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.