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.

Reading Assignments James B. Russell and J.L. Rychlik Factors that alter rumen microbial ecology. Science 292:1119 J. Miron, D. Ben-Ghedalia and M. Morrison Invited review: Adhesion mechanisms of rumen cellulolytic bacteria. J. Dairy Sci. 84:1294 Bryan A. White Bichemistry and genetics of microbial degradation of the plant cell wall. Rec. Adv. on the Nutr. Herbivores. pp J.L. Rychlik and J.B. Russell 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 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 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

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

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

Plant Carbohydrates Cell Content Cell 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

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.

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

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

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

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

Starch Amylose molecules consist of single mostly-unbranched chains with ,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.

Amylopectin Corn starch Potato starch

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

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

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 ( 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

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

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

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

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

Interaction of Lignin with
Polysaccharides Core lignin Non core lignin

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

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 6000C - Residue is ash

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 = (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.

Fiber analysis - Detergent solutions (Van Soest)
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

Neutral Detergent Soluble CHOH A calculated value:
NDSC = (%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

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)

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

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

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

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

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.

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.

Carbohydrate Fractions in Feeds

Carbohydrate Fractions in Feeds Computer 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)

The Rumen as a Fermentation Chamber Contribution 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 ( %) Temperature control ( 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

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

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

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

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

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

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

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

Cell Wall Structure of Bacteria
Gram + Gram –

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)

Cellulosome – Multienzyme Complex

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

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 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

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

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

Effects of Sugar on Cellulose Digestion Fibrobacter succinogenes Hiltner and Dehority, 1983
Added sugar was a source of readily available energy from 0 to 24 h. Subsequent drop in pH after 24 h limited the rate of cellulose digestion after 36 h.

Effect of pH on Cellulose Digestion Ruminococcus flavefaciens Hiltner and Dehority, 1983
Low pH (6.0)decreased rate of cellulose digestion, but had little effect on subsequent ability to digest cellulose. Similar results observed with Fibrobacter succinogenes.

Regulation of Rumen pH Dairy cow can produce up to 160 moles fermentation acids/d Buffers secreted in saliva Phosphate pK of 6.5 Bicarbonate pK of 6.4 Below 5.7 bicarbonate & phosphate not effective buffers At low pH VFA become most effective buffer Feeding effective fiber (forage) results in less acidic rumen Increased saliva flow – but osmotic pressures in rumen maintained close to that of blood and interstitial fluids so bicarbonate concentrations in the rumen do not vary much Only undissociated forms of VFA readily absorbed so rumen has to be acidic for an increase in VFA absorption More likely increased saliva flow increases fluid dilution rate As high as 20% per h when forages fed Compared with 5% per h when cattle fed grain Increased amounts of VFA washed out of rumen

Effects of pH Gradient Across Microbial Cell Membrane
Out In  pH XCOO— XCOO— H H+ XCOOH XCOOH ATP H+ ADP + Pi Two methods to handle Acidic pH: Use energy to pump H+ out of the cell. Anion of acid accumulates – toxic. Let intracellular pH decline to maintain a pH gradient. Enzymes have to tolerate low pH. S bovis produces lactic acid.

Hemicellulose Digesting Bacteria
Butrivibrio fibrisolvens Prevotella ruminicola Gram– non motile rod Digests starch, cellulose not digested Produces succincic, formic, acetic and some strains propionic Eubacterium ruminantium Gram+ non motil rod Ferments cellobiose, dextrins, maltose, glucose, fructose, lactose, sucrose and 5-carbon sugars Does not digest starch and cellulose Produces lactic, formic, acetic & butyric acids Ruminococcus flavefaciens Ruminococcus albus

Digestion of Forage Hemicellulose Pure cultures
Bromegrass Alfalfa Boot Bloom Prebloom Late bloom B. fibrisolvens 51.9/41.3 32.5/27.1 35.4/34.1 27.4/27.0 P. ruminicola 4.7/6.1 5.0/6.1 33.6/33.9 23.6/20.6 R. flavefaciens 56.6/23.0 34.7/17.1 44.6/10.1 23.6/0 F. succinogenes 77.3/3.0 62.0/2.4 62.1/0 28.7/0 R. albus 60.9/46.0 40.6/29.4 50.1/26.9 31.6/7.4 Degradation/Utilization

Pectin Digesting Bacteria
Lachnospira multiparus Mostly gram– motile curved rod Ferments pectin, glucose, fructose, cellobiose & sucrose Xylan, cellulose & starch not fermented Produces acetic, formic, lactic,ethanol & H2 Treponemes Anaerobic spiral organisms Ferment pectin, arabinose, inulin and sucrose Produces acetic and formic acids B. fibrosolvens P. ruminicola R. flavefaciens and R. albus can degrade pectins but not ferment the end products

Digestion of Forage Pectin Pure cultures
Bromegrass Alfalfa Boot Bloom Prebloom Late bloom B. fibrisolvens 55.3/49.7 46.7/45.3 67.5/57.3 54.4/53.1 P. ruminicola D31d 43.3/49.7 1.0/2.6 31.3/29.1 29.3/24.1 P. ruminicola 23 55.0/52.6 5.7/4.9 36.7/36.6 29.5/27.3 R. flavefaciens 71.3/29.8 35.5/8.1 70.5/30.4 54.3/26.6 L. multiparus 45.6/43.2 28.3/23.9 62.9/50.4 56.6/45.8 Degradation/Utilization

Starch Digesting Bacteria
Streptococcus bovis Gram+ spherical to ovoid in shape Hydrolyzes starch and ferments glucose Produces lactic acid, acetic, formic & ethanol 80 to 85% of CHOH fermented converted to lactic acid Tolerates low pH <5.0 and does not require low oxidation- reduction potential Rapid growth at low pH (25 to 30 min doubling time) Low numbers in the rumen of hay-fed animals & numbers remain low in grain adapted animals If too much starch is available to animals not adapted: pH drops, growth of S. bovis increases, production of lactic acid increased, further decrease in pH, loss of lactic acid utilizers (Megasphaera elsdenii), lactic acid accumulates, further decrease in pH, all resulting in acute lactic acidosis

Ruminobacter amylophilus Gram– non motile rod, some are coccoid to oval in shape Ferments starch & maltose Does not use glucose or cellobiose Produces acetic, formic, succinic & ethanol Nutritional interdependence Medium containing starch, glucose and cellobiose Inoculated with R. amylophilus, M. elsdenii & R. albus Initially only R. amylophilus grows but when growth stops cells undergo autolysis releasing amino acids M. Elsdenii require branched chain amino acids can grow M. Elsdenii produces branched chain fatty acids required by R. albus that can now grow

Succinomonas amylolytica Gram– motile rod Hydrolyzes starch and ferments dextrins, maltose & glucose Produces succinic acid and small amounts of acetic and propionic Selenomonas ruminantium Gram– motile curved rod Hydrolyzes starch and ferments soluble CHOH Produces lactic, acetic & propionic, formic, butyric & H2 Also produces an intracellular polysaccharide (glycogen) that is used when available energy is low B. fibrisolvens P. ruminicola

Sugar Utilizing Bacteria
Succinivibrio dextrinosolvens Gram – helicoidal rod Ferments sugars but does not hydrolyze starch, cellulose or xylans Produces succinic and acetic, formic & lactic Eubacterium ruminantium Gram+ non motile rod Ferments glucose, cellobiose and fructose Produces lactic, formic, acetic and butyric acids

Lactic Acid Utilizing Bacteria
Veillonella alcalescens Gram– coccus Does not ferment sugars but does ferment lactate Produces propionic and acetic acids Megasphaera elsdenii Ferments lactate, sugars, glycerol and some amino acids Produces propionic, acetic, butyric, valeric, caproic acids & H2 Increase in numbers during adaptation to grain

Methanogens CO2 + 2 H2 CH4 + 2 H2O Formic acid
Methanobrevibacter ruminantium Gram+ non motile cocobacilli Requires a low oxidation-reduction potential Methanomicrobium mobile Gram– rod Uses formic, CO2 and H2 Methanosarcina barkeri Methanobacterium formicicum Have been isolated from the rumen but thought To be of lesser importance

Acetogenic Bacteria Reduce CO2 at expense of hydrogen
2 CO2 CH3COOH + 2 H2O Bacteria present in rumen and hind gut of several species Do not compete with methanogens for hydrogen H2 threshold 100 times greater Only of significance if methanogens inhibited If active would conserve energy loss from the fermentation Fact they are present in the rumen indicates they might use other substrates

Rumen Protozoa Majority are ciliates Low numbers of flagellates
Obligate anaerobes 20 to 200 um length Very motile Not attached to feed particles Calves isolated from birth do not become faunated. Counts up to 106 cells/g – can be up to 50% of microbial mass

Rumen Protozoa Isotricha Starch, glucose, fructose, pectin Dasytricha
Starch, glucose, maltose, cellobiose Entodinium Starch, maltose Less use of cellobiose, sucrose & glucose Diplodinium Starch, pectin, maltose, glucose, sucrose Cellulose not always hydrolyzed Epidinium Starch, hemicellulose, cellobiose, sucrose, maltose Cellulose digested Ophryoscolex Pectin, starch Moderate digestion of cellulose

Role of Protozoa in the Rumen
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. Produce volatile fatty acids, CO2, H2 & NH3 Make a type of starch (amylopectin) that is digested by the animal.

Contribution Protozoa to the animal
Observations Numbers in increase when grain is added to forage diets – up to 40 to 60% concentrate Low rumen pH when high-grain diets are fed results in loss of protozoa (Numbers decline below pH 5.6) Only a slight decrease in digestion when defaunated No change in growth of the host animal Large mass Mass of protozoa might equal mass of bacteria Protein supply for animal Number of bacteria declines in faunated animals Some question how much of the protozoa mass leaves the rumen Estimates range from 50 to 85% lyses in the rumen Very sensitive to O2 and oxidation-reduction potential Digestive enzymes probably remain active in the rumen Provide nutrients for bacteria

Rumen Fungi Initially thought to be a flagellated protozoa. Later showed to contain chitin – representative of fungi Five genera have been found in the rumen: Neocallimastix Piromyces Caecomyces Orpinomyces Anaeromyces Anaerobic flagellated organisms Life cycle includes motile zoospores and non motile vegetative form Zoospores attach to feed particles followed by encystment and germination Counts range from 1.5X103 to 1.5x106 per g rumen contents

Role of Rumen Fungi Fungi can degrade cellulose, starch, xylan, hemicellulose & pectin Some evidence of esterases that free CHOH from lignin Ferments cellobiose, maltose, sucrose, glucose, fructose & xylose Digestion of wheat straw leaves in pure culture Neocall. Pirom. Caeco. DM, % 45.2 42.3 30.1 Cellulose, % 58.1 50.4 39.4 Hemicellulose, % 52.3 55.0 39.6 Pectin, % 20.5 47.3 16.3 Role of the fungi not clearly established in mixed cultures with bacteria. Bacteria seem to inhibit the fungi.

Composition of Rumen Microorganisms

Concentration of VFA in the rumen =
Energy Supply to Ruminants Contribution of the microbes to the symbiotic relationship: VFA % Microbial cells 10% Digestible unfermented feed % Concentration of VFA in the rumen = 50 to 125 uM/ml

Amino Acid Supply to Ruminants Contribution of the microbes to the symbiotic relationship
Protein in microbial mass 65% Undegraded feed proteins 30% Recycled endogenous proteins 5% Amino acid balance of microbial mass is superior to that from undegraded feed proteins when corn-based diets are fed.

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.

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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.

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