REVIEW
published: 21 October 2020
doi: 10.3389/fmicb.2020.584893
Anaerobic Fungi: Past, Present, and
Future
Edited by:
Robert Czajkowski,
University of Gdańsk, Poland
Reviewed by:
Mostafa S. Elshahed,
Oklahoma State University,
United States
Birbal Singh,
Indian Veterinary Research Institute
(IVRI), India
*Correspondence:
Matthias Hess
[email protected]
† These
authors have contributed
equally to this work
# ORCID:
Matthias Hess
orcid.org/0000-0003-0321-0380
Shyam S. Paul
orcid.org/0000-0002-9549-5741
Anil K. Puniya
orcid.org/0000-0003-0043-5330
Mark van der Giezen
orcid.org/0000-0002-1033-1335
Claire Shaw
orcid.org/0000-0002-2495-0612
Joan E. Edwards
orcid.org/0000-0003-0759-633X
Kateřina Fliegerová
orcid.org/0000-0001-9439-8168
‡ Present
address:
Joan E. Edwards,
Palital Feed Additives,
Velddriel,
Netherlands
Specialty section:
This article was submitted to
Microbial Symbioses,
a section of the journal
Frontiers in Microbiology
Received: 18 July 2020
Accepted: 29 September 2020
Published: 21 October 2020
Citation:
Hess M, Paul SS, Puniya AK,
van der Giezen M, Shaw C,
Edwards JE and Fliegerová K (2020)
Anaerobic Fungi: Past, Present,
and Future.
Front. Microbiol. 11:584893.
doi: 10.3389/fmicb.2020.584893
Matthias Hess 1* †# , Shyam S. Paul 2# , Anil K. Puniya 3# , Mark van der Giezen 4# ,
Claire Shaw 1# , Joan E. Edwards 5‡# and Kateřina Fliegerová 6†#
1
Systems Microbiology & Natural Product Discovery Laboratory, Department of Animal Science, University of California,
Davis, Davis, CA, United States, 2 Gut Microbiome Lab, ICAR-Directorate of Poultry Research, Indian Council of Agricultural
Research, Hyderabad, India, 3 Anaerobic Microbiology Lab, ICAR-National Dairy Research Institute, Dairy Microbiology
Division, ICAR-National Dairy Research Institute, Karnal, India, 4 Department of Chemistry, Bioscience and Environmental
Engineering, University of Stavanger, Stavanger, Norway, 5 Laboratory of Microbiology, Wageningen University & Research,
Wageningen, Netherlands, 6 Laboratory of Anaerobic Microbiology, Institute of Animal Physiology and Genetics, Czech
Academy of Sciences, Prague, Czechia
Anaerobic fungi (AF) play an essential role in feed conversion due to their potent fiber
degrading enzymes and invasive growth. Much has been learned about this unusual
fungal phylum since the paradigm shifting work of Colin Orpin in the 1970s, when he
characterized the first AF. Molecular approaches targeting specific phylogenetic marker
genes have facilitated taxonomic classification of AF, which had been previously been
complicated by the complex life cycles and associated morphologies. Although we now
have a much better understanding of their diversity, it is believed that there are still
numerous genera of AF that remain to be described in gut ecosystems. Recent markergene based studies have shown that fungal diversity in the herbivore gut is much like
the bacterial population, driven by host phylogeny, host genetics and diet. Since AF are
major contributors to the degradation of plant material ingested by the host animal, it is
understandable that there has been great interest in exploring the enzymatic repertoire of
these microorganisms in order to establish a better understanding of how AF, and their
enzymes, can be used to improve host health and performance, while simultaneously
reducing the ecological footprint of the livestock industry. A detailed understanding of
AF and their interaction with other gut microbes as well as the host animal is essential,
especially when production of affordable high-quality protein and other animal-based
products needs to meet the demands of an increasing human population. Such a
mechanistic understanding, leading to more sustainable livestock practices, will be
possible with recently developed -omics technologies that have already provided first
insights into the different contributions of the fungal and bacterial population in the rumen
during plant cell wall hydrolysis.
Keywords: anaerobic digestion, carbohydrate-active enzymes, food security, herbivores, methanogenesis,
Neocallimastigomycota, rumen, sustainable agriculture
INTRODUCTION
The ability of herbivorous animals to ferment recalcitrant plant fiber into utilizable forms of
energy, most cases in the form of volatile fatty acids (VFAs), has been attributed to the trillions
of microbial cells that inhabit their gastrointestinal tract (Dearing and Kohl, 2017). Importantly,
energy stored within complex plant carbohydrates is made accessible to the host animal
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to improve the feed conversion efficiency of plant-based animal
feeds. In this review, we discuss the current understanding of AF
biology, ecology and their role in livestock production, along with
future perspectives on how their true value can be realized.
only through the synergistic activity of its gut microbes
(Morais and Mizrahi, 2019).
Independent of which type of mammalian herbivore digestive
physiology is considered, all mammalian herbivores have
evolved specialized gut compartments to give home to a
complex microbial ecosystem of bacteria, anaerobic fungi (AF),
protozoa, methanogenic archaea and bacteriophages (Morgavi
et al., 2013). All three of the major types of herbivorous
mammals depend on these microbiomes and their proper
function to support their health and performance: ruminants
(e.g., cattle, goats, and sheep), pseudoruminants (e.g., camelids
and hippopotami) and hindgut herbivores (e.g., elephants,
donkeys, horses and zebras). Furthermore, in foregut fermenting
ruminants and pseudoruminants the forestomach microbes
themselves also serve as a substantial source of protein and
vitamins for the host unlike in hindgut herbivores (Mizrahi,
2013; Nagaraja, 2016). An excellent review of herbivore
gastrointestinal physiology, including detailed drawings, and
its role on microbial fermentation of plant biomass was
summarized by Dehority (2002).
Foregut fermenters are capable of an enhanced degradation
of plant biomass that is facilitated by a prolonged (60–90 h)
retention time of the feed material in the rumen, the first and
largest of three pre-gastric chambers of the foregut fermenter
digestive tract. Gastric digestion in the ruminant digestive system
occurs in the abomasum, which is the fourth chamber of the
ruminant’s foregut. Plant material in the cecum and colon of
a hindgut fermenter, on the other hand, is retained on average
only half as long (i.e., 30–40 h in equines) as it is in the
ruminant’s digestive tract and is consequently less completely
digested (Trinci et al., 1994).
Despite the recent significant advances in our understanding
of how bacteria and archaea influence the function, resilience,
and the environmental footprint of herbivorous mammals such
as ruminants (Kittelmann et al., 2014; Henderson et al., 2015; Liu
et al., 2017; Huws et al., 2018; Wallace et al., 2019), our knowledge
of AF and their influence on the host animal remains limited.
This limited knowledge is perhaps not surprising considering
that until the mid-20th century it was still believed that all
fungi required oxygen (Trinci et al., 1994). It was only in 1975
that the ground-breaking work of Colin Orpin unequivocally
confirmed the existence of AF, changing the accepted dogma of
the time. Shortly after AF were first isolated and described in
the rumen (Orpin, 1975, 1976, 1977a,b), they were also isolated
from the horse cecum (Orpin, 1981). Since then, many more AF
have been isolated from a wide range of domesticated and wild
herbivores with eighteen different genera characterized to date
(Hanafy et al., 2020).
To date, AF have been most extensively studied in ruminants,
where they are recognized as an important microbe for good
rumen function. This is primarily due to their role as highly
efficient degraders of recalcitrant plant material (Li et al., 2017;
Gilmore et al., 2019). Rumen AF are also syntrophic partners of
the methanogenic archaea (Li et al., 2017; Gilmore et al., 2019).
Additional insights into the currently understudied herbivore gut
mycobiome has the potential to expand our scientific knowledge
about life in the absence of oxygen, as well as open new avenues
Frontiers in Microbiology | www.frontiersin.org
TAXONOMY
Anaerobic fungi were first documented over 100 years ago,
when their flagellated zoospores were mistakenly identified
as flagellate protozoa (Liebetanz, 1910; Braune, 1913). After
first being incorrectly classified as Protozoa (Liebetanz, 1910;
Braune, 1913), they were reclassified, due to the significant
evidence collected over many years by Orpin, as belonging
to the fungal phylum Chytridiomycetes (Barr, 1980, 1988).
In 2007, they were acknowledged as being a distinct phylum,
the Neocallimastigomycota (Hibbett et al., 2007). Recently,
Tedersoo et al. (2018) proposed a new fungal subkingdom,
Chytridiomycota, grouping the Neocallimastigomycota
with two additional phyla, the aerobic Chytridiomycota and
Monoblepharomycota, thereby acknowledging the monophyletic
origin of the zoosporic chitinous fungi (Ebersberger et al., 2012).
The Neocallimastigomycota contains only one order
(Neocallimastigales) and one family (Neocallimastigaceae)
comprising eighteen genera; namely the monocentric rhizoidal
Neocallimastix, Piromyces, Oontomyces, Buwchfawromyces,
Pecoramyces, Liebetanzomyces, Feramyces, Agriosomyces,
Aklioshbomyces, Capellomyces, Ghazallomyces, Joblinomyces,
Khoyollomyces, and Tahromyces; the polycentric rhizoidal
Anaeromyces and Orpinomyces; and the bulbous Caecomyces and
Cyllamyces. Key morphological features of AF taxa, such as the
number of flagella on zoospores, type of thallus and rhizoids,
steps of zoosporangial development, and the shape of sporangia,
are listed in Table 1.
Although morphological features have been crucial for the
classification of AF in the past, this approach is encumbered
with difficulties due to the extensive morphological variations,
pleomorphism in sporangial and rhizoidal structures, similarities
in morphological features of monocentric/uniflagellate genera,
failure to produce sporangia, and the absence of zoosporogenesis
in some polycentric species. Hence, ribosomal RNA (rRNA)
operon-based analysis is also needed to verify and support
classification of AF. The topology of the ribosomal RNA (rrn)
operon, indicating regions that have been used for taxonomic
classification, is shown in Figure 1. This culture-independent
approach, which in many cases is based on the nucleotide
sequence of the internal transcribed spacer 1 (ITS1) region,
suggests that the digestive tract of wild and domestic herbivores
harbors several clades and genera-equivalent groups within the
Neocallimastigaceae that have not yet been cultured (Koetschan
et al., 2014; Paul et al., 2018).
Although the ITS1 region is currently the molecular
marker of choice to assign taxonomy to AF, there are some
limitations to this marker, including a high (up to 13%)
variation of the ITS1 sequence of clones from a single culture
(Callaghan et al., 2015). This variability makes classification
of new isolates specifically challenging. Furthermore, the
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TABLE 1 | Key morphological features of characterized genera of anaerobic fungi.
Genus
Morphology [zoospore (z),
thallus (t), rhizomycelium (r)]
Miscellaneous features
References
Agriosomyces
Uniflagellate (z) Monocentric (t)
Filamentous (r)
Endogenous and exogenous zoosporangial development, rhizoids are swollen below
the sporangial tightly constricted neck, swollen sporangiophores
Hanafy et al. (2020)
Aklioshbomyces
Uniflagellate (z) Monocentric (t)
Filamentous (r)
Bi or triflagellate zoospores, endogenous and exogenous zoosporangial development,
papillated sporangia, pseudo-intercalary endogenous sporangia occasionally,
unbranched sporangiophores
Hanafy et al. (2020)
Anaeromyces
Uniflagellate (z) Polycentric (t)
Filamentous (r)
Sporangia with acuminate (mucronate) apex, can be located on erect, solitary,
unbranched sporangiophore, hyphae are highly branched, often with numerous
constrictions (sausage-like appearance), sometimes with root-like appearance
Breton et al. (1990)
Buwchfawromyces
Uniflagellate (z) Monocentric (t)
Filamentous (r)
Extensive rhizoidal system with twisted rhizoids, sporangia with no apical projections,
septum can be visible, nuclei located in sporangia, but not observed in
sporangiophores or rhizoids
Callaghan et al.
(2015)
Caecomyces
Uniflagellate (z) Monocentric (t)
Bulbous (r)
Bi or quadriflagellate zoospores, vegetative stage is absent of developed branching
rhizoidal system, consists of spherical or ovoid bodies (holdfast or haustoria), tubular
sporangiophores and bulbous rhizoids, nuclei usually present both in sporangia and
vegetative cells
Gold et al. (1988)
Capellomyces
Uniflagellate (z) Monocentric (t)
Filamentous (r)
Endogenous and exogenous zoosporangial development, unbranched
sporangiophores can exhibit subsporangial swelling, zoospores released through apical
pore
Hanafy et al. (2020)
Cyllamyces
Uniflagellate (z) Polycentric (t)
Bulbous (r)
Bi or triflagellate zoospores, bulbous holdfast without rhizoids with multiple sporangia,
which can be born on a single elongate or branched sporangiophore, nuclei present in
bulbous holdfast and sporangiophores
Ozkose et al. (2001)
Feramyces
Polyflagellate (z) Monocentric (t)
Filamentous (r)
Extensive highly branched rhizoidal system with wide and narrow hyphae, wide hyphae
with constrictions at irregular intervals, single terminal sporangium per thallus with the
occasional formation of pseudo-intercalary sporangia, sporangiophores frequently
coiled or wide and flattened, often forming an apophysis-like or eggcup-like swelling
below the sporangium, both endogenous and exogenous zoosporangial development,
zoospores are released through apical pore with the sporangial wall staying intact, or
through detachment of the whole sporangium
Hanafy et al. (2018)
Ghazallomyces
Polyflagellate (z) Monocentric (t)
Filamentous (r)
Endogenous and exogenous zoosporangial development, highly branched rhizoids,
unbranched sporangiophores, pleomorphic sporangia with septum, sporangial necks
constricted with narrow port, zoospores released through apical pore
Hanafy et al. (2020)
Joblinomyces
Uniflagellate (z) Monocentric (t)
Filamentous (r)
Biflagellate zoospores, both endogenous and exogenous zoosporangial development,
sporangiophores vary in length, zoospores released through wide apical pore resulting
in empty cup-shaped sporangium
Hanafy et al. (2020)
Khoyollomyces
Uniflagellate (z) Monocentric (t)
Filamentous (r)
Endogenous and exogenous zoosporangial development, highly branched rhizoids,
intercalary swellings in broad hyphae, multisporangiate thallus, branched
sporangiophores with two to four sporangia, zoospores released through wide apical
pore
Hanafy et al. (2020)
Liebetanzomyces
Uniflagellate (z) Monocentric (t)
Filamentous (r)
Endogenous and exogenous zoosporangial development, extensive anucleate rhizoidal
system without constrictions, single terminal sporangium per thallus, sporangium with
septum on sporangiophore of variable length, sometimes forming eggcup-like structure
below the sporangium or showing cyst-like structure. Pleomorphism in sporangial and
rhizoidal structures on different substrates is typical
Joshi et al. (2018)
Neocallimastix
Polyflagellate (z) Monocentric (t)
Filamentous (r)
Rhizoid tubular or inflated below the neck of sporangia, sporangia located on
unbranched or branched sporangiophores
Heath et al. (1983)
Oontomyces
Uniflagellate (z) Monocentric (t)
Filamentous (r)
Intercalary rhizoidal swellings, sporangia never mucronated, formed terminally, long
sporangiophores can be separated from the rhizomycelium by distinct constriction
Dagar et al. (2015)
Orpinomyces
Polyflagellate (z) Polycentric (t)
Filamentous (r)
Polynucleate rhizomycelium of extensively branched hyphae, wider hyphae can have
tightly constricted points at close intervals (bead-like or sausage-like appearance)
Barr et al. (1989)
Pecoramyces
Uniflagellate (z) Monocentric (t)
Filamentous (r)
Biflagellate zoospores, both endogenous and exogenous zoosporangial development,
single terminal sporangium formed per thallus, sporangiophores unbranched, often
forming apophysis-like or eggcup-like swelling below sporangium. Extensive anucleate
rhizoidal system lacks rhizoidal swellings or constrictions
Hanafy et al. (2017)
Piromyces
Uniflagellate (z) Monocentric (t)
Filamentous (r)
Bi or quadriflagellate zoospores, both endogenous and exogenous zoosporangial
development, rhizoids with or without subsporangial swelling, septum often in mature
zoosporangia
Barr et al. (1989)
Tahromyces
Uniflagellate (z) Monocentric (t)
Filamentous (r)
Bi or triflagellate zoospores, both endogenous and exogenous zoosporangial
development, branched rhizoids, short swollen sporangiophores, sporangia with
septum, sporangial necks constricted
Hanafy et al. (2020)
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FIGURE 1 | Topology of the ribosomal RNA (rrn) operon. Genes of the small (SSU; 18S), large (LSU; 28S) and 5.8S subunit, internal transcribed spacer 1 (ITS1) and
2 (ITS2), flanked by the external transcribed spacer (ETS) regions and linked by the intergenic spacer (IGS).
the most recently described genera, are available from the AF
Network website1 .
Latest DNA sequence technologies (i.e., PacBio and Nanopore
sequencing) generating long reads appear to offer solutions
to overcome some of these challenges, specifically the
incompleteness, fragmentation and non-overlapping of extant
ribosomal data. These new sequencing techniques enable the
generation of full-length reference sequences (up to 10 kb
in length) that span several regions suitable for taxonomic
classification and intraspecies assignment, including the above
mentioned ITS1 and LSU gene. Primers targeting the whole
fungal ribosomal tandem repeat region, consisting of ETS, SSU,
ITS1, 5.8S, ITS2, LSU, and IGS (Figure 1), have already been
successfully applied to specimens of three fungal phyla, including
early diverging fungi (Wurzbacher et al., 2019). A stable and
reliable AF classification system would be enormously facilitated
by the ability to widely utilize the complete ribosomal operon
sequence as phylogenetic marker, assuming a corresponding
well-curated reference database will be available in the future.
ITS1 region itself ranges in size (Edwards et al., 2008) and
recent work of Edwards et al. (2019) indicated preferential
amplification of smaller sized ITS1 regions. PCR primer
choice is also crucial, which was highlighted by the finding
that the sequence amplified by a widely used primer (i.e.,
MN100F) is not conserved in all AF (Kittelmann et al., 2013;
Callaghan et al., 2015).
These problematic aspects of using the ITS1 as phylogenetic
marker gene, and the resulting instability of the ITS1 phylogeny,
has led to recent efforts to explore the potential of using
the sequence of the large 28S rRNA subunit (LSU) as a
phylogenetic marker instead (as the 18S rRNA gene is too
conserved in AF). It appears that the D1/D2 region of
the LSU has a taxonomic resolution similar to the ITS1
region (Wang et al., 2017). However, the more conserved
size of the D1/D2 marker and lower heterogeneity within
individual cultures enables a more stable phylogenetic backbone
for AF classification. Since it provides molecular support
for all currently accepted genera of AF, and provides a
higher resolution compared to the ITS1 region, utilization
of the D1/D2 region of the LSU as a phylogenetic marker
seems favorable (Dagar et al., 2011; Wang et al., 2017;
Hanafy et al., 2020).
A number of AF specific primers targeting the LSU region
have been published (Dollhofer et al., 2016; Nagler et al.,
2018), and employed to study AF community composition of
environmental samples (Dollhofer et al., 2017; Nagler et al.,
2019). However, a sufficiently large reference database for these
taxonomic marker genes from previously characterized taxa and
their type specimens is still lacking (Wang et al., 2017). There
is also a challenge of how to relate LSU sequences to the clades
that until now only contain “unculturable” representatives and
that were identified by environmentally derived ITS1 sequences
(Edwards et al., 2019). Despite the advances of the LSU D1/D2
region as a phylogenetic marker, ITS1 still remains the most
accepted phylogenetic marker for AF. As such, it is likely
that ITS1 will continue to be used in the near future as the
primary barcode to assess AF diversity and community structure
in environmental samples. However, it is undisputable that to
advance a more accurate and higher resolution classification
of members belonging to the AF, a continuous expansion and
curation of an AF LSU reference database is need. An alternative
option could also be a more complex database that integrates the
use of different reference taxonomic marker genes (Wurzbacher
et al., 2019). A comprehensive ITS1 sequence database and
associated taxonomy files (Koetschan et al., 2014), including
Frontiers in Microbiology | www.frontiersin.org
LIFE CYCLE
One of the additional and major challenges that contribute to
the current lack of a consistent and standardized taxonomic
classification system for AF is the morphological transformations
AF undergo throughout their corresponding life cycles (Table 1).
AF reproduce asexually and they alternate between a motile
zoospore and a non-motile vegetative stage. Flagellate zoospores,
which are released from mature sporangia, actively move toward
freshly ingested plant tissues in the rumen; a chemotactic
response triggered by soluble sugars (Orpin and Bountiff, 1978)
and phenolic acids (Wubah and Kim, 1996). Zoospore liberation
is influenced by diet, and in ruminants is induced by watersoluble haems and other porphyrins (Orpin and Greenwood,
1986; Orpin, 1994). Although zoospores are motile for several
hours (Lowe et al., 1987a), they tend to attach to plant fragments
within 30 min after being released from a sporangium (Heath
et al., 1986; Edwards et al., 2008). After attachment, the
zoospores shed their flagella and form a cyst. The encysted
fungus then germinates to produce a fungal thallus that is
composed of the sporangium and a filamentous rhizomycelium
or a bulbous holdfast.
1
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Regardless of herbivore type, the life cycle of AF has been
proposed to contain a resting phase. While resting structures are
still not fully understood, they provide a compelling explanation
for why some of the currently known AF can be cultured from
fecal material after prolonged periods of desiccation and oxygen
exposure (Milne et al., 1989; Davies et al., 1993; McGranaghan
et al., 1999; Griffith et al., 2009). To date resting cysts (Orpin,
1981), melanized resistant sporangia (Wubah et al., 1991) and
multi-chambered spore-like structures (Brookman et al., 2000)
have been described in different AF taxa. Although it may well
be the case that there is no resting structure common to all
AF, with taxon-specific structures instead, resting structures are
thought to play an important role in the inter-animal transfer
of AF. For example, it has been suggested that the survival of
AF in saliva is likely to be an important transfer mechanism
in ruminants and pseudoruminants (Lowe et al., 1987b). In
hindgut fermenters, feces may play a more important role as
a transfer mechanism between animals than saliva, particularly
as certain hindgut fermenters, like foals, exhibit coprophagic
behavior (Marinier and Alexander, 1995).
As zoospores and fungal thalli represent different parts of the
same AF life cycle, consistent and accurate enumeration of AF is
challenging. Approaches used to count exclusively free zoospores
(France et al., 1990), fungal colonies on agar strips (Ushida et al.,
1989), or both morphologies in culture supernatants (Theodorou
et al., 1990; Obispo and Dehority, 1992; Griffith et al., 2009), and
chitin measurements (Gay, 1991; Rezaeian et al., 2004b) have, in
recent years, been widely replaced by molecular quantification via
real-time PCR. This method overcomes the contrast within the
life cycle between low zoospore numbers yet high AF vegetative
biomass, as well as the paucity or absence of zoosporogenesis
observed in some polycentric axenic cultures (Ho and Bauchop,
1991). On the other hand, the real-time PCR approach to quantify
fungal biomass possesses its own challenges, such as the varying
amount of fungal biomass produced by monocentric, polycentric
and bulbous genera relative to DNA content. This makes
translating quantitative estimates derived from real-time PCR
into fungal biomass very challenging (Denman and McSweeney,
2006; Edwards et al., 2008). Estimating AF abundance by
quantifying the number of ITS1 spacer regions [(Mura et al.,
2019) or rRNA genes, i.e., 5.8S rRNA gene (Edwards et al., 2008)
or LSU (Nagler et al., 2018)] seems to be taxon independent.
However, it still remains to be determined if all AF have the same
copy number of the rrn operon.
In most of the monocentric AF, thallus development is
of the endogenous type, where on germination, the zoospore
cyst develops a germ tube that branches and grows into a
rhizoid system. The nucleus remains in the zoospore cyst, which
develops into a new sporangium and the anucleate rhizoids.
Since the zoospore cyst retains the nucleus and develops into
a sporangium, the sporangial development type is referred to
as endogenous, and since this type of development results in
a single sporangium per thallus, it is said to be monocentric.
In some of the monocentric AF, such as the Piromyces spp.,
thallus development is of the exogenous type, which involves
a two-sided germination of the zoospore cyst. During this
process, a germ tube develops into a rhizoid system during
the endogenous sporangial development, but once a substantial
rhizoid has developed, a tubular outgrowth (the sporangiophore
or sporangial stalk) emerges on the side opposite of the
main rhizoid and the sporangium develops at the apex of the
outgrowth. As the original nucleus escapes the zoospore cyst
and develops elsewhere, the sporangial development is said
to be exogenous (Barr et al., 1989). Both types are strictly
determinate. In other members of the monocentric AF, such as
the Capellomyces, both exogenous and endogenous sporangial
development takes place (Hanafy et al., 2017, 2018, 2020;
Joshi et al., 2018).
Polycentric AF display an exogenous thallus development,
during which a one-sided germination of the zoospore cyst
occurs. During the germination process, the content of the
zoospore cyst migrates into germ tube which then develops
into a nucleated branched rhizomycelium capable of developing
multiple sporangia. Currently it appears that the remaining cyst,
which has been emptied, has no further function. Development
of thalli of polycentric fungi is said to be non-determinate.
In the case of the bulbous genera, nuclei are observed
in the vegetative parts of the thallus (holdfast/branched
sporangiophores), consistent with exogenous development.
Thallus development in the bulbous genera is of a limited
polycentric type, where the encysted zoospore forms a bulbous
holdfast without rhizoids. Bulbous holdfasts give rise to single or
multiple sporangia including branched sporangiophores. Growth
in these fungi is not non-determinate like the thalli of polycentric
fungi, but not as strictly determinate as in the case of the
monocentric filamentous AF (Ozkose et al., 2001).
Our current understanding of the AF life cycle is based on
what has been learned from rumen AF, but it is likely that AF
associated with pseudoruminants pass through similar, if not
even identical, life cycles stages. However, there are numerous
aspects of AF biology that remain to be reassessed for hindgut
herbivores. The cause of zoospore release in the hindgut is
unclear, as it is not known if the known triggering compounds in
the rumen (i.e., water-soluble haems and other porphyrins) can
survive passage through the gastric stomach and small intestine.
Whereas zoospores that are released within the rumen locate
freshly ingested plant material chemotactically using soluble
sugars (Orpin and Bountiff, 1978) and phenolic acids (Wubah
and Kim, 1996), it is unclear to what extent these chemotactic
signals are available to (and used by) AF in the cecum and colon
of hindgut herbivores.
Frontiers in Microbiology | www.frontiersin.org
INFLUENCE OF HOST ON ANAEROBIC
FUNGI COMMUNITY
Although diet has a significant effect on the structure of the
AF community, host animal phylogeny has been shown to be
a more important factor (Liggenstoffer et al., 2010; Kittelmann
et al., 2012). Kittelmann et al. (2012) provided strong evidence
that both diet and ruminant species, as well as interactions
between those two parameters, affect the diversity of the AF
community by subjecting cattle, sheep and deer to three different
diets (summer pasture, winter pasture, and silage). Recent studies
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Although most gut methanogens are thought to be
hydrogenotrophic (Ferry, 2010; Borrel et al., 2013; Lyu et al.,
2018), the majority of the global microbial CH4 is produced
via acetoclastic methanogenesis (Ferry, 2010; Lyu et al., 2018).
In the rumen ecosystem, both acetate and hydrogen are
produced (Baldwin and Allison, 1983) and are available for
methanogenesis. However, since their turnover rates in the
rumen are high, the contribution of CH4 produced via the
acetoclastic pathway accounts only for a small fraction of the
overall CH4 produced in the rumen (Baldwin and Allison, 1983;
Janssen, 2010). During hydrogenotrophic CH4 synthesis H2 and
CO2 are combined to yield CH4 (Baldwin and Allison, 1983),
with contributions from the newly described methylotrophic
methanogens (Poulsen et al., 2013).
Under normal circumstances, H2 seldom reaches high
concentrations in the rumen and the dissolved H2 concentration
is usually about 0.1–50 µM, which is 0.014–6.8% of its maximum
solubility at 39◦ C and 1 atm (Hegarty and Gerdes, 1999; Janssen,
2010). The scarcity and poor water solubility of H2 limits the
access of methanogens to molecular hydrogen, necessitating the
development of close physical contact and intimate syntrophic
partnership between H2 producers and H2 metabolizers. Extreme
forms of these interspecies H2 -transfer are the methanogenic
endo- and ecto-symbionts of rumen protozoa (Embley and
Finlay, 1994). The close association seen between rumen ciliates
and methanogens (Vogels et al., 1980) seems a more general
feature of anaerobic ciliates, aimed at boosting their metabolic
rate (Rotterová et al., 2020). Although AF are not known to
have methanogenic endo- or ecto-symbionts, they do contain
modified mitochondria known as hydrogenosomes (van der
Giezen, 2009), and there is in vitro based evidence of crossfeeding (syntrophy) between hydrogenic AF and methanogenic
archaea in the herbivore gut ecosystem (Yarlett et al., 1986).
Differential centrifugation of cellular fractions revealed
that fungal hydrogenosomes convert malate or pyruvate
under anaerobic conditions into H2 , CO2 , and acetate with
the concomitant production of ATP (Yarlett et al., 1986;
Marvin-Sikkema et al., 1993). This is similar to the process
found in trichomonads, anaerobic urogenital parasites, where
hydrogenosomes were first discovered (Müller, 1993). The H2
produced is the result of the action of the oxygen-sensitive
enzyme hydrogenase (Davidson et al., 2002), which is the
terminal electron acceptor for the metabolism coming from
pyruvate. The excess H2 produced by AF can be used by
methanogens to regenerate oxidized nucleotides (e.g., NAD,
NADP) (Yarlett et al., 1986). Indeed, methanogenic archaea have
even been found attached to the surface of fungal rhizoids
and sporangia (Bauchop and Mountfort, 1981; Jin et al., 2011),
which is likely to improve interspecies hydrogen transfer. Within
the rumen microbiome, symbiotic ecto- and endosymbiotic
partnership have been reported for rumen protozoa and
methanogens (Finlay et al., 1994; Irbis and Ushida, 2004; Valle
et al., 2015). Since other fungi have been shown to accommodate
prokaryotic endosymbionts (Partida-Martinez et al., 2007), there
is the possibility that methanogenic archaea can also exist
intracellularly within AF, although such an intimate symbiotic
relationship has not been reported until today.
revealed that the genetic background of the host animal can
influence the activity of the entire rumen microbiota, including
the community of rumen AF (Roehe et al., 2016). In addition
to the genetic background, several breed-associated phenotypes,
such as eating frequency, dry matter intake, and rumen size
potentially contribute to the variations in rumen microbiota
that is observed among various breeds (Wallace et al., 2019;
Zhang et al., 2020).
Anaerobic fungi were reported to be present in domesticated
and non-domesticated equine species, with the AF community
composition in horses and ponies being more similar to zebras
than donkeys (Edwards et al., 2020b). In a separate study, AF
diversity in donkeys was shown to be higher when compared to
that of ponies and pony × donkey hybrids (Edwards et al., 2020a).
Several studies have revealed that the genus Khoyollomyces
(formerly known as AL1) is almost exclusively found in equines
(Liggenstoffer et al., 2010; Mura et al., 2019; Edwards et al.,
2020b; Hanafy et al., 2020). This may be due to the growth
or metabolic characteristics of Khoyollomyces, making it more
adapted to growth in the equine hindgut. Metabolic differences
have previously been reported for equine and rumen strains
of Piromyces, with equine strains possessing faster growth and
higher fiber degradation capacity compared to rumen isolates
(Julliand et al., 1998). This is perhaps not surprising considering
the fundamental differences between ruminants (where freshly
ingested feed directly enters the rumen), and hindgut herbivores
(where feed first passes through the stomach and small intestine
before reaching the hindgut) in terms of the main gut site
where fiber degradation primarily occurs. There is also evidence
indicating that the genus Oontomyces is specific to camelids
(Dagar et al., 2015). Further research is needed to understand key
factors that limit, or conversely broaden, the host distribution of
certain AF taxa.
ROLE OF ANAEROBIC FUNGI IN
METHANOGENESIS
Ruminants are a significant source of anthropogenic methane
(CH4 ) producing ∼90 Tg of CH4 annually (Reay et al., 2018),
with exact values differing based on the methodology employed
to quantify emissions (Capper and Bauman, 2013; Lyu et al.,
2018). Although methanogenesis occurs in the rumen, there
appears to be no direct benefit of the microbial generated
CH4 (which is released into the environment) to the animal
itself. The ruminant provides the anaerobic environment that
is necessary for the archaea, first described as archaebacteria
in the late 1970s by Woese and Fox (1977), that are capable
of CH4 production. Due to the complex nature of the
rumen microbiome and the interactions between the individual
community members and their biochemistry, the role of AF in
methanogenesis can only be understood in light of knowledge
about archaea and processes facilitated by them. There are
three different biochemical routes by which archaea can produce
CH4 : (1) acetoclastic methanogenesis, (2) hydrogenotrophic
methanogenesis, and (3) methylotrophic methanogenesis (Borrel
et al., 2013; Offre et al., 2013).
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Lee et al., 2000b). This is likely due to the broad range of enzymes
that are produced by the AF combined with their physical ability
to break open fibrous materials through their penetrating hyphal
tips. These tips have high concentrations of fibrolytic enzymes,
whose enzymatic activity subsequently also increases nutrient
access for other cellulolytic microbes (Ho et al., 1988; Ljungdahl,
2008; Haitjema et al., 2014; Dagar et al., 2015; Solomon et al.,
2016). Whilst rumen zoospore numbers are low compared to
counts of bacteria and archaea, AF have been shown to represent
up to 20% of the rumen microbial biomass (Rezaeian et al.,
2004a) and 10–16% of rRNA transcript abundance (Elekwachi
et al., 2017). The observation that some AF species are capable
of releasing up to 95% of the fermentable sugars from untreated
plant leaves during a 4-day incubation period (Sijtsma and Tan,
1996) further highlights their critical role during the rumen
digestion of fibrous plant biomass. These findings have led to
the general belief that AF have been essential in the successful
evolution of mammalian herbivores (Wang et al., 2019).
Understanding how AF and their carbohydrate-active
enzymes (CAZymes) contribute to the degradation of indigested
biomass in the herbivore gut is rather limited compared to the
knowledge of the role of bacteria. This is partially due to the
more extensive genomic resources that exist for rumen bacteria
compared to AF (Hess et al., 2011; Seshadri et al., 2018; Stewart
et al., 2019). AF genome information that is currently publicly
available is summarized in Table 2, and includes the AF strain
host isolation source and number of CAZymes identified.
One striking feature of AF besides their large repertoire and
diversity of CAZymes (Supplementary Table S1), is the ability
of their CAZymes to form cellulosomes. Cellulosomes, first
identified in anaerobic bacteria, are extracellular multi-enzyme
complexes that tether together an assortment of cellulases and
related accessory enzymes (Lamed et al., 1983). The assembly of
these AF multi-protein complexes, in some cases with individual
building blocks from different species, is facilitated by fungal
dockerins, which can directly bind to plant cell wall components
without the need for a scaffoldin (Fanutti et al., 1995). This
is in stark contrast to bacterial cellulosomes, which are highly
species-specific (Bayer et al., 2004).
Cellulosomes have been linked to the improved fitness and
biomass-degradation phenotype of both anaerobic bacteria and
AF (Bayer et al., 2004; Henske et al., 2018) by enabling the
synergistic activity of the individual biomass-degrading enzymes.
Cellulosomes have been shown to increase cellulolytic activity
over free enzymes by up to 12-fold (Krauss et al., 2012). Synthetic
cellulosomes, inspired by bacterial cellulosomes, have shown
promise for industrial applications due to outperforming free
cellulolytic enzymes when produced in recombinant systems
(Gilmore et al., 2020). Although evidence of AF cellulosomes
emerged about 20 years ago (Wilson and Wood, 1992), it is
only recent work that has provided strong support for the
hypothesis that these multi-enzyme machineries might hold
the secret to the superior biomass-degrading capability of AF.
However, cellulosomes from AF still remain poorly understood
(Haitjema et al., 2017).
Horizontal gene transfer (HGT) between the different
populations of rumen microbes appears to have played a
There have been numerous co-culture studies, particularly
with hydrogenotrophic Methanobrevibacter isolates and
representatives of the genera Piromyces, Neocallimastix,
Orpinomyces, Caecomyces, and Anaeromyces (Edwards et al.,
2017). Methane, CO2 , formate and acetate are the main products
when AF are grown in the presence of methanogens, whereas H2 ,
lactate, succinate and ethanol production is drastically decreased
compared to the corresponding pure anaerobic fungal cultures
(Bauchop and Mountfort, 1981; Marvin-Sikkema et al., 1990).
This difference between co- and pure cultures is due to the
inter-species hydrogen transfer in the methanogenic co-cultures
influencing the efficiency of anaerobic fungal fermentation. This
shifts AF product formation away from more oxidized endproducts (i.e., lactate and ethanol) and toward the production of
more reduced products (i.e., formate and acetate). Recently the
life cycle stage has also been shown to shift metabolite production
of AF grown in methanogenic co-culture (Li et al., 2019).
In exchange for the excess H2 produced by AF, methanogens
have a beneficial effect on AF growth and activity. This is
evidenced in terms of increased cellulolytic enzyme activity and
dry matter disappearance in methanogenic co-cultures compared
to anaerobic fungal cultures alone (Bauchop and Mountfort,
1981; Jin et al., 2011). The ability of AF and methanogen
co-cultures to rapidly convert lignocellulose containing plant
material into CH4 also has good potential for biotechnological
applications (Cheng et al., 2009; Jin et al., 2011; Peng et al., 2016),
particularly in terms of biogas production from lignocellulosic
waste streams. Conversely, in ruminants CH4 production is
viewed as an unfavorable outcome of the fermentation. In
ruminants, eructation of H2 in the form of large amounts of CH4
represents a loss of energy for the animal in addition to being a
significant source of anthropogenic CH4 .
Several rodent hindgut fermenters and non-ruminant foregut
fermenters also emit CH4 of a magnitude as high as ruminants,
but in contrast equids, macropods and rabbits produce much
less (Clauss et al., 2020). There has been a move in recent
years to understand why some domesticated ruminants produce
lower CH4 compared to others (Shi et al., 2014), and a
subsequent push to utilize feed-based approaches to decrease
ruminal methanogenesis (Jeyanathan et al., 2014). However,
it remains to be determined to what extent the growth and
activity of rumen AF is affected by reducing the number and/or
activity of methanogenic archaea using these approaches. Being
able to understand the interdependencies of these populations
will be important for informing a holistic understanding of
the microbiome as it pertains to ruminant function. Current
knowledge regarding interactions of AF with bacteria and
protozoa is more limited, and has been reviewed elsewhere
(Gordon and Phillips, 1993; Edwards et al., 2017).
ROLE OF ANAEROBIC FUNGI IN PLANT
CELL-WALL DEGRADATION
In vitro studies suggest that the contribution of AF to the ruminal
degradation of plant material could be more significant than the
contribution of cellulolytic rumen bacteria (Joblin et al., 1989;
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TABLE 2 | Publicly available genomes of anaerobic fungi.
Organism
Strain
Host
Genome sizea [base pairs]
Gene counta
CAZyme counta
References
Haitjema et al. (2017)
Anaeromyces robustus
S4
Sheep
71,685,009
12,832
1,766
Caecomyces churrovis
–
Sheep
165,495,782
15,009
NDb
Henske et al. (2017)
Neocallimastix californiae
G1
Goat
193,495,782
20,219
2,743
Haitjema et al. (2017)
Pecoramyces ruminantium
C1A
Cow
100,954,185
18,936
2,029
Youssef et al. (2013)
Pirfi3
Horse
56,455,805
10,992
1,463
Haitjema et al. (2017)
E2
Elephant
71,019,055
14,648
3,819
Haitjema et al. (2017)
(formerly Orpinomyces sp.)
Piromyces finnis
Piromyces sp.
a https://mycocosm.jgi.doe.gov
b Not
(Grigoriev et al., 2013).
determined.
FIGURE 2 | Carbohydrate-active enzymes employed by anaerobic fungi during biomass conversion. Anaerobic fungi (AF) deploy various strategies for the
degradation of plant biomass. It has been suggested that their ability to produce secreted free CAZymes, cell-bound multi-enzyme complexes (cellulosomes), as well
as free cellulosomes might provide the AF with the competitive advantage over the CAZyme repertoire produced by anaerobic bacteria (Henske et al., 2017).
but can be also found as free multi-enzyme complexes that are
released into the extracellular matrix (Haitjema et al., 2014).
These free cellulosomes enable an increased concentration of
enzymes that possess catalytic and carbohydrate binding sites
without the need to have them linked to and displayed on the
AF cell surface. More importantly, it has been suggested that
these free cellulosomes are non-species specific and theoretically
enable the synthesis of cellulosomes using components of
different donors (Nagy et al., 2007).
Another aspect that distinguishes AF cellulosomes from
their bacterial counterparts is the presence of modules
that have sequence signatures which are conserved within
members of glycoside hydrolase (GH) families GH3, GH6,
and GH45 (Haitjema et al., 2017). The ability to expand the
substrate repertoire of AF cellulosomes by incorporating
these GH signatures might provide additional enzymatic
capabilities not conveyed by bacterial cellulosomes, such as the
conversion of cellulose to monosaccharides by the β-glucosidase
activity of GH3.
Besides cellulosomes, free CAZymes are also produced
AF, which ultimately leaves the AF with several hydrolytic
mechanisms to attack plant cell wall polymers from multiple
directions (Haitjema et al., 2017) (Figure 2). The ability to
significant role in the evolution of the Neocallimastigomycota
(Murphy et al., 2019). HGT between rumen bacteria and rumen
AF was proposed by Garcia-Vallvé et al. (2000) as a major
mechanism that allowed rumen AF to acquire many of the
CAZymes that have now been identified in their genomes
(Supplementary Table S1). Since then, numerous other authors
have supported the hypothesis that the acquisition of CAZymes
from bacterial donors enabled the rumen AF, as well as rumen
protozoa (Ricard et al., 2006), to successfully compete for plant
carbohydrates in the herbivore gut (Duarte and Huynen, 2019;
Wang et al., 2019). Despite the numerous CAZyme families that
AF acquired via HGT, the cellulosome of AF appears to have
unique attributes that distinguish it from those that are found
in gut bacteria.
Cellulosomes, regardless of their origin, are typically
composed of several enzymes (i.e., cellulases and hemicellulases)
that contain an active site, one or several carbohydrate binding
modules and a dockerin that facilitates the “docking” of the
enzymatic multi-modular complex to one of the multiple
cohesins that are displayed by a scaffoldin. In anaerobic bacteria,
cellulosomes are anchored to the bacterial cell wall via dockerinscaffoldin modules (Fontes and Gilbert, 2010). In contrast, AF
cellulosomes are not necessarily anchored to the fungal cell wall,
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fiber (ADF) digestibility, animal growth, milk yield and VFA
concentrations (all parameters increased when AF were added)
(Dey et al., 2004; Paul et al., 2004, 2011; Tripathi et al., 2007;
Saxena et al., 2010).
Probiotic capabilities of live AF cultures for ruminants were
indicated in the 1990s when living AF cultures were added to the
rumen of cattle and sheep from which fungi had been previously
removed. Forage intake by such fungus-free early weaned calves
was 35% higher in those that had been dosed with Neocallimastix
sp. R1 (Theodorou et al., 1990), and dosing of fungus-free sheep
with Neocallimastix sp. SLl resulted in a 40% increase in intake of
a straw based diet (Gordon and Phillips, 1993).
Despite the work that has been conducted to investigate
how dosing the rumen ecosystem with AF cultures affected the
abundance of the native fungal, bacterial and even the ciliate
rumen populations (Lee et al., 2000a; Paul et al., 2004, 2011),
there has been no evidence that suggests that dosing with AF, and
therefore the increase of AF concentration in the rumen, resulted
in a decrease of bacteria or ciliate protozoa nor in a drop of feed
digestibility. In contrast to this, a positive correlation between
fungal and bacterial concentrations, most likely due to the fact
that the hyphae of AF physically open the plant tissue thereby
increasing surface area available for colonization and nutrient
access for other fibrolytic rumen microbes, has been reported
(Bauchop, 1979; Akin and Borneman, 1990). This increased
accessibility would also explain why counts of the holotrichs,
the starch degrading protozoal population, increased in response
to dosing with AF, while the overall count of the ciliate rumen
protozoa remained stable (Paul et al., 2004).
Besides the lack of evidence that an increase in AF by dosing
reduces the abundance of other rumen populations, it appears
to be noteworthy that none of the studies centering on AF
dosing has looked at potential changes of fiber-colonization
by AF after the dosing event. Considering that dosing with
AF cultures improved fiber digestion, VFA production and
animal performance related parameters, suggesting that the
altered microbiome is more efficient in the digestion of fibrous
feeds than the original community, a closer investigation of
the fiber colonization and deconstruction process post-dosing
seems warranted.
Whereas ruminal fiber digestion improved in response to
live AF, no shift in fermentation pattern was observed when
AF-derived enzymes were added to the diet (Lee et al., 2000a).
This highlights the importance of using viable cultures of AF
as ruminant feed additives (Paul et al., 2011). In contrast to
this, spent media containing AF enzymes seems to be effective
in improving monogastric livestock production (Theodorou
et al., 1996). Cell walls of cereals, major components of
swine and poultry feed, contain difficult to digest non-starch
polysaccharides, such as β-glucans in barley and wheat and
arabinoxylans in rye and oats (Sánchez-Rodríguez et al., 2012).
These polymers can have anti-nutritional effects due to their
low digestibility and tendency to form high-molecular-weight
aggregates that reduce passage rate, decrease diffusion of digestive
enzymes, promote endogenous losses, and stimulate unwanted
bacterial proliferation (Bedford and Schulze, 1998). Previous
studies suggest that recombinant GHs from AF expressed in
produce secreted free CAZymes, cell-bound cellulosomes, as
well as free cellulosomes might provide the AF with the
competitive advantage over the CAZyme repertoire produced by
the cellulolytic anaerobic bacteria also resident in the herbivore
gut (Henske et al., 2017). It has also been proposed that the
superior efficiency in biomass degradation of AF might be caused
to a great extent by the ability to simultaneously employ a
diverse set of cellulosome-bound as well as free (not cellulosome
associated) CAZymes that also display a significant functional
overlap (Couger et al., 2015).
This hypothesis, as well as the ability of AF to contribute a
complementary set of CAZymes to the ones provided by bacteria
was recently supported by genome-centric metaproteome and
metatranscriptome approaches (Hagen et al., 2020). Shotgun
metaproteome and metatranscriptome data were mapped back
on previously assembled rumen genomes from prokaryotes and
cultured AF, as well as on Metagenome Assembled Genomes
to determine the origin of the different CAZymes detected
in the microbiome that colonized recalcitrant plant material
during rumen-incubation (Hagen et al., 2020). Results from
this study revealed that the bacterial population contributed
CAZymes mostly associated with the degradation of more readily
degradable carbohydrates such as hemicellulose, whereas fungi
provided CAZymes (e.g., enzymes belonging to the GH family
GH5, GH6, GH8, and GH48) that targeted the more recalcitrant
plant cell wall components. These data also provided clear
evidence for the involvement of AF cellulosomes in the biomass
degradation process. It can be assumed that a more detailed
understanding of AF and their enzymatic repertoire, in the
context of a highly diverse and synergistically working microbial
ecosystem, will emerge as more of these complex and large-scale
meta-omics data sets are generated and analyzed.
ANAEROBIC FUNGI AS FEED ADDITIVES
TO PROMOTE ANIMAL HEALTH AND
PERFORMANCE
The capability of AF to colonize and degrade otherwise
recalcitrant plant structures via a set of highly efficient enzymes
has led to an increased interest in the application of AF, and
their enzymes, to boost the digestibility of low-quality feed and
to increase the overall feed efficiency of herbivorous animals.
Enabling the utilization of low-quality feed will be essential to
enable the production of high-quality animal proteins, especially
during a time when terrestrial areas for the production of highquality feed will become scarce due to a changing climate and
the rapid urbanization of areas that are currently being used to
grow feed crops.
Several in vivo studies indicated that continuous dosing of
ruminants with live culture of AF results in the changes of
numerous parameters indicative of enhanced feed digestibility
and feed efficiency, with benefits of AF dosing appearing to
be more pronounced in young ruminants (Sehgal et al., 2008).
Measurements performed to determine the animal’s ability
to digest feedstuff in response to AF dosing, include dry
matter (DM), neutral detergent fiber (NDF) and acid detergent
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considered when discussing AF as probiotics on a commercial
scale (Ribeiro et al., 2016).
As well as positively impacting forage intake and feed
digestibility, AF have the potential to contribute to the protein
supply of the host animal. This is both indirectly through the
production of proteolytic enzymes in the rumen and directly
as a source of microbial protein. Unlike the cellulolytic rumen
bacteria, many isolates of AF are protease positive and capable
of penetrating the proteinaceous layer of feed particles (Wallace
and Joblin, 1985; Asao et al., 1993; Michel et al., 1993; Yanke
et al., 1993). In vitro studies with defined populations of
both proteolytic and non-proteolytic rumen bacteria and a
proteolytic Neocallimastix frontalis strain have further indicated
that N. frontalis was able to contribute to rumen protein
degradation, particularly when protein was associated with feed
particles (Wallace and Munro, 1986).
Besides producing proteases, AF directly contribute to protein
supply of the host in terms of being part of the microbial
biomass that passes down to the intestines from rumen, for
subsequent digestion and absorption. Gulati et al. (1989) showed
that AF cells were composed of proteins with a well-balanced
combination of amino acids that were highly available to the
ruminant host. A high proportion of the protein components of
three monocentric AF (i.e., Neocallimastix sp. LMI, Piromyces
sp. SMI and Caecomyces sp. NMI) was digested and absorbed
in the intestine of sheep, with digestibility factors of 0.91–0.98
(Gulati et al., 1988, 1989). These high in vivo digestibility values
for AF protein compared favorably with a value of 0.77 for mixed
rumen bacteria protein measured in a similar manner (Gulati
et al., 1990). Although the amount of nutritional nitrogen derived
directly from anaerobic rumen fungi might only amount to a
small portion of the total nitrogen that is absorbed by the animal,
its importance lies in its high quality and immediate availability.
Lactobacillus reuteri maintain their fiber-degrading capability
and their resistance to bile salt and acids, while L. reuteri
itself still retained its high adhesion efficiency to mucin and
mucus (Liu et al., 2005a,b, 2007; Cheng et al., 2014), which
would explain the decomposition-promoting effect of these
recombinant proteins in the monogastric animal. Despite these
promising characteristics for recombinant AF-derived GHs, the
ability to efficiently generate significant amounts of cheap, stable,
and active recombinant enzymes will be essential for low-cost
production and large-scale application of these enzymes as a feed
additive for monogastrics.
Tannins present in many feeds and forages are inhibitory to
rumen microbes, and are an anti-nutritional issue for ruminants.
Likewise, upon anaerobic degradation of phenolic compounds
present in fibrous feeds, different phenolic monomers (i.e.,
ferulic acid, p-coumaric acid, vanillic acid, vanillin, catechol etc.)
are released into the rumen that are inhibitory to microbiota.
Therefore, attempts were made to identify rumen microbes
that had tannin or phenolic monomer tolerating or degrading
capability, so that they could be utilized as direct fed microbials
to mitigate adverse effects of these anti-nutritional factors.
The anaerobic rumen fungus Piromyces sp. FNG5, isolated
from a wild herbivore, was found to be tolerant to phenolic
monomers and its pure culture degraded p-coumaric acid (38.5–
65.1%), 65.2–74.1% ferulic acid (65.2–74.1%) and vanillic acid
(34.1–66.8%) after 14 days of incubation (Paul et al., 2003).
McSweeney et al. (2001) reported that sheep fed with condensed
tannins from Calliandra calothyrsus had reduced ruminal AF
concentration, but the inhibitory effect was less prominent
compared to rumen bacteria. Paul et al. (2006) reported that
addition of Piromyces sp. FNG5 significantly increased in vitro
degradation (12%) of condensed tannins and this AF isolate
could tolerate tannic acid concentrations up to 20 g/L. This
amount is higher than the theoretic tannic acid level expected
in the rumen of animals fed a diet composed exclusively
of high tannin content plants. Conversely, Kok et al. (2013)
found that L. leucocephala hybrid-Bahru (containing condensed
tannins) when fed to goats, significantly decreased ruminal
AF concentration. Saminathan et al. (2019) reported that high
MW fractions of condensed tannins had inhibitory effect on
ruminal AF, but relative abundance of Piromyces 4 was increased
indicating that this group of uncultured AF is likely to be
tannin resistant. The mechanisms by which some AF species
can overcome the growth inhibitory effects of condensed tannins
or phenolic monomers is unknown. Whether AF produce
tannase, or not, remains to be established; but many isolates
of AF, especially those from wild ruminants adapted to tannin
rich and fibrous diets, were shown to produce a variety of
esterases capable of degrading phenolic compounds (Paul et al.,
2003). It is possible that these esterases are directly linked the
ability of these AF to overcome growth inhibition caused by
phenolic compounds.
Although these studies highlight the potential of using
AF as probiotics to enhance digestibility of highly fibrous
or tanniferous feed, boosting ruminant livestock production,
economic aspects such as the need to repeatedly administer
oral-dosages of AF to maintain the desired response have to be
Frontiers in Microbiology | www.frontiersin.org
DIETARY MANIPULATION OF
COMMENSAL ANAEROBIC FUNGI
Rather than directly adding more AF to the ruminant animal,
there have been numerous attempts to improve the concentration
of AF in the rumen by providing animals with feed that
increases their overall concentration. It was previously believed
that increasing dietary recalcitrant fiber may increase fungal
population in rumen, as some of the plant fiber breakdown
products were shown to have a positive effect on zoosporogenesis
and chemotactic effects on fungal zoospores. Few AF were seen
in the rumen of animals that were fed lush pasture (i.e., legume
or grass when green and leafy), and the number of AF increased
when animals were fed the same pasture after it had matured
and was more recalcitrant (Bauchop, 1989; Kostyukovsky et al.,
1991). However, results later showed the opposite effect, with
an increased AF count in ruminants fed a low-lignin diet
reported compared to animals provided with more recalcitrant
feed (Gordon and Phillips, 1998). To make matters even more
complex, other studies suggested that there was no direct effect
of the hay type (with different levels of lignin content) on AF
populations (Sekine et al., 1995).
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Anaerobic Fungi in Herbivores
addition decreased the concentration and diversity of AF.
However, responses at the genus level were dependent on
concentrate/forage ratios (Tapio et al., 2017). The addition of
rapeseed oil led to a considerable decrease in the ruminal AF
population, but the mechanism was not further investigated
(Fonty and Grenet, 1994). Previously, Elliott et al. (1987)
found that feeding a supplement of sunflower meal to sheep
consuming a barley straw diet resulted in decrease of ruminal
AF concentration to below detectable levels. In another study,
the feeding of calcium salts of medium chain fatty acids (C6–
Cl2) to sheep resulted in reduced numbers of AF zoospores
in the rumen, whereas the salts of long chain fatty acids
(C ≥ 14) had no effect on AF (Ushida et al., 1992). This
indicates that the inhibitory effects of the long chain fatty acids
common in oilseed meals can be alleviated, at least partly, by
chemical pretreatment.
Anaerobic fungi are also sensitive to a shortage of nitrogen.
A low protein diet decreased rumen AF concentration in dairy
cows compared to a high protein diet (Belanche et al., 2012).
However, the AF community composition was modified by
the level of dietary protein only when cows consumed the
starch-rich diet, but not the fiber-rich diet (Belanche et al.,
2012). This highlights the potential for further complexities
when trying to determine the effects of individual dietary
components on ruminal AF.
It has been suggested that an appropriate amount of starch
or concentrate in diet may support ruminal AF growth and
stimulate zoosporogenesis (Matsui et al., 1997), but in vivo study
findings remain inconclusive (Ishaq et al., 2017). A possible
explanation for these different responses to starch rich feed
is that only some of the AF, namely species of the genera
Neocallimastix, Piromyces and Orpinomyces, have been shown
to produce amylases and, therefore, have the ability to ferment
starch grains (Phillips and Gordon, 1988; McAllister et al., 1993;
Yanke et al., 1993). More work in this area needs to be conducted
in vivo before final conclusions can be made. Furthermore,
feeding starch or concentrates tends to increase ruminal ciliate
protozoal concentrations, and protozoa are known to predate on
AF zoospores (Morgavi et al., 1994).
Beneficial effects of sulfur supplementation, specifically for
low sulfur diets, on the number of AF in sheep and their
relative contribution to fiber degradation was reported in the
early 1980s (Akin et al., 1983). This beneficial effect was further
confirmed in subsequent studies using alkali treated wheat straw
(Gordon et al., 1983; Gulati et al., 1985; Weston et al., 1988)
and other poor quality feeds (Morrison et al., 1990). Promkot
and Wanapat (2009) suggested beneficial manipulation of AF
concentrations was possible through provision of an appropriate
dietary supplement containing sulfur. For a supplement of this
type to be effective, it should ideally contain a single organic
sulfur compound which is readily utilized by the rumen AF but
not by other components of the rumen microbiota (i.e., bacteria,
archaea, and protozoa).
Two organic sulfur nutrients, mercapto-1-propionic acid
(MPA) and 3-mercapto-1-propanesulfonic acid, were tested in
cattle trials and compared to an inorganic sulfur supplement. It
was reported that the organic sulfur sources improved nitrogen
utilization and microbial protein production, but surprisingly
this was concluded to be due to a general improvement in
the efficiency of microbial fermentation of lignocellulose and
not from specific stimulation of ruminal AF (McSweeney and
Denman, 2007). Conversely, in a patent (Gordon and Phillips,
2002) it was reported that administering an effective amount
of a degradation resistant sulfur source (MPA or its functional
equivalent) promoted the growth of AF in the rumen of
animals fed low sulfur content diets. Within this patent, it
was also demonstrated that ruminal MPA infusion increased
AF zoospores concentrations, and had a strong, positive,
response on the digestive performance of sheep. However,
additional scientific literature in this field is scarce and a
sulfur supplement specific for promoting anaerobic rumen fungi
remains to be identified.
Influence of other dietary supplements on the rumen AF
community has been less studied, but some interesting findings
have been reported. Thiamine supplementation, used to attenuate
rumen metabolic disorder caused by high concentrate diet
through buffering the rumen pH, increased significantly the
proportion of ruminal AF in dairy cows (Xue et al., 2018).
Plant oils, which are attractive feed additives used to mitigate
CH4 emissions, seem to have a negative effect on AF. The
addition of soya oil significantly reduced ruminal AF diversity
in steers (Boots et al., 2013). In dairy cows, sunflower oil
Frontiers in Microbiology | www.frontiersin.org
FUTURE PERSPECTIVES AND
OPPORTUNITIES FOR ANAEROBIC
FUNGI BASED APPLICATIONS IN
ANIMAL PRODUCTION AND HEALTH
With consumer demand for affordable high-quality animal
products increasing and with a decline in natural resources,
such as farmable land area, it will be essential to create new
and refine existing strategies to improve the utilization of lowquality forages for animal feed. Such strategies will rely heavily
on approaches that render the recalcitrant fraction of the plant
material more accessible to the fermenting microorganisms that
are indigenous to the herbivore gut. AF with their ability to break
open recalcitrant plant structures will play a significant role in
these new approaches.
Whilst AF are clearly beneficial for the ruminant host, the
underlying mechanisms to boost indigenous AF populations
through standard feed components such as fiber, starch, nitrogen
and lipid are inherently complex and are still not well understood.
As such, there still remains great interest in developing a reliable
and reproducible feed-based strategy to increase AF in order
to improve animal performance and health. However, whilst
the benefits of AF for ruminants has been well established,
their value for hindgut herbivores remains to be confirmed.
The observed increase in feed intake with AF supplemented
ruminants is thought to be due to the AF causing more rapid
clearance of digesta from the rumen, due to their physical and
enzymatic disruption of fibrous plant particles (Gordon and
Phillips, 1998). If this is also the case in equines, development
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Anaerobic Fungi in Herbivores
of the time that all fungi required oxygen. Considering AF have
only been effectively studied for ∼50 years, advances made during
this time have been significant. Whereas initial work required
the refinement of isolation and cultivation techniques and was
mostly driven by morphological observations and phenotypic
characterization, more recent insights have been enabled by
novel molecular approaches. These molecular techniques have
already greatly advanced understanding of the complexity
and diversity of the AF, however, our full understanding is
still far from complete. With the ability to sequence long
genomic regions such as the entire rrn operon, it is inevitable
that a more accurate and complete understanding of the AF
phylogeny will soon emerge. Understanding the phylogenetic
relationship of individual AF will be essential to increasing
understanding of their evolutionary history, and factors that
drive their niche specialization within and between different
types of host. However, to practically develop AF for application
in livestock production, as well as other industries, functional
systems microbiology approaches will be key. Technologies
such as (meta)genomics, (meta)transcriptomics and proteomics
will enable the pinpointing of specific genes, proteins and
reactions that are employed by AF in response to extrinsic
conditions (e.g., host genotype) and changes therein (e.g., host
health status and dietary composition). Despite the significant
progress made to date, the ecological role of AF and their
quantitative contribution to host function and health still remains
to be clarified in the full range of mammalian herbivores
where they naturally reside. Furthermore, a key phase of the
AF life cycle, the resting phase, is an area of very limited
knowledge that urgently needs to be researched. Together,
newly obtained knowledge in these areas will enable utilization
of AF and their enzymes to transform the sustainability and
environmental footprint of livestock agriculture, as well as
revolutionizing biotechnological processes involving plant-based
feedstocks. Whilst we increasingly understand more about the
evolution, biology and ecology of AF, there still remains many
key “why” questions to be answered: “Why” are AF sensitive
to oxygen? “Why” do AF have the lowest GC-content among
all known microorganisms? “Why” are AF the only fungi with
polyflagellate zoospores, hydrogenosomes, and cellulosomes?
Considering that the AF phylum is currently composed of
just one family, are we only looking at the tip of an iceberg?
Or have we missed something crucial when classifying these
microorganisms? Whatever the answers are, we know for certain
that many fundamental questions still remain to be answered
before the true potential of this highly valuable and paradigm
shifting phylum of microorganisms is fully understood and
can be harnessed.
of an equine AF probiotic may enable replacement of some
of the energy dense concentrates used in horse feeds with
more bulky fibrous feeds. This will contribute to reducing the
risk of colic and dysbiosis of hindgut microbiota, which is
commonly observed in working and/or performance equines
fed high grain/concentrate diets in order to meet their higher
energy requirements (Shirazi-Beechey, 2008; Durham, 2013;
Julliand and Grimm, 2017).
Most of the studies focused on AF-based strategies for
improving animal production and health have relied on the
repeated oral-dosing of AF. This approach can only become
economically feasible if the cost of industrial scale production
of an AF probiotic is significantly less than the economic
return gained by livestock producers. Considering the significant
benefits that have been reported for live AF supplementation
to date for ruminants, the probiotic use of AF is likely to
have a significant return on investment for ruminant livestock
producers. One possibility to produce an AF probiotic could be
the use of encapsulated cultures. However, methods for largescale production of encapsulated AF are currently not available,
and would have to be developed before this approach could
become commercially feasible. An alternative approach could
be the use of AF resting structures, with their subsequent
revival into active hydrolytic AF occurring within the host.
Fundamental understanding of these structures is, however,
currently too limited for this to be practically realized in the
near future. Advances in understanding of the biology of the
AF resting phase would not only facilitate the utilization of
AF in livestock production, but also their application in other
areas where lignocellulosic plant material is used to produce
biofuels and platform chemicals. As such, characterization of
the resting phase of AF should be a high priority research area
for development.
The resulting increased efficiency of livestock production
through the application of AF will undoubtedly have
beneficial impacts in terms of the environmental footprint and
sustainability of livestock production. The impact of AF based
strategies on ruminant derived methane production remains to
be determined, however, it is clear that increased efficiency of
lignocellulosic plant material utilization will also decrease the
need for arable land for animal feed production. Recent advances
in molecular techniques enable a detailed understanding of role
of the AF and how it affects the performance and health of its
herbivorous host. Research based on state-of-the-art methods
will allow the development of more advanced and holistic
approaches to manipulate the composition and function of the
gut microbiome and ultimately the health and performance of
the host animal. Such progress will facilitate a more sustainable
livestock industry to provide affordable high-quality animal
products for a growing global population.
AUTHOR CONTRIBUTIONS
MH designed the manuscript, coordinated the co-author
contributions, and wrote the plant cell-wall degradation
section. KF wrote the habitat, life cycle, morphology and
taxonomy section and contributed to other sections. SP and AP
contributed to the feed additive section. MG contributed to the
CONCLUSION
Although AF were first observed more than hundred years ago,
it was not until Colin Orpin’s ground-breaking work in the mid
1970s that AF were correctly identified, overturning the paradigm
Frontiers in Microbiology | www.frontiersin.org
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Anaerobic Fungi in Herbivores
methanogenesis section. CS co-authored the plant cell wall
degradation section. JE wrote the section on hindgut herbivores
and contributed to other sections. All authors reviewed
the manuscript, offered critical feedback, and approved the
final version.
SUPPLEMENTARY MATERIAL
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Conflict of Interest: During the development of the manuscript, JE changed
employment from Wageningen University & Research to the company Palital Feed
Additives, Netherlands.
The remaining authors declare that the research was conducted in the absence of
any commercial or financial relationships that could be construed as a potential
conflict of interest.
Copyright © 2020 Hess, Paul, Puniya, van der Giezen, Shaw, Edwards and
Fliegerová. This is an open-access article distributed under the terms of the Creative
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