Anaerobic digestion I. The microbiology of anaerobic digestion

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<ul><li><p>Water Research Pergamon Press 1969. Vol. 3, pp. 385-416. Printed in Great Britain. </p><p>REVIEW PAPER </p><p>ANAEROBIC D IGEST ION </p><p>I. THE MICROBIOLOGY OF ANAEROBIC D IGEST ION </p><p>D. F. TOERIEN and W. H. J. HATTINGH </p><p>National Institute for Water Research, South African Council for Scientific and Industrial Research, P.O. Box 395, Pretoria, South Africa </p><p>(Received 20 November 1967) </p><p>INTRODUCTION </p><p>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. </p><p>Despite widespread use of anaerobic digestion of waste organic matter, the funda- mental 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". </p><p>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. </p><p>MICROBIOLOGICAL NATURE OF ANAEROBIC DIGESTION </p><p>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. </p><p>The terms "acid-formation" and "gas-formation" for the two stages of anaerobic </p><p>385 </p></li><li><p>386 D. F. TOERmN and W. H. J. HAa'r~Grl </p><p>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. </p><p>MICROBIOLOGY OF THE NON-METHANOGENIC PHASE </p><p>Kinds of microorganisms Not very much is known regarding the microbiology of the non-methanogenic phase </p><p>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). </p><p>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 </p><p>Comp~x organ~ molecu~| </p><p>Cellular material Non- methonogenic bacteria </p><p>Cellular material methono~llc bacteria </p><p>Methane and carbon dioxide </p><p>FIG. I. Schematic representation of anaerobic digestion. </p><p>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. </p><p>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. </p><p>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 </p></li><li><p>Anaerobic Digestion--I. The Microbiology of Anaerobic Digestion 387 </p><p>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. </p><p>TABLE 1. NON-METHANO</p></li><li><p>388 D.F. TOEa~N and W. H. J. H~TrnXGH </p><p>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. </p><p>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. </p><p>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. </p><p>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. </p><p>TOE~EN (1967a,b) isolated a wide variety of aerobic and facultative anaerobic bacteria from anaerobic digesters receiving raw sludge by direct isolation and enrich- ment culture techniques. These isolates included aerobic and facultative anaerobic bacteria and one photosynthetic bacterium (TABLE 1). </p><p>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. </p><p>Recently, considerable numbers of Bacteroides and related obligate anaerobic bacteria were found to occur in anaerobic digesters (Post et aL, 1967). </p><p>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 </p></li><li><p>Anaerobic Digestion--I. The Microbiology of Anaerobic Digestion 389 </p><p>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. </p><p>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 </p><p>TABLE 2. GENERA OF FUNGI WHICH HAVE BEEN DETECTED IN DIGESTING OR DRYING SLUDGES* </p><p>Phycomycetes </p><p>Mucor Rhizopus Syncephalastrum Zygorhynchus </p><p>Ascomycetes </p><p>Allescheria Ascophanus Eurotium Pseudoplea Sartoria Aspergillus Subbaromyces Talaromyces Thielavia </p><p>Fungi Imperfecti </p><p>Acremonium Aspergillus </p><p>f </p><p>Fungi Imperfecti (cont) </p><p>Penicillium Cephalosporium Geotrichum Gliocladium Paecilomyces Scopulariopsis Sepedonium Spicaria Trichoderma </p><p>* Trichothecium Alternaria Cladosporium harargarinomyces Memnoniella Humicola Phialophora Pullularia Stachybotrys Epicoccum Fusarium Myrothecium </p><p>* According to COOKE (1957, 1963) </p><p>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). </p><p>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. </p><p>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 </p></li><li><p>390 D.F. TOERIEN and W. H. J. HATTINGH </p><p>TABLE 3. MAGNITUDES OF NUMBERS OF NON=METHANOGENIC BACTERIA ENUMERATED DURING THE YEARS 1900-1930 </p><p>Bacterial group Numbers x 10 a ml Reference </p><p>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 </p><p>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) </p><p>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, </p><p>TABLE 4. MAGNITUDES OF NON-METHANOGENIC BACTERIA ENUMERATED SINCE 1930 </p><p>Bacterial group Feed of digester Numbers x 103 ml References </p><p>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. </p><p>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) </p></li><li><p>Anaerobic Digestion--I. The Microbiology of Anaerobic Digestion 391 </p><p>including yeasts and molds, might be taking part in the digestion process to the extent of obta...</p></li></ul>