2.1.2. Design of the Inoculation

The choice of the nutrient source was based on existing technologies. The inoculum for Scenario I is produced in a similar way to the inoculum production for tempeh as described in Wiloso et al. [28], but instead of using white rice or other grains, it is based on stale bread. Production of the inoculum for Scenario II, which is based on a similar process to cultivate yeast [29], used a synthetic medium based on molasses and ammonium chloride. Scenario III is based on glucose, and it was adapted from Jungbluth et al. [30], which describes the production of mycoprotein for Quorn™ (Table 1).

**Table 1.** Inventory analysis for the different scenarios for the production of the fermented fungal product per functional unit. The indented substances in italic refer to the inputs related to the production of the fungal biomass (inoculum) used for the inoculation.


\* Calculation based on the mass balance revealed by Wang et al. [31] and the starch, protein and moisture content in the samples.

The three submerged fermentation (SmF) processes (Scenarios I, II and III) were inoculated with 10 mL/L of the spore solution, cultivated aerobically for 48 h in cotton plugged 250 mL Erlenmeyer flasks containing 100 mL of medium autoclaved at 121 ◦C for 20 min. The initial pH of the medium was adjusted to 5.5 with 2 M HCl or NaOH. The different medium compositions are summarized in Table 1. A water bath (Grant OLS-Aqua pro, Cambridge, UK) was used to maintain the temperature with orbital shaking at 125 rpm (radius of 9 mm) for 48 h. At the end of the fermentation, the wet biomass from each shake flask was harvested by pouring the cultivation medium through a 1 mm<sup>2</sup> pore area fine mesh and washed with distilled water. The liquid fraction was collected separately and frozen for further analysis. The moisture content of the fungal biomass was measured gravimetrically and sieved wet biomass was used directly as inoculum for the solid-state fermentation step.

Scenario IV relies on the recirculation (backslopping) of part of the fermented product from a previous batch as the inoculum. The solid-state fermentation (SSF) on stale bread used as inoculum was prepared according to the SSF process below, except that 0.5 mL of spore solution was used as inoculum instead. Based on an assumption that the fungal fermented final product contained 35% fungal biomass, with a moisture content of 60%, in the substrate-fungal matrix after 5 days of solid-state fermentation [25], the amount needed for backslopping was set to 0.01% dry weight of the substrate.
