2
Upstream Processing
2.1
Inoculum Preparation
The inoculum for an SSF process will typically be vegetative cells in processes
involving bacteria or yeasts. For processes involving fungi, either vegetative
mycelium or spores may be used. Spore inocula allow greater flexibility within
the process since their dormancy enables them to be stored if necessary prior
to inoculation, although spores should not be stored for extended periods since
they gradually lose viability over time [70]. Spore inocula have the disadvantage
of an extended lag phase required for germination and induction of appropriate
extracellular enzymes. The spore inoculum may be pregerminated prior to
mixing with the solid substrate if desired.
Spore germination may have different optimal conditions to vegetative
growth or may need to be stimulated by some nutritional or environmental
factor. Inocula can be prepared either as a dry powder by SSF or as a liquid
suspension by SLF. Furthermore, liquid inocula can easily be prepared from a
sporulating solid culture by suspending the spores in a liquid medium. The
choice between inoculum preparation methods will depend partly on how
easily the inoculum can be mixed in homogeneously with the solid substrate,
although this issue has received little attention [71].
The inoculum density is of great importance. Too low a density may give insufficient biomass and permit the growth of undesirable contaminants, while
for some processes high inoculum densities may produce too much biomass
and deplete the substrate of nutrients necessary for product formation.
Germination can also be inhibited at high spore densities. Typical densities
used range from 10
4 to 10
8 spores per gram of substrate [72, 73].
On the laboratory scale, spore inocula are generally prepared by inoculating
agar slants of suitable media, incubating them, and harvesting spores by adding
3–5 ml water or 0.01% sodium lauryl sulfate and aseptically scraping or
shaking the slants. Spore density can be determined by colorimetric measurements at 540 nm, calibrated against hemocytometer counts. Typical methods
for large-scale production of spores involve SSF of grains and drying of the fermented grains for use as inocula [74]. In drying processes the maintenance of
viability of the spores is a crucial issue. The topic of drying of spores is discussed later in relation to the recovery of fungal spores as products.
2.2
Preparation of Substrates
Most substrates used in SSF are of agricultural origin and might be grains such
as rice, tubers such as cassava, processed materials such as meals (e.g., corn
meal) or byproducts such as straw. They are often relatively unprocessed,
sometimes having only been ground or chopped from larger agricultural
residues, which can give rise to particles of varying compositions. Furthermore,
Biochemical Engineering Aspects of Solid State Bioprocessing
75
Upstream Processing
2.1
Inoculum Preparation
The inoculum for an SSF process will typically be vegetative cells in processes
involving bacteria or yeasts. For processes involving fungi, either vegetative
mycelium or spores may be used. Spore inocula allow greater flexibility within
the process since their dormancy enables them to be stored if necessary prior
to inoculation, although spores should not be stored for extended periods since
they gradually lose viability over time [70]. Spore inocula have the disadvantage
of an extended lag phase required for germination and induction of appropriate
extracellular enzymes. The spore inoculum may be pregerminated prior to
mixing with the solid substrate if desired.
Spore germination may have different optimal conditions to vegetative
growth or may need to be stimulated by some nutritional or environmental
factor. Inocula can be prepared either as a dry powder by SSF or as a liquid
suspension by SLF. Furthermore, liquid inocula can easily be prepared from a
sporulating solid culture by suspending the spores in a liquid medium. The
choice between inoculum preparation methods will depend partly on how
easily the inoculum can be mixed in homogeneously with the solid substrate,
although this issue has received little attention [71].
The inoculum density is of great importance. Too low a density may give insufficient biomass and permit the growth of undesirable contaminants, while
for some processes high inoculum densities may produce too much biomass
and deplete the substrate of nutrients necessary for product formation.
Germination can also be inhibited at high spore densities. Typical densities
used range from 10
4 to 10
8 spores per gram of substrate [72, 73].
On the laboratory scale, spore inocula are generally prepared by inoculating
agar slants of suitable media, incubating them, and harvesting spores by adding
3–5 ml water or 0.01% sodium lauryl sulfate and aseptically scraping or
shaking the slants. Spore density can be determined by colorimetric measurements at 540 nm, calibrated against hemocytometer counts. Typical methods
for large-scale production of spores involve SSF of grains and drying of the fermented grains for use as inocula [74]. In drying processes the maintenance of
viability of the spores is a crucial issue. The topic of drying of spores is discussed later in relation to the recovery of fungal spores as products.
2.2
Preparation of Substrates
Most substrates used in SSF are of agricultural origin and might be grains such
as rice, tubers such as cassava, processed materials such as meals (e.g., corn
meal) or byproducts such as straw. They are often relatively unprocessed,
sometimes having only been ground or chopped from larger agricultural
residues, which can give rise to particles of varying compositions. Furthermore,
Biochemical Engineering Aspects of Solid State Bioprocessing
75