## 4 Discussion  
In this paper, bread residuals were converted into ethanol and valuable high-protein biomass for food or feed uses. According to the mass balance, the final ethanol yield ranged approx. 29.2-31.6% by weight. In previously published papers dealing with conversion of bread waste to ethanol, the reported product efficiencies were higher depending on the raw material pretreatment applied. [PERSON] and [PERSON] [21] studied conversion of bread to ethanol by _S. cerevisiae_ with the application of granular starch hydrolyzing enzyme at the same substrate concentration in the medium as applied here (150 g/L) and reported ethanol yields between 35.4-38.6% \(w/w\) with an average ethanol titer of 55 g/L. Studies on the simultaneous saccharification and fermentation of bread hydrolyses at higher solids (>30% \(w/v\)) loading by the yeast proved to be even more effective, yielding nearly 40% \(w/w\) bread to ethanol conversion efficiencies [13]. Recently, [PERSON] et al. [22] studied the comparison of acid and enzymatic hydrolysis of bread residuals on ethanol production by _S. cerevisiae_ via separate hydrolysis and fermentation. They showed that enzymatic hydrolysis results in higher ethanol yield than acid hydrolysis (176 g/Kg vs. 240 g/Kg). The use of acid as hydrolysis catalyst seemingly might be a good option for releasing glucose from starchy residuals due to short processing times needed. However, as shown by [PERSON], high processing temperature (121 \({}^{\circ}\)C) and presence of acid catalysts results in the production of high levels of toxic hydroxymethylfurral (up to 2.5 g/L) and furfural (up to 0.7 g/L), which could inhibit the fermenting microorganism as well as  
Figure 3: Mass balance for the process of bread waste valorization using edible filamentous fungi (**a**). The streams of the products obtained are contained in the dashed green line blocks. The bar graph (**b**) shows the comparison of fungal biomass and ethanol production in both fermentation stages depending on the fungal strain used. diminish the use of the fermentation by-products for food or feed use. Acid hydrolysis might, however, be an effective method for mycotoxin (namely aflat) removal from contaminated bread residuals, which further improves ethanol production in comparison to enzymatic hydrolysis [23]. Nevertheless, it is well-known that the application of _S. cerevisiae_ in the production of ethanol from starchy materials requires the use of saccharification enzymes for the hydrolysis of dextrins formed during the liquefaction stage to glucose. As analyzed by [PERSON] et al. [24], the cost of enzymes used in the production of bioethanol from corn represent up to 4.9% of the total operation costs of the ethanol plant. In this paper, it was shown that the use of _N. intermedia_ as the sole microorganism in the production of ethanol from bread residuals may be efficiently carried out without the use of any saccharification enzyme and only one-half of the recommended liquefaction enzyme dose. This would positively impact the economic indicators of the process described in the present study. It could also be speculated that the application of \(\alpha\)-amylase might be omitted, and the unpretreated bread slurry would be fermented by the fungi, which are capable of degrading starch, as shown by the carbohydrate utilization profiles in this study. This, however, might result in the generation of medium with exceptionally high viscosity and undissolved particles, the pumping and handling of which would be difficult. The use of bacterial \(\alpha\)-amylase, besides effective viscosity reduction in starchy materials, also provides the advantage of very high optimal temperature of the activity of this enzyme, which often exceeds 80 \({}^{\circ}\)C [25]. This means that the enzymatic liquefaction would serve as a process where viscosity reduction, provision of small amounts of simple sugars to initiate fungal growth and microbial stabilization would take place, thus reducing the amount of process operations and equipment needed.