Figure 2. The constituents of the carbohydrate fraction of
woody plant residues and the inherent fermentability of the basic
building blocks.
Materials and Methods
Fungal strain selection
Pleurotus ostreatus (AM-G269) and Ganoderma lucidum(AM-M177) were purchased from Aloha Medicinals (Carson City, Nevada,
USA). These strains were selected due to proven cultivation ability and
for international comparability. A further strain search was launched to
locate and isolate South African strains for assessment. A potential
newly discovered Ganoderma #1 isolate was included in this study
due to its inherent higher optimal growth temperature and only tested at
time 0 and after 18 weeks on the G2 substrate consistency. To avoid
possible spreading of spores into the environment the study was designed
to avoid fruiting cycles of Ganoderma as the mycelial state ofGanoderma does not possess the ability to effectively infect
hosts non-intentionally.
Substrate for SSF
Acacia mellifera was chosen as the preferred substrate due to
excessive volumes available in central South Africa. Substrate was
milled to three different particle sizes to elucidate possible
variations due to the available surface area of the substrate and
distinguished by G3 (rough chips, 2-5 cm), G2 (medium chips, 1-2 cm) and
G1 (fine chips, ˂1 cm). The woody substrate was allowed to ‘rest’ for
two weeks before use to circumvent any passive host-immunity to the
mushroom strains. Ganoderma #1 was included in a replicate
experiment after the completion of the benchmark strains and only G2 was
selected as the variable grind consistency.
Substrate cultivation
bags
Substrate was soaked in demineralised water for 24 hours for hydration.
The hydrated substrate was subsequently drained until no free running
water was observed. Substrate was packed into polypropylene bags with
0.45 µm pore size filter patches to contain a total of 400 g hydratedA. mellifera wood chips in the three different size categories,
i.e.,. All substrate bags were autoclaved for 90 minutes at 121 °C.
Cultivation bags were performed in duplicate for a total of 20 weeks
with sample bags being chosen at random every two weeks for analysis.
Mushroom cultures
The purchased mushroom cultures were received on Difco™ Potato Dextrose
Agar (PDA) slants. Upon reception the cultures were transferred
aseptically to fresh PDA plates and allowed to grow for seven days
before being used as spawn inoculum. All species applied were previously
characterised on genomic level using the Internal Transcribed Spacer
(ITS gene region.
Mushrooms spawn
production
Sorghum grains were soaked for 24 hours for hydration. The grains were
removed from the soaking water and surface-dried by rolling on a towel
until no longer visibly wet on the surface. The grains were then placed
in polypropylene cultivation bags with air with 0.45 µm pore size
filters and sterilised by autoclaving for 90 mins at 121 °C. After being
cooled to room temperature the bags were inoculated by adding a 1 x 1 cm
square of PDA agar covered with mushroom mycelium. Bags were then
incubated at 25 °C until fully colonised.
Substrate inoculation
Each 400 g substrate bag was inoculated with 40 g of sorghum grain
mushroom spawn and shaken to distribute inoculum. The substrate bags
were placed in a growth room at 25 °C and humidity of 85% (Figure 3).
Control samples were inoculated with the same method but immediately
placed in an oven at 80 °C to dry completely and milled to a fine
powder.