*3.1. Experimental Results*

There is a significant difference in protein content of the final fungal product after SSF for the different inoculation methods (*p* value 0.00). The inoculation scenario using molasses (Sc. II) resulted in a higher protein content after SSF compared to glucose (Sc. III), with no significant difference between Scenarios I and IV. The inoculum scenario based on glucose medium (Sc. III) also resulted in a longer lag phase and slower initial exponential growth, possibly due to change in substrate during SSF, while *N. intermedia* grown on bread (Sc. I) and molasses (Sc. III) was in a more active phase, adapted to bread as substrate and hypothetically, already producing enzymes for complex carbohydrate assimilation, when applied to the SSF step. However, considering the natural variation in biological systems, the relatively small differences between the inoculation methods do not contribute with sufficient support for choosing one scenario over the other.

As expected, the protein content increased over time for all four groups, with a significant increase over every second day (*p* value = 0.00) (Figure 2). The highest increase in protein content happened between day four and six for all inoculation methods based on SmF, varying between 38%, 49% and 51%, for Scenarios I, II and III respectively. Inoculum based on backslopping resulted in a slightly higher increase in protein content between day two and four (39% increase) compared to the other days. The linear regression equations of the four inoculation methods revealed that SmF based on molasses gave the highest average protein increase per day of 4.0, compared to SmF based on bread, backslopping and SmF based on glucose of 3.6%, 3.4% and 3.2% increase per day respectively (consider a 6.6% lack of fit of the model). By comparing the linear regression equations, it can be seen that the daily increase based on glucose medium is only slightly offset in x-axis (timescale), whereas the protein value reaches approximately the same amount as the other inoculation methods after 8 days SSF. *Fermentation* **2021**, *7*, x FOR PEER REVIEW 7 of 13

**Figure 2.** Solid-state cultivation on stale sourdough bread using *N. intermedia* after 0–10 days cultivation under light at 35 °C, 90% Rh, and 40% initial moisture content using different inoculation methods: backslopping (black circle, dotted line), submerged fermentation with synthetic medium based on glucose (grey square, solid line), submerged fermentation based on molasses (black diamonds, dotted line) and submerged fermentation based on breadcrumbs (grey triangles, solid line). Results are expressed as the mean value ± 95% confidence level. **Figure 2.** Solid-state cultivation on stale sourdough bread using *N. intermedia* after 0–10 days cultivation under light at 35 ◦C, 90% Rh, and 40% initial moisture content using different inoculation methods: backslopping (black circle, dotted line), submerged fermentation with synthetic medium based on glucose (grey square, solid line), submerged fermentation based on molasses (black diamonds, dotted line) and submerged fermentation based on breadcrumbs (grey triangles, solid line). Results are expressed as the mean value ± 95% confidence level.

The relative results for five selected impact assessment categories for the scenarios and separately for the inoculum production process are presented in Figures 3 and 4, re-

city. The results for the remaining thirteen impact categories available in the ReCiPe char-

**Figure 3.** Selected LCIA results for the scenarios for the fermented fungal product production. Sc. I uses bread as the carbon source. Sc. II uses molasses. Sc. III is based on glucose and Sc. IV relies on

I II III IV I II III IV I II III IV I II III IV I II III IV

Freshwater eutrophication

Drying Grinding Inoculum Fermentation

Marine ecotoxicity Fossil resource scarcity

Figure 3 indicates that the fermentation step and the choice of inoculum production has a large influence on the environmental burdens associated with the fermented fungal

acterization method [32] is available in Table 2.

backslopping, as described in Table 1.

Global warming Terrestrial

acidification

product.

0%

20%

40%

Relative contribution

60%

80%

100%

*3.2. Life Cycle Assessment*

The fungal morphology in the SmF based on molasses medium (Sc. II) resulted in fine, dense pellets, whereas the other SmF cultures resulted in loose filamentous mycelium. SSF based on inoculum from molasses medium gave the highest mean percentage of protein. methods: backslopping (black circle, dotted line), submerged fermentation with synthetic medium based on glucose (grey square, solid line), submerged fermentation based on molasses (black diamonds, dotted line) and submerged fermentation based on breadcrumbs (grey triangles, solid line). Results are expressed as the mean value ± 95% confidence level.

**Figure 2.** Solid-state cultivation on stale sourdough bread using *N. intermedia* after 0–10 days cultivation under light at 35 °C, 90% Rh, and 40% initial moisture content using different inoculation

0 2 4 6 8 10

Days of solid state fermentation

*Fermentation* **2021**, *7*, x FOR PEER REVIEW 7 of 13

Sc. I (SmF, bread) Sc. II (SmF, molasse) Sc. III (SmF, glucose) Sc. IV (SSF, backslopping)

#### *3.2. Life Cycle Assessment 3.2. Life Cycle Assessment*

0

10

20

30

Protein content [%]

40

50

60

The relative results for five selected impact assessment categories for the scenarios and separately for the inoculum production process are presented in Figures 3 and 4, respectively. The impact categories described in Figures 3 and 4 are global warming, terrestrial acidification, freshwater eutrophication, marine ecotoxicity and fossil resource scarcity. The results for the remaining thirteen impact categories available in the ReCiPe characterization method [32] is available in Table 2. The relative results for five selected impact assessment categories for the scenarios and separately for the inoculum production process are presented in Figures 3 and 4, respectively. The impact categories described in Figures 3 and 4 are global warming, terrestrial acidification, freshwater eutrophication, marine ecotoxicity and fossil resource scarcity. The results for the remaining thirteen impact categories available in the ReCiPe characterization method [32] is available in Table 2. *Fermentation* **2021**, *7*, x FOR PEER REVIEW 8 of 13

**Figure 3.** Selected LCIA results for the scenarios for the fermented fungal product production. Sc. I uses bread as the carbon source. Sc. II uses molasses. Sc. III is based on glucose and Sc. IV relies on backslopping, as described in Table 1. **Figure 3.** Selected LCIA results for the scenarios for the fermented fungal product production. Sc. I uses bread as the carbon source. Sc. II uses molasses. Sc. III is based on glucose and Sc. IV relies on backslopping, as described in Table 1. during the process, as shown in Table 1. The impact is the same for all scenarios, but it has a different relative contribution as shown in Figure 3. Overall, the fermentation step, drying of bread and the inoculation were identified as hotspots in the production of the fungal product (Figure 3).

**Figure 4.** Selected LCIA results for the different inoculation production processes. Sc. I uses breadcrumbs as the carbon source. Sc. II uses molasses. Sc. III is based on glucose. **Figure 4.** Selected LCIA results for the different inoculation production processes. Sc. I uses breadcrumbs as the carbon source. Sc. II uses molasses. Sc. III is based on glucose.

Figure 4 shows the detailed impact assessment for the different inoculation methods. Sc. IV has no contribution from the inoculum production since it is obtained from backslopping. This assumes that the contribution of the initial inoculum required to make the

The results indicate that the carbon source used in the inoculum production is a hotspot in the majority of impacts categories in both Scenario II, which uses molasses and Scenario III, based on glucose (Figure 4). The carbon source is responsible for 36% and 51% of the impacts on the global warming impact category for Sc. II and Sc. III, respectively. In terrestrial acidification, the contribution is higher, 74% and 69% for Sc. II and Sc. III, respectively. Scenario I, conversely, uses stale bread as the carbon source, which is

Electricity consumption has a significant contribution for both the inoculum production and the overall process. The geographical scope is Sweden, which has a relatively low impact energy system. Therefore, the results of the study are sensitive to the energy mix

modelled as waste and is, therefore, burden-free.

used.


**Table 2.** Impact assessment results for Scenarios I, II, III and IV based on the ReCiPe midpoint impact categories.

Figure 3 indicates that the fermentation step and the choice of inoculum production has a large influence on the environmental burdens associated with the fermented fungal product.

The production of inoculum had a contribution higher than 40% in 9 out of 18 impact categories assessed in both Sc. II and Sc. III. More specifically, the carbon source used for the inoculum production for these scenarios is a hotspot, as shown in more detail in Figure 4. In Scenario I the inoculation has a relatively lower contribution, ranging from 20% to 29%. The reason is that it uses stale bread as the carbon source, which is modelled as waste and therefore burden free.

The results indicate that Scenario IV, which is based on backslopping, has the lowest environmental impacts in all of the eighteen categories assessed, followed by Scenario I. Both scenarios substituted glucose or molasses for stale bread, significantly reducing the environmental impacts on the inoculum production. Scenarios II and III had the worst performance in the categories assessed. Scenario II had the worst performance in five impact categories, while Scenario III had the highest impact in 12 environmental impact categories (Table 2).

The solid-state fermentation is also a hotspot, and it contributes significantly to the majority of the impact categories. These impacts are related to the electricity consumption during the process, as shown in Table 1. The impact is the same for all scenarios, but it has a different relative contribution as shown in Figure 3. Overall, the fermentation step, drying of bread and the inoculation were identified as hotspots in the production of the fungal product (Figure 3).

Figure 4 shows the detailed impact assessment for the different inoculation methods. Sc. IV has no contribution from the inoculum production since it is obtained from backslopping. This assumes that the contribution of the initial inoculum required to make the first batch of fungi is insignificant.

The results indicate that the carbon source used in the inoculum production is a hotspot in the majority of impacts categories in both Scenario II, which uses molasses and Scenario III, based on glucose (Figure 4). The carbon source is responsible for 36% and 51% of the impacts on the global warming impact category for Sc. II and Sc. III, respectively. In terrestrial acidification, the contribution is higher, 74% and 69% for Sc. II and Sc. III, respectively. Scenario I, conversely, uses stale bread as the carbon source, which is modelled as waste and is, therefore, burden-free.

Electricity consumption has a significant contribution for both the inoculum production and the overall process. The geographical scope is Sweden, which has a relatively low impact energy system. Therefore, the results of the study are sensitive to the energy mix used.
