The role of ciliate protozoa and fungi in the rumen digestion of plant cell walls

Animal Feed Science and Technology, 10 (1983/84) 121--143 Elsevier Science Publishers B.V,, Amsterdam -- Printed in The Netherlands

121

THE ROLE OF C I L I A T E P R O T O Z O A A N D F U N G I I N THE RUMEN DIGESTION OF PLANT CELL WALLS

COLIN G. ORPIN Agricultural Research Council, Institute of Animal Physiology, Babraham, Cambridge CB2 4 A T (Gt. Britain) (Accepted for publication 3 August 1983)

ABSTRACT Orpin, C.G., 1984. The role of ciliate protozoa and fungi in the rumen digestion of plant cell walls. Anita. Feed Sc£ Teehnol., 10: 121--143. The major constituents of plant cell walls and the enzymology of cell wall degradation are reviewed. The ability of ciliates and rumen fungi to invade plant tissues and their association with these tissues are discussed. The properties of the ciliate-bacterium ecosystem are considered in relation to the enzymic degradation of the engulfed plant particles and to the production of extracellular plant cell wall degrading enzymes. Quantitative, qualitative and enzymic aspects of plant tissue fermentation in vitro by rumen phycomycete fungi are reviewed.

INTRODUCTION T h e ciliate p r o t o z o a were the first r u m e n m i c r o o r g a n i s m s t o be described and are t h e m o s t active and o b v i o u s w h e n r u m e n c o n t e n t s are observed in a light m i c r o s c o p e . F o r this reason, t h e y were c o n s i d e r e d essential t o t h e h o s t animal, b u t in r e c e n t y e a r s e x p e r i m e n t s have s h o w n t h a t r u m i n a n t s can survive and g r o w w i t h n o r u m e n ciliate p o p u l a t i o n (e.g. P o u n d e n a n d Hibbs, 1 9 5 0 ; A b o u A k k a d a and E1 Shazly, 1 9 6 4 ; Williams a n d Dinussen, 1 9 7 3 ) . H o w e v e r , the ciliates m a y c o n t a i n up to 40% o f t h e m i c r o b i a l nitrogen u n d e r f a v o u r a b l e c o n d i t i o n s and a c c o u n t for u p t o 60% o f t h e t o t a l f e r m e n t a t i o n p r o d u c t s ( H u n g a t e , 1 9 6 6 ) . U n d e r these c o n d i t i o n s t h e y are o f clear significance in r u m e n m e t a b o l i s m . T h e ciliates o f t h e r u m e n b e l o n g to the families O p h r y o s c o l e c i d a e and t h e Isotrichidae. T h e i r p o p u l a t i o n d e n s i t y is n o r m a l l y in t h e range 1 0 4 - - 1 0 6 m1-1, o f w h i c h the m a j o r i t y are m e m b e r s o f t h e O p h r y o s c o l e c i d a e . M u c h i n f o r m a t i o n on r u m e n citiates including d e s c r i p t i o n s a n d p h o t o g r a p h s can be f o u n d in H u n g a t e ( 1 9 6 6 ) , Clark ( 1 9 7 7 ) , I m a i a n d T s u n o d a ( 1 9 7 2 ) a n d Coleman (1979, 1980). T h e p h y c o m y c e t e fungi are a r e c e n t l y d i s c o v e r e d g r o u p o f r u m e n m i c r o organisms. Their life cycles consist o f an a l t e r n a t i o n o f g e n e r a t i o n s b e t w e e n 0377-8401/84/$03.00

© 1984 Elsevier Science Publishers B.V.

122 a motile zoospore stage free in rumen liquor, and a non-motile, vegetative reproductive stage which occurs on the digesta particles (Orpin, 1975, 1976, 1977a, b; Bauchop, 1979). At least three species are k n o w n and all ferment plant cell wall polysaccharides as sole sources of carbon and energy. Because of the intimate association of the rumen p h y c o m y c e t e s with the digesta particles, it has n o t y e t been possible to arrive at accurate figures for their biomass in the rumen, but it is estimated that under favourable conditions, up to 8% of the microbial biomass may consist of p h y c o m y c e t e fungi (Orpin, 1981).

ORGANISATION OF THE CELL WALLS OF HIGHER PLANTS The structure and chemical composition of plant cell walls varies considerably with the plant species concerned, the variety, the age of the plant, and the type of cell. However, three major regions of the cell wall are usually recognised; the primary wall, the secondary wall and the middle lamella. The primary wall is formed during the growth of the plant cell and the secondary wall is laid down inside the primary wall when cell growth has ceased, to form a strong, composite, cell wall. Between adjacent cells lies the middle lamella, composed of compounds which cement the cells together. The general structure of primary cell walls o f both m o n o c o t and dicot plants has been envisaged for some years to be composed of fibres of cellulose embedded in a mixture of polysaccharides, with glycoproteins present. The composition of the polysaccharide mixture in m o n o c o t s differs significantly from that found in dicots (Darvill et al., 1981), as does the extent o f the middle lamella, but the general architecture of the cell wall is considered to be similar (Albersheim, 1975). When the primary wall has ceased growing, the secondary wall is laid down, with concomitant thickening of the fibres in the primary wall and the deposition of lignin. The middle lamella is composed chiefly of pectic polysaccharides with, between mature cells, l i g n i n . Primary and secondary walls differ in their architecture. Primary walls consist of two layers, the outer containing predominantly longitudinal, the inner predominantly transverse, cellulose fibres (Rogers and Perkins, 1968), embedded in a hemicellulose matrix. The secondary wall is often thicker and more dense than the primary, with three layers of cellulose fibres embedded in a hemicellulose--lignin matrix (Wooding and Northcote, 1964). The fibres are deposited in a helical fashion around the cell axis, and can be readily distinguished by the direction of the helix and the angle of orientation (Wood, 1970). Much information on the structure and composition of plant cell walls can be found i n Rogers and Perkins (19.68), Bailey (1973), Albersheim (1975) and Darvill et al. (1981), and on cell wall synthesis in Robinson (1975).

123 STRUCTURE AND ENZYMIC DEGRADATION OF CELLULOSE Cellulose consists of linear chains of ~-l,4-1inked anhydro-D-glucopyranose units bound together by hydrogen bonding and van der Waals' forces. The precise way in which these chains are built up into larger units in the cell wall is still subject to some conjecture. The most widely accepted interpretation of experimental results suggests that the linear/3-1,4-D-glucan chains periodically form highly-ordered ribbon-like crystalline micelles consisting of about 80 glucan chains (Albersheim, 1975), with amorphous regions between the crystalline zones. These microfibrils are about 4.5 × 8 . 5 n m in cross-section (Preston, 1974), and are bound together to form fibres 25--30 nm wide; these in turn are bound together in groups of about 250 to form fibres of great tensile strength. Although cellulose molecules isolated from plant material have a degree of polymerisation of as high as 100 000 (Siegel, 1963), it is likely t h a t in vivo they have no natural ends except when the fibre is physically separated from its synthetic enzymes (Darvfll et al., 198t). The degree of crystallinity reported for cellulose depends on the source of the material, the isolation process and the m e t h o d of estimation of crystallinity (Rogers and Perkins, 1968; Wood, 1970), and may be as high as 82% in c o t t o n fibres and as low as 32% in cellulose from pine-tree pulp. Purified cellulose invariably contains traces of other sugar residues. This is probably due to the trapping (by absorption) of hemicellulose chains between cellulose fibrils in the formation of larger fibres (Darvill et al., 1981). The enzymic mechanisms involved in cellulose catabolism have been studied in m a n y micro-organisms, but in the past much confusion has arisen owing to problems with the selection and use of suitable substrates, nomenclature, estimation methods, and the use of enzymes isolated after days of existence in culture media which we now k n o w may be modified by their environment. The enzymology of cellulose catabolism has been most closely studied in species of fungi, particularly Trichoderma reesii and Sporotrichum pulverulentum. Several enzymes are involved, and these can be classified in three distinct groups, namely endo-l,4-fi-glucanases, exo-l,4-fi-glucanases and 1,4-~:glucosidases (Table I). Endo-l,4-~-glucanases attack glucosidic linkages in cellulose chains randomly, to give water-soluble cellodextrins. Exo-1,4-~-glucanases attack the cellulose chains from the non-reducing end of the molecule, splitting off cellobiose or glucose units depending on the particular enzyme. The 1, 4-~3-glucosidases then hydrolyse the cellobiose and water-soluble cellodextrins to glucose, and cellobionic acid to glucose and gluconolactone (Eriksson, 1982). Thus, for the hydrolysis of an intact cellulose fibre, it is necessary for the endo-l,4-glucanase to provide glucan chains with non-reducing end groups before the exo-l,4-glucanase can act. The 1,4-fi-glucosidases remove the cellobiose and low molecular weight

E .C.3.2.1.4 E.C.3.2.1.73 E.C.3.2.1.74 E.C.3.2.1.91 E.C.3.2.1.8 E.C.3.2.1.37 E.C.3.2.1.55 E.C.3.1.1.11 E.C.3.2.1.15 E.C.3.2.1.67 E.C.4.2.2.10

1,4-(1,3 :1,4) -/LD-glucan 4 -glucanohydrolase 1, 3-1,4-/3-D-glucan 4-glucanohydrolase 1, 4-/3-D-glucan glucohydrolase 1,4-~-D-glucan cellobiohydrolase 1,4-~-D-xylan xylanohydrolase 1,4-/3-D-xylohydrolase a-L -Arabinofuranoside arabinofuranohydrolase Pectin pectylhydrolase Poly(1,4-~-D-galacturonide)glycanohydrolase Poly(1,4-~-D-galacturonide)galacturonohydrolase Poly(methoxygalacturonide)lyase

Cellulase Lichenase Exo-1,4-/~-D-glucosidase Exocellobiohydrolase

Endo-1,4-/~-D-xylanase Exo-1,4-/3-D-xylanase a-L-Arabinofuranosidase

Pectinesterase Polygalacturonase Exopolygalacturonase Pectin lyase

Enzymes known to function in the degradation of plant cell wall polysaccharides

TABLE I

b.a t'~

125 cellodextrins from the system. To degrade crystalline cellulose, both exoand endo-l,4-/3-glucanases are required; neither can degrade it extensively alone. Amorphous cellulose is, however, attacked by both exo- and endo1,4-/3-glucanases acting alone. The precise number of enzymes required to completely hydrolyse native (crystalline) cellulose has y e t to be clearly defined. For example, eight different proteins, five with endo-l,4-fi-glucanase activity, one with exo1, 4-fi-glucanase activity and two with 1, 4-~-glucosidase activities have been isolated from culture supernatants of Sporotrichum pulverulentum growing on cellulose. A similar array of enzymes, though different in some details, is f o u n d in culture supernatants of Trichoderma reeseii growing on cellulose, although Guntzali and Brown (reported in Eriksson, 1982) found fewer enzymes were produced in short-term experiments using glucose-grown cells induced to produce cellulase-degrading enzymes after exposure to sophorose. These workers f o u n d t h a t only a single endo-l,4-/3-glucanase and two exo-1, 4-f~-glucanases were produced under these conditions, which prompted the suggestion t h a t the multiplicity of endo- and exo-l,4-/3glucanases f o u n d in the supernatants of cultures growing on cellulose may be due to partial proteolysis by proteases also present in the culture supernatant. Nakayama et al. (1976) showed that partial proteolysis of an endo1,4-glucanase from Trichoderma viride yielded enzymes with an altered substrate specificity and protein structure, so proteolysis of enzymes in culture supernatants cannot be ruled out. In addition to the synergism between e x o - a n d endo-l,4-~-glucanases in Trichoderma koningii, Wood and McCrae (1982) have shown synergism between these enzymes and two 1,4-~-glucosidases, the removal of cellobiose removing p r o d u c t inhibition of the reactions. In the rumen, Beveridge and Richards (1975) showed that crystalline and amorphous cellulose were digested at similar rates, suggesting t h a t in this complex environment, the crystallinity of the cellulose did n o t limit its rate of digestion. The situation may, however, be different within protozoal cells, where only enzymes originating from within the ciliate, and any other enzymes absorbed on the plant particle surface prior to engulfment, would be present. In addition to the enzymes described above, an additional enzyme the "sweIling f a c t o r " (see Wood, 1970, for review) may be implicated, but more evidence is required to resolve this point. It is likely that the "swelling f a c t o r " may be no more than synergism b e t w e e n the enzymes of the cellulase complex.

HEMICELLULOSES IN PLANT CELL WALLSAND THEIR ENZYMIC DEGRADATION The term "hemicellulose" was originally given to material extractable by alkali from plant tissues t h a t had previously been extracted with hot

126 and cold water. More recently, the name has been applied to material extractable b y alkali after lignin and pectin have been removed (Rogers and Perkins, 1968). Hemicelluloses are a mixture of homo- and heteroglycans, their composition varying with plant species. Some workers have classified them as neutral (hemicellulose A) or acidic (hemicellulose B). The major hemicelluloses are xyloglucans, mannans, glucomannans, arabinogalactans, arabinoxylans in dicots (Whistler and Richards, 1970) and xylans in monocots (Darvill et al., 1981). In dicots, the major hemicellulose of primary cell walls is xyloglucan. The xyloglucan consists of ~-l,4-1inked glucan chains with xylose, arabinose and xylosylgalactosylfucoside side-chains. In monocots, xyloglucans also occur, b u t the major hemicellulose is ~-l,4-xylan, This usually has 4-0-methylglucuronosyl residues attached at C2 (Aspinall and McGrath, 1966), and L-arabinofuranosyl residues or longer side-chains of D-galactosyl, D-xylosyl, L arabinosyl and glucuronosyl residues attached to C3 of xylose units in the main chain (Darvfll et al., 1981). The xylose chains in these hemicelluloses are mainly linear, b u t may be linked to other xylose chains by links other than 1-> 4ft. Phenolic acids occur ester-linked to the xylose chains and the xylose chains m a y be stabilised by water (Nieduszynski and Marchessault, 1971). A review of the hemicelluloses found in fodder plants may be found in Bailey (1973), and in primary cell walls in Darvill et al. (1981). The enzymology of hemicellulose degradation has received less attention than that of cellulose degradation and some enzymes involved are shown in Table I. Clearly, enzymes are needed to cleave the glycan chains into smaller oligosaccharides, and enzymes are needed to cleave specific glycosyl linkages and remove side-chains. Howard et al. (1960) showed two enzymes to be required for the degradation of xylans to D-xylose, and Walker (1967) purified a xylanase from mixed rumen micro-organisms which showed no xylobiase activity b u t hydrolysed higher xylo-oligosaccharides. Endo-l,4-figlucanases are known to cleave the glucan chain in xyloglucans (Darvill et al., 1981).

PECTIC POLYSACCHARIDES AND THEIR ENZYMIC DEGRADATION Pectic polysaccharides are found in all plant tissues and, as with other cell wall components, the proportion depends on the t y p e of cell, plant species and m e t h o d of estimation. Most of the pectic polysaccharides are found in the middle lamella and, in smaller quantities, in the primary cell wall. Dicot primary cell walls are, however, c o m p o s e d of a b o u t 35% pectic polysaccharides, whilst m o n o c o t s contain, at most, 8--9% (Darvill et al., 1981). Data on the m o n o c o t pectic polysaccharides are limited, and it is n o t possible to compare the chemical structures found in the two groups of plants.

127 Pectic polysaccharides of dicots are rich in galacturonosyl residues (Rogers and Perkins, 1968) and include pectin, arabinogalactan and rhamnogalacturonan (Darvill et al., 1981). The basic building unit in rhamnogalacturonan is D-galacturonic acid, in which the linear part of the molecule is (~-l,4linked polygalacturonic acid with side chains containing galactose and rhamnose residues, with rhamnose occurring in the main chain in ~-l,2linkage (Talmadge et al., 1973). Pectin, a-l,4-1inked polygalacturonic acid, with some degree of methyl esterification of C6, occurs in the middle lamella. There are two main classes of pectolytic enzymes known. These are the pectin esterases and the depolymerising enzymes (Table I). Pectin esterase hydrolyses the methyl ester linkages to convert pectin to pectic acid and methanol. The depolymerising enzymes attack the inter-galacturonic acid linkages, either hydrolysing the glycosidic bond or (the So-called "lyases") splitting it by a transeliminative reaction to yield a A4:5 unsaturated uronide product. Five classes of depolymerases are known, these are: endo- and exo-polygalacturonase, endo- and exo-low methoxy pectin lyases, and endopectin lyase (Rombouts, 1972, quoted in Prins, 1977). In order for the pectic polysaccarides to be completely hydrolysed, glyc0sidases capable of hydrolysing the linkages between the galacturonide residues and other sugar molecules must be postulated.

LIGNIN Lignin is a heterogenous polymer of substituted and dehydrogenated phenyl propanol units, the principal components being p-coumaryl, sinapyl and coniferyl alcohol. During lignin synthesis, these alcohols are dehydrogenatively polymerised by peroxidases via radical coupling and subsequent nucleophilic reactions of various nucleophiles to quinomethide intermediates, resulting in the formation of ~-~', fi-5', /~-1', fi-0-4', 5-5' and 3-0-4'a linkages (Freudenberg, 1965) between phenylpropanoid units. Lignin does not occur in primary plant cell walls, but is laid down during secondary thickening. The lignin-synthesizing enzymes are present in the cell undergoing lignification (Gross, 1980). The lignin penetrates between the polysaccharide chains within the cell wall. Recent reviews on the structure of lignin and its role in the plant cell wall can be found in Gross (1980) and Harkin (1973). Lignin is probably little metabolised by rumen microorganisms. Clearly the modification of aromatic residues by hydrogenation, dehydroxylation and demethy!ation occurs (Booth and Williams, 1963). Since cleavage of the aromatic ring usually requires molecular oxygen under aerobic conditions, or high energy to reduce the aromatic ring (Evans, 1977) under anaerobic conditions, it is unlikely that any significant ring cleavage occurs in the rumen. Aerobic degradation is reviewed by Tiguchi (1982). However, anaerobic cleavage has also been demonstrated in micro-organisms which use

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nitrate or sulphate as electron acceptors (Evans, 1977), and such reactions m a y occur in the tureen.

PROTEIN IN CELL WALLS Protein is normally present in plant cell walls, the major protein being extensin. This protein is distinct from plant cytosol proteins in that it is rich in hydroxyproline, L-arabinose and D-galactose (Lamport and Northcote, 1960; Lamport, 1965). In dicots, as much as 10% of the primary cell wall m a y consist of protein (Talmadge et al., 1973). Since carbohydrates are associated with the protein, it may possess a structural function (Albersheim, i 9 7 5 ) .

SKINS AND COVERINGS The outer walls of epidermal cells and aerial surfaces of plants are covered w i t h a protective film of wax or cutin. The c o m p o n e n t s of this material are reviewed by Martin and Juniper (1970). The outer layer is of wax which penetrates the lamella and the outer polysaccharides of the cell walls, and consists of long-chain alkanes, alcohols, ketones and fatty acids, most o f which are esterified with the alcohols. The cutin consists of polymeric fatty acids interconnected by ester, peroxide and ether linkages as a threedimensional network. H y d r o x y f a t t y acids predominate, so the polymers are mainly estolides.

LINKAGES BETWEEN CELL WALL COMPONENTS In dicots, it has been shown that the xyloglucan absorbs strongly through multiple hydrogen bonding to cellulose (Bauer et al., 1973; Keegstra et al., 1973), and each cellulose fibre is envisaged as being extensively covered with xyloglucan (Albersheim, 1975). Arabinogalactan is covalently b o u n d to the reducing end of the xyloglucan chain, and the arabinogalactan is believed to extend radially from each cellulose fibre. The arabinogalactan is covalently linked to rhamnogalacturonan, which links terminal galactose units of arabinogalactan chains attached to the same, and to adjacent, cellulose fibres, crosslinking the fibres (Talmadge et al., 1973; Keegstra et al., 1973). Calcium ions may alSO be involved in crosslinking b y chelating oxygen atoms in galacturonosyl resiclues in adjacent rhamnogalacturonan chains (Rees a n d R i c h a r d s o n , 1972; Grant et al., 1973). Less is known of the relationships between the cell wall polysaccharides in m o n o c o t s , though the overall architecture is similar despite the hemicelluloses being chemically very different (Albersheim, 1975). Indeed,

129 arabinoxylan and glucuronoarabinoxylan isolated from m o n o c o t cell wall were shown to bind reversibly to cellulose in vitro (McNeill et al., 1975, Darvill et al., 1978) probably by hydrogen bonding, and it is likely that they bind to other cell wall c o m p o n e n t s and to themselves in vivo (Darvill et al., 1981). There is evidence that m o n o c o t cell wall polysaccharides are cross-linked by diferulic acid esters (Hartley and Jones, 1976, 1977; Markwaldes and Neukom, 1976), The hydroxyproline-rich glycoprotein found in both m o n o c o t and dicot cell walls is probably b o n d e d through non-covalent interactions to the polysaccharides of the cell wall (Darvill et al., 1981). The precise relationship between lignin and cell wall carbohydrates is not k n o w n with certainty, b u t it is likely that some lignin is free and some is chemically b o n d e d to the cell wall polysaccharides (Pearl, 1967). Morrison (1974) has shown that lignin-carbohydrate complexes extracted from L o l i u m perenne contained xylan and cellulose polymers associated with each other. Acetyl and phenolic groups were linked to the complex, and three different lignin--carbohydrate linkages were identified b y their differing resistances to chemical cleavage. The digestibility of plant cell walls in ruminants is inversely related to the lignin content; this may be due to the inhibition of attachment of rumen organisms to cell walls rich in lignin (Richards, 1976), b y the protection of the polysaccharides from cellulase activity, by the presence of phenolic groups, or the physical encrustment of polysaccharides by lignin, which may prevent enzyme attack.

CILIATE-BACTERIUM ECOSYSTEM Since the rumen ciliates ingest large quantities of bacteria (Coleman, 1980), both bacteria living free in the rumen fluid and bacteria associated with plant fragments, bacteria are often seen in thin sections of ciliate protozoa when examined in the electron microscope. The intracellular b a c t e r i a m a y be present in vacuoles, where they are undergoing digestion by the p r o t o z o o n (Coleman and Hall, 1974) or in the ciliate cytoplasm surrounded b y a polysaccharide capsule. Viable bacteria have been isolated from the ciliate cell itself (White, 1969). Bacteria may also be found attached to the cuticle of the ciliate and it has been suggested that some of these are methanogenic (Vogels et al., 1980). It is not known whether any of the external bacteria have a role in the metabolism of the ciliate, b u t it is likely that cell wall degrading bacteria engulfed by the ciliate would provide the cell with some cell wall degrading enzymes, and that plant particles carry similar adsorbed enzymes into the ciliate. Whether any of these enzymes are active within the ciliate cell is not known. Some intracellular bacteria have been shown to be metabolically active (Coleman and Hall, 1974), b u t none have yet been shown to produce plant cell wall degrading enzymes. Imai and Ogimoto (1978) showed that

130 the cellulolytic bacterium Ruminococcus albus was attached to the pellicle of ciliates taken directly from the rumen, b u t there is no evidence t h a t it produces cell wall degrading enzymes which could be used by the ciliates. Where reference is made to the presence of enzymes within ciliates in this paper, it must n o t therefore b e assumed that all the enzymes come from the ciliate. In m a n y instances, experimental procedures have greatly reduced the chance t h a t the enzyme activity was associated with the bacteria, but only when the ciliates can be grown in the absence of bacteria can it be absolutely certain t h a t the enzymes examined are totally of ciliate origin.

INVASION OF PLANT TISSUES BY RUMEN CILIATES AND FUNGI When plant material is eaten by the ruminant, it is briefly chewed, mixed with saliva, pushed to the back of the m o u t h , and when sufficient has accumulated it is swallowed as a bolus. The bolus is propelled down the oesophagus and into the rumen, where r h y t h m i c contractions of the rumen wall mix it with the digesta. Chewing fresh plant tissues releases up to 50% of the cell contents (Reid et al., 1962), which diffuse into the r u m e n liquor. Components diffusing from the fresh tissues are believed to act as chemoattractants for some rumen micro-organisms, and in this way phycomycete fungal zoospores (Orpin and Bountiff, 1978) and ciliate protozoa (Orpin and Letcher, 1978; Orpin 1979a, b) locate the freshly ingested particles. The fungal zoospores attach, encyst, and germinate at regions where the tissue is fractured, and in stomata. Holotrich ciliates attach themselves by means of a specialised region of the cell surface [Isotricha spp. (Orpin and Hall, 1977; Orpin and Letcher, 1978)] or by an unidentified mechanism in Dasytricha ruminantium (Bauchop, 1980). Some of the Ophryoscolecid ciliates such as Epidinium ecaudatum and Eudiplodinium maggii attach themselves by the oral cavity to fibres projecting from the plant particle (Bauchop and Clark, 1976; Bauchop, 1980; C.G. o r p i n , personal observations, 1975) whilst others, such as Ophryoscolex caudatus and Entodinium spp., can be observed in the light microscope to scavenge chlorophyllcontaining plant fragments and starch granules from the close proximity of larger particles. Although t h e y rarely attach themselves to the large fragments, they do enter t h e particles through fractures.

CULTURAL, LIGHT AND ELECTRON MICROSCOPIC OBSERVATIONS ON THE DIGESTION OF PLANT FIBRES BY RUMEN CILIATES

It has been well established that m a n y rumen ciliates engulf plant particles in vivo and in culture in vitro. Hungate (1942, 1943) cultured rumen

131 ciliates in balanced salt solutions containing small quantities o f powdered dried grass and finely powdered cellulose. He found that these techniques s u p p o r t e d the growth of Eudiplodinium neglectum (Eremoplastron boris Dogeil), Eudiplodinium maggii and Diplodinium dentatum. Polyplastron multivesiculatum grew in the same medium, which was supplemented b y the addition of ground wheat grain. Plant fibres and cellulose fibres were observed to be ingested, and Eudiplodinium neglectum died w h e n the cellulose was omitted. E. m aggii, however, thrived on dried grass as the sole added organic substrate. Coleman et al. (1976) cultured Enoploplastron triloricatum, Eudiplo-

dinium maggii, Diploplastron affine, Epidinium ecaudatum, Diplodinium monacanthum and Diplodinium pentacanthum in vitro, in a medium containing 10% rumen fluid and dried grass as the major organic carbon source, indicating that they could probably utilize p l a n t cell walls. Both Hungate (1942, 1943) and Coleman et al. (1976) cultured the ciliates in the presence of bacteria, and there is therefore a possibility of the bacteria being implicated in the metabolism of the plant material, but cell-free extracts of these organisms centrifuged to remove bacteria were all capable of digesting cellulose, as measured b y reducing sugar prod u c t i o n (Hungate, 1942, 1943) or the release of 14C from 14C-cellulose (Coleman et al., 1976). Some ciliates, such as many Entodinium spp., require starch particles for growth in culture in vitro (Coleman, 1980). Although fragments of plant cell walls are sometimes seen in Entodinium spp. from the rumen, plant fibres cannot support their growth and consequently the degradation of plant cell wall polymers is unlikely to be important in these species. The holotrich ciliates Isotricha spp. and Dasytricha ruminantium have not been cultured in vitro for extended periods, and there is no evidence that they ingest significant quantities of plant celt walls despite their ability to attach themselves to plant fragments. They use small starch grains and soluble carbohydrates as carbon sources (Clark, 1977). Transmission electron microscope studies of thin sections of holotrichs (C.G. Orpin and F.J. Hall, unpublished observations, 1978) revealed engulfed chloroplast fragments and bacteria, b u t n o t plant cell walls. Bauchop and Clark (1976) found that in animals in which Epidinium spp. were the dominant ciliates, these ciliates became attached to the damaged regions of plant particles added to the rumen fluid. Subsequent scanning electron microscopic examination revealed that the attachment was by means of the oral cavity (Bauchop, 1980), and it appeared that these ciliates were attempting to engulf the ends of exposed plant fibres. The attachment was rapid, the fragments being heavily colonised within 15 min of placing the plant fragments in the rumen fluid. After prolonged incubation, much digestion of the mesophyll tissue occurred in the presence of antibiotics, which suppressed the activity of rumen bacteria, suggesting that these ciliates were major digesters of the mesophyll tissue.

132 Amos and Akin (1978) studied the assocaition of Epidinium spp. with forage grasses, a n d t h e y showed t h a t in leaf sections of cool-season, but n o t warm-season grasses, the mesophyll and parenchyma bundle sheath were degraded by Epidinium spp. It was suggested that the weight lost on incubating the tissues in rumen fluid containing antibiotics was due to the digestion of the plant tissues by the ciliates. Examination of thin sections in the transmission electron microscope indicated a swelling of fibrous cell walls prior to engulfment by the Epidinium spp., and the authors postulated t h a t this could be due to the release of extracellular enzymes by the ciliates. Yoder et al. (1966) f o u n d a synergism between Epidinium spp. and bacteria during the digestion of cellulose. All these workers used r u m e n fluid rich in Epidinium spp. and did n o t observe any other ciliates attempting to engulf plant fibres. J o u a n y and Senaud (1979) showed t h a t the presence of ciliated protozoa in the rumen increased the digestibility of lignocellulose by 3--10%. They suggested t h a t the ciliates participated directly in cellulose degradation when the animals were fed on a low-cellulose diet because of their predation on cellulolytic bacteria. Studies with defaunated animals indicated t h a t ciliates were responsible for 300--400 g/kg of total rumen fibre digestion, and t h a t a larger proportion of fibre was digested in the caecum and the large intestine in defaunated animals (Demeyer, 1981).

CELL WALL DEGRADING ENZYMES ASSOCIATED WITH RUMEN CILIATES Plant cell wall degrading enzymes found in preparations o f rumen ciliate p r o t o z o a are summarised in Table II. What is currently k n o w n about cell wall digestion in different genera is summarised below.

Entodinium spp. The entodinia are usually the most numerous of the ciliate protozoa. Coleman (1960) f o u n d no evidence t h a t cellulose was able to support the growth of E. caudatum, even though the organism engulfs particulate material, including cellulose, readily (Bailey and Clarke, 1963a). B o n h o m m e (1968) and Bonhomme-Florentin (1975) showed t h a t mixed entodinia from the rumen contained carboxymethylcellulase activity, although Coleman (1980), using phosphoric-acid regenerated cellulose, showed t h a t the activity of cell-free extracts of mixed entodinia (mostly E. simplex) was only 0.5% (on a per p r o t o z o o n basis) of t h a t of Eremoplastron bovis. This suggests t h a t the cellulase activity demonstrated by B o n h o m m e (1968) and Bonhomme-Florentin (1975) might have been due to either the release of reducing sugars from contaminating polysaccarides, or to contaminating bacteria. Abou Akkada and Howard (1961) were not able to demonstrate pectin

133 TABLE II Plant cell-wall polysaccharase activities found in cell-free extracts of ciliate protozoa Cellulase

Diplodinium affine Enoploplastron triloricatum Epidinium ecaudatum Eudiplodinium maggii Eremoplastron bovis Polyplastron multivesiculatum

Xylanase

Entodinium spp. Epidinium ecaudatum

Coleman et al. (1976) Coleman et al. (1976) Coleman et al. (1976) Hungate (1942, 1943) Coleman (1978) Hungate (1942, 1943) Abou Akkada et al. (1963)

Eremoplastron bovis

Bailey and Clarke (1963a) Bailey and Gaillard (1965) Bailey and Clarke (1963b)

Xylodextrinase

Epidinium ecaudatum Eremoplastron bovis

Bailey et al. (1962) Bailey and Clarke (1963b)

Arabinofuranosidase

Dasytricha ruminantium Epidinium ecaudatum

Williams(1979) Bailey and Gaillard (1965)

Pectinesterase

Isotricha :intestinalis

Abou Akkada and Howard (1961) Abou Akkada and Howard ( 1 9 6 t ) Abou Akkada et al. (1963)

L prostoma Polyplastron multivesiculatum Polygalacturonase

Isotricha prostoma I. intestinalis Dasytricha ruminantium Polyplastron multivesiculatum

Endopectate lyase

Diplodinium affine Epidinium ecaudatum Eremoplastron bovis Eudiplodinium maggii Ophryoscolex caudatus Ophryoscolex purkynei Ostracodinium obtusum

Abou Akkada and Howard (1961) Abou Akkada and Howard (1961) Abou Akkada and Howard (1961) Abou Akkada et al. (1963) Coleman et al. (1980) Coleman et al. (1980) Coleman et al. (1980) Coleman et al. (1980) Coleman et al. (1980) Mah and Hungate (1965) Coleman et al. (1980)

134

esterase or polygalacturonase activity in E. caudatum, b u t Bailey and Clarke (1963a) demonstrated cellodextrinase (possibly an exo-l,4-~-glucanase since ~-glucosidase activity was absent), endo-xylanase and arabino-oxylanase activity in a mixed entodinia population containing no E. caudatum. Coleman et al. (1980) demonstrated a low level of endopectate lyase activity in E n t o d i n i u m bursa, b u t not in E. caudatum. These results suggest that amongst the entodinia some polysaccharases exist, and have led some workers (e.g. Bailey and Clarke, 1963a) to suggest that E. caudatum may be atypical of the genus. Epidinium spp. A wide range of plant cell wall polysaccharases have been found in epidinia. Cell-free extracts of E. ecaudatum prepared under conditions to minimise contamination b y bacterial enzymes, hydrolysed wheat xylan with the release of arabinose, xylobiose and xylose (Bailey et al., 1962) and contained xylobiase activity. Subsequent work (Bailey and Gaillard, 1965) showed that three major hemicelluloses in b o t h Lolium perenne and TrifoIium pratense were hydrolysed by cell free extracts of E. ecaudatum: arabinofuranosidase, endo-l,4-~-xylanase and endo-l,4-~-xylodextrinase activity were present. The latter enzyme h y d r o x y s e d xylodextrins to xylose and xylobiose. The same extracts liberated cellobiose from linear B hemicellulose, and contained cellodextrinase activity which was not associated with the xylanase enzymes, b u t no hydrolysis of cellulose was detected. In addition ~-l,3-glucosidase activity wasJ detected. Thus, E. ecaudatum was shown to contain enzymes capable of hydrolysing many of the k n o w n glucosidic linkages in plant hemicelluloses. Coleman et al. (1976) showed that extracts of E. ecaudatum released lnc from 14C~ellulose. The enzyme was shown not to be associated with plant fragments present in the extract, b u t could possibly have come from bacteria in the endoplasm. Coleman et al. (1980) demonstrated endopectatelyase activity in extracts of the same organism. The level of endopectate-lyase in E. ecaudatum, which grows poorly on grass fragments in vitro, was only a b o u t 20--40% of that present in extracts of other ciliates such as Ostracodinium o b t u s u m which grow well when using grass f r a g m e n t s a s their major carbon source. E. ecaudatum appeared unable to utilise the products of hydrolysis of pectic substances as carbon sources. Eremoplastron bovis Cell-free extracts of Eremoplastron bovis (Hungate, 1943) contained cellulase activity, b u t this was not confirmed b y Bailey and Clarke (1963b). The latter authors did, however, identify xylanase, x y l o d e x t r i n a s e and cellodextrinase in cell-free extracts, b u t no pectin digesting enzymes, figlucosidases or fi-galactosidases. The cellodextrins were hydrolysed b y

135 the sequential removal of cellobiose units, typical of an exo-l,4-fl-glucanse. Endopectate lyase activity was detected in high concentration in cell-free extracts b y Coleman et al. (1980):

Eudiplodinium spp. E. maggii was shown by Hungate (1942, 1943) to digest cellulose, and to hydrolyse cellulose incubated in cell-free extracts. The preparation probably included bacterial products, and, as in many experiments with the cell-free extracts of ciliate protozoa, it was n o t known if any cellulase activity was due to bacterial enzymes. Coleman (1978) showed that the cellulase was n o t affected by incubation of the p r o t o z o a in antibiotics prior to breakage, and that at least 70% of the activity was of protozoal origin. He also reported the probable presence of a bacterial cellulase in suspensions of protozoa grown in vitro. The cellulase activity (measured by carboxymethylcellulose hydrolysis) present in extracts of this ciliate has been resolved into seven c o m p o n e n t s (G.S. Coleman, personal communication, 1983). In cell-free extracts [14C] cellulose was digested to [14C] cellobiose and [14C] glucose which, in whole cells, were incorporated into cellular components. High levels of endopectate lyase were found in cell-free extracts of E. maggii prepared b y Coleman et al. (1980). OTHER SPECIES OF OPHRYOSCOLECID CILIATES

Diploplastron affine, Enoploplastron triloricatum and Diplodinium monocanthum have been grown in vitro with dried grass as major carbon source (Coleman et al., 1976), and bacteria-free cell-free extracts of the first two species released [14C] from [14C]-cellulose. Cell-free extracts of Diploplastron affine also contained high levels of endo-pectate lyase activity (Coleman et al., 1980), as did Ophryoscolex caudatus and Ostracodinium obtusum, b u t Polyplastron multivesiculatum contained only low levels o f this enzyme. Ophryoscolex purkynei contained endopectate lyase and pectin esterase (Mah and Hungate, 1965). Despite these p r o t o z o a containing pectin catabolising enzymes, growth was not supported b y galacturonic acid. The reason for this is not clear, b u t it m a y be related to the high pH o p t i m u m (pH 7.7--10, Coleman et al., 1980; pH 8.7--9, Mah and Hungate, 1965) o f the enzymes. A b o u Akkada et al., (1963) showed cellulase, pectin and pectate hydrolysis and pectinesterase activity in cell-free extracts of

Polyplastron multivesiculatum. THE HOLOTRICH CILIATES None of the holotrichs have been cultured in vitro for extended periods,

136 so no information is available on which, if any, of the cell wall components will s u p p o r t their growth. In addition, w o r k with Isotricha spp. has been mostly confined to mixed suspensions of I. intestinalis and I. prostoma isolated from rumen fluid. Cell-free extracts o f mixed Isotricha spp. and Dasytricha ruminantium contained pectinesterase and polygalacturonase activity ( A b o u Akkada and Howard, 1961). The extracts converted pectin or polygalacturonic acid to oligouronides, b u t since pectinesterase was present, it is n o t known if the depolymerising enzyme specifically hydrolysed polygalacturonate (endopolygalacturonase activity) or if it could also attack pectin (endo-methyoxypolygalacturonase activity). The enzymes diffused readily o u t of the cells into the medium, as did ~-glucosidase and a-L-arabinofuranosidase in D. ruminantium (Williams, 1979). Isotricha spp. were unable to utilise the oligouronides or galacturonic acid as a carbon source ( A b o u Akkada and Howard, 1961). The reason for this was not clear, b u t it is in agreement with the results of Coleman e t al. (1980) for certain ophryoscolecid ciliates.

PLANT CELL WALL DEGRADATION BY RUMEN FUNGI The rumen p h y c o m y c e t e s were originally believed to be free-living in rumen liquor (Orpin, 1975, 1976), b u t a microscopic examination of digesta particles followed by experiments b o t h in vitro and in vivo revealed that the vegetative stages of t h e s e fungi existed in intimate association with the digesta particles in the rumen (Orpin, 1977a,b; Orpin and Letcher, 1979a). Invasion of freshly ingested plant tissues b y the zoospores of the fungi was rapid (Orpin and Bountiff, 1978) in response to soluble carbohydrates diffusing from the tissues. The zoospores located damaged regions o f the particle, cut ends and stomata, where they encysted, germinated and developed into the vegetative stage. Orpin and Letcher (1979a) and Bauchop and M o u n t f o r t (1981) showed that some rumen p h y c o m y c e t e s were cellulolytic. Microscopic examination of plant fragments removed from the rumen 22 h after the animal had eaten revealed that up to 30% of the large ( ~ 2 mm) digesta particles were infected by p h y c o m y c e t e fungi (Orpin, 1981). Fungal development on these particles was sometimes extensive (Orpin and Letcher, 1979a; Bauchop, 1979). Stained preparations showed that the rhizoids penetrated plant cell walls, indicating some capability to digest cell wall polymers. Working with pure cultures of Neocallimastrix frontalis, Piromonas c o m m u n i s and Sphaeromonas communis, Orpin and Hart (1980) showed extensive loss of cell wall c o m p o n e n t s from b o t h wheat straw and hay after incubation with these fungi. The major components lost from the plant fragments were cellulose (up to 58% loss) and hemicellulose I (a xylan) (up to 52% loss), and up to 22% of the lignin was released from the tissues

137 TABLE iII Digestion of structural components of wheat straw leaves by tureen phycomycetes Tissue composition (% dry weight) Total digestion Pectin Lignin Hemicellulose Cellulose

-8.0 13.8 33.9 44.3

Digestion (%) by : Neocallimastix frontalis

Piromonas comrnunis

Sphaeromonas communis

45.2 20.5 19.4 52.3 58.1

42.3 47.3 21.9 55.0 50.4

30.1 16.3 16.4 39.6 39.4

Cultured in vitro for 4 days with the plant particles as major carbon source (Orpin, 1981). TABLE IV Size distribution of wheat straw leaf particles after growth of phycomycetes in vitro Total DM (rag)

Control N. frontalis P. communis S. communis

880 572 534 616

Digestion (%)

-35 37 30

Particulate fractions (sieve mesh, mm 2 , as % of DM) 4--1

1--0.25

< 0.25

88 45 39 74

6 35 37 14

6 20 24 8

Cultured in vitro for 4 days; size fractions determined by wet sieving (C.G. Orpin, unpublished data, 1981). (Table III). T h e r e is no evidence to suggest t h a t a n y o f t h e lignin was used as a c a r b o n s o u r c e b y the fungi, despite its release f r o m t h e p l a n t particles. in a d d i t i o n to the digestion o f s t r u c t u r a l c o m p o n e n t s o f t h e p l a n t tissues, t h e r e was a c o n c o m i t a n t decrease in particle size a f t e r g r o w t h o f t h e p h y c o m y c e t e s (Table IV). It is likely t h a t in vivo the d i s r u p t i o n o f t h e tissues aids t h e c o l o n i s a t i o n o f t h e particles b y t h e r u m e n bacteria, w h i c h c o u l d accelerate fibre digestion in t h e r u m e n . O r p i n a n d L e t c h e r ( 1 9 7 9 a ) grew N. frontalis in v i t r o with cellulose as a m a j o r c a r b o n source. T h e f e r m e n t a t i o n p r o d u c t s were acetate, lactate, h y d r o g e n and c a r b o n d i o x i d e w i t h traces o f f o r m a t e and p y r u v a t e . T h e removal of hydrogen from the system by co-culturing a methanogenic b a c t e r i u m and a t u r e e n p h y c o m y c e t e e n h a n c e d t h e g r o w t h o f t h e fungus, increased t h e rate o f cellulolysis, and p r o v i d e d a simple s y s t e m f o r t h e m e t h a n o g e n i c f e r m e n t a t i o n o f cellulose ( B a u c h o p a n d M o u n t f o r t , 1 9 8 1 ) . Pure c u l t u r e s o f N. frontalis and P. c o m m u n i s have b e e n g r o w n in vitro w i t h l a r c h w o o d x y l a n a n d perennial ryegrass h e m i c e l l u l o s e [isolated b y

138 the procedure of Thornber and Northcote (1961)], as sole carbon source (C.G. Orpin, unpublished results, 1981). Piromonas communis grown on cellobiose has been found to release cellulase, xylanase, L-arabinofuranosidase, ~-l,4-glucosidase and ~-l,4-xylanase activity into the supernatant fluids of cultures (Table II). The nature of these enzymes is currently under examination. With Neocallimastix frontalis, cellulase, xylanase and ~-l,4glucosidase activity was detected in supernatants of cultures growing on cellobiose (Table II). Both species released much amylase and ~-l,4-glucosidase under these conditions, despite the substrate being cellobiose (~-l,4glucosylglucose). The supernatant fluids from cultures grown on glucose contained no cellulase or xylanase activity. We are currently examining the induction and repression of plant cell wall degrading enzymes in these rumen phycomycetes.

CONCLUSIONS There is now much information on the ability of the rumen ciliate protozoa to ingest fragments of plant cell walls, and on the ability of cell-free extracts prepared from the ciliates to degrade plant cell wall polysaccharides. Despite the fact t h a t ciliates isolated from the rumen are contaminated by bacteria and t h a t no rumen ciliate has been cultured axenically, the high levels of some cell-wall degrading polysaccharases present in bacteria-free cell-free extracts of some ciliates have indicated t h a t it is probable t h a t the ciliates themselves synthesise m a n y of the polysaccharases t h a t have been demonstrated. The work of Coleman (1978) with cellulose metabolism by Eudiplodinium maggii, in which incubation of the p r o t o z o a with antibiotics before breaking had no effect on the cellulase activity of cell-free extracts, is significant, but some cellulolytic activity was associated with a particulate fraction which contained contaminating bacteria. On electron microscopic evidence, Delfosse-Debousscher et al., (19.79) showed that the cytoplasm of mixed ciliates contained bacteria with a similar morphology to cellulolytic bacteria, and considered t h a t the bacteria alone were responsible for fibre digestion in the ciliates. Until the rumen ciliates are grown axenically, there will always be some d o u b t as to t h e origin of the enzymes in the ciliate--bacterium complex. How does the information of the presence of cell wall degrading enzymes in different ciliates fit in with what we k n o w of their behaviour when plant particles are added to rumen fluid? We have studied the dynamics o f plant particle invasion by rumen ciliates (Orpin, 1979a), and found t h a t the primary invaders were the holotrich ciliates. Within 5 min, 90% of the holotrichs in the rumen fluid became associated with the particles. The holotrichs utilise mainly soluble carbohydrates, and this behaviour would ensure they have m a x i m u m o p p o r t u n i t y to take up soluble carbohydrates diffusing from fresh tissues. Individual cells were seen to penetrate

139 between plant cells and in fissures. It is possible that under these circumstances extracellular pectin hydrolysing enzymes (Abou Akkada and Howard, 1961) released from the holotrichs would result in the digestion of the middle lamella and the subsequent separation of cells. This would provide access for other r u m e n micro-organisms. Dasytricha ruminantium also releases extracellular glycosidases (Williams, 1979) which would begin the hydrolysis of the hemicellulose. Within 1 0 m i n , in a B-type ciliate population (Eadie, 1962), Epidinium spp. and Eudiplodinium spp., would accumulate at the particles, attempting to engulf fibres extending into the rumen liquor, and begin to diges~ cellulose, hemicellulose and pectin intracellularly. In A-type populations, this role would be undertaken by Diploplastron affine and at a later time by

Polyplastron multivesiculatum. Soon after the peak invasion by the cellulolytic species, the Entodinium spp. and, in A-type populations, Ophryoscolex spp., accumulate around the tissues, taking up starch grains and chloroplast fragments. Whilst we know that m a n y ciliates are involved in plant cell wall degradation in the rumen, it is difficult to quantify their role. We do k n o w t h a t addition of cellulolytic ciliates to a ciliate-free animal results in increased cellulolyis (Yoder et al., 1966; J o u a n y and Senaud, 1979) and that in the absence of ciliates the rumen fluid volume increases (Orpin and Letcher, 1979b) suggesting t h a t although the ciliates are not essential for the survival of the animal, digestion of plant fibre is more rapid in their presence. One quantitative determination of the role of ciliates was reported by Demeyer (1981), who estimated t h a t t h e y could be responsible for about a third o f fibre digestion. We do n o t y e t know the full implication of the discovery of the cellulolytic rumen phycomycetes. As y e t there is no reliable m e t h o d of estimating their biomass in vivo, but estimates suggesting that they m a y represent up to 8% of the microbial biomass have been made (Orpin, 1981). Since t h e y are active in the digestion of a wide range o f plant cell wall components and their mode of growth results in fragmentation of the plant tissues, it is likely that t h e y play a significant role in plant cell wall digestion in the rumen. In addition, their numbers are higher on a high fibre-diet than on a low-fibre or high concentrate diet (Orpin, 1977a, and unpublished observations, 1982), which is contrary to findings in the Ciliate protoza. The two types of organism may therefore be complementary rather than competitive in vivo.

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