. 14:794-801. 4. COOKSON, J. T., AND N. C. BURBANK. 1965. Isolation and identification of anaerobic and facultative bacteria present in the digestion process. J. Water Pollution Control Federation 37: 822-841.
5. DIRASIAN, H. A., A. H. MOLOF, AND J. A. BORCHARDT. 1963. Electrode potentials developed during sludge digestion. J. Water Pollution Control Federation 35:424-439. 6. HEUKELEKIAN, H., AND B. HEINEMAN. 1939. Studies on methane producing bacteria. II. Enumeration in digesting sewage solids. Sewage Works J. 11:436-444. 7. HOTCHKISS, M. 1925. Factors influencing the bacterial flora of an Imhoff tank. J. Am. Public Health Assoc. 15:702-704.

The suggestions and criticisms of Paul H. Smith are gratefully acknowledged. We wish to thank Mr. Clemmons for his generous cooperation.

8. HUNGATE, R. E. 1950. The anaerobic mesophilic cellulolytic bacteria. Bacteriol. Rev. 14:1-49. 9. HUNGATE, R. E. 1960. Symposium: Selected topics in microbial ecology. I. Microbial ecology of the rumen. Bacteriol. Rev. 24:353364. 10. HUNGATE, R. E. 1966. The rumen and its microbes. Academic Press, Inc., New York. 11. MAKI, L. R. 1954. Experiments on the microbiology of cellulose decomposition in a municipal sewage plant. Antonie van Leeuwenhoek J. Microbiol. Serol. 20:185-200. 12. SMITH, P. H. 1966. The microbial ecology of sludge methanogenesis, p. 156-161. In Publication of Society for Industrial Microbiology, Developments in industrial microbiology. American Institute of Biological Sciences, Washington, D. C. 13. SMITH, P. H. 1965. Pure culture studies of methanogenic bacteria. Proc. Ind. Waste Conf., 20th, Purdue Univ., Lafayette, Ind. 20:583-588. 14. SMITH, P. H., AND R. A. MAH. 1966. Kinetics of acetate metabolism during sludge digestion. Appl. Microbiol. 14:368-371. 15. TORIEN, D. F. 1967. Direct-isolation studies on the aerobic and facultative anaerobic bacterial flora of anaerobic digesters receiving raw sewage sludge. Water Res. 1:55-59.