## 5 Liquid Mycelial Cultivation of Macrofungi: Methods and Strategies
Experimental design approaches are valuable tools for analyzing complex and multivariate processes such as fungal growth in liquid mediums because the data are analyzed in a formulated model that enables confident predictions of how certain variables might affect the experiments while discriminating true effects from random variation. [PERSON] et al. [68] claim that the biochemistry and physiology of the fungus involved should not be neglected, since they play an important role in metabolic mechanisms that might lead to better scientific understanding and faster optimization. [PERSON] et al. [91] and [PERSON] et al. [23]'s studies pinpoint that the production of bioactive compounds by macrofungi exhibits high intraspecific variability in the same cultivation conditions. Liquid cultures are divided into static cultures, agitated cultures, which are the most common, and two-stage cultures. Two-stage cultures involve a classic agitated culture as a first step, which then continues as a static culture. Researchers have reported hyperproduction of carotenoids, cordycepin, and EPS after optimization of this strategy [177; 178; 179; 180; 181; 182; 183]. There are three main strategies for executing submerged cultivation. Batch culture is the simplest and most traditional technique; all the medium components and fungus are added at the start of cultivation. Batch culture is a closed system and is characterized by a long lag phase, low productivity, and a continuous change in nutrient concentrations over time [68]. Fed-batch is a semi-closed system, where nutrients and substrate are added aseptically as the fungus is consuming the nutrients. Most of the large-scale industrial submerged cultures are performed with this method because it promotes and prolongs metabolite production and maximizes substrate utilization. Continuous culture is a constant process that follows a batch culture until the fungus grows sufficiently, after which nutrients are continually added to the bioreactor and material is removed so that the concentration of nutrients remains the same [184]. Basic research on the behavior of macrofungi in submerged cultivation is first performed in flasks in shaker incubators, followed by laboratory-scale bioreactors of various volumes and geometries, equipped with a multitude of sensors, functions, and automation. Small laboratory bioreactors serve as the first step towards the scaling up of a bioprocess that is tested for industrial application. The most common bioreactor designs used for liquid fungal cultures are stirred-tank bioreactors, which incorporate an agitator or impeller for heat and mass transfer, aeration, and mixing and airlift bioreactors which utilize inserted gas from an airlift pump for mixing and aeration [68]. Filamentous fungi are morphologically complex and versatile microorganisms exhibiting different growth morphology throughout their life cycle. Macrofungi in submerged cultures can form filamentous growth, pellet growth, or a mixture of both. The fungal morphology and metabolite production affects the rheological properties and is simultaneously affected by the type and force of mixing applied in the bioreactor, which is one of the main challenges in industrial bioprocesses [185]. A study comparing different mushroom fungi cultivated in static and agitated liquid cultures reports that most fungi form thick mycelial layers, of varying fibrosis, on the surface of the liquid substrate under static conditions. On the other hand, agitated cultures result in the production of spherical mycelial pellets, of differing sizes depending on the fungi species, with the exception of _G. applanatum_, which produced compact and very dense mycelial clumps in static flasks and loose hyphal masses in agitated culture [96]. The principal factors affecting the process and products of submerged cultures are the subject of many studies. In short, physical factors are temperature, aeration, agitation, foaming, and fluid rheology.