Weimer L Figure 6 Schematic cross-sectional view of the cell wall of two plant cell types. Abbre- ations: ML, middle lamella; Pw, primary wall; Sw, secondary wall; L, lumen, which in the living cell contains the cytoplasm but is replaced with ruminal fluid during ruminal digestion ( Left panel) Mesophyll cell, characterized by a thin, essentially unlignified pri mary cell wall that is digested rapidly from both the outer and inner(luminal) surface. The middle lamella is thin and unlignified, and is usually separated from the middle lamellae of adjacent cells by air spaces. (Right panel) Sclerenchyma cell, characterized by a thin pri- mary wall and thick, secondary walls consisting primarily of cellulose but also containing moderate amounts of hemicelluloses and lignin. Adjacent cells are separated by middle lamellae having a high lignin content. As a result, sclerenchyma cell walls are digested only from the luminal surface outward, and at a relatively slow rate and incomplete extent. extensive intrachain and interchain hydrogen bonds to form crystalline microfi- brils that in turn are bundled into larger cellulose fibers. The packing of cellulose chains within the microfibrils is so tight that even water cannot penetrate. Cellu lose fibers thus have a fairly low ratio of exposed surface to volume. Ruminal cellulose digestion appears to follow first-order kinetics with respect to cellulose concentration (i.e, the rate of cellulose digestion is limited by the availability of cellulose rather than by any inherent property of the cellulolytic microbes them- selves [Waldo et al., 1972; Van Soest, 1973) Although many species of bacteria, fungi, and protozoa have been reported to digest cellulose in vitro, only three species of bacteria--Fibrobacter(formerly Bacteroides) succinogenes, Ruminococcus flavefaciens, and R. albus-are thought to be of major importance in cellulose digestion in the rumen(Dehority, 1993). In pure culture, these three species digest crystalline cellulose as a first- order process with rate constants of 0.05-0.10 h-Ihigher than those of any cellu- lolytic microbes that grow at a similar temperature in nonruminal habitats Weimer, 1996). These relatively rapid rates of cellulose digestion derive in part from the ability of these species to attach directly to the cellulosic substrate(Fi 7)and digest the cellulose via cell-bound enzymes; this adherence appears to be a prerequisite to rapid cellulose digestion (Latham et al., 1978; Costerton et al 1987: Kudo et al., 1987). The cell-associated cellulolytic enzymes are apparently organized into supramolecular complexes resembling the cellulosome, an organ
20 Weimer Figure 6 Schematic cross-sectional view of the cell wall of two plant cell types. Abbreviations: ML, middle lamella; PW, primary wall; SW, secondary wall; L, lumen, which in the living cell contains the cytoplasm but is replaced with ruminal fluid during ruminal digestion. (Left panel) Mesophyll cell, characterized by a thin, essentially unlignified primary cell wall that is digested rapidly from both the outer and inner (luminal) surface. The middle lamella is thin and unlignified, and is usually separated from the middle lamellae of adjacent cells by air spaces. (Right panel) Sclerenchyma cell, characterized by a thin primary wall and thick, secondary walls consisting primarily of cellulose but also containing moderate amounts of hemicelluloses and lignin. Adjacent cells are separated by middle lamellae having a high lignin content. As a result, sclerenchyma cell walls are digested only from the luminal surface outward, and at a relatively slow rate and incomplete extent. extensive intrachain and interchain hydrogen bonds to form crystalline microfi- brils that in turn are bundled into larger cellulose fibers. The packing of cellulose chains within the microfibrils is so tight that even water cannot penetrate. Cellulose fibers thus have a fairly low ratio of exposed surface to volume. Ruminal cellulose digestion appears to follow first-order kinetics with respect to cellulose concentration (i.e., the rate of cellulose digestion is limited by the availability of cellulose rather than by any inherent property of the cellulolytic microbes themselves [Waldo et al., 1972; Van Soest, 1973]). Although many species of bacteria, fungi, and protozoa have been reported to digest cellulose in vitro, only three species of bacteria—Fibrobacter (formerly Bacteroides) succinogenes, Ruminococcus flavefaciens, and R. albus—are thought to be of major importance in cellulose digestion in the rumen (Dehority, 1993). In pure culture, these three species digest crystalline cellulose as a firstorder process with rate constants of 0.05–0.10 h1 higher than those of any cellulolytic microbes that grow at a similar temperature in nonruminal habitats (Weimer, 1996). These relatively rapid rates of cellulose digestion derive in part from the ability of these species to attach directly to the cellulosic substrate (Fig. 7) and digest the cellulose via cell-bound enzymes; this adherence appears to be a prerequisite to rapid cellulose digestion (Latham et al., 1978; Costerton et al., 1987; Kudo et al., 1987). The cell-associated cellulolytic enzymes are apparently organized into supramolecular complexes resembling the cellulosome, an organ-
Microbiology of the Dairy Animal Figure 7 Stereo-optic view of the adherence of the ruminal cellulolytic bacterium Fi brobacter succinogenes onto a particle of cellulose. Proper focusing of the eyes or use of a stereo-optic viewer permits a three-dimensional view of the subject. Bar represents 10m elle that has been well-characterized in the nonruminal thermophilic bacterium Clostridium thermocellum( Felix and Ljungdahl, 1993). Although cellulose di- gestion in the rumen is more rapid than in nonruminal environments, the process is slow relative to the digestion of nonstructural carbohydrates and proteins. Be- cause of this, forages, with their high rumen fill and slow digestion, must be supplemented with more rapidly digested cereal grains to adequately balance energy and protein requirements for high-producing dairy animals(Van Soest, 1994) The products of cellulose hydrolysis are cellodextrins(short water-soluble B-1,4-glucosides of two to eight glucose units) that are subject to fermentation by both cellulolytic and noncelluloytic species(Russell, 1985). Although the indi vidual cellulolytic species can compete directly for cellulose in vitro, it appears that they show differential ability to adhere to different plant cell types (Latham et al., 1978) that may indicate separate but overlapping niches in the rumen. Moreover, it appears that degradation of some plant cell types is delayed by the slow diffusion on nonmotile fibrolytic bacteria into the plant cell lumen(Wilson
Microbiology of the Dairy Animal 21 Figure 7 Stereo-optic view of the adherence of the ruminal cellulolytic bacterium Fibrobacter succinogenes onto a particle of cellulose. Proper focusing of the eyes or use of a stereo-optic viewer permits a three-dimensional view of the subject. Bar represents 10 µm. elle that has been well-characterized in the nonruminal thermophilic bacterium Clostridium thermocellum (Felix and Ljungdahl, 1993). Although cellulose digestion in the rumen is more rapid than in nonruminal environments, the process is slow relative to the digestion of nonstructural carbohydrates and proteins. Because of this, forages, with their high rumen fill and slow digestion, must be supplemented with more rapidly digested cereal grains to adequately balance energy and protein requirements for high-producing dairy animals (Van Soest, 1994). The products of cellulose hydrolysis are cellodextrins (short water-soluble β-1,4-glucosides of two to eight glucose units) that are subject to fermentation by both cellulolytic and noncelluloytic species (Russell, 1985). Although the individual cellulolytic species can compete directly for cellulose in vitro, it appears that they show differential ability to adhere to different plant cell types (Latham et al., 1978) that may indicate separate but overlapping niches in the rumen. Moreover, it appears that degradation of some plant cell types is delayed by the slow diffusion on nonmotile fibrolytic bacteria into the plant cell lumen (Wilson
Weimer and Mertens, 1995). These cell types may provide a niche for motile cellulolytic species such as Butyrivibrio fibrisolvens The three major cellulolytic species form different fermentation endprod ucts(Hungate, 1966). F succinogenes produces primarily succinate(an important precursor of propionate) with lesser amounts of acetate. R. flavefaciens produces the same acids but with acetate predominating. R. albus produces primarily ace- tate and ethanol in pure culture, but in the rumen it produces mostly acetate and ho Estimation of the relative population sizes of individual cellulolytic species based on both classic determinative schemes(van Gylswyk, 1970) and probes to 16s rRNA (Weimer et al. 1999)suggest that R. albus is the most abundant of the three species, but variations in these populations appear to be more substantial mong animals than within individual animals fed widely different diets( Fig 8). Unlike other ruminal bacteria, the ratio of fermentation endproducts formed by each of the predominant cellulolytic species changes little with growth conditions h or growth rate). It would thus seem that the relative populations of these three species might contribute to differences in the proportions of acetate and propionate in the rumen. However, because the three species typically comprise less than 4%o of the bacterial population in the rumen, their direct contribution to VFA proportions is probably modest. b. hemicelluloses Hemicelluloses. a diffuse class of structural carbo drates that may contain any of a number of monomeric units, can comprise up to one-third of plant cell wall material (Stephen, 1983). Most hemicelluloses contain a main backbone, usually having B-1, 4-glycosyl or B-1,3-glycosyl link ages; various types and degrees of branching from the main chain are frequently observed. Because of the multiplicity of hemicellulose structures present in each plant species, it is extremely difficult to isolate pure substrates of known structure, which is a fact that has severely limited the laboratory study of hemicellulose digestion. Among the most abundant of the hemicelluloses are the xylans(un- branched B-1, 4-linked polymers of xylose) and the arabinoxylans(xylans con taining pendant arabinose side chains). The latter are particularly important, because they are thought to be covalently linked to lignin via cinnamic acid deriv atives such as ferulic acid and p-coumaric acid (Hatfield, 1993) Hemicelluloses are hydrolyzed by enzymes that may be extracellular or cell-associated depending on the species(Hespell and Whitehead, 1990). The most active hemicellulose digesters among the ruminal bacterial isolates include B. fibrisolvens and the cellulolytic species R. flavefaciens, R. albus, and F. succi- nogenes; the latter can hydrolyze hemicelluloses in vitro but cannot use the hy drolytic products for growth(Dehority, 1973) c. Pectic Materials Pectins are polymers of galacturonic acids, some of which also contain substantial amounts of neutral sugars(e. g, arabinose, rham- nose,and galactose). Pectins are more abundant in leaf tissue than in stems, and
22 Weimer and Mertens, 1995). These cell types may provide a niche for motile cellulolytic species such as Butyrivibrio fibrisolvens. The three major cellulolytic species form different fermentation endproducts (Hungate, 1966). F. succinogenes produces primarily succinate (an important precursor of propionate) with lesser amounts of acetate. R. flavefaciens produces the same acids but with acetate predominating. R. albus produces primarily acetate and ethanol in pure culture, but in the rumen it produces mostly acetate and H2. Estimation of the relative population sizes of individual cellulolytic species based on both classic determinative schemes (van Gylswyk, 1970) and probes to 16S rRNA (Weimer et al. 1999) suggest that R. albus is the most abundant of the three species, but variations in these populations appear to be more substantial among animals than within individual animals fed widely different diets (Fig. 8). Unlike other ruminal bacteria, the ratio of fermentation endproducts formed by each of the predominant cellulolytic species changes little with growth conditions (pH or growth rate). It would thus seem that the relative populations of these three species might contribute to differences in the proportions of acetate and propionate in the rumen. However, because the three species typically comprise less than 4% of the bacterial population in the rumen, their direct contribution to VFA proportions is probably modest. b. Hemicelluloses Hemicelluloses, a diffuse class of structural carbohydrates that may contain any of a number of monomeric units, can comprise up to one-third of plant cell wall material (Stephen, 1983). Most hemicelluloses contain a main backbone, usually having β-1,4-glycosyl or β-1,3-glycosyl linkages; various types and degrees of branching from the main chain are frequently observed. Because of the multiplicity of hemicellulose structures present in each plant species, it is extremely difficult to isolate pure substrates of known structure, which is a fact that has severely limited the laboratory study of hemicellulose digestion. Among the most abundant of the hemicelluloses are the xylans (unbranched β-1,4–linked polymers of xylose) and the arabinoxylans (xylans containing pendant arabinose side chains). The latter are particularly important, because they are thought to be covalently linked to lignin via cinnamic acid derivatives such as ferulic acid and p-coumaric acid (Hatfield, 1993). Hemicelluloses are hydrolyzed by enzymes that may be extracellular or cell-associated depending on the species (Hespell and Whitehead, 1990). The most active hemicellulose digesters among the ruminal bacterial isolates include B. fibrisolvens and the cellulolytic species R. flavefaciens, R. albus, and F. succinogenes; the latter can hydrolyze hemicelluloses in vitro but cannot use the hydrolytic products for growth (Dehority, 1973). c. Pectic Materials Pectins are polymers of galacturonic acids, some of which also contain substantial amounts of neutral sugars (e.g., arabinose, rhamnose, and galactose). Pectins are more abundant in leaf tissue than in stems, and
Microbiology of the Dairy Animal Llnl 3 日As32 区cs32 749266136 Cow No a cS32 Cow No Figure 8 Relative populations of the cellulolytic bacteria Ruminococcus albus, Rumino- coccus flavefaciens, and Fibrobacter succinogenes and their sums in the rumens of four cows fed the same four diets. Diets were based on alfalfa silage(As)or corn silage(CS) at two different levels of fiber(24 or 32% neutral detergent fiber, analyzed after a-amylase treatment). Results are expressed as a fraction of the total bacterial RNA, determined using ligonucleotide probes on samples collected 3 h after feeding. Note differences in the scale of the ordinates. From Weimer et al., 1999; used by permission of the Americar they are also major components of some byproduct feeds(citrus pulp and fruit processing waste). Although purified pectins from forages are fairly water solu ble, they can be considered to be structural carbohydrates, because they are local ized in the plant cell wall, particularly in the middle lamellae between cells In many respects, pectins are an ideal substrate for ruminal fermentation. They are rapidly digested out of both alfalfa leaves and stems(rate constants of -0.3 h-), but unlike starch, pectins do not yield lactic acid as a fermentation
Microbiology of the Dairy Animal 23 Figure 8 Relative populations of the cellulolytic bacteria Ruminococcus albus, Ruminococcus flavefaciens, and Fibrobacter succinogenes and their sums in the rumens of four cows fed the same four diets. Diets were based on alfalfa silage (AS) or corn silage (CS) at two different levels of fiber (24 or 32% neutral detergent fiber, analyzed after α-amylase treatment). Results are expressed as a fraction of the total bacterial RNA, determined using oligonucleotide probes on samples collected 3 h after feeding. Note differences in the scale of the ordinates. (From Weimer et al., 1999; used by permission of the American Dairy Science Association.) they are also major components of some byproduct feeds (citrus pulp and fruit processing waste). Although purified pectins from forages are fairly water soluble, they can be considered to be structural carbohydrates, because they are localized in the plant cell wall, particularly in the middle lamellae between cells. In many respects, pectins are an ideal substrate for ruminal fermentation. They are rapidly digested out of both alfalfa leaves and stems (rate constants of 0.3 h1 ), but unlike starch, pectins do not yield lactic acid as a fermentation
product(Hatfield and Weimer, 1995). The acetate/propionate ratio resulting from fermentation of pectins is in the range of 6-12, which is well above those of most substrates and useful in maintaining milkfat levels in lactating dairy cows Production of these acids is accompanied by consumption of the galacturonic acid moeities of the pectin, thus assisting in the maintenance of ruminal pH Several bacterial species have been shown actively to degrade pectin, including Lachnospira multipara, B fibrisolvens, Prevotella(formerly Bacteroides)rumi nicola, some strains of the genus Ruminococcus( Gradel and Dehority, 1972), and some spirochetes(Ziolecki, 1979) d. Lignin Lignin, the third major component of the forage cell wall, is a polymer of phenylpropanoid units assembled by a random free radical conden ation mechanism during cell wall biosynthesis. Lignin is indigestible under an- aerobic conditions and constitutes the bulk of the indigestible material leaving the digestive tract. Moreover, the covalent linkages between lignin(or phenolic acids) and hemicelluloses reduce the digestibility of these forage components (Hatfield, 1993). Electron microscopic studies clearly reveal the recalcitrance of lignified tissues to ruminal digestion(Akin, 1979) 2. Nonstructural Carbohydrates Nonstructural carbohydrates are those carbohydrates in plant cells that are con tained in the cytoplasm or in storage vacuoles. The most abundant of these are the starches(the linear amylose and the branched amylopectin), which are major components of cereal grains(e. g, corn) that comprise much of the diet of high producing dairy cows. a. Starch Starches are depolymerized fairly rapidly by extracellular en- zymes(amylases and pullulanases) that produce maltodextrins(a-1, 4-oligomers of glucose), which are easily converted by other a-glucosidases to glucose and maltose-substrates utilizable by almost all of the carbohydrate-fermenting mi crobes in the rumen(Hungate, 1966). Consequently, starches have the potenti to be completely digestible, although the form of the starch is an important deter minant of the rate of digestion. Wheat and barley starch are digested more rapidly than is that of high-moisture corn, which in turn is digested more rapidly than are those of dried corn or dried sorghum. The more rapidly digesting starches have first-order rate constants of digestion of -0 25 h-l or above Several bacterial species are important in starch digestion, including Rumi- nobacter(formerly Bacteroides)amylophilus, B fibrisolvens, P ruminicola, Suc cinomonas amylolytica, Succinivibrio dextrinosolvens, and Streptococcus bovis The latter species can grow extremely rapidly, particularly on glucose(minimum doubling time is 13 min), and it is the causative agent of lactic acidosis(see Sec V.D. 1). As noted above, some protozoa actively engulf starch granules but do
24 Weimer product (Hatfield and Weimer, 1995). The acetate/propionate ratio resulting from fermentation of pectins is in the range of 6–12, which is well above those of most substrates and useful in maintaining milkfat levels in lactating dairy cows. Production of these acids is accompanied by consumption of the galacturonic acid moeities of the pectin, thus assisting in the maintenance of ruminal pH. Several bacterial species have been shown actively to degrade pectin, including Lachnospira multipara, B. fibrisolvens, Prevotella (formerly Bacteroides) ruminicola, some strains of the genus Ruminococcus. (Gradel and Dehority, 1972), and some spirochetes (Ziolecki, 1979). d. Lignin Lignin, the third major component of the forage cell wall, is a polymer of phenylpropanoid units assembled by a random free radical condensation mechanism during cell wall biosynthesis. Lignin is indigestible under anaerobic conditions and constitutes the bulk of the indigestible material leaving the digestive tract. Moreover, the covalent linkages between lignin (or phenolic acids) and hemicelluloses reduce the digestibility of these forage components (Hatfield, 1993). Electron microscopic studies clearly reveal the recalcitrance of lignified tissues to ruminal digestion (Akin, 1979). 2. Nonstructural Carbohydrates Nonstructural carbohydrates are those carbohydrates in plant cells that are contained in the cytoplasm or in storage vacuoles. The most abundant of these are the starches (the linear amylose and the branched amylopectin), which are major components of cereal grains (e.g., corn) that comprise much of the diet of highproducing dairy cows. a. Starch Starches are depolymerized fairly rapidly by extracellular enzymes (amylases and pullulanases) that produce maltodextrins (α-1,4-oligomers of glucose), which are easily converted by other α-glucosidases to glucose and maltose—substrates utilizable by almost all of the carbohydrate-fermenting microbes in the rumen (Hungate, 1966). Consequently, starches have the potential to be completely digestible, although the form of the starch is an important determinant of the rate of digestion. Wheat and barley starch are digested more rapidly than is that of high-moisture corn, which in turn is digested more rapidly than are those of dried corn or dried sorghum. The more rapidly digesting starches have first-order rate constants of digestion of 0.25 h1 or above. Several bacterial species are important in starch digestion, including Ruminobacter (formerly Bacteroides) amylophilus, B. fibrisolvens, P. ruminicola, Succinomonas amylolytica, Succinivibrio dextrinosolvens, and Streptococcus bovis. The latter species can grow extremely rapidly, particularly on glucose (minimum doubling time is 13 min), and it is the causative agent of lactic acidosis (see Sec. V.D.1). As noted above, some protozoa actively engulf starch granules but do