Bioprocessing of Agricultural Residues as Substrates and Optimal Conditions for Phytase Production of Chestnut Mushroom, Pholiota adiposa, in Solid State Fermentation

Phytase is an enzyme that breaks down phytates to release phosphorus in an available form. This enzyme plays an important role in animals, especially monogastric animals. It serves to improve phytate digestion along with phosphorus absorption, which are required for optimal growth performance and health. In this study, five mushroom species (Amauroderma rugosum SDBR-CMU-A83, Ganoderma mastoporum SDBR-CMU-NK0244, Marusmius sp.1 SDBR-CMU-NK0215, Pholiota adiposa SDBR-CMU-R32 and Piptoporellus triqueter SDBR-CMU-P234) out of 27 mushroom species displayed positive phytase production by agar plate assay. Consequently, these five mushroom species were selected for determination of their potential ability to produce phytase under solid-state fermentation using five agricultural residues (coffee parchment, oil palm empty fruit bunches, rice bran, sawdust, and water hyacinth) as substrates. The highest yield of phytase production (17.02 ± 0.92 units/gram dry substrate) was obtained after one week of fermentation. Optimization for phytase production was determined by statistical approaches using a Plackett–Burman design to screen ten parameters of relevant substrate components. Two significant parameters, the amount of water hyacinth and the moisture content, were found to affect the production process of phytase. Furthermore, the optimal temperature, pH value, and fermentation period were evaluated. The results indicated that the highest degree of phytase production at 53.66 ± 1.68 units/gram dry substrate (3.15-fold increase) was obtained in water hyacinth containing 85% moisture content by addition with a suitable basal liquid medium at a pH value of 6.5 after being incubated at 30 °C for seven days. The crude phytase of P. adiposa was precipitated and the precipitated extract was then used to determine partial characterizations. The precipitated extract displayed high activities after exposure to conditions of 42 °C and pH 5.0. Furthermore, Fe2+ enhanced phytase activity and precipitated extract displayed the best stability at a pH value of 8.0 and a temperature of 4 °C.

The selection of phytase producing strains was performed using phytase screening agar (PSA), containing (gram per litter) sodium phytate 4, glucose 20, CaCl 2 ·2 H 2 O 2, NH 4 NO 3 5, KCl 0.5, MgSO 4 ·7H 2 O 0.5, FeSO 4 ·7H 2 O 0.01, MnSO 4 .7H 2 O 0.01, agar 15; pH 6.0 [18,25]. A mycelial plug (0.5 cm in diameter) obtained from an agar plate of each fungi cultivated on PDA was inoculated onto the modified PSA. The presence of a clear zone around the colony was considered a positive indicator of phytase production. Three replications were generated. Positive phytase producing strains were selected to evaluate the production of phytase under SSF using different agricultural residues as substrates.
Five agricultural residues, including coffee parchment, oil palm empty fruit bunches, rice bran, sawdust, and water hyacinth, were collected from Chiang Mai, Thailand and used in this study. Five grams of each dried substrate were placed in 125 mL Erlenmeyer flasks. The moisture content of the substrate was adjusted to approximately 80% (w/v) using deionized water. The flasks were then autoclaved at 121 • C for 15 minutes. After allowing the flasks to cool at room temperature for 24 h, five mycelium plugs (0.5 mm diameter) from 5-day-old PDA culture were inoculated and the inoculated flasks were incubated at 30 • C for 7 days. After fermentation, crude phytase was extracted and measured for phytase activity through the process described below. All experiments were performed in triplicate.

Extraction and Determination of Crude Phytase Atcivity
Crude phytase obtained from fermented agricultural residue with mushroom strains were extracted following the method described by Salmon et al. [18] and Ajith et al. [26] with some modifications. Each fermented agricultural residue was extracted using 25 mL of 0.2 M sodium acetate buffer (pH 5.5) and filtrated through sterilized Whatman's No.1 paper. The filtrated solution was centrifuged at 6000 rpm at 4 • C for 20 min. The clear supernatant was then collected and used as a crude enzyme. The extraction process was performed under cool conditions (4 • C).
Phytase activity was determined using the colorimetric assay according to the method described by Harland and Harland [27] with some modifications. In brief, 20 µL of crude enzyme solution was mixed with 110 µL of 6.82 mM sodium phytate in 200 mM sodium acetate buffer (pH 5.5) at 37 • C for 60 min. The enzyme reaction was stopped by adding 500 µL of 10% (w/v) trichloroacetic acid. The development of a blue color was indicative of a positive result for phytase activity. Subsequently, the absorbance was measured at 660 nm and phytase activity was expressed as units per gram dry substrate (U/gds) [28].

Effect of Carbon and Nitrogen Sources for Phytase Production
A suitable mushroom strain (P. adiposa SDBR-CMU-R32) and suitable agricultural residue (water hyacinth) were selected in order to optimize the effects of the nutrient sources for phytase production. Fermentation was conducted using the following process. The moisture content of the substrate was adjusted to approximately 80% (w/v) using basal liquid medium (20 g glucose, 2 g CaCl 2 ·2H 2 O, 0.5 g KCl, 0.5 g MgSO 4 ·7H 2 O, 5 g NH 4 NO 3 , 0.01 g FeSO 4 ·7H 2 O, 0.01 g MnSO 4 .7H 2 O, 15 g agar, 1000 mL deionized water, pH 6.0) [18,25]. Deionized water and basal liquid medium in absence of each carbon or nitrogen sources were used as the negative control. Five different carbon sources, including sucrose, fructose, maltose, and corn starch, were used at concentrations of 2.0% by replacing the glucose in the basal liquid medium as has been described above. Moreover, different organic (peptone, tryptone, malt extract, and yeast extract) and inorganic (urea and ammonium sulfate) nitrogen sources at concentrations of 0.5% were used to replace the ammonium nitrate in the basal liquid medium.
After seven days of fermentation, the crude enzyme was extracted and phytase activity was measured. Each treatment was performed in three replications. Consequently, suitable carbon and nitrogen sources were selected for further experimentation.

Optimization of Substrate Component
A Plackett-Burman design (PBD) was used to determine the suitable substrate component for phytase production with a selected mushrooms strain. Glucose (suitable carbon source) and ammonium nitrate (suitable nitrogen source) were used together with other components for the PBD study. A total of ten parameters; glucose (A), ammonium nitrate (B), MgSO 4 ·7H 2 O (C), KCl (D), MnSO 4 ·H 2 O (E), FeSO 4 ·7H 2 O (F), CaCl 2 (G), water hyacinth (H), moisture content (I), and inoculum (J), were selected and tested as independent variables. The substances were examined at three levels: −1, 0 and +1 for low, medium, and high levels, respectively ( Table 1). The maximum levels of A, B, C, D, E, F, G, H, and J were set at 25, 6, 0.1, 0.1, 0.1, 0.1, 2, and 5 g, respectively. The minimum levels of A, B, C, D, E, F, G, H, and J were set at 2, 0.1, 0.1, 0.01, 0.01, 0.01, 0.1, and 0 g, respectively. The maximum and minimum levels of I were set at 5 and 25 mL, respectively. The maximum and minimum levels of J were set at 8 and 12 mycelium plugs, respectively. This experimental design consisted of 20 experiments and 3 central points, totaling 23 experiments. Fermentation was conducted using the process previously described using P. adiposa. The enzyme was extracted after 7 days of fermentation at 30 • C and phytase activity was measured. The parameters for which the p-value was less than 0.05 were determined to have a significant effect on phytase activity and were chosen for further optimization by respond surface methodology.

Respond Surface Methodology (RSM)
The results from PBD indicated that water hyacinth and moisture content were significant positive factors (p < 0.05) on phytase production by P. adiposa. In this experiment, the optimum levels were estimated by the CCD of RSM. A CCD consisting of 13 experimental runs with five replications at the center point was employed to determine the effects of the independent variables (Table 2). Fermentation, enzyme extraction and determination of phytase activity were conducted by following the method previously described.
2.5.3. Determination of pH Value of Basal Liquid Medium, Temperature, and Fermentation Period for Phytase Production A suitable substrate component from previous experiments was used in this experiment for the phytase production of SSF from P. adiposa. The pH values of the basal liquid medium were adjusted to 4.0, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, and 9.0 using 0.5 M HCI and 0.25 M NaOH. Each designated pH value was added into a suitable substrate component according to the value of the moisture content in each fermented flask before the flasks were autoclaved. After inoculation, the flasks were incubated at 30 • C for 7 days. The pH value that presented the highest yield of phytase activity was selected for further experimentation.
The effect of temperature on phytase production was determined. In this experiment, the suitable components and pH values of the basal liquid medium that had been obtained from previous experiments were used. SSF was performed at different temperatures (20,25,30,35,40, and 45 • C). The crude enzyme was extracted and phytase activity was then measured. The suitable value of SSF for phytase production was selected and used for further experimentation.
The effect of the fermentation period on phytase production was determined. Suitable substrate component, pH value of the basal liquid medium, and optimum temperature obtained from previous experiments were used. Fermentation was performed for 2 weeks. The crude enzyme was extracted and phytase activity was measured every day. Three replications were prepared for each treatment.  Phytase in the crude extract was obtained from SSF of P. adiposa under the optimal conditions established in previous experiments. The phytase was then precipitated by saturated ammonium sulfate according to the method described in previous studies [29,30]. The concentration of ammonium sulfate was used in the range of 40%-70%. The precipitation process was performed under cool conditions (4 • C). After 1 h, the precipitate was collected by centrifugation at 6000 rpm at 4 • C for 20 min and stored at -20 • C before being used in further experiments. All treatments in each experiment were carried out in triplicate.

Determination of Optimal Temperature and pH on Phytase Activity
The precipitated extract obtained from a previous experiment was used to determine the optimal temperature for phytase activity. The precipitated extract was dissolved in 0.2 M sodium acetate buffer (pH 5.5) [27]. The enzyme reaction consisted of 20 µL precipitated extract solution and 110 µL of 6.82 mM sodium phytate in 0.2 M sodium acetate buffer (pH 5.5). The enzyme reaction was incubated at different temperatures ranging from 30 to 80 • C. After 1 h of incubation, phytase activity was measured following the method described above. Each treatment was tested in triplicate. In this experiment, one phytase unit was defined as the amount of enzyme to catalyze the release of 1 µmol of inorganic phosphorus per minute under the defined reaction conditions. The amount of phytase activity recorded at different pH values was determined. Furthermore, 20 µL of the precipitated extract was mixed with 110 µL of 6.82 mM sodium phytate in 0.2 M of different buffering solutions; KCl/HCl buffer for pH 2.0, sodium acetate buffer for pH 3.0-5.5, citrate-phosphate buffer for pH 6.0-7.0, and Tris/HCl buffer for pH 8.0-9.0. The enzyme reaction mixture was incubated at the optimal temperature that had been established in a previous experiment.

Effect of Cations and Potential Inhibitors on Phytase Activity
The effect of different cations (K + , Ca 2+ , Mg 2+ , Mn 2+ , Zn 2+ , Cu 2+ , Fe 2+ , Co 2+ , Na + ) and potential inhibitors (EDTA, tartrate, citrate, and molybdate) on phytase activity at concentrations of 1 and 5 mM were investigated following the method of Salmon et al. [18]. The activity assayed in the absence of cations and inhibitors was defined as the control. The phytase activity was calculated and expressed as a percent degree of relative activity [31].

Determination of pH and Temperature Stability
The effect of pH on enzyme stability was determined following the modified method of Karthik et al. [23] and Ajith et al. [26]. The precipitate extract was mixed with different buffering solutions; KCl/HCl buffer for pH 2.0, sodium acetate buffer for pH 3.0-5.5, citrate-phosphate buffer for pH 6.0-7.0, Tris/HCl buffer for pH 8.0-9.0, NaHCO 3 /NaOH buffer for pH 10-11, and KCl/NaOH for pH 12 at ratio 1:1 (v/v). The mixtures were incubated at 4 • C. The relative enzyme activity was determined after 12 h and 24 h of incubation.
The effect of temperature on enzyme stability was determined following the method described by Salmon et al. [18] and Spier et al. [19]. The enzyme solution was then stored at different conditions; room temperature (26 ± 2 • C), cooling temperature (4 • C), and freezing temperature (−20 • C). The phytase activity was measured every 2 days until 50 days. The percentage of relative enzyme activity was then calculated.

Statistical Analysis
Data were analyzed by one-way analysis of variance (ANOVA) using the SPSS version 17.0 for Windows (Chicago, IL, USA). Duncan's multiple range test was used to determine significant differences between treatments with a p-value of less than 0.05. The Design Expert Software version 7.0.0 (Minnesota, USA) was used for statistical analysis of PDB and CCD. The resulting values were then used to design the experiments. The regression and graphical analyses of the experimental data were subsequently obtained.

Selection of Phytase Producing Mushroom Strains and Agricultural Residue for Phytase Production under SSF
Phytase production was detected on PSA containing sodium phytate as sole source of phosphorus. Among the twenty-seven mushroom strains, only five strains, namely Am. rugosum, G. mastoporum, Marasmius sp.1, P. adiposa (chestnut mushroom), and Pi. triqueter, were positive for phytase production by the formation of a clear zone (hydrolysis zone) around the colonies (Table 3, Figure 1). Thus, these five mushroom strains were selected for the investigation of phytase production under SSF using different agricultural residues. Table 3. The list of mushroom strains screened for phytase activity in this study.
J. Fungi 2020, 6, x FOR PEER REVIEW 8 of 22 Trametes cubensis An-L1−5 MW267646 -Xylaria sp. NK0216 MW267256 -"+" = positive phytase production and "-" = negative phytase production. Five selected mushroom strains were fermented with five different agricultural residues for phytase production under SSF. It was found that the phytase production of each selected mushroom strain was significantly affected by the agricultural residue that was used. The highest phytase activity of 17.02 ± 1.13 U/gds was achieved with water hyacinth fermented with P. adiposa, followed by oil palm empty fruit bunch fermented with Am. rugosum (8.28 ± 0.26 U/gds), and coffee parchment fermented with Am. rugosum (7.81 ± 0.28 U/gds) ( Figure 2). Notably, the lowest phytase activity was obtained from coffee parchment fermented with Marasmius sp.1 (0.28 ± 0.05 U/gds). On the basis of the highest phytase activity, P. adiposa was selected for further investigation. Five selected mushroom strains were fermented with five different agricultural residues for phytase production under SSF. It was found that the phytase production of each selected mushroom strain was significantly affected by the agricultural residue that was used. The highest phytase activity of 17.02 ± 1.13 U/gds was achieved with water hyacinth fermented with P. adiposa, followed by oil palm empty fruit bunch fermented with Am. rugosum (8.28 ± 0.26 U/gds), and coffee parchment fermented with Am. rugosum (7.81 ± 0.28 U/gds) ( Figure 2). Notably, the lowest phytase activity was obtained from coffee parchment fermented with Marasmius sp.1 (0.28 ± 0.05 U/gds). On the basis of the highest phytase activity, P. adiposa was selected for further investigation.

Effect of Carbon and Nitrogen Sources in Basal Liquid Medium for Phytase Production
This experiment was performed to examine suitable carbon and nitrogen sources of the basal liquid medium for phytase production from P. adiposa under SSF. Water hyacinth was used as a substrate based on the suitable substrate for its phytase production. The results revealed that the highest degree of phytase activity was found in glucose at an average of 23.24 ± 0.37 U/gds, followed by fructose (22.68 ± 0.66 U/gds), maltose (20.07 ± 2.15 U/gds), and starch (19.83 ± 1.17 U/gds) ( Figure 3A). The basal liquid medium containing glucose was replaced with different organic and inorganic nitrogen sources. The highest phytase activity was obtained from ammonium nitrate at 24.48 ± 0.3 U/gds, followed by peptone (17.66 ± 0.63 U/gds), and ammonium sulfate (17.04 ± 1.48 U/gds), whereas the lowest phytase activity was found in malt extract (9.42 ± 0.87 U/gds) ( Figure 3B). Therefore, glucose and ammonium nitrate were selected and used in the basal liquid medium for further experimentation.
J. Fungi 2020, 6, x FOR PEER REVIEW 9 of 22 Figure 2. Phytase production during SSF using different agricultural residues by five selected mushroom strains. Five different substrates were tested but only three substrates were found to display phytase activity. The results are means ± SD. Different letters above each bar in the same parameter indicate the significant difference (p < 0.05).

Figure 2.
Phytase production during SSF using different agricultural residues by five selected mushroom strains. Five different substrates were tested but only three substrates were found to display phytase activity. The results are means ± SD. Different letters above each bar in the same parameter indicate the significant difference (p < 0.05).

Figure 2.
Phytase production during SSF using different agricultural residues by five selected mushroom strains. Five different substrates were tested but only three substrates were found to display phytase activity. The results are means ± SD. Different letters above each bar in the same parameter indicate the significant difference (p < 0.05).

Optimization of Substrate Components
In this experiment, the PBD experimentation included 10 parameters, while 3 levels with a total of 23 experiments were used to select and investigate the optimal level of the variables that influenced the substrate components in phytase production by P. adiposa. The values of Prob > F of less than 0.05 indicated that the model terms were significant. In this study, water hyacinth (p = 0.001) and moisture (p = 0.003) were found to be significant variables that influenced the substrate component for phytase production, whereas other variables indicated no significant effects (Table 4). Therefore, the noninfluential factors involved in the substrate component on phytase production were used at each minimum level. For future experiments, water hyacinth and moisture were used to find the optimal amounts for the highest level of phytase production using the response surface methodology.

Optimization of Substrate Components
In this experiment, the PBD experimentation included 10 parameters, while 3 levels with a total of 23 experiments were used to select and investigate the optimal level of the variables that influenced the substrate components in phytase production by P. adiposa. The values of Prob > F of less than 0.05 indicated that the model terms were significant. In this study, water hyacinth (p = 0.001) and moisture (p = 0.003) were found to be significant variables that influenced the substrate component for phytase production, whereas other variables indicated no significant effects (Table 4). Therefore, the non-influential factors involved in the substrate component on phytase production were used at each minimum level. For future experiments, water hyacinth and moisture were used to find the optimal amounts for the highest level of phytase production using the response surface methodology.

Respond Surface Methodology (RSM)
The RSM experiment was designed to find the optimal levels of water hyacinth and moisture to achieve the highest yield of phytase production obtained from P. adiposa. The final regression equation of the model employing two variables for the evaluation of the highest phytase activity is given in terms of the actual factors in the following model: Phytase activity (U/gds) = −43.14 + 58.26A where A is the quantity of water hyacinth (g) and B is the quantity of moisture content (%). After analysis of variance, the quadratic model for phytase activity was found to be significant at p < 0.0001 with a reliability rating of R 2 = 0.9711, while the lack of fit was found not to be significant ( Table 5). The interactive effect of water hyacinth and moisture content in the 3D plot showed the optimum level and the maximum yield of the enzyme (Figure 4). According to RSM, the maximum predicted amount of phytase was 40.45 U/gds using 2 g of a suitable substrate component and 85% moisture content, and the produced amount of phytase under this condition was 40.45 ± 2.52 U/gds. The activity was increased by approximately 2.36-fold. Thus, this condition was used for further experimentation.  The phytase production obtained from P. adiposa fermented with a suitable substrate component was investigated under different conditions including an initial pH value of basal liquid medium, selected temperature for incubation, and the appropriate fermentation period. The effect of pH variation of the basal liquid medium for phytase production is shown in Figure 5A. It was found that

Determination of pH Value of Basal Liquid Medium, Temperature, and Fermentation Period for Phytase Production of P. adiposa
The phytase production obtained from P. adiposa fermented with a suitable substrate component was investigated under different conditions including an initial pH value of basal liquid medium, selected temperature for incubation, and the appropriate fermentation period. The effect of pH variation of the basal liquid medium for phytase production is shown in Figure 5A. It was found that P. adiposa had the ability to produce phytase at pH values ranging from 4.0 to 9.0. A pH of 6.5 was determined to be the optimal pH value of the basal liquid medium at which this mushroom strain produced the highest amount of phytase.
The results indicated that P. adiposa grew at tested temperatures ranging from 20-35 • C. The statistical analysis of the data revealed that 30 • C was the best temperature for phytase production and that the highest phytase activity was obtained at 51.98 ± 2.90 U/gds ( Figure 5B). However, phytase activity was not observed at 40 and 45 • C due to the fact that this mushroom strain did not grow under that condition.
The effect of fermentation period on phytase production was determined for this mushroom strain. The results showed that the highest amount of phytase activity (53.66 ± 1.68 U/gds) was observed 7 days after incubation at 30 • C and it then slowly decreased ( Figure 5C). Therefore, the optimal condition for phytase production of P. adiposa was observed in the suitable substrate components (water hyacinth and nutrients) supplemented with basal liquid medium (containing glucose as a carbon source and ammonium nitrate as a nitrogen source) pH 6.5, containing 85% moisture content, and at 30 • C for seven days. It was found that the selected statistical approach increased phytase activity to 3.15-fold.

Determination of Optimal Temperature and pH on Phytase Activity
The obtained precipitate extract after precipitation of the crude enzyme by ammonium sulfate was used for the characterization of phytase activity and stability. It was found that temperatures significantly affected phytase activity. The results indicated that the highest phytase activity (1.55 ± 0.07 U/mL) in the precipitated extract was found at 42 °C ( Figure 6A). The activity gradually decreased at a temperature higher than 42 °C. The effect of pH on phytase activity was determined at pH values ranging from 2.0-9.0. The results indicated that pH value affected phytase activity. The highest significant phytase activity (1.93 ± 0.02 U/mL) was found at a pH value of 5.0 ( Figure 6B). Notably, phytase activity was not observed at pH values of 7.0, 8.0, and 9.0.

Determination of Optimal Temperature and pH on Phytase Activity
The obtained precipitate extract after precipitation of the crude enzyme by ammonium sulfate was used for the characterization of phytase activity and stability. It was found that temperatures significantly affected phytase activity. The results indicated that the highest phytase activity (1.55 ± 0.07 U/mL) in the precipitated extract was found at 42 • C ( Figure 6A). The activity gradually decreased at a temperature higher than 42 • C. The effect of pH on phytase activity was determined at pH values ranging from 2.0-9.0. The results indicated that pH value affected phytase activity. The highest significant phytase activity (1.93 ± 0.02 U/mL) was found at a pH value of 5.0 ( Figure 6B). Notably, phytase activity was not observed at pH values of 7.0, 8.0, and 9.0. letters above each bar in the same parameter indicate the significant difference (p < 0.05).

Determination of Optimal Temperature and pH on Phytase Activity
The obtained precipitate extract after precipitation of the crude enzyme by ammonium sulfate was used for the characterization of phytase activity and stability. It was found that temperatures significantly affected phytase activity. The results indicated that the highest phytase activity (1.55 ± 0.07 U/mL) in the precipitated extract was found at 42 °C ( Figure 6A). The activity gradually decreased at a temperature higher than 42 °C. The effect of pH on phytase activity was determined at pH values ranging from 2.0-9.0. The results indicated that pH value affected phytase activity. The highest significant phytase activity (1.93 ± 0.02 U/mL) was found at a pH value of 5.0 ( Figure 6B). Notably, phytase activity was not observed at pH values of 7.0, 8.0, and 9.0.

Effect of Cations and Potential Inhibitors on Phytase Activity
The relative activity of the precipitate extract in different cations and inhibitors is presented in Table 6. It was found that Cu 2+ , Zn 2+ , phosphate, and molybdate strongly decreased phytase activity (relative activity value of less than 50%) at concentrations of both 1 and 5 mM. In addition, Mn 2+ , K + , Ca 2+ , Na 2+ , Co 2+ , Na + , EDTA, tartrate, and citrate exhibited an inhibition effect on phytase activity. However, stimulation of the relative activity of the enzyme was found at concentrations of both 1 and 5 mM of Fe 2+ .

Determination of pH and Temperature Stability
The effect of pH stability on the precipitate extract of P. adiposa phytase was determined in pH values ranging from 2.0-12 at 4 • C for 12 and 24 h. The relative activities of the precipitate extract at different pH values are shown in Figure 7A. It was found that the pH value affected phytase activity. The results indicate that the precipitate extract displayed the highest stability at a pH value of 8.0 followed by pH values of 7.0, and 6.0, respectively. The pH values of 2.0 and 9.0 showed a strong decrease in enzyme activity. Notably, the pH values of 11.0 and 12.0 were not observed to have influenced phytase activity.
The temperature stability of the phytase enzyme in the precipitate extract was performed at three different temperatures, namely room temperature (26 ± 2 • C), cooling temperature (4 • C), and freezing temperature (−20 • C), during 50 days of incubation. The results indicated that the relative activity decreased when the length of the incubation period increased ( Figure 7B). The degree of relative activity of less than 50% at room temperature, cool temperature, and freezing temperature was observed after incubation for 12, 18, and 14 days, respectively. The results revealed that enzyme activity was lost after incubation at room temperature, freezing temperature, and cool temperature at 24, 32, and 42 days, respectively.

Discussion
The bioprocess for phytase production by fungi, especially within the genera Aspergillus, Mucor, Penicillium, Rhizopus, and Thermomyces, using different agricultural residues had been previously studied and reported on by a number of researchers [20,21,23,24,[32][33][34][35][36][37]. Nevertheless, only a limited number of published reports are available with regard to the production of phytase obtained from mushroom cultures using agricultural residue through SSF. This present study found that five selected mushrooms strains, namely Am. rugosum, G. mastoporum, Marasmius sp1., P. adiposa, and Pi. triqueter, effectively produced phytase in cultures on PSA and agricultural residues under SSF. Specifically, the amount of phytase production that was obtained ranged from 1.47 to 17.02 U/gds. Our results indicate that the chestnut mushroom, P. adiposa, possessed the greatest phytase producing strains, while water hyacinth was found to be the most suitable substrate for phytase production. These results are similar to those of previous studies [18,38,39] which reported that pure cultures of mushrooms could produce phytase after being cultured with agricultural residues under SSF. Notably, the efficiency of phytase production varied among different mushroom species and strains and was dependent upon the type of agricultural residues being used [18,19,[38][39][40][41]. A study conducted by Spier et al. [19] found that the phytase production of S. commune DRL−01, T. versicolor DRL−18, G. applanatum DRL−56, and G. stipitatum DRL−70 on wheat bran under SSF ranged from 9.15

Discussion
The bioprocess for phytase production by fungi, especially within the genera Aspergillus, Mucor, Penicillium, Rhizopus, and Thermomyces, using different agricultural residues had been previously studied and reported on by a number of researchers [20,21,23,24,[32][33][34][35][36][37]. Nevertheless, only a limited number of published reports are available with regard to the production of phytase obtained from mushroom cultures using agricultural residue through SSF. This present study found that five selected mushrooms strains, namely Am. rugosum, G. mastoporum, Marasmius sp1., P. adiposa, and Pi. triqueter, effectively produced phytase in cultures on PSA and agricultural residues under SSF. Specifically, the amount of phytase production that was obtained ranged from 1.47 to 17.02 U/gds. Our results indicate that the chestnut mushroom, P. adiposa, possessed the greatest phytase producing strains, while water hyacinth was found to be the most suitable substrate for phytase production. These results are similar to those of previous studies [18,38,39] which reported that pure cultures of mushrooms could produce phytase after being cultured with agricultural residues under SSF. Notably, the efficiency of phytase production varied among different mushroom species and strains and was dependent upon the type of agricultural residues being used [18,19,[38][39][40][41]. A study conducted by Spier et al. [19] found that the phytase production of S. commune DRL−01, T. versicolor DRL−18, G. applanatum DRL−56, and G. stipitatum DRL−70 on wheat bran under SSF ranged from 9.15 to 192.42 U/gds. Salmon et al. [18] found that S. commune LPB 101 produced phytase at 113.7 U/gds by SSF using wheat bran. Our findings represent the first report on phytase production by Am. rugosum, G. mastoporum, P. adiposa, and Pi. triqueter. Moreover, certain previous studies found that some filamentous fungi in the genera Aspergillus, Mucor, Neurospora, Pennicillium, Humicola, and Rhizopus produced phytase in a range from 16 to 200 U/gds using different agricultural residues under SSF [19][20][21]23,24,42].
The supplementation of different carbon and nitrogen sources had a significant effect on the phytase production of P. adiposa under SSF. Our results revealed that glucose and ammonium nitrate can improve the phytase production of P. adiposa using water hyacinth. This result was supported by the findings of several previous studies which found that the phytase production of fungi can be influenced by the supplementation of various carbon and nitrogen sources. Our results are similar to those of previous studies [43][44][45][46] which found that glucose and ammonium nitrate supported phytase production under SSF. However, the suitable carbon and nitrogen sources for optimum phytase production were dependent upon both fungal species and strain. Awad et al. [37] found that the highest phytase productivity of Pen. purpurogenum GE1 on mixed corn cob and corn bran as substrates was obtained when the specimen was supplemented with glucose and peptone as carbon and nitrogen sources, respectively. Salmon et al. [18] and Gunashree and Venkateswaran [47] studied the effects of different carbon sources for phytase production by S. commune LPB 101 and A. niger CFR 335 on wheat bran and found that the maximal phytase activity was obtained when using sucrose as a carbon source and yeast extract as a nitrogen source. Furthermore, glucose and ammonium sulfate were indicated as suitable carbon and nitrogen sources for the phytase production of A. ficuum PTCC 5288 [24], A. tubingensis SKA [43], and Sporotrichum thermophile TLR50 [48] through SSF using wheat straw, wheat bran, and sesame oil cakes as substrates, respectively. While Bala et al. [42] found that SSF using wheat bran and sucrose as carbon sources, and ammonium sulfate as a nitrogen source, gave the highest yield of phytase production from H. nigrescens BJ82.
A number of factors such as the agricultural residue, nutrient content, the amount of inoculum, and the moisture content can significantly influence the substrate components in the production of fungal enzymes during fermentation [49][50][51][52]. Therefore, the application of various statistical experimental methods by PBD has been used to determine an optimal substrate in order to increase the efficiency of the production of various enzymes by fungi in several previous studies [53][54][55]. According to the statistical results of PDB, the substrate (water hyacinth) and moisture content used in this study were determined to be the primary significant factors on the substrate of phytase production by P. adiposa through SSF. This result is similar to previous studies that reported that both substrates and moisture were significant influential factors on the substrate for phytase production by Aspergillus species after application of PBD [56][57][58]. Notably, other factors, such as KH 2 PO 4 , (NH 4 ) 2 SO 4 , MgSO 4 , FeSO 4 , yeast extract, casein, Tween 80, and glucose, were also found to be influential factors on substrate composition for the phytase production of fungi through SSF using agricultural residues [59][60][61][62][63]. Consequently, the optimal conditions for the phytase production of P. adiposa were estimated by CCD using response surface methodology (RSM). Subsequently, the phytase enzyme production increased by 2.36-fold. This finding is supported by the findings of previous studies that indicated that RSM could significantly increase the phytase production of several fungi species under SSF using different agricultural residues [34,64]. For example, Salmon et al. [18] found that the optimal conditions obtained from RSM could significantly increase phytase production in S. commune LPB101 using wheat bran as a substrate at an increasing value of 2.85-fold. Phytase production by A. ficuum NRRL 3135 and Ganoderma sp. MR−56 in wheat bran increased by 1.67-fold and 5.8-fold, respectively after using RSM when compared to non-optimal conditions [39,63]. Spier et al. [58] found that RSM could improve the phytase production of A. niger FS3 in citric pulp by increasing enzyme production by 4.3-fold. A study conducted by Wang et al. [57] on phytase production from A. ficuum NTG−23 in waste vinegar residues on RSM increased the degree of enzyme production by 7.34-fold. Furthermore, an improved degree of phytase production by RSM was also found in M. racemosus NRRL 1994 [62] and Sp. thermophile BJTLR50 [59] at increasing values of 1.85-fold and 11.6-fold, respectively.
In this study, the optimum pH value of the supplemented basal liquid medium, temperature, and fermentation period for the highest yield of phytase production from P. adiposa were obtained after 7 days of fermentation in culture supplemented with basal liquid medium at a pH value of 6.5 and at 30 • C. This result is supported by the findings of several previous studies that found that phytase production yields were greatly influenced by fermentation conditions (pH value of basal liquid medium, temperature, fermentation period, mineral, aeration, and type of fermentation), while the optimum conditions for phytase production were not found to be homologous for fungal species and strain [65]. The optimum conditions for phytase production from S. commune LPB 101 were achieved at three days of fermentation using wheat bran basal liquid medium at a pH value of 7.0 after being incubated at 33 • C [18]. Moreira et al. [66] reported on the effective phytase production of A. japonicus URM 5633 in wheat bran using basal liquid medium at a pH value of 6.0 after being incubated at 30 • C for three days. Qasim et al. [41] found that the SSF of A. tubingensis SKA in wheat bran supplemented with basal liquid medium at a pH value of 5.0 after being incubated at 30 • C for 4 days resulted in the highest yield of phytase production. Additionally, the yield of phytase production by Ganoderma sp. MR−56 was found at four days in wheat bran using basal liquid medium at a pH value of 6.0 and an incubation temperature of 30 • C by submerged fermentation (SmF) [39]. Haritha and Sambasivarao [67] found that the maximum yield of phytase production by R. oligosporus MTCC556 was obtained in wheat bran supplemented with basal liquid medium at a pH value of 6.0 after incubation at 30 • C for eight days. Additionally, several previous studies have suggested that the optimal substrate component and physical conditions for phytase fermentation could significantly improve the yields of phytase production on an industrial scale [37,41,68].
Ammonium sulfate precipitation is the method that is the most widely used for the partial purification of phytase from microbial sources [26,69]. Temperature is one of the external factors that plays a significant role in microbial phytase activity. In the present study, the optimal phytase activity of the precipitate extract obtained from P. adiposa was achieved at 42 • C. Our findings agree with the results of a number of previous studies which found that the optimal temperature for phytase activity from filamentous fungi occurred within temperatures ranging from 33-50 • C depending upon the fungal species as well as the fungal strain [26,45,47,[70][71][72]. However, some previous studies have reported that the optimal temperature for the phytase activity of several Aspergillus species, Ceriporia sp. CBS 100231, Mucor hiemalis, Peniophora lycii CBS 686.96, and Penicillium simplicissimum W46 was 55 • C [35,[73][74][75][76]. Studies on phytase activity involving A. niger ATCC 9142 [77] and A. ficuum NTG−23 [78] indicated that the optimal temperatures were 65 and 67 • C, respectively. Additionally, the phytases of the thermophilic fungi, Sp. thermophile and [48] Rhizopus microsporus var. microsporus [79], and R. pusillus [80] were optimally active at temperatures of 60, 65, and 70 • C, respectively. Furthermore, pH value had a significant effect on the phytase activity of P. adiposa. Its phytase activity was achieved at pH values ranging from 2.0 to 6.0, of which 5.0 proved to be the optimal pH value. This result was in accordance with the findings of several previous studies that had reported that the optimal pH value for phytase activity for most fungi was in the acidic range (4.0-5.5). Sanni et al. [70] reported that the optimal phytase activity from A. fumigatus was obtained at a pH value of 6.0. Nevertheless, pH values of 7.0 (neutral condition) and 9.5 (basic condition) were found to be optimally active for phytase obtained from A. flavus ITCC 6720 [71] and R. microsporus var. microsporus [79], respectively.
Effect of metal ions and inhibitors on fungal phytase activity was investigated [22,35,81]. In the present study, the activity of phytase from P. adiposa decreased in the presence of Ca 2+ , Co 2+ , Cu 2+ , K + , Mg 2+ , Mn 2+ , Na + , and Zn 2+ at concentrations of 1 and 5 mM and increased in the presence of Fe 2+ . Studies on the effect of metal ions on phytase activity from Malbranchea sulfurea [82] and Thermomyces lanuginosus IMI 096218 [22] reported that Fe 2+ at a concentration of 1 mM increased enzyme activity. Previous reports indicated that metal ions and inhibitors affected fungal phytase activity depending upon the specific type and concentration of the metal ion and inhibitor, as well as the type of fungal phytase [35,83,84]. For example, Soni et al. [83] found that the addition of Hg 2+ and Cu 2+ to phytase from A. niger NCIM 563 decreased the degree of enzyme activity, whereas Mg 2+ increased the level of enzyme activity. The activity of partially purified phytase from A. niveus was reported to have strongly decreased in 1 mM Mg 2+ , Mn 2+ , Hg 2+ , Cd 2+ , Zn 2+ , and Fe 2+ , but the activity was enhanced by the presence of 1 mM Fe 3+ [82]. Zn 2+ and Cu 2+ at concentrations of 2 mM and inhibited phytase activity from A. japonicas BCC18313 and A. niger BCC18018 [85]. Phytase activity from A. fumigatus PPF−6 was decreased by the addition of 1mM Ca 2+ , Co 2+ , Cu 2+ , Mg 2+ , and Na + was strongly decreased by the addition of 1 mM Ag 2+ , Hg 2+ , Zn 2+ , and Mn 2+ [86]. The phytase activity from A. niger 7A−1 was inhibited by Ca 2+ [35]. Salmon et al. [18] investigated the effect of metal ions on phytase production by S. commune and reported that Ca 2+ , Co 2+ , Cu 2+ , Fe 2+ , Fe 3+ , K + , Mg 2+ , Mn 2+ , Ni 2+ , and Zn 2+ at concentrations of 1 mM increased enzyme activity. Our results indicate that EDTA, tartrate, and citrate decreased the activity of phytase from P. adiposa, while molybdate significantly inhibited enzyme activity. This result was in accordance with published findings that revealed that the phytase activity from S. commune LPB101 and R. oligosporus DSMZ 1964 were decreased by the presence of molybdate [18,87]. Notably, a study conducted by Azeke et al. [87] and Vats and Banerjee [73] on phytase activity from Aspergillus species found that fluoride strongly inhibited enzyme activity. In contrast, 1 mM EDTA has been reported to have increased the activity of partially purified phytase obtained from several strains of A. niger [73,83].
This study found that the degree of pH stability of the precipitated extract obtained from P. adiposa was highly stable within a pH range of 6.0-8.0 and at the low temperature of 4 • C. This result was supported by several previous studies that reported the stability of phytase produced from fungi was influenced by certain conditions such pH and temperature and was dependent upon the fungal species [18,66,88]. Salmon et al. [18] reported that phytase produced from S. commune was stable at pH values in a range of 5.0 to 6.0 and at 4 • C. A study conducted by Karaman et al. [88] on the pH stability of phytase obtained from A. niger found that it was highly stable at a range of pH values of 5.0 to 6.0. The phytase production from Monascus sp., Neurospora crassa CICIM F00021, and Thermomyces lanuginosus IMI 096218 was highly stable at pH values ranging from 5.0 to 7.0, 6.0 to 7.0, 5.0 to 7.0, and 6.0 to 6.5, respectively. Moreira et al. [66] found that phytase produced by A. japonicus URM 5633 could be stable in a pH range of 3.0 to 8.0. Notably, studies on the stability of phytase produced by Aspergillus sp. L117, G. applanatum DRL−56, G. stipitatum DRL−70, S. commune DRL−01, S. commune LPB101, and T. versicolor DRL−18 found them to be stable at 4 • C. The stability of fungal phytase toward different processing conditions, pH values, and temperatures make up the major factors affecting the section and application of fungal phytase [89,90].

Conclusions
Fungi as a source of phytase production can be a candidate for beneficial applications in biotechnological processes that would facilitate the reduction and valorization of agricultural residues. This would be achieved as a result of the eco-friendly conversion of low-value by-products into potentially beneficial resources and value-added products. In this study, five selected mushroom strains, namely P. adiposa, G. mastoporum, Pi. triqueter, Am. rugosum, and Marasmius sp.1, produced phytase in various agricultural residues under SSF. Our study obtained high yields of phytase production from P. adiposa using water hyacinth under SSF. The optimization for its phytase production was determined by various statistical approaches. The highest phytase yield was observed in the suitable substrate component of water hyacinth containing 85% moisture content along with the addition of a suitable basal liquid medium at a pH value of 6.5 after being incubated at 30 • C for seven days. Enzyme extracts revealed the highest activity at 42 • C and a pH of value of 5.0. Moreover, Fe 2+ enhanced phytase activity and the precipitated extract displayed the stability at a pH value of 8.0 and a temperature of 4 • C. Further studies on phytase obtained from this mushroom are required to fully process and evaluate its purification and kinetic potential. Additionally, phytase production from selected agricultural residues should be conducted on a larger scale to confirm their potential use in various industries like those associated with the production of food additives and animal feeds, as well as for use in the medical field.