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

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.

Enhanced phytase production by Aspergillus niger mutants in solid state fermentation

The present research work was conducted to improve the phytase production by genetic alteration of Aspergillus niger with induced mutagenesis using solid state fermentation. Strain improvement was carried out in the presence of ultra violet (UV) irradiation and ethylmethane sulphonate (EMS) [0.5% v/v] treatments for various time intervals. We reported an improved strain of Aspergillus niger designated as UV-3 mutant producing a zone of hydrolysis of about 40 mm, in comparison to wild strain (26 mm). The highest enzyme activity was found to be 547.64 IU/g for UV-3 mutant followed by EMS-4 mutant (492.23 IU/g)compared to wild strain which showed 406.45 IU/g of enzyme activity. There was 1.35 fold increase in phytase production after mutation studies of Aspergillus niger. Phytase was applied as poultry feed additive and given to broiler chickens for 5 weeks. The results exhibited that there was increase in body weight gain (BWG) of chicks for experimental group (2028 g) in comparison to control group (1903 g). Thus, physical and chemical mutagenesis was proved as an effective technique for the improvement of strain and ultimately for enhanced and economical phytase production for different industrial applications.

Production of Phytase by three thermophilic fungi

Production of phytase by three thermophilic fungi, Thermomyces lanuginosus, Talaromyces luteus and Rhizomucor pusillus under different cultural conditions was assessed. Temperature of 45°C, pH-6.0 were optimum for phytase production by the all three fungi under investigation . Carbon and nitrogen sources for production of phytases by the three thermophilic fungi varied with the fungus. When T. lanuginosus opted for D-glucose followed by D-fructose, T. luteus preferred D-glucose, D-mannose and mannitol for production a phytase. On the other hand, R. pusillus produced maximum phytase during its growth on mannitol and maltose as carbon source. L- asparagine, L- arginine and L-asparatic acid were preferred nitrogen sources for production of phytase by T. lanuginosus. On the other hand T. luteus, opted for L- asparagine, L-glutamic acid and L- glycine for the activity of phytase. R. pusillus produced maximum phytase in medium containing L-argine, L-asparagine and L- asparatic acid.

Effects of Nitrogen Sources in Phytase Production on Bacterial Strains Isolated from Malaysia’s Hot Spring

Efficient strategies for phytase production gained increasing importance as more applications require high amounts of phytase for the market. Four phytase-producing bacterial strains isolated from Malaysia’s hot springs were used in this study to determine the effect of nitrogen sources on phytase production. All of the strains were screened out by applying halozone method which shows all of the strains were definitely positive phytase producer. Phytase Screening Medium (PSM) with soybean extract as substrate was used as a cultivation medium. Optimised condition with 1.0 % (w/v) of glucose (as carbon source), pH 5.5 and 37°C temperature was applied. Yeast extract and peptone were used to identify optimum nitrogen source in maximum phytase production. Quantitative analysis observed were optical density, colony forming unit, pH values and phytase activity to identify the effect of nitrogen source in phytase production. The finding was bacterial strain L3 as the best producer in producing maximum phytase (0.2162 U/mL) with optimised condition using yeast extract as nitrogen source. Findings in this study proved that yeast extract act as the optimum nitrogen source which contribute to maximum phytase production as supported by previous studies. This study can provide an efficient strategy to produce maximum phytase as few studies stated that phytase is an application tool in functional food production that consists of myo-inositol phosphates that is believed to have important pharmacological effects.

Effect of Carbon Sources on Different Strains of Phytase-Producing Bacteria Isolated from Malaysia’s Hot Spring

Animal feed from cereal grains and oilseed meals mainly containing phytic acid which has adverse effects on animal nutrition and its environment. Ruminants can easily digest the phytic acid as they have fungi and bacteria in their guts which can produce phytase to degrade the phytic acid. Meanwhile, phytic acid in non-ruminant animals is poorly digested due to the lack of sufficient phytase in their guts. Thus, the feed must be supplemented with inorganic phosphate to ensure it can absorb adequate nutrients. This study aimed to determine the effects of using different carbon sources to the growth of different strains of phytase producing bacteria based on optical density (OD), colony forming unit (CFU), and their phytase production. All four strains of potentially producing-phytase bacteria have been isolated from several hot springs in Malaysia. The bacteria were grown in modified Phytase Screening Medium (PSM) with glucose and lactose as a carbon source and under optimum culture conditions (pH 5.5, 37˚C, 200 rpm) for 72 hours. For quantitative screening of phytase production, the bacterial cultures were harvested to obtain the supernatants that were used to measure the amount of inorganic phosphorus released by the bacterial strains. Among these carbon sources, glucose has shown consistency between their CFU counts and the observed ODs whereas lactose shown inconsistency. Meanwhile, the maximum phytase activity was recorded for all strains in the presence of glucose in which bacteria strain L3 (0.0404 U/mL), RT (0.0359 U/mL), B9 (0.0262 U/mL), and A (0.0263 U/mL). As for the overall, strain L3 (Labis, Johor) gave a promising rate of inorganic phosphate released with optimum phytase activity value of 0.0404 U/mL in presence of glucose and lactose. The optimisation of the fermentation medium can contribute to more economical production of industrial enzyme as phytase has the potential to produce feed additives for poultry feeding.

Statistical Optimization, Partial Purification, and Characterization of Phytase Produced from Talaromyces purpureogenus NSA20 Using Potato Peel Waste and its Application in Dyes De-colorization

In this study, Talaromyces purpureogenus NSA20 as phytase-producing marine fungus was isolated and identified morphologically and genetically and deposited in Gene Bank with accession number MW031769.1. One factor at a time (OFAT) optimization was performed, where the result revealed that potato peel waste (1.5%) as a substrate was the highest for phytase production at 6 days, where the maximum activity of phytase was 138.4 U/ml. Moreover, Box–Behnken design as response surface methodology was carried out for statistical optimization of phytase production by T. purpureogenus NSA20. Statistical optimization illustrated that the optimized medium for phytase production increased 1.57 fold compared to the OFAT optimized medium. Partial purification of phytase was carried out, where the enzyme after precipitation with ammonium sulfate (80%) was 2.6-fold purified phytase, and the yield was 39.8 %., the specific activity was 31.19 U/mg proteins. Additionally, partially purified phytase was characterized; the maximum activity of phytase at Fe++ 0.1% and pH 5.5 at 37 oC was 350 U/ml. Eventually, phytase was applied for crystal violet and methyl red decolorization, where decolorization percentages of crystal violet and methyl red were 85.5% and 75% at 120 min, respectively.

Enhanced Phytase Production by Bacillus subtilis subsp. subtilis in Solid State Fermentation and its Utility in Improving Food Nutrition

Background: Phytic acid acts as anti-nutritional factor in food and feed ingredients for monogastric animals as they lack phytases. Objective: Phytase production by Bacillus subtilis subsp. subtilis JJBS250 was studied in solid state fermentation and its applicability in dephytinization of food Methods: Bacterial culture was grown in solid state fermentation using wheat bran and various culture conditions were optimized using ‘One variable at a time’ (OVAT) approach. Effects of different substrates (wheat bran, wheat straw, sugarcane bagasse), incubation time (24, 48, 72 and 96 h), incubation temperatures (25, 30, 35 and 40 oC), pH (4.0, 5.0, 6.0, 7.0 and 8.0) and moisture content (1:1.5, 1:2.0, 1:2.5 and 1:3) were studied on phytase production. Bacterial phytase was used in dephytinization of food samples. Results: Optimization of phytase production was studied in solid state fermentation (SSF) using ‘One variable at a time’ (OVAT) approach. Bacillus subtilis subsp. subtilis JJBS250 grew well in various agroresidues in SSF and secreted high enzyme titres using wheat bran at 30 oC and pH 5.0 after incubation time of 48 h with substrate to moisture ratio of 1:3. Glucose and ammonium sulphate supplementation to wheat bran further enhanced phytase production in SSF. Optimization of phytase production resulted in 2.4-fold improvement in phytase production in solid state fermentation. The enzyme resulted in dephytinization of wheat and rice flours with concomitant release of inorganic phosphate, reducing sugar and soluble protein. Conclusion: Optimization resulted in 2.34-fold enhancement in phytase production by bacterial culture that showed dephytinization of food ingredients with concomitant release of nutritional components. Therefore, phytase of B. subtilis subsp. subtilis JJBS250 could find application in improving nutritional quality of food and feed of monogastric animals.