Copyright as part of the March 1977, Part 1, JOURNAL WATER POLLUTION CONTROL FEDERATION, Washington, D. C. 20016Printed in U. S. A.

Bacterial populations and end products during anaerobic sludge fermentation of glucose
David P. Chynoweth University of North Carolina, Chapel Hill Robert A. Mah University of California, Los Angeles

Waste treatment of domestic wastewater sludge by anaerobic digestion is effected by a mixture of organisms which eventually convert the organic compounds to methane and carbon dioxide. The starting substrates include carbohydrates, fats, long chain organic acids, proteins, and other nitrogenous compounds which are initially degraded to small molecules of organic acids, neutral products, hydrogen, and carbon dioxide. A separate group of methanogenic bacteria dissimilates certain of these fermentation products, yielding methane as a major product. Although approximately 70 percent of the methane in this fermentation is produced from acetate,l,2 the actual substrates for acetate formation and organisms responsible for its production have not been thoroughly investigated. Formation of acetate from carbohydrates, fats, and proteins was examined by previous investigators only after prolonged enrichment periods on the appropriate substrate.!' 3-5, 11 This procedure leads to selection of organisms which best metabolize the compound being fed and does not give a true evaluation of the unaltered fermentation. To avoid this selection, unenriched sludge must be examined. If a given substrate normally served as an important source of acetate in the unenriched system, addition of that substrate at high concentrations should result in the accumulation of acetate, provided it is formed at a rate faster than its conversion to methane. Determination of the types of end products (particularly acetate) and their rates of formation would permit

an evaluation of the organisms and substrates responsible for their production. In the following study the anaerobic sludge fermentation of glucose was investigated to examine changes in bacterial populations and patterns of fermentation products during adaptation to that substrate. MATERIALS AND METHODS Digester sludge. The source of digester sludge was a laboratory digester inoculated with a sample of anaerobic sludge from the primary digester of a domestic treatment system, Third Fork Sewage Plant, Durham, N. C. It was maintained on a raw sludge feed under conditions previously described by Mah and Sussman. 6 Anaerobic culture techniques. Strict anaerobic procedures were employed throughout the course of this investigation. Sludge samples were flushed with oxygen-free 70 percent N 2 -30 percent CO 2 .2 Anaerobic culture methods of Hungate 7 and Mah and Sussman 6 were used for enumeration and isolation of anaerobic bacteria. Media. The inorganic salts solution contained the following compounds (final percent w/v): NaCI, 0.1; NH 4CI, 0.05; MgCI 2'6H 20, 0.005; CaCI 2, 0.005; KH 2P0 4, 0.005; CoCI2 ' 6H 20, 0.001; (NH4)6Mo7024'4H20, 0.001; NaHC0 3 , 0.5. Cysteine and sodium sulfide were added as reducing agents at respective concentrations (w/v) of 0.05 and 0.025 percent. 8 A 33 percent sludge supernatant medium 6 was used in estimating the anaerobic, viable compounds. Selective counts for acidMarch 1977 405

Chynoweth and Mah

700

SUCROSE GLUCOSE CELLOBIOSE

600

Pressure
mmH9 500

400

200

100

ENOOGENOUS

10

12

14

16

18

20

22

samples were centrifuged to remove sludge solids before analysis. Radioisotope measurement of glucose metabolism. The rates of conversion of glucoseU-l4C to l4C02 were followed after trapping the l4C02 in hyamine according to the following procedure. An empty vial was placed in the side arm of the Warburg vessels (as above); the appropriate sludge sample containing glucose-U_HC was placed in the main chamber. At appropriate sampling times, 1.5 ml of hyamine solution was injected through the serum cap into the vial. At the same time, the vessels were placed in an ice bath and the reaction stopped by injection of 1 ml concentrated H 2 S04 , After 60 min, the hyamine solution was transferred to a scintillation vial, 10 ml scintillation fluid 10 added, and the sample counted in a liquid scintillation spectrometer. RESULTS Fermentation of glucose, cellobiose, and sucrose, was measured manometrically and the results are shown in Figure 1. The endogenous control represents the rate of gas production without added substrates. All substrates were metabolized, but vigorous gas production occurred only after a 10 to 12-h lag; gas was

HOURS

FIGURE 1.

Fermentation of soluble sugars. Symbols: O-glucose; t::.-cel lobiose; X-sucrose; D-endogenous.

producing anaerobes were made in indicator medium containing the basal medium plus 0.5 percent glucose, 0.005 percent brom-thymol blue, and 1.0 percent precipitated CaCOa. Aerobic counts were made on plate count agar and eosin methylene blue agar. Manometric procedures. Fermentation rates of various substrates were measured manometrically by the method of Smith and Mah. 8 Speciall00-ml Warburg vessels 8 contained 25 ml sludge. Test substrates were added tq digesting sludge at a final concentration of 0.5 percent (w/v) by injection through a side arm fitted with a serum cap. Analysis of fermentation products. Volatile fatty acids and ethanol were analyzed by flame ionization gas chromatography. A 1.8 m X 3.3 mm (6-ft X 0.13-in.) stainless steel column packed with 50/80 mesh Porapak Q was used. The column temperature was 185C and the injector and detector 220C. The carrier gas was N 2 at a flow rate of 20 ml/min. The H 2 How rate was 20 ml/min and air 400 ml/min. Prior to analysis, samples were acidified with 20 percent metaphosphoric acid (0.15 ml/ml sample) and centrifuged. The injection volume was 2 pl. Glucose assay. Glucose was determined enzymatically by the Glucostat method. All
(> (>

700

GLUCOSE PRESSURE
mm Hg

400

10

12

14

16

18

20

22

HOURS

FIGURE 2.

Worthington

Biochemical Corp.,

Freehold,

N.

J.
JournalWPCF

Fermentation of glucose. Symbols: O-glucose; t::.-raw sludge; D-endogenous.

406

Bacterial Population then produced exponentially for a 3-h period, . finally leveling off after 20 h. Such a long lag would probably not be indicative of induction in resident organisms present in high numbers but rather of growth and selection of organisms initially present at low concentrations. Figure 2 shows a comparison of gas production from glucose with that from raw sludge, the normal substrate for the digester, which contains other carbon sources in addition to carbohydrates. Gas production from the raw sludge occurred at a steady and initially higher rate than from glucose, indicating that substrates other than glucose were being fermented. However, the glucose vessel again exhibited an exponential increase in gas production after a 10-h lag; its subsequent rate and total gas production far exceeded that of the raw sludge vessel. This observation was repeated on a number of occasions using different sludge samples. Further examination of the glucose fermentation indicated that gas production. was accompanied by the expected disappearance of glucose and an increase in certain fermentation products. Figure 3 shows that acetic acid,
ACETATE

1<1

30
VOLATILE ACIDS
~M/ml

ETHANOL

--~

PROPIONATE

12

II

10

24

HOURS
4 6 8
~

HOURS

FIGURE 4.

Aerobic and anaerobic viable counts.

100 90
GAS

80

PERCENT GLUCOSE REMAINING

70 60 50 40 30 20 10

HOURS

FIGURE 3.

End product formation and glucose disappearance. Symbols, upper graph: O-acetate; O-ethanol; 6-propionate. Lower graph: 6-glucose; 0 gas.

propionic acid, and ethanol were the main products and the increase in their production corresponded to the time of rapid use of glucose and increase in gas production. Other organic acids and alcohols which are separated by the procedures used were not detected. Analyses for formic and lactic acids were not made. To determine if the growth of glucose-using organisms could account for the sudden increase in the rate of glucose metabolism, viable counts were made under both aerobic and anaerobic conditions at various times after addition of glucose to digesting sludge. Figure 4 shows that the total anaerobic count increased only lO-fold and thus could not account for the sudden increase in gas production and accumulation of fermentation products March 1977 407

Chynoweth and Mah TABLE I. Comparison of Aerobic and Anaerobic Counts in Endogenous and GlucoseEnriched Sludge. Hours after adding 0.5% glucose thymol blue CaCO g indicator medium. Assuming acid-producers were mainly euryoxic, such anaerobic counts should reveal any increase in those organisms capable of producing acid products from glucose and provide another measurement of the increase in euryoxic bacteria. Colonies were selectively counted in the highest dilution exhibiting growth at the beginning and during the peak of gas formation. The results (Table I) showed that the increase in numbers during the enrichment period was similar to that of the aerobic counts (Figure 4). Thus, the total anaerobic count (Figure 4) included the euryoxic bacteria, and the smaller increase in total anaerobic numbers can be accounted for mainly by an increase in this acid-producing population. Several organisms were isolated from anaerobic roll tubes (10- 8 dilution) inoculated with the glucose-enriched sludge. Characteristics of these isolates are shown in Table II. Three isolates (6, 11, 12) were obligate anaerobes and produced acetate and propionate when grown on glucose. The remaining eight isolates (2, 3, 4, 5, 7, 8, 9, 10) were euryoxic and produced acetate and ethanol when grown on glucose. These products corresponded to those produced by the mixed sludge population (Figure 3). A second series of organisms was isolated as follows: 1. From normal sludge which had not received glucose: Group 1. Isolation of representative colonies from the highest dilution

o
Aerobic plate counts (numbers/ml) Selective anaerobic counts (numbers/ml) Total anaerobic counts (numbers/ml)

10-12

6.5 X 10& 4.0 X 107 2.0 X 10 4 2.6 X 107

2.7 X 107

3.5 X 10 7

depicted in Figure 3. However, the total aerobic count increased rapidly (1 OOO-fold) after an incubation time corresponding to the rapid increase in production of gas and other products indicating that euryoxic organisms could account for the rapid increase in glucose fermentation. Theoretically, the aerobic count should not exceed the anaerobic count since the latter should support growth of facultative The and obligately anaerobic organisms. higher aerobic counts depicted here may be caused by sampling variations. When both an aerobic and anaerobic sample were examined simultaneously, the agreement between samples was close at appropriate times. The exponential growth period exhibited by the aerobic counts corresponded closely to the time of rapid gas production. Selective anaerobic counts of glucose-fermenting organisms were estimated by inoculation into bromTABLE

n.

Characteristics of Isolates from Glucose-Enriched Sludge. Characteristics Glucose endproducts

Isolate
2 3 4 5 6

Aerobic

EMB*

Ethanol

Acetate

Propionate

,1

7
8 9 10
11 12

+ + + + + + + +

+ + + + + + + +

+ + + + + + + +

+ + + + + + + + + + +

+ + +

* EMB-Eosin methylene blue agar selective for coliforms.

408

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Bacterial Population

TABLE

m.

Number and Designation of Organisms Isolated.

Strict anaerobes Euryoxic Euryoxic, growing better anaerobically

Group 1 Group 2

8: 52, 53, 54, 55, 56, 57, 59, 510 3: 52-6, R1, R6

o
7: R8, RlO, 52-4 52-5,52-7, 52-8,52-9 9: GI, G3, G5, G7 G8, G9, G2-1 G2-4, G2-9

o
I: R2

Group 3

3: G4; G2-6 G2-7

6: G2, GIO; G2-3,52-5 G2-8, G2-1O

(l0-7) of habitat-simulating medium showing growth. Group 2. Acid-producing colonies from the highest dilution (10- 3 ) of glucose indicator medium showing such colonies. 2. From a sample of the same sludge incubated with glucose for 14 h: Group 3. Acid-producing colonies from the highest dilution (10- 7) of indicator medium showing such colonies. A summary of the organisms isolated is shown in Table III. Most of the strict anaerobic isolates were identified to the genus Bacteroides; further characterization to the species level was not made. Some characteristics pertinent to general classification of euryoxic organisms are given in Table IV. All were gram-negative motile rods of the enteric type. Group A had the general properties of the Providence group 9; Group C organisms resembled members of the genus Escherichia. Group B isolates were also apparently related to the Genus Escherichia with the notable exceptions of a delayed fermentation of lactose and peptonization of litmus milk. Since addition of soluble sugars to unenriched digesting sludge did not immediately result in manometrically detectable gas production above the endogenous control (Figure 1), glucose-U_14C fermentation was measured as a method of detecting low dissimilation rates. Formation of 14C02 was measured at 10-min intervals and the rate of glucose dissimilation was calculated on the assumption that two moles of 14C02 were produced per mole of glucose dissimilated. The rate of glucose dissimilation after 12-h enrichment was also determined by the same procedure. Aerobic counts and selective (acid-producing)

anaerobic counts were made in unenriched and 12-h glucose-enriched sludge. The glucose dissimilation rates and bacterial counts are presented in Table IV. Glucose was used immediately in normal and glucose-enriched sludge at rates of 1.3 X 10- 3 and 3.4 X 10-2 f-LMIml min respectively. This represents a 20-fold higher dissimilation rate in the glucose-enriched sludge. Aerobic, acid-producing anaerobic, and total anaerobic counts in this particular experiment were respectively 160, 75, and 48 times higher in the glucose-enriched sludge.

DISCUSSION Addition of the soluble sugars glucose, cellobiose, and sucrose to actively fermenting digester sludge initially resulted in a fermentation rate only slightly greater than the endogenous control but less than the normal raw sludge substrate fermenter which receives a mixture of lipids, carbohydrates, and proteins. 10 However, after a 10 to 12-h period, the rate of sugar fermentation was exponential and greatly exceeded the endogenous or raw sludge rates. This increase in gas production was accompanied by a corresponding decrease in glucose and an increase in concentration of acetate, propionate, and ethanol. Since the number of bacteria present in sludge is approxiinately 10 9Iml, 8 either enzyme induction in this population was necessary to metabolize soluble sugars, or, more likely, growth and selection for sugar-metabolizing organisms took place during the lag period. Viable counts of bacteria taken through the experimental period disclosed that the latter was the case. Examination of aerobic plate counts for euryoxic organisms shows an exponential increase in numbers corresponding to the period of maximum gas production. These organisms increased as much as 1 OOO-foid. Anaerobic
March 1977 409

Chynoweth and Mah TABLE IV. Characteristics of Euryoxic Isolates. Group A GI, G3, G5, G7, G8,G9 Litmus milk Gelatin Urea Indol Methyl red Voges-Proskauer Citrate HtS Phenylalanine deaminase Adonitol Cellobiose Dulcitol Esculin Galactose Glucose Glycerol Inositol Lactose Maltose Mannose Mannitol Raffinose Rhamnose Salicin Sorbitol Sucrose Starch Trehalose Note: Acid & peptonization after 10 days Group C 52-4; 52-5; 52-7; 52-8; 52-9; G2-1; G2-4; G2-9 Acid coagulation

Group B R8, RIO Acid & peptonization after 10 days + + +

-\-

+ + +

-()

+G +G +G +G +G + after 10 days +G +G +G +G + after 10 days +G +G (+G) +G +G (+G) +G +G +G +G +G (+G) +G +G

+ + + +

-()

+ after 2 weeks + +
-()

+ after 2 weeks
-(:;I::)

+ + +

= negative; + = growth; G = gas; ( ) = delayed.

indicator medium, which permitted identifica- and not result in selection for those initially tion 'of glucose-metabolizing acid-producing present at low dilutions. Although all isolates examined were obtained colonies, yielded similar results. On the other hand, the total anaerobic count on non-selective from anaerobic roll tubes, 31/41 acid-produchabitat-simulating media, which includes eury- ing organisms were euryoxic. All 8 isolates oxic organisms, yielded only a lO-fold increase from the highest dilution of unenriched sludge were obligate anaerobes (Table III, Group 1). in numbers throughout the entire period. The euryoxic and acid-producing organisms Acid-producing colonies were not observed at had a generation time of less than 20 min and the highest dilutions of unenriched sludge; were responsible for the sudden increase in however, they were present and isolated from accumulation of fermentation products result- the 10- 3 dilution (Table III, Group 2). Of ing from glucose dissimilation. They initially these, 8/11 were euryoxic. Colonies isolated accounted for about 2.4 percent of the total from the highest dilution of glucose-enriched anaerobic count but after a 10 to 12-h enrich- sludge, and selected because of acid-producing ment period on glucose were equivalent in properties (Table III, Group 3), contained numbers to the anaerobic counts. This change 15/18 euryoxic isolates. All of the organisms in Table III were isoin composition of the bacterial population also showed that soluble sugars were not in excess lated from the same sludge sample before and during the normal fermentation since any sig- after incubation with glucose. Members of the nificant sludge substrates should be quickly genus Escherichia and the Providence group metabolized by resident predominant organisms .were among the organisms which predominated 410 Journal WPCF

Bacterial Population after glucose enrichment. Organisms of the Providence group were not isolated prior to glucose enrichment, and members of the genus Escherichia were obtained only from the 10- 3 dilution. Organisms of the Providence group must also have been present at a concentration of 10 3/ml or less. It is clear that the growth rates of members of these two groups were sufficient to permit their establishment as one of the predominating types after only a 10 to 12-h enrichment period on glucose. The acid fermentation products produced by these pure cultures were the same as those found in the sludge containing glucose which suggests that enrichment for these organisms during the dissimilation of glucose by the mixed fermentation occurred. If production of volatile acids is not balanced by their conversion to methane and carbon dioxide, the eventual result is complete inhibition of the overall methane fermentation. When substrates such as glucose enter a digester at concentrations higher than can be readily metabolized by the resident organisms, a shift in the bacterial population may occur, and the result is an enrichment for organisms which rapidly convert the substrate to acid end products. The frequent failure of domestic wastewater digesters may be caused by such selective pressures which elicit development of unwanted organisms which leads to suppression of desired organisms. In this study, the rate of conversion of glucose to acetate, propionate, and ethanol by euryoxic bacteria exceeded the rate of conversion of these end products to methane and carbon dioxide. Organisms responsible for the methanogenic conversion were apparently unable to grow rapidly enough to accommodate the rapid production of these products. Accumulation of ethanol was, in fact, unexpected; it has not been reported as an intermediate in the sludge fermentation. Its formation here may simply reflect its role as a terminal electron acceptor product of the glucose fermentation. In the balanced methane fermentation, methane bacteria serve as mediators of the terminal electron-accepting reaction via reduction of carbon dioxide to methane. During glucose enrichment, the rate of reduction of carbon dioxide to methane was apparently inadequate for the continued dissimilation of glucose. Other electron-accepting reactions (such as the production of propionate and ethanol) replace the methanogenic role of electron removal. The present findings illustrate that digesters "acclimatized" to glucose,l, 4, 11 or other substrates 1, 3, 5, 11 may support a large population of bacteria not present in significant numbers in normal sludge digesters. In the case of glucose-enriched digesters, a shift in the bacterial population can occur within a 10 to 12-h period, and it would be incorrect to assume 4 that only slow-growing strict anaerobes were present in such "acclimatized" digesters. Although laboratory digesters receiving pure substrates may support a vigorous methane fermentation, some caution should be exercised in their examination in view of the intimate physiological association (s) 12 which may exist between a methane bacterium and its nonmethanogenic partner. Determination of the rate of formation of 14C02 from glucose-U_14C permitted a more sensitive measurement of glucose dissimilation during the initial fermentation period. The 20-fold increase in the rate of 14C02 evolution after enrichment corresponded exactly to the period of logarithmic growth exhibited by the euryoxic population. Although the results might also be explained by an increase in the predominating anaerobic bacteria, it is difficult to establish whether such organisms increased in numbers sufficient to account for these changes during the short enrichment period. The growth rates for such anaerobes may indeed be too slow 6 to effect any significant population changes within this time. However, it should be noted that the 20-fold increase in fermentation rate does not correspond to the 100 to 1 ODD-fold increase in euryoxic bacteria; that is, these organisms cannot alone account for glucose dissimilation in unenriched sludge. Apparently, strict anaerobes present in the normal sludge (unenriched) also used glucose, but because of their slow growth rates were unable to compete for glucose with the faster-growing euryoxic organisms. The anaerobic sludge digestion process may thus be regarded as a heterogenous population of opportunists each of which can gain predominance because of favorable selective pressures. The balanced activity of these organisms makes the sludge fermentation a practical treatment process for disposal of organic sludges via the methane fermentation.

SUMMARY AND CONCLUSIONS Addition of glucose to unenriched (unacclimatized) digesting sludge resulted in no apparent response for 8 to 10 h. At this time a sudden and rapid increase in gas production
March 1977 411

Chynoweth and Mah and accumulation of acetate, propionate, and ethanol was observed. This shift in the fermentation pattern was shown to result from the sudden selective growth of euryoxic bacteria normally present in low numbers in the unenriched fermentation. Several isolates from the enriched digester sludge were characterized to the genus Esoherichia and the Providence group. These isolates produced fermentation products identical to those found in the enriched sludge in high concentrations. The growth and activities observed for these organisms clearly illustrate the imbalance that may occur upon altering the substrates added to anaerobic digesters. The continued imbalance of bacterial populations in the fermentation would lead to accumulation of toxic fermentation products and the eventual cessation of decomposition. This research further demonstrates that acclimatization of digester sludge to pure substrates results in a major change in the inherent bacterial populations. In view of this, knowledge gained in the study of acclimatizeddigester sludge may not be applicable in understanding the sludge fermentation receiving wastewater sludge. ACKNOWLEDGMENTS Credits. The technical assistance of Lena Meck and James R. Williams III is gratefully acknowledged. This investigation was supported in part by Public Health Service grant WP-00921-03. Authors. At the time of this paper, David P. Chynoweth was Graduate Student, Department of Environmental Sciences and Engineer,ing, University of North Carolina, Chapel Hill. He is currently Assistant Professor, Department of Environmental and Industrial Health, The University of Michigan, Ann Arbor. Robert A. Mah is Professor, Division of Environmental Science and Nutritional Biochemistry, University of California at Los Angeles. REFERENCES
1. Jeris, J. S., and McCarty, P., "The Biochemistry of Methane Fennentation using HC Tracers." Jour. Water Poll. Control Fed., 37, 178 (1965). 2. Smith, P. H., and Mah, R. A., "Kinetics of Acetate Metabolism during Sludge Digestion." Appl. Microbiol., 14, 368 (1966). 3. Andrews, J. F., and Pearson, E. A., "Kinetics and Characteristics of Volatile Acid Production in Anaerobic Fennentation Processes." Int. Jour. Air Water Pol., 9, 439(1965). 4. Jeris, J. S., and Cardenas, P. R., "GluC9se disappearance in biological treatment systems." Appl. Microbiol., 14, 857 (1966). 5. Kotze, J. P., et al., "A Biological and Chemical Study of Several Anaerobic Digesters." Water Res., 2, 195 (1968). 6. Mah, Robert A., and Sussman, Carol, "Microbiology of Anaerobic Sludge Fennentation. I. Enumeration of the Nonmethanogenic anaerobic bacteria." Appl. Microbiol., 16, 358 (1967). 7. Hungate, R. E., "A Roll Tube Method for Cultivation of Strict Anaerobes." In Norris, J. R., and Ribbons, D. W. (eds.). "Methods in Microbiology." Academic Press, New York, pp. 117-132. (1969). 8. Smith, P. H., "Pure Culture Studies of Methanogenic Bacteria." Proc. 20th Purdue Ind. Waste Cont., Purdue Univ. pp. 583588 (1966). 9. Hormaeche, E., Chainnan, Enterobacteriaceae Subcommittee. "Report of the Nomenclature Committee of the International Association of Microbiological Societies." Int. Bull. Bacteriol. Nom. and Tax, 8, 25 (1958). 10. Chynoweth, D. P., and Mah, R. A., "Volatile Acids Fonnation in Sludge Digestion." In Pohland, F. G. (ed.) "Anaerobic Biological Treatment Processes." Advances in Chern. Ser. 105., Am. Chern. Soc., pp. 41-54, (1971). 11. McCarty, P. L., et al., '1ndividual Volatile Acids in Anaerobic Treatment." Jour. Water Poll. Control Fed., 35, 1501 (1963). 12. Bryant, M. P., et al., "Methanobacillus amelianskii, a symbiotic association of two species of bacteria." Arch. Mikrobiol., 59, 20 (1967).

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