1. Groups of Bacteria in the Rumen 1. Free-living in the liquid phase 2. Loosely associated with feed particles 3. Firmly adhered to feed particles 4. Associated with rumen epithelium 5. Attached to surface of protozoa and fungi

2. 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

3. 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

4. Attachment of Bacteria to Fibers Adherent cell Nonadherent cell Glycocalyx Cellulose CellCell Digested and fermented Cellodextrins by adherent and nonadherent cells

5. Mechanisms of Bacterial Adhesion 1. Large multicomponent complexes Cellulosomes 2. Filamentous extracellular material Pili-protein complex 3. Carbohydrate epitopes of bacterial glycocalyx 4. Enzyme binding domains

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

7. Microbiology of the Rumen Role of Protozoa Digestion and fermentation –Carbohydrates and proteins Ingest bacteria and feed particles More of a digestive process. Engulf feed particles and digest CHOH, proteins and fats.

8. Protozoa Contribution to the animal? Disappear when high grain diets are fed if pH not controlled Large mass Protein Produce some volatile fatty acids and NH 3 Make a type of starch that is digested by the animal. Some question how much of the protozal mass leaves the rumen.

9. Rumen Microorganisms Nutritional Requirements CO 2 Energy –End products from digestion of CHOH Fermentation of sugars Nitrogen –Ammonia (Majority of N needs) –Amino acids (nonstructural CHOH digesters) Minerals –Co, S, P, Na, K, Ca, Mg, Mn, Fe, Zn, Mo, Se

10. Rumen Microorganisms Nutritional Requirements - Continued Vitamins –None required in mixed cultures Nutrient requirements of pure cultures more complex

11. Energy Supply to Ruminants VFA70% Microbial cells10% Digestible unfermented feed20% Concentration of VFA in the rumen = 50 to 125 uM/ml

12. Rumen Digestion CelluloseHemicellulosePectinStarch Uronic acids Galactose CellobiosePentosesPentose Dextrose pathway Maltose Glucose

13. Fermentation in the Rumen Mostly fermentation of sugars from polysaccarides Rumen is an anaerobic habitat Disposal of reducing equivalents is a critical feature of anaerobic fermentation - Production of lactic acid and ethanol not extensively used in the rumen ­ Production of VFA major pathway ­ Hydrogenases produce hydrogen gas from reduced cofactors ­ Methanogens use hydrogen to produce methane

14. Rumen Digestion and Fermentation CO 2 VFA Degradable Rumen Microbial cells Feed microbes NH 3 CH 4 Heat Long-chain fatty acids H 2 S

15. ADP ATP NADP + NADPH Sugars Catabolism Biosynthesis Growth Maintenance Transport Microbial Metabolism VFA CO 2 CH 4 Heat

16. Microbial Interactions Secondary Fermentations CelluloseFibrobacter Cellulose fragments succinogenes Succinate + Acetate + Formate Selenomonas ruminantium Lactic acid + Propionate + Acetate + Formate + H 2 Megasphaera elsdenii Propionate + Acetate +H 2

17. Fermentation of Six Carbon Sugars (Glycolysis or Embden-Meyerhof- Parnas) Glucose Fructose Starch Glu-1-PGlu-6-P Fru-6-P Fru-1,6-bisP Dihydroxyacetone-P PhospoenolpyruvateGlyceraldehyde-3-P PyruvateGlycerol Predominant pathway for six carbon sugars (2 ATP + 2 NADH 2 )/Glucose 6 carbon Fructose bisphosphate aldolase 3 carbon

18. An Alternate Pathway of Glucose Metabolism (Entner-Doudoroff & Pentose) Gucose Glu-6-P 6-P-Guconolactone Ribulose-5-P + CO 2 6-P-gluconate Ribose-5-P 2-Keto-3-deoxy-6-P-gluconate PyruvateGlyceraldehyde-3-P Pyruvate 1 ATP, 1NADPH/Glucose Source of five carbon sugars NADP NADPH

19. Fermentation of Sugars Hexose Monophosphate Pathway Gucose Glu-6-P 6-P-Guconolactone Ribulose-5-P + CO 2 Xylulose-5-P Glyceraldehyde-3-P Ribose-5-P Acetyl-P Pyruvate Phosphoketolase Acetyl CoA Acetate Major pathway for five carbon sugars Source of five carbon sugars for biosynthesis 2 ATP, 2 NADPH, 1 NADH/Glucose NADP + NADPH

20. Acetic Acid 1. Pyruvate-formate lyase PyruvateAcetyl COAAcetate Formate 6HCH 4 + 2H 2 O 2. Pyruvate oxidoreductase (Most common pathway) FD FDH 2 (Flavin adenine dinucleotide) PyruvateAcetyl COAAcetate CO2 3 carbon2 carbon

21. Acetic Acid AcetylCoAAcetyl-P ADP Phosphotransacetylase Acetate kinase ATP Acetate

22. Butyric Acid PyruvateAcetyl COA Acetaldyhyde CO2COA Acetoacetyl CoA Ethanol Malonyl COA NADH+H Acetyl CoA NAD COAB-hydroxybutyryl COA Crotonyl COA NADH+H Butyryl COA NAD Acetate Butyrate Butyrate-PAcetyl COA FDFDH 2 CO 2 3 carbon 4 carbon ATP ADP

23. Propionic Acid 1. Succinate or dicarboxylic acid pathway Accounts for about 60% of propionate production ATP PyruvateOxaloacetateMalate CO2ADP Fumarate NADH+H Propionly COASuccinate NAD Propionate Methylmalonly COASuccinyl COA CoVit B 12 Pyruvate carboxylase Uses H 3 carbon

24. Propionic Acid 2. Acrylate pathway (mostly by Megasphaera elsdinii) NADH NAD PyruvateLactic acid Acrylyl COA NADH+H Propionate NAD Propionyl COA This pathway becomes more important when ruminants adjusted to high starch diets Uses H

25. Methane CO 2 + 4 H 2 CH 4 + 2H 2 O The above is the overall reaction. There are a number of enzymes and cofactors involved in combining CO 2 and H 2 to form CH 4 Formate + 3 H 2 CH 4 + 2H 2 O CO 2 + 2 H 3H 2 Methane is the predominant hydrogen sink in the rumen Methanogens use H 2 as a source of energy Methanogenic bacteria Methanobacterium ruminantium Vibrio succinogenes LyasePreferred pathway

26. Fermentation of Glucose and Other Sugars Glucose PyruvateCO 2 FormateLactateOxaloacetate 2H Acetyl-CoAMalate AcrylateFumarate Acetoacetyl CoA Succinate Methane AcetateButyrate Propionate Succinyl CoA Propionyl CoA Methylmalonyl CoA CoVit B12

27. Fermentation Balance Low Acetate (High grain) Glucose2 Acetate + 2 CO 2 + 8 H GlucoseButyrate + 2 CO 2 + 4 H Glucose2 Propionate + 2 [O] CO 2 + 8 H CH 4 + 2 H 2 O

28. Fermentation Balance High Acetate (High forage) 3 Glucose6 Acetate + 6 CO 2 + 24 H GlucoseButyrate + 2 CO 2 + 4 H Glucose2 Propionate + 2 [O] 3 CO 2 + 24 H 3 CH 4 + 6 H 2 O

29. Fermentation Low Acetate Net: 3 Glucose2 Acetate + Butyrate + 2 Propionate + 3 CO 2 + CH 4 + 2 H 2 O (Acetate:Propionate = 1 Methane:glucose =.33) High Acetate Net: 5 Glucose6 Acetate + Butyrate + 2 Propionate + 5 CO 2 + 3 CH 4 + 6 H 2 O (Acetate:Propionate = 3 Methane:Glucose =.60)

30. Energetic Efficiency VFA Production Heat of combustion kcal/mole kcal/mole of % of of acid glucose fermentedglucose Acetate 209.4 418.862.2 Propionate 367.2 734.4 109.1 Butyrate 524.3 524.377.9 Glucose 673.0

31. Effect of Diet VFA Ratios Forage:Grain-----Molar ratios----- Acetate PropionateButyrate 100:0 71.4 16.0 7.9 75:25 68.2 18.1 8.0 50:50 65.3 18.4 10.4 40:60 59.8 25.9 10.2 20:80 53.6 30.6 10.7

32. Branched-Chain Fatty Acids Propionyl CoA + Acetyl CoA Valerate ValineIsobutyrate + NH 3 + CO 2 LeucineIsovalerate + NH 3 + CO 2 Isoleucine2-methylbutyrate + NH 3 + CO 2 Fiber digesting bacteria have a requirement for branched-chain fatty acids.

33. Rumen Acidosis Animals gorge on grain Streptococcus bovis usually not present in high numbers (10 7 /ml) Grow very fast if sufficient glucose is present Double numbers within 12 min (up to 10 9 /ml) Produce lactic acid Lactobacillus ruminis & L. vitulinus also Produce lactic acid Methanobacter ruminantium in rumen (2 x 10 8 /ml) Sensitive to pH below 6.0 Have no capacity to utilize more H + Excess H + accumulates Some formation of ethanol Most is used to produce lactic acid

34. Rumen Acidosis Increased production of lactic acid Lactic acid poorly absorbed from rumen compared with other VFAs Lactic acid is a relatively strong acid pK: Lactic acid 3.08 A, P, & B 4.75 - 4.81 Very low rumen pH Might be pH 5.5 or less Both D and L isomers produced – D is poorly metabolized in the body Results in metabolic acidosis

35. Acidosis Subacute acidosis Decreased fiber digestion Depressed appetite Diarrhea Liver abscess Feedlot bloat Decreased milk fat Acute acidosis Laminitis Death

36. Acidosis Liver abscess Rumen epithelium not protected by mucous Acid causes inflammation and ulceration (rumenitis) Lactate promotes growth of Fusobacterium necrophorum Fus. necrophorum infects ruminal ulcers If Fus. necrophorum pass from rumen to blood, they colonize in the liver causing abscesses Incidence of liver abscess in feedlot cattle fed high concentrate diets (60+ % grain) ranges from 10 to 50+%. Feeding antibiotic Tylosin (10 g/ton of feed) reduces incidence of liver abscess in feedlot cattle.

37. Acidosis Laminitis (founder) If rumen pH is chronically acidic Epithelium releases metalloproteinases Cause tissue degradation If enter the blood stream causes inflammation of laminae above the hoof Feedlot bloat Starch fermenting bacteria secrete polysaccharides Produce a foam Gas trapped in foam Sudden death If large amounts of starch escape the rumen Overgrowth of Clostridium perfringens in the intestine Produce enterotoxin that might cause death

38. Acidosis Diarrhea Can be caused by some diseases Often related to the diet Extensive fermentation in the hind gut Produces acids Absorbed but might cause damage to gut wall Mucin secreted Mucin casts can be observed in feces Retention of water Produces gas Gas bubbles in feces

39. Managing Acidosis 1. Allow time for adjustment to diets with grain Gradually increase grain in the diet Program “step up” rations Limit intake until adjusted 2. Feed adequate roughage Effective fiber (eNDF) 3. Manage feed consumption Prevent gorging of high starch feeds “Read bunks” System for knowing when to change amount of feed offered 4. Feed ionophores

40. Adaptation to Grain Diets Two to Four Weeks Allow lactic acid utilizers to increase in numbers Megasphaera elsdenii Rarely present in rumen of hay fed animals Selenomonas ruminantium Propionibacter spp. Not major populations in the rumen Commercial preparations available Maintain protozoa (lost at low pH, <5.5) Ingest starch Engulf bacteria producing lactic acid Use glucose to make polysaccaride Maintain methanogens Use hydrogen Growth of rumen papillae Increased absorption of VFA

41. Action of Ionophores Transmembrane Flux OutIN (High Na +, low K + ) (High K +, low Na + ) ATP H + H + ADP + P i H + H + K + K + Na + Na + H + H + M M Uses energy

42. Gram Negative Ionophores Excluded M M Gram - positive Gram-negative

43. Effect of Ionophores Carbohydrates Sensitive toResistant toionophoreProduce more acetate & Hpropionate & less acetate CH 4

44. Ionophores - Continued Inhibit Result Rumminococcus albus Decreased acetate, Ruminococcus flavefaciens formate and CH 4 Butrivibrio fibrisolvens Increase Bacteroides succinogenes Increased propionate Bacteroides ruminicola Selanomonas ruminantium Also inhibit Streptococci Decreased lactate Lactobacilli production No effect Megasphaera Utilize lactate Selenomonas

45. Ionophores Monensin sodium ( Rumensin) 10 to 30 g per ton of 90% DM feed Feedlot: 27 to 28 g per ton Lasalosid ( Bovatec) 10 to 30 g per ton of 90% DM feed Feedlot: 30 g per ton Laidlomycin propionate ( Cattlyst) 5 to 10 g per ton of 90% DM feed Feedlot: 10 g per ton

46. Effects of Rumensin on Rumen Propionate Propionate production moles/day Roughage 5.96 Roughage + Rumensin 8.91 Concentrate 6.89 Concentrate + Rumensin 12.15

47. Predominant Microbial Populations 1. pH Fiber digesters less competitive in acid environment - Active pH >6.2 2. Ionophores Inhibits Gram + organisms 3. Rate of passage With increased rate of passage, organisms with longer generation time tend to be lost Protozoa Fungi