Ruminal anaerobic fungi: Ruminal anaerobic fungi:

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

Mahdi bayat

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

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

Identified anaerobic fungi in sheep rumen, each of which Identified anaerobic fungi in sheep rumen, each of which

had a motile stage (the zoospore) and a non-motile had a motile stage (the zoospore) and a non-motile

zoosporangiumzoosporangium

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

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

enzymesenzymes

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

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

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

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))

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

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

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

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

Zoospores are produced infrequently or zoosporogenesis Zoospores are produced infrequently or zoosporogenesis

is even absentis even absent

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

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

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

used to estimate fungal populations used to estimate fungal populations in vitroin vitro and and in vivoin vivo

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..

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

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

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

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.

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; (Mountfort et al, 1982;

Akin and Benner, 1988)Akin and 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 (Orpin, 1983; Gordon

et al, 1985)et 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. .

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

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

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

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

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 of methanogenic bacteria as a result of their high production of

hydrogen hydrogen

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 of biomass and exhibit an increase in both the rate and extent of

cellulose degradationcellulose degradation

Cellulolytic activity appeared to be inhibited, when combined Cellulolytic activity appeared to be inhibited, when combined

in coculture with the cellulolytic ruminococciin coculture with the cellulolytic ruminococci

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

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

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

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)

THE ENDTHE END