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, Abstract
Oklahoma State University, Department of
Microbiology and Molecular Genetics, 1110 S Anaerobic fungi (phylum Neocallimastigomycota) inhabit the gastrointestinal
Innovation Way Dr., Stillwater, OK 74074, tract of mammalian herbivores, where they play an important role in the deg-
USA. Tel.: +1 (405) 744 3005; radation of plant material. The Neocallimastigomycota represent the earliest
fax: +1 (405) 744 1112; diverging lineage of the zoosporic fungi; however, understanding of the rela-
e-mail: Mostafa@okstate.edu
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
Received 9 June 2014; revised 3 July 2014;
accepted 7 July 2014. Final version published
identification and classification, and recent evidence suggests the presence of
online 11 August 2014. 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
DOI: 10.1111/1574-6941.12383 rumen, but not elsewhere in the ruminant alimentary tract. Greater under-
MICROBIOLOGY ECOLOGY

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

FEMS Microbiol Ecol 90 (2014) 1–17 ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
2 R.J. Gruninger et al.

features in common with mitochondria (van der Giezen, et al., 2006a; Powell & Letcher, 2012). The phylum Neo-
2002) and are thought to be derived from them (Embley callimastigomycota currently comprises six genera, each
et al., 1997, 2003; Voncken et al., 2002; Muller et al., distinguishable by morphological features: thallus mor-
2012). Hydrogenosomes contain hydrogenase; producing phology (rhizoidal vs. bulbous) and zoospore flagellation
H2, CO2, formate and acetate as metabolic waste products (monoflagellate vs. polyflagellate) (Ho & Barr, 1995; Ozk-
(Brul & Stumm, 1994; Theodorou et al., 1996; Muller ose et al., 2001). These features are summarised in
et al., 2012). These, along with lactate and ethanol, are Table 1, along with some illustrative microscopy images
the main fermentation end products produced from the (Fig. 1). Although conclusive genus identification of
anaerobic fungal degradation and fermentation of a anaerobic fungi using microscopic approaches can be
variety of plant cell wall polysaccharides. challenging, assignment of isolates into bulbous (Caec-
Anaerobic fungi degrade recalcitrant lignocellulosic omyces, Cyllamyces), hyphael monocentric (Neocallimastix
material both by invasive rhizoidal growth and the associ- and Piromyces) and hyphael polycentric (Orpinomyces and
ated production of a range of powerful polysaccharide- Anaeromyces) by direct examination of colonies/cultures
degrading enzymes. The recently published genome is reasonably straightforward (Griffith et al., 2009). How-
sequence of Orpinomyces sp. strain C1A has provided ever, difficulties in observing zoospore release (Ho &
valuable insight into the diversity of these carbohydrate Bauchop, 1991), the pleomorphic growth form and vari-
active enzymes (Youssef et al., 2013), many of which able sporangial morphology of some isolates (Ho & Barr,

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appear to have been acquired by horizontal gene transfer 1995; Brookman et al., 2000a; Leis et al., 2013) can make
from rumen bacteria. The cellulolytic machinery of anaer- further identification challenging. This has led to dis-
obic fungi consists of both free enzymes, as well as high agreement as to the validity and distinctiveness of some
molecular weight extracellular multi-enzyme complexes taxa (Wubah et al., 1991; Ho & Barr, 1995). Prior to the
(Wilson & Wood, 1992) called cellulosomes (Krause advent of DNA barcoding, the difficulty of transporting
et al., 2003; Joblin et al., 2010). These potent enzymes viable cultures and the absence of any established reposi-
have received much attention in recent years, particularly tory for these fungi also made interlab comparisons very
in terms of their biotechnological application. This review difficult.
covers the key advances that have been made in this area Application of DNA bar coding has served to highlight
over the last decade, as well as numerous other areas of these problems; however, sharing of data via the GenBank
anaerobic fungal research, and highlights the challenges repository has also provided a route towards a more
and opportunities that currently exist within this research robust reappraisal of these fungi. Genotypic analysis was
field. 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
Taxonomy
of the anaerobic fungi. However, the 18S region is highly
Anaerobic fungi belong to the phylum Neocallimastigomy- conserved in Neocallimastigomycota making it difficult to
cota, the earliest diverging lineage unequivocally assigned determine relationships between more closely related taxa
to kingdom Fungi and are closely related to the chytrids (Dagar et al., 2011). Subsequent genetic classification has
(phylum Chytridiomycota) (James et al., 2006a,b; Hibbett mostly focused on the more variable internal transcribed
et al., 2007). While they share key morphological features spacer (ITS) region of the rRNA locus, now formally
with their chytrid relatives, they possess a distinctive agreed as the primary DNA barcode region for all fungi
anaerobic physiology and flagellar apparatus. Further- (Schoch et al., 2012).
more, genetic analyses have consistently shown that the PCR amplification of the ITS region relies on primers
Neocallimastigomycota form a distinct, well-supported which bind to the highly conserved flanking 18S and 28S
clade basal to the chytrids (Fliegerova et al., 2004; James (large subunit; LSU) regions, yielding an amplicon of

Table 1. Morphological classification of the currently recognised anaerobic fungal genera

Genus Thallus Rhizoids Flagella per zoospore

Neocallimastix Monocentric Filamentous Polyflagellate (7–30)
Piromyces Monocentric Filamentous Uniflagellate, sometimes bi- or quadriflagellate
Caecomyces Monocentric Bulbous Uniflagellate, sometimes bi- or quadriflagellate
Orpinomyces Polycentric Filamentous Polyflagellate (14–24)
Anaeromyces Polycentric Filamentous Uniflagellate
Cyllamyces Polycentric Bulbous Uniflagellate, sometimes bi- or triflagellate

ª 2014 Federation of European Microbiological Societies. FEMS Microbiol Ecol 90 (2014) 1–17
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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 sequencing (Liggenstoffer et al., 2010; Kittelmann et al.,
(ITS1 and ITS2) split by the 5.8S rRNA subunit (159 bp 2012, 2013). Furthermore, ITS1-based approaches have
long), the latter providing an additional region for design also been used for the quantification of anaerobic fungi
of conserved primers and amplification of ITS1 or ITS2 in environmental samples by qPCR (Denman & McSwee-
separately. To date, the ITS1 region has been the more ney, 2006; Edwards et al., 2008; Sekhavati et al., 2009;
widely used amplicon for comparison of different genera McDonald et al., 2010; Lwin et al., 2011; Kittelmann
and species of anaerobic fungi (Li & Heath, 1992; Brook- et al., 2012; Marano et al., 2012). The utilisation of these
man et al., 2000a; Fliegerova et al., 2004; Tuckwell et al., approaches has clearly demonstrated that the scope of
2005). These analyses consistently support the close rela- diversity of anaerobic fungi is significantly wider than
tionship between the two genera which form polyflagel- previously implied by culture-based studies (see Ecology).
late zoospores (Neocallimastix, Orpinomyces) and also the Despite the widespread use of ITS1 as the formal fungal
two genera which form bulbous holdfasts (Caecomyces barcode (Schoch et al., 2012), it is apparent that its use
and Cyllamyces). However, the phylogenetic relatedness of for anaerobic fungi is problematic. High sequence vari-
the rhizoidal genera with monoflagellate zoospores (Pir- ability can cause difficulties in ITS1 sequence alignment,
omyces and Anaeromyces) is less clear, and it seems likely while intragenomic variation within the ITS region causes
that the genus Piromyces is polyphyletic and in need of problems for direct sequencing of PCR products (Li &
reappraisal (Brookman et al., 2000a; Hausner et al., 2000; Heath, 1992; Brookman et al., 2000a; Hausner et al.,
Fliegerova et al., 2004). 2000; Ozkose et al., 2001; Fliegerova et al., 2004; Nichol-
In addition to its application in taxonomic studies, the son et al., 2005, 2010; Chen et al., 2007; Edwards et al.,
ITS1 locus has been widely used in culture-independent 2008). These problems have also been found for some
studies to assess fungal diversity and community struc- other groups of fungi (O’Donnell & Cigelnik, 1997; Ko &
ture. Various techniques utilising ITS-based PCR ampli- Jung, 2002; Nilsson et al., 2008). Lastly, the misidentifica-
cons have been employed in such studies, including tion of sequences in NCBI database has caused confusion
DGGE (Kittelmann et al., 2012), ARISA (Edwards et al., when attempting to classify novel sequences (Bidartondo,
2008; Cheng et al., 2009; Sundset et al., 2009), clone 2008; Kittelmann et al., 2012). To start addressing this
library sequencing (Fliegerova et al., 2006; Denman et al., issues, a revised taxonomic framework for the anaerobic
2008; Nicholson et al., 2010) and next-generation 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.

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).

permission from Koetschan et al., 2014) in the light of induction of sporangia by haem and other related por-
the large amounts of next-generation sequencing data that phyrins (Fig. 3), which are released from ingested plant
has being generated, and the discovery of many novel material (Orpin & Greenwood, 1986).
candidate genera that remain to be cultivated (Liggenstof- The locomotion of released Neocallimastix frontalis
fer et al., 2010; Kittelmann et al., 2012; Koetschan et al., zoospores is provided by beating of up to 8–17 flagella
2014; see also Ecology). A website has also been created which form a locomotory organelle, the activity of which
to allow for researchers to use the secondary structure has been described previously (Lowe et al., 1987a,b,c).
prediction approach used by Koetschan et al. (2014) to Motile zoospores locate plant material for colonisation
incorporate new ITS1 sequences from anaerobic fungi via chemotactic responses to soluble sugars (Orpin &
into this classification scheme (https://www.anaerobicfun- Bountiff, 1978) and/or phenolic acids (Wubah & Kim,
gi.biocommons.org.nz). 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-
Life cycle
uble sugars leaking from these and other areas of dam-
Anaerobic fungi reproduce through the asexual produc- aged tissues creating a gradient towards which zoospores
tion of flagellated zoospores from sporangia (Heath et al., are preferentially attracted (Orpin, 1975, 1977b; Orpin &
1986), with no sexual reproductive life stage identified to Bountiff, 1978). The high sensitivity of anaerobic fungal
date. Zoospores may be posteriorly monoflagellate or zoospores to soluble sugars means that freshly ingested
polyflagellate, with the subsequently developed thallus food is quickly colonised, prior to or at the same time as
being either monocentric (single reproductive body i.e. other rumen microorganisms (Orpin & Bountiff, 1978;
one sporangium from single zoospore) or polycentric Edwards et al., 2008). As soluble carbohydrates are gener-
(many sporangia from a single zoospore) (Ho & Barr, ally depleted 2–3 h after feeding in sheep, rapid colonisa-
1995). In the rumen, zoospores are released from anaero- tion of plant material by anaerobic fungi is crucial in the
bic fungal sporangia in response to ingestion of food, competitive environment of the rumen (Edwards et al.,
with the timing of peak zoospore density being reached 2008).
within 30–60 min (Orpin, 1975, 1976, 1977b; Orpin & Following attachment to plant material, the zoospores
Joblin, 1997). Ruminal zoospore differentiation and sub- shed their flagella to form a cyst, although amoeboid
sequent maturation are thought to occur through the movement across the plant surface has occasionally been

ª 2014 Federation of European Microbiological Societies. FEMS Microbiol Ecol 90 (2014) 1–17
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Anaerobic fungi: the who, what, why, where and how useful 5

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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 However, the development of these thalli, while not as
involve the thickening of the cell wall and production of strictly determinate as the monocentric/rhizoidal Neocalli-
a germ tube from the polar end opposite from where the mastix and Piromyces, is clearly more limited than the
flagella originated (Orpin, 1975, 1994). Cyst development polycentric/rhizoidal Anaeromyces/Orpinomyces.
varies depending on whether the fungus is mono- or The anaerobic fungal rhizomycelium physically pene-
poly-centric (Fig. 3; Table 1). In monocentric taxa, cyst trates and enzymatically digests plant material while pro-
germination is termed endogenous, as the nucleus viding an anchor for the production of an external
remains within the cyst which enlarges to form a zoospo- sporangium (monocentric) or multiple sporangia (poly-
rangium. Thus the rhizoids remain anucleate. In contrast centric) (Lowe et al., 1987b; Orpin & Joblin, 1997). The
polycentric taxa exhibit exogenous development, with rhizomycelium is categorised as being either filamentous
migration of nuclei into the more extensive rhizoidal sys- or bulbous (Table 1, Fig. 1), the latter possessing spheri-
tem and thereby enabling the formation of multiple spo- cal holdfasts (Ozkose et al., 2001; Chen et al., 2007). The
rangia on each thallus (Trinci et al., 1994; Orpin & developing rhizoids are capable of physically penetrating
Joblin, 1997). The terminology above is less clear when rigid, undamaged plant cell walls using an appressorium-
applied to the two genera which form bulbous holdfasts like structure (Ho et al., 1988a,b). This process opens up
(Caecomyces/Cyllamyces), as discussed by Ozkose et al. internal plant tissues to enzymatic breakdown, providing
(2001). In both cases nuclei are observed in the holdfast, nutrients that enable the development and maturation of
consistent with exogenous development, and in the case the multinucleate sporangia (Fig. 3). These mature spo-
of Cyllamyces, also in the branched sporangiophores. rangia may produce a few (1 or 2) to many (50–80)

FEMS Microbiol Ecol 90 (2014) 1–17 ª 2014 Federation of European Microbiological Societies.
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6 R.J. Gruninger et al.

zoospores (Heath et al., 1983; Lowe et al., 1987a). In the mammals lacking foregut and hindgut fermentation
presence of suitable inducers, the mature sporangium chambers such as the Panda (Milne et al., 1989), presum-
then undergoes zoospore differentiation, followed by the ably due to the simplicity of their alimentary tract.
release of its zoospores by the dissolution of the sporan- Within the reptiles (Class Reptilia), microscopic (Mac-
gial wall. kie et al., 2004) and molecular identification of anaerobic
The fact that it is very difficult to maintain host ani- fungi (Liggenstoffer et al., 2010) (Table S2) have been
mals free of anaerobic fungi (Becker, 1929) attests to the reported in the family Iguanidae; however, they have not
efficient dispersal of anaerobic fungi between hosts, pre- been identified in other herbivorous reptiles (i.e. Tor-
sumably via the formation of aerotolerant survival struc- toises) (Liggenstoffer et al., 2010). The microscopic evi-
tures. Several studies have demonstrated that anaerobic dence of structures resembling chytrid thalli (similar to
fungi can be cultured from faecal material following air- Caecomyces spp.) and zoospores in the gut of a burrowing
drying, freezing and even from cow dung left for many irregular sea urchin (Echinocardium cordatum), however,
months under field conditions (Lowe et al., 1987c; Milne is truly intriguing; potentially representing the first docu-
et al., 1989; Davies et al., 1993a; McGranaghan et al., mentation of anaerobic fungi in a nonherbivorous (detri-
1999; Griffith et al., 2009). Of the resistant structures that tivore) marine invertebrate (Thorsen, 1999). The
have been observed to date, probably the most convincing detection of Neocallimastigomycota DNA in soil (Lockhart
are the 2–4 chambered spores formed by some Anaeromy- et al., 2006), and estuarine sediments (Devon and Marti-

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ces cultures (Brookman et al., 2000b; Ozkose, 2001) ny, 2011), is also consistent with their ability to disperse
although the processes whereby these form and later ger- efficiently between hosts (Life cycle). However, the failure
minate are currently unknown. of most efforts to isolate anaerobic fungi from nongut
habitats (various personal communications; Wubah &
Kim, 1995) suggests that it is only the aerotolerant propa-
Ecology
gules of these fungi that remain viable in the wider envi-
Multiple reports on the isolation of anaerobic fungi from ronment.
various herbivores have been published, collectively con- The geographical distribution of anaerobic fungi is
firming their detection in 24 different host genera repre- wide, for example, Cyllamyces (first identified in the UK)
senting eight different animal families (Supporting has since been found to be widely distributed in domestic
Information, Table S1). On the basis of these reports, it and wild animals in Africa, Australia, Czech Republic,
appears that the establishment of anaerobic fungi in the India and the USA (Sridhar et al., 2007; Fliegerova et al.,
gut of herbivores has two main prerequisites: a digestive 2010; Liggenstoffer et al., 2010; Nicholson et al., 2010;
process involving long resident times for ingested plant Sirohi et al., 2013b). On this basis, it seems more likely
material and a dedicated digestive chamber (e.g. rumen, that factors such as host phylogeny, gut type and diet are
forestomach or caecum) with a relatively neutral pH. more important than geographical location in determin-
Anaerobic fungi appear to be present in all foregut ing populations of anaerobic fungi in the herbivore gut.
fermenters (i.e. those where the majority of plant fermen- From the broad range of studies performed with domestic
tation occurs prior to gastric digestion) including: ruminants, it is apparent that diet is one of the key fac-
ruminants (families Bovidae and Cervidae), pseudorumi- tors affecting this population (Denman et al., 2008; Khej-
nants (i.e. those with a three-chambered stomach, e.g. ornsart & Wanapat, 2010; Belanche et al., 2012;
hippopotamus, camels, llamas, alpaca and vicuna) and Kittelmann et al., 2012; Boots et al., 2013; Sirohi et al.,
foregut nonruminants (animals possessing an enlarged 2013a), although many of these studies focussed primarily
forestomach, e.g. marsupials) (Table S1). Anaerobic fungi on quantitation of anaerobic fungi. In contrast to the
have also been detected in multiple hindgut fermenters effect of diet, systematic studies of the effect of host phy-
(i.e. those where the majority of plant fermentation logeny and/or gut type on anaerobic fungi have been
occurs postgastric digestion in the caecum and large more limited (Liggenstoffer et al., 2010).
intestine, e.g. elephants, horses and rhinoceros) where Association patterns between specific animal hosts and
they appear to be a normal part of the gut microbial anaerobic fungi can be elucidated from the large body of
community (Table S1). They have also been identified in literature describing the isolation of these microorganism.
some larger herbivorous rodents such as the Mara Culture-dependent isolation surveys collectively suggest a
(Dolichotis patagonum); Teunissen et al., 1991), but not pattern in which Piromyces and Caecomyces are the most
in other small animals with a hindgut fermentation (pre- prominent genera in the alimentary tract of hindgut fer-
sumably due to the shorter duration time of ingested menters, although the isolation of Anaeromyces from
plant material in their smaller caecum). Interestingly, mules has also been reported (Table S1 and refer-
anaerobic fungi appear to be absent in strict herbivorous ences within). No concrete evidence for the recovery of

ª 2014 Federation of European Microbiological Societies. FEMS Microbiol Ecol 90 (2014) 1–17
Published by John Wiley & Sons Ltd. All rights reserved
Anaerobic fungi: the who, what, why, where and how useful 7

Neocallimastix, Orpinomyces or Cyllamyces from hindgut In addition to the ecological insight obtained from
fermenters is currently available. While the communities these recent sequence-based surveys, these studies have
of anaerobic fungi within the alimentary tract of hindgut made substantial progress in furthering our understand-
fermenters appear to be dominated by a subset of the ing of the global genus-level diversity that exists within
currently described genera, this does not appear to be the the Neocallimastigomycota. The existence of new taxa is
case in ruminants. Ruminal anaerobic fungi are more perhaps unsurprising (Orpin, 1994; Fliegerova et al.,
diverse, with representatives of all six described genera 2010; Nicholson et al., 2010), although it is unclear
having been isolated from domesticated and wild rumi- whether these novel candidate genera are uncultivable
nants. Isolates belonging to the genus Neocallimastix using current methods or whether their occurrence has
appear to be the most commonly recovered, followed by merely been overlooked due to microscopic resemblance
members of the genus Piromyces. Orpinomyces and Anaer- to known genera. The relative lack of overlap between
omyces have also been isolated from several domesticated novel lineages identified in recent studies is striking
and wild ruminants. In contrast, members of the genus (Fig. 2, Koetschan et al., 2014) and suggests that addi-
Cyllamyces appear to have a fairly limited host distribu- tional, yet-unknown novel candidate genera may exist in
tion, having only been recovered from domesticated cattle nature.
to date. Finally, although more commonly encountered in
hindgut fermenters, Caecomyces have also been isolated

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Significance for other gut
from domesticated cattle (See Table S2 and references
microorganism and the host
within). Although there appears to be a distinct distribu-
tion of anaerobic fungi within various host animals, In axenic culture, anaerobic fungi produce a variety of
transfer of anaerobic fungi between different animal spe- metabolic end products including acetate, formate, lac-
cies in nature appears plausible. Indeed, anaerobic fungi tate, ethanol, hydrogen and carbon dioxide (Bauchop &
have been successfully transplanted from horse and rein- Mountfort, 1981; Cheng et al., 2009). In the rumen, how-
deer to sheep (Orpin, 1989). ever, this metabolic profile shifts due to interspecies
The increasing use of cultivation-independent hydrogen transfer with physically associated methanogens
approaches in recent years has given additional insight (Orpin & Joblin, 1997; Voncken et al., 2002), resulting in
into the diversity and community structure of anaerobic the energetically favourable disposal of electrons via
fungi in the herbivorous gut and enabled the identifica- methanogenesis (Cheng et al., 2009). Enhanced anaerobic
tion of sixteen different putative novel lineages (Candi- fungal enzyme production and fibre dedgradation have
date genera) within the Neocallimastigomycota (Table S2). been reported to occur as a consequence of this favour-
Liggenstoffer et al. (2010) used a pyrosequencing-based able interaction (Bauchop & Mountfort, 1981; Mountfort
approach to survey populations of anaerobic fungi in a et al., 1982; Teunissen et al., 1992; Cheng et al., 2009);
large number of diverse zoo animals; concluding that ani- however, interactions with other rumen microorganism
mal host phylogeny appeared to be the most important are not always so mutually beneficial (Gordon & Phillips,
factor in determining anaerobic fungal community struc- 1998). Incubation of anaerobic fungi with protozoa inhib-
ture and diversity. The findings of this study are also in its fungal-mediated plant cell wall degradation (Lee et al.,
agreement with the observation that Piromyces and Caec- 2000a); presumably due to protozoal predation of zoosp-
omyces are the most prevalent genera cultivated from ores and potential damage caused to fungal cell walls by
hindgut fermenters (Table S1). Interestingly, the majority protozoal enzymes (Morgavi et al., 1994; Miltko et al.,
of sequences obtained from horses represented novel taxa 2014). The mechanical and enzymatic degradation of
(Table S2). Within foregut fermenters, genus-level diver- plant material by anaerobic fungi provide an increased
sity was generally higher relative to hindgut fermenters, surface area for bacterial colonisation (Ho et al., 1988a,b;
with Neocallimastix and Piromyces being the most preva- Orpin & Joblin, 1997), resulting in an increase in the deg-
lent known genera (Liggenstoffer et al., 2010). The domi- radation of plant cell walls (Lee et al., 2000a). It has been
nance of Neocallimastix and Piromyces has also been seen reported; however, that some rumen bacteria can have a
in subsequent studies that extensively sampled a narrower negative impact on anaerobic fungi (Gordon & Phillips,
range of foregut herbivores (sheep, deer and cows) over 1998).
different seasons (Kittelmann et al., 2012, 2013). Addi- In contrast to the large number of rumen-based stud-
tional studies have found that Orpinomyces can also be ies, little is known about the role and microbial interac-
abundant in the foregut (Sirohi et al., 2013b). Similar to tions of anaerobic fungi in the other gut organs of
the currently described genera of anaerobic fungi, several ruminants. Anaerobic fungi have been found to be pres-
of the novel candidate genera also show evidence of dis- ent throughout the alimentary tract of cattle and sheep
tinct host distribution patterns (Table S2). (Davies et al., 1993b; Rezaeian et al., 2004), with the

FEMS Microbiol Ecol 90 (2014) 1–17 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 have investigated the biohydrogenation potential of
and then decreasing exponentially further down the tract anaerobic fungi, with seemingly contrasting findings
after the abomasum (Davies et al., 1993b). Anaerobic reported regarding their enzymatic capabilities and activi-
fungal communities sampled in the rumen, duodenum ties (Kemp et al., 1984; Maia et al., 2007; Nam & Garns-
and rectum of domesticated cattle have been shown to be worthy, 2007a,b,c). Like proteolysis, however, the rate
similar (Jimenez et al., 2007), suggesting that the higher and extent of biohydrogenation activity also appears to
proportion of anaerobic fungi with a resistance phenotype vary greatly among anaerobic fungal isolates (Nam &
in the ruminant hindgut (Davies et al., 1993a) is due to Garnsworthy, 2007a). In general, anaerobic fungi appear
an altered physiological state. The implications that this to play a less active role in biohydrogenation than rumen
has for the functionality of anaerobic fungi in the rumi- bacteria, suggesting that their contribution to this process
nant hindgut, as well as for nonruminants, are currently is limited. Anaerobic fungi themselves are also unlikely to
unclear. serve as a source of polyunsaturated fatty acids to the
In the rumen, anaerobic fungi are considered to con- host, as N. frontalis only contains saturated (48%) and
tribute significantly to the overall metabolism of their monounsaturated (52%) fatty acids (Body & Bauchop,
host by playing a major role in the degradation of ligni- 1985).
fied plant tissues (Akin et al., 1990). Experiments, where
anaerobic fungi were either absent or eliminated from the

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Biotechnological applications
rumen, have provided insight into the contribution of
anaerobic fungi to fibre digestion, feed intake, rumen fer- The numerous benefits demonstrated from the presence
mentation and overall rumen metabolism. Removal of of anaerobic fungi within the rumen have led to an
anaerobic fungi from the rumen results in a decrease in increasing interest in their use as a probiotic over the last
voluntary feed intake and dry matter degradation, indi- decade. The application of anaerobic fungi as a direct-fed
cating digestion of feed is impaired in these animals microbial supplement has been investigated, in both
(Akin et al., 1989; Morrison et al., 1990; Gordon & Phil- ruminant and nonruminant livestock production, as a
lips, 1998). Furthermore, the elimination of anaerobic means to improve utilisation of low-quality forages.
fungi from the rumen of sheep also significantly reduced Inclusion of cultures of anaerobic fungi in the diets of
the degradation of dry matter, neutral detergent fibre, various ruminants has been investigated and demon-
acid detergent fibre and the activity of carboxymethylcel- strated to improve feed intake, animal growth rate, feed
lulase (Ford et al., 1987; Gordon & Phillips, 1993; Gao efficiency and milk production (Lee et al., 2000b; Dey
et al., 2013). et al., 2004; Paul et al., 2004, 2011; Tripathi et al., 2007;
Although the importance of anaerobic fungi in carbo- Samanta et al., 2008; Sehgal et al., 2008; Mamen et al.,
hydrate digestion is well established, the role they play in 2010; Saxena et al., 2010; Gao et al., 2013). The benefits
protein metabolism in the rumen is not clear as the pro- observed being even more marked in young ruminants
tease activity of anaerobic fungal isolates has been found (Theodorou et al., 1990; Sehgal et al., 2008) and sheep
to vary significantly (Michel et al., 1993; Yanke et al., devoid of anaerobic fungi (Elliott et al., 1987; Gordon &
1993). Anaerobic fungi produce extracellular proteases Phillips, 1993). Collectively these studies illustrate that the
that display catalytic activity comparable to the highly application of anaerobic fungi as a direct-fed microbial
active proteases produced by numerous rumen bacteria, can be used to improve in vivo digestibility by enhancing:
the majority of which are most active at typical rumen rumen fermentation characteristics (pH, VFA, ammonia-
pH (Wallace & Joblin, 1985; Asao et al., 1993; Bonnemoy N) rumen microbial populations and cellulolytic enzyme
et al., 1993; Michel et al., 1993). Anaerobic fungal prote- activities. In contrast, the inclusion of enzymes secreted
ases provide amino acids for fungal growth, as well as by anaerobic fungi alone does not alter rumen fermenta-
aiding the penetration of plant material. In addition to tion, highlighting the importance of using viable cultures
producing enzymes, anaerobic fungi themselves contrib- as a ruminant feed additive (Lee et al., 2000b).
ute to the protein supply of the host by serving as a In contrast to the findings with ruminants, anaerobic
source of high-quality microbial protein, that is synthes- fungal enzymes alone appear to be more effective than
ised in the rumen, passing to the abomasum and intes- viable cultures in monogastric animal (swine and poultry)
tines for subsequent digestion and absorption by the host production (Theodorou et al., 1996). It is speculated that
(Kemp et al., 1985; Gulati et al., 1989; Atasoglu & Wal- this is likely to be due to the inability of anaerobic fungi
lace, 2002). to establish in the gastrointestinal tract of these animals.
In recent years, there has been great interest in the bio- In swine and poultry diets, cereals containing difficult to
hydrogenation of lipids in the rumen, particularly the for- digest nonstarch polysaccharides (b-glucan in barley and
mation of conjugated linoleic acid. Numerous studies wheat, and arabinoxylans in rye and oats), constitute the

ª 2014 Federation of European Microbiological Societies. FEMS Microbiol Ecol 90 (2014) 1–17
Published by John Wiley & Sons Ltd. All rights reserved
Anaerobic fungi: the who, what, why, where and how useful 9

main recalcitrant component of these feedstuffs. These plant cell wall-degrading enzymes is greater than some
polymers also have well-known antinutritive effects that currently used commercial enzyme mixtures. Extracellular
are associated with the propensity of these molecules to enzyme preparations from a Piromyces strain and
form high molecular weight, viscous aggregates that N. patriciarum have been found to be highly stable and
reduce intestinal passage rate, decrease diffusion of diges- exhibited a higher capacity to degrade microcrystalline
tive enzymes, promote endogenous losses and stimulate cellulose than the commercial enzyme products derived
unwanted bacterial proliferation (Bedford & Schulze, from Trichoderma reesei (Celluclast: cellulase preparation)
1998). Dietary inclusion of anaerobic fungal glycoside and Aspergilus niger (Novozyme: b-glucosidase prepara-
hydrolases (GH) has been found to increase the growth tion, Novo-Nordisk, Denmark) (Dijkerman et al., 1997).
of broiler chickens by 25%, by aiding the breakdown of In the paper industry, anaerobic fungal cellulases and xy-
these polymers (Azain et al., 2002). lanases provide environmentally friendly methods to treat
The most significant challenge for the biotechnological paper pulp. As paper processing and pulp bleaching uti-
use of anaerobic fungi, however, as a feed additive or lises harsh conditions (high temperatures and pH),
otherwise, is the difficulty in setting up continuous-flow rational enzyme engineering using error-prone PCR has
cultures for the efficient production of anaerobic fungal been used to develop a xylanase, derived from Neocalli-
biomass and/or enzymes. For monogastric application, mastix, which is more stable during these processes (Liu
this has led to attempts to genetically engineer the bacte- et al., 1999; Wang et al., 2011). Perhaps one of the most

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rium Lactobacillus reuteri, a natural component of broiler exciting research areas for the application of anaerobic
gut Microbial communities, to express anaerobic fungal fungi, however, is that of biofuel production.
xylanases and cellulases (Liu et al., 2005a,b, 2007, 2008). There has recently been a push towards developing
This type of approach, however, is not feasible for rumi- renewable fuels from the fermentative production of eth-
nant production systems and other applications where anol from lignocellulosic agricultural residues. This pro-
viable anaerobic fungi are needed. Currently, cultivation cess involves both the breakdown of the lignocellulose
of anaerobic fungi requires repeated batch cultures with by carbohydrate active enzymes and the conversion of
frequent transfers that are often difficult to maintain, as these hydrolysis products into fermentable sugars. Efforts
well as being time-consuming and expensive. Processes to incorporate fibrolytic enzymes from anaerobic fungi
using immobilised growing cells seem to be more promis- have focused on expressing a range of carbohydrate
ing than traditional fermentations with free cells. Studies active enzymes into a number of aerobic fungal expres-
examining the immobilisation of monocentric and poly- sion strains (Li et al., 2007; Tsai & Huang, 2008; van
centric fungi have been reported (McCabe et al., 2001, Wyk et al., 2010; O’Malley et al., 2012). Currently, the
2003; Nagpal et al., 2009; Sridhar & Kumar, 2010), how- dominant microbial strain for industrial ethanol produc-
ever, none of these technologies were feasible for com- tion is Saccharomyces cerevisiae, however, the wild-type
mercial use in their current form. The use of anaerobic strain of this yeast cannot metabolise xylose and arabi-
fungi as a direct-fed microbial for ruminants will also nose: two important pentoses released during hydrolysis
require the development of a suitable means of applying of hemicellulose. This has focused efforts on genetically
and storing cultures to ensure maintenance of their via- engineering Sacharomyces cerevisiae to facilitate the con-
bility. Progress has already been made identifying a more version of xylose to xylulose by incorporating a xylose
suitable strain by isolating a strain of Neocallimastix, that isomerase into this organism. Strains of Sacharomyces
is more tolerant of oxygen, changes in culture conditions cerevisiae expressing xylose isomerase from Piromyces
and requires fewer transfers for maintenance (Leis et al., and/or Orpinomyces have been developed (Kuyper et al.,
2013). The recent identification of anaerobic fungi in 2005; van Maris et al., 2007; Madhavan et al., 2009),
novel hosts (Ecology) and improved understanding of the and at this time represents the most promising option
production of resistant states (Life cycle) in the future, for industrial production of ethanol (Bellissimi et al.,
however, may also enable other opportunities to over- 2009). Another approach that has only recently been
come current challenges. Meanwhile, a large amount of investigated is the use of anaerobic fungi to break down
research effort has instead focused specifically on the lignocellulose while simultaneously fermenting the result-
exploitation of anaerobic fungal enzymes in various ing sugars to ethanol (Youssef et al., 2013). This
industries. approach was used with the recently sequenced Orpino-
Anaerobic fungi produce a broad range of potent poly- myces sp. strain C1A and was found to break down up
saccharide-degrading enzymes making them of particular to 62.3% of dry weight of corn stover while yielding
interest to several industries: brewing, food, textile, paper 0.045–0.096 mg ethanol per mg biomass. Although this
and biofuel production. The cellulolytic and hemicellulo- is a relatively minor amount of ethanol, this work shows
lytic capacity conferred to the anaerobic fungi by their that simultaneous saccharification and fermentation is

FEMS Microbiol Ecol 90 (2014) 1–17 ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
10 R.J. Gruninger et al.

possible, and future efforts can now be directed towards 1.home.html), it has not been described in the literature
enhancing ethanol yield. to date. More recently, Youssef et al. (2013) reported on
A promising source of renewable, environmentally the genome sequencing of Orpinomyces sp. strain C1A,
friendly energy is the production of biogas from the where they used a combined strategy that utilised both
anaerobic digestion of organic waste. Currently used bior- single molecule real time (SMRT) and Illumina sequenc-
eactors display somewhat low degradation of organic ing approaches. This recently conceived approach (Koren
material (40–60%) (Proch
azka et al., 2012) thus, technol- et al., 2012) utilises the short read high-accuracy data
ogies that can improve this efficiency are needed. The obtained by Illumina sequencing to correct errors
biological pretreatment of crop residues with white and encountered in long reads produced by SMRT sequenc-
brown rot fungi has been shown to be effective at ing. A comparison of the sequencing of the two currently
improving biogas production (Ghosh & Bhattacharyya, available anaerobic fungal genomes is shown in Table 2,
1999). However, as biogas production is an anaerobic although it should be noted that neither of these genomes
process, the inclusion of an aerobic pretreatment step have been closed. Despite this, however, genome analysis
increases the overall cost of biogas production. In con- has still provided valuable insights into anaerobic fungal
trast, the direct incorporation of anaerobic fungi into genomic features, metabolic capabilities and cellular pro-
these bioreactors would eliminate the requirement of an cesses. In particular, two key factors appear to have
aerobic predigestion. Incorporation of anaerobic fungi in shaped the genome of Orpinomyces sp. strain C1A: its

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bioreactors improved biogas yield for up to 10 days post- position as a basal fungal lineage and its unique habitat
inoculation, enhancing yield by 4–22% depending on the within the gut of herbivores.
substrate and fungal species used (Fliegerova et al., 2012; The position of anaerobic fungi as a basal fungal line-
Proch
azka et al., 2012). Unfortunately, the inability of age is reflected in their genome characteristics that are
anaerobic fungi to survive long term in fermenters, how- also present in other early-branching fungal lineages and/
ever, makes the application of anaerobic fungi in com- or nonfungal Opisthokonts, but are absent in the Dikarya
mercial full-scale biogas production systems unfeasible (Ascomycetes and Basidiomycetes) genomes. These charac-
using current technologies (Proch
azka et al., 2012). teristics include possession of genes indicating the capa-
bility for post-translational fucosylation, production of a
complete axoneme and intraflagellar trafficking machinery
-omic based studies
proteins, production of a near-complete focal adhesion
Anaerobic fungal genomes have an extremely high AT machinery, production of a near-complete c-secretase
content (Brownlee, 1989), particularly in their noncoding complex and the production of extracellular protease
regions (Nicholson et al., 2005; Youssef et al., 2013). The inhibitors that have not been previously encountered in
first study to describe genomic sequences from an anaero- Dikarya. These features are thought to have evolved prior
bic fungus (Orpinomyces sp. OUS1) identified multiple to fungal separation from an Opisthokonta ancestor and
skeletal genes, secretory pathways, transporters, as well as appear to have subsequently been lost during the evolu-
numerous genes encoding for central metabolic pathways tion of Dikarya (Youssef et al., 2013). In contrast, multi-
(e.g. Pyruvate formate lyase and malate dehydrogenase) ple features observed in the Orpinomyces sp. strain C1A
and enzymes for biopolymer degradation (e.g. peptidases genome appear to be unique to anaerobic fungi.
and xylanases) (Nicholson et al., 2005). Since this study,
the development of high throughput sequencing
Table 2. Comparison of the genome sequencing of Orpinomyces sp.
approaches has provided new tools and opportunities for
strain C1A and Piromyces sp. strain E2
sequencing anaerobic fungal genomes, although their
application has been challenging. The use of pyrosequenc- Piromyces Orpinomyces
ing technology alone is unfeasible, due to the high ade- sp. E2* sp. C1A†
nine–thymine (AT) content of anaerobic fungal genomes Sequencing technologies Illumina and Sanger Illumina and PacBio
and their prevalence of homopolymeric A and T repeats. Contigs 17 217 32 574
Furthermore, the proliferation of simple sequence repeats Scaffolds 1656 32 574
Genome size (Mbp) 71.02 100.95
also complicates the assembly of anaerobic fungal
AT content (%) 78.1 83
genomes generated using Illumina sequencing technologies. Genes predicted 14 648 16 347
Using a combination of Illumina Hi-seq and Sanger AT content of ORFs (%) 70.7 73.9
sequencing approaches, however, the Joint Genome Insti- Standard Deviation‡ 5.1 5.8
tute was the first to generate a draft anaerobic fungal gen- *http://genome.jgi.doe.gov/PirE2_1/PirE2_1.info.html.
ome. Despite the Piromyces sp. E2 genome being available †
http://www.ncbi.nlm.nih.gov/pubmed/23709508.
‡
online though (http://genome.jgi.doe.gov/PirE2_1/PirE2_ Standard deviation in open reading frame (ORF) AT content.

ª 2014 Federation of European Microbiological Societies. FEMS Microbiol Ecol 90 (2014) 1–17
Published by John Wiley & Sons Ltd. All rights reserved
Anaerobic fungi: the who, what, why, where and how useful 11

Many of the unique features of the anaerobic fungal substrates to stimulate expression of cellulases, and the
genome could be considered a consequence of their evo- transcriptome then sequenced using a combination of 454
lution in the herbivore gut over hundreds of millions of and Illumina sequencing technologies. A total of 219 gly-
years. Several genomic features observed in the Orpinomy- coside hydrolases from 25 different GH families were
ces sp. strain C1A genome are characteristically associated identified, with a number of these enzymes displaying
with the process of genetic drift; a process that impacts novel cellulase activities (Wang et al., 2011).
the genomes of microbial lineages experiencing low- A meta-transcriptome approach has since been used to
effective population sizes, bottlenecks in vertical transmis- examine the activity of rumen eukaryotic microorganism
sion and an asexual life style. Genetic drift is character- in Musk-oxen (Ovibos moschatus), through targeted
ised by the expansion of genome size, accumulation of sequencing of the poly-adenylated mRNA extracted from
repeats, and gene duplications (Lynch & Conery, 2003; rumen solids (Qi et al., 2011). This approach detected
Kelkar & Ochman, 2012), and an increase in the rate of significantly more cellulases (28% of contigs) than that
nonlethal mutations, which tends to be biased towards previously found in a bovine rumen metagenome (8.5%
adenine or thymine mutations such as cytosine deamina- of contigs) (Qi et al., 2011). The lack of detection of
tion or guanine oxidation (McCutcheon & Moran, 2012). anaerobic fungal genomic sequences in recent rumen
Orpinomyces sp. strain C1A has a large genome relative to metagenomic studies is likely a contributing factor to this
other fungal genomes sequenced to date, the presence of observation (Brulc et al., 2009; Hess et al., 2011) and

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large intergenic regions, high (83.0%) AT content, and a highlights the need for targeted analysis of anaerobic
high level of gene duplication and microsatellite repeats fungi when using sequencing based approaches for
(Youssef et al., 2013). ‘omics’ analysis.
In addition to genetic drift, the anaerobic fungal gen-
ome shows evidence of multiple adaptations to improve
Conclusions and future directions
its fitness in the anaerobic, prokaryote-dominated envi-
ronment of the herbivore gut. These adaptations include The recent application of next-generation sequencing
the dependence on a mixed acid fermentation pathway techniques to identify anaerobic fungi has provided a
for pyruvate metabolism and energy production, the sub- great deal of insight into the phylogenetic diversity and
stitution of ergosterol (which requires molecular oxygen host distribution of these microorganism. The identifica-
for its biosynthesis) with tetrahymanol and the acquisi- tion of a number of putative uncultured genera should
tion of many genes from bacterial donors (Youssef et al., encourage the development of novel culturing techniques,
2013). Of these adaptations, the latter appears to have in order to attempt to isolate anaerobic fungi representa-
been perhaps most important in improving the plant bio- tive of these taxa. It is likely that these efforts will further
mass degradation capacities of this fungus. A large pro- emphasise the need for an overhaul of the taxonomy of
portion of the genes encoding carbohydrate active the Neocallimastigomycota. Increased efforts to understand
enzymes (CAZYmes) in the Orpinomyces sp. strain C1A the functional diversity of these microorganism are begin-
genome originate from known bacterial inhabitants of the ning to suggest that our current view of anaerobic fungi
rumen and hindgut of herbivores (Youssef et al., 2013). in the rumen is too simplistic. Efforts to study more
This gene acquisition strategy appears to have evolved anaerobic fungal genera using a combination of ‘-omics’
Orpinomyces sp. strain C1A from an ancestor with limited and traditional techniques will enhance our understand-
cellulolytic capability to a robust cellulolytic and hemicel- ing of their functional diversification which is likely to
lulolytic organism (Youssef et al., 2013). have evolved as a means of avoiding niche competition,
To circumvent the challenges posed by whole genome as previously proposed (Griffith et al., 2009). Addition-
assembly, transcriptome based approaches have also ally, attempts to genetically engineer and isolate robust
recently been applied to examine the metabolic and func- strains of anaerobic fungi will result in wider biotechno-
tional capacity of the anaerobic fungal genome. Transcri- logical application of anaerobic fungi and/or their
ptomics gives an unbiased perspective of gene enzymes in the future: making it more feasible to work
transcription in situ providing a truer reflection of meta- with these fungi on a commercial scale and/or in contin-
bolic activities than genome-based analysis (Sorek & uous culture.
Cossart, 2010). The first paper to use this type of
approach employed a combination of transcriptomics and
Acknowledgements
proteomics techniques to identify carbohydrate active
enzymes that were expressed and secreted by Neocallimas- MSE & NY would like to acknowledge financial support
tix patriciarum W5 (Wang et al., 2011). Neocallimastix from the US National Science foundation (NSF EPSCoR
patriciarum was grown on a number of recalcitrant award EPS 0814361) and the US Department of

FEMS Microbiol Ecol 90 (2014) 1–17 ª 2014 Federation of European Microbiological Societies.
Published by John Wiley & Sons Ltd. All rights reserved
12 R.J. Gruninger et al.

Transportation (Sun Grant Initiative award number rumen microbial community and on the representativeness
DTOS59-07-G-00053). RJG, RF and TAM would like to of bacterial fractions used in the determination of microbial
acknowledge support from Agriculture and Agri-foods protein synthesis. J Anim Sci 90: 3924–3936.
Canada and Genome Alberta. AT would like to acknowl- Bellissimi E, Van Dijken JP, Pronk JT & Van Maris AJ (2009)
edge support from Genome Canada and Genome Quebec. Effects of acetic acid on the kinetics of xylose fermentation
TMC would like to gratefully acknowledge funding from by an engineered, xylose-isomerase-based Saccharomyces
cerevisiae strain. FEMS Yeast Res 9: 358–364.
the Aberystwyth Postgraduate Research Studentship. JE
Bidartondo MI (2008) Preserving accuracy in GenBank. Science
received funding from the Biotechnology and Biological
319: 1616.
Sciences Research Council. SSD and AKP would like to Body DR & Bauchop T (1985) Lipid composition of an
acknowledge funding to visit Aberystwyth University obligately anaerobic fungus Neocallimastix frontalis isolated
(UK) from the Stapledon Memorial Trust, UK; DBT- from a bovine rumen. Can J Microbiol 31: 463–466.
CREST award and Network Project of VTCC – Rumen Bonnemoy F, Fonty G, Michel V & Gouet P (1993) Effect of
Microbes (ICAR). KF would like to acknowledge funding anaerobic fungi on the ruminal proteolysis in gnotobiotic
form ‘Ruminomics (project no. 289319 of EC’s 7th lambs. Reprod Nutr Dev 33: 551–555.
Framework Programme: Food, Agriculture, Fisheries and Boots B, Lillis L, Clipson N, Petrie K, Kenny DA, Boland TM
Biotechnology)’. & Doyle E (2013) Responses of anaerobic rumen fungal
diversity (phylum Neocallimastigomycota) to changes in

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bovine diet. J Appl Microbiol 114: 626–635.
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Brookman JL, Ozkose E, Rogers S, Trinci APJ & Theodorou
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