## 5 Liquid Mycelial Cultivation of Macrofungi: Methods and Strategies
\begin{table}
\begin{tabular}{l l l l} \hline
**Mushroom Species** & **Bioactive Compound** & **Fruiting Body** & **Mycelium** \\ \hline _Pleurotus ostreatus_ & Crude protein & 36\% & 40.1\% \\  & Total lipids & 2\% & 3\% \\  & Non-polar lipids & 1.50\% & 4\% \\  & Fat & 1.90\% & 2.6\% \\ _Pleurotus ostreatrosseus_ & Free sugars & 26 mg/g & 58 mg/g \\  & Total organic acids & 212 mg/g & 91 mg/g \\ _Pleurotus_ spp. & Proteins & 24.30\% & 23.3\% \\  & Carbohydrates & 27.70\% & 70.4\% \\  & Fatty acids & 1.60\% & 1.5\% \\ _Pleurotus sajor-\(\alpha\)ju_ & Soluble carbohydrates & 19.55\% & 4.1\% \\  & Proteins & 36.36\% & 32.1\% \\  & Total fat & 2.30\% & 10.2\% \\ _Agaricus campestris_ & Proteins & 46.30\% & 44.4\% \\  & Fat & 2.80\% & 2.8\% \\ _Agaricus bisporus_ & Proteins & 45.90\% & 47.1\% \\  & Fat & 2.70\% & 5.8\% \\ _Lentinus eddes_ & Proteins & 23–24\% & 17\% \\ _L. eddes_ & Lipids & 4\% & 20\% \\ _L. eddes_ & Proteins & 26.50\% & 52.8\% \\ _Cordyceps militaris_ & Proteins & 18.47\% & 21.1\% \\  & Total amino acids & 18.57\% & 28.7\% \\ _Tuber sirense_ & Total umami amino acids & 1.5 mg/g & 9.0 mg/g \\  & Sterol content & 1883 \(\mathrm{\SIUnitSymbolMicro g}\)/g & 5740.4 \(\mathrm{\SIUnitSymbolMicro g}\)/g \\ _Tuber indigum_ & Total umami amino acids & 5.3 \(\mathrm{\SIUnitSymbolMicro g}\)/g & 12.6 \(\mathrm{\SIUnitSymbolMicro g}\)/g \\  & Sterol content & 2204.2 \(\mathrm{\SIUnitSymbolMicro g}\)/g & 6638.1 \(\mathrm{\SIUnitSymbolMicro g}\)/g \\ _Tuber aestivum_ & Total umami amino acids & 7.1 mg/g & 11.6 mg/g \\  & Sterol content & 3239.9 \(\mathrm{\SIUnitSymbolMicro g}\)/g & 5792.5 \(\mathrm{\SIUnitSymbolMicro g}\)/g \\ \hline \end{tabular}
\end{table} 
Table 2: Comparison of fruiting body and mycelium biomass content in bioactive compounds (%, \(w\)/\(w\); mg/g; \(\mathrm{\SIUnitSymbolMicro g}\)/g) in different mushroom species (adapted from [PERSON] et al. [118]). * High yield potential;
* Mixing and stirring allow for very good diffusion of nutrients;
* Temperature control is precise and easier because of the high-water content and stirring;
* Existing kinetic and transfer information of bioprocess can guide the design and operation of bioreactors;
* A Variety of online sensors are available, and more are being developed;
* Sensors allow for the automatic addition of the proper reagents to control the ongoing process;
* Easy manipulation of environmental conditions, growth factors, and nutritional requirements;
* Short overall time of cultivation compared to solid state cultivation of fruit bodies that takes several months, a large volume of substrate and space, and the quality is not always reliable. Disadvantages of liquid mycelial cultivation of macrofungi [177; 178; 180]. The raw material must be sterilized because the high-water activity creates the risk of contamination. In addition,
* Crude medium ingredients may need processing and/or solubilization and characterization which increases the cost of the process. * Downstream processing of the products requires removal of large water volumes and can be more expensive;
* Media dilution leads to lower volumetric productivity;
* High substrate concentrations may cause rheological problems;
* Gas transfer from the gas to liquid requires high air pressure and may be slow and limiting;
* Scaling up from flasks to bioreactors can create technical problems that need to be tackled, such as increased broth viscosity and sufficient oxygen supply;
* Submerged cultivation can be more demanding in energy, water, and labor compared to solid-state fermentation;
* Optimization is necessary. Optimization of submerged cultivation to produce specific metabolites can be costly, difficult, and time-consuming, especially if the classical empirical process of \"change one-factor-at-a-time\" is used. It is highly advised to employ experimental design such as principal component analysis to efficiently optimize the process of submerged cultivation to achieve specific goals (increase EPS production) while studying several contributing factors at the same time.