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Kenealy et al. (20)

Fibrobacter succinogenes

Fibrobacter succinogenes step in the anaerobic breakdown of organic matter under sulfate-limiting conditions. The substrate range for meth-anogenesis is limited to carbon dioxide, formate, carbon monoxide, methanol, methylamines, and acetate, but no single isolate is able to utilize all these carbon sources (21). The metabolic pathways of methane formation are unique and involve a number of enzymes and coenzymes that occur only in methanogens. A primary Na+ gradient and H+ gradients are formed by interesting and unique membrane-bound enzymes, and the reactions by which these ion-motive forces are generated are also novel (22). The organisms and the methanogenic reactions (21,23), and the pathways of energy conservation, have been extensively reviewed (22).

In recent years, bioconversion of agricultural and industrial wastes to methane has been considered important from two perspectives, as a waste-disposal method for residual organic matter and as a source of energy. Anaerobic digestors for treatment of sewage, agricultural and animal waste, and industrial effluent are being used worldwide. More recently, methanogenic microbial communities have been found to be very efficient in treating toxic organic wastes. For example, transformation of C1 and C2 halocar-bons was observed in the early 1980s and, as one of the mechanisms responsible for these transformations, was proposed as a biologically mediated reductive dechlorina-

tion (24-26). Anaerobic dechlorination and degradation of chlorinated aromatic compounds such as chlorophenols, chlorobenzenes, and polychlorinated biphenyls (PCBs) occurs in a wide variety of environments. Methanogenic mi-crobial consortia that dechlorinate tetrachloroethylenes (PCE) and trichloroethylenes (TCE), and dechlorinate, degrade, and mineralize chlorophenols and PCBs have been developed (27-31). The potential of these consortia in bio-remediating the contaminated soil and sediment systems is being examined. DDE [1,1-dichloro-2,2-bis(p-chloro-phenyl)ethane], a metabolite of the pesticide DDT, has been observed to reductively dechlorinate to DDMU at a relatively high rate under methanogenic conditions as compared to sulfidogenic conditions (32).

The catabolic reactions carried out by methanogens yield very little energy compared to those of aerobes. For example, use of H2 to reduce CO2 to CH4 has a AG0' of — 34 kJ/mol H2, whereas the corresponding energy value for an aerobe oxidizing H2 using O2 as an electron acceptors is — 237 kJ/mol H2. Most of the known species of methanogens can use H2 to reduce CO2 to CH4. Many of the hydro-genotrophic methanogens also utilize formate. In contrast, only species belonging to the genera Methanosarcina and Methanosaeta use acetate (acetotrophic) and convert it to methane. Acetate is an important end product of many fermentative anaerobes, and is a primary methanogenic sub strate in anaerobic digestors. The methylotrophic meth-anogens can utilize several simple methylated compounds. These methanogens include Methanosarcina, which can also use acetate and usually H2—CO2, and Methanolobus and Methanococcoides, which are only known to grow on methylated compounds. Carbon monoxide is also converted to methane by methanogenic species. Methanoge-nesis has been observed at low, moderate, and high temperatures, and in neutral to mildly acidic or alkaline conditions. In summary, methane is produced under a variety of environmental conditions and is considered a byproduct of the anaerobic treatment of industrial wastes.

Solventogenesis. Ethanol production is a very well-known fermentation process since it has been an outgrowth of the alcoholic beverages industry. During World War I, shortages of acetone in the manufacture of munitions led to the development of an acetone-butanol-ethanol process involving the fermentation of starch by Clostridium acetobutylicum. This fermentation gave approximately 2 parts butanol to 1 part acetone and 1 part ethanol. During the 1940s and 1950s, lower costs of petrochemical processes stopped butanol production by fermentation routes in the U.S. and Europe. The process was also stopped in South Africa, but continued in China into the early 1990s.

The present market for ethanol is met by yeast fermentation, but the industrial market for isopropanol, acetone, and butanol could be met by other anaerobic fermentations. Biomass-derived solvents produced by fermentation can enter into the current petrochemical synthetic pathways through a number of reactions. The most important of these is the dehydration of alkanols to alkene to form ethylene, propylene, butylene, and butadiene. Therefore, production of chemical feedstocks from biomass via fermentation is becoming increasingly attractive because biomass production costs are not as tightly bound to energy costs.

A great diversity exists among the solvent-producing microorganisms. These organisms span several groups of yeasts and a broad range of both mesophilic and thermo-philic, and aerobic and anaerobic, bacteria. By far, the ethanol-producing organisms are the most abundant sol-ventogens. However, industrial use is mainly limited to strains of yeasts, but great potential exists for recombinant Zymomonas mobilis. Several clostridia are capable of producing butanol and either acetone or isopropanol with eth-anol as a byproduct. The C. acetobutylicum and Clostridium beijerinckii strains are of prime importance for these industrial fermentations.

Clostridium thermocellum, Thermoanaerobacter ethan-olicus, Clostridium thermosaccharolyticum, Thermoan-aerobacterium thermosulfurigenes, Thermoanaerobacter brockii, and Thermobacteroides acetoethylicus, use a wide range of substrates, from polymeric carbohydrates such as cellulose, pectin, xylan, and starch, to mono- and disaccha-rides such as glucose, cellobiose, xylose, and xylobiose. The primary fermentation product is ethanol, with the production of acetate, lactate, carbon dioxide, and hydrogen in various ratios. T. acetoethylicus has not been shown to produce lactic acid. At present, one of the major limitations in the use of thermoanaerobes is the variability of end-product ratios, yield, and low ethanol concentration. These are affected by species, enzyme complement, and environmental conditions. Certain strains of T. ethanolicus have the best conversion of carbohydrates to ethanol, forming 1.6-1.9 mol/mol of glucose fermented (33). High ethanol and hydrogen concentrations also reduce the yield of eth-anol (33) owing to the flexibility of the carbon and electron pathways, which may possess many reversible enzyme systems (33,34).

In addition to ethanol, several saccharolytic clostridia produce butanol and acetone besides volatile fatty acids and gaseous products from carbohydrate fermentation. In some cases acetone is further reduced to isopropanol. In most species, the production of solvents only occurs late in the fermentation cycle, following a shift from the pathways leading to acetate and butyrate production. The production of butanol and ethanol is usually associated with the uptake and reutilization of acids, and the production of acetone or butanol. The ability to produce solvents is influenced by the type and concentration of substrate, the pH and the buffering capacity of the culture medium, and the environmental conditions. Several strains that have the ability to produce solvents undergo degenerative changes, resulting in the loss of their ability to produce solvents. The solvent-producing clostridia are found in a wide variety of natural habitats (35); however, the strains of C. acetobutylicum and Clostridium beijerinckii are the most frequently studied species. C. acetobutylicum, which characteristically produces butanol, acetone, and ethanol in the ratio of 6:3:1, has been used extensively for the industrial production of solvents. Most butanol producers are mesophiles with a temperature optima for fermentation between 30 and 37 °C. Recently, solvent yield was shown to increase at lower temperatures and acetone but not butanol yield to decrease at elevated temperatures (36; B.K. Soni and M.K. Jain, unpublished data 1991).

At present, many ethanol- and butanol-producing strains of Clostridium remain poorly classified, and there is still no accepted standard classification for the clostridia group as a whole. Strains of C. beijerinckii (formerly classified as C. butylicum) constitute a second group of solvent producers that do not show DNA homology with the C. acetobutylicum group. This group contains both high- and low-solvent-producing strains that produce either acetone or isopropanol in addition to butanol. The strains of Clostridium aurantibutyricum group also include butanol-producing strains that produce both acetone and isopro-panol (37). Clostridium tetanomorphum produces butanol and ethanol, but not acetone or isopropanol. As obligate anaerobes, butanol producers require anaerobic conditions. However, vegetative cells of C. acetobutylicum, for example, survived several hours exposure to oxygen and formed fruiting-body-like structures on agar plates when exposed to oxygen (38).

Acetogenesis. The term acetogen is commonly applied to a bacterium that forms acetate, whether the acetate is produced by a catabolic process such as fermentation or by an autotrophic-type synthesis. Acetogenic bacteria are ubiquitous in anaerobic ecological systems. Homoaceto-

gens have the ability to grow well on H2 plus CO2 and poorly on CO, and to form acetyl CoA by a CO-dependent pathway involving CO dehydrogenase (39). Clostridium thermoaceticum and Clostridium thermoautotrophicum are homoacetogens and synthesize acetate from C1 compounds by the recently established Wood pathway of acetyl CoA synthesis (40). This pathway was established by studies with C. thermoaceticum that can grow on CO or CO2— H2, utilizing them as carbon and energy sources (41).

In industrial fermentations, the formation of a single product is advantageous because its recovery would be simplified. In this respect, homoacetogenic bacteria essentially form only acetate in the fermentations of hexoses and pentoses. They appear to be ideal for the microbial production of acetate. Energy for cell growth is obtained by the reduction of CO2 to acetate via the Wood pathway. The homoacetogens also grow on other one-carbon compounds, including CO, formate, and methanol with acetate as the product. In the acetyl CoA pathway for synthesis of acetate, acetyl CoA is the first two-carbon intermediate of the autotrophic fixation of CO2, and is either used for the synthesis of cell carbon or converted to acetate.

Butyribacterium methylotrophicum is an acetogenic anaerobe that can grow on multicarbon compounds as well as on one-carbon compounds (e.g., CO or methanol). It produces acetate or butyrate using the Wood pathway, but ace-tyl CoA can be condensed and reduced to butyrate. Higher levels of NADH were found in butyrate-producing than in acetate-producing cells. In B. methylotrophicum, butyrate production is regulated by the carbon source and is dependent on cellular NADH/NAD ratios, and the levels and direction of ferredoxin- and NAD-linked oxidoreductases

(42). Also, the growth pH regulates both hydrogenase and FD-NAD oxidoreductase activities such that, at acid pH, more intermediary electron flow was directed towards bu-tyrate synthesis than H2 production.

Acidogenesis. The acidogenic bacteria include those bacteria that only form acetate by fermentation. These bacteria invariably produce other acids in addition to acetate. The acidogenic bacteria grow heterotrophically on a variety of carbohydrates, producing a mixture of acids such as lactic, propionic, butyric, succinic, and caproic. The pathways for production of these acids have been reviewed

(43). A generalized scheme for anaerobic production of fermentative organic acids and alcohols is presented in Figure 1. The acidogens differ from the acetogens in that they do not synthesize acetate from CO2 or other C1 compounds by the Wood autotrophic pathway. Many of the species can utilize hexoses such as glucose, fructose, galactose, and mannose; disaccharides including cellobiose, lactose, and maltose; pentoses represented by xylose; and other substrates such as glycerol, mannitol, sorbitol, inositol, and polymeric carbohydrates. For example, Clostridium butyr-icum, the type strain of the genus Clostridium, is sacchar-olytic in nature and metabolizes glucose, producing butyrate, acetate, CO2, and H2 as fermentation products. Glucose is converted to pyruvate by the Embden-Meyerhof-Parnas glycolytic pathway. Pyruvate is then simultaneously decarboxylated and oxidized by the enzyme complex pyruvate-ferredoxin oxidoreductase to yield ace-

tyl CoA, CO2, and reduced ferredoxin. The reduced ferre-doxin is reoxidized in several reactions, the most important of which involves H2 evolution catalyzed by hydrogenase. Acetyl CoA is condensed and reduced to bu-tyrate.

In some other acidogens, phosphoenolpyruvate (PEP) serves as a branching point in the pathway producing py-ruvate and oxaloacetate. The PEP-oxaloacetate step involves CO2 fixation mediated by PEP carboxykinase. Depending upon the species, products such as succinate and propionate are produced as fermentation products with acetate, formate, or H2. For example, oxaloacetate is converted to propionate in species like Propionispira arboris

(44), whereas, oxaloacetate is further converted to succinate in species like Anaerobiospirillum succiniciproducens

(45). Lactic acid bacteria such as Lactobacillus acidophilus convert pyruvate to lactic acid, whereas Clostridium klu-vyri produces caproic acid from ethanol and acetate (43).

Sulfidogenesis. Sulfate, a chemically inert, nonvolatile, and nontoxic compound, is widespread in rocks, soil, and water. In contrast, hydrogen sulfide, because of its chemical properties and physiological effects, is a far more conspicuous substance and is chemically reactive. The presence of black-colored sediments due to the formation of ferrous sulfide from iron-containing materials, accompanied by a distinctive smell, is indicative of H2S production. Hydrogen sulfide acts as a reductant, and is toxic to plants, animals, and humans. It is produced by reduction of sulfate by sulfate-reducing bacteria, which are present in ecological niches where oxygen has no access. In the absence of oxygen, the oxidized form of sulfur (SO2-) is used as an electron acceptor by sulfate-reducing bacteria (sulfate reducers). Since reduction of the inorganic compound serves for energy conservation, the process is distinguished as dis-similatory reduction from the assimilatory reduction of sulfate in plants and bacteria.

Under anaerobic, reduced conditions, hydrogen sulfide is the energetically stable form of sulfur, as sulfate is the stable form under aerobic conditions. The most important reducers of elemental sulfur in nature are probably special anaerobes that may be designated as true sulfur-reducing bacteria; these carry out the dissimilatory reduction of sulfur as their primary or even obligate metabolic reaction during oxidation of organic substrates. The true sulfur-reducing bacteria do not reduce sulfate, and only some reduce other oxygen-containing sulfur compounds. The majority of the eubacterial sulfur reducers are mesophilic, as are most sulfate reducers. In contrast, all described ar-chaebacterial sulfur reducers are hyperthermophiles with temperature optima (up to 110 °C) the highest known in the living world.

Sulfate-reducing bacteria include a rather heteroge-nous assemblage of microorganisms having in common merely dissimilatory sulfate metabolism and obligate an-aerobiosis. The genus Desulfovibrio includes species that are still the best-studied sulfate reducers. These are non-sporing, having curved motile cells and growing on a relatively limited range of organic substrates, preferably lac-tate or pyruvate, which are incompletely oxidized to acetate and H2S. Spore-forming sulfate-reducing bacteria with a similar metabolism were classified within the genus

Glucose Glycerol

Glucose Glycerol

Acetoacetyl CoA

Fumarate

Succinate

Crotonyl CoA

Succinyl CoA

Butyrl coa — Butyraldehyde-

Methylmalonyl CoA

Butyrate

Propionyl CoA - CoA

Propionate

Butanol

Figure 1. Generalized scheme for anaerobic fermentative production of common organic acids and alcohols.

Desulfotomaculum. Other types of sulfate-reducing bacteria differ markedly physiologically and morphologically from the known Desulfovibrio and Desulfotomaculum species. The reactions by which sulfate-reducing bacteria are involved in anaerobic degradation are indicated by their metabolic capacities. As far as tested, these bacteria use low molecular weight compounds as electron donors and therefore depend on fermentative bacteria that degrade the original polymers from biomass. Thus sulfate reducers are terminal degraders and their role is analogous to that of methanogenic bacteria that form methane and carbon dioxide as final anaerobic products. The recognition of dis-similatory sulfate reduction and methanogenesis as two alternative terminal carbon degradation processes has contributed a great deal to our understanding of anaerobic mineralization.

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