Identification of isolated white-rot fungi
WRF were preliminary identified based on morphological characteristics. All tested white-rot fungal isolates belonged to the Basidiomycetes phylum. The WRF strains, designed as Fomes fomentarius TMF2, Bjerkandera adusta TMF1 and Schizophyllum commune TMF3 were identified as Fomes fomentarius, Bjerkandera adusta, and Schizophyllum commune based on the internal transcribed spacer region (ITS) sequence, located between the 18S and 5.8S rRNA coding genes. This region is widely used for analyzing fungal diversity in environmental samples . The obtained sequences were deposited in the NCBI-GenBank database under the following accession numbers: MW327505 for B. adusta, MW327506 for S. commune and MW327504 for F. fomentarius.
Detection of enzyme activity using the API ZYM
The examination of the hydrolytic enzyme activities of all tested isolates was done using the API ZYM test (Table 1). This test is simple, rapid and reliable and is widely used for identifying various hydrolytic enzymes present in novel strains .
All tested WRF showed a broad range of extracellular enzyme activities. Beta glucosidase activity was very high for these isolates, indicating that these fungi have a good potential for cellulose degradation . Acid phosphatase was negative for F.fomentarius TMF2 and S. commune TMF3 but strong for B. adusta TMF1. F.fomentarius TMF2 and S. commune TMF3 produced α- and β-galactosidase, and mannosidase, suggesting potential for enzymatic hydrolysis of different carbohydrates.EsteraseC4, esterase lipase C8, leucinearyl amidase, valinearyl amidase were present at medium levels at B. adusta TMF1 while cystinearyl amidase was found at a low level at F.fomentarius TMF2 and B. adusta TMF1.
Very high levels of lipase C14 were detected in F. fomentarius TMF2 and S. commune TMF3.
The API ZYM test confirmed that the enzymatic system of the isolated WRF is diverse.
Detection of enzyme activity using a qualitative test for hydrolases and laccase
F.fomentarius TMF2, B.adusta TMF1 and S.commune TMF3 were able to grow on CMC, Avicel, pectin, starch, xylan and guiacol agar plates. For F.fomentarius TMF2 and S. commune TMF3, extracellular hydrolytic enzyme activities were observed already after 2 days of mycelia growth.
The occurrence of a halo zone around mycelium indicated the area of the substrate hydrolysis and thus the presence of the specific enzyme (Fig. 1). These strains were able to utilize both types of cellulose, CMC as a soluble form of cellulose and Avicel as microcrystalline cellulose, because of their synergistic endoglucanases and exoglucanases systems, which is typical for WRF . F. fomentarius TMF2 and B. adusta TMF1 mycelia were very dense and within the mycelia growth was the inner clearance zone that indicated amylase, pectinase and xylanase activity. A similar zone of clearance for Fomes sp. was reported by Hadda and coworkers . The very dense mycelium of B. adusta TMF1 on starch, pectin and xylan substrates made it impossible to identify the zone of clearance (Fig. 1), but after removing the mycelium, the amylase, pectinase and xylanase activities could be detected as very clear zone throw the entire growth surface (Fig. 1).
Laccase activity was noticed after 24h of mycelia incubation for F. fomentarius TMF2 and B. adusta TMF1 and after 48 h of incubation for S. commune TMF3 using guaiacol as an oxidizing reagent. The brown color under mycelia indicated the presence of laccase (Fig. 1).
Solid-state fermentation (SSF) and enzymes production
In SSF, the most important step is the selection of appropriate lignocellulosic substrate for WRF growth and enzyme synthesis . Literature data showed that various lignocellulosic waste substrates, such as sugarcane bagasse, wheat straw, wheat bran, or corn stover could be applied for the enzyme production by WRF . Among them, wheat bran is the most used substrate for WRF enzyme induction. For example, wheat bran was used for cellulase, xylanase and laccase production by Fomes sp. [22, 23], and for cellulase production by S. commune , while wheat straw was used for cellulase synthesis by B. adusta .
The literature data about the simultaneous production of laccase and hydrolytic enzymes on BSG, SCR, SFM and SBM by WRF is still very scarce. In this study, the production of cellulases (CMCase and Avicelase), amylase, pectinase, xylanase and laccase by three tested isolates on these lignocellulosic waste substrates was evaluated. The results showed that all used substrates could support WRF growth and enzyme synthesis. However, some differences were observed in terms of the suitability of the substrates to stimulate the synthesis of specific enzymes by each WRF tested (Tables 2-4).
For F. fomentarius TMF2, SFM seemed to be the most potent lignocellulosic waste substrate for induction of both laccase and hydrolases production (Table 2). This substrate is usually used for microbial amylase production by genus Bacillus , but it was also used as a substrate for the simultaneous production of various hydrolases (cellulase, amylase, pectinase and xylanase) by actinomycetes Streptomyces fulvisimuss CKS7, isolated in our group . Thermophilic fungus Humicola lanuginose was used for cellulase production during SSF of SFM . It has not been shown until this study, that a strain of F.fomentarius could grow on sunflower meal and synthesize extracellular laccase. Compared with Trametes versicolor laccase, produced in submerged fermentation using 2% (w/v) sunflower stems , higher laccase activity was reported in our study (35 IU/L vs. 2.45 IU/g). Slightly lower F. fomentarius TMF2 laccase activity was obtained on BSG (2.28 IU/g), while activities on SBM and SCR were significantly lower.
F. fomentarius TMF2 showed also the highest amylase activity (27.29 IU/g) on SFM, compared to other tested substrates. Pectinase and xylanase produced by F. fomentarius TMF2 on SFM reached activity of 10.86 IU/g and 16.84 IU/g, respectively, and these values were also the highest among all tested substrates. However, these values were not significantly higher than the pectinase activity on SBM (9.34 IU/g) and xylanase activity obtained on BSG (15.88 IU/g) suggesting these three substrates were suitable for pectinase and xylanase production by F. fomentarius TMF2.
Spent coffee waste, as lignocellulosic material, induced a very low enzyme activity, especially for laccase and pectinase (0.21 IU/g and 1.02 IU/g, respectively). However, SCR showed to be a suitable substrate for CMCase production by F. fomentarius TMF2 with 1.32 IU/g, which was not significantly lower than values obtained on SFM and BSG (1.42 IU/g and 1.49 IU/g, respectively). Keeping in mind its composition, these results were expected.
Literature data showed that coffee pulp, coffee husk and spent coffee grounds were mainly used as substrates for WRF growth [8, 29, 30]. The different chemical composition of these coffee by-products affects the fungal growth and the absorption of nutrients necessary for enzyme synthesis . The most studied WRF, with the potential to grow on different coffee waste substrates, belong to genus Pleurotus [29, 30]. However, there is a lack of literature data about Basidiomycetes growth on coffee waste substrates.
B. adusta TMF1 and S. commune TMF3 showed a similar pattern of response to substrates used for stimulation of different enzyme synthesis (Tables 3 and 4). The laccase activities of 1.44 IU/g and 2.79 IU/g produced by B. adusta TMF1 and S. commune TMF3, respectively, obtained on BSG as a substrate were significantly higher than laccase activities on other waste substrates. The literature data generally reports lower laccase activities produced by these WRF species. For example, testing the ligninolytic potential of B. adusta under different growth conditions, Tripathi and coworkers  found laccase activity of 64 U/L only in nutrient-rich medium, after 20 days of growth under static conditions. These authors suggested that laccase activity was inducible in this WRF. There are studies that reported no laccase activity in B. adusta. [32, 33].
Pariatamby and Nithiya reported laccase activity ~ 1.0 IU/g for S. commune after 7 days growth on BSG . In the study of Zhu and coworkers no laccase activity was detected for S. commune during 30 days SSF on Jerusalim artichoke stalk . However, literature data also reported higher values of laccase activity under optimized conditions .
The most suitable substrate for amylase production by B. adusta TMF1 and S. commune TMF3 was BSG with measured activities of 11.47 IU/g and 10.98 IU/g, respectively. Moderate amylase activities were obtained on SFM and SBM, while SCR was the least appropriate substrate for inducing amylase production. It is interesting to note that the enzymatic potential of B. adusta was mainly associated with the decomposition of lignocellulose biomass, while in this study we showed, for the first time, that amylases produced by this species could degrade starch to simple sugars. Shimazaki and coworkers  reported amylase activities after 9 days of cultivation S. commune under shaking conditions from 0.69 IU/mL to 30.0 IU/mL, depending on the carbon source. The highest activity was obtained with 5% wheat bran as a carbon source and it was higher than the highest activity obtained in our study (30.0 IU/mL vs. 10.98 IU/g). Starch content in wheat bran varies from 9-26% , and this is higher content than present in all different substrates used in our study. Therefore, even higher amylase activities produced by S. commune TMF3 could be expected after cultivation on a richer substrate.
The most suitable substrate for pectinase production by B. adusta TMF1 and S. commune TMF3 was SBM (Tables 3 and 4). This could be expected given that the pectin content in SBM is higher than in the other tested substrates [38, 39].
Ganbarow and coworkers  studied pectinase production of five fungi belonging to the genus Bjerkandera on wheat bran during SSF. Two of these five fungi (B. adusta 41 and B. fumosa 97) had a lower pectinase activity, then pectinase activity obtained in our study (22.1 IU/g and 10.3 vs. 23.73 IU/g), while higher pectinase activities were reported at B. adusta 1 (25.4 IU/g), B. adusta 40 (54.1 IU /g) and B. fumosa 22 (44.7 IU/g) strains.
Within this study, 9.25 IU/g for pectinase activity of S. commune TMF3 was noted on the SBM. Mehmood et al.  reported a higher value of produced pectinase (480.45 IU/g) than in our study, by growing S. commune on optimized solid medium citrus waste - mosambi peels (sweet limetta). Interestingly, during SSF on Jerusalim artichoke stalk, pectinase activity for S. commune was not detectable . It is important to note that enzyme activity in our study was determined only after 6 days of growth and no optimization has been performed, and that could contribute to their latively lower observed pectinase activity.
The highest levels of xylanase activities of 18.39 IU/g and 17.47 IU/g produced by B. adusta TMF1 and S. commune TMF3 respectively, were measured on BSG as substrate (Tables 3 and 4). A high level of xylanase activity by S. commune was also reported in the work of Gautam and coworkers . Another study showed that S. commune could produce xylanase in submerged fermentation, while growing on bamboo, sugarcane bagasse and banana stem, for 15 days. The obtained xylanase activity was very low on bamboo and banana stem, while on sugarcane bagasse this activity reached the value of ~1.2 IU/mL.
A literature survey showed that studies about Bjerkandera sp. xylanase production on waste substrates are extremely scarce. Only Qirouz-Castaneda and coworkers  reported xylanase production by this WRF on several waste substrates with maximum xylanase specific activity of 0.4 IU/mg of proteins obtained using oak dust for B. adusta growth.
S. commune TMF3 and B. adusta TMF1 produced a maximum of cellulase activity (2.51 IU/g and 3.71 IU/g respectively) during the growth on SFM. Literature data about cellulase production by S. commune during SSF are very rare with exception of the work of Zhu and coworkers . In their study, S. commune was grown on a Jerusalem artichoke stalk (mixed with Mandels’ salt solution) during 30 days and produced cellulases. Maximum of endoglucanase activity 15.2 IU/g was reached after 20 days of incubation. In the study of Qirouz-Castaneda  B. adusta was grown on a solid agar plate with 2% wheat straw and produced maximum CMCase activity of 2.4 IU/mg proteins. It is important to note that all of these results are difficult to compare due to the different methods for fungal growth and due to the different reported expression of cellulase activities.