Ruminal anaerobic fungi:

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
Genus Species Source(s) Reference(s)
Caecomyces C. communis, C. equi Sheep Horse Gold et al, 1988 Gold et al, 1988
Piromyces P. Communis P. mae P. dumbonica P. rhizinflata P. Minutus P. Spiralis P. citronii Sheep Cow Horse Elephant Ass Deer Goat Horse Donkey Gold et al, 1988 Julliand et al, 1998 Li et al, 1990 Li et al, 1990 Breton et al, 1991 Ho et al, 1993 Ho et al, 1993 Gaillard-Martinie et al, 1995 Julliand et al, 1998
Neocallimastix N. frontalis N. patriciarum N. hurleyensis N. variabilis Sheep Sheep Sheep Cow Heath et al, 1983 Orpin and Munn, 1986 Webb and Theodorou, 1991 Ho et al, 1993
Anaeromyces A. elegans A. mucronatus Cow Sheep Ho et al, 1993 Breton et al, 1990
Orpinomyces O. joyonii O. intercalaris Sheep Cow Breton et al, 1989 Ho et al, 1994
Cyllamyces C. aberensis Cow Ozkose et al, 2001
11
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)

12
Teunissen and Op den Camp, 1993 Harhangi, 2002
13
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)

14
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)

15
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)

16
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

17
Identification

• Genus identification
• Number of flagella per zoospore
• Rhizomycelium
• Shape of sporangium
• (Breton et al, 1990 Asao et al, 1993)

contd
18

• 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)

19
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)

20
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)

21
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)

22
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)

23
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)

24

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

25
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

26
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)

27
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)

28
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)

29
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)

30
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

31
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)