APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1987, p.

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0099-2240/87/102414-06$02.00/0 Copyright 1987, American Society for Microbiology

Vol. 53, No. 10

Difference in Sporogenous Bacterial Populations in Thermophilic (55C) and Mesophilic (35C) Anaerobic Sewage Digestion
MIN CHEN Wadsworth Center for Laboratories and Research, New York State Health Department, Albany, New York 12201
Received 10 March 1987/Accepted 23 June 1987

Spores, sporeforming vegetative cells, and asporogenous populations were enumerated in two semicontinuous anaerobic fermentors digesting municipal primary sludge at 35 and 55C for more than 87 days. In the 35C fermentor, the anaerobic total population was 312.5 x 106/ml, with 25.0 x 106/ml being sporogenous. The populations that digest casein, starch, pectin, and cellulose were 23.1 x 106, 59.2 x 106, 26.2 x 106, and 7.3 x 106/ml, respectively, with 2.8 x 106, 6.7 x 106, 3.4 x 106, and 1.5 x 106/ml being sporogenous, respectively. The sporeformers accounted for 8.0 to 20.0% of each of the respective populations. In the 55C fermentor, the anaerobic total population was 512.5 x 106/ml, with 336.6 x 106/ml being sporogenous. The populations that digest casein, starch, pectin, and cellulose were 97.7 x 106, 190.7 x 106, 75.8 x 106, and 11.2 x 106/ml, respectively, with 47.8 x 106, 110.6 x 106, 43.3 x 106, and 5.1 x 106/ml, respectively, being sporogenous. The sporeformers represented 45.5 to 65.7% of each of the respective populations. The numbers of thermophilic sporeforming vegetative cells in the 55C fermentor were 9.0 to 19.8 times higher than their counterparts in the 35C fermentor. Most sporeformers were in the vegetative state in the 35 and 55C fermentors. After 18 days of fermentation at 55C, sporeformers carried out most of the digestion; however, the digestion was shared by both sporeformers and asporogenous bacteria after 87 days of fermentation. In the 35C fermentor, asporogenous bacteria digested most of the sludge. During the 18- and 87-day experimental periods, sporeformers were never predominant.

Bacterial populations in anaerobic municipal sludge digested at moderate temperatures (25 to 35C) have been enumerated by several workers (10, 19, 21), who have found that the percentage of sporeformers in the total population is low. Cooney and Wise (7) measured the DNA content in a 35C and a 65C fermentor and found less DNA in the 65C fermentor, which produced more methane. This fact suggests a marked difference in the microbial activities of these two fermentors. Very little is known about the difference between bacterial populations selected by the moderate and high temperature ranges in sewage sludge. Since thermophilic digestion usually begins with wastes that have been long exposed at moderate temperatures, the bacteria must be able to survive at moderate temperatures. Knowledge of the difference between these bacterial populations will be beneficial to workers using anaerobic digestion. I therefore undertook a study by establishing a 35C and a 55C fermentor to investigate the bacterial populations in the digested sludge. The objective of the present study was to determine the difference in numbers of total anaerobic bacteria and the bacterial cells that degraded casein, starch, pectin, and cellulose in these two fermentors. The numbers of spores, sporeforming vegetative cells, and asporogenous cells that degraded the specific carbon sources were also determined.
MATERIALS AND METHODS

Primary sludge. The primary anaerobic sludge was collected from the Albany County Sewage Treatment Plant, Albany, N.Y. Approximately 50 liters was collected and dispensed into 500-ml screw-cap plastic bottles under N2, and the bottles were stored immediately at -20C. One day
2414

before use, a bottle was thawed and moved to storage at 4C. Each day, approximately 60 ml of the sludge was blended at high speed in a blender (Oster Corp., Milwaukee, Wis.) under N2 for 3 min, equilibrated to near 35 or 55C, and then placed in the respective fermentor. Fermentors. The fermentation vessel was a 1-liter Pyrex (Corning Glass Works, Corning, N.Y.) aspirator bottle fitted with a butyl rubber stopper containing two glass tubes (inner diameter, 5 mm). The stopper was tightened with stove wire and sealed with silicone sealant (Dow Corning Corp., Midland, Mich.). One of the glass tubes (22 cm in length with butyl rubber tubing on the open end) was closed with a hemostat clamp. The sludge was syringed into and out of the fermentor through this tubing. The other tube was 4.5 cm in length with rubber tubing, and the open end of the tubing was connected to a Y-tube. Two 60-ml plastic syringes with stainless steel one-way Perfektum stopcocks (Popper & Sons, New York Park, N.Y.) were inserted into the rubber tubing of the Y-tube to collect gas and measure gas production. Two nails were fixed near the finger grip of the barrel of each syringe to prevent the plunger from popping out and to ensure proper closure of the syringes. The fermentors each contained 500 ml of sludge sample. The sludge in the moderate-temperature fermentor was incubated at 35C for 10 days without feeding. The drop in pH was adjusted daily with NaOH to maintain the sample at pH 7.0 + 0.2; a total of 8 mmol of NaOH was used. The high-temperature fermentor was established 10 days before the 35C one. The initial temperature was 50C for 10 days and was elevated to 55C for 10 days; no feeding took place. A total of 12.8 mmol of NaOH was used to adjust the pH during the establishment period. After day 21 (day 11 of the 35C fermentor), 28 ml of the fermentor contents was removed daily from each fermentor,

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and an equivalent volume of sludge was added daily to both fermentors (turnover time, 18 days; loading rate, 0.68 g of volatile solid per day). The pH did not change after the establishment period. Both fermentors produced methane consistently. Each day the fermentors were thoroughly shaken by hand immediately before and after the feeding and at least twice thereafter. Unless otherwise stated, bacteriological and chemical analyses were started after 87 days of feeding (approximately five turnovers) and ended on day 108

m z m < '- ' 3 X

0. 3 .2 .3 1+ 1+ + 00 > o00
,

Media. The media were prepared by the modified Hungate n serum bottle technique (15). Total anaerobic bacteria wereo 0 enumerated in SMS agar medium (3), which contained theoo o 1+1+ I 1+ 0 following (per liter): glucose, 5 g; Trypticase soy broth (BBL 0 Microbiology Systems, Cockeysville, Md.), 5 g; yeast extract (Difco Laboratories, Detroit, Mich.), 2 g; K2HPO4 and o o KH2PO4, 0.24 g each; (NH4)2SO4, 1.24 g; NaCl, 0.48 g; 0 0 MgSO4 7H2O, 0.1 g; CaCl2 2H20, 60 mg; FeSO4 7H20, ,, 4 mg; Na2CO3, 4.0 g; dithioerythritol, 15.4 mg; hydrated 1+ 00 0 L-cysteine hydrochloride, 0.875 g; Na2S- 10H20, 0.375 g; isobutyric, 2-methylbutyric, n-valeric, and isovaleric acids, 3 ,x oo o_ 2 0.1 ml each; hemin, thiamine hydrochloride, calcium-DH pantothenate, nicotinamide, riboflavin, and pyridoxine hydrochloride, 2 mg each; biotin and resazurin, 1 mg each; Z p-aminobenzoic acid, 100 ,ug; cyanocobalamin, 20 ,ug; folic I+ . acid, 50 ,ug; folinic acid, 10 ,ug; and Bacto-Agar (Difco), 20 g. The gas phase was 100% CO2. The pH was adjusted to 7.0. Bacteria that utilized specific carbon substrates were enumerated in a medium containing the following (per liter): 0 o K2HPO4, KH2PO4, and (NH4)2SO4, 0.3 g each; NH4Cl, 1.0 0 g; NaCl, 0.6 g; MgSO4- 7H20, 0.2 g; CaCl2 2H20, 20 mg; It1+ I+ 0 z MnSO4. 2H20, 5 mg; FeSO4- 7H20, CoSO4, ZnSO4, and o resazurin, 1 mg each; biotin and folic acid, 20 ,ug each; thiamine hydrochloride, riboflavin, nicotinic acid, p-aminobenzoic acid, lipoic acid, DL-calcium pantothenate, and oo hemin, 50 jig each; cyanocobalamin, 1 ,ug; casein hydrolysate (BBL), 1.0 g; DL-tryptophan, 0.05 g; NaHCO3, 5.0 g; 3 0+ 0t o 0t Z 3 0o a hydrated L-cysteine hydrochloride, 0.88 g; Na2S- 10H20, 0.38 g; sludge filtrate, 100 ml; and Bacto-Agar, 20 g. The gas o o3 phase was 100% CO2. The pH was adjusted to 7.0. The specific carbon source added was 5.0 g of starch or pectin Z 0 ~ (Sigma Chemical Co., St. Louis, Mo.), 50 ml of skim milk, or o. 1+1+ 1+ 4.0 g of cellulose (no. 1 filter paper [Whatman, Inc., Clifton, N.J.] ground wet in a pebble mill). The pectin was prepared o o Z as described previously (17). The cellulose was prepared by previously published methods (14). Enumeration of total bacteria. At 18 h after the previous addition of the feedstock, 0.5 ml of the contents was withdrawn from each fermentor and syringed into a 10-ml serum bottle, which contained 4.5 ml of anaerobic dilution solution (2). This dilution was mixed, swirled, and further diluted as P', described previously (3). Triplicate or quadruplicate roll o o + tubes were prepared with 0.1 ml of each 10-fold serial I W. 0 dilution from each of the fermentors and incubated at 35 or o o0 55C. Total colonies were counted on SMS agar medium and on the individual carbon sources (casein, starch, or pectin) after 4 weeks. Cellulolytic colonies were counted after 8 to oco \0 0 10 weeks. Only the colonies that cleared the translucent 1+ 1+ 1+ 0 0 0 casein, pectin, and cellulose media were counted as specific substrate degraders. Colonies surrounded by clear zones . . after iodine solution was added were counted as starch degraders. ll Enumeration of spores. Another 0.5 ml of the contents was 0o withdrawn from each fermentor, diluted, and mixed as 1+ 1+ 1+ "A 9: described above. The dilutions were then heated in a water bath at 85 + 1C for 10 min and chilled in cold water for 5 min
-

(six turnovers).

o >

1+

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APPL. ENVIRON. MICROBIOL.

Fermentor

TABLE 2. Percent of sporeformers (spores plus vegetative cells) in the total population after 87 days of digestion % of sporeformers (no. of sporeformers/total no. of colonies) degrading:
Casein Starch Pectin
Cellulose

temp

(0C)

Total

35 55

12.2 (5/41) 48.9 (22/45)

11.4 (5/44) 58.0 (29/50)

12.8 (5/39) 57.1 (20/35)

20.0 (2/10) 45.5 (5/11)

8.0 (6/75) 65.7 (44/67)

described previously (11). The heated dilutions were incubated on the agar medium containing the specific carbon substrates, casein, starch, pectin, or cellulose. Colonies were counted after 4 weeks of incubation as spore populations, except for cellulolytic colonies, which were counted after 8 to 10 weeks. Enumeration of asporogenous populations and sporogenous vegetative cells. Colonies were isolated from the agar media containing the specific carbon source, and each was suspended in the anaerobic dilution solution described previously (2). Appropriate dilutions were distributed among agar tubes containing medium with specific substrate. After two such purifications, a colony was isolated and transferred to SMS-glucose broth. After 14 days of incubation, the cultures were heated to test their ability to form spores. A 0.2-ml sample of the heated culture was then transferred to 4.8 ml of SMS-glucose broth for further incubation. The optical density of these cultures was examined as A,%O (light path, 1.5 cm). A culture with an optical density reading equivalent to or greater than 0.5 was scored as a sporeformer. Cultures showing no growth after 4 weeks were scored as asporogenous bacteria. A ratio of sporogenous population to asporogenous population was thus obtained. The total sporogenous population (spores plus vegetative cells) was obtained by subtracting the asporogenous population from the total population. Subtraction of the spore population from the total sporogenous population gave the sporeforming vegetative-cell population. Analytical procedures. Dry matter and Kjeldahl nitrogen were determined in triplicate from each fermentor as described previously (1). Total organic carbon was analyzed by acid persulfate digestion, and CO2 was determined by infrared spectroscopy (1). Ammoniacal N was determined by the Nessler test (1). Total phosphorus was digested by acid persulfate and determined as described previously (22).
as

Soluble protein was measured with the Folin phenol reagent (12). Methane was determined by gas chromatography as described previously (4). Volatile fatty acids were determined by procedures described previously (5). The Eh was measured by using a combination redox electrode consisting of a platinum electrode, a silverlsilver chloride reference electrode, and a KCI-agar salt bridge in an anaerobic glove box. Both Eh and pH were measured with a digital lonalyzer (no. 701A; Orion Research, Inc., Cambridge, Mass.). The Eh was standardized with an equimolar solution of Fe(II) and Fe(III) chloride in 0.1 N HCl. Statistical evaluation. The t values comparing the mesophilic with the thermophilic populations were calculated from the common logarithms of the populations by procedures described previously (18).

RESULTS The constituents of the raw and fermented sludges are shown in Table 1. After fermentation, a decrease in dry matter, organic carbon, organic acids, protein, and nonammoniacal N was observed in both fermentors. The 55C fermentor, however, produced more methane and organic acids and less dry matter, and the redox potential was lower than that of the 35C fermentor. Table 2 shows the number of pure cultures isolated from the sludges after 87 days of digestion and the number of sporeformers in the isolates. The percentages of sporeformers in each population were therefore determined. In the 35C sludge, sporeformers constituted 8% of the total population. Approximately 12% of the populations degraded casein, starch, and pectin, and 20% of the population degraded cellulose. In the 55C sludge, sporeformers constituted 65.7% of the total population, with 45.5 to 58% of the populations degrading the tested carbon sources.

TABLE 3. Sporogenous and asporogenous populations utilizing different substrates in the fermented sludges after 87 days of digestion Population (106/ml)' Substrate Temp (C) Sporogenous

Casein (skim milk)

Starch
Pectin

35 55

Spores 0.8 0.6 (3.5) 5.3 1.9 (5.4)

Vegetative cells

2.0 0.2 (8.7) 42.5 10.8 (43.6)

20.3 1.6 (87.9) 49.8 13.6 (51.0)

23.1 1.8 97.7 26.7

35 55 35 55 35 55
35 55

1.9 1.3 (3.3) 26.3 2.5 (13.8) 0.2 0.03 (0.8) 6.5 1.1 (8.6) 1.0 0.3 (13.7) 0.6 0.2 (5.3)
6.3 1.2 (2.0) 129.6 88.4 (25.3)

4.8 1.5 (8.1) 84.3 23.1 (44.2) 3.2 0.5 (12.2) 36.8 2.3 (48.5) 0.5 0.02 (7.0) 4.5 0.9 (40.2)
18.7 3.8 (6.0) 207.0 10.1 (40.4)

52.5 17.0 (88.7) 80.1 22.0 (42.0) 22.8 + 3.2 (87.0) 32.5 2.0 (42.9) 5.8 0.2 (79.5) 6.2 1.2 (55.5)
287.5 + 57.9 (92.0) 175.9 8.6 (34.3)

59.2 19.0 190.7 52.3 26.2 3.7 75.8 4.6 7.3 0.3 11.2 2.1
312.5 62.9 512.5 + 25.0

Cellulose SMS-glucose

a Mean + standard deviation. Numbers in parentheses are the percentages of the total population.

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TABLE 4. Student t test values for pair comparison of thermophilic versus mesophilic populations, and the ratio (TIM) of thermophilic to mesophilic populations' Total Asporogenous cells Sporogenous vegetative cells Sporesb
Substrate
t

TIM

TIM

TIM

TIM

Casein Starch Pectin Cellulose Total

3.57 7.97 35.02 -0.30 8.32

S S S NS S

6.6 13.8 32.5 0.6 20.6

23.38 24.47 26.95 20.95 20.89

S S S S S

19.8 17.6 11.5 9.0 11.0

7.12 4.00 3.99 0.47 -6.29

S S S NS -S

2.5 1.5 1.4 1.1 0.6

11.62 10.90 16.06 4.04 6.63

S S S S S

4.2 3.2 2.9 1.5 1.6

" Abbreviations: S, thermophilic significantly higher than mesophilic population at 99.9% level; -S. mesophilic significantly higher than thermophilic population at 99.9% level; NS, not significant at 50% level. b The degrees of freedom were as follows: spores, 4; vegetative and asporogenous cells, 3.

The concentrations of sporeformers (subdivided into and sporeforming vegetative cells), asporogenous bacteria, and total bacterial populations capable of degrading the tested organic polymers in these two sludges after 87 days of fermentation are listed in Table 3. In the 35C sludge the total anaerobic population was 312.5 x 106/ml, with 25.0 x 106/ml being sporogenous. The populations that digested casein, starch, pectin, and cellulose were 23.1 x 106, 59.2 x 106, 26.2 x 106, and 7.3 x 106/ml, respectively, with 2.8 x 106, 6.7 x 106, 3.4 x 106, and 1.5 x 106/ml being sporogenous, respectively. In the 55C sludge the anaerobic population was 512.5 x 106/ml, with 336.6 x 106/ml being sporogenous. The populations that digested casein, starch, pectin, and cellulose were 97.7 x 106, 190.7 x 106, 75.8 x 106, and 11.2 x 106/ml, respectively, with 47.8 x 106, 110.6 106, 43.3 x 106, and 5.1 x 106/ml, respectively, being sporogenous. The asporogenous bacteria constituted 79.5 to 92.0% of the populations in the 35C sludge and 34.3 to
spores
X

1o0

E2 E

Casein
Starch

9 Cellulose
a Total * Spore

2 Pectin

9z
350C
35

OC

55

OC

55.5% in the 55C sludge. With the exception of cellulose degraders grown at 35C, sporeforming vegetative cells outnumbered spore populations approximately 2- to 16-fold in both sludges. Differences between the concentration of thermophilic degraders of organic polymers and their mesophilic counterparts were evaluated statistically. The t values listed in Table 4 indicate that the concentration of spores, sporeforming vegetative cells, asporogenous bacteria, and total bacteria degrading the tested organic polymers in the 55C sludge was significantly higher than in the 35C sludge at a 99.9% confidence level. The sporeforming vegetative cells in the 55C sludge outnumbered their 35C counterparts 9- to 19.8-fold, and total bacterial populations were 1.5 to 4.2 times more numerous than their 35C counterparts. The concentrations of spores and asporogenous cellulose degraders in these two sludges were similar, and the total asporogenous bacteria in the 35C sludge was somewhat higher than in the 55C sludge. Common-logarithmic concentrations of spores and total bacterial populations in the raw and the 18-day-fermented sludge are shown in Fig. 1 (unfortunately, I do not have results for the sporeforming vegetative cells). Although the populations increased after fermentation, the percentages of spores in the sludge digesting casein, starch, pectin, or cellulose ranged from 5.5 to 9.7% (Table 5), which were quite similar to the percentages of their counterparts in the raw sludge (1.2 to 12.4%; Table 5) and in the sludge that had been fermented for 87 days (2.0 to 13.7%; Table 3). The raw sludge contained fewer than 2.5 x 103 thermophilic casein, starch, pectin, and cellulose degraders per ml and fewer than 1.25 x 104 total anaerobic bacteria per ml at 55C. After an 18-day fermentation, a large number of thermophilic bacteria capable of degrading the tested organic polymers were present (Fig. 1), and 47 to 86.6% of them were spores (Table 5). These percentages not only were

2bi

TABLE 5. Percentage of spores in the raw sludge and in the sludges after 18 days of digestion at 35 and 55C % of spores" at:
Substrate
Raw

35C
18 days

55C, 18 days

RAW
FIG. 1. Concentrations of spores and polymer degraders in sludge and in sludges digested at 35 and 55C for 18 days.
raw

2.2 Casein 2.4 Starch 3.3 Pectin 12.4 Cellulose 1.2 Total " The population in raw sludge

9.7 5.7 5.5 6.9 7.6

at

73.6 86.6 56.6 76.0 47.6

55C was too small to be detected.

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APPL. ENVIRON. MICROBIOL.

significantly higher than the 5.4 to 25.3% of spores (Table 3) but also were higher than the sum of spores and sporeforming vegetative cells (Table 2) in the sludge that had been fermented for 87 days. DISCUSSION The dry-solid portion of most municipal sludges contains approximately 19% protein, 18 to 53% carbohydrate, 5% pectin, and 25 to 36% cellulose (9, 14, 16). The rate-limiting steps in the anaerobic digestion of municipal sludge are those involving hydrolysis of organic polymers (3, 8). Sludge fermented at 55C was degraded more thoroughly, with the production of more methane and organic acids, than sludge fermented at 35C (Table 1). The degree of degradation appeared to be directly related to bacterial concentrations. Previous reports (19, 21) indicate that sporeforming bacteria constitute less than 10% of the total bacterial population in anaerobic municipal sludges fermented at moderate temperatures. This is consistent with the present study, in which sporogenous bacteria represented 8% of the total anaerobic populations (Table 2). There is a severe lack of information regarding the concentration of sporeformers in municipal sewage sludge digesting at high temperatures. However, Zeikus (23) noticed that most gram-negative coccoidal bacteria undergoing digestion at moderate temperatures are replaced with rodshaped bacteria at high temperatures, and clostridia have been isolated from the high-temperature sludge. That study indicates that clostridia may be present at high levels in other municipal sludges digested at high temperatures. Whether they will reach 45.5 to 65.7% of the total bacterial population, as found in the present study, remains to be confirmed. The fact that sporeforming vegetative cells in both sludges outnumbered spores suggests that the sporeformers are actively degrading the polymers. Since 79.5 to 92% of the bacterial populations were asporogenous bacteria in the 35C fermentor, hydrolysis of the organic polymers is believed to be carried out mainly by asporogenous bacteria. Since 34.3 to 55.5% of the bacteria were asporogenous and most sporeformers were in the form of vegetative cells in the 55C fermentor, hydrolysis of the organic polymers is believed to be carried out by both spores and asporogenous bacteria. When 209 mesophilic isolates were tested for obligate anaerobiosis by being grown aerobically on the specific agar medium from which they were isolated, only 6 strains were able to grow aerobically, and 1 of these was a sporeformer. Of the 208 thermophilic isolates tested, 5 were facultative anaerobes and 3 of these were sporeformers. The overwhelming numbers of obligately anaerobic bacteria isolated in the present study are quite different from results of early studies (6, 20), which indicates that bacteria in anaerobic sewage sludge are mostly aerobic and facultatively anaerobic. The bacteria are probably similar to those reported by Mah and Sussman (13) and Toerien (19), who found that anaerobic bacteria considerably outnumber aerobic and facultatively anaerobic bacteria. High-temperature digestion usually begins with wastes which have been exposed to moderate temperatures over a long period. The bacteria therefore must be able to survive at temperatures unfavorable to them. Sporeformers obviously are capable of doing so. However, the asporogenous bacteria are temperature sensitive. The raw sludge may contain a considerably higher number of thermophilic sporeformers than asporogenous bacteria. The outnumbered sporeformers in the raw sludge may have caused sporeformers to predom-

inate at an early stage of fermentation at 55C (Fig. 1; Table 5). When the fermentation is prolonged, asporogenous bacterial populations increase, many perhaps through adaptation (3, 8) and a few through multiplication of the indigenous thermophilic populations in the sludge.
ACKNOWLEDGMENTS
I thank R. W. Toombs for support and M. F. Mahoney, M. H. Poskanzer, and D. A. Sculco for technical help.

LITERATURE CITED 1. American Public Health Association. 1985. Standard methods for the examination of water and wastewater, 16th ed., p. 96, 379, 408, 508. American Public Health Association, Inc., Washington, D.C. 2. Bryant, M. P., and L. A. Burkey. 1953. Cultural methods and some characteristics of some of the more numerous groups of bacteria in the bovine rumen. J. Dairy Sci. 36:205-217. 3. Chen, M. 1983. Adaptation of mesophilic anaerobic sewage fermentor populations to thermophilic temperatures. Appl. Environ. Microbiol. 45:1271-1276. 4. Chen, M., and M. J. Wolin. 1977. Influence of CH4 production by Methanobacterium ruminantium on the fermentation of glucose and lactate by Selenomonas ruminantium. Appl. Environ. Microbiol. 34:756-759. 5. Chen, M., and M. J. Wolin. 1981. The influence of heme and vitamin B12 on growth and fermentation of Bacteroides species. J. Bacteriol. 145:466-471. 6. Cookson, J. T., and N. C. Burbank, Jr. 1965. Isolation and identification of anaerobic and facultative bacteria present in the digestion process. J. Water Pollut. Control Fed. 37:822-841. 7. Cooney, C. L., and D. L. Wise. 1975. Thermophilic anaerobic digestion of solid waste for fuel gas production. Biotechnol. Bioeng. 17:1119-1135. 8. Hobson, P. N., S. Bousfield, and R. Summers. 1974. Anaerobic digestion of organic matter. Crit. Rev. Environ. Control 4:131-191. 9. Hunter, J. V., and H. Heckelekian. 1965. The composition of domestic sewage fractions. J. Water Pollut. Control Fed. 37: 1142-1163. 10. Kirsch, E. J. 1969. Studies on the enumeration and isolation of obligate anaerobic bacteria from digesting sewage sludge. Dev. Ind. Microbiol. 21:170-176. 11. Lord, T. H. 1962. Determinative bacteriology laboratory manual, p. 16. Burgess Publishing Co., Minneapolis. 12. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 13. Mah, R. A., and C. Sussman. 1968. Microbiology of anaerobic sludge fermentation. I. Enumeration of the nonmethanogenic anaerobic bacteria. Appl. Microbiol. 16:358-361. 14. Maki, L. R. 1954. Experiments on the microbiology of cellulose decomposition in a municipal sewage plant. Antonie Van Leeuwenhoek J. Microbiol. 20:185-200. 15. Miller, T. L., and M. J. Wolin. 1974. A serum bottle modification of the Hungate technique for cultivating obligate anaerobes. Appl. Microbiol. 27:985-987. 16. Pfeffer, J. T., and L. C. Liebman. 1976. Energy from refuse by bioconversion, fermentation and residue disposal processes. Resour. Recovery Conserv. 1:295-313. 17. Schink, B., J. C. Ward, and J. G. Zeikus. 1981. Microbiology of wetwood: importance of pectin degradation and Clostridium species in living trees. Appl. Environ. Microbiol. 42:526-532. 18. Snedecor, G. W., and W. G. Cochran. 1967. Statistical methods, 6th ed., p. 59 and 549. Iowa State University Press, Ames. 19. Toerien, D. F. 1970. Population description of the nonmethanogenic phase of anaerobic digestion. I. Isolation, characterization and identification of numerically important bacteria. Water Res. 4:129-148. 20. Toerien, D. F., and W. H. J. Hattingh. 1969. Anaerobic diges-

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tion. I. The microbiology of anaerobic digestion. Water Res. 3:385-416. 21. Ueki, A., E. Miyagawa, H. Minato, R. Azuma, and T. Suto. 1978. Enumeration and isolation of anaerobic bacteria in sewage digestor fluids. J. Gen. Appl. Microbiol. 24:317-332. 22. U.S. Environmental Protection Agency. 1979. Methods for chem-

ical analysis of water and wastes. Method 365. 1-3, EPA 600/4-79-020. U.S. Environmental Protection Agency, Cincinnati. 23. Zeikus, J. G. 1979. Microbial populations in digestors, p. 61-89. In D. A. Stafford, B. I. Wheatley, and D. E. Hughes (ed.), Anaerobic digestion, vol. 4. Applied Science Publishers Ltd., London.