Citrus trees fibers and cooking oil waste as a substrate for fungal growth

In this study, alkaline pretreatment of CTF was carried out due to its strong pretreatment effect and relatively simple process scheme, also it can remove lignin without degrading carbohydrates, and increases porosity and surface area, thereby enhancing enzymatic hydrolysis (Kim et al. 2016). Fungal strains; R. oryzae, R. microsporus, A. niger, A. fumigatus, A. flavus and A. terreus were grown on pretreated CTF with and without COW through SSF as shown in Table 1. Results illustrated that A. flavus is the best for fungal growth on both alkaline pretreated CTF with and without COW among other fungal strains. The high growth of A. flavus may be due to its secrets high amount of cellulases, hemicellulases, lignin peroxidase, laccase as well as lipases which degrade cellulose, hemicellulose, lignin and COW. Therefore, A. flavus was selected for further experiments. For knowledge, these enzymes which produced by A. flavus were estimated in both alkaline pretreated CTF with and without COW at different pH in the next experiment.

Table 1 Fungal growth of different fungal strains on pretreated CTF with and without COW

Enzyme evaluation of the extracted culture supernatant

In order to investigate the citrus trees fibers degradation enzymes which directly contribute in the improvement of CTF characteristics, we initially determined the xylinolytic, cellulolytic and ligninolytic enzyme activities in both CTF and it’s supplemented with COW culture media. In this regard, the enzymes Avicelase, CMCase, α-glucosidase and β-glucosidase activities were used to evaluate the cellulase activity, and xylanase, mannanase activities were used to evaluate hemicellulase activity, laccase, and lignin peroxidase activities were corresponding to the ligninolytic enzyme activity (Table 2). Also, the lipolytic activity reflects the COW utilization as supplement carbon source and that appeared via degradation process by A. flavus.

Table 2 Screening of different enzymes resulting from growth of A. flavus on CTF

After incubation time of the A. flavus culturing under solid-state fermentation, the enzyme mixture were isolated from the solid cultures using different buffers system (ranging from 5 to 9 for 2 h. at 180 rpm and 30 °C) in order to determine the change of fibers by the enzymatic action. As can be seen in Table 2, the maximum extraction of cellulase enzymes was found at pH. 7, while the highest isolation of hemicellulase enzymes was obtaining at pH. 5. Significantly, enzyme activities in CTF supplemented with COW cultures was considerably higher than in CTF cultures in terms of cellulase enzymes. In contrast, the hemicellulase enzymes were significantly superior under CTF culture medium than in CTF supplemented with COW cultures. The CTF supplemented with COW had a much higher cellulase activities compared with hemicellulase, especially CMCase enzyme activity. It suggests that cellulolytic enzymes were preferably induced during cultivation with CTF supplemented with COW culture medium. Similarly, the ligninolytic enzyme activities were found induced in the COW–citrus trees fibers culture medium in terms of lignin peroxidase. In addition, the maximum lipase production was obtained in the CTF supplemented with COW culture medium (22.46 ± 1.26 U g−1), which becomes a greater than tenfold increase in activity if compared to the citrus trees fibers culture medium (2.26 ± 0.57 U g−1). In addition, the mannan-degrading enzyme activity of citrus trees fibers supplemented with COW culture was close to that of citrus trees fibers culture, and the extraction of enzyme was found to be active across the different pH buffers.

As shown in Table 2, the CTF supplemented with COW culture significantly induced higher levels of β, α-glucosidase activity than CTF only. This lower activity of β, α-glucosidase implies accumulation of cellobiose, which is a strong inhibitor of cellobiohydrolase and endoglucanase activities during cellulose hydrolysis (Fujii et al. 2009; Sehnem et al. 2006). In fact, our studies demonstrated that the fungal hydrolyzed cellulosic materials more slowly when cultivating with CTF only. However, the supplementations of CTF with COW generally produce higher levels of lipase activity which contribute in the lipid formation leading to higher levels of cellulase enzymes. In this study, the culture supernatant derived from CTF supplemented with COW culture was proved to display a slight decrease of xylan-hydrolyzing activity than that derived from CTF culture and this result might be related to the differences in culture composition. This suggests that the genes related to xylan-hydrolyzing performance of A. flavus may have been suppressed, thus resulting in a reduction in xylanase activity (Fujii et al. 2009). Maximum hemicellulases activities are known to improve when cultivated with carbon sources are rich in hemicellulose like rice straw and bagasse (Kogo et al. 2017).

Accordingly, the enhancement of the CTF under fungal enzymes influence along with COW in order to obtaining a highest heating process is a new method could open a promising way in the alternative energy. Interestingly, selection of alternative lower cost agriculture residues to treat by solid-state fermentation plays a prominent role in the green eco-friendly system, since it benefits in two ways. One of them, providing a possibility to the production of several microbial enzymes with an elevated yield and a lower cost in comparison to the most used culture media (de Cassia Pereira et al. 2015; Rodrigues et al. 2017; Singhania et al. 2010). The other way, the fiber characteristics in these alternative substrates would be improved as a result of the synergistic action of microbial enzymes along with the hydrolyzing COW, thus yielding maximum heating capacity yield.

Indeed, the significant activities of the cellulose-hydrolytic enzymes by fungal cells under solid-state condition have been extensively investigated in the previous reports. It is well evident that Aspergillus species have been frequently in these studies as a promising candidate to produce cellulases and hemicellulases complexes (Gomes et al. 2016; Santos et al. 2015). Otherwise, the cellulolytic and hemicellulytic enzyme induction was also dependent on the nature of substrate, since sugarcane bagasse (SCB) and wheat bran (WB) was found to be an excellent substrates for xylanase production by Aspergillus fumigates M.7.1 with higher activity (1040 U g−1) after 6 days (Moretti et al. 2012).

In accordance of our results, lower productivity of β-glucosidase (3 U g−1) by Aspergillus niger NS-2 strain using Sugarcane Bagasse (SCB) under SSF condition was also reported by (Bansal et al. 2012; Santos et al. 2015). In addition, induction of β-glucosidase, β-xylosidase and xylanase by the Aspergillus niger SCBM3 strain using SCB and Wheat Bran (WB) as substrates was studied by (Bajar et al. 2020), they also investigated  the cellulase and xylanase enzyme production by A. heteromorphus using anaerobically treated distillery spent wash (ADSW) and rice straw (RS) and revealed that the highest exoglucanase, xylanase and endoglucanase enzyme activities under optimum conditions were 6.3 IU/mL, 11.6 IU/mL and 8.1 IU/mL, respectively.

It is noteworthy to mention that, the higher activities of CMCase, Avicelase, α-glucosidase, and β-glucosidase in the presence of COW can be suggested by the increasing of the metabolic activity of A. flavus, in which COW exhibited as a growth promoter encourage the biosynthesis of these enzymes. Where, it can be noted a plausible lipolytic activity in the presence of COW, which can contribute in the increased of the fungal metabolic activity (Hashem et al. 2022). On contrast, the presence of COW was found to correlated with the decrease of xylanase and mannanase which may be attributed to the inhibition effect of COW on the production of these enzymes by accumulation of some fatty acids resulted by COW degradation (Abdelraof et al., 2019). In addition, the lower activities of β-glucosidase and α-glucosidase in both culture media may be due to the intracellular formation nature of these enzymes by fungal strains, which was released at the end of incubation period (i.e., stationary phase) by the autolysis of cells as reported previously (Shahriarinour et al. 2011; Umikalsom et al. 1998).

Based on these comparisons, it can be concluded that A. flavus evaluated in the present study was good candidate of cellulolytic enzymes, showing a superior enzymes production leading to enhancement of fiber characteristics along with COW generating high heating energy. To the best of our knowledge, this is the first time that this idea has been reported.

Fibers analysis

The fibers chemical analysis is tabulated in Table 3 which included Klason`s lignin, cellulose, hemicellulose, ash and wax/resin. The fibers analysis contained raw CTF, tCTF and tCTF/COW. The lignin content was affected by the attachment of lignin peroxidases in both fermentation conditions (fiber with and without oil). The fiber analysis confirmed that the lignin content is changed by non-significant values in both treatment conditions. Otherwise, the cellulose as well as hemicellulose content was affected by a significant value where cellulose was decreased in raw CTF and CTF and COW samples by about 12 and 27%, respectively (Abdelraof et al. 2020; Elleboudy et al. 2021). Additionally, hemicellulose contents were decreased by around 41 and 25%, respectively (Hasanin et al. 2019, 2020). These results were in a good agreement with enzyme evaluation of the extracted culture supernatant part. Herein, fibers composition might be affect the heating value.

Table 3 Main chemical compositions of CTF, tCTF and tCTF and COW

Fiber characterization


FTIR spectroscopy is the useful technique used to assign the functional groups as well as keep track of changes in main functional groups. Raw CTF, COW, tCTF and tCTF and COW FTIR spectra are presented in Fig. 1. On comparing raw CTF and tCTF, a significant change in raw CTF after fermentation process is observed. The band of OH stretching vibration assigned at 3586 in raw CTF was shifted to low frequency at 3273 cm−1. In addition, the band of CH stretching vibration shifted to low frequency at 2912 cm−1. This shifting referred to decrease in cellulose ratio as well as hemicellulose. On the other hand, the bands of lignin at around 1589 and 1765 cm−1 illustrated a non-significant change. Moreover, the band at 1024 cm-1 which referred to b-glycosidic linkage appeared as sharp band at the same position approximately (Hasanin et al. 2019; Hashem et al. 2020; Youssef et al. 2019). Otherwise, the COW spectrum was assigned the 3012, 2925, 2848, 1737, 1659, 1371 and 979 cm−1 which due to OH stretching vibration, =C–C–H, C=C, –CH=CH2, respectively (Qiao et al. 2019). In this context, the tCTF/COW spectrum appeared as combination between fiber and oil with main changes in the bands position of fibers as showed in the tCTF. Herein, the tCTF/COW spectrum assigned the significant changes in the COW bands where the main bands were shifted to 3000, 2919 and 1716 cm−1. In addition, the band at 979 cm-1 disappeared. These results affirmed that the simplification fibers and oil was observed may be induced the heating value of the tCTF/COW. However, both materials are maintained their microstructure that observed with minor changes.

Fig. 1
figure 1

FTIR spectra of raw CTF, tCTF, COW and tCTF/COW


The topography study of the surface morphology of raw CTF, tCTF and CTF/COW is illustrated in Fig. 2. The raw CTF (Fig. 2A), was observed as overlapped fibers collected together as a smooth surface in many points. In addition, the tCTF appeared with less overlapping as well as disappearing of collections and the surface observed as fibers structure morphology. However, the tCTF/COW sample image was illustrated the surface of fibers as pours surface that may be according to the role of oil that penetrated the fibers and induced the fungal growth to attach fibers as well as oil. These observations emphasized that the fungal treatment affected the fibers inter- and intra-molecular structures in present and absent of COW. These observations may be affected the heating value.

Fig. 2
figure 2

Topography analysis of raw CTF (A), tCTF (B) and tCTF/COW (C)

Thermal stability

Figure 3 shows the thermal analysis (TGA and DTGA) of the raw CTF, tCTF and tCTF/COW. The thermal behavior of each sample was related and illustrated the heating value as well as the starting burring attitude. The first decomposition peak was closely with the flashing point of burring. Herein, the COW was recorded the first decomposition peak at 409 °C as a single stage of decomposition. In contrast, all other samples were recorded two decomposition stages. Moreover, the CTF/COW was recorded the lowest decomposition temperature in the first stage of decomposition. Additionally, the second stage of CTF/COW was observed the sharper peak at 350 oC. Otherwise, the raw CTF and tCTF are recorded the first stage decomposition peaks at 290 and 300 °C. These results confirmed that the thermal behavior of untreated CTF and COW was effected significantly after mixing and fermented together. Additionally, these observations affirmed that the fungal enzymes made the coal simple to burn with low flashing point.

Fig. 3
figure 3

Thermal analysis of samples (TGA (upper) and DTGA (lower))

Heating value

Calorific value is the amount of heat energy present in the fuel, and determined by the complete combustion of a specified quantity at constant pressure and in normal conditions. It is also called calorific power. The measured calorific values of CTF, CTF/COW, tCFF, tCTF/COW, and COW processes were 18,214,18,497,14,200,43,422 and 39,823 kJ/kg, respectively, as shown in Fig. 4. COW is an edible oil that has formerly been used for frying in restaurants and hotels, and no longer be used for similar purposes. In most towns in developing countries including Egypt, waste cooking oil is simply dumped into the environment. COW used to produce solid fuel is environmentally friendly for it recycles waste cooking oil and gives renewable energy with lower pollution. It substitutes some amount of petrochemical oil import and also lowers the cost of waste management. The COW provides alternative energy with a high calorific value to producing solid fuels from biomass for various uses, COW has a high calorific value of 39,823 kJ/kg compared to CFT 18,214 kJ/kg. The screw press was used to increase the density by compressing the fibers in a small volume. This led to an increase in the heating value. COW added to CTF increased the calorific value for the oil fiber mix because of the higher calorific value of oil. A. flavus decreased grain size and increased the mixing between the fibers and oil. It led to an increased calorific value of tCTF/COW. The calorific value of fuel determines the availability of heat to produce the power. Therefore, calorific values are important in the choice of alternative fuel for coal for higher performance. The high calorific value of oil and good mixture by A. flavus, finally high density by compressed mix using a screw press caused to the higher calorific value of tCTF/COW.

Fig. 4
figure 4

Calorific value of each substrate before and after fungal enzyme treatment

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