(Fig. 17.4b; such as cellobiose, xylan) as a carbon source. A step-by-step description of the
procedure can be found in Peng et al. (2018).
In brief, the fecal sample is mixed to a slurry (ca. 10%
w/v) in liquid medium that contains antibiotics to prevent growth of prokaryotes. Commonly used antibiotics include a mix of penicillin G (or Kanamycin),
ampicillin,and streptomycin C (effective against bacteria), and/or chloramphenicol for suppressing both bacteria and methanogens. The slurry is serially diluted
(1:10, 1:100, and 1:1000), and 1 ml aliquots are used to
inoculate 9 ml of liquid culture medium containing
plant fiber. Fungal growth is monitored daily and is
identified by the occurrence of floating mats of fiber,
which occur due to gas production (Fig. 17.4a). For
bulbous species that are less effective in producing
floating mats, positive growth is detected using a
microscope. Anaerobic fungal enrichments are further
purified by several passages through agar containing
roll-tubes (Fig. 17.4c) from which single colonies are
picked (Fig. 17.4d) to inoculate fresh liquid culture
medium (Haitjema et al. 2014).
Subsequent routine sub-culturing is performed in
either complex liquid medium containing up to 15% of
rumen fluid or chemically defined medium (Caldwell
and Bryant 1966; Davies et al. 1993; Lowe et al. 1985).
Cultivation is typically done in small (50–100 mL) gastight serum bottles or Hungate tubes (10–20 mL), under
100% CO 2 or a N 2 /CO 2 (70%/30%) atmosphere and in
the dark at 39
C (Haitjema et al. 2014; Lowe et al. 1985).
The most common substrates that are used
to cultivate anaerobic fungi in laboratory conditions vary highly in their lignin, cellulose, and
hemicellulose contents (Table 17.1). NonetheFig. 17.4 Cultivation of anaerobic fungi (a) growing on
wheat straw forming a floating mat; (b) aggregation of
rhizoid in medium without fibers; (c) agar roll-tube
with fungal colonies; (d) picking a single colony from
a roll-tube for purification
Table 17.1 Composition of lignocellulosic substrates
used for anaerobic fungal growth
Substrate
Cellulose
(% DM)
Hemicellulose
(%DM)
Lignin
(%DM)
Corn stover 44
29
22
Corncob
36–45
35–41
14–15
Grass silage 25–40
26–50
9–30
Maize silage 24
19
3
Rice straw
32–47
18–28
5.5–24
Ryegrass
43
28
7
Wheat straw 33–45
20–32
8–22
Filya (2004); Hassan et al. (2018); Jin et al. (2011)
17 The Biotechnological Potential of Anaerobic Gut Fungi
417
procedure can be found in Peng et al. (2018).
In brief, the fecal sample is mixed to a slurry (ca. 10%
w/v) in liquid medium that contains antibiotics to prevent growth of prokaryotes. Commonly used antibiotics include a mix of penicillin G (or Kanamycin),
ampicillin,and streptomycin C (effective against bacteria), and/or chloramphenicol for suppressing both bacteria and methanogens. The slurry is serially diluted
(1:10, 1:100, and 1:1000), and 1 ml aliquots are used to
inoculate 9 ml of liquid culture medium containing
plant fiber. Fungal growth is monitored daily and is
identified by the occurrence of floating mats of fiber,
which occur due to gas production (Fig. 17.4a). For
bulbous species that are less effective in producing
floating mats, positive growth is detected using a
microscope. Anaerobic fungal enrichments are further
purified by several passages through agar containing
roll-tubes (Fig. 17.4c) from which single colonies are
picked (Fig. 17.4d) to inoculate fresh liquid culture
medium (Haitjema et al. 2014).
Subsequent routine sub-culturing is performed in
either complex liquid medium containing up to 15% of
rumen fluid or chemically defined medium (Caldwell
and Bryant 1966; Davies et al. 1993; Lowe et al. 1985).
Cultivation is typically done in small (50–100 mL) gastight serum bottles or Hungate tubes (10–20 mL), under
100% CO 2 or a N 2 /CO 2 (70%/30%) atmosphere and in
the dark at 39
C (Haitjema et al. 2014; Lowe et al. 1985).
The most common substrates that are used
to cultivate anaerobic fungi in laboratory conditions vary highly in their lignin, cellulose, and
hemicellulose contents (Table 17.1). NonetheFig. 17.4 Cultivation of anaerobic fungi (a) growing on
wheat straw forming a floating mat; (b) aggregation of
rhizoid in medium without fibers; (c) agar roll-tube
with fungal colonies; (d) picking a single colony from
a roll-tube for purification
Table 17.1 Composition of lignocellulosic substrates
used for anaerobic fungal growth
Substrate
Cellulose
(% DM)
Hemicellulose
(%DM)
Lignin
(%DM)
Corn stover 44
29
22
Corncob
36–45
35–41
14–15
Grass silage 25–40
26–50
9–30
Maize silage 24
19
3
Rice straw
32–47
18–28
5.5–24
Ryegrass
43
28
7
Wheat straw 33–45
20–32
8–22
Filya (2004); Hassan et al. (2018); Jin et al. (2011)
17 The Biotechnological Potential of Anaerobic Gut Fungi
417