Use of Plackett-Burman design for enhanced phytase production by Williopsis saturnus NCIM 3298 for applications in animal feed and ethanol production.

Distiller-dried grain solid (DDGS), a co-product of alcohol production, contains cereal grain residues, proteins, and yeast metabolites, which make it suitable in poultry feeding. However, high phytate content of DDGS limits its applicability in poultry feed. In this study, Plackett-Burman design was used to improve cell-bound phytase production by Williopsis saturnus NCIM 3298, and we achieved an enzyme activity of 269 IU/g of dry-wet biomass. The effect of this enhanced phytase-displaying yeast strain on hydrolysis of corn phytate and subsequently on ethanol production and DDGS quality was then investigated. Results of saccharification in the presence of phytase showed that reducing sugar content of liquefied mash increased by 11%, which subsequently improved the ethanol production by 18% (w/v) (p < 0.01) compared with the control. Notably, phytase treatment decreased the phytate content of corn by 70% (p < 0.01) compared with the control, thereby improving the availability of free phosphate in fermentation broth and DDGS. Thus, the results obtained suggest that the addition of W. saturnus NCIM 3298 strain has the potential of providing a new source of phytase that would be useful in the feed and ethanol industries.

Dephytinizing and Probiotic Potentials of Saccharomyces cerevisiae (NCIM 3662) Strain for Amelioration of Nutritional Quality of Functional Foods.

Increase of undigested complexes of phytic acid in food is gaining serious attention to overcome nutritional challenges due to chelation effects. We investigated soil-borne yeast phytase from Saccharomyces cerevisiae (NCIM 3662) for dephytinization of foods, probiotic properties, and process development. The strain produced 45 IU/DCG by cell-bound phytase in an unoptimized medium was increased fourfold (164 IU/DCG) in 12 h using statistical media optimization. The process was scaled-up up to 10-L fermenter scale with increased phytase productivity of 6.4 IU/DCG/h as compared to the lab scale. The strain displayed probiotic characteristics like tolerance to artificial gastric acid conditions, hydrophobicity, autoaggregation, coaggregation, and bile salt hydrolase (BSH) activity. Further, it could dephytinize (removal of phytic acid; an anti-nutritional factor) functional foods like ragi (finger millet) flour, soya flour, chickpea flour, and poultry animal feed. A combination of cell-bound dephytinizing phytase and nutrition-ameliorating probiotic traits of S. cerevisiae (NCIM 3662) presents profound applications in food technology sector.

Characterization of mineral phosphate solubilizing and plant growth promoting bacteria from termite soil of arid region.

Five highly efficient phosphate solubilizing bacteria, viz., Pantoea sp. A3, Pantoea sp. A34, Kosakonia sp. A37, Kosakonia sp. B7 and Bacillus sp. AH9 were isolated from termitorial soils of Sanjivani island of southern Maharashtra, India. These isolates were characterized and explored for phosphate solubilization and plant growth promotion. Among these, Bacillus sp. AH9 showed highest phosphate solubilization index (3.5) and solubilization efficiency (250%) on Pikovskaya agar. Interestingly, Pantoea sp. A34 displayed maximum mineral phosphate solubilization (1072.35 mg/L) in liquid medium and during this period the pH dropped to 3.13. All five isolates had highest P solubilization at 48 h after inoculation. During mineral phosphate solubilization, both gluconic acid and 2-keto gluconic acid were produced by Kosakonia and Bacillus isolates, while only 2-keto gluconic acid was detected in Pantoea isolates. Highest organic acid (39.07 ± 0.04 g/L) production was envisaged in Bacillus sp. AH9, while Pantoea sp. A34 produced the least amount (13.00 ± 0.01 g/L) of organic acid. Seed bacterization with Pantoea sp. A3 and Kosakonia sp. A37 resulted in ~ 37% and ~ 53% increase in root length of tomato seedlings, respectively, while Pantoea sp. A34 and Kosakonia sp. B7 had deleterious effects on root length as well as overall growth of the seedlings. To our knowledge, this is the first report of plant growth promoting potential of microorganisms isolated from termitorial soil of Sanjivani island, which is a drought-prone area. Therefore, such efficient growth promoting P solubilizers can offer an effective solution for sustainable agriculture in arid, dryland farming and drought-prone regions.

Phytase production by Aspergillus niger NCIM 563 for a novel application to degrade organophosphorus pesticides.

The production of phytase using Aspergillus niger NCIM 563 under submerged fermentation conditions was studied using protein rich chickpea flour as substrate. Employing a hybrid statistical media optimization strategy of Plackett-Burman and Box-Behnken experimental designs in shake-flasks gave an increased phytase activity from an initial 66 IU/mL in 216 h to 160 IU/mL in a reduced time of 132 h. Productivity, thus increased by 3.97 times from 7.3 to 29 IU/mL/day. Using the optimized media, the production was successfully scaled-up further and improved up to 164 IU/mL in 96 h by studies carried out employing 2 and 10-L fermenters. The enzyme supernatant was recovered using centrifugal separation of biomass and the stability of the produced phytase was tested for animal feed applications under gastric conditions. In vitro degradation studies of water soluble monocrotophos, methyl parathion and water insoluble chlorpyrifos, pesticides used extensively in agriculture was carried out. It was observed by HPLC analysis that phytase could degrade 72% of chlorpyrifos at pH 7.0, 35 °C. Comparable results were obtained with monocrotophos and methyl parathion. With chlorpyrifos at higher temperature 50 °C as much as 91% degradation could be obtained. The degradation of chlorpyrifos was further validated by spraying phytase on harvested green chilli (Capsicum annuum L) under normal conditions of pH 7.0, 35 °C and the degradation products obtained analyzed by LCMS. Thus, the present study brings out a potentially novel application of phytase for biodegradation of organophosphorus pesticides.

Phytase production by solid-state fermentation of groundnut oil cake by Aspergillus niger: A bioprocess optimization study for animal feedstock applications.

This investigation deals with the use of agro-industrial waste, namely groundnut oil cake (GOC), for phytase production by the fungi Aspergillus niger NCIM 563. Plackett-Burman design (PBD) was used to evaluate the effect of 11 process variables and studies here showed that phytase production was significantly influenced by glucose, dextrin, distilled water, and MgSO4 · 7H2O. The use of response surface methodology (RSM) by Box-Behnken design (BBD) of experiments further enhanced the production by a remarkable 36.67-fold from the original finding of 15 IU/gds (grams of dry substrate) to 550 IU/gds. This is the highest solid-state fermentation (SSF) phytase production reported when compared to other microorganisms and in fact betters the best known by a factor of 2. Experiments carried out using dried fermented koji for phosphorus and mineral release and also thermal stability have shown the phytase to be as efficient as the liquid enzyme extract. Also, the enzyme, while exhibiting optimal activity under acidic conditions, was found to have significant activity in a broad range of pH values (1.5-6.5). The studies suggest the suitability of the koji supplemented with phytase produced in an SSF process by the "generally regarded as safe" (GRAS) microorganism A. niger as a cost-effective value-added livestock feed when compared to that obtained by submerged fermentation (SmF).

Charge-switchable gold nanoparticles for enhanced enzymatic thermostability.

This study illustrates a facile strategy for efficient immobilization of enzymes on a metal nanoparticle surface. The strategy proposed here enables the enzymatic activity to be retained while increasing the enzyme thermostability. It is demonstrated that the use of a zwitterionic amino acid tyrosine as a reducing and capping agent to synthesise gold nanoparticles allows efficient immobilization of phytase enzyme through charge-switchable electrostatic interactions. The detailed kinetic and thermodynamic studies reveal that the proposed enzyme immobilization strategy improves the overall quality of phytase by reducing the activation energy required for substrate hydrolysis and broadening the temperature window in which immobilized enzyme is able to operate. The outcomes of this study indicate that the underlying zwitterionic nature of 20 natural amino acids along with significant variability in their isoelectric points and hydropathy indices as well the ability of some of the amino acids to reduce metal ions is likely to offer significant opportunities for tailoring nano-bio interfaces in a rational manner for a range of biological applications.

Effect of phytase from Aspergillus niger on plant growth and mineral assimilation in wheat (Triticum aestivum Linn.) and its potential for use as a soil amendment.

BACKGROUND: Extensive use of non-replenishable phosphate reserves as phosphate supplements in agriculture and animal feed poses a threat for environmental pollution and necessitated a search for alternative phosphate sources. Unlocking the phytate phosphorus using microbial phytase can provide an ecofriendly solution in agriculture. The aim of the present work was to evaluate the effect of phytase from Aspergillus niger NCIM 563 in degradation of phytate phosphorus to benefit plant nutrition and soil amendment in comparison with chemical fertilizers. RESULTS: An enzyme dose of 12 IU was sufficient to hydrolyze phytate and increase assimilation of phosphorus by about 74%. Phytase supplementation leads to increase in shoot:total length ratio by about 200%, indicating its growth-promoting effect. Consistency in phytase-induced growth was reflected at pot and tray levels, wherein shoot:total length ratio was observed to be 2.01 and 2.12 respectively. Mineral assimilation due to phytase was more efficient as compared to chemical fertilizers, thus overcoming the constraints of practicability and economics in the agriculture industry. Phytase was efficient in reducing the phytic acid content of soil by about 30% while simultaneously increasing the phytate phosphate availability by 1.18-fold. CONCLUSION: Phytase from A. niger showed improvement in phytate phosphorus and mineral availability. Besides a plant growth-promoting effect, reduction in use of chemical fertilizers and soil improvement could be achieved simultaneously for maintaining the sustainability of agriculture.

Strain improvement and up scaling of phytase production by Aspergillus niger NCIM 563 under submerged fermentation conditions.

Combination of physical and chemical mutagenesis was used to isolate hyper secretory strains of Aspergillus niger NCIM 563 for phytase production. Phytase activity of mutant N-1 and N-79 was about 17 and 47% higher than the parent strain. In shake flask the productivity of phytase in parent, mutant N-1 and N-79 was 6,181, 7,619 and 9,523 IU/L per day, respectively. Up scaling of the fermentation from shake flask to 3 and 14 L New Brunswick fermenter was studied. After optimizing various fermentation parameters like aeration, agitation and carbon source in fermentation medium the fermentation time to achieve highest phytase activity was reduced considerably from 14 days in shake flask to 8 days in 14 L fermenter. Highest phytase activity of 80 IU/ml was obtained in 1% rice bran-3.5% glucose containing medium with aeration 0.2 vvm and agitation 550 rpm at room temperature on 8th day of fermentation. Addition of either bavistin (0.1%), penicillin (0.1%), formalin (0.2%) and sodium chloride (10%) in fermented broth were effective in retaining 100% phytase activity for 8 days at room temperature while these reagents along with methanol (50%) and ethanol (50%) confer 100% stability of phytase activity at 4 degrees C till 20 days. Among various carriers used for application of phytase in feed, wheat bran and rice bran were superior to silica and calcium carbonate. Thermo stabilization studies indicate 100% protection of phytase activity in presence of 12% skim milk at 70 degrees C, which will be useful for its spray drying.

Utilization of molasses sugar for lactic acid production by Lactobacillus delbrueckii subsp. delbrueckii mutant Uc-3 in batch fermentation.

Efficient lactic acid production from cane sugar molasses by Lactobacillus delbrueckii mutant Uc-3 in batch fermentation process is demonstrated. Lactic acid fermentation using molasses was not significantly affected by yeast extract concentrations. The final lactic acid concentration increased with increases of molasses sugar concentrations up to 190 g/liter. The maximum lactic acid concentration of 166 g/liter was obtained at a molasses sugar concentration of 190 g/liter with a productivity of 4.15 g/liter/h. Such a high concentration of lactic acid with high productivity from molasses has not been reported previously, and hence mutant Uc-3 could be a potential candidate for economical production of lactic acid from molasses at a commercial scale.

Production of lactic acid from cellobiose and cellotriose by Lactobacillus delbrueckii mutant Uc-3.

Lactobacillus delbrueckii mutant Uc-3 utilizes both cellobiose and cellotriose efficiently, converting it into L(+) lactic acid. The enzyme activities of cellobiose and cellotriose utilization were determined for cell extracts, whole cells, and disrupted cells. Aryl-beta-glucosidase activity was detected only for whole cells and disrupted cells, suggesting that these activities are cell bound. The mutant produced 90 g/liter of lactic acid from 100 g/liter of cellobiose with 2.25 g/liter/h productivity.