Harnessing the Phytase Production Potential of Soil-Borne Fungi from Wastewater Irrigated Fields Based on Eco-Cultural Optimization under Shake Flask Method

Naila Sadaf,Aishah Alatawi,Naeem Iqbal,Muyassar H Abualreesh,Muhammad Zulqurnain Haider

doi:10.3390/agriculture12010103

Abstract

Indigenous fungi present in agricultural soils could have synchronized their inherent potentials to the local climatic conditions. Therefore, the fungi resident in the untreated wastewater irrigated agricultural field might develop their potential for producing various enzymes to handle the induced full organic load from domestic wastewater and toxic chemicals from the textile industry. Around 53 various fungal isolates were grown and separated from the soil samples from these sites through soil dilution, soil-culture plate, and soil-culture plate methods. All the purified fungi were subjected to a phosphatase production test, and only 13 fungal strains were selected as phosphatase producers. Among them, only five fungi identified as Aspergillus niger, Aspergillus flavus, Aspergillus fumigatus, Penicillium purourogenum, and Mucor rouxii based on morphological similarities, showing higher phosphate solubilizing indices, were utilized for eco-cultural fine-tuning to harness their full production potential under shake flask (SF) method. Among various media, orchestral tuning, 200 µM sodium phytate as substrate with 1.5 mL of inoculum size of the fungi, pH 7, temperature 30 °C, glucose, and ammonium nitrate as carbon and nitrogen additive with seven days of incubation were found to be the most appropriate cultural conditions to harness the phytase production potential of the selected fungi. Aspergillus niger and Aspergillus flavus showed initial phytase activity (5.2 Units/mL, 4.8 Units/mL) and phytase specific activity (2.85, 2.65 Units/mL per mg protein) during screening to be enhanced up to 17 ± 0.033 (Units/mL), 16 ± 0.033 (Units/mL) and (13 ± 0.012), 10 ± 0.066 (Units/mL per mg protein), respectively, with the above-mentioned conditions. The phytase enzyme produced from these fungi were found to be almost stable for a wide range of pH (4–8); temperature (20–60 °C); insensitive to Ca2+ and Mg2+ ions, and EDTA, Ni2+, and Ba2+ inhibitors but highly sensitive to Mn2+, Cu2+, and Zn2+ ions, and Co2+, Cr3+, Al3+, Fe2+ and Ag1+ inhibitors. It was suggested that both phytase-producing strains of A. niger and A. flavus or their crude phytase enzymes might be good candidates for application in soils to release phosphates from phytate and a possible valuable substitute of phosphate fertilizers.

Highlights

Phytases are an enzyme that hydrolyze phytic acid into two sub group myo-inositol, Hexakisphosphate, or phytate
All purified fungal isolates were plated on the Pikovskaya agar medium (PKV) supplemented with 0.5% tri-calcium phosphate (TCP) to estimate their phosphate solubilizing potential
The results showed that phytase activity and specific phytase activity by A. niger and A. flavus were highest at pH 7, representing its highest stability (Figure 5a,b)

Summary

Introduction

Phytases are an enzyme that hydrolyze phytic acid into two sub group myo-inositol, Hexakisphosphate, or phytate (salt form). Phytate is a primary storage form of phosphorus in plant tissues, such as cereal grains, pollen, and oilseed; these are sources of phosphorus and are known as macro elements for both plants and animals [1,2,3]. Phytic acid/phytate is the predominant/primary form of phosphorus (60–90%) present in plants [4] and is not available to plants for direct utilization. Phosphate supplementation is required for optimum growth of plants in the form of fertilizers. It is considered as an antinutrient responsible for phosphorus pollution present in animal manure [5,6]. Plants, along with monogastric animals, could not utilize phytate–phosphorus, enhancing the utilization of phytase as a supplement would be useful to tap this important phosphorus source [9]

Methods

Results

Conclusion