Ruminal anaerobic fungi: Ruminal anaerobic fungi:

The potential plant-fiber The potential plant-fiber degraders in the rumen degraders in the rumen

__________________________Ravinder Nagpal

Dairy Microbiology Division,National Dairy Research Institute,

Karnal 132001 (Haryana) India.

IntroductionIntroduction

Importance of ruminants to mankind has led to a great Importance of ruminants to mankind has led to a great deal of research into the digestion of plant fibers in deal of research into the digestion of plant fibers in order to improve the production efficiency.order to improve the production efficiency.

Majority of livestock subsist on poor quality fibrous Majority of livestock subsist on poor quality fibrous crop residues and agro-industrial wastescrop residues and agro-industrial wastes

Attempts are being made to enhance the digestibility of Attempts are being made to enhance the digestibility of poor quality feeds by various feed additivespoor quality feeds by various feed additives

contd…

Ruminal anaerobic fungi, an emerging group of Ruminal anaerobic fungi, an emerging group of

animal probiotics, account for upto 8% of the animal probiotics, account for upto 8% of the

microbial biomass in rumen and actively microbial biomass in rumen and actively

colonize plant cell-wallscolonize plant cell-walls

Gain access to plant materials that is Gain access to plant materials that is

unavailable to other rumen microorganismsunavailable to other rumen microorganisms

contd…

Rhizoids of vegetative thalli penetrate plant tissues better Rhizoids of vegetative thalli penetrate plant tissues better

than bacteria and protozoathan bacteria and protozoa

Help in access of other rumen microbes to the secondary Help in access of other rumen microbes to the secondary

cell wall of feed particlescell wall of feed particles

More rapid and complete degradation of forage entering More rapid and complete degradation of forage entering

the rumen through proficient fibre degrading enzymesthe rumen through proficient fibre degrading enzymes

Development of direct-fed microbials (DFM) for Development of direct-fed microbials (DFM) for

improved rumen performance appears to be a pre-improved rumen performance appears to be a pre-

requisite for the sustainable animal production.requisite for the sustainable animal production.

Rumen anaerobic fungiRumen anaerobic fungi

Observed in the rumen as early as 1910, but were believed to Observed in the rumen as early as 1910, but were believed to be flagellate protozoabe flagellate protozoa

(Liebetanz et al, 1910; Braune et al, 1913)(Liebetanz et al, 1910; Braune et al, 1913)

Confirmed to be a true fungus by the presence of chitin in its Confirmed to be a true fungus by the presence of chitin in its cell wall and designated as cell wall and designated as Neocallimastix frontalisNeocallimastix frontalis

(Orpin, 1975)(Orpin, 1975)

Identified anaerobic fungi in sheep rumen, each of which had Identified anaerobic fungi in sheep rumen, each of which had a motile stage (the zoospore) and a non-motile a motile stage (the zoospore) and a non-motile zoosporangiumzoosporangium (Orpin, 1975)(Orpin, 1975)

Found in the rumen and other parts of the gastro-Found in the rumen and other parts of the gastro-

intestinal tract of herbivorous animalsintestinal tract of herbivorous animals (Williams et al, 1987; Rezaeian et al, 2004)(Williams et al, 1987; Rezaeian et al, 2004)

Play an active and positive role in fibre degradation as Play an active and positive role in fibre degradation as evidenced by the presence of different hydrolytic evidenced by the presence of different hydrolytic enzymes enzymes

(Williams et al, 1987; Samanta et al, 2001; Paul et al, 2003)(Williams et al, 1987; Samanta et al, 2001; Paul et al, 2003)

There is a significant reduction in There is a significant reduction in in-vitroin-vitro gas production gas production and degradation of fibrous feeds, indicating a positive and degradation of fibrous feeds, indicating a positive

role played by fungi in fibre degradationrole played by fungi in fibre degradation (Kamra et al, 2004; Lee et al, 2004; Dey et al, 2004)(Kamra et al, 2004; Lee et al, 2004; Dey et al, 2004)

The fibre-based diets stimulate the fungal growth in The fibre-based diets stimulate the fungal growth in

the rumen of buffalo in comparison to diets rich in the rumen of buffalo in comparison to diets rich in

easily fermentable carbohydrateseasily fermentable carbohydrates

(Kamra et al, 2003)(Kamra et al, 2003)

These fungi prefer to get attached to the most These fungi prefer to get attached to the most

lignified tissues of plant feed material by producing lignified tissues of plant feed material by producing

variety of enzymesvariety of enzymes

(Akin et al, (Akin et al,

1987)1987)

ClassificationClassification Based on ultrastructural characteristics of the zoospores, Based on ultrastructural characteristics of the zoospores,

anaerobic fungi were assigned to the order of anaerobic fungi were assigned to the order of Spizellomycetales and in the family, Spizellomycetales and in the family, NeocallimasticaceaeNeocallimasticaceae

(Barr et al, 1988)(Barr et al, 1988) Suggested the subdivision of this family into three genera Suggested the subdivision of this family into three genera

containing monocentric species, containing monocentric species, Neocallimastix, PiromycesNeocallimastix, Piromyces (previously (previously PiromonasPiromonas) and ) and CaecomycesCaecomyces (previously (previously SphaeromonasSphaeromonas))

(Gold et al(Gold et al, , 1988)1988) Three polycentric genera have been described, Three polycentric genera have been described, OrpinomycesOrpinomyces

(Barr et al, 1989)(Barr et al, 1989), , AnaeromycesAnaeromyces (Breton et al, 1990)(Breton et al, 1990) and and CyllamycesCyllamyces (Ozkose et al, 2001)(Ozkose et al, 2001). .

Division:Division: EumycotaEumycotaSubdivision:Subdivision: MastigomycotinaMastigomycotinaClass:Class: ChytridiomycetesChytridiomycetesOrder:Order: SpizellomycetalesSpizellomycetalesFamily:Family: NeocallimasticaceaeNeocallimasticaceaeGenera:Genera: Monocentric:Monocentric:

CaecomycesCaecomyces:: zoospores with one or two flagella; zoospores with one or two flagella; thallus thallus with a globular rhizoidwith a globular rhizoid

NeocallimastixNeocallimastix:: zoospore with four to twenty zoospore with four to twenty flagella; flagella; thallus with filamentous branching rhizoidsthallus with filamentous branching rhizoids

PiromycesPiromyces:: zoospore with one to four flagella and zoospore with one to four flagella and thallus with filamentous branching rhizoidsthallus with filamentous branching rhizoidsPolycentric:Polycentric:OrpinomycesOrpinomyces:: multiflagellate zoosporemultiflagellate zoosporeAnaeromycesAnaeromyces:: zoospore with one flagellumzoospore with one flagellumCyllamycesCyllamyces:: zoospore with one to two flagella with zoospore with one to two flagella with thalloid branched sporangiophorethalloid branched sporangiophore

Genus Genus Species Species Source(s) Source(s) Reference(s) Reference(s)

CaecomycesCaecomyces C. communis, C. communis, C. equiC. equi

Sheep Sheep Horse Horse

Gold Gold et alet al, 1988, 1988Gold Gold et alet al, 1988 , 1988

PiromycesPiromyces P. CommunisP. Communis P. maeP. maeP. dumbonicaP. dumbonicaP. rhizinflataP. rhizinflataP. Minutus P. Minutus P. SpiralisP. SpiralisP. citroniiP. citronii

SheepSheepCowCowHorseHorseElephantElephantAssAssDeerDeerGoatGoatHorseHorseDonkey Donkey

Gold Gold et alet al, 1988, 1988Julliand Julliand et alet al, 1998, 1998Li Li et alet al, 1990, 1990Li Li et alet al, 1990, 1990Breton Breton et alet al, 1991, 1991Ho Ho et alet al, 1993, 1993Ho Ho et alet al, 1993, 1993Gaillard-Martinie Gaillard-Martinie et alet al, 1995, 1995Julliand Julliand et alet al, 1998 , 1998

NeocallimastixNeocallimastix N. frontalisN. frontalisN. patriciarumN. patriciarumN. hurleyensisN. hurleyensisN. variabilisN. variabilis

SheepSheepSheepSheepSheepSheepCow Cow

Heath Heath et alet al, 1983, 1983Orpin and Munn, 1986Orpin and Munn, 1986Webb and Theodorou, 1991 Webb and Theodorou, 1991 Ho Ho et alet al, 1993 , 1993

AnaeromycesAnaeromyces A. elegansA. elegansA. mucronatusA. mucronatus

CowCowSheep Sheep

Ho Ho et alet al, 1993, 1993Breton Breton et alet al, 1990 , 1990

OrpinomycesOrpinomyces O. joyoniiO. joyoniiO. intercalarisO. intercalaris

SheepSheepCow Cow

Breton Breton et alet al, 1989 , 1989 Ho Ho et alet al, 1994 , 1994

CyllamycesCyllamyces C. aberensisC. aberensis CowCow Ozkose Ozkose et alet al, 2001 , 2001

LIFE CYCLELIFE CYCLE Life cycle lasts about 23-32 hoursLife cycle lasts about 23-32 hours

(Joblin 1981; Bauchop 1983; Lowe (Joblin 1981; Bauchop 1983; Lowe et alet al., 1987)., 1987)

The life cycle of monocentric fungi consists of an alteration The life cycle of monocentric fungi consists of an alteration between a motile, zoosporic stage and a vegetative, between a motile, zoosporic stage and a vegetative, zoosporangial stage zoosporangial stage

Flagellate zoospores are released from a sporangium and Flagellate zoospores are released from a sporangium and encyst by shedding their flagella encyst by shedding their flagella

The cyst germinates to produce a germ tube, which later The cyst germinates to produce a germ tube, which later develops into rhizoidsdevelops into rhizoids (Orpin et al, 1977)(Orpin et al, 1977)

Teunissen and Op den Camp, 1993; Harhangi, 2002

contd…contd…

The development of zoospores from young sporangia may The development of zoospores from young sporangia may occur within 8 hours after encystment under appropriate occur within 8 hours after encystment under appropriate conditionsconditions

(Orpin et al, (Orpin et al,

1977)1977)

Polycentric fungi have indeterminate life cycles and are Polycentric fungi have indeterminate life cycles and are not dependent upon the formation of zoospores for their not dependent upon the formation of zoospores for their continued survivalcontinued survival

(Ho & Bauchop, 1991)(Ho & Bauchop, 1991)

Zoospores are produced infrequently or zoosporogenesis Zoospores are produced infrequently or zoosporogenesis is even absentis even absent (Phillip et al, (Phillip et al, 1989)1989)

DistributionDistribution

First isolation in the UK from the rumen of sheepFirst isolation in the UK from the rumen of sheep

(Orpin, 1975)(Orpin, 1975)

Have been found on almost all the continents and in all Have been found on almost all the continents and in all of the geographic regions, where there have been sought of the geographic regions, where there have been sought

Ubiquitous among ruminants such as cattle, buffalo, Ubiquitous among ruminants such as cattle, buffalo,

goatgoat (Singhal et al, 2000; Dey et al, 2004; Thareja et al, (Singhal et al, 2000; Dey et al, 2004; Thareja et al,

2006)2006)

Red deer and impalaRed deer and impala

(Bauchop et al, 1979; Singhal et al, 2000)(Bauchop et al, 1979; Singhal et al, 2000) Grey kangaroo, wallaroo and swamp wallabyGrey kangaroo, wallaroo and swamp wallaby

(Breton et al, 1989)(Breton et al, 1989) Fecal samples of hindgut fermenters such as ass, horse, Fecal samples of hindgut fermenters such as ass, horse,

elephant and zebraelephant and zebra

(Breton et al, 1990; Li et al, 1990)(Breton et al, 1990; Li et al, 1990)

Isolated from fecal and rumen samples of wild NeelgaiIsolated from fecal and rumen samples of wild Neelgai (Paul et al, 2004; Tripathi et al, 2007)(Paul et al, 2004; Tripathi et al, 2007)

contd…contd…

IsolationIsolation Overlayering with partially molten agar with filtered Overlayering with partially molten agar with filtered

rumen fluidrumen fluid (Orpin, 1975)(Orpin, 1975)

Plate culture technique for anaerobic fungi from rumen Plate culture technique for anaerobic fungi from rumen digesta of sheep and cattle digesta of sheep and cattle (Lowe et al, (Lowe et al,

1985)1985)

Roll-bottle method involving inoculating a dilution series Roll-bottle method involving inoculating a dilution series

of molten agar medium with filtered rumen fluidof molten agar medium with filtered rumen fluid

(Joblin, 1981)(Joblin, 1981)

Penicillin, Streptomycin, Neomycin and Chloramphenicol are added to thePenicillin, Streptomycin, Neomycin and Chloramphenicol are added to the

isolation media to suppress the bacterial growthisolation media to suppress the bacterial growth

IdentificationIdentification

Genus identificationGenus identification::

• Number of flagella per zoosporeNumber of flagella per zoospore

• RhizomyceliumRhizomycelium

• Shape of sporangiumShape of sporangium

(Breton et al, 1990; Asao et al, 1993)(Breton et al, 1990; Asao et al, 1993)

contd…contd…

Species are delimited on the basis of zoospore Species are delimited on the basis of zoospore

ultrastructureultrastructure

(Ho & Barr, (Ho & Barr,

1995)1995) 18 species in six genera have been classified in the 18 species in six genera have been classified in the

literatureliterature

Species identification by PCR-amplification and Species identification by PCR-amplification and

sequencing of sequencing of ITS1ITS1 and and ITS2ITS2

(Brookman et al, 2000; Fliegerova et al, 2004)(Brookman et al, 2000; Fliegerova et al, 2004)

Enumeration Enumeration Counts of individual zoospores and zoosporangia have Counts of individual zoospores and zoosporangia have

been used to estimate fungal populations been used to estimate fungal populations in vitroin vitro and and in in vivovivo

(Joblin, 1981; Ushida et al, 1989)(Joblin, 1981; Ushida et al, 1989)

Used colony-forming units per gram dry weight of feces as Used colony-forming units per gram dry weight of feces as the basis for quantifying species of the basis for quantifying species of PiromycesPiromyces..

(Breton et al, 1991)(Breton et al, 1991) Procedure based on the technique of most probable Procedure based on the technique of most probable

numbers, was developed to enumerate rumen fungi as numbers, was developed to enumerate rumen fungi as thallus-forming unitsthallus-forming units

(Theodorou et al, 1990)(Theodorou et al, 1990)

Role of anaerobic fungi in fibre digestionRole of anaerobic fungi in fibre digestion

Role of rumen fungi in the degradation of plant fibre has Role of rumen fungi in the degradation of plant fibre has been examined extensivelybeen examined extensively

(Lee et al, 2000; 2004; Samanta et al, 2001; Dey et al, 2004, Paul et al, 2004; (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)Thareja et al, 2006; Tripathy et al, 2007)

These fungi are better at penetrating plant tissue than These fungi are better at penetrating plant tissue than

are bacteria and protozoaare bacteria and protozoa (Orpin and Joblin, 1988)(Orpin and Joblin, 1988)

Such penetration leads to faster and more complete Such penetration leads to faster and more complete degradation of forage that enters the rumen.degradation of forage that enters the rumen.

(Bauchop and Mountfort, 1981)(Bauchop and Mountfort, 1981)

Degradation of lignin-containing walls of plant cells is an Degradation of lignin-containing walls of plant cells is an

important characteristic of rumen fungiimportant characteristic of rumen fungi (Mountfort et al, 1982; Akin and (Mountfort et al, 1982; Akin and

Benner, 1988)Benner, 1988)

Rumen fungi dissolve small amounts of phenolic Rumen fungi dissolve small amounts of phenolic

compounds from plant cell wallscompounds from plant cell walls (Orpin, 1983; Gordon et (Orpin, 1983; Gordon et

al, 1985)al, 1985)

Zoospores of many species appear to colonize the lignin-Zoospores of many species appear to colonize the lignin-containing tissues preferentially and to establish colonies containing tissues preferentially and to establish colonies

localized on sclerenchyma and xylem cellslocalized on sclerenchyma and xylem cells . . (Akin et al, 1986)(Akin et al, 1986)

contd…contd…

Anaerobic fungi penetrate the cuticle, a barrier that Anaerobic fungi penetrate the cuticle, a barrier that other microorganisms cannot cross. other microorganisms cannot cross.

Rumen fungi attack recalcitrant plant cell walls by Rumen fungi attack recalcitrant plant cell walls by weakening the textural strength of the residueweakening the textural strength of the residue

(Akin et al, 1989, 1990)(Akin et al, 1989, 1990)

The greater ability of rumen fungi to weaken forage The greater ability of rumen fungi to weaken forage fibre may be important in enhancing forage utilization fibre may be important in enhancing forage utilization by the host animalby the host animal

(Borneman and Akin, 1990)(Borneman and Akin, 1990)

contd…contd…

Increased digestibility of straw with use of different Increased digestibility of straw with use of different anaerobic fungi viz., anaerobic fungi viz., Orpinomyces, PiromycesOrpinomyces, Piromyces and and

AnaeromycesAnaeromyces was observed was observed (Manikumar et al(Manikumar et al,, 2002; Sehgal et al 2002; Sehgal et al, , 2002; Tripathy et al, 2002; Tripathy et al,

2007)2007) 7-12% increase in voluntary intake of straw based diet 7-12% increase in voluntary intake of straw based diet

was reported when the sheeps were dosed with cultures of was reported when the sheeps were dosed with cultures of

monocentric fungimonocentric fungi (Gordon and Phillips, 1998)(Gordon and Phillips, 1998)

Fungal culture increased Cellulose degradation by 26% Fungal culture increased Cellulose degradation by 26% under under in vitroin vitro environment. environment.

(Lee et al, 2004)(Lee et al, 2004)

contd…contd…

Fungi degrades Fungi degrades plant cell wall::

Xylem and mestome bundle sheath in Xylem and mestome bundle sheath in

leaves

Schlerenchyma ring in stemSchlerenchyma ring in stem

Cuticular barrier of leavesCuticular barrier of leaves

(Bauchop (Bauchop et alet al., 1989)., 1989)

While rumen protozoa and bacteria have been shown to While rumen protozoa and bacteria have been shown to

play a role in plant fibre degradationplay a role in plant fibre degradation

(Williams, 1988; Akin and Benner, 1988)(Williams, 1988; Akin and Benner, 1988)

Rumen fungi display a somewhat greater potential for Rumen fungi display a somewhat greater potential for

the degradation of more heavily lignified plant tissuesthe degradation of more heavily lignified plant tissues

(Akin et al, 1988)(Akin et al, 1988)

To degrade and utilize plant cell walls, anaerobic fungi To degrade and utilize plant cell walls, anaerobic fungi

produce a wide range of hydrolytic enzymes including:produce a wide range of hydrolytic enzymes including:

Hydrolytic EnzymesHydrolytic Enzymes

contd…contd…

Cellulases Cellulases (Barichievich and Calza, 1990; Yanke et al, 1993; Paul et al, (Barichievich and Calza, 1990; Yanke et al, 1993; Paul et al,

2004)2004)

HemicellulasesHemicellulases (Lowe et al, 1987; Mountfort and Asher, 1989)(Lowe et al, 1987; Mountfort and Asher, 1989)

ProteasesProteases (Wallace and Joblin, 1985; Michel et al, 1993)(Wallace and Joblin, 1985; Michel et al, 1993)

Amylases, AmyloglycosidasesAmylases, Amyloglycosidases (Mountfort and Asher, 1988; Paul et al, 2004)(Mountfort and Asher, 1988; Paul et al, 2004)

Feruloyl and p-coumaryl esterases Feruloyl and p-coumaryl esterases

(Borneman et al, 1990; 1991; 1992; Paul et al, 2004)(Borneman et al, 1990; 1991; 1992; Paul et al, 2004)

Various disaccharidasesVarious disaccharidases

(Hebraud and Fevre, 1988; Chen et al, 1994)(Hebraud and Fevre, 1988; Chen et al, 1994)

pectinases pectinases

(Gordon and Phillips, 1992)(Gordon and Phillips, 1992)

Exonucleases or avicelasesExonucleases or avicelases

(Cabe, 1998)(Cabe, 1998)

contd…contd…

Interaction with other rumen microorganismsInteraction with other rumen microorganisms

Anaerobic fungi form quite stable cocultures with rumen Anaerobic fungi form quite stable cocultures with rumen methanogenic bacteria as a result of their high production methanogenic bacteria as a result of their high production of hydrogen of hydrogen

(Fonty and Joblin, 1991; Orpin and Joblin, 1997)(Fonty and Joblin, 1991; Orpin and Joblin, 1997)

These cocultures produce an increased amount of fungal These cocultures produce an increased amount of fungal biomass and exhibit an increase in both the rate and extent biomass and exhibit an increase in both the rate and extent of cellulose degradation of cellulose degradation

(Bernalier (Bernalier et alet al, 1989; 1991; Joblin , 1989; 1991; Joblin et alet al, ,

1989)1989) Cellulolytic activity appeared to be inhibited, when Cellulolytic activity appeared to be inhibited, when

combined in coculture with the cellulolytic ruminococcicombined in coculture with the cellulolytic ruminococci

(Bernalier (Bernalier et alet al, 1992; Roger , 1992; Roger et alet al, 1993), 1993)

Growth of the rumen fungi was found to be markedly Growth of the rumen fungi was found to be markedly inhibited in cocultures with rumen bacteriainhibited in cocultures with rumen bacteria

(Dehority and Tirabasso, 2000)(Dehority and Tirabasso, 2000)

Coincubation of protozoa with fungi has shown that the Coincubation of protozoa with fungi has shown that the protozoa are able to both ingest and digest fungiprotozoa are able to both ingest and digest fungi

(Orpin and Joblin, 1997)(Orpin and Joblin, 1997)

Chitinase activity in samples of mixed rumen protozoa Chitinase activity in samples of mixed rumen protozoa account for their predatory activity on the rumen fungiaccount for their predatory activity on the rumen fungi

(Joblin, 1990; Williams et al, 1994; Morgavi et al(Joblin, 1990; Williams et al, 1994; Morgavi et al ,, (1994) (1994)

contd…

Potential benefits of ruminal anaerobic fungi Potential benefits of ruminal anaerobic fungi for improved animal nutrition and productivityfor improved animal nutrition and productivity

Improved fibre digestion and nutrient Improved fibre digestion and nutrient

utilization utilization

More feed intake and feed efficiencyMore feed intake and feed efficiency

Increased body weightIncreased body weight

Improved milk productionImproved milk production

Prospective applications of Prospective applications of ruminal anaerobic fungiruminal anaerobic fungi

Could be exploited as:Could be exploited as:

Direct-fed microbialsDirect-fed microbials

Animal feed additivesAnimal feed additives

Novel silage inoculantsNovel silage inoculants

For large scale production of enzymes For large scale production of enzymes

(e.g. cellulase)(e.g. cellulase)