Solid-state fermentation of each fungal strain was carried out on UV-sterilized and non-sterilized cheese whey powder, with three different substrate moisture contents (40, 50 and 60%). UV sterilization was achieved by spreading 10 grams of cheese whey powder into a petri dish as a thin film and then exposing it to UV for 20 min. The substrate in each plate was then aseptically inoculated with 2 mL of spore suspension and then incubated in a climate cabinet (Memmert, Germany) at 35°C for 10 days (90% RH, 35% Light). All plates were photographed on days 4, 7 and 10 of the fermentation. The biomass on plates was dried using an oven at 50°C for 24 h to obtain dry biomass. The dried biomass samples were powdered with a grinder (Bosch Coffee Grinder, Germany) and the powdered samples were stored for use in protein analysis.

Fungal cultivation under SmF was conducted in 250 mL cotton plugged shake flasks containing 100 mL of 3% (w/v) cheese whey powder prepared with distilled water. The prepared substrates were sterilized before fungal inoculation in an autoclave at 121°C for 20 min (VX-95, Systec, Linden, Germany). In submerged fermentation, fungal growth was assessed at two distinct pH levels, namely initial pH (5.70, non-controlled pH) and controlled pH (5.0), for each microorganism. Each flask was then inoculated with a 2 mL fungal spore suspension, and cultivation was performed at 35°C in a water bath shaker (Grant Instruments Ltd., Cambridge, UK) at 125 rpm. After the 6, 24, 30, and 48 h of incubation, the substrate pH was adjusted to 5.0 with 2 M H₂SO₄. The wet fungal biomass was harvested by sieving through a screen after 48 h of cultivation. The harvested biomass was washed with water, and dried in a freeze dryer (Labconco, United States).

Cultivation of two fungal strains (A. oryzae and N. intermedia) was carried out in 4.5 L bench-scale bubble column reactors (Belach Bioteknik, Stockholm, Sweden). The bioreactors were filled with 3 L of substrate that contained 3% (w/v) of cheese whey powder (initial pH adjusted to 5.0 with 2 M H₂SO₄ [Sigma-Aldrich, Darmstadt, Germany)] and were sterilized at 121°C for 20 min. Then, the media were inoculated with 20 mL/L of fungal spore suspension, and the cultivation was set at 35°C with an aeration rate of 1.0 vvm (volume of air per volume of medium per minute). The pH was continuously adjusted by pumping either 2 M H₂SO₄ or 2 M NaOH solutions using peristaltic pumps. The biomass was harvested after 48 h and subsequently subjected to freeze-drying at 0.05 bar and − 50°C prior to analysis.

Initially, three fungal strains were cultivated in both UV-sterilized and non-UV-sterilized whey powder, and varying moisture contents, for 10 days,. Photographs of all petri plates were taken throughout the incubation period (days 4, 7, and 10; Figure 1). No visual growth differences were observed between UV-sterilized and non-UV-sterilized petri plates for all fungal species. Optimum growth for N. intermedia occurred with whey powder substrate containing 40% moisture, while the ideal moisture level for A. oryzae and R. oryzae was 60%. Visually, an increase in the incubation period corresponded to enhanced microbial growth for all fungal strains. The growth period of R. oryzae was 7 days, and that of A. oryzae and N. intermedia was 10 days. However, it was determined that all these factors did not increase the substrate’s protein content (Table 2). Gmoser et al. (2020) reported that the observed increase in protein content after SSF was attributed to the metabolic activity of the fungi during growth and the decomposition of the substrate into H₂O and CO₂. The lack of an increase in protein content in the current study may be attributed to fungus’ insufficient access to nutrients in the substrate for its growth. To solve this problem, blending whey powder with porous products, such as bread, could be more suitable for SSF (Gmoser et al., 2020). Moreover, according to the results of this study, growth was observed on the petri-dish surface for all tested fungal species. Therefore, whey powder has the potential to serve as an alternative to commercial culture media used for fungi, such as potato dextrose agar and malt extract agar. Similarly, Macwan et al. (2018) also reported that the use of cheese whey as a culture medium for biomass production by lactic acid bacteria offers a cost-effective alternative compared to commercial media.

During submerged fungal cultivation, the growth at two parameters, namely non-controlled pH (pH 5.7) and controlled pH (pH 5.0) was evaluated for A. oryzae, R. oryzae, and N. intermedia. After 48 h of cultivation under non-controlled pH conditions, the lowest pH (6.73) was observed with N. intermedia (Figure 2A). For A. oryzae (8.02) and R. oryzae (7.97), the final pH of the medium was similar at the end of the cultivation. Similarly, under controlled pH conditions, the lowest pH at the end of cultivation was measured in N. intermedia (5.49), while the final pH values for A. oryzae and R. oryzae were 7.19 and 6.77, respectively (Figure 2B). The low pH observed at the 48 h of cultivation in N. intermedia might be associated with the formation of organic acids such as pyruvic acid and acetic acid, which contribute to the biosynthesis of various compounds, including bioethanol (Restiawaty et al., 2023).

The biomass production at both pH parameters exhibited statistical differences among the fungal species (Figure 3A). The highest fungal biomass amount (based on dry weight) after the cultivation was obtained under controlled pH conditions for all test fungal species. The highest amount of biomass was observed for A. oryzae under controlled pH (5.29 g/L, p < 0.05) and non-controlled pH (3.25 g/L, p < 0.05) conditions.

In the current study, the high biomass yield harvested from the controlled pH substrate of Aspergillus oryzae can be correlated with high enzyme activity. In fungal biomass cultivation with A. oryzae, pH varies depending on the substrate used. For example, pH 5.2 in olive oil mill wastewater (OOMW) (Sar et al., 2020a) and pH 5.0 in pomegranate peel (Braho et al., 2024), while pH 6.5 in fish processing wastewater (Sar et al., 2021) and vinasse (Karimi et al., 2019) increased biomass yield. Nair et al. (2016) observed that pH is a significant factor in the pellet formation of the N. intermedia strain and determined that the biomass yield increased at the optimum pH of 5.5. It was also observed that when the R. delemar was grown on potato protein liquor, higher amounts of fungal biomass were obtained at acidic pH values, with higher protein yield at pH levels of 4.0–4.5 (Sar et al., 2022b). It can be stated that slightly acidic conditions (pH 5.0), as obtained in this study, can be considered ideal for fungal biomass production for cheese whey. Although A. oryzae and N. intermedia are both members of the Ascomycetes, they exhibited differences in biomass yield under the same conditions. A similar trend was also observed in OOMW with 8.02 g/L biomass obtained by cultivating A. oryzae, while 2.1 g/L biomass was obtained with N. intermedia (Sar et al., 2020a). Differences were also observed between A. oryzae and N. intermedia grown on pomegranate juice and peel (Braho et al., 2024) and dairy by-products (Mahboubi et al., 2017a). In a study investigating the growth of different filamentous fungal strains in pure olive oil, A. oryzae showed superior performance compared to N. intermedia and R. oryzae in terms of biomass growth (Nazir et al., 2022). In another study, 19 g/L and 14 g/L biomass were obtained from A. oryzae and N. intermedia grown in thin stillage, respectively (Ferreira et al., 2014). Although N. intermedia yields a lower amount of biomass, it contributes to the development of bioprocesses with high protein yield and ethanol production (Ferreira et al., 2014; Parchami et al., 2021).

The hydrolysis of lactose into glucose and galactose in fungi is associated with galactosidase activity (Tonelotto et al., 2014; Kamran et al., 2017). Mahboubi et al. (2017a) proposed that galactosidase activity may vary within the pH range among Ascomycetes strains. According to their study, galactosidase produced by A. oryzae demonstrated higher expression/activity at pH <5, while the galactosidase produced by N. intermedia exhibited similar expression/activity within the pH range of 4–6. Various studies also reported the galactosidase activity of R. oryzae (Battaglia et al., 2011; Gajdhane et al., 2016). However, in the study conducted by Seyis and Aksoz (2004), it was determined that the galactosidase activity of Rhizopus sp. was low (less than 10 U/mL) during submerged fermentation on a substrate containing lactose. The low R. oryzae biomass obtained in this study may be related to the low galactosidase enzyme activity of the strain. Another factor affecting biomass production is pH changes, especially pH increases, during the cultivation. Souza Filho et al. (2017) reported that high ammonia concentration ammonia resulting from the final pH being alkaline (7.0–8.0) stopped the growth of R. oryzae. In this study, pH reached 6.97 after 24 h and 7.80 after 48 h of incubation for R. oryzae in whey substrate (Figure 2A). Although lower pH increases were observed in R. oryzae under controlled pH conditions, pH exceeded 6.50 after 48 h (Figure 2B). Additionally, Ibarruri and Hernández (2019) explained the decrease in biomass yield despite increasing whey concentration by proposing that the high concentration of lactoferrin and derived peptides in whey (> 1 g/L of whey) could potentially lead to the inhibition of R. oryzae.

In this study, the protein contents of fungal biomasses derived from A. oryzae, R. oryzae, and N. intermedia were investigated under varying pH conditions. Notable differences in protein levels among the strains were revealed by finding results. Under controlled pH conditions, the protein content of A. oryzae biomass was found to be 299.20 g/kg, while R. oryzae exhibited a slightly lower content at 265.31 g/kg. In contrast, a significantly higher protein content was displayed by N. intermedia under the same conditions. Further analysis indicated that biomass with higher protein levels was consistently produced by N. intermedia compared to the other strains, regardless of pH conditions. Specifically, both controlled pH (379.26 g/L) and non-controlled pH (385.23 g/L) conditions resulted in elevated protein levels for N. intermedia (p < 0.05). However, no significant difference in protein levels between controlled and non-controlled pH conditions was observed for A. oryzae and R. oryzae. Regarding biomass production, the highest yield was demonstrated by A. oryzae at 5.29 g/L, while N. intermedia showed the most significant increase in protein yield, reaching a 4.89-fold increase compared to initial levels. Overall, the potential of utilizing cheese whey powder through SmF to enhance protein content in fungal biomass is highlighted by our study. This increase is attributed to the production of protein-rich fungal biomass and efficient nutrient utilization from the substrate (Kumitch et al., 2020).

Among three different filamentous fungi, the highest biomass and highest protein levels in shake-flasks were obtained by A. oryzae and N. intermedia, respectively (Figure 3). Therefore, the fungal biomass and crude protein content of A. oryzae and N. intermedia were compared under controlled pH conditions in both Erlenmeyer flasks (250 mL capacity) and bubble column reactors (4.5 Liter capacities) (Figures 4, 5). Furthermore, pH changes were examined throughout the cultivation period of both fungal species in both flasks and bioreactor systems (Figure 6). Throughout the cultivation period, pH measured lower in the bioreactor scale than in Erlenmeyer-scale for fungal species. However, the final pH for Neurospora increased to 5.49 in the Erlenmeyer-scale, while it decreased to 4.63 in the bioreactor scale (Figure 6B).

The biomass yield harvested from the Erlenmeyer flasks for N. intermedia, which was 3.63 g/L, was increased to 6.39 g/L at the reactor scale. For A. oryzae, the biomass level increased from 5.29 g/L to 7.05 g/L (Figure 7A). In a study conducted by Mahboubi et al. (2017a), 0.27 g biomass/g substrate by Aspergillus oryzae and 0.12 g biomass /g substrate by N. intermedia were obtained from cheese whey. In this study, the biomass yield of A. oryzae obtained from cheese whey powder (0.23 g biomass/g substrate) was in accordance with that reported by Mahboubi et al. (2017a), while a higher biomass yield of N. intermedia (0.21 g biomass/g substrate) was obtained compared to same study. In the present study, fermentation was conducted at pH 5, whereas Mahboubi et al. (2017a) studied within the pH range of 4–7. Therefore, the difference in N. intermedia biomass yield efficiency may be attributed to the fermentation pH. It was determined that the protein values of the biomass obtained from bioreactor-scale were lower (30.19% for A. oryzae, 43.50% for N. intermedia) than those obtained from Erlenmeyer-scale (Figure 7B). This phenomenon can be attributed to the rapid fungal growth leading to decrease in crude protein levels under aeration conditions (Sar et al., 2020b, 2022a). It is also suggested that the aeration rate could play a significant role in yields of biomass and protein (Ferreira et al., 2015).