anaerobic digestion i. the microbiology of anaerobic digestion

Water Research Pergamon Press 1969. Vol. 3, pp. 385-416. Printed in Great Britain.

REVIEW PAPER

ANAEROBIC D IGEST ION

I. THE MICROBIOLOGY OF ANAEROBIC D IGEST ION

D. F. TOERIEN and W. H. J. HATTINGH

National Institute for Water Research, South African Council for Scientific and Industrial Research, P.O. Box 395, Pretoria, South Africa

(Received 20 November 1967)

INTRODUCTION

THE RAVIn increase in urbanization and industrialization necessitates the prevention of pollution of vital and limited water resources by providing adequate treatment of liquid wastes emanating from domestic and industrial sources. Waste pollutants are mostly dissolved and suspended organic materials. Biological processes, such as anaerobic digestion, are frequently used in the stabilization of such wastes (LAWRENCZ and McCARrY, 1967). Anaerobic digestion, a process also occurring widely in nature, can be defined as a biological process in which organic matter, in the absence of oxygen, is converted to methane and carbon dioxide.

Despite widespread use of anaerobic digestion of waste organic matter, the fundamental microbiology and biochemistry of this process is still poorly understood and at an elementary level (AGARDY et al., 1963; WUHRMAN, 1964 ; LAWRENCE and MCCARTY, 1967), and McK_rNNEY (1962) has stated that "anaerobic digestion is the unchartered wilderness of sanitary engineering". WUHRMAN (1964) observed that the use of biological processes in water pollution abatement was characterized by "too much engineering and too little microbiology".

An attempt is made in this paper to present the current state of knowledge on the microbiology of anaerobic digestion, to discuss important findings, and to delineate areas in which fruitful research may be undertaken.

MICROBIOLOGICAL NATURE OF ANAEROBIC DIGESTION

Anaerobic digestion is usually considered to be a two-stage process consisting of acid-formation (liquefaction) and gas-formation (gasification) (FIo. 1). LAWRENCE and MCCARTY (1967) preferred to divide the process into three stages namely hydrolysis, acid-formation and gas-formation. At least two large, physiologically different, bacterial populations must be present for the overall conversion of organic matter to methane and carbon dioxide to occur. In the first stage, a heterogeneous group of microorganisms convert proteins, carbohydrates and lipids mainly into fatty acids by hydrolysis and fermentation (McCARTY, 1963). In the second stage, the end-products of the metabolism of the microorganisms of the first stage are converted to methane and carbon dioxide by a physiologically unique group of strict obligate anaerobic bacteria termed the methanogenic bacteria.

The terms "acid-formation" and "gas-formation" for the two stages of anaerobic

385

386 D. F. TOERmN and W. H. J. HAa'r~Grl

digestion are misnomers, since not only acids are produced as metabolic end- products during the first stage, and not all the gas formed during anaerobic digestion is derived from the second stage. Acid-formation or liquefaction should therefore be named the non-methanogenic phase and gas-formation or gasification should be the methanogenic phase. The microbiology of these two phases will be discussed separately.

MICROBIOLOGY OF THE NON-METHANOGENIC PHASE

Kinds of microorganisms Not very much is known regarding the microbiology of the non-methanogenic phase

of anaerobic digestion. However, the presence of different physiological groups of microorganisms in this phase has been demonstrated and some pure culture studies have been carried out (TABLES 1, 3, 4).

Bacteria. The presence of coliforms, the proteus group, denitrifying, lipolytic and cellulolytic bacteria was reported by O'Shaughnessy in 1914. He also established that poorly digesting sewage sludge contained comparatively fewer denitrifying and

Comp~x organ~ molecu~|

Cellular material Non- methonogenic bacteria

Cellular material methono~llc bacteria

Methane and carbon dioxide

FIG. I. Schematic representation of anaerobic digestion.

lipolytic bacteria. GAUB (1924) isolated 16 aerobic and 5 facultative anaerobic bacteria, mostly intestinal types, from anaerobic sludge. HOTCHKISS (1924) indicated the presence of denitrifying, albumen digesting and H2S producing bacteria in the sludges from Imhoff tanks. SOPPELAND (quoted by RUCI-mOFT et aL, 1930) indicated the presence of different types of proteolytic bacteria, such as gelatin liquefiers and protein digesters.

HUNGArr (1950) and MAKI (1954) obtained strains of cellulolytic bacteria in pure culture during enumeration and isolation studies on cellulolytic bacteria that occurred in anaerobic digesters. The isolates of HUNGATE (1950) were gram-negative motile rods and diplorods, extremely variable in size and slightly curved, and some showed internal granules, usually at each end of a cell. The cells were non-sporeforming and the production of hydrogen and carbon dioxide clearly distinguished them from Bacteroides succinogenes, one of the predominant cellulolytic bacteria of the rumen. MArd (1954) isolated 10 cellulolytic pure cultures, one of which was a spore-former. The motile species were all peritrich flagellated. He suggested that the non-sporeforming and sporeforming types were probably related.

BUCK et aL (1953) isolated the facultative anaerobe Streptococcus diploidus and K ~ et al. (1953a,b) studied several aspects of the effect of mass inoculation of this organism into anaerobic digesters. They found that the bacterium functioned synergistically with other bacteria in the liquefaction and gasification of volatile sludge

Anaerobic Digestion--I. The Microbiology of Anaerobic Digestion 387

solids. BUCK et al. (1954) isolated a bacterium from digesting sludge for which they proposed the name Bacillus endorhythmos. The classification of this bacterium in the genus Bacillus requires further investigation because of its vigorous anaerobic growth (HARKNESS, 1966). However, Bergey's Manual (BREED et aL, 1957) provides the inclusion of facultative anaerobic, catalase-positive sporeforming bacteria in the genus Bacillus.

TABLE 1. NON-METHANO

388 D.F. TOEa~N and W. H. J. H~TrnXGH

MCCARTY et al. (1962) isolated aerobic and facultative anaerobic bacteria such as Escherichia coli and Micrococcus varians from several digesters and they also tentatively identified Pseudomonas reptilivora, Sarcina lutea, Neisseria catarrhalis, Alcaligenes viscolactis and Alcaligenes faecalis.

ROEDmER (1960) suggested that the non-methanogenic bacteria were facultative anaerobes, and MCKINNEY (1962) commented that "the acid-formers are made up predominantly of facultative bacteria, with a few strict anaerobes. The ease of growth of the facultative bacteria give them an edge over the strict anaerobes". This concept was recently questioned by TOERIEN et al. (1967) who were of the opinion that aerobic and facultative anaerobic bacteria account for only a small proportion of the total bacterial population of anaerobic digesters.

COOKSON and BURBANK (1965) and BURBANK et al. (1966) developed anaerobic analytical techniques for the isolation of facultative anaerobic and obligate anaerobic bacteria from anaerobic digesters. COOKSON and BURBANK (1965) reported the culture of four bacteria, two of which were present in considerable numbers. Their four isolates included Escherichia coli, Clostridium carnofoetidum and two other bacterial species for which they proposed the names, Sarcina cooksonii and Bacillus knefelkampi. In a review of the bacteria of sewage treatment processes, H~a~KNESS (1966) suggested that Sarcina cooksonii was probably a facultative anaerobe and pointed out that the classification of Bacillus knefelkampi in the genus Bacillus was wrong because of its anaerobic nature and gram-negative reaction. COOKSON and BURBANK (1965) and BURBANK et al. (1966) reported that their Clostridium carnofoetidum isolate was able to produce methane under certain culturing conditions. This observation was in contrast to the general belief that methane production is characteristic of the specialized methanogenic bacteria only. The observed methane production suggested that the authors were probably not working with an absolutely pure culture since Pgrvrw and FREDm'rZ (1966) did not list methane production as a characteristic of Clostridium carnofoetidum.

In addition to Escherichia coli, Sarcina cooksonii, Bacillus knefelkampi and Clostri- dium carnofoetidum, BURBANK et al. (1966) isolated Pseudomonas denitrificans, other pseudomonads, a Klebsiella sp., a member of the Neisseriaceae and Serratia indicans.

TOE~EN (1967a,b) isolated a wide variety of aerobic and facultative anaerobic bacteria from anaerobic digesters receiving raw sludge by direct isolation and enrichment culture techniques. These isolates included aerobic and facultative anaerobic bacteria and one photosynthetic bacterium (TABLE 1).

Aerobic isolates from a digester acclimatized to a synthetic substrate, included Bacillus cereus, B. cereus var. mycoides, B. megaterium, B. pantothenticus, B. pumilus as well as several Pseudomonas spp. (HATnNGH et al., 1967). Species of the genera Pseudomonas, Aeromonas, Micrococcus, Bacillus, Alcaligenes and Escherichia, as well as some Actinomycetes were isolated by KOTZ~ et al. (1968) from digesters that received different substrates.

Recently, considerable numbers of Bacteroides and related obligate anaerobic bacteria were found to occur in anaerobic digesters (Post et aL, 1967).

The bacteria concerned with the non-methanogenic phase of anaerobic digestion include a wide range of physiological groups, from chemolithotrophic bacteria to chemo-organotrophic bacteria and photo-organotrophic bacteria, but whether all the different isolates obtained by various workers are physiologically significant in the


digestion process still remains to be determined. Some of these bacteria, such as the obligate aerobic nitrifying bacteria detected by HOTCRrdSS (1924), may have been "contaminating" organisms introduced via the digester input, and were probably present in a resting state.

Protozoa. Protozoa have frequently been observed in anaerobic digesters, but do not occur in large numbers, a fact which precludes a significant role in normal anaerobic digestion (LAcKeY, 1949). LACKEY (1925) and LW.BMAN (1936) indicated that about 18 species of protozoa may be observed in anaerobic digestion, consisting of roughly

TABLE 2. GENERA OF FUNGI WHICH HAVE BEEN DETECTED IN DIGESTING OR DRYING SLUDGES*

Phycomycetes

Mucor Rhizopus Syncephalastrum Zygorhynchus

Ascomycetes

Allescheria Ascophanus Eurotium Pseudoplea Sartoria Aspergillus Subbaromyces Talaromyces Thielavia

Fungi Imperfecti

Acremonium Aspergillus

f

Fungi Imperfecti (cont)

Penicillium Cephalosporium Geotrichum Gliocladium Paecilomyces Scopulariopsis Sepedonium Spicaria Trichoderma

* Trichothecium Alternaria Cladosporium harargarinomyces Memnoniella Humicola Phialophora Pullularia Stachybotrys Epicoccum Fusarium Myrothecium

* According to COOKE (1957, 1963)

equal numbers of flagellates, ciliates and amoebae. Flagellates belonging to the genera Trepomonas, Tetramitus and Trigomonas, amoebae belonging to the genera Vahlkamp- fia and Hartmanella, and ciliates belonging to the genera Metopus, Trimyena and Saprodinium were observed (LACKEY, 1949).

Fungi. The occurrence of a wide range of fungi (molds and yeasts) in polluted waters, and in sewage and sewage treatment systems has been demonstrated by COOKE (1954, 1957, 1959, 1961, 1963, 1965a, b). The most important genera from which species were isolated from digesting sludge, scum of digesting sludge or drying sludge are presented in TABLE 2.

CooI~ (1965b) reported that large populations of filamentous fungi and yeasts were added to his digestion system with each batch of settled sewage, slurry of activated sludge and fish-meal, or slurry or unsterilized fish-meal. As the sludge passed through the digester the original inoculum of filamentous fungi and yeast cells was not killed

390 D.F. TOERIEN and W. H. J. HATTINGH

TABLE 3. MAGNITUDES OF NUMBERS OF NON=METHANOGENIC BACTERIA ENUMERATED DURING THE YEARS 1900-1930

Bacterial group Numbers x 10 a ml Reference

Coli group 7 Coli group 29-60 Proteus 100 Denitrifiers 0'5 Denitrifiers 223 Ammoniafying I000 Lipolytic bacteria 10 Proteolytic (gelatin liquefiers) 1000 Proteolytic (gelatin liquefiers) 1500 Proteolytic (albumen digesters) I000 Proteolytic (albumen digesters) 19 Proteolytic (protein digesters) 2800 Cellulolytic 100 Cellulolytic 0-22 Total counts (aerobic) 130,000 Total counts (aerobic) 2400-6400 Total counts (aerobic) 65,000 Total counts (aerobic) 1000-60,000 Nitrite formers 0.1 Nitrite formers 3 Nitrate formers 0-1 Nitrate formers 2 Sulfate reducers 1000 Sulfate reducers 21 H2S producers (from protein) 140

O'SEmuGHNESSY (1914) GAUB (1924) O'SHAUGHNESSY (1914) O'SrtAUGHNESSY (1914) HOTCHKISS (1924) GAtm (1924) O'SHAuGHNESSY (1914) GAUB (1924) SOPPELAND quoted by RLrCHHOI~r et al. (1930) GAtm (1924) HOTCHKISS (1924) SOPPELAND quoted by RucI-mor'r et al. (1930) O'SHAuGH,'WeSSY (1914) SOPPELAND quoted by Rucrmor'r et al. (1930) O'SI-IAUOHNESSY (1914) GAUB (1924) SOPPELAND quoted by Rucm-IOrT et al. (1930) RUDOLFS et al. (1926) GAtm (1924) HOTCHKISS (1924) GAtm (1924) HOTCI-mlSS (1924) GAtm (1924) HOTCRKISS (1924) HOTCHKISS (1924)

and some species even multiplied. In a digester which received a sterile fish-meal feed, some fungi were still active after seven complete digester replacements, but others did not survive the process. COOKE (1965b) found that fermentative yeasts were the first to disappear during these experiments but the filamentous species persisted, although incapable of fermenting sugars and being non-cellulolytic. Cooke concluded that fungi,

TABLE 4. MAGNITUDES OF NON-METHANOGENIC BACTERIA ENUMERATED SINCE 1930

Bacterial group Feed of digester Numbers x 103 ml References

Total counts (aerobic) Total counts (aerobic) Total counts (microscopic) Total counts (aerobic) Total counts (anaerobic) Total counts (aerobic) Proteolytic bacteria (aerobic) Lipolytic bacteria (aerobic) Cellulolytic bacteria (anaerobic) Cellulolytic bacteria (anaerobic) Leptospira sp.

Fatty acids 2000-20,000 McC~tTYetal . (1962) Carbohydrate 15,000-350,000 McCARTYetaI.(1962) Sewage sludge 60,000 B ~ et a1.(1966) Carbohydrates and wastes 3000-300,000 KOTZ~ et al. (1968) Synthetic substrate 390,000-15,000,000 Tom~.mN et aL (1967) Synthetic substrate 800-100,000 To~ et al. (1967) Carbohydrates and wastes 100-9000 KOT~ et al. (1968) Carbohydrates and wastes 20-160 KOT~ et al. (1968) Sewage sludge 0-8-2.0 HU~GATE (1950) Sewage sludge 16-970 MA~ (1954) Sewage sludge 1 M.~d (1954)


including yeasts and molds, might be taking part in the digestion process to the extent of obtaining nutrients for growth, rather than being merely present in a resting state.

Numbers of microorganisms o f t he non-methanogenic phase In quantitative ecological studies of the microbiology of an ecosystem such as an

anaerobic digester, the numbers in which different species or physiological groups occur are very important. Although it is sometimes very difficult to arrive at definite conclusions from reported bacterial numbers, these numbers serve to elucidate certain characteristics of the anaerobic digestion systems. Enumeration studies on the non- methanogenic phase are summarized in TABLES 3 and 4.

RtrOOLFS et al. (1926) found that the bacterial population (estimated by aerobic techniques) of sewage sludge digestion decreased from 60 x 106 bacteria per ml to 1-2 x 106 bacteria per ml within 48 hr after addition of fresh substrate, but RUCHHOFT et al. (1930) observed a peak in numbers during the initial 10-14 days after which the numbers decreased rapidly. RUDOLFS et al. (1926) indicated that their counts remained at a low level for a further five months and suggested that coliforms participated actively during the initial stages of anaerobic digestion of domestic sludges.

RucrtHOrr et al. (1930) cited work from Winslow and Belcher which indicated that few obligate anaerobic bacteria occurred in sewage and that their numbers declined rapidly. The results of Winslow and Belcher must be considered as doubtful since the incidence of large numbers of obligate anaerobic bacteria in sewage and sewage treatment processes has been demonstrated by modern culture techniques (ZUBRYCKI and SPAULDING, 1962; SEELIGER and WERNER, 1963; POST et aL, 1967; VAN HOUTE and GIBBONS, 1966 ; and TOERmN et al., 1967). Obligate anaerobes constitute 80-95 per cent of the bacteria that may be cultivated from the adult faecal flora, while E. coli and enter- ocoeci average 1-5 per cent of this ~tora (WERNER, 1966, VAN HOLrrE and GIBBONS, 1966).

HUNGATE (1950) and MAKI (1954) respectively found from 0.8 x 103 to 2 x 103 and from 16 x 103 to 970 x 103 anaerobic cellulolytic bacteria per ml in sludge digestion. MAKI (1954) also indicated the presence of 1 x 103 Spirochetes (Leptospira) per ml of sludge. Aerobic bacterial counts from 2 x 106 to 350 x 106 bacteria per ml on tryptone glucose agar were obtained by MCCARTY et al. (1962). The bacterial counts of digesters that received fatty acids, alcohol and proteins ranged from 2 x 106 to 20 x 106 bacteria per ml, and the counts of digesters that received carbohydrates ranged from 15 x 106 to 350 x 106 bacteria per ml (McCAgrY et al., 1962). With longer hydraulic retention times the bacterial numbers appeared to have increased in digesters that received glucose and nutrient broth but not in a digester that received octanoic acid. MCCARTY et aL (1962) also found that a digester, at a hydraulic retention time of 10 days, contained 9 x l0 6 bacteria per ml. Echerichia coli accounted for 55 per cent and Micro- coccus varians for 22 per cent of the total population. The coliforms, however, dis- appeared after several months of operation. HEUKELEKIAN (1958) reported that the observed increase of coliform organisms during the first few days of digester operation was followed by a decrease in numbers. Coliforms appeared to play a role only during the initial stages of digestion (BURBANK et al., 1966). TOERIEN (1967a) was unable to isolate coliforms directly from digesters after ten months of digester operation but could isolate coliforms after an enrichment procedure was used. Coliforms therefore do not seem to play an active role during the later part of anaerobic digestion.

The direct microscopic count of bacteria (Breed count) was modified by BURBANK

392 D.F. TOEIU~.'q and W. H. J. HATTINGH

et al. (1966) who obtained figures approximating 60 x 106 bacteria per ml in anaerobic digesters. These authors claimed to have been able to distinguish microscopically between a Clostridium sp. and a Bacillus sp. This claim is questionable since both bacteria were rod-shaped, spore-bearing cells (according to the genera in which they were classified) and were therefore too similar to be distinguished microscopically. Microscopic counts would only serve to distinguish between widely different morphological shapes, but even then results should be interpreted with caution. An excellent review on the limitations and requirements of the Breed count method has been given by WILSON (1935) which should be consulted before the Breed method is used. A tendency to determine bacterial species and physiological abilities from microscopic morphological appearance is evident in the work of a number of authors in the field of anaerobic digestion. This is a most unreliable procedure and should never be used.

KOTZ~ et al. (1968) reported on the total aerobic, aerobic proteolytic and aerobic lipolytie bacteria in several laboratory and large-scale digesters. Total counts varied from 3 x 106 to 300 x 106 bacteria per ml, while proteolytic counts varied from 1 105 to 90 x 105 bacteria per ml and the lipolytic counts varied from 2 x 104 to 16 x i04 bacteria per ml. The total aerobic count of a digester receiving a sterile synthetic substrate was less than 8 x 105 bacteria per ml, although this digester contained the highest amount of DNA per ml. KOTZ~ et al. (1968) also used the DNA contents of the digesters to predict the probable number of cells per unit volume. Ratios of numbers of total aerobic counts to predicted total numbers were calculated and these results indicated that a much larger number of cells must have been present than were accounted for by the aerobic counts. Korz_A et aL (1968) concluded that the non- methanogenic bacterial population probably consisted mainly of obligate anaerobes.

A method developed by TOERtEN and SmBERT (1967), based on the cultivation methods used in rumen microbiology, was used to enumerate obligate anaerobic non- methanogenic bacteria in digesters. TOE~UZN et aL (1967) reported 39 107 to 15 109 obligate anaerobic non-methanogenic and 8 x 105 to 1 x 10 a aerobic and facultative anaerobic bacteria per ml in several digesters. The anaerobic counts were usually 100 or more times greater than the aerobic counts. MAKI (1954) and POST et al. (1967) also indicated the presence of a large obligate anaerobic bacterial population in anaerobic digestion. To~ et al. (1967) obtained highly significant correlation coefficients (P = 0.01) between the following pairs of observations:

1. Obligate anaerobic non-methanogenic bacterial numbers and DNA. 2. Obligate anaerobic non-methanogenic bacterial numbers and volatile suspended

solids (VSS). The following pairs of results were not highly significantly correlated. 1. Obligate anaerobic non-methanogenic bacterial numbers and aerobic and

facultative anaerobic bacterial numbers. 2. Aerobic bacterial numbers and DNA. 3. Aerobic bacterial numbers and VSS. These authors concluded that the obligate anaerobic non-methanogenic bacterial

group formed a numerically important and constant part of the total populations of their digesters but the aerobic and facultative anaerobic bacteria were not numerically important or constant part of the total population. The suggestion by MCKINNEY (1962) that acid formation is mainly carried out by facultative bacteria seems to be incorrect in the light of these results.

Anaerobic DigestionmI. The Microbiology of Anaerobic Digestion 393

Further information on the nature of the non-methanogenic populations was provided by THroE et al. (1968). A wide range of chemical, biochemical and microbiological characteristics were determined on three similar digesters and correlation coefficients calculated between all of these observations. The obligate anaerobic non- methanogenic bacteria were again more numerous (about 100 times) than the aerobes and facultative anaerobes. Both the numbers of the aerobic and facultative anaerobic bacterial group and the obligate anaerobic non-methanogenic bacterial group were significantly (P = 0.05) or highly significantly (P = 0.01) correlated with a wide range of the characteristics determined. For instance both these groups were significantly or highly significantly correlated with chemical parameters such as pH, volatile fatty acid content, alkalinity and biochemical analyses such as activities of enzymes of the glycolytic pathway and the tricarboxylic acid cycle. These two bacterial groups were also significantly correlated with each other, in contrast to the findings of TOERIEN et aL (1967). The significant correlations of the aerobic and facultative anaerobic population with many parameters of anaerobic digestion indicated that this bacterial group, although numericallyless important, formed a constant portion of the total population.

At this stage it is still not clear whether the aerobic and facultative anaerobic bacterial population plays an important part in anaerobic digestion and studies should be undertaken to elucidate the relative roles of aerobic and facultative anaerobic and obligate anaerobic bacteria in anaerobic digestion.

Literature indicates that the knowledge of the microbiology of the non-methanogenie phase of anaerobic digestion is still very empiric in nature. Although some information has been obtained, virtually no bacteriological studies have been attempted on a quantitative ecological basis which is the only logical basis for studies of ecosystems such as anaerobic digestion (see later). The relative roles of facultative anaerobes and obligate anaerobes in the digestion process are therefore still uncertain. The roles of fungi and protozoa are also rather obscure, but it seems probable that they do not play significant roles in the degradation of organic matter. A complete bacterial ecological analysis of anaerobic digestion, involving studies to determine the growth rates, metabolic activities, kinds and amounts of end-products produced, nutrient requirements, effect of environmental factors etc. on both the facultative anaerobic, as well as obligate anaerobic bacteria, has yet to be carried out. However, studies of this type will, no doubt, be carried out in due course since the facultative anaerobic bacteria present few problems in their cultivation and the difficulties to the exacting anaerobic requirements of the obligate anaerobes can be overcome with methods developed by MAKI (1954), MYLROIE and HUNGATE (1954), SMITH (1965) and by TOERIEN and SIEBERT (1967).

Metabolism of the non-methanogenic population

The outcome of the metabolism of the non-methanogenic bacterial population is the degradation of complex organic matter to substrates which can be used by the methanogenic bacteria and the synthesis of cellular material (FIG. 1). The complete degradation of the organic matter requires many different types of bacteria to bring about the multitude of reactions required to degrade complex substrates. The presence of different bacterial physiological groups in anaerobic digesters has been indicated by O'SHAUGHNESSY (1914); GAUB (1924); HOTCHKISS (1924); RUDOLFS et al. (1926); RUCHHOFT et al. (1930); KOTZ~ et al. (1968).

394 D.F. TOERIEN and W. H. J. HATTINGH

The presence of several extracellular enzymes such as cellobiase, pretense and amylase have been established and measured in anaerobic digesters (AGARDY et al., 1963; THIEL and HAYnNGI-I, 1967; Ko'rz~ et al., 1968). Extra-cellular enzymes degrade complex organic molecules to smaller units and it may be assumed that cellulase and lipase would be present in digesters receiving substrates rich in cellulose and lipids. HATTINGH et al. (1967) demonstrated that the activities of protease, ceUobiase and amylase determined under optimal analytical conditions, were capable of hydrolyzing much more substrate than was present in the daily feeds to the digesters. KOTZ~ et al. (1968) concluded that hydrolytic activity did not appear to be a rate-limiting step during anaerobic digestion, the surface of the complex organic molecules exposed to enzyme activity might, however, be rate-limiting.

Catabolism of lipids. The degradation of lipids in anaerobic digesters probably proceeds through the initial break-down of fats by lipase. The long-chain fatty acids are degraded by r-oxidation as shown with ~4C tracers using octanoic and palmitic acids (MCCARTY et al., 1962). The glycerol portion of the lipids is probably degraded via glyceraldehyde phosphate and pyruvic acid (LACKEY and HENDRICKSON, 1958). McCARTY (1964) stated that the terminal electron accepter for r-oxidation of long- chain fatty acids under anaerobic conditions was carbon dioxide. This statement implies that the anaerobic lipolytic bacteria are able to regenerate their reduced co- enzymes from r-oxidation by reaction with CO2. Carbon dioxide reduction may lead to the formation of methane by the methanogenic bacteria or to acetate by bacteria such as Clostridium aceticum. Methanogenic bacteria do not seem to be able to utilize fatty acids other than acetate and formate (Bryant, personal communication), therefore the lipolytic bacteria appear to be unable to produce methane during r-oxidation of long-chain fatty acids, suggesting that if CO: is used as terminal electron accepter then substances such as acetate must be formed. Anaerobic lipolytic bacteria may regenerate their reduced co-enzymes by the liberation of hydrogen for which no terminal electron accepter is necessary. Although the presence of hydrogen during digestion of lipids has not been demonstrated, the liberation of hydrogen is not excluded since the methanogenic bacteria would utilize the hydrogen together with carbon dioxide.

Catabolism of proteins. The degradation of proteins probably proceeds via the extracellular hydrolysis of the proteins into polypeptides and amino acids by pretense (LACKEY and HENDRICKSON, 1958). Thereafter the amino acids may be degraded by several different mechanisms depending on the organisms involved (LACKEY and HENDRICKSON, 1958; MCCARTY, 1964). The end-products of protein degradation are organic acids (MCCARTY et al., 1962).

The presence and activity of extracellular protease were demonstrated by AGARDY et aL (1963) and HATTINGH et al. (1967). High activities for the enzyme glutamate oxaloacetate transaminase (GOT) were determined in both laboratory-scale and large-scale anaerobic digesters receiving several different substrates (HATTtNGH et aL, 1967; KOTZ~ et al., 1968; THIEL et aL, 1968). High activities for the enzyme glutamate dehydrogenase (Glut-DH) (TJ, aEL et aL, 1967) and for glutamate pyruvate transaminase (GPT) (KoTZ~, personal communication) have also been established in anaerobic digesters. The high Glut-DH activities indicated a high rate of fixation or release of ammonia (KOTZI~, personal communication). The high activities of GOT and GPT indicated a rapid turnover in the amino acid metabolism in digesters.


Catabolism of carbohydrates. The catabolism of carbohydrates in digesters has been studied in considerable detail in recent years (McC/u~TY et al., 1962; HATT~GH et al., 1967; KOTZ~, 1967; KOTZ~ et aL, 1968; T~m~L et aL, 1968).

Through the use of C 14 tracers, MCCARTY et al. (1962)concluded that both glycolysis (Embden-Meyerhof-Parnass pathway) and the pentose phosphate cycle (hexose monophosphate shunt) probably occurred in digesters receiving carbohydrates and that carbon dioxide fixation also might have taken place. KOTZ~ (1967) developed analytical techniques to demonstrate and determine activities of intermediary metabolic enzymes (FIG. 2) and HATTINGH et al. (1967); KOTZ~ et al. (1968) and Tm~L et al. (1968) reported on the activities of a wide range of intermediary metabolic enzymes during anaerobic digestion. Enzyme activities altered during the adaptation of a raw sewage sludge digester to a synthetic substrate, and glycolytic and certain tricarboxylic acid cycle (TCA-cycle) enzymes were active in synthetic substrate as well as raw sewage sludge digesters (HATT~GH et aL, 1967). The activity of fructose-6-phosphate kinase (F-6-PK), a normal rate-limiting enzyme in glycolysis, was found to increase proportionally with the increase of gas production until 6 weeks after the start of the adaptation to the new substrate after which the F-6-PK activity increased more rapidly than gas production (HATTINGH et al., 1967). Phosphoglucomutase (P-GluM) activity which HATTINGH et al. (1967) considered as a measure of cell synthesis and other intermediary enzyme activities, were still increasing after 60 days of adaptation, indicating that a steady state in digester behaviour had not occurred during this period.

A comparison in the activities of intermediary metabolic enzymes in several laboratory-scale and large-scale digesters indicated that differences in the enzyme patterns existed (KoTz~ et al., 1968). All the digesters contained enzymes of glycolysis and the glyoxylie acid pathway, but only the digester that received synthetic substrate contained indications of an active hexose monophosphate shunt (activity of glucose- 6-phosphate dehydrogenase, G-6-PDH). In the two digesters tested, namely the digesters receiving respectively synthetic substrate and raw sewage sludge, indications of an active TCA cycle or at least part thereof, was found, as indicated by high malate dehydrogenase (MDH) activities. SIMPSON (1960) stated that oxaloacetic acid metabolism could not proceed in anaerobic systems such as anaerobic digesters. However, the presence of high GOT activities in digesters indicated an active participation of oxaloacetate in amino acid metabolism. The MDH activity probably indicated the participation of oxaloacetate in at least a part of the TCA cycle in anaerobic digestion. The presence of malic enzyme, catalyzing the reaction:

pyruvate + CO2 ~ malate

has also been demonstrated in anaerobic digesters (Kotz6, personal communication). In another anaerobic habitat, the rumen of herbivours, succinate is formed by a number of the important rumen bacteria, probably through the formation of oxaloacetate from pyruvate (HtmOA~, 1966). Wrn~ et al. (1962) demonstrated the presence of a cytochrome-linked fumarie reductase which was possibly a source of additional ATP formation during the production of succinate by a rumen bacterium, Bacteroides ruminicola.

TmEL et al. (1968) studied the biological and chemical changes taking place in three similar laboratory-scale digesters, which were operated in such a way as to induce

396 D.F. TOERIEN and W. H. J. HATrINOH

digester failure. Calculation of correlation coefficients between all the parameters indicated that the activities of most of the enzymes tested were significantly correlated t o each other as well as to chemical and bacteriological parameters. The enzymes determined, included enzymes of glycolysis, the hexose monophosphate shunt, the TCA cycle and amino acid metabolism.

It may be possible to develop activity parameters in anaerobic digestion systems from enzyme studies since changes in the ratio's of some of the intermediary metabolic enzymes such as

P-GluM GOT Hexokinase or

F-6-PK ' F-6-PK F-6-PK '

might be indicative of unbalanced conditions (KoTzt et aL, 1968). Such observations require to be verified under controlled conditions in the laboratory.

Although progress has been made in the study of the intermediary metabol ism of carbohydrate util ization, similar studies will have to be carried out on protein and lipid degradations as well as on the metabol ism of pure cultures isolated from anaerobic digesters in order to obtain a more complete understanding of the whole non-methanogenic phase of anaerobic digestion. Such studies will contribute towards a fuller understanding of the whole non-methanogenic population and its physiological abilities.

Metabolic end-products of the non-methanogenic population. The end-products of the non-methanogenic populat ion are saturated fatty acids, carbon dioxide and ammonia

FIG. 2. Intermediary metabolic enzymes (thick arrows) demonstrated in anaerobic digestion by Kotz6 (1967).

Abbreviations Enzymes. ACON -- aconitase; ALD = aldolase; DHF-R = dihydrofolic reductas;

ENOL = enolase; F-1-PK = fructose-l-phosphate kinase; F-6-PK = fructose-6-phosphate kinase; FDH = formic dehydrogenase; FUM = fumarase; GAPDH = glyceraldehyde-3- phosphate dehydrogenase; G-6-PDH = glucose-6-phosphate dehydrogenase; GDH = glycerol-l-phosphate dehydrogenase; GlutDH = glutamic dehydrogenase; GOT = glutamate- oxaloacetate transaminase; GPT = glutamate-pyruvate transaminase; GR = glyoxylate reductase; HK = hexokinase; ICDH = isocitric dehydrogenase; ICL = isocitric lyase; a-KGDH = a-ketoglutarate dehydrogenase; LDH = lactate dehydrogenase; MDH = malate dehydrogenase; ME = malic enzyme; PGAK = phosphoglycerate kinase; PGluM = phosphoglucomutase; PGI = phosphoglucose isomerase; PGM = phosphoglycerate mutase; PK = pyruvate kinase; 6-PGADH = phosphogluconic acid dehydrogenase; SDH = succinic dehydrogenase; TIM = those phosphate isomerase; UDPG-PP = uridine diphosphate pyrophosphorylase.

Other Abbreviations. AcCoA = acetyl-coenzyme A; AcP = acetyl-phosphate; DAP = dihy- droxyacetone phosphate; DHF = dihydrofolic acid; F-1-P = fructose-l-phosphate; F-6-P = fructose-6-phosphate; F-1,6-P2 = fructose-l,6-diphosphate; GAP = D-glyceraldehyde 3- phosphate; GDP = guanine diphosphate; GTP = guanine triphosphate; G-I-P = glucose- 1-phosphate; G-6-P = glucose-6-phosphate; NAD+= nicotinamide adenine dinucleotide (oxidized); NADH = nicotinamide adenine dinucleotide (reduced); NADP + = nicotinamide adenine dinucleotide phosphate (oxidized); NADPH = nicotinamide adenine dinucleotide phosphate (reduced); PEP = phosphoenol pyruvate; P.P. shunt = pentose phosphate shunt; 2-PGA = 2-phosphoglycerate; 3-PGA = 3-phosphoglycerate; 1,3-DPGA = 1,3-diphospho- glycerate; 6-PGA = 6-phosphogluconic acid; THF = tetrahydrofolic acid; UDPG = uridine diphosphate glucose; UTP = uridine triphosphate.


D- glucose I HK UTP PP

G ~ G-6-P ' ' G-t-PX~"~/UDPG " carbohydrates PG, PG, M UOPG-PP

/ F -6 - P " D-ribulose-5-P / I F6PK

~\ / FIPK " D - #ructose / F- I ,6-P ~ : F - I -P

\P.R shunt / ~LD / ~" ~ GDH

~GAp F j . 'DAP ~ : glycerol-l-phosphate TIM

GAPDH glycerol

I, :3 D PGA

I PGAK

3 - PGA glycolysis I PGM

2-PGA

ENOL

PEP COz+ GDP/ tp K

+CO2 I/~N~C#~ ' LDH // ~ ' ' Pyruvate , . lactate

l DHF -F NADPH

~U/ I AcCoA - AcP ~ acetate ~aCtyocids~THF + NADP + / ~ , \ _ ~ormate ' C -THF- - - - - H. . . I C

I I [rlcarboxyhc acid cycle IFDH GTP / /~L T

J, ' / / ~ NADH + CO 2 / \ oxoloacetote / -citrate

. DP / \, malate ~ c's-oconitgte

/ ACON FUM/ glyoxylate ICL ~ pyruvote fumaraf te ~ iso-citrate / /~ glutamate

~SDH ~ IlCDH succ'nafe ~ / ...~" GPT ' 2-oxoglutarate alanine

a -KGDH/ succinyl CoA ~ ] / . _ _ - - -~-aspar fa fe

NAD~ Glut DH ~, , ,. . . . . . . .

glutamate oxo loacetate

398 D.F. TOEP.~N and W. H. J. HATrm68

derived from amino acid catabolism (McCAgTy, 1963). LEVIh~E (1938) stated that the non-methanogenic population yielded end-products such as fatty acids, alcohols and ketones. However, HEUKELEK~N and BERGEg (1951) failed to obtain significant con- centrations of alcohols, acetone and acetaldehyde during the anaerobic digestion of sewage solids and industrial wastes. Inoculation of sterile sewage sludge with Clostri- dium acetobutylicum, a known vigorous producer of alcohols and acetone, failed to produce large quantities of any of these compounds. HEUKELEKIAN and BEGGER (1951) concluded that sewage sludge did not provide a suitable substrate for the organisms capable of producing these compounds. It is, however, possible that alcohol-producing bacteria may occur in anaerobic digestion and that the failure to demonstrate alcohols during digestion was due to a high turnover rate of alcohols and ketones. The inability of Clostridium acetobutylicum to grow and produce alcohols and acetone in sterile raw sludge was only an indication that this particular organism was unable to grow under the conditions provided and it does not necessarily follow that all other alcohol- producing bacteria would be unable to produce alcohols during active digestion. HUNGATE (1950) and MAKI (1954), for example, demonstrated that ethanol was an end-product of the metabolism of pure anaerobic cellulolytic bacteria isolated from digesting sludge. Further studies to detect the possible production of alcohols and other neutral compounds during anaerobic digestion remain to be carried out.

The present state of knowledge suggests that hydrogen must also be a major end- product in the metabolism of the non-methanogenic population, or of an intermediary population which metabolizes fatty acids. This question will be discussed in more detail in a subsequent section.

The production of volatile fatty acids in anaerobic digestion has been demonstrated by SYMONS and BUSWELL (1933); TARVIN and BUSWELL (1934); KAPLOVSK (1951, 1952); MUELLER et al. (1959); HINDIN and DUNSTAN (1960); MCCARTY et al. (1962, 1963); POHLAND and BLOODC, OOD (1963); JERIS and ~ICCARTY (1965). These acids were found to be acetic, propionic and butyric acids, with lesser amounts of formic, lactic and valeric acids (HEUKELEKIAN and KAPLOVSKY, 1949; KAPLOVSKY, 1951, 1952 ; GOLEUKE, 1958 ; MUELLER et al., 1959; HIVrOIN and DUNSTAN, 1959, 1960; MCCARTY et al., 1962, 1963; JERIS and MCCARTY, 1965).

MCCARTY et al. (1962, 1963) established that sludges adapted to a range of fatty acids, all utilized formic acid. They concluded that formic acid was either an important intermediate in methanogenesis or that sludges adapted to other substrates were simultaneously adapted to formate. The importance of formic acid as end-product was not further studied.

McCAgTY et al. (1962, 1963) concluded that acetic acid was the most plentiful and therefore the most important intermediate, followed by propionic acid, found in carbohydrate, protein and fat metabolism. Acetic acid was also found in the methane fermentation of propionic and butyric acids. Acetic and propionic acids were also found to be the major acids present during unbalanced digestion conditions.

These studies indicate that the main end-products formed by the metabolism of the non-methanogenic bacteria were acetic, propionic and butyric acids, and perhaps formic and lactic acids. Succinate may also be produced in digesters but is most probably rapidly decarboxylated to propionic acid, as observed in the rumen (HUN- GATE, 1966).

The end-products of the metabolism of the non-methanogenic population are

Anaerobic DigestionmI. The Microbiology of Anaerobic Digestion 399

important in anaerobic digestion since these molecules are the energy sources for subsequent bacterial growth, and will therefore govern the secondary populations developed in digesters. McChg,'i~" et aL (1962); McChgTv (1963) and Jm~Is and McCARrV (1965) postulated that variations in the end-products of the non-methanogenie phase might cause shifts in the methanogenic population with resultant upset in digester behaviour. Studies of pure cultures of non-methanogenic bacteria may be useful to determine the end-products of the metabolism of the acid-forming phase, and to gain an insight in the way in which various environmental factors may influence it. The pronounced effect of environmental changes on end-product composition has been demonstrated for Escherichia coli and Clostridium spp. (DOBROC, OSZ, 1966; Moo~ et aL, 1966).

MICROBIOLOGY OF THE METHANOGENIC PHASE

One of the major end-products of the anaerobic digestion of organic wastes is the gas methane, which is produced from the end-products of the non-methanogic phase by the methanogenic bacteria (McC~a~a~, 1963).

Types of methanogenic bacteria Since the methanogenic bacteria occurring in sludge are presumably similar to those

occurring in other habitats, available literature on all methanogenic bacteria will be reviewed.

The methanogenic bacteria and their characteristics are still poorly defined, although studies on methanogenic bacteria have been carried out for many years. B&hamp indicated in 1868 that methane was probably formed by a microbiological process and he called his "ferment" Microzyma cretae (BARKER, 1956). Popoff, Hoppe-Seyler and other workers in the latter half of the 19th century confirmed the microbial nature of methanogenesis (BAR~, 1936a). Fundamental investigations by S~hngen served to elucidate the morphological appearance and characteristics of the methanogenic bacteria (BARm~R, 1936a). Omelianski, Maz6 and Groenewege also contributed towards a better understanding of these organisms (BARKER, 1936a,b). For more comprehensive surveys of the earliest studies on methanogenesis, readers are referred to papers by BARKER (1936a,b; 1956) and by BUSWELL and HATFmLD (1936).

Early work on the methanogenic bacteria was carried out on enrichment cultures since these organisms were exceedingly di~icult to isolate (BARKER, 1936b, 1956). In 1906 Srhngen described two organisms which fermented lower fatty acids to methane (BARKER, 1936b). One was a gram-negative immotile rod-shaped bacterium and the other a gram-negative, large conspicious sarcina. Several subsequent workers claimed to have observed S6hngen's sarcina in acetate enrichment cultures and sewage sludge (BARKER, 1936b). In 1916 Omelianski described a non-sporeforming methane-producing bacterium which fermented ethyl alcohol (BARKER, 1936b). BhRI(~ (1936b) considered this bacterium to have been responsible for the fermentation of acetone as reported by Mazr. GROENEWEGE (1920)described an alcohol-fermenting methanogenie bacterium (BARKER, 1936b) as a small micrococcus of which the individual cells usually clumped in large masses. Coccus-shaped methanogenic bacteria were also described by MAZE (1903, 1915) quoted by BAg_g~ (1936b). COOLI~,hS (1928) described

B W

400 D.F . TO~R~'N and W. H. J. HAI~'nqGH

a thermophilic methanogenic bacterium which fermented some of the lower fatty acids. BaggER (1936b) doubted whether this bacterium differed from the other methanogenic bacteria described at that time.

STEPHENSON and ST:CKLAND (1933) isolated a pure culture of a methanogenic bacterium which utilized formate with the production of methane and carbon dioxide but BaggER (1936b) doubted the purity of this isolate. BARKER (1936b) obtained purified methanogenic cultures of a sarcina, for which he proposed the name Methano- sarcina methanica, a coccus, for which he proposed the name Methanococcus mazei, and two rod-shaped bacteria, for which he proposed the names Methanobacterium si~hngenii and Methanobacterium omelianskii. Several years later BaggER (1940) suc- ceeded in isolating M. omelianskii in what he believed to be a pure culture. This organism was later named Methanobacillus omelianskii because of the formation of endospores of low heat resistance (BaggER, 1956). As the name Methanobacterium omelianskii is still used in the seventh edition of Bergey's Manual (BREED et al., 1957), the latter name will be used throughout this review. Methanobacterium omelianskii has been used extensively in the study of the physiology of methanogenesis (BARKER, 1940, 1941 ; PINE and BARKER, 1954; JOHNS and BARKER, 1960; WOL:N et al., 1963a, b; WOOD et al., 1965, 1966; WOOD and WOLFE, 1965, 1966a,b; LEZIUS and BARKER, 1965)but BRYANT et al. (1967) showed that M. omelianskii was a symbiotic association of two bacterial species.

SCmqELLEN (1947) isolated two pure cultures of methanogenic bacteria from sewage sludge and river mud and named them Methanobacterium formicicum and Methano- sareina barkerii. Methanobacterium formicieum was rod-shaped, and produced methane both from formate and by carbon dioxide reduction. Methanosarcina barkerii, was a sarcina producing methane from methyl alcohol, acetate and by carbon dioxide reduction.

STAnTMAN and BaggER (1951a) isolated a pure culture of a methanogenic formate- utilizing bacterium from black mud. This organism was an actively motile medium- sized coccus, with fragile cell walls. The pH ~ange for growth was between 7.4 and 9.2 and for this bacterium the authors proposed the name Methanoeoecus vannielii.

During tracer studies on fatty acid oxidation with enriched cultures, STADTMAN and BARKER (1951b) obtained two purified (but not pure) cultures of methanogenic bacteria. One was a small, thin, bent rod, sluggishly motile which fermented caproic, valeric and butyric acids with the production of acetate and methane. For this bacterium STADTMAN and BaggER (195 lb) proposed the name Methanobaeterium suboxydans. The other organism utilized propionate and was a motile short thick rod-shaped to coccoid bacterium, for which the name Methanobacteriumpropionicum was proposed.

SMITH and Ht~GATE (1958) obtained pure cultures of a methanogenic rod-shaped bacterium from the rumen of fistulated cattle and sheep. The wide geographic dis- tribution of this bacterium was indicated by its occurrence in the rumen contents of an African Zebu steer. The name Methanobacterium ruminantium was proposed by SMITH and Ht~GATE (1958) for this bacterium.

MYLROIE and HLrNGATE (1954) isolated Methanobacterium formicicum and SMITH (1961) isolated M. ruminantium from digesting sludge. SMITH (1966) in a quantitative microbial ecological study of the methanogenic bacteria of digesting sludge, isolated members of Methanobacterium, Methanococcus and Methanosareina from high sludge dilutions. All these isolates utilized hydrogen as an oxidizable substrate. The isolates

Anaerobic Digestion--I, The Microbiology of Anaerobic Digestion 401

included Methanobacterium formicicum, M. ruminantium, Methanosarcina barkerii, two new Methanobacterium spp. and a new Methanococcus sp. Other methanogenic bacteria were also observed by SMn'ri (1966) but efforts to isolate and maintain them in pure culture were unsuccessful, SMrra (1966) was unable to verify the observations of LAmP~T (1945) that Clostridium perfringens formed methane in the presence of potassium iodide under certain culture conditions.

BRYANT et aL (1967) described a further methanogenic bacterium which was the methanogenic partner of the symbiotic association formerly known as Methano- bacterium omelianskii. The bacterium was rod-shaped, could not utilize ethanol but reduced carbon dioxide with the formation of methane. The other partner of the symbiotic association was also rod-shaped and utilized ethanol with the production of acetate and hydrogen. The presence of hydrogen was inhibitory for the growth of this bacterium. When the hydrogen-producing bacterium was grown in the presence of ethyl alcohol and carbon dioxide, together with the methanogenic partner or with Methanobacterium ruminantium (both were unable to utilize ethanol) excellent growth was obtained. The presence of two types of DNA was established in cultures of Methanobacterium omelianskii but only one type of DNA was found in cultures of the methanogenic partner. BRYANT et al. (1967) concluded that Methanobacterium omelianskff was not a pure culture, and if this is so it is evident that the results of studies on the physiology of M. omelianskii must be reappraised.

The occurrence of methanogenic bacteria in the rumen has also been studied. In addition to Methanobacterium ruminantium, the formate-utilizing M. formicicum (OPPERMAN et aL, 1957) acetate-fermenters, including a Methanosarcina sp. (BELIER, 1952) and a species resembling Methanobacterium s6hngenii (OPPERMAN et al., 1957). and a valerate and butyrate fermenting species resembling M. suboxydans (NELSON et aL, 1958) have been isolated or were obtained in a highly purified culture from rumen contents.

Methanogenic bacteria are apparently very widespread in nature and can be found in ordinary garden soil, black mud (BARKER, 1936b), the rumen of herbivorous animals (SMITH and HUNGATE, 1958; BRYA~n" 1964, 1965), marshes, ponds, lakes (BARKER, 1956) and in sewage and sewage treatment processes (MCCARTY, 1963).

Numbers of methanogenic bacteria HEUKELErdAN and HEINEMANN (1939a) developed a most probable number technique

for the enumeration of methanogenic bacteria. HEUKELEKIAN and HEINEMA~CS (1939b) reported on the enumeration of methanogenic bacteria of several aspects of sludge digestion. They found from 25 x 102 to 25 x 10 6 acetate fermenters per ml, 6 x 102 to 25 X 10 6 "butyrate fermenters" per ml and 25 x 102 to 25 x 10 s "ethyl alcohol fermenters" per ml in digesting sludge.

MYr_ROIE and HUNGATE (1954) developed an agar culture method for the determination of methanogenic bacterial numbers. This method was largely based on the method of HU~GATE (1950) which has been widely used in rumen microbiology. MYLROIE and HUNGATE (1954) provided hydrogen and carbon dioxide as substrates and used palladium as a reduction catalyst. Culture counts of methanogenic bacteria ranging from 105 to 108 per ml were obtained. These experiments formed an interesting approach to the study of methanogenic bacteria in sludge digestion and it is unfortun- ate that further work does not appear to have been carried out.

402 D.F. TOERI~ and W. H. J. HATnNGH

SMrrH and HUNGATE (1958), using a modification of the method of HUNGATE (1950), enumerated and isolated methanogenic bacteria from the rumen. Culture counts as high as 2 x l0 s methanogenic bacteria per ml of rumen contents were obtained. These observations were later extended and counts ranging from 107 to 109 per ml were obtained with rumen contents (BRYANT, 1965).

SMITH (1965), using a further modification of the method of litiGATE (1950), studied the methanogenic bacteria of sludge. Pure cultures of methanogenic bacteria were obtained from growth in 10-6 dilution. SMITH (1966) extended his quantitative ecological studies on the methanogenic bacteria of sludge digestion and isolated six methanogenic bacteria in pure culture. The numbers in which they were present were as follows:

Methanobacteriumformicicum exceeding 1 x 10 7 per ml, M. ruminantium exceeding 1 x 10 7 per ml, Methanosarcina barkerii exceeding 1 x 10 6 per ml, a long rod-shaped Methanobacterium sp. exceeding 1 x 108 per ml; a Methanococcus sp. exceeding I x 106 per ml, another Methanobacterium sp. exceeding 1 x 107 per ml. Numbers of methanogenic bacteria obtained with a highest dilution method on several sewage sludge digesters, indicated the following: hydrogen-utilizers 1 x 107 per ml, acetate utilizers 1 x 105 to 1 x 10 6 per ml, "propionate utilizers" 1 x 10 6 to 1 x 10 7 per ml, "butyrate utilizers" 1 x 106 to I x 10 s per ml and "ethanol utilizers" 1 x 107 per ml.

SIEBERT and HAr'rtNGH (1967) used a most probable number method for the estimation of the methanogenic bacterial numbers in anaerobic digesters, but THIEL et al. (1968) concluded that this method was not entirely satisfactory.

The above studies indicate that methanogenic bacteria may be present in anaerobic digesters in numbers of 106 to 108 per ml. When these numbers are compared with the values of 106 to 108 recorded for obligate anaerobic non-methanogenic bacteria in digesters (TOERmN et aL, 1967), it appears that the numbers of the bacteria of the non- methanogenic phase and those of the methanogenic phase are nearly equal. The significance of these findings is not clear at this stage and more information is needed on the bacterial numbers of the different phases to evaluate their roles in the equilibria of anaerobic digesting systems.

Possibly the best approach to the enumeration of methanogenic bacteria in sludge would be the use of methods which simulates conditions in digesters and which are based on Hungate's technique (HtmGAT~, 1950). This approach has been used with good results by SMITH (1965, 1966) in quantitative microbial ecological studies.

Nutritional requirements of the methanogenic bacteria BARKER (1940, 1956) indicated that the nutritional requirements of methanogenic

bacteria appeared to be simple. Methanobacterium omelianskii grew satisfactorily in growth media containing the usual nutritive salts, carbon dioxide, a reducing agent, a single oxidizable compound and ammonia as source of nitrogen (BARKER, 1940). M. omelianskii was also able to utilize nitrogen gas (PINE and BARKER, 1954). How- ever, BRYANT et al. (1967) indicated that M. omelianskii was not a pure culture. The nutritional requirements of the respective partners of the symbiotic association which was called M. omelianskii have not yet been determined.

H~UKm~raAN and Hml, a~Mhr~ (1939a) reported enhanced growth of methanogenie bacteria on the addition of yeast extract to their growth media. Wour~ et al. (1963a) reported that growth of Methanobacterium omelianskii was more reproducible after the addition of a vitamin solution to their mineral medium. SPF~CE and MCCARrV


(1964) reported more complex nutritional requirements of methanogenic bacteria than that reported by BARKER (1956).

SMITH (1959), found that Methanobacterium ruminantium required growth factors present in rumen fluid which were not found in several ingredients commonly used as sources for growth factors for bacteria. BRYANT (1965) reported on unpublished work of Bryant and Robinson (1964) which extended the observations of SMITh (1959) in that it was found that M. ruminantium required three different growth factors, one of which was acetate and was required in large amounts. Of the two other factors present in rumen fluid one could be separated by ether extraction at pH 2.0. The ether- extractable factor was found to be one or more volatile acids that distilled at rates similar to butyric or longer-chained fatty acids, during Duclaux distillations. Branched- chained fatty acids, especially 2-methylbutyric acid were most active. The third factor was not absolutely identified, but was found to be dialysable and was not detroyed by autoclaving at 121C in 0.86 M HC1 (pH 0.2). It could be precipitated with 90~ ethyl alcohol or 80~ acetone and it was not a cation (BRYANT, 1965). BRYANT and NAL- nANDOV (1966) showed that the third factor necessary for the growth of M. ruminantium was also present in sewage sludge but not in various other crude materials, and they determined that this factor was a highly polar acidic compound which was produced by other microorganisms, possibly from an unknown precursor present in yeast extract.

The information presently available indicates that all methanogenic bacteria require a low redox potential for growth. SMITH and HUNGATE (1958) and BRYANT (1965) for example, have shown that a redox potential approaching - 300 mV is required for the growth of M. ruminantium in pure culture.

Since the methanogenic bacteria play an important role in the anaerobic digestion of organic matter an understanding of the nutritional requirements of these bacteria is obviously necessary to ensure that a digestion process does not fail due to lack of these materials.

Metabolism of the methanogenic bacteria Substrates used by methanogenic bacteria. Evidence that lower fatty acids could be

quantitatively converted to methane and carbon dioxide was obtained by S6hngen (BARKER, 1936b). StShngen also found that a mixture of hydrogen and carbon dioxide could be utilized to produce methane (BARKER, 1936a).

Other investigations extended the list of organic substrates which could be decom- posed with the formation of methane. Omelianskii studied the methane fermentation of cellulose and ethyl alcohol, Maz6 studied the fermentation of acetone, and Groene- wege demonstrated the formation of methane from lower alcohols, while Bach and Sierp obtained methane from the fermentation of proteinaceous materials (BARKER, 1936a). COOLHAAS (1928) studied the thermophilic fermentation of various fatty acids. BUSWELL and NEAVE (1930), SYMONS and BUSWELL (1933), TARVIN and BUSWELL (1934) and BUSWELL and HATFIELD (1936) found that a wide range of organic compounds could be degraded to methane during anaerobic digestion. BARKER (1936a) commented on the two-stage process in the production of methane from complex organic compounds and suggested that the lower fatty acids, alcohols and ketones would probably be directly fermented by methanogenic bacteria.

BUSWELL and NEAVE (1930) developed a general theory of methane fermentation.

404 D.F. TOERIEN and W. H. J. HA'rrn~OH

They assumed water to act as an oxidizing agent for part of a substrate molecule whilst the remainder of the same molecule was reduced by acceptance of hydrogen atoms from water. This suggestion implied that some of the carbon atoms of the substrate were liberated as methane, but the theory did not explain the occurrence of methane as the sole reduction product of the substrate (BARKER, 1936a). FISCHER et aL (1932) and STEPrmNSON and STICKLAND (1933) confirmed the reduction of carbon dioxide to methane.

A carbon dioxide reduction theory was subsequently developed by van Niel which stated that methane was in all circumstances derived from carbon dioxide reduction (BARKER, 1936a), according to the reaction:

CO2 + 4H 2 --~ CH 4 + 2H20. (1)

Methane production by enriched cultures, resulted from the reduction of carbon dioxide during the degradation of ethyl and butyl alcohol and probably also during the degradation of butyric acid (BARKER, 1936a). He generalized these reactions as follows:

4H 2 A + CO2-, 4A + CH, + 2H20 (2)

H2 A represented any organic or inorganic compound which could be activated by methanogenic bacteria. BARKER (1936a) represented the methane fermentation of acetic acid as a dehydrogenation of the acid accompanied by a reduction of carbon dioxide as follows:

CH3COOH + 2H20 + CO 2"-''2CO2 + CH, + 2H20. (3)

BARKER (1941) and STADTMAN and BARKER (1949) suggested that M. omelianskii oxidized ethyl alcohol almost quantitatively to acetate as follows:

2CH3CH2OH + CO2 ~ 2CHaCOOH + CH4. (4)

BARKER (1941) found that this reaction was dependent on the supply of carbon dioxide and that the reaction did not proceed in its absence. JOHNS and BARKER (1960) concluded that in the absence of carbon dioxide the following reaction occurred:

CHaCH2OH + H20~ CHaCOOH + 2H:. (5)

Reaction (4) was shown by BRYANT et al. (1967) to be the product of the action of two bacterial species, one of which used carbon dioxide in substrate amounts. The growth of the other bacterium was inhibited by the production of hydrogen (6, 7).

Bacterium A 2CH3CH2OH+2H20--~2CH3COOH+4H2 (6)

Bacterium B CO2 + 4H2-~CH4 + 2H20. (7)

The reduction of carbon dioxide to methane (1) was substantiated with Methano- bacterium omelianskii by BARKER (1943). The carbon dioxide reduction took place as depicted in (1). SCHNELLEN (1947) confirmed the above reaction in pure cultures of Methanobacterium formicicum, Methanosarcina barkerii and Methanobacterium omelianskii. The same reaction was found in M. ruminantium (SMITH and HUNGATE, 1958) and in three new methanogenic bacteria isolated from sludge digestion (SMITH, 1966) and in the methanogenic partner of the Methanobacterium omelianskii symbiotic association (BRYANT et al., 1967). A rapid exchange of carbon between carbon dioxide and formate during tracer studies on Methanococcus vannielii prevented confirmation of the presence of carbon dioxide reduction (STADTMAN and BARKER, 1951a).


KLUYVeR and SCHN~LLEN (1947) studied the reduction of carbon monoxide to methane by pure cultures of Methanobacterium formicicum, M. ornelianskii and Methanosarcina barkerii. They obtained evidence that this reaction proceeded as suggested by FISCHER et al. (1931, 1932) according to the following reactions:

4CO + 4H20--.4CO 2 + 4H 2

CO2 + 4H2--* CH4 + 2HzO 4CO + 2H20~3CO 2 + CH4" (8)

STADTMAN and B~a~K~ (1951b) showed that the fermentation of butyrate was similar to the fermentation of ethyl alcohol, as follows:

2CHsCH2CHzCOOH + 2H:O + CO2--* 4CHsCOOH + CH4. (9)

STADTMAN and BARKER (1951b) also showed that Methanobacterium propionicum probably utilized propionate according to the reactions:

4CHsCH:COOH + 8H20~4CH3COOH + 4CO2 + 24H (10)

3CO2 + 24H~ 3CH; + 6H20 (11)

4CHaCH2COOH + 2H20-*4CH3COOH + CO2 + 3CH4" (12)

The carbon dioxide formed during reaction (10) was the precursor of most of the methane formed in reaction (11). Tracer studies by BUSWELL et al. (1951) on enrichment cultures receiving propionate indicated that carbon dioxide reduction took place and that all three carbon atoms of propionate were converted to methane or carbon dioxide in varying degrees. BARKER (1956) in discussing the results of BUSWELL et al. (1951) pointed out that ff a secondary decomposition of acetate took place, the results of BUSWELL et al. (1951), did not differ from those of STADTMAN and BARKER (1951b). BARKER (1956) also considered that an additional reaction would have been required to account for the preferential conversion of the c~-carbon of propionate to methane and fl-carbon to CO2 and he suggested that these results could have been caused by the formation of a symmetrical intermediate, such as succinate, from propionate, which would then allow randomization of the ~- and fl-carbons of propionate and would therefore account for the similarity of their behaviour.

The carbon dioxide reduction theory of van Niel (BARKER, 1936a) was shown not to apply in the conversion of methyl alcohol and acetate to methane. SCmC~LLEN (1947) considered that Methanosarcina barkerii readily fermented methyl alcohol as follows:

4CH3OH~3CH4 + CO2 +2H20. (13)

Tracer studies by STAD~N and BARKER (1951C) and PIr~E and VISrIN~ (1957) excluded the possibility that methanol was oxidized to carbon dioxide which was then reduced to methane since they found that the methane was formed from the methyl moiety of the methyl alcohol. PiNE and BARKER (1954) confirmed these results with the use of deuterium isotopes and suggested the reaction:

D20 4CH3OH---*3CHaD + CO2. (14)

In van Niel's carbon dioxide reduction theory, acetate would be fermented by complete oxidation to carbon dioxide followed by carbon dioxide reduction (3). BUSWELL

406 D.F. TOERIEN and W. H. J. HATrlNGH

and SOLLO (1948) tested this mechanism by the use of 14CO2 and concluded that virtually no methane was derived from CO2 reduction. STADTMAN and BARKER (1949, 1951C), also using tracer methods, found that methane was formed entirely from the methyl group and carbon dioxide exclusively from the carboxyl carbon of acetate:

C*H3C +OOH~C*H4 + C+O2 . (15)

Using acetate labelled with deuterium in the methyl group, PINE and BARKER (1956) showed that:

HzO CDaCOOH--~ CDaH + CO2

D20 CH3COOH--~CH3D + CO2 (16)

Formate conversion to methane apparently occurred via carbon dioxide and hydrogen (DOEa'SCn et al., 1953; CARROLL and HU,'qGATE, 1955). Tracer experiments showed that enrichment cultures of formate-decomposing methanogenic bacteria produced methane by direct reduction of formate to methane without initial splitting of the formate into carbon dioxide and hydrogen (FXNA et al., 1960). The direct reduction of formate to methane accounted for only a part of the total methane production and a large proportion of the methane was formed by reduction of carbon dioxide. A negligible exchange of carbon between formate and carbon dioxide occurred, in contrast to the findings of STADTMAN and BARKER (1951 a) with Methano- coccus vannielii.

The studies summarized above showed that lower fatty acids and alcohols could be fermented to methane and carbon dioxide, a fact which is of primary importance in digestion of organic wastes. However, recent work by BRYANT et al. (1967) has shown that the described fermentation of ethyl alcohol was not brought about by a pure methanogenic bacterium. It is now thought that methanogenic bacteria are not able to utilize substrates other than formate, acetate, methyl alcohol and carbon dioxide and hydrogen (Bryant, personal communication). If this conclusion is correct it will be of far-reaching importance since it implies that:

1. Methanogenic bacteria such as M. suboxydans and M. propionicum which are said to directly convert substrates other than formate, acetate, methanol and carbon dioxide to methane probably do not exist.

2. Since substrates such as propionate, butyrate, valerate, caproate, alcohols, are produced in digesters (McCARTY et al., 1962, 1963; McCARTY, 1964) by non-methanogenic bacteria, but no accumulation during well-balanced conditions occurs, the presence of an intermediary population for the conversion of these substrates must exist. This intermediary population probably produce hydrogen during the conversion of these substances. These reactions could occur according to a general equation:

AHz~A + H2 + energy (17)

where AH2 may be propionate, valerate, butyrate, caproate and alcohols other than methyl alcohol etc. This new bacterial group can probably be characterized by the formation of hydrogen and oxidized compounds.

3. If such a hydrogen-producing bacterial population is present, no knowledge concerning its kinds, numbers, nutrition or metabolism exists--a knowledge of which

Anaerobic Digestion I. The Microbiology of Anaerobic Digestion 407

could be of the utmost importance in understanding the behaviour of methane- producing systems, and for kinetic studies on anaerobic digestion.

4. Large amounts of extraceUular hydrogen must be produced in anaerobic digesters MCCARTY et al. (1962, 1963); McCARTY (1964) and SMITH and MAIl (1966) have concluded that approximately 70 per cent of the methane from various digesters originated from acetate, whilst about 30 per cent of the methane originated from carbon dioxide reduction.

The considerable quantity of hydrogen involved can be demonstrated by consider- ation of the case of a digester producing 30 1. gas per day with a gas composition of 50 per cent methane and 50 per cent carbon dioxide, of which 4-5 1. methane (30 per cent of the total methane volume) originated from carbon dioxide reduction. Since only one volume of methane is produced from 4 volumes of hydrogen and one volume of carbon dioxide according to (I)

CO2 + 4H2--* CH, + 2H20

then, 18 1. of hydrogen must have been involved. If none of the methanogenic bacteria are able to dehydrogenate substrates such as Ca and higher fatty acids, alcohols, ketones etc. in their cells, then all the hydrogen involved in the reduction of carbon dioxide to methane must have been present as extraceUular hydrogen. If this hydrogen had not been further metabolized, then a gas composition of approximately 41~ CO2, 24~ CH4 and 3570 H2 would have been expected. Since hydrogen is normally either not detected or is present in minute quantities it appears that there must be a very rapid utilization of hydrogen produced.

5. Since it was observed by BRYANT et al. (1967), that extracellular hydrogen inhibited growth of the hydrogen-producing partner of the symbiotic association Methanobacterium omelianskii it may also be true for the postulated hydrogen- producing bacterial population and the rapid removal of hydrogen by methanogenic bacteria may stimulate growth of the hydrogen-producing population.

In view of these possibilities it is evident that further information is urgently needed on the ability of pure cultures of methanogenic bacteria to utilize substrates other than formate, acetate, methyl alcohol and carbon dioxide, and on the existence of a hydrogen-producing intermediary population in anaerobic digestion and its characteristics. It may be noted in passing that since BAUCHOP (1967) obtained inhibition of rumen methanogenesis with methane analogues it may be that a similar procedure would elucidate the role of hydrogen in methanogenesis during anaerobic digestion. Thiel (personal communication) obtained inhibition of methanogenesis of anaerobic digestion by methane analogues, while hydrogen was concomitantly produced.

The pathway of methane formation. The pathway(s) of methane formation is still rather uncertain. BARKER (1956) proposed a general scheme for methane formation involving a hypothetical one-carbon carrier. He postulated that carbon dioxide com- bined with an unidentified organic compound (XJ-I) to form a carboxylated derivative of X. This derivative of X was considered to be reduced by successive steps to a methyl derivative of .V, which, on further reduction, yielded methane and the regenerated carbon dioxide acceptor (XH). Methanol and acetate were postulated to react with XH yielding the methyl derivative of X present in the carbon dioxide reduction pathway.

408 D.F. TOERI~N and W. H. J. HATTINGH

PINE and V IS~C (1957) proposed the following scheme for the intermediary metabolism of acetate and methanol fermentations.

CH30H CH3C0 z H (exogenous) [

HCOzH =- =- AHz

+ HzO

I coz r

i

b i

I

i

cH3x + .~ I

I cH~ / . . . . j

C0z COz

Since 1963 several reports on the formation of methane in cell-free extracts of methanogcgJc bacteria have appeared. WOI.IN et al. (1963a) demonstrated that precursors of methane formation by cell-free extracts of Methanobacterium omelianskff included carbon dioxide, pyruvate and serine. BLAYLOCK and STADTMAN (1963, 1964a) showed that the methyl moiety of methylcobalamin served as precursor of methane in cell-free extracts of Methanosarcina barkerii. Cell-free extracts of Methanobacterium omelianskii formed methane stoichiometdcally from methylcobalamin and this reaction was dependent on ATP (WOLIN et al., 1963b). The formation of methane in cell-free extracts of M. omelianskii proceeded in a phosphate buffer (pH 7.0) when hydrogen, carbon dioxide, coenzyme A and ATP were components of the reaction mixture. Pyruvate, serine or O-phosphoserine could substitute for ATP and carbon dioxide.

WOLIN et al. (1964) presented evidence that the cobalamin product formed in extracts of M. omelianskii had the spectral properties of vitamin B12 , and could be alkylated with methyl iodide. These studies suggested that methylcobalamin might be an intermediate in the formation of methane from carbon dioxide, pyruvate and serine.

WOOD and WOLFE (1965) suggested that 5N-methyl tetrahydrofolate was an important intermediate in the formation of methane from carbon dioxide, pyruvate or serine and that methylcobalamin might not be an obligatory intermediate. WOOD et al. (1965) showed the presence and discussed the importance of L-serinetranshydroxy-. methylase which must be one of the key enzymes in C1 transfer leading to methane:

+ ATP -- ATP

CH4 ~ H 4- foto le X HCOOH

L- serin /H 4 - folole ,~

N - formyl-H4-folote

x grycine f 5,10-CHa- Ha- folo~e )


formation. They postulated the following routes for C1 transfer in the presence and absence of ATP. Methylcobalamin seemed not to be a true intermediate for methane formation in M. omelianskii (WOOD et al., 1965). The participation of methylcobalamin as an actual intermediate of methanogenesis in Methanosarcina barkerii was not established by BLAYLOCK and STADTMAN (1964b) but evidence was obtained that some type of cobamide derivative was functional.

Inhibition of an enzyme in the methane-forming system of M. omelianskii was obtained through alkylation (WOOD and WOLFE, 1966a). WOOD et al. (1966) studied the formation of methane in cell-free extracts of M. omelianskii from methyl factor B and methyl factor IIL The methyl-cobalt ligands of both substrates were approximately as active as that of methylcobalamin. ATP was required for the formation of methane from these ligands. WOOD et al. (1966) postulated that the lack of specificity of methyl-Bl~ analogs as methyl donors in biological systems rested in their ability to chemically alkylate reduced corrinoid-containing enzymes. LEZIUS and BARKER (1965) reported on the presence of corrinoid compounds in Methanobacterium omelianskff.

WOOD and WOLFE (1966b) demonstrated a requirement for co-factors after partial purification of the methane-forming system in ceil-free extracts of M. omelianskii. They found that two protein fractions, ATP, Mg 2+ and a reduced ravin adenine dinucleotide-generating system were necessary for optimal conditions of the formation of methane by ceil-free extracts.

STADTMAN and BLAYLOCK (1966) discussed the role of B~2 compounds in methanogenesis and considered that several lines of evidence suggested a direct participation of B:2 compounds in methane biosynthesis. Tetrahydrofolate also appears to be implicated in methane formation and may function, at least partially, as the unknown C1 carrier (X) of Barker's scheme (GAUDY and GAUDY, 1966).

By the extension of these enzymatic studies on cell-free extracts of methanogenic bacteria the sequence of reactions in methanogenesis still remains to be determined.

PERSPECTIVES AND HORIZONS

Anaerobic digestion is a biological process which is extensively used for the stabilization of potentially polluting wastes. This fact should never be neglected by the microbiologist who should seek to contribute information which could improve the practical applications of anaerobic digestion. For this purpose, quantitative and not qualitative ecological information is likely to be the most valuable, as previously suggested by PIPES (1966), for activated sludge. This approach to the study of microbial ecosystems is based on principles such as van Niel's modifications of Koch's postulates (GIBsoN, 1957) namely:

I. An organism must always be present when a relevant chemical process occurs 2. The organism must be cultivated in pure culture 3. The chemical change should follow when a suitable culture medium is inoculated

with a pure culture of this organism 4. Re-isolation of the organism from the culture liquid should be possible at the end

of growth.

HUNGATE (1960, 1962) provides an excellent discussion of the ecology of bacteria and the problems related to ecological studies. His requirements and those of Kistner

410 D.F. TOERIEN and W. H. J. HATTn~GH

(personal communication) for a quantitative ecological study of microbial ecosystem can be summarized as follows:

1. Analyses of the ecosystem (a) knowledge of the nature of the system (chemical, biochemical etc.) (b) knowledge of the kinds and numbers of microorganisms occurring in the

system (c) determination of the physiological activities of the microorganisms

2. Knowledge of the factors which determine a specific equilibrium between kinds of bacteria (a) competition for limiting substrates (b) synergism and antagonism.

The above-mentioned principles should be employed in bacteriological studies in anaerobic digestion.

When microbiological studies on anaerobic digestion are undertaken on a quantitative ecological basis, these studies would serve to supplement kinetic studies. WUHR- MAN (1964) stated that, "the application of the mathematical approach to the descrip- tion of mixed fermentation systems stands or falls upon detailed knowledge on the ecology and physiology of the organisms involved". The kinetic studies of STEWART (1958); SPEV.CE and MCCARTY (1964); AGARDY et al. (1963); ANDREWS et al. (1964); and LAWRENCE and MCCARTY (1967) on various aspects of anaerobic digestion must be supplemented with quantitative ecological studies to obtain a complete understanding of the whole process of anaerobic digestion.

Many microbiological aspects of both the non-methanogenic and methanogenic phases must still be investigated. In the non-methanogenic phase the predominant bacterial population seems to be obligate anaerobic, but little information on the kinds of bacteria, their numbers, growth, nutritional requirements, metabolism, factors which influence their metabolism, synergistic effects etc. has been obtained as yet. This knowledge is necessary for a full understanding of the non-methanogenic phase.

The presence of a hydrogen-producing bacterial population has been postulated. It is absolutely necessary that this postulate should be examined and if such a population is found, that its characteristics should be determined on a quantitative ecological basis.

The quantitative ecological studies on methanogenic bacteria of SMXrH (1965, 1966) should be confirmed and extended for the digestion of other substrates. The role of individual volatile fatty acids and alcohols in methanogenesis during digestion, as determined by McCARTY et al. (1962, 1963) and SMITH and MAH (1966), should be confirmed and extended. It should be determined whether methanogenic bacteria are able to utilize substrates other than formate, acetate, methyl alcohol and carbon dioxide and hydrogen. The nutritional requirements of the sludge methanogenic bacteria, their growth characteristics, steady state requirements etc. should also be determined.

Anaerobic digestion presents the microbiologist with ample opportunity for the study of obligate anaerobic bacteria in particular. The study of kinds of bacteria, their numbers, growth, nutrition, metabolism and factors that influence it, in relation to anaerobic digestion, is a challenging and stimulating problem that depends upon the microbiologist for its resolution.

Anaerobic Digestion--I. The Microbiology of Anaerobic Digestion 41I

CONCLUSIONS

1. Many aspects of the microbiology of anaerobic digestion still remain to be investigated.

2. Such studies are essential for a full understanding of the whole process and for its useful applications.

3. Anaerobic digestion presents microbiologists with ample opportunity for the study of complex microbial populations.

Acknowledgements--The encouragement, suggestions and criticisms of Dr. G. J. STANDER, Dr. W. A. PRETOVaUS, Mr. J. HEMENS and our other colleagues are greatfully acknowledged.

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anaerobic digestion i. the microbiology of anaerobic digestion

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