Microbiology of the Dairy Animal not appear to produce lactate, thus sequestering these granules from serving as substrates for bacterial lactate production Even though diets high in grain content are usually preferred for high producing cows because of their greater energy density, the presence of an ade quate level of fiber in the diet is important for several reasons(Van Soest, 1994) Fiber promotes the long-term health of the ruminant animal by providing a mod est rate of carbohydrate digestion and by stimulating rumination and salivation, all of which aid in maintaining ruminal pH within a range desirable for balanced microbial activity. Moreover, fiber in the diet helps the animal avoid milkfat depression, a syndrome resulting primarily from a relative deficiency in acetic acid(a precursor of short chain fatty acids in milk triglycerides) and a relative excess of propionate, which inhibits mobilization of body fat(a precursor of long chain fatty acids in milk triglycerides ). b Soluble Sugars and Oligomers Many ruminal carbohydrate-fermentin bacteria can utilize most of the different monosaccharides that comprise the vari ous plant polysaccharides(Hungate, 1966): D-glucose, D-xylose, D-galactose, L-arabinose, and D-or L-rhamnose. Many can also use at least some oligosac- charides that are released from the plant cytoplasm by cell wall breakage or that are produced by enzymatic hydrolysis of plant polysaccharides. The latter in- clude cellodextrins(Russell, 1985)and xylooligosaccharides(Cotta, 1993)hav ing seven or fewer glycosyl residues. Concentrations of soluble sugars and their oligomers are maintained at very low levels in the rumen; indicating that biopoly mer hydrolysis is the rate-limiting step in digestion and that competition for solu ble carbohydrates is probably an important determinant of species composition in the rumen(Russell and Baldwin, 1979a). In the few cases that have been systematically examined, sugar fermenters have shown dramatic changes in fermentation product ratios with changes in growth rate. Both S. bovis(Russell and Hino, 1985)and Selenomonas rumi nantium(Melville et al., 1988)carry out mixed acid fermentations at low growth rates but nearly homolactic fermentations at growth rates near their maxima. 3. Conversion of Fermentation Intermediate Compounds to Volatile Fatty Acids Microbial fermentation of both structural and nonstructural polysaccharides pro- duces a mixture of VFAs(usually acetic with some butyric)and other fermenta- tion acids(succinic, lactic, and formic) that are further metabolized by other ruminal microbes. Most of these bacteria require additional growth factors such as amino acids, peptides, and vitamins. Succinate is decarboxylated to propionate (see Fig. 5) by several ruminal species, including the metabolically versatile Sele- nomonas ruminantium and the metabolically specialized Succiniclasticum rum inis(van Gylswyk, 1995). Lactate is converted to propionate by several bacterial
Microbiology of the Dairy Animal 25 not appear to produce lactate, thus sequestering these granules from serving as substrates for bacterial lactate production. Even though diets high in grain content are usually preferred for highproducing cows because of their greater energy density, the presence of an adequate level of fiber in the diet is important for several reasons (Van Soest, 1994). Fiber promotes the long-term health of the ruminant animal by providing a modest rate of carbohydrate digestion and by stimulating rumination and salivation, all of which aid in maintaining ruminal pH within a range desirable for balanced microbial activity. Moreover, fiber in the diet helps the animal avoid milkfat depression, a syndrome resulting primarily from a relative deficiency in acetic acid (a precursor of short chain fatty acids in milk triglycerides) and a relative excess of propionate, which inhibits mobilization of body fat (a precursor of long chain fatty acids in milk triglycerides). b. Soluble Sugars and Oligomers Many ruminal carbohydrate-fermenting bacteria can utilize most of the different monosaccharides that comprise the various plant polysaccharides (Hungate, 1966): D-glucose, D-xylose, D-galactose, L-arabinose, and D- or L-rhamnose. Many can also use at least some oligosaccharides that are released from the plant cytoplasm by cell wall breakage or that are produced by enzymatic hydrolysis of plant polysaccharides. The latter include cellodextrins (Russell, 1985) and xylooligosaccharides (Cotta, 1993) having seven or fewer glycosyl residues. Concentrations of soluble sugars and their oligomers are maintained at very low levels in the rumen; indicating that biopolymer hydrolysis is the rate-limiting step in digestion and that competition for soluble carbohydrates is probably an important determinant of species composition in the rumen (Russell and Baldwin, 1979a). In the few cases that have been systematically examined, sugar fermenters have shown dramatic changes in fermentation product ratios with changes in growth rate. Both S. bovis (Russell and Hino, 1985) and Selenomonas ruminantium (Melville et al., 1988) carry out mixed acid fermentations at low growth rates but nearly homolactic fermentations at growth rates near their maxima. 3. Conversion of Fermentation Intermediate Compounds to Volatile Fatty Acids Microbial fermentation of both structural and nonstructural polysaccharides produces a mixture of VFAs (usually acetic with some butyric) and other fermentation acids (succinic, lactic, and formic) that are further metabolized by other ruminal microbes. Most of these bacteria require additional growth factors such as amino acids, peptides, and vitamins. Succinate is decarboxylated to propionate (see Fig. 5) by several ruminal species, including the metabolically versatile Selenomonas ruminantium and the metabolically specialized Succiniclasticum ruminis (van Gylswyk, 1995). Lactate is converted to propionate by several bacterial
Weimer species, particularly S ruminantium, Megasphaera elsdenii, Veillonella parvula Anaerovibrio lipolytica, and some Propionibacterium spp(Mackie and Heath, 1979). Formate is produced in abundance in the rumen both from carbohydrate fermentation and from reduction of carbon dioxide. Formate is rapidly turned over to methane and rarely accumulates(Hungate et al., 1970) 4. H2 Consumption and Interspecies Hydrogen Transfer Anaerobic metabolism requires that electrons(reducing equivalents) generated from biological oxidations be transferred to terminal electron acceptors other than oxygen. Most anaerobes that ferment carbohydrates dispose of these electrons by transfer to one or more organic intermediate compounds in the catabolic path way such as pyruvate(producing lactate), acetyl coenzyme A and acetaldehyde (producing ethanol), and carbon dioxide(producing formate)(see Fig. 5).An alternative electron acceptor is the protons present in all aqueous environments, resulting in production of hydrogen gas(H2). Disposal of electrons as H2 is partic- ularly advantageous in that it does not consume carbon-containing intermediate compounds that may be used as biosynthetic precursors. However, production of H2 is thermodynamically unfavorable unless its production is coupled to its continuous removal by Hr-consuming reactions. This spatial and temporal cou pling of H, production with H2 use, referred to as interspecies H, transfer, is one of the most important processes in the ecology of anaerobic habitats(Oremland, 1988; Wolin, 1990). Interspecies H2 transfer benefits both the H2 consumer, which directly receives its energy source, and the H] consumer, which can channel more of its substrate into the ATP-yielding production of acetate as a fermentation endproduct (Table 6) The dominant ha-consuming reaction in the rumen is the reduction of bon dioxide to methane gas 4H2+CO2→CH4+2HO This reaction is carried out by a specialized group of organisms, the methanogens These organisms are classified with the Archaea, a phylogenetically distinct group that represents an early evolutionary lineage distinct from both eubacteria (true bacteria) and eukaryotes(Woese and Olsen 1986). Methanogens are highly specialized metabolically. Most are restricted in their catabolism to reduction of carbon dioxide to methane using H, as an electron donor, whereas a few have the ability to convert one or more simple organic compounds(methanol, methyl- amine, formate, or acetate)to methane(Oremland, 1988). Methanol may be peri dically available in the rumen from deesterification of pectins. Formate, al though not a major ruminal fermentation product, is probably produced by carbon dioxide reduction in amounts sufficient to contribute slightly to ruminal methano- genesis. Acetate, although abundant in the rumen, does not support growth of
26 Weimer species, particularly S. ruminantium, Megasphaera elsdenii, Veillonella parvula, Anaerovibrio lipolytica, and some Propionibacterium spp. (Mackie and Heath, 1979). Formate is produced in abundance in the rumen both from carbohydrate fermentation and from reduction of carbon dioxide. Formate is rapidly turned over to methane and rarely accumulates (Hungate et al., 1970). 4. H2 Consumption and Interspecies Hydrogen Transfer Anaerobic metabolism requires that electrons (reducing equivalents) generated from biological oxidations be transferred to terminal electron acceptors other than oxygen. Most anaerobes that ferment carbohydrates dispose of these electrons by transfer to one or more organic intermediate compounds in the catabolic pathway such as pyruvate (producing lactate), acetyl coenzyme A and acetaldehyde (producing ethanol), and carbon dioxide (producing formate) (see Fig. 5). An alternative electron acceptor is the protons present in all aqueous environments, resulting in production of hydrogen gas (H2). Disposal of electrons as H2 is particularly advantageous in that it does not consume carbon-containing intermediate compounds that may be used as biosynthetic precursors. However, production of H2 is thermodynamically unfavorable unless its production is coupled to its continuous removal by H2-consuming reactions. This spatial and temporal coupling of H2 production with H2 use, referred to as interspecies H2 transfer, is one of the most important processes in the ecology of anaerobic habitats (Oremland, 1988; Wolin, 1990). Interspecies H2 transfer benefits both the H2 consumer, which directly receives its energy source, and the H2 consumer, which can channel more of its substrate into the ATP-yielding production of acetate as a fermentation endproduct (Table 6). The dominant H2-consuming reaction in the rumen is the reduction of carbon dioxide to methane gas: 4H2 CO2 → CH4 2H2O (1) This reaction is carried out by a specialized group of organisms, the methanogens. These organisms are classified with the Archaea, a phylogenetically distinct group that represents an early evolutionary lineage distinct from both eubacteria (true bacteria) and eukaryotes (Woese and Olsen 1986). Methanogens are highly specialized metabolically. Most are restricted in their catabolism to reduction of carbon dioxide to methane, using H2 as an electron donor, whereas a few have the ability to convert one or more simple organic compounds (methanol, methylamine, formate, or acetate) to methane (Oremland, 1988). Methanol may be periodically available in the rumen from deesterification of pectins. Formate, although not a major ruminal fermentation product, is probably produced by carbon dioxide reduction in amounts sufficient to contribute slightly to ruminal methanogenesis. Acetate, although abundant in the rumen, does not support growth of
Microbiology of the Dairy Animal Table 6 Fermentation Products from Cellulose in ruminococcus albus monocultures and r. albus/Methanobreveibacter smithii Cocultures Illustrating Changes Caused by terspecies Transfer of H, to the Methanogen mmol/100 mmol Glucose equivalents consumed R. albus Product R albus alone Ethanol 22 Formate 84854 151 H CH Mean values from continuous culture trials conducted at five different dilution rates Equivalent to 300 mmol H] consumed (at a stoichiome- try of 4 mol H, consumed per mol CH4 formed). Source: Paviostathis et al. 1990. aceticlastic" methanogens, whose growth rates even under ideal conditions are well below dilution rates of both liquids and solids in the rumen. Most methano gens are also autotrophs; that is, they can obtain all of their cell carbon from carbon dioxide. Thus, they can produce microbial protein for the ruminant host without consumption of otherwise useful organic matter. The energy associated with the reduction of the abundant ruminal carbon dioxide to methane is sufficient to permit both growth of the methanogens and thermodynamic displacement or"pulling"of the reduction of protons to H2. A: a result, the concentration of H2 in ruminal fluid is very low--normally near 1 UM with only occasional excursions to approximately 20 HM for a few minutes postfeeding(Smolenski and Robinson, 1988). Thus, ruminal methanogenesis which is viewed unfavorably by nutritionists as a loss of -8% of the metaboliz- able energy of the feed, in fact has an important thermodynamic function that permits an adequate rate and extent of carbohydrate fermentation Representatives of another group of bacteria, the carbon dioxide-reducing homoacetogens, have been isolated from the rumen and appear to be present at low cell densities. Like the methanogens, these eubacteria can reduce carbon dioxide with H2, but according to the stoichiometry 4H,+ 2C0,->CH: COOH 2H2O
Microbiology of the Dairy Animal 27 Table 6 Fermentation Products from Cellulose in Ruminococcus albus Monocultures and R. albus/Methanobreveibacter smithii Cocultures Illustrating Changes Caused by Interspecies Transfer of H2 to the Methanogen mmol/100 mmol Glucose equivalents consumeda R. albus Product R. albus alone M. smithii Ethanol 81 22 Formate 14 0 Acetate 89 151 CO2 156 98 H2 140 0 CH4 0 75b a Mean values from continuous culture trials conducted at five different dilution rates. b Equivalent to 300 mmol H2 consumed (at a stoichiometry of 4 mol H2 consumed per mol CH4 formed). Source: Pavlostathis et al., 1990. ‘‘aceticlastic’’ methanogens, whose growth rates even under ideal conditions are well below dilution rates of both liquids and solids in the rumen. Most methanogens are also autotrophs; that is, they can obtain all of their cell carbon from carbon dioxide. Thus, they can produce microbial protein for the ruminant host without consumption of otherwise useful organic matter. The energy associated with the reduction of the abundant ruminal carbon dioxide to methane is sufficient to permit both growth of the methanogens and thermodynamic displacement or ‘‘pulling’’ of the reduction of protons to H2. As a result, the concentration of H2 in ruminal fluid is very low—normally near 1 µM with only occasional excursions to approximately 20 µM for a few minutes postfeeding (Smolenski and Robinson, 1988). Thus, ruminal methanogenesis, which is viewed unfavorably by nutritionists as a loss of 8% of the metabolizable energy of the feed, in fact has an important thermodynamic function that permits an adequate rate and extent of carbohydrate fermentation. Representatives of another group of bacteria, the carbon dioxide–reducing homoacetogens, have been isolated from the rumen and appear to be present at low cell densities. Like the methanogens, these eubacteria can reduce carbon dioxide with H2, but according to the stoichiometry 4H2 2CO2 → CH3COOH 2H2O (2)
Weimer The homoacetogens have attracted interest as potential competitors of the metha nogens in that they could, in principle, remove fermentatively produced H2 while at the same time producing acetic acid, an energy source and biosynthetic precur sor that the ruminant is well equipped to use(Mackie and Bryant, 1994). Unfortu- nately, numerous in vitro studies have shown that the acetogens are ineffective ompetitors of the methanogens because of the latters superior affinity for low concentrations of H2. The actual role of the acetogens in the rumen is presently unclear; because this metabolically diverse group is capable of sugar fermentation and removal of methoxyl groups from some feed constituents, its members may fill several niches A third group of H, utilizers, the sulfate-reducing bacteria, can couple the oxidation of H, or certain organic compounds such as lactate to reduction of sulfate(Odom and Singleton, 1993) 4H,+ 2H+ SO,-H,S 4H,O Sulfate-reducing bacteria have an affinity for H, that even surpasses that of the methanogens; indeed, sulfate reduction is the dominant means of disposal of ex- cess electrons in a sulfate-rich environment (e.g, ocean sediments). Sulfate- reducing bacteria have the unusual capacity to act as H2 consumers when sulfate is abundant or as H2 producers(from lactate) when sulfate is absent(Bryant et al., 1977). In the latter situation, the sulfate reducers may be maintained in the rumen by a symbiotic interaction with methanogens wherein the sulfate reducers oxidize lactate to H2, whose concentration is kept low by methanogenic activity 5. Nitrogen Metabolism in the Rumen a. Protein Degradation Availability of protein to the ruminant is deter- mined by the amount of protein in the feed, its loss in the rumen from microbial fermentation, and the efficiency of microbial protein synthesis that occurs in the rumen. It is estimated that approximately 35-80%o of the protein of most forages and grains is degraded by ruminal fermentation and is thus not directly available for intestinal absorption(National Research Council, 1985). Hydrolysis of protein depends on several factors--particularly solubility, which determines both availability to ruminal microbes and its rate of escape from the rumen. The gener alized scheme of protein degradation(see Fig. 9)suggests some similarities to polysaccharide degradations. Proteins are hydrolyzed extracellularly or at the mi crobial cell surface to produce soluble oligomers that serve as the actual growth substrates. Major proteolytic species in the rumen are B. fibrisolvens, S. bovis, and P. ruminicola. These species also have important roles in carbohydrate fer mentation The fermentation of amino acids and peptides released from protein hydro sis is carried out by a number of ruminal species. The most active appear to
28 Weimer The homoacetogens have attracted interest as potential competitors of the methanogens in that they could, in principle, remove fermentatively produced H2 while at the same time producing acetic acid, an energy source and biosynthetic precursor that the ruminant is well equipped to use (Mackie and Bryant, 1994). Unfortunately, numerous in vitro studies have shown that the acetogens are ineffective competitors of the methanogens because of the latter’s superior affinity for low concentrations of H2. The actual role of the acetogens in the rumen is presently unclear; because this metabolically diverse group is capable of sugar fermentation and removal of methoxyl groups from some feed constituents, its members may fill several niches. A third group of H2 utilizers, the sulfate-reducing bacteria, can couple the oxidation of H2 or certain organic compounds such as lactate to reduction of sulfate (Odom and Singleton, 1993): 4H2 2H SO4 → H2S 4H2O (3) Sulfate-reducing bacteria have an affinity for H2 that even surpasses that of the methanogens; indeed, sulfate reduction is the dominant means of disposal of excess electrons in a sulfate-rich environment (e.g., ocean sediments). Sulfatereducing bacteria have the unusual capacity to act as H2 consumers when sulfate is abundant or as H2 producers (from lactate) when sulfate is absent (Bryant et al., 1977). In the latter situation, the sulfate reducers may be maintained in the rumen by a symbiotic interaction with methanogens wherein the sulfate reducers oxidize lactate to H2, whose concentration is kept low by methanogenic activity. 5. Nitrogen Metabolism in the Rumen a. Protein Degradation Availability of protein to the ruminant is determined by the amount of protein in the feed, its loss in the rumen from microbial fermentation, and the efficiency of microbial protein synthesis that occurs in the rumen. It is estimated that approximately 35–80% of the protein of most forages and grains is degraded by ruminal fermentation and is thus not directly available for intestinal absorption (National Research Council, 1985). Hydrolysis of protein depends on several factors—particularly solubility, which determines both its availability to ruminal microbes and its rate of escape from the rumen. The generalized scheme of protein degradation (see Fig. 9) suggests some similarities to polysaccharide degradations. Proteins are hydrolyzed extracellularly or at the microbial cell surface to produce soluble oligomers that serve as the actual growth substrates. Major proteolytic species in the rumen are B. fibrisolvens, S. bovis, and P. ruminicola. These species also have important roles in carbohydrate fermentation. The fermentation of amino acids and peptides released from protein hydrolysis is carried out by a number of ruminal species. The most active appear to
Microbiology of the Dairy Animal SOLUBLE INSOLUBLE PROTEINS PROTEINS OLIGOPEPTIDES AMINO ACIDS DIPEPTIDES > VFAs Figure 9 Generalized scheme of protein degradation in the rumet acteria and protozoa participate in the process. a-Keto acids may be used intraco as anabolic intermediate compounds, or decarboxylated to VFAs, which are the be Clostridium aminophilum, C sticklandii, and Peptostreptococcus anaerobius. Classic proteolytic species such as P. ruminicola appear to be important in protein hydrolysis (Wallace et al., 1999), but they are probably less important in amino acid fermentations, as their rates of ammonia production from amino acids in vitro are one or two orders of magnitude lower. Both C. sticklandii and P anaero- bius are monensin-sensitive, which may explain the protein-sparing effect ob- served on inclusion of monensin in ruminant diets(Krause and Russell, 1995) Because the concentrations of peptides and free amino acids in the rumen are very low, competition for these substrates among both proteolytic and nonproteolytic microbes is probably intense b. Protein Synthesis Whereas the ruminal microflora is responsible for this extensive loss of feed protein, they also contribute up to half of the nitrogen requirements of the animal through synthesis of microbial cell protein, which is hydrolyzed in the abomasum and is subsequently available to the animal(Orskov, 1982). Protein synthesis by ruminal bacteria occurs primarily from ammonia and organic acids. Indeed, most ruminal bacteria will grow in vitro on ammonia as the sole nitrogen source, and many species cannot incorporate significant amounts of amino acids or peptides. Ruminal ammonia is supplied either as a direct prod- uct of the ruminal degradation of feed proteins or from urea recycled back into
Microbiology of the Dairy Animal 29 Figure 9 Generalized scheme of protein degradation in the rumen. Both bacteria and protozoa participate in the process. α-Keto acids may be used intracellularly as anabolic intermediate compounds, or decarboxylated to VFAs, which are then exported. be Clostridium aminophilum, C. sticklandii, and Peptostreptococcus anaerobius. Classic proteolytic species such as P. ruminicola appear to be important in protein hydrolysis (Wallace et al., 1999), but they are probably less important in amino acid fermentations, as their rates of ammonia production from amino acids in vitro are one or two orders of magnitude lower. Both C. sticklandii and P. anaerobius are monensin-sensitive, which may explain the protein-sparing effect observed on inclusion of monensin in ruminant diets (Krause and Russell, 1995). Because the concentrations of peptides and free amino acids in the rumen are very low, competition for these substrates among both proteolytic and nonproteolytic microbes is probably intense. b. Protein Synthesis Whereas the ruminal microflora is responsible for this extensive loss of feed protein, they also contribute up to half of the nitrogen requirements of the animal through synthesis of microbial cell protein, which is hydrolyzed in the abomasum and is subsequently available to the animal (Ørskov, 1982). Protein synthesis by ruminal bacteria occurs primarily from ammonia and organic acids. Indeed, most ruminal bacteria will grow in vitro on ammonia as the sole nitrogen source, and many species cannot incorporate significant amounts of amino acids or peptides. Ruminal ammonia is supplied either as a direct product of the ruminal degradation of feed proteins or from urea recycled back into