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The rumen

The rumen. The rumen. A fermentation vat converting plant materials to VFA’s, CH 4 , CO 2 NH 3 and microbial cells 38 to 42 o C pH normally 5.5 to 6.5 maintained by phosphate and bicarbonate ions in saliva rapid absorption of VFA’s and ammonia

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The rumen

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  1. The rumen

  2. The rumen • A fermentation vat converting plant materials to VFA’s, CH4, CO2 NH3 and microbial cells • 38 to 42oC • pH normally 5.5 to 6.5 maintained by • phosphate and bicarbonate ions in saliva • rapid absorption of VFA’s and ammonia • Bacteria growth is limited mainly by availability of energy yielding substrates and factors such as pH

  3. Rumen Characteristics • Rumen DM is 10-18% • Osmolarity is usually <400 mOsmol/kg • Oxidation-reduc pot is -0.35V • Gas phase 65% CO2 27% CH4 7% N2 0.6% O2 0.2% H2 0.01% H2S

  4. Rumen Characteristics Volatile fatty acids 66 mM Ac 23 mM Pr 10 mM But 2 mM higher c

  5. Why rumen envir. reduced • Oxygen taken in with the food and water is rapidly metabolized by aerobic organism • CO2 and CH4 displces O2 from the fluid • Rumen bact produces reducing substances such as sulfide

  6. Establishment of rumen microorganisms Days after birth 0 5 10 Fungi Caecomyces Piromyces Neocallimastix Facultative anaerobes Microaerophilic Cellulolytic population 15 20 25 50 Protozoa, Mycoplasma Entodinia Diplodinia Holotrichs

  7. Rumen Microbial Symbionts PROVIDE • EnergyVFA can provide up to 80% of the energy needs • Proteinmicrobes convert NPN into high quality protein • Vitaminssynthesis of B-complex and K vitamins • Detoxifying functions

  8. 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

  9. 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

  10. Substrate specificity • The basis for role assignation for bacteria; tremendous variation • Bulk of knowledge based on studies with cultivated species • poor representation ? • evolution during propagation ? • Polymer utilisers and utilisers of hydrolysis and fermentation products • specialists • generalists

  11. filamentous Bacterial forms rods cocci Spirochete budding and appendaged

  12. pairs groups chains Cocci may be found in different arrangements single

  13. Fibrobacter succinogenes • Gram - none motile rods but can become coccoid or oval on culture

  14. Fibrobacter succinogenes • Reportedly most widespread cellulolytic bacteria; about 5 % of all rumen isolates • Most extensive lignocellulosic degradation in vitro; ferment cellobiose and glucose • Do not hydrolyse xylan and make limited use of pentoses liberated from fiber digestion

  15. Fibrobacter succinogenes • Some strains hydrolyse starch, pectin and lactose • Not proteolytic; utilises NH3, amino acids and di-peptides as nitrogen (N) sources • Major fermentation products are A and S; but may also produce F, P and isovalerate (iV) • H2 and CO2 not produced • Sensitive to low pH; relatively resistant to antibiotics

  16. Ruminococcus species • Gram + non motile cocci • 2 cellulolytics: R. albus and R. flavefaciens • Most active degraders of plant fiber

  17. Ruminococcus species • Both exhibit a glycocalyx (attachment) and cell surface protrubances (enzyme complexes ?) • R. albus more numerous but not all strains are cellulolytic; produces yellow pigment when grown on cellulose • Both degrade xylan and ferment cellobiose but only R. albus ferments glucose

  18. Ruminococcus species • Not proteolytic; require NH3 for growth • Major fermentation products are A, and A, S for R. albus and R. flavefaciens respectively • H2 and CO2 also produced • Sensitive to monensin and low pH • Third species, R. Bromii an important starch digester

  19. Ruminobacter amylophilus • Oval to long Gram - rods • Dominant starch digesters hence, prevalent on grain diets

  20. Ruminobacter amylophilus • Specialists; utilise mainly starch and maltose • Intracellular starch hydrolysis • Highly proteolytic • Major fermentation products A, S, F (L) • H2 and CO2 not produced

  21. Streptococcus bovis • Gram + non motile oval to coccoid cells • Strict anaerobe and aero-tolerant strains; prevalent on grain diets • Most rapidly acting degraders of starch; ferment a wide range of hydrolysis products of plant polymers • Major fermentation product is L, (F,A) • Co2 produced

  22. Streptococcus bovis • Capable of growing at low pH (<5.0) • Play a major role in development of lactic acidosis • Proteolytic

  23. Succinomonas amylolytic • Gram + straight rods or coccobacilli motile by single flagellum • Specialists. Predominantly starch digesters; ferment glucose and maltose • Fermentation products mainly S, (A, P) • H2 and CO2 not produced

  24. Butyrivibrio fibrisolvens • Gram - B producing bacteria • Genetically very diverse; taxonomy undergoing re-definition • Occur singly, in pairs or chains; motile by means of polar flagellum; posses capsular material • Generalists. Important role in degradation of starch, xylan and pectin hence dominant on diets ranging from grain to alfalfa hay

  25. Butyrivibrio fibrisolvens • Proteolytic • Cellulolytic isolates; but ability not present in lab cultures • Major fermentation products: B, F and A (L and S) • H2 and CO2 also produced

  26. Lachnospira multipara • Pectin degrading Gram + curved rods • Motile by means of 1 lateral flagellum • Capsular material similar in composition to B. fibrisolvens • High concentrations on forage legumes • Major fermentation products: F, A and L. • H2 and CO2 also produced • Proteolytic

  27. Prevotella species • Gram + rods or cocci; numerous • 4 separate species now identified

  28. Prevotella species • Generalists. Degrade starch, xylan and pectins but not cellulose • Proteolytic and play critical role in uptake and fermentation of peptides • Produce A, F, S, P, isobutyrate (iB), other minor FA’s too

  29. Succinivibrio dextrinosolvens • Gram - helically twisted rods; polar flagellum

  30. Succinivibrio dextrinosolvens • Hydrolyse dextrin (starch diets) and grass levans • Some strains ferment end products of plant cell wall degradation e.g. cellobiose • Major fermentation products are A, S and F (L)

  31. Anaerovibrio lipolyptica • Gram - rods motile normally by single polar flagellum; some isolates have multiple flagella • Specialists. 3 key properties • hydrolyse lipids, utilise lactate and ferment fructose • Fermentation products: P, S, A (L) • H2 and CO2 also produced

  32. Utilisers of hydrolysis products

  33. Selenomonas ruminantium • Distinctive Gram - curved rods; linear array of upto 16 flagella on concave side

  34. Selenomonas ruminantium • Prevalent on cereal grain diets • Proteolytic • Utilises mainly sugars; some strains hydrolyse starch; most strains are unable to degrade pectins and xylans • Some strain utilise lactate for growth • Fermentation products: either L or P and A when grown on high and low concentrations of glucose, respectively • H2 and CO2 also produced

  35. Megasphaera elsdenii • Gram - non motile cocci occurring in pairs and chains of upto 20 cells • Young animals and animals of high grain diets • Utilises wide range of degradation products such as sugars but not polymers • Wide range of fermentation products depending on substrate

  36. Megasphaera elsdenii • Ferments L to mainly B, P, iB, V • Ferments glucose to mainly caproate and F with some A, P, B and V • H2 and CO2 also produced • Important roles include production of branched chain fatty acids from amino acids

  37. Methanogenesis • Exclusively in anaerobic environments; 5 to 110oC; fresh water to salt water; • Terminal step in carbon flow • Methanogens: archaea not bacteria • Strict anaerobes: most difficult rumen microbes to culture in vitro • O2 conc of 10-56 required for CH4 production and growth • require simple molecules for growth

  38. Rumen methanogens • Convert 800 L H2 to 200 L CH4 in a 500 kg dairy cow in a day • 9 to 25 % associated with protozoa • Maintain low H2 partial pressures in rumen and thus allow for reduced cofactors in metabolic pathways (NADH) to be oxidised (NAD) • VFA’s are the main products of this process

  39. Rumen methanogens • Methanobrevibacter ruminantium* Methanobacterium formicicum and Methanomicrobium mobile (rods) • CH4 from H2, CO2 and formate • Methanosarcina barkeri (cocci) • CH4 from H2, CO2 (slow growth) but mainly from acetate, methanol and methylamines

  40. Isolation of rumen bacteria • Usually total and cellulolytic bacteria • amylolytic, xylanolytic and saccharolytic e.t.c. • Sampling • Cannulated animals • Multi-site sampling • Minimize headspace • Homogenise under CO2; filter through cheese cloth into pre-warmed (ca 39oC) thermos

  41. Viable cell count 1 ml 1.dilute sample 9 ml 107 106 105 104 1000 100 10 cell no/ml 2. plate out 0.1 ml

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