The microbial communities in anaerobic operations are primarily procaryotic. with members of both the Bacteria and the Archaea being involved. Although fungi and protozoa have been observed under some circumstances, the importance of eucary-otic organisms is questionable. N Thus, the emphasis here will be on the complex and important interactions between the Bacteria and the Archaea that are fundamental to the successful functioning of methanogenic communities. Because those intcrac-
tions occur in both suspended and attached growth systems, no distinctions will be made between the two.
General Nature of Anaerobic Operations. The multistep nature of anaerobic biochemical operations is depicted in Figure 2.3. Before insoluble organic materials can be consumed, they must be solubilized, just as was necessary in aerobic systems. Furthermore, large soluble organic molecules must be reduced in size to facilitate transport across the cell membrane. The reactions responsible for solubilization and size reduction are usually hydrolytic and are catalyzed by extracellular enzymes produced by bacteria. They are all grouped together as hydrolysis reactions (reaction 1) in Figure 2.3, but in reality many enzymes are involved, such as cellulases, amylases, and proteases. They are produced by the fermentative bacteria that are an important component of the second step, acidogenesis.
Acidogenesis is carried out by members of the domain Bacteria. Amino acids and sugars are degraded by fermentative reactions (reaction 2) in which organic compounds serve as both electron donors and acceptors. The principal products of reaction 2 are intermediary degradative products like propionic and butyric acids and the direct methane precursors, acetic acid and H:. The H: production from fermentative reactions is small and originates from the dehydrogenation of pyruvate by mechanisms that are different from the production of the bulk of the H produced.1'" In contrast, most of the H; produced comes from oxidation of volatile and long chain fatty acids to acetic acid (reactions 3 and 4) and arises from the transfer of electrons from reduced carriers directly to hydrogen ions, in a process called anaerobic oxidation."' Because of the thermodynamics of this reaction, it is inhibited by high partial pressures of H:, whereas the production of H: from pyruvate is not.
The production of H- by anaerobic oxidation is very important to the proper functioning of anaerobic processes. First, H, is one of the primary substrates from which methane is formed. Second, if no were formed, acidogenesis would not result in the oxidized product acetic acid being the major soluble organic product. Rather, the only reactions that could occur would be fermentative, in which electrons released during the oxidation of one organic compound are passed to another organic compound that serves as the electron acceptor, yielding a mixture of oxidized and reduced organic products. Consequently, the energy level of the soluble organic matter would not be changed significantly because all of the electrons originally present would still be in solution in organic form. When H, is formed as the reduced product, however, it can escape from the liquid phase because it is a gas, thereby causing a reduction in the energy content of the liquid. In actuality, the H: docs not escape. It is used as a substrate for methane production, but because methane is removed as a gas, the same thing is accomplished. Finally, if H; formation did not occur and reduced organic products were formed, they would accumulatc in the liquid because they cannot be used as substrates for methane production. Only acetic acid. H-, methanol, and methylamines can be used. As shown by reaction 5, some of the H: can be combined with carbon dioxide by H:-oxidizing acetogens to form acetic acid,*'' but since the acetic acid can serve as a substrate for methanogens, the impact of this reaction is thought to be small.
The products of the acidogenic reactions, acetic acid and H,, are used by methanogens, which are members of the domain Archaea, to produce methane gas. Two groups are involved: (1) aceticlastic methanogens, which split acetic acid into methane and carbon dioxide (reaction 6), and (2) H;-oxidizing methanogens. which reduce
carbon dioxide (reaction 7). It is generally accepted that about two-thirds of the methane produced in anaerobic digestion of primary sludge is derived from acetic acid, with the remainder coming from H2 and carbon dioxide."1 With the exception of the electrons incorporated into the cell material formed, almost all of the energy removed from the liquid being treated is recovered in the methane. Chemical oxygen demand (COD),"7 a common measure of pollutant strength, is a measure of the electrons available in an organic compound, expressed in terms of the amount of oxygen required to accept them when the compound is completely oxidized to carbon dioxide and water. One mole of methane requires two moles of oxygen to oxidize it to carbon dioxide and water. Consequently, each 16 grams of methane produced and lost to the atmosphere corresponds to the removal of 64 grams of COD from the liquid.4" At standard temperature and pressure, this corresponds to 0.34 nr of methane for each kg of COD stabilized.41
Microbial Groups and Their interactions. The hydrolytic and fermentative bacteria comprise a rather diverse group of facultative and obligately anaerobic Bacteria. Although facultative bacteria were originally thought to be dominant, evidence now indicates that the opposite is true,11 at least in sewage sludge digesters where the numbers of obligate anaerobes have been found to be over 100 times greater. This does not mean that facultative bacteria are unimportant, because their relative numbers can increase when the influent contains large numbers of them,"" or when the bioreactor is subjected to shock loads of easily fermentable substrates.1h Nevertheless, it does appear that most important hydrolytic and fermentative reactions are performed by strict anaerobes, such as Bacteroides, Clostridia, and Bifidobacteria although the nature of the substrate will determine the species present.
As mentioned previously, the role of H, as an electron sink is central to the production of acetic acid as the major end product of acidogenesis. Reactions leading from long chain fatty acids, volatile acids, amino acids, and carbohydrates to acetic acid and H: are thermodynamically unfavorable under standard conditions, having positive standard free energies.^ Thus, when the H. partial pressure is high, these reactions will not proceed and instead, fermentations occur, with the results discussed above. Under conditions in which the partial pressure of H: is 10 4 atmospheres or less, however, the reactions are favorable and can proceed, leading to end products (acetic acid and H:) that can be converted to methane. This means that the bacteria that produce H2 are obligately linked to the methanogens that use it. Only when the methanogens continually remove H: by forming methane will the H: partial pressure be kept low enough to allow production of acetic acid and H; as the end products of acidogenesis. Likewise, methanogens are obligately linked to the bacteria performing acidogenesis because the latter produce the substrates required by the former. Such a relationship between two microbial groups is called obligate syntrophy.
While the organisms responsible for the fermentative reactions are reasonably well characterized, less is known about the H2-producing acetogenic bacteria. This is due in part to the fact that the enzyme system for H: production is under very strict control by H2.hX As a consequence, early studies which attempted to enumerate the H2-forming bacteria underestimated them by allowing H, to accumulate during testing. However, because H: partial pressures are kept low in anaerobic biochemical operations,'1 H2-forming bacteria play an important role, and thus they have been the subject of more intensive research in recent years. Several species have been identified and studied, including members of the genus Syntrophomonas. which oxidize fatty acids, and the genus Syntrophohacler, which oxidize propionate.
As mentioned previously, the major nuisance organisms in anaerobic operations are the sulfate-reducing bacteria, which can be a problem when the wastewater contains significant concentrations of sulfate. Sulfate-reducing bacteria are all obligate anaerobes of the domain Bacteria. They are morphologically diverse, but share the common characteristic of being able to use sulfate as an electron acceptor. Group I sulfate reducers can use a diverse array of organic compounds as their electron donor, oxidizing them to acetate and reducing sulfate to sulfide. A common genus found in anaerobic biochemical operations is Desulfovibrio. Group II sulfate reducers specialize in the oxidation of fatty acids, particularly acetate, to carbon dioxide, while reducing sulfate to sulfide. An important genus in this group is Desulfobacter.
The H,-oxidizing methanogens are classified into three orders within the domain Archaea: (1) Methanobacteriales, (2) Methanococcales, and (3) Melhanomi-crobiiiles. ' A wide variety of these microorganisms have been cultured from anaerobic digesters, including the genera Methanobrevibuctcr and Methanobacterium from the first order, and the genera Methanospirillum and Methanogeriium from the third/ They are all strictly obligate anaerobes which obtain their energy primarily from the oxidation of H, and their carbon from carbon dioxide. Because of this autotrophic mode of life, the amount of cell material synthesized per unit of H used is low. During their metabolism they also use carbon dioxide as the terminal electron acceptor, 1 forming methane gas in the process.
Their range of electron donors is very restricted, usually being limited to H. and formate."' In some cases, short chain alcohols can also be used.'
In spite of the importance of the aceticlastic route to methane (reaction 6). fewer aceticlastic methanogens have been cultured and identified. All are of the order Methunosurcinak's. which contains two families, Methanosarcinuceae and Melhan-osactuceac.1 Methanosarctna. of the first family, can be cultivated from anaerobic operations'1 and is among the most versatile genera of methanogens known, being able to use H, and carbon dioxide, methanol, methylamines, and acetic acid as substrates."'When acetic acid is the substrate, it is cleaved, with all of the methyl carbon ending up as methane and all of the carboxyl carbon as carbon dioxide
Mi'thanosarcitm grows relatively rapidly at high acetic acid concentrations, although it is very sensitive to changes in that concentration. Furthermore, H. exerts a regulatory effect on acetic acid utilization, shutting it down as the H. partial pressure increases. The family Melhanosaetaceae contains a single genus, Methanosaeta (formerly Methunothrix), the members of which can use only acetic acid as their electron and carbon donor.' They grow much more slowly than Methanosarcina at high acetic-acid concentrations, but are not influenced as strongly by that concentration and can compete effectively when it is low. As a consequence, the manner in which an anaerobic operation is designed and operated will determine the predominant aceticlastic methanogen.
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