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  1. Reading Assignments James B. Russell and J.L. Rychlik. 2001. Factors that alter rumen microbial ecology. Science 292:1119 J. Miron, D. Ben-Ghedalia and M. Morrison. 2001. Invited review: Adhesion mechanisms of rumen cellulolytic bacteria. J. Dairy Sci. 84:1294 Bryan A. White. 1991. Bichemistry and genetics of microbial degradation of the plant cell wall. Rec. Adv. on the Nutr. Herbivores. pp 217-225 J.L. Rychlik and J.B. Russell. 2002. Bacteriocin-like activity of Butyrivibrio fibrisolvens JL5 and its effect on other ruminal bacteria and ammonia production. Appl. And Environ. Microbiol. 68:1040 H. Krajcaraski-Hunt, J.C. Plaizier, J.-P. Walton, R. Spratt and B.W. McBride. 2002. Short communication: Effect of subacute ruminal acidosis on in situ fiber digestion in lactating dairy cows. J. Dairy Sci. 85:570 A.L. Oliver, R.J. Grant, J.F. Pedersen and J.O. O’Rear. 2004. Comparison of brown midrib-6 and -18 forage sorghum with conventional sorghum and corn silage in diets of lactating dairy cows. J. Dairy Sci. 87:637

  2. Carbohydrates • Importance • Make up 60% to 70% of diet • Major source of energy • 1. Microbes • Energy for microbes • Metabolism, Growth, Protein synthesis • 2. Animal • End products of the fermentation • Digestible CHOC escaping the rumen • Classification • Nonstructural (NSC) • Cell contents - storage • Structural (SC) • Cell walls

  3. Chemistry of Feed Dry Matter • Organic • Carbohydrates • Fiber • Cellulose, hemicellulose • Soluble fiber • Pectin, fructans, β-glucans • Starch • Free sugars • Lignin and other phenolics • Proteins • Lipids • Inorganic

  4. Plant Carbohydrates Cell ContentCell Wall Organic acids Pectins Sugars β-glucans Starches Hemicelluloses Fructans Cellulose Mammalian enzymes will digest starch and sucrose (limited in ruminants) Microbes digest the plant polysaccharides

  5. Plant Cell Walls Many plant cells have a primary cell wall, which accommodates the cell as it grows, and a secondary cell wall that develops inside the primary wall after the cell has stopped growing. The primary cell wall is thinner and more pliant than the secondary cell wall. A specialized region of the cell walls of plants is the middle lamella. Rich in pectins, the middle lamella is shared by neighboring cells and cements them firmly together. Secondary cell wall would develop The main chemical components of the primary cell wall include cellulose and two groups of branched polysaccharides, the pectins and cross-linking glycans (hemicellulose). The secondary plant cell wall, which is often deposited inside the primary cell wall as a cell matures, contains lignin in addition to cellulose, but less hemicellulose and pectin.

  6. Carbohydrates • Monosaccharides - one sugar molecule • Hexoses - 6 carbons • Glucose Fructose Galactose Mannose • Pentoses - 5 carbons • Arabinose Xylose Ribose • Disaccharides - two sugar molecules • Maltose = glucose + glucose • Cellobiose = glucose + glucose • Sucrose = glucose + fructose • Lactose = glucose + galactose

  7. Carbohydrates - Continued 3. Polysaccharides - polymers of sugar molecules - Starch - polymer of glucose (plants) • Alpha 1- 4 linkages, branch at alpha 1-6 • Amylose (unbranched) 20 to 30% of starch in grain • Amylopectin (branched) 70 to 80% of starch in grain - Glycogen - polymer of glucose (animals) • Alpha 1- 4 linkages, branch at alpha 1- 6 - Cellulose - polymer of glucose (plants) • Beta 1- 4 linkages

  8. Cellulose Cellulose: A polymer of glucose units in β – 1,4 linkages. Cellulose is a linear molecule consisting of 1,000 to 10,000 β-D-glucose residues with no branching. Neighboring cellulose chains may form hydrogen bonds leading to the formation of microfibrils with partially crystalline parts. Hydrogen bonding among microfibrils can form microfibers and microfibers react to form cellulose fibers. Cellulose fibers usually consist of over 500,000 cellulose molecules. β-1,4 linkage

  9. Starch Starch: A polymer of α-D-glucose in α-1, 4 linkages. Starch consists of two types of molecules, amylose and amylopectin. Amylose is a single chain of glucose units whereas in amylopectin at about every twenty glucose units there is a branch with an α-1, 6 linkage. The relative proportions of amylose to amylopectin depend on the source of the starch, e.g. normal corn contains over 50% amylose whereas 'waxy' corn has almost none (~3%). Amylose has lower molecular weight with a relatively extended shape, whereas amylopectin has large but compact molecules. Partial structure of amylose Partial structure of amylopectin

  10. Starch Amylose molecules consist of single mostly-unbranched chains with 500-20,000 α-(1, 4)-D-glucose units with a few α-1, 6 branches. Amylose can form an extended shape. Hydrogen bonding occurs between aligned chains. The aligned chains may form double stranded crystallites that are resistant to amylases. Amylopectin is formed by non-random α-1, 6 branching of the amylose-type α-(1, 4)-D-glucose structure. This branching is determined by branching enzymes that leave each chain with up to 30 glucose residues. Each amylopectin molecule contains one to two million residues, about 5% of which form the branch points, in a compact structure forming granules. The molecules are oriented radially in the starch granule and as the radius increases so does the number of branches required to fill the space, resulting in concentric regions of alternating amorphous and crystalline structure.

  11. Amylopectin Corn starch Potato starch

  12. Carbohydrates - Continued • Polysaccharides - Pentosans - polymers of 5-carbon sugars - Fructans – Water soluble chains of fructose β-2-6 with β-2-1 branching Found in temperate grasses β-2-1 Found in Jerusalem artichokes - β-Glucans – Soluble chains of glucose β-1-3 and β-1-4 chains not linear like cellulose Found in oats & barley

  13. Carbohydrates - Continued • Mixed polysaccharides • Hemicellulose • Branched polysaccharides that are structurally homologous to cellulose because they have a backbone composed of β-1, 4 linked sugar residues – Most often xylans, no exact structure • Hemicellulose is abundant in primary walls but is also found in secondary walls • Various side chains : arabinose, glucuronic acid, manose, glucose, 4-0-methylglucuronic acid – varies among species • In plant cell walls: • Close association with lignin – linkages to coumaric and ferulic acids • Xylan polymers may be crosslinked to other hemicellulose backbones • Bound to cellulose in plant cell wall • Ratio of cellulose to hemicellulose ranges from 0.8:1 to 1.6:1

  14. Mixed Polysaccharides - Continued • Pectins • Pectins have a complex and not exact structure. Backbone is • most often α-1- 4 linked D-galacturonic acid • Rhamnose might be interspersed with galacturonic acid with • branch-points resulting in side chains (1 - 20 residues) of • mainly L-arabinose and D-galactose • Also contain ester linkages with methyl groups and sidechains • containing other residues such as D-xylose, L-frucose, D- • glucuronic acid, D-apiose, 3-deoxy-D-manno-2-octulosonic acid • and 3-deoxy-D-lyxo-2-heptulosonic acid attached to poly-α-(1, • 4)-D-galacturonic acid regions • Proteins called extensins are commonly found associated • with pectin in the cell wall • Commonly form crosslinkages and entrap other polymers • Composition varies among plants and parts of plants • Citrus pulp, beet pulp, soybean hulls have • high concentrations • Alfalfa intermediate concentrations of pectin • Grasses low concentrations of pectin

  15. Structural Carbohydrates in Plants Pectins less in grass than legumes. Hemicellulose greater in grass than legumes. Hemicellulose and cellulose increase with maturity.

  16. Lignin Lignin Monomers • Not a carbohydrate – does not contain sugars • Large phenolic three-dimensional polymers in secondary cell walls • The monomers are polymerized phenylpropane units, predominantly coumaryl alcohol [with an OH-group in position 4 of the phenyl ring], coniferyl alcohol (OH-group in position 4, -OCH3 in position 3) and sinapyl alcohol (OH-group in position 4, -OCH3 group in positions 3 and 5). • The side groups of the monomers are reactive forming poorly defined structures that are heavily cross linked. • Attach with hemicellulose and pectins • Not digested in the rumen

  17. Relation of Lignin to Digestibility of Cell Walls • 1. A negative relationship usually observed • Encrustation of cell wall polysaccharides • Enzymes can not digest polysaccharides • However lignin content related to maturity • rather than digestibility of cell walls • 2. Ratio of monomers varies among plants • High concentrations of syringyl unit (sinapyl) • less digestible • However ratio of monomers not always • related to digestibility of cell walls • 3. Hydroxycinnamic acids (acid forms of monomers) • can form cross links among polysaccarides and • link polysaccarides with lignin

  18. Lignin and Digestibility of Cell Walls • Cross links • Ferulic acid (acid form of coniferyl alcohol) is first • product synthesized • The ferulates (hydroxycinnamic acids) • 1. Can react with polysaccharides of cell wall • Reduces digestibility of cell wall polysaccharides • 2. Can link polysaccharides in cell wall with lignin • More dramatic reduction in digestibility of cell walls • Form early in the plant and become diluted with • maturity so negative relationship not always • apparent

  19. Interaction of Lignin with Polysaccharides Core lignin Non core lignin

  20. Tannins • Not carbohydrate – do not contain sugars • Polyphenolic compounds of diverse nature • 1. Hydrolysable tannins • Residues of gallic acid that are • linked to glucose via glycosidic • bonds • 2. Condensed tannins (nonhydrolyzable) • Biphenyl condensates of phenols • Anti-nutrient effects • Combine with proteins, cellulose, • hemicellulose, pectin and minerals • Can inhibit microorganisms and enzymes • In plants • Most domesticated plants have been selectively bred for • low concentrations of tannins – bird resistant milo exception • Many warm season legumes and browses contain tannins • Colored seed coats indicative of tannins - Acorns

  21. Feed Evaluation - Chemical • Sample feed • Need representative sample • Proximate analysis (Weende procedure) • Moisture - Residue is dry matter • Oven dry Volatile components will be lost Overheating causes reactions of carbohydrates with proteins and changes solubility of carbohydrates • Freeze dry • Distill with toluene – Best for fermented feeds • Determine water with Karl Fischer reagent • Organic matter • Burn @ 6000C - Residue is ash

  22. Feed Evaluation - Continued • Crude protein • Kjeldahl N x 6.25 • Ether extract • Lipids, waxes, pigments, fat soluble vitamins • Extract with ether or hexane • Crude fiber • Cellulose, hemicellulose, lignin • Boil in dilute acid and then dilute alkali, dry, weigh, ash (Wt loss is crude fiber) • Nitrogen-free extract Starch & Sugars + Other NFE = 100 - (moisture + ash + crude fiber + protein + ether extract) Acid and sodium hydroxide used for crude fiber dissolve some cellulose, hemicellulose and lignin in cell walls which then are included in NFE.

  23. Forage (Neutral detergent solution) Soluble Insoluble Cell contents Cell walls (NDF) Starch & Sugars Hemicellulose (Pectin, β-glucans Cellulose & fructans) Lignin Soluble proteins Insoluble proteins Lipids Insoluble minerals (dirt) Organic acids Fiber analysis - Detergent solutions (Van Soest)

  24. Neutral Detergent Soluble CHOH • A calculated value: • NDSC = 100 - (%NDF+%CP+%Fat+%Ash) • NDF corrected for protein • 98% potentially digestible in the rumen • Rapidly fermented in the rumen • Diverse group and not easily measured • directly in feeds • Not all digested by mammalian enzymes

  25. Neutral Detergent Soluble CHOH • Includes: Organic acids, sugars, disaccharides, • oligosaccharides, starches, fructans, pectins, β-glucans • Rate and extent of digestion of each will vary • Organic acids provide no energy to rumen microbes • Sugars rapidly fermented in rumen • Starch digestion varies with source, processing and • other dietary components • ND soluble fiber usually rapidly fermented, but not at • low rumen pH • Want to estimate: • 1. Digestibility of the feed (available energy) • 2. Microbial growth (microbial protein)

  26. Neutral Detergent Soluble Fiber • Pectins • Galactans • β -glucans • Fructans – some lactic acid • Not digested by mammalian enzymes • Rapidly fermented in the rumen • 20 to 40% per hour • Produces mostly acetic acid – no lactate • Some byproduct feeds high in these soluble • fibers will be more rapidly fermented than • predicted from starch and free sugars

  27. Fiber analysis - NDF • NDF (insoluble residue) of high starch • feeds may be contaminated with starch • if not predigested with -amylase • Treat sample with heat stable -amylase • Pectin is associated with cell walls • However soluble in NDF solution • Pectin insoluble in ADF solution • Extract samples high in pectin with • NDF solution before ADF extraction

  28. Fiber analysis – (Van Soest) NDF (Insoluble residue) (Acid detergent solution) SolubleInsoluble (ADF) Hemicellulose Cellulose Protein Lignin Cutin Insoluble minerals (soil) Acid detergent insol N (ADIN) ADIN is unavailable protein - not digested in rumen or intestines

  29. Lignin Assays Klason Procedure (wood) Feed (72% H2SO4) Lignin Cellulose dissolved Residue contains more than lignin Protein, smaller molecular weight phenolics, cutin Acid Detergent (proteins removed) ADF (KMnO4) Lignin measured as weight loss (Includes tannins complexed with protein) Cellulose, Cutin, minerals as residue ADF (72% H2SO4) Cellulose measured as weight loss Lignin, cutin, minerals as residue

  30. Limitations of Fiber Analysis NDF and ADF should be done sequentially on the same sample. Not done this way in most commercial labs. Pectin solubilized in ND soln, but not soluble in the AD soln. Should report NDF and ADF on an organic basis. Minerals, especially soil, are not solubilized in the detergent solns. Detergent system developed to measure fiber fractions in plant materials, not animal derived feeds. Keratin proteins insoluble in ND soln. Add Na sulfite to dissolve keratinized proteins but also attacks lignin. Lipids interfere with NDF determination in feeds containing more than 10% lipids. ND is lipid soluble, so results in high NDF values.

  31. Starch Analysis Starch and cellulose both contain glucose. 1. Extract free sugars from the feed 2. Use enzymes specific for -linkage to digest starch. (Amylase and Amyloglucosidase) 3. Measure glucose released 4. Starch = glucose x .9 Release of glucose following treatment of grain with amyloglucosidase provides an indication of availability starch in the rumen.

  32. Carbohydrate Fractions in Feeds

  33. Carbohydrate Fractions in Feeds

  34. Carbohydrate Fractions in FeedsComputer Models • Available fiber = NDF – NDF protein – (lignin*2.4) • Sugars = NFC (nonfiber) – (starch + pectin) • NFC = NDSC • CHOH fractions • CHO A = sugars • CHO B1 = starch & pectin • CHO B2 = available fiber • CHO C = unavailable fiber (lignin*2.4)

  35. The Rumen as a Fermentation ChamberContribution of the animal to the symbiotic relationship: • Open and continuous system Open for inoculation from feed and water Continuous passage • Constant supply of nutrients Feed intake and feed retained in rumen and reticulum • Mixing of contents (Motility of rumen and reticulum) • Low oxygen concentration Oxidation reduction potential –150 to –350 mv • Control of moisture content (85 - 90%) • Temperature control (38 - 40 Co) • pH control (5.5 – 7.0) Saliva NaHCO3, VFA, less from HPO4= at rumen pH • Removal of end products (though acid concentrations are high) Eructation of gases and absorption of end products

  36. Microbiology of the Rumen • Relative stable population for a given feed (substrate) • Microorganisms adapted to rumen environment • Mostly obligate anaerobes • Bacteria - 1010 to 1011 cells/g • Protozoa - 105 to 106 cells/g • Fungi - 103 to 105 zoospores/ml

  37. Groups of Bacteria in the Rumen Habitats in the Rumen • Free-floating in the liquid phase • Maybe up to 50% of bacteria in rumen are free floating • Probably daughter cells of attached bacteria • Feed on solubles released by attached cells • 2. Associated with feed particles • Loosely associated with feed particles • Firmly adhered to feed particles • Up to 75% of bacteria associated with feed particles • Do most of the initial digestion of feed particles • 3. Associated with rumen epithelium • Similarities and differences from bacteria in the • rumen fluid • Suggested functions • Scavenging O2, tissue recycling, digest urea • 4. Other • Attached to surface of protozoa and fungi • Engulfed in protozoa

  38. Bacteria Associated with Feed Particles • Groups 2 and 3 • 75% of bacterial population in rumen • 90% of endoglucanase and xylanase activity • 70% of amylase activity • 75% or protease activity

  39. Adherence of mixed rumen bacteria to plant material. Protuberances from cells probably are binding factors.

  40. Bacterial Adhesion to Plant Tissues 1. Transport of bacteria to fibrous substrate Low numbers of free bacteria & poor mixing 2. Initial nonspecific adhesion Electrostatic, hydrophobic, ionic On cut or macerated surfaces 3. Specific adhesion to digestible tissue Ligands or adhesins on bacterial cell surface 4.Proliferation of attached bacteria Allows for colonization of available surfaces

  41. Mechanisms of Bacterial Adhesion • Cellulosome paradigm 2 MDa • 1. Large multicomponent complexes • Multifunctional, multienzyme • Polycellulosomes up to 100 MDa • 1. Form protuberances on cell surface • 2. Cellulose binding proteins • 3. Enzyme binding domains

  42. Attachment of Bacteria to Fibers Adherent cell Nonadherent cell Glycocalyx (on outer membrane of cell) Cellulose Cell Cell Digested and fermented Cellodextrins by adherent and nonadherent cells

  43. Cell Wall Structure of Bacteria Gram + Gram –

  44. Carbohydrate epitopes of bacterial glycolcalyx • Slime layer surrounding bacteria composed of • glycoproteins • Proteins and carbohydrates involved in adhesion • Ruminococcus flavefaciens, Fibrobacter succinogenes • Cellulose-binding domains of cellulolytic • enzymes • Cellulase has two functional domains • Catalytic domain - hydrolysis of glycosidic bonds • Binding domain - binds enzyme to cellulose • Fibrobacter succinogenes • Ruminococcus flavefaciens (maybe)

  45. Cellulosome – Multienzyme Complex

  46. Benefits of Bacterial Attachment • If attachment prevented or reduced digestion • of cellulose greatly reduced • Brings enzymes and substrate together in • a poorly mixed system • Protects enzymes from proteases in the rumen • Allows bacteria to colonize on the digestible • surface of feed particles • Retention in the rumen to prolong digestion • Reduces predatory activity of protozoa

  47. Cellulose Digesting Bacteria Predominant: Ruminococcus flavefaciens Gram+ cocci, usually in chains Ferments cellulose, cellobiose & glucose Produces acetic, formic, succinic, some lactic & H2 Fibrobacter succinogenes Gram– rod Ferments cellulose, cellobiose & glucose Produces acetic, formic & succinic Ruminococcus albus Gram– cocci Ferments cellulose, cellobiose, usually not sugars Produces acetic, formic, lactic, ethanol & H2 Strict anaerobes Tolerate narrow pH range (pH 6 to 7) Attach to feed particles

  48. Cellulose Digesting Bacteria • Secondary: • Eubacterium cellulosolvens Numbers usually low in rumen • Gram– rod • Ferments cellulose & soluble sugars • Produces mostly lactic acid • Butyrivibrio fibrisolvens Several strains in rumen • Gram– curved rod • Ferments cellulose (slow) & starch • Produces formic, butyric & lactic acids, ethanol & H2 • Strict anaerobes • Tolerate narrow pH range (pH 6 to 7) • Attach to feed particles

  49. Nutrient Requirements of Cellulose Digesters • Carbohydrates (source of energy) • Branched chain volatile fatty acids • Isobutyric, isovaleric, 2-methylbutyric • Needed for: • Synthesis of branched chain amino acids • Synthesis of branched chain fatty acids (phospholipids) • CO2 • Minerals (PO4, Mg, Ca, K, Na, probably other trace minerals) • Nitrogen • Mostly NH3 rather than amino acids • Biotin is stimulatory in pure cultures