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The rumen. yA fermentation vat converting plant materials to VFA’s, CH 4, CO 2 NH 3 and microbial cells y38 to 42 o C ypH normally 5.5 to 6.5 maintained.

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Presentation on theme: "The rumen. yA fermentation vat converting plant materials to VFA’s, CH 4, CO 2 NH 3 and microbial cells y38 to 42 o C ypH normally 5.5 to 6.5 maintained."— Presentation transcript:

1 The rumen

2 yA fermentation vat converting plant materials to VFA’s, CH 4, CO 2 NH 3 and microbial cells y38 to 42 o C ypH normally 5.5 to 6.5 maintained by xphosphate and bicarbonate ions in saliva xrapid absorption of VFA’s and ammonia yBacteria growth is limited mainly by availability of energy yielding substrates and factors such as pH

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6 Rumen Characteristics zRumen DM is 10-18% zOsmolarity is usually <400 mOsmol/kg zOxidation-reduc pot is -0.35V zGas phase 65% CO 2 27% CH 4 7% N 2 0.6% O 2 0.2% H 2 0.01% H 2 S

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

9 Why rumen envir. reduced z Oxygen taken in with the food and water is rapidly metabolized by aerobic organism z CO 2 and CH 4 displces O 2 from the fluid z Rumen bact produces reducing substances such as sulfide

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11 Establishment of rumen microorganisms 05 152025 10 Days after birth Facultative anaerobes Microaeroph ilic Celluloly tic populati on Fungi Caecomyces Piromyces Neocallimastix Protozoa, Mycoplasma EntodiniaDiplodiniaHolotrichs 50

12 Rumen Microbial Symbionts PROVIDE zEnergy VFA can provide up to 80% of the energy needs zProtein microbes convert NPN into high quality protein zVitamins synthesis of B-complex and K vitamins zDetoxifying functions

13 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

14 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

15 Substrate specificity yThe basis for role assignation for bacteria; tremendous variation yBulk of knowledge based on studies with cultivated species xpoor representation ? xevolution during propagation ? yPolymer utilisers and utilisers of hydrolysis and fermentation products xspecialists xgeneralists

16 Bacterial forms cocci rods Spirochete filamentous budding and appendaged

17 Cocci may be found in different arrangements single pairs groups chains

18 Fibrobacter succinogenes zGram - none motile rods but can become coccoid or oval on culture

19 Fibrobacter succinogenes zReportedly most widespread cellulolytic bacteria; about 5 % of all rumen isolates zMost extensive lignocellulosic degradation in vitro; ferment cellobiose and glucose zDo not hydrolyse xylan and make limited use of pentoses liberated from fiber digestion

20 Fibrobacter succinogenes ySome strains hydrolyse starch, pectin and lactose yNot proteolytic; utilises NH 3, amino acids and di-peptides as nitrogen (N) sources yMajor fermentation products are A and S; but may also produce F, P and isovalerate (iV) yH 2 and CO 2 not produced ySensitive to low pH; relatively resistant to antibiotics

21 Ruminococcus species zGram + non motile cocci z2 cellulolytics: R. albus and R. flavefaciens zMost active degraders of plant fiber

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

23 Ruminococcus species zNot proteolytic; require NH 3 for growth zMajor fermentation products are A, and A, S for R. albus and R. flavefaciens respectively yH 2 and CO 2 also produced zSensitive to monensin and low pH zThird species, R. Bromii an important starch digester

24 Ruminobacter amylophilus zOval to long Gram - rods zDominant starch digesters hence, prevalent on grain diets

25 Ruminobacter amylophilus zSpecialists; utilise mainly starch and maltose zIntracellular starch hydrolysis zHighly proteolytic zMajor fermentation products A, S, F (L) yH 2 and CO 2 not produced

26 Streptococcus bovis zGram + non motile oval to coccoid cells zStrict anaerobe and aero- tolerant strains; prevalent on grain diets zMost rapidly acting degraders of starch; ferment a wide range of hydrolysis products of plant polymers zMajor fermentation product is L, (F,A) yCo 2 produced

27 Streptococcus bovis zCapable of growing at low pH (<5.0) zPlay a major role in development of lactic acidosis zProteolytic

28 Succinomonas amylolytic zGram + straight rods or coccobacilli motile by single flagellum zSpecialists. Predominantly starch digesters; ferment glucose and maltose zFermentation products mainly S, (A, P) yH 2 and CO 2 not produced

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

30 Butyrivibrio fibrisolvens zProteolytic zCellulolytic isolates; but ability not present in lab cultures zMajor fermentation products: B, F and A (L and S) yH 2 and CO 2 also produced

31 Lachnospira multipara zPectin degrading Gram + curved rods zMotile by means of 1 lateral flagellum zCapsular material similar in composition to B. fibrisolvens zHigh concentrations on forage legumes zMajor fermentation products: F, A and L. yH 2 and CO 2 also produced zProteolytic

32 Prevotella species zGram + rods or cocci; numerous z4 separate species now identified

33 Prevotella species zGeneralists. Degrade starch, xylan and pectins but not cellulose zProteolytic and play critical role in uptake and fermentation of peptides zProduce A, F, S, P, isobutyrate (iB), other minor FA’s too

34 Succinivibrio dextrinosolvens zGram - helically twisted rods; polar flagellum

35 Succinivibrio dextrinosolvens zHydrolyse dextrin (starch diets) and grass levans zSome strains ferment end products of plant cell wall degradation e.g. cellobiose zMajor fermentation products are A, S and F (L)

36 Anaerovibrio lipolyptica zGram - rods motile normally by single polar flagellum; some isolates have multiple flagella zSpecialists. 3 key properties yhydrolyse lipids, utilise lactate and ferment fructose zFermentation products: P, S, A (L) yH 2 and CO 2 also produced

37 Utilisers of hydrolysis products

38 Selenomonas ruminantium zDistinctive Gram - curved rods; linear array of upto 16 flagella on concave side

39 Selenomonas ruminantium yPrevalent on cereal grain diets yProteolytic yUtilises mainly sugars; some strains hydrolyse starch; most strains are unable to degrade pectins and xylans ySome strain utilise lactate for growth yFermentation products: either L or P and A when grown on high and low concentrations of glucose, respectively xH 2 and CO 2 also produced

40 Megasphaera elsdenii zGram - non motile cocci occurring in pairs and chains of upto 20 cells zYoung animals and animals of high grain diets zUtilises wide range of degradation products such as sugars but not polymers zWide range of fermentation products depending on substrate

41 Megasphaera elsdenii yFerments L to mainly B, P, iB, V yFerments glucose to mainly caproate and F with some A, P, B and V xH 2 and CO 2 also produced zImportant roles include production of branched chain fatty acids from amino acids

42 Methanogenesis yExclusively in anaerobic environments; 5 to 110 o C; fresh water to salt water; yTerminal step in carbon flow yMethanogens: archaea not bacteria yStrict anaerobes: most difficult rumen microbes to culture in vitro xO 2 conc of 10 -56 required for CH 4 production and growth xrequire simple molecules for growth

43 Rumen methanogens zConvert 800 L H 2 to 200 L CH 4 in a 500 kg dairy cow in a day z9 to 25 % associated with protozoa zMaintain low H 2 partial pressures in rumen and thus allow for reduced cofactors in metabolic pathways (NADH) to be oxidised (NAD) yVFA’s are the main products of this process

44 Rumen methanogens zMethanobrevibacter ruminantium* Methanobacterium formicicum and Methanomicrobium mobile (rods) yCH 4 from H 2, CO 2 and formate zMethanosarcina barkeri (cocci) yCH 4 from H 2, CO 2 (slow growth) but mainly from acetate, methanol and methylamines

45 Isolation of rumen bacteria zUsually total and cellulolytic bacteria yamylolytic, xylanolytic and saccharolytic e.t.c. zSampling yCannulated animals yMulti-site sampling yMinimize headspace zHomogenise under CO 2 ; filter through cheese cloth into pre- warmed (ca 39 o C) thermos

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50 Viable cell count 1.dilute sample 1 ml 9 ml 10 7 10 6 10 5 10 4 1000 10010 cell no/ml 2. plate out 0.1 ml

51 Isolation of bacteria ySolid associated microorganisms may be detached using methyl cellulose (or tween-80 followed by chilling for 6 - 8 h) zDilute sample in a 10 fold dilution series using an anaerobic diluent zRole tube method employed for all but cellulolytic zAnaerobic and sterile procedures

52 Enumeration of bacteria xFor cellulolytics: MPN procedure. 1 ml from 10 -6 to 10 -8 dilutions inoculated in triplicate into 4 ml anaerobic growth media (39 o C) in Hungate tubes, containing a strip of filter paper as sole C source. Incubate at 39 o C for up to 2 weeks Numbers determined statistically based on numbers of tubes containing digested filter paper at each dilution xFor other groups, Same procedure but inoculate (10 -6 to 10 -9 ) into media containing 1.8 % agar(47 o C) and either a mixture of energy sources for total counts, or just the specific substrate for each sub group. Spin tubes in ice slurry to solidify agar Incubate at 39 o C for 2 to 3 days and count colonies (/ml rumen fluid)

53 Water + nutrients energy source;organic, inorganic or light carbon, nitrogen source Agar can be added to make the medium semi-solid and poured into Petri dishes Culture media 1. Biochemical requirements for growth

54 2. Physical and environmental requirements for growth oxygen concentration pH (hydrogen ion concentration) temperature

55 Growth media yRumen fluid containing Clarified autoclaved rumen fluid (10 to 20 %) Mineral solution 1: KH 2 PO 4 Mineral solution 2: NaCl, (NH 4 ) 2 SO 4, CaCl 2, MgSO 4 and microelements VFA and Vitamin solutions Resazurin Energy substrate(s) as required Casein hydrolysate (amino acids and peptides) pH adjusted to 6.5 xBoil; replace headspace with stream of CO 2 Reducing agent and bicarbonate Dispense in to tubes, seal and autoclave

56 Growth media zChemically defined media yNo rumen fluid; nutrient requirements usually met by rumen fluid must be supplied e.g phenylpropanoic acid for R. albus and 1, 4-napthoquinone for S. dextrinosolvens.

57 Anaerobic glove box yPermits growth of anaerobes on Petri dishes, hence the use of techniques such as replica plating rapid identification systems such as strip tests xavoids exposure of bacteria to molten agar (47 o C) ySufficient CO 2 in atmosphere to xmaintain medium pH xfor nutrition of microbes requiring CO 2 y95 % CO 2 / 5% H 2 ok for most rumen bacteria; for methanogens, 80% H 2 /20% CO 2

58 An anaerobic glove box

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60 Maintenance of cultures zStorage xat 4 o C can retain viability for up to 1 month (storage usually for up to 1 week) xof 12 to 24 h cultures in 20 % glycerol for up to 1 year xunder liquid nitrogen (-196 o C); followed by rapid thawing in water at 32 to 35 o C. Very successful. xFreeze drying. Reports of viability for up to 5 years

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64 Conversion of carbohydrates to pyruvate

65 Methanogenesis yThree major catabolic pathways xCO 2 reduction* CO 2 and H 2 to form CH 4 ; electrons mainly from free H2 but also formate xMethyltrophic pathway reduce CH 3 groups from compounds methanol, trimethylamine xAceticlastic pathway Split acetic acid and reduce CH 3 to CH 4


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