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Direct-isolation studies on the aerobic and facultative anaerobic bacterial flora of anaerobic digesters receiving raw sewage sludge
Water Research, Pergamon Press 1967. Vol. 1, pp. 55-59. Printed in Great Britain.
DIRECT-ISOLATION STUDIES ON THE AEROBIC AND FACULTATIVE ANAEROBIC BACTERIAL FLORA OF ANAEROBIC DIGESTERS RECEIVING RAW SEWAGE SLUDGE D. F. TOERIEN National Institute for Water Research, Pretoria, South Africa (Received 12 August 1966)
Abstract-Identification of bacterial cultures isolated from anaerobic digesters receiving raw sewage sludge, showed that the aerobic and facultative anaerobic bacteria belonged mainly to the genera Bacillus, Pseudomonas and Micrococcus. Coliforms were present in very small numbers. INTRODUCTION
ANAEROBIC digestion of complex organic matter takes place in two phases (TARVIN and BUSWELL,1934; BARKER,1956). In the first phase the organic matter is broken down into fatty acids (KAPLOVSKY,1951 and 1952) by a heterogenous bacterial flora (MCCARTY, 1963). In the second phase the acids formed during the first phase are converted by the methane bacteria to carbon dioxide and methane (BARKER,1956). Although the methane bacteria have received a considerable amount of study (BARKER,1936 and 1940; SCHNELLEN,1947 ; STADTMAN and BARKER,1951a, b), the bacteria of the first phase have received but little attention. Studies on the bacteria of the acid-forming phase were carried out by GAUB(1924), MCCARTYet al. (1962) and BURBANKet al. (1964). This study was undertaken to determine by direct isolation of cultures, which of the aerobic and facultative anaerobic bacteria occur in anaerobic digesters receiving raw sewage sludge. EXPERIMENTAL
PROCEDURES
Three anaerobic digesters were started using the vessels (capacity 14 1.) of a New Brunswick F14 fermentation apparatus. Each digester was provided with a feed inlet, a gas inlet, a gas outlet, a mechanical stirrer and sampling outlet, and was seeded with 10 1. of digested primary sludge obtained from the local municipal sewage treatment plant. This sludge was first screened through a 1 mm sieve and then transferred to the digesters. Filtered high purity nitrogen (African Oxygen Ltd.) was led through each digester for 3 min at a rate of 5 l/min to obtain anaerobic conditions. The digesters were then acclimatized to a feed of raw sewage sludge over a period of 41 days, during which routine chemical analyses were carried out. These determinations included volatile fatty acid content (MONTGOMERY et al., 1962), pH, alkalinity (STANDARD METHODS, 1960) and volume of gas produced per day. Bacteriological studies were not begun until chemical analyses showed efficient digestion. Samples of the digester liquor for analyses were withdrawn 24 hr after the previous feeding time and centrifuged at 1200 g for 10 min. The analyses were performed on the 55
56
D. F.
TOWEN
supematant liquor. For bacteriological purposes samples of the digester liquor from all three digesters were mixed in equal proportions. Cultures of bacteria were isolated by streaking mixed samples from digesters on nutrient agar (Difco) or tryptone dextrose agar (Difco). Single colonies were picked at random and transferred to nutrient agar slopes and, when the necessity arose, were further purified by repeated streaking on nutrient agar. The pure cultures obtained were studied microscopically under phase contrast to determine morphology and mobility. For culture identification the flagella stain (LEMON, 1960) and Hucker’s modification of the Gram stain (SOCIETY OF AMERICAN BACTERIOLOGISTS, 1957) were used when necessary. Specific tests for the identification of cultures were carried out as described by the SOCIETYOF AMERICANBACTERIOLOGISTS (1957) and by SKERMAN (1959). For the basic classification Bergey’s Munuul (BREEDet al., 1957) was used. Although no coliforms were obtained by direct isolational procedures it proved possible to demonstrate their presence by inoculating mixed samples of digester liquor into tubes containing brilliant green broth (Difco) and observing any gas production. Samples from tubes giving positive reactions were streaked on Endo agar (Difco) and the different colony types which developed, were purified and then identified. RESULTS Good growth was obtained 24 to 36 hr after streaking of samples. Single colonies were picked at random for further purification. In this way 46 different bacterial cultures were obtained and identified. The results are presented in TABLE1. TABLE1. BACTERIA ISOIA~ PROM LAB~RA~-ORY-S~ALEANAEROBIC DKiEpTws REEIVING RAW SEWAGE SLUDQB AS FEED
Group Gram positive spore-fomhg rods
Gram positive cob Gram nqative pohrly flagellated rods
Species
No. of CUltUES
Bacillus cereus
6
Bacilk cereIi.9var. mycoidcs
2
Bacillus megaterium
Bacillw circrrlans
1 1
Bacillus subtilis Microunxus ureae
2
M&roc~cus mrians Microwccus cadaks
4 1
PselKbmo~ aerI@nosa PsacrdomonaspscrsrlbmJhi Pse~monas ribojlavina Psedmonas okovorans Pseuhntvnae ambigua PseM. not
2
c4xnplctclyidcn~
1
3
11
3 1 8
Brilliant green broth cultures which gave positive tests for gas production yielded three different colony types upon streaking on Endo agar, namely: typical coiiform colonies with purple colour and copper sheen, large pink colonies and small white colonies. Transfers of these colony types to lactose fermentation broth showed that
Dicatt-Isolation Studies on the Aerobic and Facultativc Anaaobii
Bectaial Flora
57
organisms of the typical coliform colonies fermented lactose with production of acid and gas. The organisms of the pink colonies fermented lactose wrth the produo tion of acid but no gas. The organisms of the small white colonies did not ferment lactose at all. The results of the identification of the organisms of the different colony types are presented in TALSU2. the
TABLE2. BAalmY
WLA.~~ PROMUcroSe
-ATION
BROTH
BacmialqKes
type
Plnplish with copper sheen Large pink mlonica Snlall white c4Aonies
Acroboctera4wogenzs a?zwherichiaitltewnedia
Pscrdomonruau~
DISCUSSION The cultures obtained by direct isolational procedures were members of the following groups : (a) Gram positive cocci ; (b) Gram positive spore-forming rods ; (c) Gram negative polarly flagellated rods. The Gram positive cocci were all members of the genus h4zi~ococmr. The presence of Micrococcus varkansin anaerobic digesters was reported by MCCARTYet al. (1962). During this investigation, M. varians as well as hf. ureae and M. candi&.r were detected. No Badus spp. were detected by MCCARTYet al. (1962), but BURBANKel al. (1964) reported the isolation of a Bacillus species. The presence of this group. of organisms was suspected from the occasional microscopical observations of bacterial endospores in digester liquor. Gf the Bacillus spp. detected, B. cereus, B. cereus var. mycoides and B. circulans are facultative anaerobes (BREEDet al., 1957) and may be physiologically active in anaerobic digesters. Since B. megaterium and most strains of B. subtilis are obligate aerobes (BREEDet al., 1957), they were probably not physiologically active under the conditions which prevailed in the anaerobic digesters and may have been introduced via the feed received by the digesters. D&ulty in the identification of Pseuabmonas cultures using reference works such as Bergey’s Manual (BRBBDer al., 1957) have been reported by PRmoRIus (1964), DE LEY (1964) and BOARD(1965). The same problem was encountered during this study. However, despite this di5culty, several Pseuabnonas spp. (TABLE1) were completely identified. No cultures of P. reptilivora (MCCARTYet al., 1962) were detected. Some of the isolated Pseuabmonas spp. are facultative anaerobes (BREEDet al., 1957) and may have been physiologically active in the anaerobic digesters. Alcaligenes spp., Staphylococcus epidennidis, Sarcina lutea and Neisseria catarrhalis were detected by MCCARTYet al. (1962). In this investigation no Alcaligenes spp., staphylococci, sarcinae or Gram negative cocci were detected. HEURBLBBLUN (1957) and MCCARTYet al. (1962) reported that Escherichia coli occurd in large numbers during the early stages of digestion but that their numbers subsequently deiAined. E. coli were isolated from anaerobic digesters by BUIUANK et al. (1964). During this investigation the presence of coliforms was established, but their numbers were so small that they could not be detected by direct isolational
58
D. F. TOER~EN
procedures. The coliform organisms present were found to be Aerobacter aerogenes and Escherichia intermedia. Escherichia coli was not detected. CONCLUSIONS I. Aerobic and facultative anaerobic bacteria were found to be present in anaerobic digesters fed with raw sewage sludge.
2. Most of these bacteria were of the genera: Micrococcus, Bacillus and Pseudomonas. 3. Coliforms were present in the anaerobic digesters used for this study, but their numbers appeared to decline during active digestion when no coliforms were introduced via the feed.
Acknowledgemerzts-The helpful comments and suggestions received from his colleagues is gratefully acknowledged by the author. This paper is published with the permission of the South African Council for Scientific and Industrial Research, P.O. Box 395, Pretoria.
REFERENCES BARKER H. A. (1936) Studies upon the methane-producing bacteria. Arch. Mikrobiol. 7, 420-438. BARKER H. A. (1940) Studies upon the methane fermentation. IV. The isolation and culture of Methanobacterium omelianskii. Antonie van Leeuwenhoek 6, 201-220. BARKERH. A. (1956) Bacterial Fermentations. John Wiley, New York. BOARDR. G. (1965) The properties and classification of predominant bacteria in rotten eggs. J. appl. Bact. 28,437-453. BREEDR. S., MURRAYE. G. D. and Snmn N. R. (1957) Bergey’s Manual of Determinative Bacteriology 7th ed. Williams and Wilkins, Baltimore. BURBANKN. C., CooKso~ J. T., GOEPPNERJ. and BR~~MAND. (1964) Isolation and identification of anaerobic and facultative bacteria present in the digestion process. Proceedings of the 19th lnricrstrial Waste Conference, Purdue University. pp. 552-577. DE LEY J. (1964) Pseudomona and related genera. Ann. Rev. Microbial. 18, 17-46. GAUB W. H. (1924) A bacteriologcial study of a sewage disposal plant. New Jers. Agric. Expt Sta. Bull. 394, l-24. HEUKELEKIANH. (1957) Basic principals of sludge digestion. In Biological Treatment of Sewage and Industrial Wastes. Vol. 2. Anaerobic Digestion and Solids-Liquid Separation. pp. 25-43. (Ed. MCCABEJ. and EXXENFELDER W. W.). Reinhold, New York. KAPLOV~KYA. J. (1951) Volatile acid production during digestion of seeded, unseeded and limed fresh solids. Sewage ind. Wastes 23,713-721. KAPL~VSKYA. J. (1952) Volatile acid production during digestion of several industrial wastes. Sewage ind. Wastes 24,194-201. LEIMN E. (1960) Atlas of Bacterial Flagellation. Academic Press, New York. MCCARTYP. L. (1963) The methane fermentation. Paper presented at Rudolfs Research Conference on June 7, 1963, at Rutgers, The State University, New Brunswick. McCann P. L., JERISJ. S., MCKINNEY R. E., REED K. and VA~H C. A. (1962) Report on: Microbiology of anaerobic digestion. Sedgwick Laboratories of Sanitary Science, Department of Civil Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts. MONTGOMERY H. A. C., D~klocxc J. F. and THOMN. S. (1962) The rapid calorimetric determination of orgariic acids and their salts in sewage-sludge liquor. Analyst 87, 949-955. PRETOIUUSW. A. (1964) An ecological study of the aerobic proteolytic, free-living nitrogen fixing, and spiral-form bacteria in stabilization ponds. D.Sc. (Agric)-Thesis, University of Pretoria. SCHNELLENC. J. T. P. (1947) Onderzoekingen over de methaangisting. Thesis, Technische Hoogeschool, Delft.
Direct-Isolation Studies on the Aerobic and Facultative Anaerobic Bacterial Flora S-N
59
V. B. D. (1959) A Gui& to the Zden~ifcurionofthe Generu ofBacteria. Williams and Wilkins, Baltimore. Socnrtv OF AMERICAN BACIQUOL~~I~~(1957) Manual of Microbiological Methods. McGraw-Hill, New York. ST~TMAN T. C. and BARKERH. A. (19%) Studies on the methane fermentation. IX. The origin of methane in the acetate fermentation by Methunosarcina. J. Duct. 61,81-86. STADTMAN T. C. and BARKWH. A. (19516) Studies on the methane fermentation. X. A new formatedecomposing bacterium, Methanococcus vannielii. J. Bact. 62, 269-280. STANDARDMETHODSFORTEE EXAMINATION OF WATERAND WASTEWATER(1960) 11th ed. American Public Health Association, New York. TARVIND. and BU~WELL A. M. (1934) The methane fermentation of organic acids and carbohydrates. J. Am. them. Sot. 56,1751-1775.
DIRECT-ISOLATION STUDIES ON THE AEROBIC AND FACULTATIVE ANAEROBIC BACTERIAL FLORA OF ANAEROBIC DIGESTERS RECEIVING RAW SEWAGE SLUDGE D. F. TOERIEN National Institute for Water Research, Pretoria, South Africa (Received 12 August 1966)
Abstract-Identification of bacterial cultures isolated from anaerobic digesters receiving raw sewage sludge, showed that the aerobic and facultative anaerobic bacteria belonged mainly to the genera Bacillus, Pseudomonas and Micrococcus. Coliforms were present in very small numbers. INTRODUCTION
ANAEROBIC digestion of complex organic matter takes place in two phases (TARVIN and BUSWELL,1934; BARKER,1956). In the first phase the organic matter is broken down into fatty acids (KAPLOVSKY,1951 and 1952) by a heterogenous bacterial flora (MCCARTY, 1963). In the second phase the acids formed during the first phase are converted by the methane bacteria to carbon dioxide and methane (BARKER,1956). Although the methane bacteria have received a considerable amount of study (BARKER,1936 and 1940; SCHNELLEN,1947 ; STADTMAN and BARKER,1951a, b), the bacteria of the first phase have received but little attention. Studies on the bacteria of the acid-forming phase were carried out by GAUB(1924), MCCARTYet al. (1962) and BURBANKet al. (1964). This study was undertaken to determine by direct isolation of cultures, which of the aerobic and facultative anaerobic bacteria occur in anaerobic digesters receiving raw sewage sludge. EXPERIMENTAL
PROCEDURES
Three anaerobic digesters were started using the vessels (capacity 14 1.) of a New Brunswick F14 fermentation apparatus. Each digester was provided with a feed inlet, a gas inlet, a gas outlet, a mechanical stirrer and sampling outlet, and was seeded with 10 1. of digested primary sludge obtained from the local municipal sewage treatment plant. This sludge was first screened through a 1 mm sieve and then transferred to the digesters. Filtered high purity nitrogen (African Oxygen Ltd.) was led through each digester for 3 min at a rate of 5 l/min to obtain anaerobic conditions. The digesters were then acclimatized to a feed of raw sewage sludge over a period of 41 days, during which routine chemical analyses were carried out. These determinations included volatile fatty acid content (MONTGOMERY et al., 1962), pH, alkalinity (STANDARD METHODS, 1960) and volume of gas produced per day. Bacteriological studies were not begun until chemical analyses showed efficient digestion. Samples of the digester liquor for analyses were withdrawn 24 hr after the previous feeding time and centrifuged at 1200 g for 10 min. The analyses were performed on the 55
56
D. F.
TOWEN
supematant liquor. For bacteriological purposes samples of the digester liquor from all three digesters were mixed in equal proportions. Cultures of bacteria were isolated by streaking mixed samples from digesters on nutrient agar (Difco) or tryptone dextrose agar (Difco). Single colonies were picked at random and transferred to nutrient agar slopes and, when the necessity arose, were further purified by repeated streaking on nutrient agar. The pure cultures obtained were studied microscopically under phase contrast to determine morphology and mobility. For culture identification the flagella stain (LEMON, 1960) and Hucker’s modification of the Gram stain (SOCIETY OF AMERICAN BACTERIOLOGISTS, 1957) were used when necessary. Specific tests for the identification of cultures were carried out as described by the SOCIETYOF AMERICANBACTERIOLOGISTS (1957) and by SKERMAN (1959). For the basic classification Bergey’s Munuul (BREEDet al., 1957) was used. Although no coliforms were obtained by direct isolational procedures it proved possible to demonstrate their presence by inoculating mixed samples of digester liquor into tubes containing brilliant green broth (Difco) and observing any gas production. Samples from tubes giving positive reactions were streaked on Endo agar (Difco) and the different colony types which developed, were purified and then identified. RESULTS Good growth was obtained 24 to 36 hr after streaking of samples. Single colonies were picked at random for further purification. In this way 46 different bacterial cultures were obtained and identified. The results are presented in TABLE1. TABLE1. BACTERIA ISOIA~ PROM LAB~RA~-ORY-S~ALEANAEROBIC DKiEpTws REEIVING RAW SEWAGE SLUDQB AS FEED
Group Gram positive spore-fomhg rods
Gram positive cob Gram nqative pohrly flagellated rods
Species
No. of CUltUES
Bacillus cereus
6
Bacilk cereIi.9var. mycoidcs
2
Bacillus megaterium
Bacillw circrrlans
1 1
Bacillus subtilis Microunxus ureae
2
M&roc~cus mrians Microwccus cadaks
4 1
PselKbmo~ aerI@nosa PsacrdomonaspscrsrlbmJhi Pse~monas ribojlavina Psedmonas okovorans Pseuhntvnae ambigua PseM. not
2
c4xnplctclyidcn~
1
3
11
3 1 8
Brilliant green broth cultures which gave positive tests for gas production yielded three different colony types upon streaking on Endo agar, namely: typical coiiform colonies with purple colour and copper sheen, large pink colonies and small white colonies. Transfers of these colony types to lactose fermentation broth showed that
Dicatt-Isolation Studies on the Aerobic and Facultativc Anaaobii
Bectaial Flora
57
organisms of the typical coliform colonies fermented lactose with production of acid and gas. The organisms of the pink colonies fermented lactose wrth the produo tion of acid but no gas. The organisms of the small white colonies did not ferment lactose at all. The results of the identification of the organisms of the different colony types are presented in TALSU2. the
TABLE2. BAalmY
WLA.~~ PROMUcroSe
-ATION
BROTH
BacmialqKes
type
Plnplish with copper sheen Large pink mlonica Snlall white c4Aonies
Acroboctera4wogenzs a?zwherichiaitltewnedia
Pscrdomonruau~
DISCUSSION The cultures obtained by direct isolational procedures were members of the following groups : (a) Gram positive cocci ; (b) Gram positive spore-forming rods ; (c) Gram negative polarly flagellated rods. The Gram positive cocci were all members of the genus h4zi~ococmr. The presence of Micrococcus varkansin anaerobic digesters was reported by MCCARTYet al. (1962). During this investigation, M. varians as well as hf. ureae and M. candi&.r were detected. No Badus spp. were detected by MCCARTYet al. (1962), but BURBANKel al. (1964) reported the isolation of a Bacillus species. The presence of this group. of organisms was suspected from the occasional microscopical observations of bacterial endospores in digester liquor. Gf the Bacillus spp. detected, B. cereus, B. cereus var. mycoides and B. circulans are facultative anaerobes (BREEDet al., 1957) and may be physiologically active in anaerobic digesters. Since B. megaterium and most strains of B. subtilis are obligate aerobes (BREEDet al., 1957), they were probably not physiologically active under the conditions which prevailed in the anaerobic digesters and may have been introduced via the feed received by the digesters. D&ulty in the identification of Pseuabmonas cultures using reference works such as Bergey’s Manual (BRBBDer al., 1957) have been reported by PRmoRIus (1964), DE LEY (1964) and BOARD(1965). The same problem was encountered during this study. However, despite this di5culty, several Pseuabnonas spp. (TABLE1) were completely identified. No cultures of P. reptilivora (MCCARTYet al., 1962) were detected. Some of the isolated Pseuabmonas spp. are facultative anaerobes (BREEDet al., 1957) and may have been physiologically active in the anaerobic digesters. Alcaligenes spp., Staphylococcus epidennidis, Sarcina lutea and Neisseria catarrhalis were detected by MCCARTYet al. (1962). In this investigation no Alcaligenes spp., staphylococci, sarcinae or Gram negative cocci were detected. HEURBLBBLUN (1957) and MCCARTYet al. (1962) reported that Escherichia coli occurd in large numbers during the early stages of digestion but that their numbers subsequently deiAined. E. coli were isolated from anaerobic digesters by BUIUANK et al. (1964). During this investigation the presence of coliforms was established, but their numbers were so small that they could not be detected by direct isolational
58
D. F. TOER~EN
procedures. The coliform organisms present were found to be Aerobacter aerogenes and Escherichia intermedia. Escherichia coli was not detected. CONCLUSIONS I. Aerobic and facultative anaerobic bacteria were found to be present in anaerobic digesters fed with raw sewage sludge.
2. Most of these bacteria were of the genera: Micrococcus, Bacillus and Pseudomonas. 3. Coliforms were present in the anaerobic digesters used for this study, but their numbers appeared to decline during active digestion when no coliforms were introduced via the feed.
Acknowledgemerzts-The helpful comments and suggestions received from his colleagues is gratefully acknowledged by the author. This paper is published with the permission of the South African Council for Scientific and Industrial Research, P.O. Box 395, Pretoria.
REFERENCES BARKER H. A. (1936) Studies upon the methane-producing bacteria. Arch. Mikrobiol. 7, 420-438. BARKER H. A. (1940) Studies upon the methane fermentation. IV. The isolation and culture of Methanobacterium omelianskii. Antonie van Leeuwenhoek 6, 201-220. BARKERH. A. (1956) Bacterial Fermentations. John Wiley, New York. BOARDR. G. (1965) The properties and classification of predominant bacteria in rotten eggs. J. appl. Bact. 28,437-453. BREEDR. S., MURRAYE. G. D. and Snmn N. R. (1957) Bergey’s Manual of Determinative Bacteriology 7th ed. Williams and Wilkins, Baltimore. BURBANKN. C., CooKso~ J. T., GOEPPNERJ. and BR~~MAND. (1964) Isolation and identification of anaerobic and facultative bacteria present in the digestion process. Proceedings of the 19th lnricrstrial Waste Conference, Purdue University. pp. 552-577. DE LEY J. (1964) Pseudomona and related genera. Ann. Rev. Microbial. 18, 17-46. GAUB W. H. (1924) A bacteriologcial study of a sewage disposal plant. New Jers. Agric. Expt Sta. Bull. 394, l-24. HEUKELEKIANH. (1957) Basic principals of sludge digestion. In Biological Treatment of Sewage and Industrial Wastes. Vol. 2. Anaerobic Digestion and Solids-Liquid Separation. pp. 25-43. (Ed. MCCABEJ. and EXXENFELDER W. W.). Reinhold, New York. KAPLOV~KYA. J. (1951) Volatile acid production during digestion of seeded, unseeded and limed fresh solids. Sewage ind. Wastes 23,713-721. KAPL~VSKYA. J. (1952) Volatile acid production during digestion of several industrial wastes. Sewage ind. Wastes 24,194-201. LEIMN E. (1960) Atlas of Bacterial Flagellation. Academic Press, New York. MCCARTYP. L. (1963) The methane fermentation. Paper presented at Rudolfs Research Conference on June 7, 1963, at Rutgers, The State University, New Brunswick. McCann P. L., JERISJ. S., MCKINNEY R. E., REED K. and VA~H C. A. (1962) Report on: Microbiology of anaerobic digestion. Sedgwick Laboratories of Sanitary Science, Department of Civil Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts. MONTGOMERY H. A. C., D~klocxc J. F. and THOMN. S. (1962) The rapid calorimetric determination of orgariic acids and their salts in sewage-sludge liquor. Analyst 87, 949-955. PRETOIUUSW. A. (1964) An ecological study of the aerobic proteolytic, free-living nitrogen fixing, and spiral-form bacteria in stabilization ponds. D.Sc. (Agric)-Thesis, University of Pretoria. SCHNELLENC. J. T. P. (1947) Onderzoekingen over de methaangisting. Thesis, Technische Hoogeschool, Delft.
Direct-Isolation Studies on the Aerobic and Facultative Anaerobic Bacterial Flora S-N
59
V. B. D. (1959) A Gui& to the Zden~ifcurionofthe Generu ofBacteria. Williams and Wilkins, Baltimore. Socnrtv OF AMERICAN BACIQUOL~~I~~(1957) Manual of Microbiological Methods. McGraw-Hill, New York. ST~TMAN T. C. and BARKERH. A. (19%) Studies on the methane fermentation. IX. The origin of methane in the acetate fermentation by Methunosarcina. J. Duct. 61,81-86. STADTMAN T. C. and BARKWH. A. (19516) Studies on the methane fermentation. X. A new formatedecomposing bacterium, Methanococcus vannielii. J. Bact. 62, 269-280. STANDARDMETHODSFORTEE EXAMINATION OF WATERAND WASTEWATER(1960) 11th ed. American Public Health Association, New York. TARVIND. and BU~WELL A. M. (1934) The methane fermentation of organic acids and carbohydrates. J. Am. them. Sot. 56,1751-1775.