ABSTRACT
Hungate, 1958; Hungate, 1950; Zeikus, 1979; Gunsalus and Wolfe, 1977). Barker was first able to obtain highly enriched cultures of several methane-producing strains which were not an axenic culture (Barker, 1956). It was further inferred that the methane bacteria could utilize a specific number of low-molecular-weight end products of metabolism, such as formate, ethanol, acetate, propionate, and butyrate, obtained by fermentation, besides hydrogen (Gunsalus and Wolfe, 1977). However, the isolation of a pure strain of methane producers was reported by Smith and Hungate (1958). Pohland and Ghosh (1974) demonstrated anaerobic digestion as a two-stage process, namely acid formation and methane generation. Two physiologically different digesting organisms, acid formers (acidogens) and methane formers (methanogens), are involved in this process (Pohland and Ghosh, 1970; 1971; 1974). Acidogens consist of a number of organisms which can convert proteins, carbohydrates, and lipids mainly to a volatile fatty acid (VFA) by hydrolysis and fermentation (Pohland and Ghosh, 1974). These bacteria are Ruminococcus albus, R. flavefaciens, Butvrivibrio fibrisolvens, Bacteriodes succinogens, Cellobacterium cellulosolvens, Eubacterium ruminantium, B. amylophilus, B. ruminicola, Anaerovibrio lipolytica, etc. (Smith and Hungate, 1958; Hungate, 1950; Zeikus, 1979; Gunsalus and Wolfe, 1977). Populations of 108-109 hydrolytic bacteria per milliliter of mesophilic sewage sludge have been reported by several investigators (Gunsalus and Wolfe, 1977). Studies on hydrolytic bacteria that utilize specific carbon substrates demonstrated 107 proteolytic and 105 celluloytic bacteria per milliliter of sewage sludge (Das, 1985). The end products of the metabolism of acidogens are convert-ed to methane and carbon dioxide in the presence of methano-gens, which are obligate anaerobes. Different species of methaneproducing bacteria were identified. They consisted of Methanobacterium omelianskii, Methanobacterium bryanti, Methanobacterium formicicum, Methanobacterium thermoautotrophicum, Methanobrevibacter arboriphilus, Methanobrevibacter ruminantium, Methanobrevibacter smithi, Methanobrevibacter vannielli, Methanobacterium mobile, Methanoqenium cariaci, Methanospirillum hungatei, Methanosarcina barkeri, Metnanococcus
mazei, Methanobacterium sohuqenii, Methanobacterium suboxydans, Methanobacterium propionicum, Methanococcus thermolithotrophicus, etc. (Hungate, 1950; Zeikus, 1979; Gunsalus and Wolfe, 1977). Various studies indicate that methanogenic bacteria are present in anaerobic digestion in a number of 106 to 108 per milliliter (Das, 1985). The characteristics of methanogenic bacteria are given in Table 5.1. Methanosarcina barkeri and Methanococcus mazei are of special
interest due to their versatility with respect to the utilization of various substrates (Das, 1985; Sahm, 1984). Methanogenic bacteria are strict anaerobes. They require a lower redox potential (–330 mV, which corresponds to a concentration of 1 molecule of oxygen in about 1056 L of water) for growth compared to most other anaerobic bacteria. Oxygen is a potent inhibitor of methanogenesis. These bacteria can be gram-positive or gram-negative, and they have quite different cell shapes (Table 5.1). Balch et al. proposed a new taxonomy for 13 species of methanogens (Balch et al., 1979). Later several new strains were isolated. The more interesting thermophilic strains are Methanosarcina species, Methanobacterium soehngenii, Methanococcus mazei, and Methanothermus fervidus (Bryant, 1963; Dehority, 1971). Among these methanogens, Methanothermus fervidus is able to grow near the boiling point of water. Methanogenesis also occurs in nature at 0°C, but most pure strains of methanogens have optimum growth around 40°C (mesophiles) and at 65° to 75°C (thermophiles) (Colleran et al., 1982). 5.3 BiochemistryThe biochemistry of anaerobic digestion has been studied on sewage, farm animal wastes, or small-scale digestions using artificial mixtures of feed constituents, for example, cellulose, hemicellulose, protein, lipid, and lignin (Hungate and Stack, 1982). Carbohydrates play a major role in digester reactions and their breakdown. It is an important rate-controlling step in the digestion (Das, 1985). These carbohydrates are mainly cellulose and hemicellulose. Cellulose degradation takes place in the presence of extracellular enzymes produced by cellulolytic bacteria. These bacteria differ from each other in the relative amounts of cellulases (endoglucanase and exoglucanase) and the ability to attack different forms of cellulose (Hobson, 1976). The absolute rate at which cellulose is attacked depends on its physical form, for example, the degradation of domestic sewage is more compared to agricultural residues (Zeikus, 1980). Lignin, which is in complex association with the cellulosic structure, acts as a barrier to bacterial attack on the cellulose
molecule (Stadtman, 1967). It prevents cellulolytic bacteria from adhering to the plant fibers. This is a prerequisite for optimum bacterial attack. Lignin was also shown to be almost entirely undegraded in anaerobic digesters (Bryant et al., 1967). However, a number of compounds of the same types which make up the lignin polymer are themselves degraded to methane and carbon dioxide by the mixture of bacteria from a domestic sewage sludge digester (Kluyver and Schnellen, 1947). The hemicellulose in the digester does not require the complex enzyme system of the cellulases for its hydrolysis. Hemicellulase was found to degrade hemicellulose (Das, 1985). The primary metabolic products of cellulolytic bacteria include aliphatic acids (formic, acetic, propionic, butyric, valeric, etc.), lactic acid, succinic acid, ethanol, carbon dioxide, and hydrogen (Das, 1985). Lactic and succinic acids are rapidly fermented to acetic and propionic acids. The primary breakdown of sugars in fermentation is usually to pyruvic acid with the simultaneous liberation of hydrogen in the form of hydrogen carrier complex. The degradation of proteins proceeds via extracellular hydrolysis of proteins into peptides and amino acids by protease. Several mechanisms are involved in the degradation of amino acids in different bacteria (Hobson et al., 1981). Organic acids and ammonia are the end products of protein hydrolysis. The presence of extracellular proteases was found by several researchers (Hungate and Stack, 1982). The degradation of lipids in anaerobic digestion proceeds through the initial breakdown of fats by lipase. The long fatty acids are degraded by β-oxidation, as shown with the 14C tracer, using octanoic and palmitic acids (Das, 1985). The biochemical qualities of methanogenic bacteria are different from other bacteria in the following manner: ∑ A very restricted range of oxidizable substrates coupled to the biosynthesis of methane ∑ Synthesis of an unusual range of cell wall components ∑ Synthesis of biphytanyl glycerol ethers as well as high amounts of squalene ∑ Synthesis of unusual coenzymes and growth factors
∑ Synthesis of rRNA that is distantly related to that of typical bacteria ∑ Possession of a genome size (DNA) approaching 1/3 that of
Escherichia coli Barker and coworkers (Barker, 1956; Das, 1985; Hobson et al., 1981) established the mechanism of methane formation. They showed that carbon dioxide, methanol, or acetate can be used as a precursor of methane by methanogenic bacteria (Fig. 5.1). It was further proved that the methyl group of acetate or methanol was transferred intact and was reduced, accepting one proton from the medium (Wolfe, 1982). The acetate system has been studied extensively as a major substrate of methanogens compared to hydrogen and carbon dioxide (and formate) (Wolfe, 1982).
