**5. Conclusions**

The current analysis, which included different production pathways for the valorisation of stale bread into fungal biomass according to the environmental impacts, protein content, lag phase and morphology, indicated the potential use of the fungal biomass as a substitute for traditional food products. The technology proposed uses an abundant waste flow and has the potential to be implemented in supermarkets, small and medium-sized bakeries and industrial bakeries on its returned flows.

Technical and environmental analyses were performed to determine potential tradeoffs of four proposed scenarios to valorise stale bread into a fungal based food product rich in protein. The results from the hotspot analysis indicate that the choice of the medium used in the inoculum production has a large influence on the environmental and technical performance. Inoculum production using molasses or glucose as medium, Sc. II and III respectively, are not preferred from an environmental perspective. Sc. I and Sc. IV, which uses surplus bread and backslopping, showed the lowest environmental impacts in all categories studied here, while maintaining important characteristics such as high protein content. Therefore, it is recommended that such alternatives should be further investigated for the technical feasibility of the process. Moreover, the hotspot analysis indicated that for all scenarios assessed, the fermentation step and drying of bread have a significant impact on the environmental burdens. Therefore, improvements aimed at environmental gains in the fungal biomass production should focus on alternatives for decreasing the fermentation time and consequently the electricity consumption, and on the development of alternative processes in which the drying of bread is not required.

None of the scenarios performed best in all of the parameters analysed. Therefore, it is not possible to draw a simple conclusion regarding the preferred scenario. The decision will ultimately be made according to the technical, environmental or economic agenda of the decision-makers. The contribution of this study is to highlight the trade-offs inherently involved in the decision process within the product development and to provide guidance for further development of the process.

Bread waste has the potential to be retained in the food chain by applying a SSF process using the edible fungus *N. intermedia*. The study has integrated environmental considerations into the early stages of the development of a fungal food product, showing which scenario has the best environmental performance and highlighting trade-offs and the parts of the process that are hotspots and should, thereby, be in focus when optimizing the process. This approach is suggested to contribute to a sustainable way to handle otherwise wasted bread, consistent with a circular economy, and it provides a broader base for the developers of the technology to make sustainable decisions during process optimisation.

**Author Contributions:** Conceptualization, P.B. and R.G.; methodology, P.B., R.G., K.B. and M.J.T.; validation, P.B., R.G., K.B. and M.J.T.; formal analysis, P.B., R.G., K.B. and M.J.T.; investigation, P.B. and R.G.; resources, K.B. and M.J.T.; data curation, P.B. and R.G.; writing—original draft preparation, P.B. and R.G.; writing—review and editing, P.B., R.G., K.B. and M.J.T.; visualization, P.B. and R.G.; supervision, K.B. and M.J.T.; project administration, M.J.T.; funding acquisition, M.J.T. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Swedish Agency for Economic and Regional Growth (Tillväxtverket) via the European Union European Regional Development Fund within the project "Ways2Taste".

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Not applicable.

**Conflicts of Interest:** The authors declare no conflict of interest.
