Enhanced submerged Aspergillus ficuum phytase production by implementation of fed-batch fermentation

Phytase is an important feed and food additive, which is both used in animal and human diets. Phytase has been used to increase the absorption of several divalent ions, amino acids, and proteins in the bodies and to decrease the excessive phosphorus release in the manure to prevent negative effects on the environment. To date, microbial phytase has been mostly produced in solid-state fermentations with insignificant production volumes. There are only a few studies in the literature that phytase productions were performed in submerged bench-top reactor scale. In our previous studies, growth parameters (temperature, pH, and aeration) and important fermentation medium ingredients (glucose, Na-phytate, and CaSO₄) were optimized. This study was undertaken for further enhancement of phytase production with Aspergillus ficuum in bench-top bioreactors by conducting fed-batch fermentations. The results showed that addition of 60 g of glucose and 10 g of Na-phytate at 96 h of fermentation increased phytase activity to 3.84 and 4.82 U/ml, respectively. Therefore, the maximum phytase activity was further enhanced with addition of glucose and Na-phytate by 11 and 40 %, respectively, as compared to batch phytase fermentations. It was also reported that phytase activity increased higher in early log stage additions than late log stage additions because of higher microbial activity. In addition, the phytase activity in fed-batch fermentation did not drop significantly as compared to the batch fermentation. Overall, this study shows that fungal phytase can be successfully produced in submerged fed-batch fermentations.     

Optimization of Fermentation Conditions for Phytase Production by Two Strains of Bacillus licheniformis (LF1 and LH1) Isolated from the Intestine of Rohu, Labeo rohita (Hamilton)

The culture conditions for extracellular production of phytase by two strains of Bacillus licheniformis (LF1 and LH1) isolated from the proximal and distal intestine of rohu (Labeo rohita) were optimized to obtain maximum level of phytase. Both the strains were cultured TSA broth for 24 h at 37 ± 2 °C, when average viable count of 9.75 × 10⁷ cells ml^?1 culture broth was obtained. This was used as the inoculum for the production medium. Sesame (Sesamum indicum) oilseed meal was used as the source of phytic acid (substrate). The effects of moisture, pH, temperature, fermentation period, inoculum size, different nitrogen sources, vitamins and surfactants on phytase production by these two strains were evaluated. Phytase yield was highest (1.87 U in LF1 and 1.57 U in LH1) in solid-state fermentation. Enzyme production in both the isolates increased in an optimum pH range of 5.5–6.5. Minimum phytase production was observed at 50 °C, while maximum production was obtained at 40 °C. To standardize the fermentation period for phytase production, production rate was measured at 12-h intervals up to 120 h. Enzyme production increased for 72 h of fermentation in both strains, and decreased thereafter. The enzyme production increased with increased inoculum size up to 3.0 percentage points for the strain LF1 and up to 2.0 % for the strains LH1. Ammonium sulphate as the nitrogen source was most effective in LF1, while beef extract proved useful to maximize enzyme production by LH1.     

Phytase production byAspergillus ficuum on semisolid substrate

Summary Phytase production byAspergillus ficuum was studied using solid state cultivation on several cereal grains and legume seeds. The microbial phytase was used to hydrolyze the phytate in soybean meal and cotton seed meal. Wheat bran, soybean meal, cottonseed meal and corn meal supported good fungal growth and yielded a high level of phytase when an adequate amount of moisture was present. The level of phytase production on solid substrate was higher than that obtained by submerged liquid fermentation. Higher levels of phosphorus (more than 10 mg Pi/100 g substrate) in the growth medium (static culture) inhibited phytase synthesis, and the degree of phosphorus inhibition was less apparent in semisolid medium than in liquid medium. A static cultivation on semisolid substrate produced a higher level of phytase (2-20-fold) than that obtained by agitated cultivation. The minimal amount of water required for growth and enzyme production on those substrates was about 15%, while the optimum level for phytase production was between 25 and 35% and that for cell growth was above 50%. Optimum pH for phytase production was between 4 and 6.A ficuum grew well on raw (unheated) substrate containing a minimal amount of water and produced as much phytase as on heated substrate. About half of the phytic acid in soybean meal and cottonseed meal was hydrolyzed by treatment withA. ficuum phytase.     

OPTIMIZATION OF SOLID-STATE FERMENTATION FOR PHYTASE PRODUCTION BY Thermomyces lanuginosus USING RESPONSE SURFACE METHODOLOGY

A strain of Thermomyces lanuginosus, isolated from hot spring water in Turkey, was studied for optimization of phytase production using solid-state fermentation. Effects on fermentation of different production parameters such as substrate type, moisture, culture time, and inoculum size were investigated using a one-factor-at-a-time approach. Central composite design (CCD) of response surface methodology was applied for the optimization of four factors (culture temperature, initial pH, aeration area, age of seeding culture) that were affecting phytase production by Thermomyces lanuginosus in rice bran. Maximum phytase activity was achieved by using rice bran. The optimum levels of variables that supported maximum enzyme activity were moisture 70%, culture time 7 days, inoculum size 40%, culture temperature 55°C, initial pH 7.5, aeration area 30%, age of seeding culture 5 days, sucrose 1%, and ZnSO₄ 2.5 mM. An overall 10.83-fold enhancement in phytase activity (0.30 to 3.248 U) was attained due to the optimization.     

Monitoring fermentation parameters during phytase production in column-type bioreactor using a new data acquisition system

Fermentation parameters for phytase production in column-type bioreactor were monitored using a new data acquisition system. There are a number of studies reporting phytase production in flasks, but a lack of data about microorganism respiration behaviour during phytase production using column bioreactor. The objectives of this work were the monitoration of fermentation parameters during phytase production and its relation with fungal growth and forced air. Phytase production by A. niger FS3 increased with forced air. The O₂ consumption and CO₂ production during solid-state fermentation were monitored by sensors (in the bottom and top of the columns) linked to controllers, recorded by acquisition software and processed by Fersol2^® software tool. Phytase synthesis was associated with fungal growth. Therefore, phytase could be used to estimate FS3 biomass formed in citric pulp degradation.     

Culture conditions for a new phytase-producing fungus

Extracellular phytase produced by Aspergillus sp. 5990 showed a 5-fold higher activity in liquid culture when compared with cultures of Aspergillus ficuum NRRL 3135. The optimum fermentation conditions were determined to be 35 °C, neutral pH, and 4 days incubation. The phytase had a higher optimum temperature for its activity than the commercial enzyme, Natuphos, from Aspergillus ficuum NRRL 3135.     

Combinatorial approach of statistical optimization and mutagenesis for improved production of acidic phytase by Aspergillus niger NCIM 563 under submerged fermentation condition

Combination of statistical optimization and mutagenesis to isolate hypersecretory strains is studied to maximize phytase production from Aspergillus niger NCIM 563 under submerged fermentation. The overall results obtained show a remarkable 5.98-fold improvement in phytase production rates when compared to that using basal medium. Optimization of culture conditions from parent strain is studied first by the Plackett–Burman technique to evaluate the effects of 11 variables for phytase production. The results showed that glucose, MgSO₄, KCl, incubation period, and MnSO₄ are the most significant variables affecting enzyme production. Further optimization in these variables, using a central composite design technique, resulted in 3.74-fold increase in the yield of phytase production to 254,500 U/l when compared with the activity observed with basal media (68,000 U/l) in shake flask. Our experiments show that the phytase from A. niger NCIM 563 exhibits desirable activity in simulated gastric fluid conditions with low pH and also improved thermostability when compared to commercial phytase. The improved yield demonstrates the potential applicability of phytase enzyme as a source of phytase supplement for phosphorus nutrition and environmental protection in animal feed industry. Physical and chemical mutagenesis experiments were carried out in parallel to isolate hypersecretory mutants that could possibly further enhance the enzyme production. Using optimized media conditions of the parent strain, our results show that mutant strain A. niger NCIM 1359 increased the phytase activity by another 1.6-fold to 407,200 U/l.     

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).     

A bioprocess for the production of phytase from Schizophyllum commune: studies of its optimization, profile of fermentation parameters, characterization and stability

Schizophyllum commune produces phytase through solid-state fermentation using different agroindustrial residues. After optimization of phytase production, a maximal level of phytase (113.7 Units/gram of dry substrate) was obtained in wheat bran based medium containing 5% sucrose, 50% humidity, 7.5% of biomass at 33 °C pH 7.0 during 72 h and a 285% improvement in enzyme titre was achieved. Analysis of fermentation parameters profile for phytase production showed the highest productivity (1.466 Units/gram of dry substrate/hour) in 66 h of fermentation. Phytase has an optimal pH of 5.0, an optimal temperature of 50 °C and K (m) and V (max) values of 0.16 mM and 1.85 μmol mL(-1) min(-1), respectively. Phytase activity was stimulated essentially in the presence of K(+), Ca(2+), Mg(2+), Mn(2+), Zn(2+), Cu(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), acetate and citrate at concentrations of 1 mM. Phytase had the best shelf life when stored at a cooling temperature, maintaining 38% of its initial activity after 112 days of storage, and still presenting enzymatic activity after 125 days of storage. Stability studies of phytase performed in aqueous enzyme extracts showed satisfactory results using polyethyleneglycol 3350, carboxymethylcellulose, methylparaben, mannitol and benzoic acid in concentrations of 0.25, 0.025, 0.025, 0.25, and 0.0025%, respectively. PEG 3350 was shown to be the best stabilizing agent, resulting in 109% of phytase activity from the initial crude extract remaining activity in after 90 days.     

Strain improvement and up scaling of phytase production by <Emphasis Type="Italic">Aspergillus niger</Emphasis> 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°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°C, which will be useful for its spray drying.     

Screening of phenylpyruvic acid producers and optimization of culture conditions in bench scale bioreactors

Alpha keto acids are deaminated forms of amino acids that have received significant attention as feed and food additives in the agriculture and medical industries. To date, their production has been commonly performed at shake-flask scale with low product concentrations. In this study, production of phenylpyruvic acid (PPA), which is the alpha keto acid of phenylalanine was investigated. First, various microorganisms were screened to select the most efficient producer. Thereafter, growth parameters (temperature, pH, and aeration) were optimized in bench scale bioreactors to maximize both PPA and biomass concentration in bench scale bioreactors, using response surface methodology. Among the four different microorganisms evaluated, Proteus vulgaris was the most productive strain for PPA production. Optimum temperature, pH, and aeration conditions were determined as 34.5 °C, 5.12, and 0.5 vvm for PPA production, whereas 36.9 °C, pH 6.87, and 0.96 vvm for the biomass production. Under these optimum conditions, PPA concentration was enhanced to 1,054 mg/L, which was almost three times higher than shake-flask fermentation concentrations. Moreover, P. vulgaris biomass was produced at 3.25 g/L under optimum conditions. Overall, this study demonstrated that optimization of growth parameters improved PPA production in 1-L working volume bench-scale bioreactors compared to previous studies in the literature and was a first step to scale up the production to industrial production.     

Production,characteristics and applications of phytase from a rhizosphere isolated <Emphasis Type="Italic">Enterobacter</Emphasis> sp. ACSS

Optimization of process parameters for phytase production by Enterobacter sp. ACSS led to a 4.6-fold improvement in submerged fermentation, which was enhanced further in fed-batch fermentation. The purified 62 kDa monomeric phytase was optimally active at pH 2.5 and 60 °C and retained activity over a wide range of temperature (40–80 °C) and pH (2.0–6.0) with a half-life of 11.3 min at 80 °C. The kinetic parameters K _m, V _max, K _cat, and K _cat/K _m of the pure phytase were 0.21 mM, 131.58 nmol mg^?1 s^?1, 1.64 × 10³ s^?1, and 7.81 × 10⁶ M^?1 s^?1, respectively. The enzyme was fairly stable in the presence of pepsin under physiological conditions. It was stimulated by Ca⁺², Mg⁺² and Mn⁺², but inhibited by Zn⁺², Cu⁺², Fe⁺², Pb⁺², Ba⁺² and surfactants. The enzyme can be applied in dephytinizing animal feeds, and the baking industry.     

Optimization of phytase production by solid substrate fermentation

The production of phytase by three feed-grade filamentous fungi (Aspergillus ficuum NRRL 3135, Mucor racemosus NRRL 1994 and Rhizopus oligosporus NRRL 5905) on four commonly used natural feed ingredients (canola meal, cracked corn, soybean meal, wheat bran) was studied in solid substrate fermentation (SSF). A. ficuum NRRL 3135 had the highest yield [15 IU phytase activity/g dry matter (DM)] on wheat bran. By optimizing the supplementation of wheat bran with starch and (NH₄)₂SO₄, phytase production increased to 25 IU/g DM. Optimization was carried out by Plackett-Burman and central composite experimental designs. Using optimized medium, phytase, phosphatase, alpha-amylase and xylanase production by A. ficuum NRRL 3135 was studied in Erlenmeyer flask and tray SSF. By scaling up SSF from flasks to stationary trays, activities of 20 IU phytase activity/g DM were reproducibly obtained. Electronic Publication     

Bioconversion of tea polyphenols to bioactive theabrownins by Aspergillus fumigatus

Theabrownins (TB) are water-soluble phenolic compounds associated with the various health benefits of Pu-erh tea, a post-fermented Chinese dark tea. This work reports on the production of theabrownins from infusions of sun-dried green tea leaves using a pure culture of Aspergillus fumigatus isolated from a solid-state Pu-erh tea fermentation. A theabrownins yield of 158 g kg^?1 sun-dried green tea leaves was obtained in 6 days at 45 °C in an aerobic fermentation. In a 2 l fermenter, the yield of theabrownins was 151 g kg^?1 sun-dried green tea leaves in 48 h of aerobic culture (45 °C, 1 vvm aeration rate, 250 rpm agitation speed). Extracellular polyphenol oxidase and peroxidase of A. fumigatus contributed to this bioconversion. Repeated batch fermentation process was used for producing theabrownins but was less productive than the batch process.     

Citrobacter amalonaticus Phytase on the Cell Surface of Pichia pastoris Exhibits High pH Stability as a Promising Potential Feed Supplement

Phytase expressed and anchored on the cell surface of Pichia pastoris avoids the expensive and time-consuming steps of protein purification and separation. Furthermore, yeast cells with anchored phytase can be used as a whole-cell biocatalyst. In this study, the phytase gene of Citrobacter amalonaticus was fused with the Pichia pastoris glycosylphosphatidylinositol (GPI)-anchored glycoprotein homologue GCW61. Phytase exposed on the cell surface exhibits a high activity of 6413.5 U/g, with an optimal temperature of 60°C. In contrast to secreted phytase, which has an optimal pH of 5.0, phytase presented on the cell surface is characterized by an optimal pH of 3.0. Moreover, our data demonstrate that phytase anchored on the cell surface exhibits higher pH stability than its secreted counterpart. Interestingly, our in vitro digestion experiments demonstrate that phytase attached to the cell surface is a more efficient enzyme than secreted phytase.     

Overexpression and Biochemical Characterization of a Thermostable Phytase from Bacillus subtilis US417 in Pichia pastoris

The overexpression of the native gene encoding the thermostable Bacillus subtilis US417 phytase using Pichia pastoris system is described. The phytase gene, in which the sequence encoding the signal peptide was replaced by that of the α-factor of Saccharomyces cerevisiae, was placed under the control of the methanol-inducible promoter of the alcohol oxidase 1 gene and expressed in Pichia pastoris. Small-scale expression experiments and activity assays were used to screen positive colonies. A recombinant strain was selected and produces 43 and 227 U/mL of phytase activity in shake flasks and in high-cell-density fermentation, respectively. The purified phytase was glycosylated protein and varied in size (50–65 kDa). It has a molecular mass of 43 kDa when it was deglycosylated. The purified r-PHY maintains 100 % of its activity after 10 min incubation at 75 °C and pH 7.5. This thermostable phytase, which is also active over broad pH ranges, may be useful as feed additives, since it can resist the temperature used in the feed-pelleting process.     

Purification and Biochemical Characterization of Phytase Enzyme from <Emphasis Type="Italic">Lactobacillus coryniformis</Emphasis> (<Emphasis Type="Italic">MH121153</Emphasis>)

Phytase (myo-inositol hexaphosphate phosphohydrolase) belongs to phosphatases. It catalyzes the hydrolysis of phytate to less-phosphorylated inorganic phosphates and phytate. Phytase is used primarily for the feeding of simple hermit animals in order to increase the usability of amino acids, minerals, phosphorus and energy. In the present study, phytase isolation from the Lactobacillus coryniformis strain, isolated from Lor cheese sources, phytase purification and characterization were studied. The phytase was purified in simple three steps. The enzyme was obtained with 2.60% recovery and a specific activity of 202.25 (EU/mg protein). The molecular mass of the enzyme was determined to be 43.25 kDa with the sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) method. The optimum temperature and pH for the enzyme were found as 60 °C and 5.0 and respectively. To defined the substrate specificity of the phytase, the hydrolysis of several phosphorylated compounds by the purified enzyme was studied and sodium phytate showed high specificity. Furthermore, the effects of Ca²⁺, Ag⁺, Mg²⁺, Cu²⁺, Co²⁺, Pb²⁺, Zn²⁺ and Ni²⁺ metal ions on the enzyme were studied.     

EVALUATION OF BIOETHANOL PRODUCTION FROM CAROB PODS BY Zymomonas mobilis AND Saccharomyces cerevisiae IN SOLID SUBMERGED FERMENTATION

Bioethanol production from carob pods has attracted many researchers due to its high sugar content. Both Zymomonas mobilis and Saccharomyces cerevisiae have been used previously for this purpose in submerged and solid-state fermentation. Since extraction of sugars from the carob pod particles is a costly process, solid-state and solid submerged fermentations, which do not require the sugar extraction step, may be economical processes for bioethanol production. The aim of this study is to evaluate the bioethanol production in solid submerged fermentation from carob pods. The maximum ethanol production of 0.42 g g^?1 initial sugar was obtained for Z. mobilis at 30°C, initial pH 5.3, and inoculum size of 5% v/v, 9 g carob powder per 50 mL of culture media, agitation rate 0 rpm, and fermentation time of 40 hr. The maximum ethanol production for S. cerevisiae was 0.40 g g^?1 initial sugar under the same condition. The results obtained in this research are comparable to those of Z. mobilis and S. cerevisiae performance in other culture mediums from various agricultural sources. Accordingly, solid submerged fermentation has a potential to be an economical process for bioethanol production from carob pods.     

Isolation and characterization of phytase isozymes produced by Aspergillus oryzae

A mutant strain (KL-38) of Aspergillus oryzae was obtained by UV irradiation. Phytase activity of KL-38 in molded rice (koji rice) was about 2.7-fold of that obtained from the parent strain (BP-1). Phytase activity of KL-38 in the submerged culture was similar to that of BP-1. Two types of phytase were produced from koji culture: phytase I (Phy I) was produced during incubation of both koji and submerged cultures, and phytase II (Phy II) was obtained only from koji culture. Phy II production was increased in KL-38 compared with BP-1, whereas the production of Phy I was similar for both KL-38 and BP-1. This finding indicates that A. oryzae has at least two types of phytase isozyme.     

Comparing submerged and solid-state fermentation of agro-industrial residues for the production and characterization of lipase by Trichoderma harzianum

Lipase production by Trichoderma harzianum was evaluated in submerged fermentation (SF) and solid-state fermentation (SSF) using a variety of agro-industrial residues. Cultures in SF showed the highest activity (1.4 U/mL) in medium containing 0.5 % (w/v) yeast extract, 1 % (v/v) olive oil and 2.5 C:N ratio. This paper is the first to report lipase production by T. harzianum in SSF. A 1:2 mixture of castor oil cake and sugarcane bagasse supplemented with 1 % (v/w) olive oil showed the best results among the cultures in SSF (4 U/g ds). Lipolytic activity was stable in a slightly acidic to neutral pH, maintaining 50 % activity after 30 min at 50 °C. Eighty percent of the activity remained after 1 h in 25 % (v/v) methanol, ethanol, isopropanol or acetone. Activity was observed with vegetable oils (olive, soybean, corn and sunflower) and long-chain triacylglycerols (triolein), confirming the presence of a true lipase. The results of this study are promising because they demonstrate an enzyme with interesting properties for application in catalysis produced by fermentation at low cost.