Clostridium sticklandii Pep, AA Ac But. BCVFAs NH3 Peptostreptococcus anaero- Pep, AA Ac But. BCVFAs NH3 Hydrogen consum Acetitomaculum ruminis H,+ CO,. Hx Probably not an important o H, consumer in the rumen Desulfovibrio ruminis H,+ SO", EtOH, Lac H,S: Ac+H Produces h, from etoh言 and Lac in presence of Methanobrevibacter bryant H2+ CO, Methanosarcina barkeri H,+ CO,. MeOH CH4 Autotrophic, methylotrophic Wolinella succinogenes fumarate H formate, or Suc Can also reduce inorganic nitro compounds(e. g, Other nutritional specialists Acidaminococcus fermentans Glu. Cit. TAA Ac. But, H, Detoxifies TAA Anaerovibrio lipolytica TG. GoL. F. Rib Pro, Ac, But, Suc, H2, CO Oxalobacter formigenes Oxalate For CO Detoxifies oxalate Succiniclasticum numinis Pro co Synergists jonesii Arg. His, DHP Ac. Pro. H Detoxifies mimosine -d Lac Gol AA, amino acids: Arg, arginine; Cd, cellodextrins(except where ethanol; F, fructose; For, formate: G, glucose; Gz, cellobiose: Glu Lac, lactate: MeOH, methanol: P, pectin; Pep, peptides; Prot, prot lu, glutamate glucose also fermented): DHP, 2, 3- and 3. 4-dihydroxypyridinediols: EtOH, ate; Gol, glycerol; His, histidine; Hx, most common hexose sugars; L, lactose: triglycerides; X, xylose; Xd, xylodextrins: Xn, xylan. b Ac, acetate; BCVFAs, branch-chain volatile fatty acids(isobutyrate 2-methylbutyrate); But, butyrate; EtOH, ethanol; For, formate; Lac. lactate: Pro, propionate; Suc, succinate Includes C. cellobioparum, C. chartatabidium, C. lochheadii, C. longisporum, C. polysaccharolyticum. A few of these species also produce ethanol Stain gram negative but phylogenetically related to gram positive eubacteria. Abundant in ovine rumen but not bovine rumen
Microbiology of the Dairy Animal 15 Clostridium sticklandii Pep, AA Ac, But, BCVFAs, NH3, CO2 Peptostreptococcus anaero- Pep, AA Ac, But, BCVFAs, NH3, bius CO2 Hydrogen consumers Acetitomaculum ruminis H2 CO2, Hx Ac Probably not an important H2 consumer in the rumen Desulfovibrio ruminis H2 SO4, EtOH, Lac H2S; Ac H2 Produces H2 from EtOH and Lac in presence of methanogens Methanobrevibacter bryantii H2 CO2 CH4 Autotrophic Methanosarcina barkeri H2 CO2, MeOH CH4 Autotrophic, methylotrophic Wolinella succinogenes fumarate H2, formate, or Suc Can also reduce inorganic H2S nitro compounds (e.g., NO3) Other nutritional specialists Acidaminococcus fermentans d Glu, Cit, TAA Ac, But, H2 Detoxifies TAA Anaerovibrio lipolytica TG, Gol, F, Rib Pro, Ac, But, Suc, H2, CO2 Oxalobacter formigenes Oxalate For, CO2 Detoxifies oxalate Succiniclasticum ruminis Suc Pro, CO2 Synergistes jonesii Arg, His, DHP Ac, Pro, H2 Detoxifies mimosine Veillonella parvulae d Lac, Gol Ac, Pro, H2, CO2 a AA, amino acids; Arg, arginine; Cd, cellodextrins (except where indicated, glucose also fermented); DHP, 2,3- and 3,4-dihydroxypyridinediols; EtOH, ethanol; F, fructose; For, formate; G, glucose; G2, cellobiose; Glu, glutamate; Gol, glycerol; His, histidine; Hx, most common hexose sugars; L, lactose; Lac, lactate; MeOH, methanol; P, pectin; Pep, peptides; Prot, protein; TG, triglycerides; X, xylose; Xd, xylodextrins; Xn, xylan. b Ac, acetate; BCVFAs, branch-chain volatile fatty acids (isobutyrate, isovalerate, 2-methylbutyrate); But, butyrate; EtOH, ethanol; For, formate; Lac, lactate; Pro, propionate; Suc, succinate. c Includes C. cellobioparum, C. chartatabidium, C. lochheadii, C. longisporum, C. polysaccharolyticum. A few of these species also produce ethanol. d Stain gram negative but phylogenetically related to gram positive eubacteria. e Abundant in ovine rumen but not bovine rumen
Cellulose or Starch and Pectins 2 ADP 2ATP Oxaloacetate ACCOA Butyrate Acetyl-P Acetaldehyde (2H] CH Acetate Succin Propionate Figure 5 Generalized pathway of carbohydrate fermentations in the rumen Fermentation products in dark bor- dered boxes are maintained in substantial concentrations in the normal rumen. Fermentation products in light bordered boxes are produced and excreted by some organisms but do not accumulate under normal conditions. Abbreviations: [2H], pairs of reducing equivalents: ADP and ATP, adenosine di-or triphosphate; GDP and GTP. guanosine di- and triphosphate; PEP, phosphoenolpyruvate: AcCoA, acetyl coenzyme A Reactions coded by a circled letter are restricted to a few species, as follows: A, fibrolytic or amylolytic microbes; B, lactate utilizers. albus, S. ruminantium, Streptococcus bovis; E, homoacetogenic bacteria(e.g, Acetitomaculum ruminis); F, sulfate educing bacteria; G, methanogenic archaea; h, S. ruminantium and Succiniclasticum rumini
16 Weimer Figure 5 Generalized pathway of carbohydrate fermentations in the rumen. Fermentation products in dark bor- dered boxes are maintained in substantial concentrations in the normal rumen. Fermentation products in light bordered boxes are produced and excreted by some organisms but do not accumulate under normal conditions. Abbreviations: [2H], pairs of reducing equivalents; ADP and ATP, adenosine di- or triphosphate; GDP and GTP. guanosine di- and triphosphate; PEP, phosphoenolpyruvate; AcCoA, acetyl coenzyme A. Reactions coded by a circled letter are restricted to a few species, as follows: A, fibrolytic or amylolytic microbes; B, lactate utilizers, particularly Selenomonas ruminantium and Megasphaera elsdenii; C, Butyrivibio fibrisolvens; D, Ruminococcus albus, S. ruminantium, Streptococcus bovis; E, homoacetogenic bacteria (e.g., Acetitomaculum ruminis); F, sulfatereducing bacteria; G, methanogenic archaea; H, S. ruminantium and Succiniclasticum ruminis
Microbiology of the Dairy Animal 2. Protozoa Because of their large size(100 um or more in length), protozoa are readily observed microscopically and thus were first described in 1843. Many species of ruminal protozoa have been identified, primarily based on morphological criteria Hungate, 1966). These can be classified into flagellates and ciliates Flagellates dominate the ruminal protozoan population of young animals, but they are gradu- ally displaced by the ciliates with aging. The ciliates contain two main groups the relatively simple holotrichs(e.g, Isotricha or Dasytricha) or the structurally more complex oligotrichs(e. g, Entodinium and Diplodinium). The populations of protozoa in the rumen vary widely, but they are usually in the range of 10- 10/mL. These densities are much lower than those of the bacteria: however because of their large size, the protozoa may in fact represent up to half of the microbial biomass in the rumen(Van Soest, 1994; Jouany and Ushida, 1999 All of the ruminal protozoa appear to have a strictly fermentative metabo- lism. Relative to the bacteria, much less is known regarding the physiology and biochemistry of the protozoa for two reasons. First, the protozoa are rather diffi cult to cultivate in the laboratory(Coleman et al. 1963); ruminal protozoa gene ally die within hours of transferring mixed rumen microflora into most laboratory culture environments. Second, many protozoa in a variety of habitats contain intracellular or surface-attached bacterial symbionts that engage in syntrophic interactions with their hosts(Fenchel et al, 1977; Vogels et al., 1980).Thus,even when"pure" cultures of protozoa(i.e, single protozoal species in the absence of free-living bacteria) are established and maintained, it is difficult to evaluate the potential contribution of the associated bacteria to the metabolic activities of the protozoa. Some continuous culture systems have successfully maintained proto- zoa by including a floating-mat matrix that allows the protozoa to resist washout from the vessel at fluid dilution rates similar to those operating in the rumen (Abe and Kurihara, 1984), and it is likely that ruminal protozoa associate in vivo with the ruminal mat or the ruminal wall in a similar manner. Populations of different protozoal species vary among individual animals and within the same animal fed different diets(Faichney et al., 1997) Because their relatively large size permits microscopic identification of s cies and behavioral examination, much of our knowledge of these organisms has come from study of samples withdrawn directly from the rumen itself, particu- larly for comparisons of faunated animals (i.e, those having a natural protozoal population) and defaunated animals (i.e, those whose protozoal populations have been nearly or completely removed, usually by treatment with chemical agents such as 1, 2-dimethyl-5-nitroimidazole or dioctyl sodium sulfosuccinate) The holotrich appear to be adapted to growth purely on soluble carbohy drates. On the other hand, microscopic observations have revealed that the entodi morphs can engulf plant particles or can attach to the cut ends of plant fiber and
Microbiology of the Dairy Animal 17 2. Protozoa Because of their large size (100 µm or more in length), protozoa are readily observed microscopically and thus were first described in 1843. Many species of ruminal protozoa have been identified, primarily based on morphological criteria (Hungate, 1966). These can be classified into flagellates and ciliates. Flagellates dominate the ruminal protozoan population of young animals, but they are gradually displaced by the ciliates with aging. The ciliates contain two main groups: the relatively simple holotrichs (e.g., Isotricha or Dasytricha) or the structurally more complex oligotrichs (e.g., Entodinium and Diplodinium). The populations of protozoa in the rumen vary widely, but they are usually in the range of 102 – 106/mL. These densities are much lower than those of the bacteria; however, because of their large size, the protozoa may in fact represent up to half of the microbial biomass in the rumen (Van Soest, 1994; Jouany and Ushida, 1999). All of the ruminal protozoa appear to have a strictly fermentative metabolism. Relative to the bacteria, much less is known regarding the physiology and biochemistry of the protozoa for two reasons. First, the protozoa are rather diffi- cult to cultivate in the laboratory (Coleman et al. 1963); ruminal protozoa generally die within hours of transferring mixed rumen microflora into most laboratory culture environments. Second, many protozoa in a variety of habitats contain intracellular or surface-attached bacterial symbionts that engage in syntrophic interactions with their hosts (Fenchel et al., 1977; Vogels et al., 1980). Thus, even when ‘‘pure’’ cultures of protozoa (i.e., single protozoal species in the absence of free-living bacteria) are established and maintained, it is difficult to evaluate the potential contribution of the associated bacteria to the metabolic activities of the protozoa. Some continuous culture systems have successfully maintained protozoa by including a floating-mat matrix that allows the protozoa to resist washout from the vessel at fluid dilution rates similar to those operating in the rumen (Abe and Kurihara, 1984), and it is likely that ruminal protozoa associate in vivo with the ruminal mat or the ruminal wall in a similar manner. Populations of different protozoal species vary among individual animals and within the same animal fed different diets (Faichney et al., 1997). Because their relatively large size permits microscopic identification of species and behavioral examination, much of our knowledge of these organisms has come from study of samples withdrawn directly from the rumen itself, particularly for comparisons of faunated animals (i.e., those having a natural protozoal population) and defaunated animals (i.e., those whose protozoal populations have been nearly or completely removed, usually by treatment with chemical agents such as 1,2-dimethyl-5-nitroimidazole or dioctyl sodium sulfosuccinate). The holotrichs appear to be adapted to growth purely on soluble carbohydrates. On the other hand, microscopic observations have revealed that the entodiniomorphs can engulf plant particles or can attach to the cut ends of plant fiber and
Weimer can obtain their nutrition from engulfed starches and apparently some structural polysaccharides as well. Despite the observed associations of protozoa and partic- ulate feeds, it is widely held that the primary ecological role of the entodinio- morph protozoa is the grazing of bacteria( Clarke, 1977; Hobson and wallace, 1982). Using phase-contrast microscopy, these protozoa can be observed rapidly to ingest free bacteria (i.e, those not attached to plant fiber), and bacterial cell concentrations are approximately 10-fold higher in rumen samples from defau- nated than faunated animals Numerous studies(reviewed by Hobson and wal- lace, 1982) have thus far not identified any specific predatory relationships be- tween particular species of protozoa and bacteria. Protozoal grazing of bacteria can reduce the availability of microbial protein to ruminants, which is a notion reflected by lower weight gain in faunated than in defaunated cattle and lambs when tests were conducted with protein-deficient diets-an effect that disappears at higher levels of feed protein On the other hand, protozoa do appear to provide some benefits to the ruminal microflora (Jouany and Ushida, 1999). By engulfin starch granules and fermenting them more slowly than do bacteria, and by converting lactic acid to the weaker propionic acid, protozoa can help attenuate acidosis and thereby maintain fibrolytic activity of pH-sensitive cellulolytic bac- terra Protozoa are not the only agents that control bacterial numbers; the rumen maintains substantial populations of bacteriophages(viruses that infect bacteria) Characterization of phage DNAs from rumen contents by pulsed-field electropho- esis(Swain et al., 1996) has revealed that individual animals harbor their own unique populations of phages. Regardless of these differences among host ani- mals, phage populations(as measured by total phage DNA)follow diurnal popu- maxima at approximately 2 h and 10-12 h postfeeding, respective/y ima and lation cycles related to the populations of the bacterial hosts, with mi 3. Fungi Orpin(1975)demonstrated that several microorganisms originally thought to be flagellated protozoa were actually the zoospore stage of anaerobic fungi. These fungi alternate between a freely motile zoospore stage and a particle-associated thallus. Fungal populations in rumen contents range from 10 to 10 thallus-form ing units per gram of ruminal fluid(Theodorou et al., 1990). Approximately 24 species of these fungi have now been identified on the basis of morphology and 16S rRNA sequences (Trinci et al., 1994). Much of our understanding of the metabolic capabilities of the ruminal fungi has been derived from a single species Neocallimastix frontalis Ruminal fungi are strictly anaerobic and have a catabolism based on fer mentation of carbohydrate. All described hemicelluloses via extracellular enzymes that are produced in low titer but have very high specific activities (Wood et al., 1986). The major products of carbohy
18 Weimer can obtain their nutrition from engulfed starches and apparently some structural polysaccharides as well. Despite the observed associations of protozoa and particulate feeds, it is widely held that the primary ecological role of the entodiniomorph protozoa is the grazing of bacteria (Clarke, 1977; Hobson and Wallace, 1982). Using phase-contrast microscopy, these protozoa can be observed rapidly to ingest free bacteria (i.e., those not attached to plant fiber), and bacterial cell concentrations are approximately 10-fold higher in rumen samples from defaunated than faunated animals. Numerous studies (reviewed by Hobson and Wallace, 1982) have thus far not identified any specific predatory relationships between particular species of protozoa and bacteria. Protozoal grazing of bacteria can reduce the availability of microbial protein to ruminants, which is a notion reflected by lower weight gain in faunated than in defaunated cattle and lambs when tests were conducted with protein-deficient diets—an effect that disappears at higher levels of feed protein. On the other hand, protozoa do appear to provide some benefits to the ruminal microflora (Jouany and Ushida, 1999). By engulfing starch granules and fermenting them more slowly than do bacteria, and by converting lactic acid to the weaker propionic acid, protozoa can help attenuate acidosis and thereby maintain fibrolytic activity of pH-sensitive cellulolytic bacteria. Protozoa are not the only agents that control bacterial numbers; the rumen maintains substantial populations of bacteriophages (viruses that infect bacteria). Characterization of phage DNAs from rumen contents by pulsed-field electrophoresis (Swain et al., 1996) has revealed that individual animals harbor their own unique populations of phages. Regardless of these differences among host animals, phage populations (as measured by total phage DNA) follow diurnal population cycles related to the populations of the bacterial hosts, with minima and maxima at approximately 2 h and 10–12 h postfeeding, respectively. 3. Fungi Orpin (1975) demonstrated that several microorganisms originally thought to be flagellated protozoa were actually the zoospore stage of anaerobic fungi. These fungi alternate between a freely motile zoospore stage and a particle-associated thallus. Fungal populations in rumen contents range from 104 to 105 thallus-forming units per gram of ruminal fluid (Theodorou et al., 1990). Approximately 24 species of these fungi have now been identified on the basis of morphology and 16S rRNA sequences (Trinci et al., 1994). Much of our understanding of the metabolic capabilities of the ruminal fungi has been derived from a single species, Neocallimastix frontalis. Ruminal fungi are strictly anaerobic and have a catabolism based on fermentation of carbohydrate. All described species can digest cellulose and/or hemicelluloses via extracellular enzymes that are produced in low titer but have very high specific activities (Wood et al., 1986). The major products of carbohy-
Microbiology of the Dairy Animal drate fermentation are acetate. formate. and h, with lesser amounts of lactate (primarily the D isomer), CO2, and traces of succinate(Borneman et al., 1989) H2 production occurs via hydrogenosomes, which are intracellular organelles con- taining high levels of the enzyme hydrogenase In pure culture, the amounts of luble and gaseous fermentation products essentially equal the amount of carbo- hydrate consumed(Borneman et al., 1989); suggesting that the yield of fungal mycelia is very small. This notion is in accord with direct measurements that indicate the ruminal fungi contribute little to the total microbial biomass in the rumen(Faichney et al., 1997). However, the ruminal fungi appear to have specific roles not readily duplicated by bacteria. For example, there is considerable evi- dence that fungi can attach to and physically disrupt plant tissue(particularly the more recalcitrant tissues such as sclerenchyma and vascular bundles) during growth by penetration through cell walls and expansion into the pit fields between cells(Akin et al., 1989). This physical disruption is thought to make the plant material more easily broken apart during rumination and thus more available to bacteria, which are more efficient at digesting the individual plant cell compo- nents such as cellulose. Fungal populations are highest in animals fed diets high in fibrous stem materials; perhaps because of the latter's long ruminal retention time that coincides with the slow growth rate of the fungi D. Microbial Fermentations in the Rumen 1. Structural Carbohydrates Plant cell walls(the fibrous component of most forages) are composed primarily of cellulose, hemicellulose, pectin, and lignin. These polymers are differentially localized into the different layers of the cell wall(Fig. 6). The architecture of he plant cell wall varies greatly with cell type(Harris, 1990). Some cell types such as mesophyll and collenchyma are thin walled and essentially unlignified and thus are easily digested. Other cell types such as sclerenchyma and xylem tracheary elements display more complex architectures with clearly distinct struc- tures. Groups of these cell types are separated from one another by a middle lamella, which is a highly lignified region that is also rich in pectin. Interior to the middle lamella is the primary wall, the region where wall growth initiates it is composed primarily of xyloglucans and other hemicelluloses as well as various wall-associated proteins. The secondary wall is laid down later in de- velopment and is very thick in mature plants. This region, which contains mostly cellulose with smaller amounts of hemicelluloses and lignin, can be further differentiated into layers(Sl, S2, S3) based on the orientation of the cellulose microbe a Cellulose Cellulose is the major component of forage fiber, comprising 35-50% of dry weight. Individual cellulose molecules are linear polymers of B- 1. 4-linked D-glucose molecules. These chainlike molecules are assembled via
Microbiology of the Dairy Animal 19 drate fermentation are acetate, formate, and H2 with lesser amounts of lactate (primarily the D isomer), CO2, and traces of succinate (Borneman et al., 1989). H2 production occurs via hydrogenosomes, which are intracellular organelles containing high levels of the enzyme hydrogenase. In pure culture, the amounts of soluble and gaseous fermentation products essentially equal the amount of carbohydrate consumed (Borneman et al., 1989); suggesting that the yield of fungal mycelia is very small. This notion is in accord with direct measurements that indicate the ruminal fungi contribute little to the total microbial biomass in the rumen (Faichney et al., 1997). However, the ruminal fungi appear to have specific roles not readily duplicated by bacteria. For example, there is considerable evidence that fungi can attach to and physically disrupt plant tissue (particularly the more recalcitrant tissues such as sclerenchyma and vascular bundles) during growth by penetration through cell walls and expansion into the pit fields between cells (Akin et al., 1989). This physical disruption is thought to make the plant material more easily broken apart during rumination and thus more available to bacteria, which are more efficient at digesting the individual plant cell components such as cellulose. Fungal populations are highest in animals fed diets high in fibrous stem materials; perhaps because of the latter’s long ruminal retention time that coincides with the slow growth rate of the fungi. D. Microbial Fermentations in the Rumen 1. Structural Carbohydrates Plant cell walls (the fibrous component of most forages) are composed primarily of cellulose, hemicellulose, pectin, and lignin. These polymers are differentially localized into the different layers of the cell wall (Fig. 6). The architecture of the plant cell wall varies greatly with cell type (Harris, 1990). Some cell types such as mesophyll and collenchyma are thin walled and essentially unlignified, and thus are easily digested. Other cell types such as sclerenchyma and xylem tracheary elements display more complex architectures with clearly distinct structures. Groups of these cell types are separated from one another by a middle lamella, which is a highly lignified region that is also rich in pectin. Interior to the middle lamella is the primary wall, the region where wall growth initiates; it is composed primarily of xyloglucans and other hemicelluloses as well as various wall-associated proteins. The secondary wall is laid down later in development and is very thick in mature plants. This region, which contains mostly cellulose with smaller amounts of hemicelluloses and lignin, can be further differentiated into layers (S1, S2, S3) based on the orientation of the cellulose microfibrils. a. Cellulose Cellulose is the major component of forage fiber, comprising 35–50% of dry weight. Individual cellulose molecules are linear polymers of β- 1,4-linked D-glucose molecules. These chainlike molecules are assembled via