less, anaerobic fungi are able to degrade many
of these materials with comparable efficiency,
even when composed of high levels of phenolic
compounds inhibitory to other fungi (Hooker
et al. 2018; Skyba et al. 2013). To date, milled
wheat straw has been the preferred substrate to
grow anaerobic fungi in laboratory conditions,
but other types of agricultural waste (such as,
maize silage, grass silage, grasses, rice straw,
hay, reed canary grass, ryegrass at earing
stage, maize stems, soybean hulls, and palm
press fiber) also promote anaerobic fungal
growth (Griffith et al. 2009; Mountfort and
Orpin 1994; Trinci et al. 1994; Zhu et al. 1996).
Young forages, rich in leaf and poor in stems
(such as, ryegrass at the leafy stage) that contain low amounts of crude fibers and high
amounts of pectin and starch, were found to
be unfavorable for anaerobic fungal growth
(Mountfort and Orpin 1994).
Long-term viability of gut fungal cultures
requires routine sub-culturing every 3–14 days
(dependent on the species) (Haitjema et al.
2014; Peng et al. 2018). For long-term storage
and to avoid genetic drift by repeated subculturing, anaerobic fungi can be cryopreserved (Solomon et al. 2016b). However,
the cryo-preservation methodology has not
been standardized and is not always successful
(Callaghan et al. 2015; Haitjema et al. 2014;
Peng et al. 2018; Solomon et al. 2016b). The
culture methodology may be improved by
establishing techniques that allow continuous
cultivation that better resembles the conditions
in the rumen ecosystem. Semi-continuous cultivation of anaerobic fungi has been performed
in continuous-flow cultures containing up to
8% (w/v) fibrous substrate (Zhu et al. 1996;
Zhu et al. 1997). Teunissen et al. (1992) also
used a semi-continuous culture for harvesting
anaerobic fungal enzymes. In these systems,
effluent is usually removed discretely or continuously from the culture vessel and replaced by
an equivalent amount of fresh culture medium.
Anaerobic fungi showed higher enzyme production and increased degradation of plantfibers in continuous cultivation systems, possibly due to the removal of inhibitory fermentation end products (see Sect. II.A) in culture
effluents (Zhu et al. 1996).
C. Physiology and Metabolism
Anaerobic fungi have evolved strategies to
thrive in oxygen-free habitats where they
degrade a wide range of substrates: (1) They
possess a large arsenal of lignocellulolytic
enzymes that are secreted either freely or
organized in multi-protein complexes called
fungal cellulosomes. (2) Energy production
occurs in hydrogenosomes instead of mitochondria. Anaerobic fungi rely on substratelevel phosphorylation (mixed-acid fermentation), rather than oxidative phosphorylation
(respiration) (Mountfort and Orpin 1994). (3)
Their cell membranes contain tetrahymanol
rather than sterols (ergosterol in most fungi),
since synthesis of these requires oxygen in the
oxidation of the squalene precursor.
Tetrahymanol, a pentacyclic sterol-like compound, is
formed directly by the cyclisation of squalene via squalene hydro-lyase, a process similar to the synthesis of
the pentacyclic hopanoids in bacteria (Youssef et al.
2013).
1. Some Biomass-Degrading Enzymes Are
Organized in Cellulosomes
The hydrolytic strategy of anaerobic fungi is
based on the extensive production of carbohydrate-active enzymes (CAZymes) that they
secrete to the surrounding environment.
CAZymes include all enzymes involved in carbohydrate assembly (glycosyltransferases) and
breakdown (glycoside hydrolases, polysaccharide lyases, carbohydrate esterases) (Lombard
et al. 2014). Each carbohydrate polymer
requires a certain set of CAZymes (Fig. 17.5).
Whether anaerobic fungi have evolved strategies to enzymatically depolymerize and/or
catabolize lignin remains unclear but their
ability to solubilize linkages in the ligninpolysaccharide complex has been reported
(Joblin and Naylor 1989), rendering cellulose
and hemicellulose accessible for degradation.
For information on the individual enzymatic
reactions, see Theodorou et al. (1996b).
Notably, whereas aerobic fungi typically
secrete enzymes into the environment, anaerobic fungi are unique among the fungal kingdom
418
V. Flad et al.
of these materials with comparable efficiency,
even when composed of high levels of phenolic
compounds inhibitory to other fungi (Hooker
et al. 2018; Skyba et al. 2013). To date, milled
wheat straw has been the preferred substrate to
grow anaerobic fungi in laboratory conditions,
but other types of agricultural waste (such as,
maize silage, grass silage, grasses, rice straw,
hay, reed canary grass, ryegrass at earing
stage, maize stems, soybean hulls, and palm
press fiber) also promote anaerobic fungal
growth (Griffith et al. 2009; Mountfort and
Orpin 1994; Trinci et al. 1994; Zhu et al. 1996).
Young forages, rich in leaf and poor in stems
(such as, ryegrass at the leafy stage) that contain low amounts of crude fibers and high
amounts of pectin and starch, were found to
be unfavorable for anaerobic fungal growth
(Mountfort and Orpin 1994).
Long-term viability of gut fungal cultures
requires routine sub-culturing every 3–14 days
(dependent on the species) (Haitjema et al.
2014; Peng et al. 2018). For long-term storage
and to avoid genetic drift by repeated subculturing, anaerobic fungi can be cryopreserved (Solomon et al. 2016b). However,
the cryo-preservation methodology has not
been standardized and is not always successful
(Callaghan et al. 2015; Haitjema et al. 2014;
Peng et al. 2018; Solomon et al. 2016b). The
culture methodology may be improved by
establishing techniques that allow continuous
cultivation that better resembles the conditions
in the rumen ecosystem. Semi-continuous cultivation of anaerobic fungi has been performed
in continuous-flow cultures containing up to
8% (w/v) fibrous substrate (Zhu et al. 1996;
Zhu et al. 1997). Teunissen et al. (1992) also
used a semi-continuous culture for harvesting
anaerobic fungal enzymes. In these systems,
effluent is usually removed discretely or continuously from the culture vessel and replaced by
an equivalent amount of fresh culture medium.
Anaerobic fungi showed higher enzyme production and increased degradation of plantfibers in continuous cultivation systems, possibly due to the removal of inhibitory fermentation end products (see Sect. II.A) in culture
effluents (Zhu et al. 1996).
C. Physiology and Metabolism
Anaerobic fungi have evolved strategies to
thrive in oxygen-free habitats where they
degrade a wide range of substrates: (1) They
possess a large arsenal of lignocellulolytic
enzymes that are secreted either freely or
organized in multi-protein complexes called
fungal cellulosomes. (2) Energy production
occurs in hydrogenosomes instead of mitochondria. Anaerobic fungi rely on substratelevel phosphorylation (mixed-acid fermentation), rather than oxidative phosphorylation
(respiration) (Mountfort and Orpin 1994). (3)
Their cell membranes contain tetrahymanol
rather than sterols (ergosterol in most fungi),
since synthesis of these requires oxygen in the
oxidation of the squalene precursor.
Tetrahymanol, a pentacyclic sterol-like compound, is
formed directly by the cyclisation of squalene via squalene hydro-lyase, a process similar to the synthesis of
the pentacyclic hopanoids in bacteria (Youssef et al.
2013).
1. Some Biomass-Degrading Enzymes Are
Organized in Cellulosomes
The hydrolytic strategy of anaerobic fungi is
based on the extensive production of carbohydrate-active enzymes (CAZymes) that they
secrete to the surrounding environment.
CAZymes include all enzymes involved in carbohydrate assembly (glycosyltransferases) and
breakdown (glycoside hydrolases, polysaccharide lyases, carbohydrate esterases) (Lombard
et al. 2014). Each carbohydrate polymer
requires a certain set of CAZymes (Fig. 17.5).
Whether anaerobic fungi have evolved strategies to enzymatically depolymerize and/or
catabolize lignin remains unclear but their
ability to solubilize linkages in the ligninpolysaccharide complex has been reported
(Joblin and Naylor 1989), rendering cellulose
and hemicellulose accessible for degradation.
For information on the individual enzymatic
reactions, see Theodorou et al. (1996b).
Notably, whereas aerobic fungi typically
secrete enzymes into the environment, anaerobic fungi are unique among the fungal kingdom
418
V. Flad et al.