Wild-cultivated medicinal mushroom Ganoderma lucidum was morphologically identified and sequenced using phylogenetic software. In submerged-liquid fermentation (SLF), biomass, exopolysaccharide (EPS) and intracellular polysaccharide (IPS) production of the identified G. lucidum was optimised based on initial pH, starting glucose concentration and agitation rate parameters using response surface methodology (RSM). Molecularly, the G. lucidum strain QRS 5120 generated 637 base pairs, which was commensurate with related Ganoderma species. In RSM, by applying central composite design (CCD), a polynomial model was fitted to the experimental data and was found to be significant in all parameters investigated. The strongest effect ( p < 0.0001) was observed for initial pH for biomass, EPS and IPS production, while agitation showed a significant value ( p < 0.005) for biomass. By applying the optimized conditions, the model was validated and generated 5.12 g/L of biomass (initial pH 4.01, 32.09 g/L of glucose and 102 rpm), 2.49 g/L EPS (initial pH 4, 24.25 g/L of glucose and 110 rpm) and 1.52 g/L of IPS (and initial pH 4, 40.43 g/L of glucose, 103 rpm) in 500 mL shake flask fermentation. The optimized parameters can be upscaled for efficient biomass, EPS and IPS production using G. lucidum .

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The pellet morphology and diameter range (DR) of Ganoderma lucidum were observed in a repeated-batch fermentation (RBF) for the trio total production of biomass, exopolysaccharide (EPS) and endopolysaccharide (ENS). Two factors were involved in RBF; broth replacement ratio (BRR: 60%, 75% and 90%) and broth replacement time point (BRTP: log, transition and stationary phase) in days. In RBF, 34.31 g/L of biomass favoured small-compact pellets with DR of 20.67 µm< d < 24.00 µm (75% BRR, day 11 of BRTP). EPS production of 4.34 g/L was prone to ovoid-starburst pellets with DR of 34.33 µm< d <35.67 µm (75% BRR, day 13 of BRTP). Meanwhile, the highest 2.43 g/L of ENS production favoured large-hollow pellets with DR of 34.00 µm< d < 38.67 µm (90% BRR, day 13 of BRTP). In addition, RBF successfully shortened the biomass-EPS–ENS fermentation period (31, 33 and 35 days) from batch to 5 days, in seven consecutive cycles of RBF. In a FTIR detection, β-glucan (BG) from EPS and ENS extracts were associated with β-glycosidic linkages (2925 cm −1 , 1635 cm −1 , 1077 cm −1 , 920 cm −1 and 800 cm −1 wavelengths) with similar 1 H NMR spectral behaviour (4.58, 3.87 and 3.81 ppm). Meanwhile, 4 mg/L of BG gave negative cytotoxic effects on normal gingival cell line (hGF) but induced antiproliferation (IC 50 = 0.23 mg/mL) against cancerous oral Asian cellosaurus cell line (ORL-48). Together, this study proved that G. lucidum mycelial pellets could withstand seven cycles of long fermentation condition and possessed anti-oral cancer beta-glucan, which suits large-scale natural drug fermentation.

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There is a pressing demand for new sustainable eco-friendly approaches to producing green energy worldwide. This study represents the novel production of biodiesel feedstock from the medicinal mushroom Ganoderma lucidum QRS 5120 using state-of-the-art biotechnology tools. Response surface methodology (RSM) was used to enhance G. lucidum production in a repeated-batch fermentation strategy. By referring to the broth replacement ratio (BRR) and broth replacement time point (BRTP), RSM that was formulated using a central composite design (CCD) resulted in a significant model for all tested variables, which are exopolysaccharide (EPS), endopolysaccharide (ENS) and biomass, with BRR (%) of 60, 75 and 90, and BRTP (days) of 11, 13 and 15. The model was validated using the optimised conditions, and the results showed 4.21 g/L of EPS (BRR 77.46% and BRTP 12 days), 2.44 g/L of ENS (BRR 60% and BRTP 12.85 days), and 34.32 g/L of biomass (BRR 89.52% and BRTP 10.96 days) were produced. Biomass produced from the G. lucidum was subsequently used as feedstock for biodiesel production. Approximately 20.36% of lipid was successfully extracted from the dried G. lucidum biomass via a solvent extraction and subsequently converted to Ganodiesel through a transesterification process. The Ganodiesel produced fulfilled most of the international standards, i.e., US (ASTM D6751-08) and EU (EN 14214). Overall, this study demonstrates the optimised G. lucidum production and its lipid production as a new biodiesel feedstock.