MINIREVIEW

Anaerobic fungi (phylum Neocallimastigomycota): advances in

understanding their taxonomy, life cycle, ecology, role and
biotechnological potential
Robert J. Gruninger1, Anil K. Puniya2, Tony M. Callaghan3, Joan E. Edwards3, Noha Youssef4, Sumit
S. Dagar5, Katerina Fliegerova6, Gareth W. Griffith3, Robert Forster1, Adrian Tsang7, Tim McAllister1
& Mostafa S. Elshahed4
1
Agriculture and Agri-Food Canada, Lethbridge, AB, Canada; 2National Dairy Research Institute, Karnal, India; 3IBERS, Aberystwyth University,
Aberystwyth, Wales, UK; 4Oklahoma State University, Stillwater, OK, USA; 5Agharkar Research Institute, Pune, Maharashtra, India; 6Institute of
Animal Physiology and Genetics, Czech Academy of Sciences, Prague, Czech Republic; and 7Concordia University, Montreal, QC, Canada

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Correspondence: Mostafa S. Elshahed,
Oklahoma State University, Department of
Microbiology and Molecular Genetics, 1110 S
Innovation Way Dr., Stillwater, OK 74074,
USA. Tel.: +1 (405) 744 3005;
fax: +1 (405) 744 1112;
e-mail: Mostafa@okstate.edu
Received 9 June 2014; revised 3 July 2014;
accepted 7 July 2014. Final version published
online 11 August 2014.
DOI: 10.1111/1574-6941.12383
MICROBIOLOGY ECOLOGY
Abstract
Anaerobic fungi (phylum Neocallimastigomycota) inhabit the gastrointestinal
tract of mammalian herbivores, where they play an important role in the deg-
radation of plant material. The Neocallimastigomycota represent the earliest
diverging lineage of the zoosporic fungi; however, understanding of the rela-
tionships of the different taxa (both genera and species) within this phylum is
in need of revision. Issues exist with the current approaches used for their
identification and classification, and recent evidence suggests the presence of
several novel taxa (potential candidate genera) that remain to be characterised.
The life cycle and role of anaerobic fungi has been well characterised in the
rumen, but not elsewhere in the ruminant alimentary tract. Greater under-

standing of the ‘resistant’ phase(s) of their life cycle is needed, as is study of

Editor: Gerard Muyzer their role and significance in other herbivores. Biotechnological application of
anaerobic fungi, and their highly active cellulolytic and hemi-cellulolytic
Keywords
enzymes, has been a rapidly increasing area of research and development in the
gut fungi; herbivore; biotechnology; rumen
last decade. The move towards understanding of anaerobic fungi using –omics
fungi; fungal genomics.
based (genomic, transcriptomic and proteomic) approaches is starting to yield
valuable insights into the unique cellular processes, evolutionary history, meta-
bolic capabilities and adaptations that exist within the Neocallimastigomycota.

rumen fungi was complicated; however, due to the long-

Introduction
Mammalian herbivores do not produce cellulolytic or
hemi-cellulolytic enzymes to degrade ingested plant mate-
rial; instead they rely on symbiotic associations with
microorganism (i.e. anaerobic fungi, bacteria, methano-
genic archaea and protozoa) that reside within their gut.
Within this microbial consortium, anaerobic fungi are
known to be key players in the degradation of lignocellu-
losic plant fibre in the rumen (Akin et al., 1988, 1989,
1990; Lee et al., 2000a). Zoospores of anaerobic fungi in
the rumen were originally classified as protozoa (Liebet-
anz, 1910), until it was recognised that these ‘flagellates’
represented the dispersal phase of a zoosporic fungus
(Orpin, 1975). The initial acceptance of these novel
held belief that all fungi were obligate aerobes. However,
Orpin (1977a) showed that the cell wall of these organ-
isms contained chitin, confirming their correct placement
in kingdom fungi. Since this time, research has started to
uncover the basis for the novel mechanisms that enable
these fungi to live in the absence of oxygen.
Known adaptations of anaerobic fungi to their strict
anaerobic lifestyle include the absence of mitochondria,
cytochromes and other biochemical features of the oxida-
tive phosphorylation pathway (Yarlett et al., 1986; You-
ssef et al., 2013). Instead, anaerobic fungi possess
specialised organelles called hydrogenosomes, which cou-
ple the metabolism of glucose to cellular energy produc-
tion without the need for oxygen. These organelles have

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2 R.J. Gruninger et al.
features in common with mitochondria (van der Giezen,
2002) and are thought to be derived from them (Embley
et al., 1997, 2003; Voncken et al., 2002; Muller et al.,
2012). Hydrogenosomes contain hydrogenase; producing
H2, CO2, formate and acetate as metabolic waste products
(Brul & Stumm, 1994; Theodorou et al., 1996; Muller
et al., 2012). These, along with lactate and ethanol, are
the main fermentation end products produced from the
anaerobic fungal degradation and fermentation of a
variety of plant cell wall polysaccharides.
Anaerobic fungi degrade recalcitrant lignocellulosic
material both by invasive rhizoidal growth and the associ-
ated production of a range of powerful polysaccharide-
degrading enzymes. The recently published genome
sequence of Orpinomyces sp. strain C1A has provided
valuable insight into the diversity of these carbohydrate
active enzymes (Youssef et al., 2013), many of which
appear to have been acquired by horizontal gene transfer
from rumen bacteria. The cellulolytic machinery of anaer-
obic fungi consists of both free enzymes, as well as high
molecular weight extracellular multi-enzyme complexes
(Wilson & Wood, 1992) called cellulosomes (Krause
et al., 2003; Joblin et al., 2010). These potent enzymes
have received much attention in recent years, particularly
in terms of their biotechnological application. This review
covers the key advances that have been made in this area
over the last decade, as well as numerous other areas of
anaerobic fungal research, and highlights the challenges
and opportunities that currently exist within this research
field.
Taxonomy
Anaerobic fungi belong to the phylum Neocallimastigomy-
cota, the earliest diverging lineage unequivocally assigned
to kingdom Fungi and are closely related to the chytrids
(phylum Chytridiomycota) (James et al., 2006a,b; Hibbett
et al., 2007). While they share key morphological features
with their chytrid relatives, they possess a distinctive
anaerobic physiology and flagellar apparatus. Further-
more, genetic analyses have consistently shown that the
Neocallimastigomycota form a distinct, well-supported
clade basal to the chytrids (Fliegerova et al., 2004; James
Table 1. Morphological classification of the currently recognised anaerobic fungal genera
Genus Thallus
Neocallimastix Monocentric
Piromyces Monocentric
Caecomyces Monocentric
Orpinomyces Polycentric
Anaeromyces Polycentric
Cyllamyces Polycentric
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
et al., 2006a; Powell & Letcher, 2012). The phylum Neo-
callimastigomycota currently comprises six genera, each
distinguishable by morphological features: thallus mor-
phology (rhizoidal vs. bulbous) and zoospore flagellation
(monoflagellate vs. polyflagellate) (Ho & Barr, 1995; Ozk-
ose et al., 2001). These features are summarised in
Table 1, along with some illustrative microscopy images
(Fig. 1). Although conclusive genus identification of
anaerobic fungi using microscopic approaches can be
challenging, assignment of isolates into bulbous (Caec-
omyces, Cyllamyces), hyphael monocentric (Neocallimastix
and Piromyces) and hyphael polycentric (Orpinomyces and
Anaeromyces) by direct examination of colonies/cultures
is reasonably straightforward (Griffith et al., 2009). How-
ever, difficulties in observing zoospore release (Ho &
Bauchop, 1991), the pleomorphic growth form and vari-
able sporangial morphology of some isolates (Ho & Barr,
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1995; Brookman et al., 2000a; Leis et al., 2013) can make
further identification challenging. This has led to dis-
agreement as to the validity and distinctiveness of some
taxa (Wubah et al., 1991; Ho & Barr, 1995). Prior to the
advent of DNA barcoding, the difficulty of transporting
viable cultures and the absence of any established reposi-
tory for these fungi also made interlab comparisons very
difficult.
Application of DNA bar coding has served to highlight
these problems; however, sharing of data via the GenBank
repository has also provided a route towards a more
robust reappraisal of these fungi. Genotypic analysis was
initiated by Dore & Stahl (1991), who used partial
sequence of the ribosomal RNA (rRNA) small subunit
(SSU; 18S; c. 1800 bp in total) to confirm the monophyly
of the anaerobic fungi. However, the 18S region is highly
conserved in Neocallimastigomycota making it difficult to
determine relationships between more closely related taxa
(Dagar et al., 2011). Subsequent genetic classification has
mostly focused on the more variable internal transcribed
spacer (ITS) region of the rRNA locus, now formally
agreed as the primary DNA barcode region for all fungi
(Schoch et al., 2012).
PCR amplification of the ITS region relies on primers
which bind to the highly conserved flanking 18S and 28S
(large subunit; LSU) regions, yielding an amplicon of
Rhizoids Flagella per zoospore
Filamentous Polyflagellate (7–30)
Filamentous Uniflagellate, sometimes bi- or quadriflagellate
Bulbous Uniflagellate, sometimes bi- or quadriflagellate
Filamentous Polyflagellate (14–24)
Filamentous Uniflagellate
Bulbous Uniflagellate, sometimes bi- or triflagellate
FEMS Microbiol Ecol 90 (2014) 1–17
Anaerobic fungi: the who, what, why, where and how useful 3

(a) (b) (c)

(d) (e) (f)

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Fig. 1. Phase contrast (a–d) and bisbenzimide stained fluorescence (e and f) microscopy images of various anaerobic fungal morphological
features (scale bar, 50 lm): (a) free polyflagellated zoospores in a mixed culture; (b) germination and rhizoidal development of a monocentric,
filamentous Piromyces sp.; (c) Polycentric sporangia of an Anaeromyces sp.; (d) bulbous rhizoidal system of Caecomyces sp.; (e) nuclear migration
to rhizoids (white arrow) in a polycentric isolate; (f) nucleated rhizoids of Orpinomyces joyonii.

600–700 bp. The ITS region comprises two type I introns
(ITS1 and ITS2) split by the 5.8S rRNA subunit (159 bp
long), the latter providing an additional region for design
of conserved primers and amplification of ITS1 or ITS2
separately. To date, the ITS1 region has been the more
widely used amplicon for comparison of different genera
and species of anaerobic fungi (Li & Heath, 1992; Brook-
man et al., 2000a; Fliegerova et al., 2004; Tuckwell et al.,
2005). These analyses consistently support the close rela-
tionship between the two genera which form polyflagel-
late zoospores (Neocallimastix, Orpinomyces) and also the
two genera which form bulbous holdfasts (Caecomyces
and Cyllamyces). However, the phylogenetic relatedness of
the rhizoidal genera with monoflagellate zoospores (Pir-
omyces and Anaeromyces) is less clear, and it seems likely
that the genus Piromyces is polyphyletic and in need of
reappraisal (Brookman et al., 2000a; Hausner et al., 2000;
Fliegerova et al., 2004).
In addition to its application in taxonomic studies, the
ITS1 locus has been widely used in culture-independent
studies to assess fungal diversity and community struc-
ture. Various techniques utilising ITS-based PCR ampli-
cons have been employed in such studies, including
DGGE (Kittelmann et al., 2012), ARISA (Edwards et al.,
2008; Cheng et al., 2009; Sundset et al., 2009), clone
library sequencing (Fliegerova et al., 2006; Denman et al.,
2008; Nicholson et al., 2010) and next-generation
sequencing (Liggenstoffer et al., 2010; Kittelmann et al.,
2012, 2013). Furthermore, ITS1-based approaches have
also been used for the quantification of anaerobic fungi
in environmental samples by qPCR (Denman & McSwee-
ney, 2006; Edwards et al., 2008; Sekhavati et al., 2009;
McDonald et al., 2010; Lwin et al., 2011; Kittelmann
et al., 2012; Marano et al., 2012). The utilisation of these
approaches has clearly demonstrated that the scope of
diversity of anaerobic fungi is significantly wider than
previously implied by culture-based studies (see Ecology).
Despite the widespread use of ITS1 as the formal fungal
barcode (Schoch et al., 2012), it is apparent that its use
for anaerobic fungi is problematic. High sequence vari-
ability can cause difficulties in ITS1 sequence alignment,
while intragenomic variation within the ITS region causes
problems for direct sequencing of PCR products (Li &
Heath, 1992; Brookman et al., 2000a; Hausner et al.,
2000; Ozkose et al., 2001; Fliegerova et al., 2004; Nichol-
son et al., 2005, 2010; Chen et al., 2007; Edwards et al.,
2008). These problems have also been found for some
other groups of fungi (O’Donnell & Cigelnik, 1997; Ko &
Jung, 2002; Nilsson et al., 2008). Lastly, the misidentifica-
tion of sequences in NCBI database has caused confusion
when attempting to classify novel sequences (Bidartondo,
2008; Kittelmann et al., 2012). To start addressing this
issues, a revised taxonomic framework for the anaerobic
fungi has recently been proposed (Fig. 2; reprinted with

FEMS Microbiol Ecol 90 (2014) 1–17 ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
4 R.J. Gruninger et al.
permission from Koetschan et al., 2014) in the light of
the large amounts of next-generation sequencing data that
has being generated, and the discovery of many novel
candidate genera that remain to be cultivated (Liggenstof-
fer et al., 2010; Kittelmann et al., 2012; Koetschan et al.,
2014; see also Ecology). A website has also been created
to allow for researchers to use the secondary structure
prediction approach used by Koetschan et al. (2014) to
incorporate new ITS1 sequences from anaerobic fungi
into this classification scheme (https://www.anaerobicfun-
gi.biocommons.org.nz).
Life cycle
Anaerobic fungi reproduce through the asexual produc-
tion of flagellated zoospores from sporangia (Heath et al.,
1986), with no sexual reproductive life stage identified to
date. Zoospores may be posteriorly monoflagellate or
polyflagellate, with the subsequently developed thallus
being either monocentric (single reproductive body i.e.
one sporangium from single zoospore) or polycentric
(many sporangia from a single zoospore) (Ho & Barr,
1995). In the rumen, zoospores are released from anaero-
bic fungal sporangia in response to ingestion of food,
with the timing of peak zoospore density being reached
within 30–60 min (Orpin, 1975, 1976, 1977b; Orpin &
Joblin, 1997). Ruminal zoospore differentiation and sub-
sequent maturation are thought to occur through the
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
Fig. 2. Profile Neighbour Joining tree of
Neocallimastigomycota ITS1 sequences based
on 575 unique ITS1 sequences across the
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Neocallimsatigomycota. The predicted
secondary structure of the ITS1 region was
modelled to generate an improved alignment
of sequences. In addition to the six named
genera, it is apparent that ten or more clades,
which at present are unnamed, also exist. This
figure has been reprinted with permission
from Koetschan et al. (2014).
induction of sporangia by haem and other related por-
phyrins (Fig. 3), which are released from ingested plant
material (Orpin & Greenwood, 1986).
The locomotion of released Neocallimastix frontalis
zoospores is provided by beating of up to 8–17 flagella
which form a locomotory organelle, the activity of which
has been described previously (Lowe et al., 1987a,b,c).
Motile zoospores locate plant material for colonisation
via chemotactic responses to soluble sugars (Orpin &
Bountiff, 1978) and/or phenolic acids (Wubah & Kim,
1996). Flagellated zoospores of Piromyces communis and
N. frontalis show chemotaxis in vivo towards stomata and
lateral spikes on ingested plant material, with certain sol-
uble sugars leaking from these and other areas of dam-
aged tissues creating a gradient towards which zoospores
are preferentially attracted (Orpin, 1975, 1977b; Orpin &
Bountiff, 1978). The high sensitivity of anaerobic fungal
zoospores to soluble sugars means that freshly ingested
food is quickly colonised, prior to or at the same time as
other rumen microorganisms (Orpin & Bountiff, 1978;
Edwards et al., 2008). As soluble carbohydrates are gener-
ally depleted 2–3 h after feeding in sheep, rapid colonisa-
tion of plant material by anaerobic fungi is crucial in the
competitive environment of the rumen (Edwards et al.,
2008).
Following attachment to plant material, the zoospores
shed their flagella to form a cyst, although amoeboid
movement across the plant surface has occasionally been
FEMS Microbiol Ecol 90 (2014) 1–17
Anaerobic fungi: the who, what, why, where and how useful
Fig. 3. Summary of the anaerobic fungal life
cycle. The stages in the life cycle where
‘resistant’ structures (that have been reported
to date) may be formed are also indicated (*).
observed (Orpin, 1975). Cyst formation and germination
involve the thickening of the cell wall and production of
a germ tube from the polar end opposite from where the
flagella originated (Orpin, 1975, 1994). Cyst development
varies depending on whether the fungus is mono- or
poly-centric (Fig. 3; Table 1). In monocentric taxa, cyst
germination is termed endogenous, as the nucleus
remains within the cyst which enlarges to form a zoospo-
rangium. Thus the rhizoids remain anucleate. In contrast
polycentric taxa exhibit exogenous development, with
migration of nuclei into the more extensive rhizoidal sys-
tem and thereby enabling the formation of multiple spo-
rangia on each thallus (Trinci et al., 1994; Orpin &
Joblin, 1997). The terminology above is less clear when
applied to the two genera which form bulbous holdfasts
(Caecomyces/Cyllamyces), as discussed by Ozkose et al.
(2001). In both cases nuclei are observed in the holdfast,
consistent with exogenous development, and in the case
of Cyllamyces, also in the branched sporangiophores.
FEMS Microbiol Ecol 90 (2014) 1–17
5
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However, the development of these thalli, while not as
strictly determinate as the monocentric/rhizoidal Neocalli-
mastix and Piromyces, is clearly more limited than the
polycentric/rhizoidal Anaeromyces/Orpinomyces.
The anaerobic fungal rhizomycelium physically pene-
trates and enzymatically digests plant material while pro-
viding an anchor for the production of an external
sporangium (monocentric) or multiple sporangia (poly-
centric) (Lowe et al., 1987b; Orpin & Joblin, 1997). The
rhizomycelium is categorised as being either filamentous
or bulbous (Table 1, Fig. 1), the latter possessing spheri-
cal holdfasts (Ozkose et al., 2001; Chen et al., 2007). The
developing rhizoids are capable of physically penetrating
rigid, undamaged plant cell walls using an appressorium-
like structure (Ho et al., 1988a,b). This process opens up
internal plant tissues to enzymatic breakdown, providing
nutrients that enable the development and maturation of
the multinucleate sporangia (Fig. 3). These mature spo-
rangia may produce a few (1 or 2) to many (50–80)
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
6 R.J. Gruninger et al.
zoospores (Heath et al., 1983; Lowe et al., 1987a). In the
presence of suitable inducers, the mature sporangium
then undergoes zoospore differentiation, followed by the
release of its zoospores by the dissolution of the sporan-
gial wall.
The fact that it is very difficult to maintain host ani-
mals free of anaerobic fungi (Becker, 1929) attests to the
efficient dispersal of anaerobic fungi between hosts, pre-
sumably via the formation of aerotolerant survival struc-
tures. Several studies have demonstrated that anaerobic
fungi can be cultured from faecal material following air-
drying, freezing and even from cow dung left for many
months under field conditions (Lowe et al., 1987c; Milne
et al., 1989; Davies et al., 1993a; McGranaghan et al.,
1999; Griffith et al., 2009). Of the resistant structures that
have been observed to date, probably the most convincing
are the 2–4 chambered spores formed by some Anaeromy-
ces cultures (Brookman et al., 2000b; Ozkose, 2001)
although the processes whereby these form and later ger-
minate are currently unknown.
Ecology
Multiple reports on the isolation of anaerobic fungi from
various herbivores have been published, collectively con-
firming their detection in 24 different host genera repre-
senting eight different animal families (Supporting
Information, Table S1). On the basis of these reports, it
appears that the establishment of anaerobic fungi in the
gut of herbivores has two main prerequisites: a digestive
process involving long resident times for ingested plant
material and a dedicated digestive chamber (e.g. rumen,
forestomach or caecum) with a relatively neutral pH.
Anaerobic fungi appear to be present in all foregut
fermenters (i.e. those where the majority of plant fermen-
tation occurs prior to gastric digestion) including:
ruminants (families Bovidae and Cervidae), pseudorumi-
nants (i.e. those with a three-chambered stomach, e.g.
hippopotamus, camels, llamas, alpaca and vicuna) and
foregut nonruminants (animals possessing an enlarged
forestomach, e.g. marsupials) (Table S1). Anaerobic fungi
have also been detected in multiple hindgut fermenters
(i.e. those where the majority of plant fermentation
occurs postgastric digestion in the caecum and large
intestine, e.g. elephants, horses and rhinoceros) where
they appear to be a normal part of the gut microbial
community (Table S1). They have also been identified in
some larger herbivorous rodents such as the Mara
(Dolichotis patagonum); Teunissen et al., 1991), but not
in other small animals with a hindgut fermentation (pre-
sumably due to the shorter duration time of ingested
plant material in their smaller caecum). Interestingly,
anaerobic fungi appear to be absent in strict herbivorous
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mammals lacking foregut and hindgut fermentation
chambers such as the Panda (Milne et al., 1989), presum-
ably due to the simplicity of their alimentary tract.
Within the reptiles (Class Reptilia), microscopic (Mac-
kie et al., 2004) and molecular identification of anaerobic
fungi (Liggenstoffer et al., 2010) (Table S2) have been
reported in the family Iguanidae; however, they have not
been identified in other herbivorous reptiles (i.e. Tor-
toises) (Liggenstoffer et al., 2010). The microscopic evi-
dence of structures resembling chytrid thalli (similar to
Caecomyces spp.) and zoospores in the gut of a burrowing
irregular sea urchin (Echinocardium cordatum), however,
is truly intriguing; potentially representing the first docu-
mentation of anaerobic fungi in a nonherbivorous (detri-
tivore) marine invertebrate (Thorsen, 1999). The
detection of Neocallimastigomycota DNA in soil (Lockhart
et al., 2006), and estuarine sediments (Devon and Marti-
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ny, 2011), is also consistent with their ability to disperse
efficiently between hosts (Life cycle). However, the failure
of most efforts to isolate anaerobic fungi from nongut
habitats (various personal communications; Wubah &
Kim, 1995) suggests that it is only the aerotolerant propa-
gules of these fungi that remain viable in the wider envi-
ronment.
The geographical distribution of anaerobic fungi is
wide, for example, Cyllamyces (first identified in the UK)
has since been found to be widely distributed in domestic
and wild animals in Africa, Australia, Czech Republic,
India and the USA (Sridhar et al., 2007; Fliegerova et al.,
2010; Liggenstoffer et al., 2010; Nicholson et al., 2010;
Sirohi et al., 2013b). On this basis, it seems more likely
that factors such as host phylogeny, gut type and diet are
more important than geographical location in determin-
ing populations of anaerobic fungi in the herbivore gut.
From the broad range of studies performed with domestic
ruminants, it is apparent that diet is one of the key fac-
tors affecting this population (Denman et al., 2008; Khej-
ornsart & Wanapat, 2010; Belanche et al., 2012;
Kittelmann et al., 2012; Boots et al., 2013; Sirohi et al.,
2013a), although many of these studies focussed primarily
on quantitation of anaerobic fungi. In contrast to the
effect of diet, systematic studies of the effect of host phy-
logeny and/or gut type on anaerobic fungi have been
more limited (Liggenstoffer et al., 2010).
Association patterns between specific animal hosts and
anaerobic fungi can be elucidated from the large body of
literature describing the isolation of these microorganism.
Culture-dependent isolation surveys collectively suggest a
pattern in which Piromyces and Caecomyces are the most
prominent genera in the alimentary tract of hindgut fer-
menters, although the isolation of Anaeromyces from
mules has also been reported (Table S1 and refer-
ences within). No concrete evidence for the recovery of
FEMS Microbiol Ecol 90 (2014) 1–17
Anaerobic fungi: the who, what, why, where and how useful
Neocallimastix, Orpinomyces or Cyllamyces from hindgut
fermenters is currently available. While the communities
of anaerobic fungi within the alimentary tract of hindgut
fermenters appear to be dominated by a subset of the
currently described genera, this does not appear to be the
case in ruminants. Ruminal anaerobic fungi are more
diverse, with representatives of all six described genera
having been isolated from domesticated and wild rumi-
nants. Isolates belonging to the genus Neocallimastix
appear to be the most commonly recovered, followed by
members of the genus Piromyces. Orpinomyces and Anaer-
omyces have also been isolated from several domesticated
and wild ruminants. In contrast, members of the genus
Cyllamyces appear to have a fairly limited host distribu-
tion, having only been recovered from domesticated cattle
to date. Finally, although more commonly encountered in
hindgut fermenters, Caecomyces have also been isolated
from domesticated cattle (See Table S2 and references
within). Although there appears to be a distinct distribu-
tion of anaerobic fungi within various host animals,
transfer of anaerobic fungi between different animal spe-
cies in nature appears plausible. Indeed, anaerobic fungi
have been successfully transplanted from horse and rein-
deer to sheep (Orpin, 1989).
The increasing use of cultivation-independent
approaches in recent years has given additional insight
into the diversity and community structure of anaerobic
fungi in the herbivorous gut and enabled the identifica-
tion of sixteen different putative novel lineages (Candi-
date genera) within the Neocallimastigomycota (Table S2).
Liggenstoffer et al. (2010) used a pyrosequencing-based
approach to survey populations of anaerobic fungi in a
large number of diverse zoo animals; concluding that ani-
mal host phylogeny appeared to be the most important
factor in determining anaerobic fungal community struc-
ture and diversity. The findings of this study are also in
agreement with the observation that Piromyces and Caec-
omyces are the most prevalent genera cultivated from
hindgut fermenters (Table S1). Interestingly, the majority
of sequences obtained from horses represented novel taxa
(Table S2). Within foregut fermenters, genus-level diver-
sity was generally higher relative to hindgut fermenters,
with Neocallimastix and Piromyces being the most preva-
lent known genera (Liggenstoffer et al., 2010). The domi-
nance of Neocallimastix and Piromyces has also been seen
in subsequent studies that extensively sampled a narrower
range of foregut herbivores (sheep, deer and cows) over
different seasons (Kittelmann et al., 2012, 2013). Addi-
tional studies have found that Orpinomyces can also be
abundant in the foregut (Sirohi et al., 2013b). Similar to
the currently described genera of anaerobic fungi, several
of the novel candidate genera also show evidence of dis-
tinct host distribution patterns (Table S2).
FEMS Microbiol Ecol 90 (2014) 1–17
7
In addition to the ecological insight obtained from
these recent sequence-based surveys, these studies have
made substantial progress in furthering our understand-
ing of the global genus-level diversity that exists within
the Neocallimastigomycota. The existence of new taxa is
perhaps unsurprising (Orpin, 1994; Fliegerova et al.,
2010; Nicholson et al., 2010), although it is unclear
whether these novel candidate genera are uncultivable
using current methods or whether their occurrence has
merely been overlooked due to microscopic resemblance
to known genera. The relative lack of overlap between
novel lineages identified in recent studies is striking
(Fig. 2, Koetschan et al., 2014) and suggests that addi-
tional, yet-unknown novel candidate genera may exist in
nature.
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Significance for other gut
microorganism and the host
In axenic culture, anaerobic fungi produce a variety of
metabolic end products including acetate, formate, lac-
tate, ethanol, hydrogen and carbon dioxide (Bauchop &
Mountfort, 1981; Cheng et al., 2009). In the rumen, how-
ever, this metabolic profile shifts due to interspecies
hydrogen transfer with physically associated methanogens
(Orpin & Joblin, 1997; Voncken et al., 2002), resulting in
the energetically favourable disposal of electrons via
methanogenesis (Cheng et al., 2009). Enhanced anaerobic
fungal enzyme production and fibre dedgradation have
been reported to occur as a consequence of this favour-
able interaction (Bauchop & Mountfort, 1981; Mountfort
et al., 1982; Teunissen et al., 1992; Cheng et al., 2009);
however, interactions with other rumen microorganism
are not always so mutually beneficial (Gordon & Phillips,
1998). Incubation of anaerobic fungi with protozoa inhib-
its fungal-mediated plant cell wall degradation (Lee et al.,
2000a); presumably due to protozoal predation of zoosp-
ores and potential damage caused to fungal cell walls by
protozoal enzymes (Morgavi et al., 1994; Miltko et al.,
2014). The mechanical and enzymatic degradation of
plant material by anaerobic fungi provide an increased
surface area for bacterial colonisation (Ho et al., 1988a,b;
Orpin & Joblin, 1997), resulting in an increase in the deg-
radation of plant cell walls (Lee et al., 2000a). It has been
reported; however, that some rumen bacteria can have a
negative impact on anaerobic fungi (Gordon & Phillips,
1998).
In contrast to the large number of rumen-based stud-
ies, little is known about the role and microbial interac-
tions of anaerobic fungi in the other gut organs of
ruminants. Anaerobic fungi have been found to be pres-
ent throughout the alimentary tract of cattle and sheep
(Davies et al., 1993b; Rezaeian et al., 2004), with the
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
8 R.J. Gruninger et al.
quantity of anaerobic fungi highest in the rumen/omasum
and then decreasing exponentially further down the tract
after the abomasum (Davies et al., 1993b). Anaerobic
fungal communities sampled in the rumen, duodenum
and rectum of domesticated cattle have been shown to be
similar (Jimenez et al., 2007), suggesting that the higher
proportion of anaerobic fungi with a resistance phenotype
in the ruminant hindgut (Davies et al., 1993a) is due to
an altered physiological state. The implications that this
has for the functionality of anaerobic fungi in the rumi-
nant hindgut, as well as for nonruminants, are currently
unclear.
In the rumen, anaerobic fungi are considered to con-
tribute significantly to the overall metabolism of their
host by playing a major role in the degradation of ligni-
fied plant tissues (Akin et al., 1990). Experiments, where
anaerobic fungi were either absent or eliminated from the
rumen, have provided insight into the contribution of
anaerobic fungi to fibre digestion, feed intake, rumen fer-
mentation and overall rumen metabolism. Removal of
anaerobic fungi from the rumen results in a decrease in
voluntary feed intake and dry matter degradation, indi-
cating digestion of feed is impaired in these animals
(Akin et al., 1989; Morrison et al., 1990; Gordon & Phil-
lips, 1998). Furthermore, the elimination of anaerobic
fungi from the rumen of sheep also significantly reduced
the degradation of dry matter, neutral detergent fibre,
acid detergent fibre and the activity of carboxymethylcel-
lulase (Ford et al., 1987; Gordon & Phillips, 1993; Gao
et al., 2013).
Although the importance of anaerobic fungi in carbo-
hydrate digestion is well established, the role they play in
protein metabolism in the rumen is not clear as the pro-
tease activity of anaerobic fungal isolates has been found
to vary significantly (Michel et al., 1993; Yanke et al.,
1993). Anaerobic fungi produce extracellular proteases
that display catalytic activity comparable to the highly
active proteases produced by numerous rumen bacteria,
the majority of which are most active at typical rumen
pH (Wallace & Joblin, 1985; Asao et al., 1993; Bonnemoy
et al., 1993; Michel et al., 1993). Anaerobic fungal prote-
ases provide amino acids for fungal growth, as well as
aiding the penetration of plant material. In addition to
producing enzymes, anaerobic fungi themselves contrib-
ute to the protein supply of the host by serving as a
source of high-quality microbial protein, that is synthes-
ised in the rumen, passing to the abomasum and intes-
tines for subsequent digestion and absorption by the host
(Kemp et al., 1985; Gulati et al., 1989; Atasoglu & Wal-
lace, 2002).
In recent years, there has been great interest in the bio-
hydrogenation of lipids in the rumen, particularly the for-
mation of conjugated linoleic acid. Numerous studies
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
have investigated the biohydrogenation potential of
anaerobic fungi, with seemingly contrasting findings
reported regarding their enzymatic capabilities and activi-
ties (Kemp et al., 1984; Maia et al., 2007; Nam & Garns-
worthy, 2007a,b,c). Like proteolysis, however, the rate
and extent of biohydrogenation activity also appears to
vary greatly among anaerobic fungal isolates (Nam &
Garnsworthy, 2007a). In general, anaerobic fungi appear
to play a less active role in biohydrogenation than rumen
bacteria, suggesting that their contribution to this process
is limited. Anaerobic fungi themselves are also unlikely to
serve as a source of polyunsaturated fatty acids to the
host, as N. frontalis only contains saturated (48%) and
monounsaturated (52%) fatty acids (Body & Bauchop,
1985).
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Biotechnological applications
The numerous benefits demonstrated from the presence
of anaerobic fungi within the rumen have led to an
increasing interest in their use as a probiotic over the last
decade. The application of anaerobic fungi as a direct-fed
microbial supplement has been investigated, in both
ruminant and nonruminant livestock production, as a
means to improve utilisation of low-quality forages.
Inclusion of cultures of anaerobic fungi in the diets of
various ruminants has been investigated and demon-
strated to improve feed intake, animal growth rate, feed
efficiency and milk production (Lee et al., 2000b; Dey
et al., 2004; Paul et al., 2004, 2011; Tripathi et al., 2007;
Samanta et al., 2008; Sehgal et al., 2008; Mamen et al.,
2010; Saxena et al., 2010; Gao et al., 2013). The benefits
observed being even more marked in young ruminants
(Theodorou et al., 1990; Sehgal et al., 2008) and sheep
devoid of anaerobic fungi (Elliott et al., 1987; Gordon &
Phillips, 1993). Collectively these studies illustrate that the
application of anaerobic fungi as a direct-fed microbial
can be used to improve in vivo digestibility by enhancing:
rumen fermentation characteristics (pH, VFA, ammonia-
N) rumen microbial populations and cellulolytic enzyme
activities. In contrast, the inclusion of enzymes secreted
by anaerobic fungi alone does not alter rumen fermenta-
tion, highlighting the importance of using viable cultures
as a ruminant feed additive (Lee et al., 2000b).
In contrast to the findings with ruminants, anaerobic
fungal enzymes alone appear to be more effective than
viable cultures in monogastric animal (swine and poultry)
production (Theodorou et al., 1996). It is speculated that
this is likely to be due to the inability of anaerobic fungi
to establish in the gastrointestinal tract of these animals.
In swine and poultry diets, cereals containing difficult to
digest nonstarch polysaccharides (b-glucan in barley and
wheat, and arabinoxylans in rye and oats), constitute the
FEMS Microbiol Ecol 90 (2014) 1–17
Anaerobic fungi: the who, what, why, where and how useful
main recalcitrant component of these feedstuffs. These
polymers also have well-known antinutritive effects that
are associated with the propensity of these molecules to
form high molecular weight, viscous aggregates that
reduce intestinal passage rate, decrease diffusion of diges-
tive enzymes, promote endogenous losses and stimulate
unwanted bacterial proliferation (Bedford & Schulze,
1998). Dietary inclusion of anaerobic fungal glycoside
hydrolases (GH) has been found to increase the growth
of broiler chickens by 25%, by aiding the breakdown of
these polymers (Azain et al., 2002).
The most significant challenge for the biotechnological
use of anaerobic fungi, however, as a feed additive or
otherwise, is the difficulty in setting up continuous-flow
cultures for the efficient production of anaerobic fungal
biomass and/or enzymes. For monogastric application,
this has led to attempts to genetically engineer the bacte-
rium Lactobacillus reuteri, a natural component of broiler
gut Microbial communities, to express anaerobic fungal
xylanases and cellulases (Liu et al., 2005a,b, 2007, 2008).
This type of approach, however, is not feasible for rumi-
nant production systems and other applications where
viable anaerobic fungi are needed. Currently, cultivation
of anaerobic fungi requires repeated batch cultures with
frequent transfers that are often difficult to maintain, as
well as being time-consuming and expensive. Processes
using immobilised growing cells seem to be more promis-
ing than traditional fermentations with free cells. Studies
examining the immobilisation of monocentric and poly-
centric fungi have been reported (McCabe et al., 2001,
2003; Nagpal et al., 2009; Sridhar & Kumar, 2010), how-
ever, none of these technologies were feasible for com-
mercial use in their current form. The use of anaerobic
fungi as a direct-fed microbial for ruminants will also
require the development of a suitable means of applying
and storing cultures to ensure maintenance of their via-
bility. Progress has already been made identifying a more
suitable strain by isolating a strain of Neocallimastix, that
is more tolerant of oxygen, changes in culture conditions
and requires fewer transfers for maintenance (Leis et al.,
2013). The recent identification of anaerobic fungi in
novel hosts (Ecology) and improved understanding of the
production of resistant states (Life cycle) in the future,
however, may also enable other opportunities to over-
come current challenges. Meanwhile, a large amount of
research effort has instead focused specifically on the
exploitation of anaerobic fungal enzymes in various
industries.
Anaerobic fungi produce a broad range of potent poly-
saccharide-degrading enzymes making them of particular
interest to several industries: brewing, food, textile, paper
and biofuel production. The cellulolytic and hemicellulo-
lytic capacity conferred to the anaerobic fungi by their
FEMS Microbiol Ecol 90 (2014) 1–17
9
plant cell wall-degrading enzymes is greater than some
currently used commercial enzyme mixtures. Extracellular
enzyme preparations from a Piromyces strain and
N. patriciarum have been found to be highly stable and
exhibited a higher capacity to degrade microcrystalline
cellulose than the commercial enzyme products derived
from Trichoderma reesei (Celluclast: cellulase preparation)
and Aspergilus niger (Novozyme: b-glucosidase prepara-
tion, Novo-Nordisk, Denmark) (Dijkerman et al., 1997).
In the paper industry, anaerobic fungal cellulases and xy-
lanases provide environmentally friendly methods to treat
paper pulp. As paper processing and pulp bleaching uti-
lises harsh conditions (high temperatures and pH),
rational enzyme engineering using error-prone PCR has
been used to develop a xylanase, derived from Neocalli-
mastix, which is more stable during these processes (Liu
et al., 1999; Wang et al., 2011). Perhaps one of the most
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exciting research areas for the application of anaerobic
fungi, however, is that of biofuel production.
There has recently been a push towards developing
renewable fuels from the fermentative production of eth-
anol from lignocellulosic agricultural residues. This pro-
cess involves both the breakdown of the lignocellulose
by carbohydrate active enzymes and the conversion of
these hydrolysis products into fermentable sugars. Efforts
to incorporate fibrolytic enzymes from anaerobic fungi
have focused on expressing a range of carbohydrate
active enzymes into a number of aerobic fungal expres-
sion strains (Li et al., 2007; Tsai & Huang, 2008; van
Wyk et al., 2010; O’Malley et al., 2012). Currently, the
dominant microbial strain for industrial ethanol produc-
tion is Saccharomyces cerevisiae, however, the wild-type
strain of this yeast cannot metabolise xylose and arabi-
nose: two important pentoses released during hydrolysis
of hemicellulose. This has focused efforts on genetically
engineering Sacharomyces cerevisiae to facilitate the con-
version of xylose to xylulose by incorporating a xylose
isomerase into this organism. Strains of Sacharomyces
cerevisiae expressing xylose isomerase from Piromyces
and/or Orpinomyces have been developed (Kuyper et al.,
2005; van Maris et al., 2007; Madhavan et al., 2009),
and at this time represents the most promising option
for industrial production of ethanol (Bellissimi et al.,
2009). Another approach that has only recently been
investigated is the use of anaerobic fungi to break down
lignocellulose while simultaneously fermenting the result-
ing sugars to ethanol (Youssef et al., 2013). This
approach was used with the recently sequenced Orpino-
myces sp. strain C1A and was found to break down up
to 62.3% of dry weight of corn stover while yielding
0.045–0.096 mg ethanol per mg biomass. Although this
is a relatively minor amount of ethanol, this work shows
that simultaneous saccharification and fermentation is
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
10
possible, and future efforts can now be directed towards
enhancing ethanol yield.
A promising source of renewable, environmentally
friendly energy is the production of biogas from the
anaerobic digestion of organic waste. Currently used bior-
eactors display somewhat low degradation of organic
material (40–60%) (Proch
azka et al., 2012) thus, technol-
ogies that can improve this efficiency are needed. The
biological pretreatment of crop residues with white and
brown rot fungi has been shown to be effective at
improving biogas production (Ghosh & Bhattacharyya,
1999). However, as biogas production is an anaerobic
process, the inclusion of an aerobic pretreatment step
increases the overall cost of biogas production. In con-
trast, the direct incorporation of anaerobic fungi into
these bioreactors would eliminate the requirement of an
aerobic predigestion. Incorporation of anaerobic fungi in
bioreactors improved biogas yield for up to 10 days post-
inoculation, enhancing yield by 4–22% depending on the
substrate and fungal species used (Fliegerova et al., 2012;
Proch
azka et al., 2012). Unfortunately, the inability of
anaerobic fungi to survive long term in fermenters, how-
ever, makes the application of anaerobic fungi in com-
mercial full-scale biogas production systems unfeasible
using current technologies (Proch
azka et al., 2012).
-omic based studies
Anaerobic fungal genomes have an extremely high AT
content (Brownlee, 1989), particularly in their noncoding
regions (Nicholson et al., 2005; Youssef et al., 2013). The
first study to describe genomic sequences from an anaero-
bic fungus (Orpinomyces sp. OUS1) identified multiple
skeletal genes, secretory pathways, transporters, as well as
numerous genes encoding for central metabolic pathways
(e.g. Pyruvate formate lyase and malate dehydrogenase)
and enzymes for biopolymer degradation (e.g. peptidases
and xylanases) (Nicholson et al., 2005). Since this study,
the development of high throughput sequencing
approaches has provided new tools and opportunities for
sequencing anaerobic fungal genomes, although their
application has been challenging. The use of pyrosequenc-
ing technology alone is unfeasible, due to the high ade-
nine–thymine (AT) content of anaerobic fungal genomes
and their prevalence of homopolymeric A and T repeats.
Furthermore, the proliferation of simple sequence repeats
also complicates the assembly of anaerobic fungal
genomes generated using Illumina sequencing technologies.
Using a combination of Illumina Hi-seq and Sanger
sequencing approaches, however, the Joint Genome Insti-
tute was the first to generate a draft anaerobic fungal gen-
ome. Despite the Piromyces sp. E2 genome being available
online though (http://genome.jgi.doe.gov/PirE2_1/PirE2_
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
R.J. Gruninger et al.
1.home.html), it has not been described in the literature
to date. More recently, Youssef et al. (2013) reported on
the genome sequencing of Orpinomyces sp. strain C1A,
where they used a combined strategy that utilised both
single molecule real time (SMRT) and Illumina sequenc-
ing approaches. This recently conceived approach (Koren
et al., 2012) utilises the short read high-accuracy data
obtained by Illumina sequencing to correct errors
encountered in long reads produced by SMRT sequenc-
ing. A comparison of the sequencing of the two currently
available anaerobic fungal genomes is shown in Table 2,
although it should be noted that neither of these genomes
have been closed. Despite this, however, genome analysis
has still provided valuable insights into anaerobic fungal
genomic features, metabolic capabilities and cellular pro-
cesses. In particular, two key factors appear to have
shaped the genome of Orpinomyces sp. strain C1A: its
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position as a basal fungal lineage and its unique habitat
within the gut of herbivores.
The position of anaerobic fungi as a basal fungal line-
age is reflected in their genome characteristics that are
also present in other early-branching fungal lineages and/
or nonfungal Opisthokonts, but are absent in the Dikarya
(Ascomycetes and Basidiomycetes) genomes. These charac-
teristics include possession of genes indicating the capa-
bility for post-translational fucosylation, production of a
complete axoneme and intraflagellar trafficking machinery
proteins, production of a near-complete focal adhesion
machinery, production of a near-complete c-secretase
complex and the production of extracellular protease
inhibitors that have not been previously encountered in
Dikarya. These features are thought to have evolved prior
to fungal separation from an Opisthokonta ancestor and
appear to have subsequently been lost during the evolu-
tion of Dikarya (Youssef et al., 2013). In contrast, multi-
ple features observed in the Orpinomyces sp. strain C1A
genome appear to be unique to anaerobic fungi.
Table 2. Comparison of the genome sequencing of Orpinomyces sp.
strain C1A and Piromyces sp. strain E2
Piromyces Orpinomyces
sp. E2* sp. C1A†
Sequencing technologies Illumina and Sanger Illumina and PacBio
Contigs 17 217 32 574
Scaffolds 1656 32 574
Genome size (Mbp) 71.02 100.95
AT content (%) 78.1 83
Genes predicted 14 648 16 347
AT content of ORFs (%) 70.7 73.9
Standard Deviation‡ 5.1 5.8
*http://genome.jgi.doe.gov/PirE2_1/PirE2_1.info.html.
†
http://www.ncbi.nlm.nih.gov/pubmed/23709508.
‡
Standard deviation in open reading frame (ORF) AT content.
FEMS Microbiol Ecol 90 (2014) 1–17
Anaerobic fungi: the who, what, why, where and how useful
Many of the unique features of the anaerobic fungal
genome could be considered a consequence of their evo-
lution in the herbivore gut over hundreds of millions of
years. Several genomic features observed in the Orpinomy-
ces sp. strain C1A genome are characteristically associated
with the process of genetic drift; a process that impacts
the genomes of microbial lineages experiencing low-
effective population sizes, bottlenecks in vertical transmis-
sion and an asexual life style. Genetic drift is character-
ised by the expansion of genome size, accumulation of
repeats, and gene duplications (Lynch & Conery, 2003;
Kelkar & Ochman, 2012), and an increase in the rate of
nonlethal mutations, which tends to be biased towards
adenine or thymine mutations such as cytosine deamina-
tion or guanine oxidation (McCutcheon & Moran, 2012).
Orpinomyces sp. strain C1A has a large genome relative to
other fungal genomes sequenced to date, the presence of
large intergenic regions, high (83.0%) AT content, and a
high level of gene duplication and microsatellite repeats
(Youssef et al., 2013).
In addition to genetic drift, the anaerobic fungal gen-
ome shows evidence of multiple adaptations to improve
its fitness in the anaerobic, prokaryote-dominated envi-
ronment of the herbivore gut. These adaptations include
the dependence on a mixed acid fermentation pathway
for pyruvate metabolism and energy production, the sub-
stitution of ergosterol (which requires molecular oxygen
for its biosynthesis) with tetrahymanol and the acquisi-
tion of many genes from bacterial donors (Youssef et al.,
2013). Of these adaptations, the latter appears to have
been perhaps most important in improving the plant bio-
mass degradation capacities of this fungus. A large pro-
portion of the genes encoding carbohydrate active
enzymes (CAZYmes) in the Orpinomyces sp. strain C1A
genome originate from known bacterial inhabitants of the
rumen and hindgut of herbivores (Youssef et al., 2013).
This gene acquisition strategy appears to have evolved
Orpinomyces sp. strain C1A from an ancestor with limited
cellulolytic capability to a robust cellulolytic and hemicel-
lulolytic organism (Youssef et al., 2013).
To circumvent the challenges posed by whole genome
assembly, transcriptome based approaches have also
recently been applied to examine the metabolic and func-
tional capacity of the anaerobic fungal genome. Transcri-
ptomics gives an unbiased perspective of gene
transcription in situ providing a truer reflection of meta-
bolic activities than genome-based analysis (Sorek &
Cossart, 2010). The first paper to use this type of
approach employed a combination of transcriptomics and
proteomics techniques to identify carbohydrate active
enzymes that were expressed and secreted by Neocallimas-
tix patriciarum W5 (Wang et al., 2011). Neocallimastix
patriciarum was grown on a number of recalcitrant
FEMS Microbiol Ecol 90 (2014) 1–17
11
substrates to stimulate expression of cellulases, and the
transcriptome then sequenced using a combination of 454
and Illumina sequencing technologies. A total of 219 gly-
coside hydrolases from 25 different GH families were
identified, with a number of these enzymes displaying
novel cellulase activities (Wang et al., 2011).
A meta-transcriptome approach has since been used to
examine the activity of rumen eukaryotic microorganism
in Musk-oxen (Ovibos moschatus), through targeted
sequencing of the poly-adenylated mRNA extracted from
rumen solids (Qi et al., 2011). This approach detected
significantly more cellulases (28% of contigs) than that
previously found in a bovine rumen metagenome (8.5%
of contigs) (Qi et al., 2011). The lack of detection of
anaerobic fungal genomic sequences in recent rumen
metagenomic studies is likely a contributing factor to this
observation (Brulc et al., 2009; Hess et al., 2011) and
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highlights the need for targeted analysis of anaerobic
fungi when using sequencing based approaches for
‘omics’ analysis.
Conclusions and future directions
The recent application of next-generation sequencing
techniques to identify anaerobic fungi has provided a
great deal of insight into the phylogenetic diversity and
host distribution of these microorganism. The identifica-
tion of a number of putative uncultured genera should
encourage the development of novel culturing techniques,
in order to attempt to isolate anaerobic fungi representa-
tive of these taxa. It is likely that these efforts will further
emphasise the need for an overhaul of the taxonomy of
the Neocallimastigomycota. Increased efforts to understand
the functional diversity of these microorganism are begin-
ning to suggest that our current view of anaerobic fungi
in the rumen is too simplistic. Efforts to study more
anaerobic fungal genera using a combination of ‘-omics’
and traditional techniques will enhance our understand-
ing of their functional diversification which is likely to
have evolved as a means of avoiding niche competition,
as previously proposed (Griffith et al., 2009). Addition-
ally, attempts to genetically engineer and isolate robust
strains of anaerobic fungi will result in wider biotechno-
logical application of anaerobic fungi and/or their
enzymes in the future: making it more feasible to work
with these fungi on a commercial scale and/or in contin-
uous culture.
Acknowledgements
MSE & NY would like to acknowledge financial support
from the US National Science foundation (NSF EPSCoR
award EPS 0814361) and the US Department of
ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
12
Transportation (Sun Grant Initiative award number
DTOS59-07-G-00053). RJG, RF and TAM would like to
acknowledge support from Agriculture and Agri-foods
Canada and Genome Alberta. AT would like to acknowl-
edge support from Genome Canada and Genome Quebec.
TMC would like to gratefully acknowledge funding from
the Aberystwyth Postgraduate Research Studentship. JE
received funding from the Biotechnology and Biological
Sciences Research Council. SSD and AKP would like to
acknowledge funding to visit Aberystwyth University
(UK) from the Stapledon Memorial Trust, UK; DBT-
CREST award and Network Project of VTCC – Rumen
Microbes (ICAR). KF would like to acknowledge funding
form ‘Ruminomics (project no. 289319 of EC’s 7th
Framework Programme: Food, Agriculture, Fisheries and
Biotechnology)’.
Authors’ contribution
R.J.G., A.K.P., T.M.C., J.E.E. and M.S.E. contributed
equally to this work.
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Supporting Information
Additional Supporting Information may be found in the
online version of this article:
Table S1. Anaerobic fungi detected using enrichment and
isolation-based approaches.
Table S2. Culture-independent studies identifying anaero-
bic fungal diversity and community structure in gut eco-
systems.
ª 2014 Federation of European Microbiological Societies.
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