**4. Discussion**

The environmental impacts of a product are often determined by the decisions made at the early stages of development. For this reason, the development of the product should integrate environmental considerations to minimize the environmental impacts throughout its life cycle without compromising essential characteristics such as function, cost and quality. The estimation of environmental impact at this early stage is challenging due to the limited amount of information available, for example, the energy consumption in the processes. In contrast, the later stages in the product development provide more quantitative data but fewer opportunities to change the system.

Table 3 summarizes and ranks the main technical and environmental differences between the four scenarios studied here, namely the environmental impacts, protein content, lag phase and morphology. These aspects are critical to support the decision of the more suitable method for fungal biomass production using bread as substrate. The environmental impact ranking in Table 3 is based on the single score from ReCiPe endpoint (H) as available in the SimaPro software version 9.1.

**Table 3.** Summary of technical and environmental indicators for the scenarios assessed. The protein content refers to SSF after 6 days.


Filamentous fungi grow in different morphological forms in submerged cultures, including freely suspended filamentous mycelia and pellets depending on the genotype of the strain and culture conditions [36]. The growth mode affects the rheological properties of the cultivation medium, and consequently, the overall process performance and final product yields. Generally, the excessive growth of free filamentous mycelia is connected to practical and technical difficulties, such as lower oxygen diffusion, laborious harvesting, high medium viscosity and a relative lower yield of products [37]. These problems associated with the filamentous morphology can be overcome when the fungi grow in the form of pellets, which also have a higher potential for cell reuse and higher productivity due to the possibility of using high-density cultivations [38]. However, including in the pellet form, critical characteristics such as size and compactness versus fluffiness can influence oxygen and substrate transfer rates. The trend in research works has been towards the production of small fluffy pellets. Among the scenarios assessed, only Scenario II grew in the pellet morphology. The slightly shorter lag phase in Scenario II compared to Scenario III further promote this inoculation method since it promotes a faster initiation of the subsequent SSF step. Nevertheless, the environmental impact of Sc. II is significantly higher in comparison with Sc. I and IV (Table 3).

Scenario IV had the lowest environmental impact, and the protein content was sufficient for the intended use. However, one potential drawback with this method is the higher risk of contamination since it is more problematic to sustain a sterile condition at a larger scale when backslopping part of the final product as inoculum compared to inoculum using pure fungal biomass from SmF. Thus, microbiology analysis of the final product and industrial trials need to be assessed before this method can be applied.

Considering the weighted results at Table 3, Scenario I had an environmental impact that is 28% higher in comparison with Scenario IV, but the results indicate a lower impact in comparison with Scenarios II and III. Moreover, the main advantage of this

scenario in comparison to Scenario IV is that it is less sensitive to contamination. Scenario I also yields filamentous mycelia morphology. However, further development of this scenario by changes in cultivation conditions such as pH and aeration can potentially alter the morphology.

The fermentation time is another important aspect influencing both the protein content and the environmental impacts of the final product. The results showed that the protein content for all scenarios reached similar values after 8 days of SSF. However, based on previous studies [6] on the visual appearance and smell of the final product, the SSF step should not be longer than a maximum of eight days and preferably six days if the protein content is sufficient for the intended purpose as food. A direct comparison of the environmental performance of this fungal product with established food products based on mycoprotein requires special attention due to scale-up issues and technology uncertainties inherent in the majority LCAs of products in an early stage of development [18]. Moreover, it is expected that the technology under assessment will need further development in relation to technical aspects and consumer's acceptance.

Jungbluth et al. [30] reported an electricity consumption of 1.17 kWh and a heat consumption of 13 MJ for the industrial production of 1 kg of mycoprotein at plant. Smetana et al. [9] reported 6 kWh/kg of mycoprotein ready to eat. The electricity consumption in this study ranged from 2.61 to 2.64 kWh/functional unit for the different scenarios. Nevertheless, it is important to note that a direct comparison is not possible, due to differences in the system boundaries of each study. The system boundaries in this study and Jungbluth et al. [30] end at the plant gate, while Smetana et al. [9] consider the system boundaries until the consumer's plate. There are also discrepancies in the technology readiness level between this study and Jungbluth et al. [30] in which inventory is based on an operational plant.

A huge advantage is that we now have an LCA model that can be used to get updated environmental impacts when the scenarios are adjusted to improve factors such as protein content, lag time and morphology. This allows for environmental impacts to be assessed throughout the development process.
