The effects of nutrients and physical conditions on phytase production were investigated with a recently isolated strain of Aspergillus tubingensis SKA under solid state fermentation on wheat bran. The nutrient factors investigated included carbon source, nitrogen source, phosphate source and concentration, metal ions (salts) and the physical parameters investigated included inoculum size, pH, temperature and fermentation duration. Our investigations revealed that optimal productivity of phytase was achieved using wheat bran supplemented with: 1.5% glucose. 0.5% (NH 4) 2SO 4, 0.1% sodium phytate. Additionally, optimal physical conditions were 1 × 10 5 spore/g substrate, initial pH of 5.0, temperature of fermentation 30°C and fermentation duration of 96 h. Overall, a 34% improvement in phytase activity was achieved by using the optimal conditions.
Phytic acid (C6H18O24P6) (myo-inositol hexakisphosphate (IP6), or myo-inositol polyphosphate), also known as phytate when in salt form, is the principle storage form of phosphorus in most plant tissues, and has been found to be a nearly ubiquitous component in cereals, legumes and oilseeds crops, constituting 1% - 5% of their weight [
Phytases (myo-inositol hexakisphosphate phosphohydrolases) are a group of phosphatases which catalyze the hydrolysis of phytate to less phosphorylated myo-inositol derivatives and in some cases to free myo-inositol and inorganic phosphate (Pi) [
Production of enzymes including phytase by solid-state fermentation (SSF) has advanced, due to the advantages of this method of fermentation with respect to economic and practical perspectives. These included: better product recovery, low-technology cultivation equipment, high product concentration and lower plant operation cost [
Improving the production of microbial phytases is achieved either by optimizing nutritional and environmental conditions, or by genetic improvements of the producing isolate [
Equipment sources were: pH meter (Sartorius, USA), water bath and spectrophotometer (Thermo Fisher Scientific, USA), incubator (Memmert, Germany), and shaking incubator (Eppendorf, Germany). Materials sources were: wheat bran (Bob’s Red Mill, USA), beef extract, yeast extract, and Whatman No. 4 filter paper (Fisher Scientific), carbohydrates (Sigma-Aldrich, USA), cornstarch (purchases locally), all other materials and salts were purchased from Sigma-Aldrich (USA), Fisher Scientific (USA) and Bio-Rad (USA).
Isolate Aspergillus tubingensis SKA was isolated according to Qasim et al., 2016. Thereafter, a phenotypic/morphologic and molecular characterization of the isolate was conducted by (Fungus Testing Laboratory/University of Texas- Health Science Center, USA) to accurately classify this organism as a new strain of Aspergillus tubingensis.
A. tubingensis SKA was cultivated in malt extract media slants for 5 days at 30˚C and all the slants were stored at 4˚C for further use. For SSF, A. tubingensis SKA was cultivated in 250 ml Erlenmeyer flasks containing 10 g of wheat bran as the fermentation substrate (support). After the flasks were autoclaved at 121˚C for 15 min, they were cooled before supplementation with (1:1) (w/v) sterilized modified phytase screening broth (PSB) containing 15.0 g/L D-glucose, 3.0 g/L sodium phytate, 5.0 g/L NH4NO3, 0.5 g/L MgSO4∙7H2O, 0.5 g/L KCl, 0.01 g/L FeSO4∙7H2O, 0.01 g/L MnSO4∙4H2O and adjusted to pH 5.5. Flasks were inoculated with the pre-prepared fungal spores at a density of 1 × 106 spore/g and then incubated at 30˚C for 5 days (120 hrs). In order to reach the optimal conditions for the production of the extracellular phytase from the isolate A. tubingensis SKA, the variables that affect the production/growth; carbon, nitrogen, phosphate and metal sources were studied. The physical conditions including pH, temperature, size of inoculum and duration of fermentations were also investigated. In all the conducted experiments, conditions of fermentation were retained as they mentioned above (growth in PSB) at 30˚C for 5 days, except for the studied factor, which varied, according to requirements of the experiment. All experiments were replicated 3 times and analyzed in triplicate. T-tests were performed to test for statistical significance (a = 0.05) using SAS 9.4.
Fermentation was carried out with different carbon sources including, sucrose, maltose, fructose, galactose, cornstarch and lactose at a concentration of 1.5%, replacing the glucose in the PSB. Fermentation was carried out with different nitrogen sources including, (NH4)2SO4, (AmSu) NaNO3, NH4Cl, peptone (Pep), yeast extract (YE) and beef extract (BE) at concentrations of 0.5% replacing the NH4NO3, (AmNi) in the PSB. Fermentation was carried out with different phosphate sources including, KH2PO4, Na2HPO4 replacing the sodium phytate in the PSB. All these sources were used at concentrations of 0.3%, and a control sample with the absence of any source of phosphate was conducted. Fermentation was carried out with different concentrations of sodium phytate (0.05, 0.1, 0.3, 0.5 and 1.0 %). In order to study the effect of metal salts one of these salts (MgSO4∙7H2O, KCl, FeSO4∙7H2O or MnSO4∙4H2O) was eliminated at time while the others were used at the concentrations stated above in PSB. The control was all 4 salts as stated in the PSB.
Fermentation was carried out with different sizes of inoculum, (1 × 102, 1 × 103, 1 × 104, 1 × 105, 1 × 106, and 2 × 106) spore/g substrate with 1 × 106 as the control. Fermentation was carried out at different periods of incubation, (48, 72, 96, 120, 144, 168, and 192 h) with 120 hrs as the control. PSB was prepared at different pH values (2.0, 3.0, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 8.0), using HCl or NaOH with pH 5.5 as the control. Fermentation was carried out at different temperatures, (25˚C, 30˚C, 35˚C, 40˚C, 45˚C) with 30˚C as the control.
Phytase was produced under the optimum conditions: 1.5% glucose, 0.5% (NH4)2SO4, 0.1% sodium phytate, 0.5% MgSO4∙7H2O, 0.5% KCl, 0.1% MnSO4∙4H2O, with an incubation temperature of 30˚C, at pH 5.0, with an inoculum size of 1 × 105 spore/g substrate and an incubation time of 96 h (4 days). These conditions were compared with the fermentation conditions using PSB as described above for 120 hrs (5 days) at pH 5.5, at 30˚C with 1 × 106 spore/g substrate.
Crude enzyme (phytase) was collected at the end of incubation period by the addition of 50 ml acetate buffer (0.2 M, pH 5.5) to the samples in the 250 ml Erlenmeyer flasks. The flasks were shaken in a rotary shaker at 200 rpm for 1 h at room temperature. The mixture was separated from solid biomass by filtration through filter paper (Whatman No. 4), then the filtrate was centrifuged at 10,000 rpm for 10 min at 4˚C. The supernatant was used as the source of crude enzyme for phytase assay.
Phytase activity was assayed by determining by measuring the amount of liberated inorganic phosphate as described by Awad et al. [
A. tubingensis SKA was isolated as described by Qasim et al. [
A series of experiments were conducted to examine the best nutrient sources for the production of phytase from A. tubingensis SKA under SSF. Wheat bran was chosen as a substrate for the production since it has a high nutritional value (a rich source of carbon, protein and other nutrients) and low cost. For such characteristics, wheat bran has aroused the attention of researchers over years as
an acceptable nutrient source for use in the production of microbial enzymes via SSF [
Optimum conditions for the production of phytase from A. tubingensis SKA were studied and for the effect of carbon sources, six carbon sources were added at concentrations of 1.5% to the fermentation medium (
Glucose is considered a simple carbon source commonly utilized by microorganisms, leading to an increase in the fungal biomass with a high yield of phytase [
For the optimum source of nitrogen, 0.5% of various sources of nitrogen were used in the fermentation medium (
yielded significantly higher phytase activity than the use of NH4Cl (ammonium chloride). The phytase activity with (NH4)2SO4 is similar to the findings of Santos [
Fungi need a phosphorus source for their growth and production of metabolites. Inorganic sources include phosphate salts while an organic source is phytate. Among the various phosphate sources used in this study (
Phosphorus acts as a regulator for phytase production, therefore the type and concentration of the phosphate in the medium is one of the important factors in the production of microbial phytases [
The above results are similar to those of Lata et al. [
For the effect of metal salts, four metal salts were added to the fermentation medium and given as the control in
Physical parameters for the production of phytase were investigated. For the effect of inoculum size (
production is reduced due to the competition among the fungal population for the nutrients in the growth medium, such as carbon and nitrogen sources, which lead to exhaustion of nutrients resulting in a decrease in enzyme activity. On the other hand, low levels of the inoculum resulted in low phytase activity due to the small number of fungal cells [
In order to determine the optimum incubation period for the maximal phytase production by SSF, isolate A. tubingensis SKA was incubated at various times (
The effect of different pH values of the SSF on phytase activity is shown (
A specific trend can be seen for the influence of incubation temperature on phytase activity (
We then compared phytase activity from A. tubingensis SKA under optimal conditions. The changes to the PSB buffer were the use of ammonium sulfate in place of ammonium nitrate and the use of sodium phytate at 0.3%. The physical parameter changes included using 1 × 105 spores/gram substrate and an incubation time of 4 days (96 hrs). Overall, a 34% improvement in phytase activity was achieved by using the optimal conditions, with an enzymatic activity value that reached 60.42 unit/ml, compared to 45.02 unit/ml activity under original conditions.
Optimizing the conditions which affected phytase production has been the goal of many studies. Rani et al. [
The ultimate aim for optimization is to create an idealistic condition for the production of an enzyme by using straightforward procedures and cost-effective materials that would be feasible to implement on a large scale. To improve any bioprocess it is very important to optimize all the parameters that affect the production of any bio products. Here we showed that we could increase the phytase activity from A. tubingensis SKA by 34% by determining the optimum nutrient and physical conditions.
This project was partially supported by the Utah State University Utah Agricultural Experiment Station and approved as journal paper number 9015.
Qasim, S.S., Shakir, K.A., Al-Shaibani, A.B. and Walsh, M.K. (2017) Optimization of Culture Conditions to Produce Phytase from Aspergillus tubingensis SKA. Food and Nutrition Sciences, 8, 733-745. https://doi.org/10.4236/fns.2017.87052