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Ruminal anaerobic fungi: The potential plant-fiber degraders in the rumen

Ruminal anaerobic fungi: The potential plant-fiber degraders in the rumen . __________________________ Ravinder Nagpal Dairy Microbiology Division, National Dairy Research Institute, Karnal 132001 (Haryana) India. Introduction.

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Ruminal anaerobic fungi: The potential plant-fiber degraders in the rumen

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  1. Ruminal anaerobic fungi: The potential plant-fiber degraders in the rumen __________________________ Ravinder Nagpal Dairy Microbiology Division, National Dairy Research Institute, Karnal 132001 (Haryana) India.

  2. Introduction • Importance of ruminants to mankind has led to a great deal of research into the digestion of plant fibers in order to improve the production efficiency. • Majority of livestock subsist on poor quality fibrous crop residues and agro-industrial wastes • Attempts are being made to enhance the digestibility of poor quality feeds by various feed additives contd…

  3. Ruminal anaerobic fungi, an emerging group of animal probiotics, account for upto 8% of the microbial biomass in rumen and actively colonize plant cell-walls • Gain access to plant materials that is unavailable to other rumen microorganisms contd…

  4. Rhizoids of vegetative thalli penetrate plant tissues better than bacteria and protozoa • Help in access of other rumen microbes to the secondary cell wall of feed particles • More rapid and complete degradation of forage entering the rumen through proficient fibre degrading enzymes • Development of direct-fed microbials (DFM) for improved rumen performance appears to be a pre-requisite for the sustainable animal production.

  5. Rumen anaerobic fungi • Observed in the rumen as early as 1910, but were believed to be flagellate protozoa (Liebetanz et al, 1910; Braune et al, 1913) • Confirmed to be a true fungus by the presence of chitin in its cell wall and designated as Neocallimastix frontalis (Orpin, 1975) • Identified anaerobic fungi in sheep rumen, each of which had a motile stage (the zoospore) and a non-motile zoosporangium(Orpin, 1975)

  6. Found in the rumen and other parts of the gastro-intestinal tract of herbivorous animals (Williams et al, 1987; Rezaeian et al, 2004) • Play an active and positive role in fibre degradation as evidenced by the presence of different hydrolytic enzymes (Williams et al, 1987; Samanta et al, 2001; Paul et al, 2003) • There is a significant reduction in in-vitro gas production and degradation of fibrous feeds, indicating a positive role played by fungi in fibre degradation (Kamra et al, 2004; Lee et al, 2004; Dey et al, 2004)

  7. The fibre-based diets stimulate the fungal growth in the rumen of buffalo in comparison to diets rich in easily fermentable carbohydrates (Kamra et al, 2003) • These fungi prefer to get attached to the most lignified tissues of plant feed material by producing variety of enzymes(Akin et al, 1987)

  8. Classification • Based on ultrastructural characteristics of the zoospores, anaerobic fungi were assigned to the order of Spizellomycetales and in the family, Neocallimasticaceae (Barr et al, 1988) • Suggested the subdivision of this family into three genera containing monocentric species, Neocallimastix, Piromyces (previously Piromonas) and Caecomyces (previously Sphaeromonas) (Gold et al, 1988) • Three polycentric genera have been described, Orpinomyces(Barr et al, 1989), Anaeromyces(Breton et al, 1990)and Cyllamyces(Ozkose et al, 2001).

  9. Division: Eumycota Subdivision: Mastigomycotina Class: Chytridiomycetes Order: Spizellomycetales Family: Neocallimasticaceae Genera:Monocentric: Caecomyces:zoospores with one or two flagella; thallus with a globular rhizoid Neocallimastix:zoospore with four to twenty flagella; thallus with filamentous branching rhizoids Piromyces:zoospore with one to four flagella and thallus with filamentous branching rhizoids Polycentric: Orpinomyces:multiflagellate zoospore Anaeromyces:zoospore with one flagellum Cyllamyces:zoospore with one to two flagella with thalloid branched sporangiophore

  10. LIFE CYCLE • Life cycle lasts about 23-32 hours(Joblin 1981; Bauchop 1983; Lowe et al., 1987) • The life cycle of monocentric fungi consists of an alteration between a motile, zoosporic stage and a vegetative, zoosporangial stage • Flagellate zoospores are released from a sporangium and encyst by shedding their flagella • The cyst germinates to produce a germ tube, which later develops into rhizoids(Orpin et al, 1977)

  11. Teunissen and Op den Camp, 1993; Harhangi, 2002

  12. contd… • The development of zoospores from young sporangia may occur within 8 hours after encystment under appropriate conditions (Orpin et al, 1977) • Polycentric fungi have indeterminate life cycles and are not dependent upon the formation of zoospores for their continued survival (Ho & Bauchop, 1991) • Zoospores are produced infrequently or zoosporogenesis is even absent(Phillip et al, 1989)

  13. Distribution • First isolation in the UK from the rumen of sheep(Orpin, 1975) • Have been found on almost all the continents and in all of the geographic regions, where there have been sought • Ubiquitous among ruminants such as cattle, buffalo, goat(Singhal et al, 2000; Dey et al, 2004; Thareja et al, 2006)

  14. contd… • Red deer and impala (Bauchop et al, 1979; Singhal et al, 2000) • Grey kangaroo, wallaroo and swamp wallaby(Breton et al, 1989) • Fecal samples of hindgut fermenters such as ass, horse, elephant and zebra (Breton et al, 1990; Li et al, 1990) • Isolated from fecal and rumen samples of wild Neelgai(Paul et al, 2004; Tripathi et al, 2007)

  15. Isolation • Overlayering with partially molten agar with filtered rumen fluid(Orpin, 1975) • Plate culture technique for anaerobic fungi from rumen digesta of sheep and cattle (Lowe et al, 1985) • Roll-bottle method involving inoculating a dilution series of molten agar medium with filtered rumen fluid (Joblin, 1981) Penicillin, Streptomycin, Neomycin and Chloramphenicol are added to the isolation media to suppress the bacterial growth

  16. Identification • Genus identification: • Number of flagella per zoospore • Rhizomycelium • Shape of sporangium • (Breton et al, 1990; Asao et al, 1993) contd…

  17. Species are delimited on the basis of zoospore ultrastructure (Ho & Barr, 1995) • 18 species in six genera have been classified in the literature • Species identification by PCR-amplification and sequencing of ITS1 and ITS2 (Brookman et al, 2000; Fliegerova et al, 2004)

  18. Enumeration • Counts of individual zoospores and zoosporangia have been used to estimate fungal populations in vitro and in vivo (Joblin, 1981; Ushida et al, 1989) • Used colony-forming units per gram dry weight of feces as the basis for quantifying species of Piromyces. (Breton et al, 1991) • Procedure based on the technique of most probable numbers, was developed to enumerate rumen fungi as thallus-forming units (Theodorou et al, 1990)

  19. Role of anaerobic fungi in fibre digestion • Role of rumen fungi in the degradation of plant fibre has been examined extensively (Lee et al, 2000; 2004; Samanta et al, 2001; Dey et al, 2004, Paul et al, 2004; Thareja et al, 2006; Tripathy et al, 2007) • These fungi are better at penetrating plant tissue than are bacteria and protozoa(Orpin and Joblin, 1988) • Such penetration leads to faster and more complete degradation of forage that enters the rumen. (Bauchop and Mountfort, 1981)

  20. contd… • Degradation of lignin-containing walls of plant cells is an important characteristic of rumen fungi (Mountfort et al, 1982; Akin and Benner, 1988) • Rumen fungi dissolve small amounts of phenolic compounds from plant cell walls(Orpin, 1983; Gordon et al, 1985) • Zoospores of many species appear to colonize the lignin-containing tissues preferentially and to establish colonies localized on sclerenchyma and xylem cells. (Akin et al, 1986)

  21. contd… • Anaerobic fungi penetrate the cuticle, a barrier that other microorganisms cannot cross. • Rumen fungi attack recalcitrant plant cell walls by weakening the textural strength of the residue(Akin et al, 1989, 1990) • The greater ability of rumen fungi to weaken forage fibre may be important in enhancing forage utilization by the host animal (Borneman and Akin, 1990)

  22. contd… • Increased digestibility of straw with use of different anaerobic fungi viz., Orpinomyces, Piromyces and Anaeromyces was observed(Manikumar et al, 2002; Sehgal et al, 2002; Tripathy et al, 2007) • 7-12% increase in voluntary intake of straw based diet was reported when the sheeps were dosed with cultures of monocentric fungi(Gordon and Phillips, 1998) • Fungal culture increased Cellulose degradation by 26% under in vitro environment. (Lee et al, 2004)

  23. Fungi degrades plant cell wall: • Xylem and mestome bundle sheath in leaves • Schlerenchyma ring in stem • Cuticular barrier of leaves (Bauchop et al., 1989)

  24. Hydrolytic Enzymes • While rumen protozoa and bacteria have been shown to play a role in plant fibre degradation (Williams, 1988; Akin and Benner, 1988) • Rumen fungi display a somewhat greater potential for the degradation of more heavily lignified plant tissues (Akin et al, 1988) • To degrade and utilize plant cell walls, anaerobic fungi produce a wide range of hydrolytic enzymes including:

  25. contd… • Cellulases (Barichievich and Calza, 1990; Yanke et al, 1993; Paul et al, 2004) • Hemicellulases (Lowe et al, 1987; Mountfort and Asher, 1989) • Proteases (Wallace and Joblin, 1985; Michel et al, 1993) • Amylases, Amyloglycosidases (Mountfort and Asher, 1988; Paul et al, 2004)

  26. contd… • Feruloyl and p-coumaryl esterases (Borneman et al, 1990; 1991; 1992; Paul et al, 2004) • Various disaccharidases (Hebraud and Fevre, 1988; Chen et al, 1994) • pectinases (Gordon and Phillips, 1992) • Exonucleases or avicelases (Cabe, 1998)

  27. Interaction with other rumen microorganisms • Anaerobic fungi form quite stable cocultures with rumen methanogenic bacteria as a result of their high production of hydrogen (Fonty and Joblin, 1991; Orpin and Joblin, 1997) • These cocultures produce an increased amount of fungal biomass and exhibit an increase in both the rate and extent of cellulose degradation (Bernalier et al, 1989; 1991; Joblin et al, 1989) • Cellulolytic activity appeared to be inhibited, when combined in coculture with the cellulolytic ruminococci (Bernalier et al, 1992; Roger et al, 1993)

  28. contd… • Growth of the rumen fungi was found to be markedly inhibited in cocultures with rumen bacteria (Dehority and Tirabasso, 2000) • Coincubation of protozoa with fungi has shown that the protozoa are able to both ingest and digest fungi (Orpin and Joblin, 1997) • Chitinase activity in samples of mixed rumen protozoa account for their predatory activity on the rumen fungi (Joblin, 1990; Williams et al, 1994; Morgavi et al, (1994)

  29. Potential benefits of ruminal anaerobic fungi for improved animal nutrition and productivity • Improved fibre digestion and nutrient utilization • More feed intake and feed efficiency • Increased body weight • Improved milk production

  30. Prospective applications of ruminal anaerobic fungi Could be exploited as: • Direct-fed microbials • Animal feed additives • Novel silage inoculants • For large scale production of enzymes (e.g. cellulase)

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