Optimization of xylanase production using inexpensive agro-residues by alkalophilic Bacillus subtilis ASH in solid-state fermentation

Alkalophilic Bacillus subtilis ASH produced high levels of xylanase using easily available inexpensive agricultural waste residues such as wheat bran, wheat straw, rice husk, sawdust, gram bran, groundnut and maize bran in solid-state fermentation (SSF). Among these, wheat bran was found to be best substrate. Xylanase production was highest after 72 h of incubation at 37 °C and at a substrate to moisture ratio of 1:2 (w/v). The inoculum level of 15% resulted in maximum production of xylanase. The enzyme production was stimulated by the addition of nutrients such as yeast extract, peptone and beef extract. In contrast, addition of glucose and xylose repressed the production of xylanase. The extent of repression by glucose (10%, w/v) was 81% and it was concentration-dependent. Supplementation of the medium with 4% xylose caused 59% repression. Under optimized conditions, xylanase production in SSF (8,964 U of xylanase/g dry wheat bran) was about twofold greater than in submerged fermentation. Thus, B. subtilis produced a very high level of xylanase in SSF using inexpensive agro-residues, a level which is much higher than that reported by any other bacterial isolate. Furthermore, the enzyme was produced at room temperature and with tap water without the addition of any mineral salt in SSF, leading to a marked decrease in the cost of xylanase production, which enhances its industrial potential.     

Statistical Optimization of Medium and Fermentation Conditions of Recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis> for the Production of Xylanase

Recombinant xylanase (rPcXynC) from Pichia pastoris was produced on large-scale by optimizing production-medium composition using statistical experimental methods. Production medium was optimized through the use of statistical methods such as one factor at a time (OFAT), Plackett-Burman design, fractional factorial design (FFD), steepest ascent method (SAM), and response surface methodology (RSM). The optimum medium composition was established to be (g/L); wheat bran 11.62, yeast extract 30, Tween 60.5, DL-β-Phenylalanine 0.5, Thiamine 0.5, FeSO₄ 0.01, KH₂PO₄ 0.66, and KHSO₄ 0.09. The optimum medium composition yielded 3,051 mU/mL of xylanase activity which was three times higher than that obtained from the initial medium composition. Finally, fermentation conditions were examined using the optimized production medium in a laboratory bioreactor. The optimal fermentation conditions were found to be 25ºC, pH 6, 170 rpm and 1 vvm with intermittent feeding of methanol (67.5 mL) and the xylanase activity was 3,683 mU/mL. In repeated-batch fermentation using optimized production medium and fermentation condition, the xylanase activity was 3,680 mU/mL at the first cycle of 96 h harvesting time using 90% of the culture solution. The activity was similarly maintained until the last cycle of 264 h.     

High-level xylanase production by alkaliphilic Bacillus pumilus ASH under solid-state fermentation

Bacillus pumilus ASH produced a high level of an extracellular and thermostable xylanase enzyme when grown using solid-state fermentation (SSF). Among a few easily available lignocellulosics tested, wheat bran was found to be the best substrate (5,300 U/g of dry bacterial bran). Maximum xylanase production was achieved in 72 h (5,824 U/g). Higher xylanase activity was obtained when wheat bran was moistened with deionized water (6,378 U/g) at a substrate-to-moisture ratio of 1:2.5 (w/v). The optimum temperature for xylanase production was found to be 37°C. The inoculum level of 15% was found to be the most suitable for maximum xylanase production (7,087 U/g). Addition of peptone stimulated enzyme production followed by yeast extract and mustard oil cake, whereas glucose, xylose and malt extract greatly repressed the enzyme activity. Repression by glucose was concentration-dependent, repressing more than 60% of the maximum xylanase production at a concentration of 10% (w/v). Cultivation in large enamel trays yielded a xylanase titre that was slightly lower to that in flasks. The enzyme activity was slightly lower in SSF than in SmF but the ability of the organism to produce such a high level of xylanase at room temperature and with deionized water without addition of any mineral salts in SSF, could lead to substantial reduction in the overall cost of enzyme production. This is the first report on production of such a high level of xylanase under SSF conditions by bacteria.     

Improved xylanase production from a haloalkalophilic Staphylococcus sp. SG-13 using inexpensive agricultural residues

The production of an alkali-stable xylanase, with dual pH optima, from haloalkalophilic Staphylococcus sp. SG-13 has been enhanced using agro-residues in submerged fermentation and a biphasic growth system. The agro-residues such as wheat bran, sugarcane bagasse, corncobs and poplar wood when used as sole carbon source, improved the xylanase yield by five-fold as compared to xylose and xylan. Staphylococcus sp. SG-13 also produced equally good amounts of xylanase when grown simply in deionized water (pH 8.0) supplemented with agro-residues as sole carbon source. In the biphasic growth system (lower layer containing agricultural residue set in agar medium with liquid medium above it), the prime substrate, wheat bran (1% w/v), resulted in maximum xylanase production of 4525 U l^–1 (pH 7.5) and 4540 U l^–1 (pH 9.2) at an agar: broth ratio of 4.0 after 48 h of incubation at 37 °C under static conditions. In general, the cost-effective agro-residues were found to be more suitable inducers for xylanase production over expensive substrates like xylan.     

Optimization of nutrient medium containing agricultural waste for xylanase production by Bacillus pumilus B20

We aimed to optimize a nutrient medium containing agricultural waste for xylanase production by Bacillus pumilus B20. Xylanase production with lignocellulosic material was optimized in two steps using DeMeo’s fractional factorial design. A 3.4-fold increase in xylanase production (313.3 U/mL) was achieved using the optimized culture medium consisting of (g/L): K₂HPO₄, 2; MgSO₄·7H₂O, 0.3; CaCl₂·2H₂O, 0.01; NaCl, 2; peptone, 5 yeast extract, 4; and wheat bran, 50. _{B. pumilus} B20 produced a high level of xylanase, which may have potential industrial application.     

Extracellular xylanase production by Fusarium species in solid state fermentation

Fusarium sp. has been shown to be a promising organism for enhanced production of xylanases. In the present study, xylanase production by 21 Fusarium sp. isolates (8 Fusarium culmorum, 4 Fusarium solani, 6 Fusarium verticillioides and 3 Fusarium equiseti) was evaluated under solid state fermentation (SSF). The fungal isolate Fusarium solani SYRN7 was the best xylanase producer among the tested isolates. The effects of some agriculture wastes (like wheat straw, wheat bran, beet pulp and cotton seed cake) and incubation period on xylanase production by F. solani were optimized. High xylanase production (1465.8 U/g) was observed in wheat bran after 96 h of incubation. Optimum pH and temperature for xylanase activity were found to be 5 and 50 degrees C, respectively.     

Improvement of xylanase production in solid-state fermentation by alkali tolerant Aspergillus versicolor MKU3

AIMS: To optimize the media components for xylanase production by Aspergillus versicolor MKU3 in solid-state fermentation (SSF). METHODS AND RESULTS: Medium optimization was carried out using De Moe's fractional factorial design with seven components. Maximum production of xylanase 3249.9 U g(-1) was obtained in SSF with an optimized medium containing (g l(-1)): NaNO(3), 20; K(2)HPO(4), 20; MgSO(4), 10; FeSO(4), 0.001; KCl, 1; peptone, 10 and yeast extract, 10. Four components namely NaNO(3), MgSO(4), peptone and K(2)HPO(4) significantly increased the xylanase production by A. versicolor MKU3. CONCLUSIONS: Fractional factorial design was used to optimize the seven components in the fermentation medium for SSF. The optimized media increased xylanase production by 3.4-fold. SIGNIFICANCE AND IMPACT OF THE STUDY: Aspergillus versicolor MKU3 produced maximum xylanase after two steps of media optimization under alkaline condition. This medium will be significant value for xylanase production in SSF.     

Glucoamylase production by solid-state fermentation using rice flake manufacturing waste products as substrate

Glucoamylase production has been investigated by solid-state fermentation of agro-industrial wastes generated during the processing of paddy to rice flakes (categorized as coarse, medium and fine waste), along with wheat bran and rice powder by a local soil isolate Aspergillus sp. HA-2. Highest enzyme production was obtained with wheat bran (264 +/- 0.64 U/gds) followed by coarse waste (211.5 +/- 1.44 U/gds) and medium waste (192.1 +/- 1.15 U/gds) using 10(6) spores/ml as inoculum at 28 +/- 2 degrees C, pH 5. A combination of wheat bran and coarse waste (1:1) gave enzyme yield as compared to wheat bran alone. Media supplementation with carbon source (0.04 g/gds) as sucrose in wheat bran and glucose in coarse and medium waste increased enzyme production to 271.2 +/- 0.92, 220.2 +/- 0.75 and 208.2 +/- 1.99 U/gds respectively. Organic nitrogen supplementation (yeast extract and peptone, 0.02 g/gds) showed a higher enzyme production compared to inorganic source. Optimum enzyme activity was observed at 55 degrees C, pH 5. Enzyme activity was enhanced in the presence of calcium whereas presence of EDTA gave reverse effect.     

Production of alkali tolerant cellulase free xylanase in high levels by Bacillus pumilus SV-205

The fermentation conditions were optimized for hyper production of xylanase from Bacillus pumilus SV-205. The bacterium secretes high levels (7382.7±1200 IU/mL) of cellulase-free xylanase using wheat bran led to 21.63 fold increase in activity. A combination of yeast extract and peptone stimulated highest xylanase production (2448.0 IU/mL) as compared to other combinations. The most important characteristic of the enzyme is its high pH stability (100%) over a broad pH range of 6-11 for 24h. Thermostability studies revealed that enzyme retained 65% activity after an incubation of 2h at 60°C. The level of production is remarkable as compared to earlier reports.     

Optimization of nutrient medium containing agricultural wastes for xylanase production by Aspergillus niger B03 using optimal composite experimental design

The xylanase biosynthesis is induced by its substrate - xylan. The high xylan content in some of the wastes like corn cobs and wheat bran makes them an accessible and cheap source of inducers. Nutrient medium for xylanase biosynthesis in submerged cultivation of Aspergillus niger B03 has been optimized. The optimization process was analyzed using optimal composite experimental design and response surface methodology. The predicted by the regression model optimum components of nutrient medium are as follows (g/l): (NH(4))(2)HPO(4) 2.6, urea 0.9, corn cobs 24.0, wheat bran 14.6 and malt sprout 6.0. Five parallel experiments have been carried out, at definite, optimum components concentrations of the nutrient medium, and a mean value of the activity Y=996.30 U/ml has been obtained. The xylanase activity, obtained with the optimized nutrient medium is 33% higher than the activity, achieved with the basic medium.     

Production of xylanase under solid-state fermentation by <Emphasis Type="Italic">Aspergillus tubingensis</Emphasis> JP-1 and its application

The production of extracellular xylanase by a locally isolated strain of Aspergillus tubingensis JP-1 was studied under solid-state fermentation. Among the various agro residues used wheat straw was found to be the best for high yield of xylanase with poor cellulase production. The influence of various parameters such as initial pH, moisture, moistening agents, nitrogen sources, additives, surfactants and pretreatment of substrates were investigated. The production of the xylanase reached a peak in 8 days using untreated wheat straw with modified MS medium, pH 6.0 at 1:5 moisture level at 30 °C. Under optimized conditions yield as high as 6,887 ± 16 U/g of untreated wheat straw was achieved. Crude xylanase was used for enzymatic saccharification of agro-residues like wheat straw, rice bran, wheat bran, sugarcane bagasse and industrial paper pulp. Dilute alkali (1 N NaOH) and acid (1 N H₂SO₄) pretreatment were found to be beneficial for the efficient enzymatic hydrolysis of wheat straw. Dilute alkali and acid-pretreated wheat straw yielded 688 and 543 mg/g reducing sugar, respectively. Yield of 726 mg/g reducing sugar was obtained from paper pulp after 48 h of incubation.     

Production and optimization of cellulase-free, alkali-stable xylanase by Bacillus pumilus SV-85S in submerged fermentation

This paper reports the production of a cellulase-free and alkali-stable xylanase in high titre from a newly isolated Bacillus pumilus SV-85S using cheap and easily available agro-residue wheat bran. Optimization of fermentation conditions enhanced the enzyme production to 2995.20 ± 200.00 IU/ml, which was 9.91-fold higher than the activity under unoptimized basal medium (302.2 IU/ml). Statistical optimization using response-surface methodology was employed to obtain a cumulative effect of peptone, yeast extract, and potassium nitrate (KNO₃) on enzyme production. A 2³ central composite design best optimized the nitrogen source at the 0 level for peptone and yeast extract and at the −α level for KNO₃, along with 5.38-fold increase in xylanase activity. Addition of 0.1% tween 80 to the medium increased production by 1.5-fold. Optimum pH for xylanase was 6.0. The enzyme was 100% stable over the pH range from 5 to 11 for 1 h at 37°C and it lost no activity, even after 3 h of incubation at pH 7, 8, and 9. Optimum temperature for the enzyme was 50°C, but the enzyme displayed 78% residual activity even at 65°C. The enzyme retained 50% activity after an incubation of 1 h at 60°C. Characteristics of B. pumilus SV-85S xylanase, including its cellulase-free nature, stability in alkali over a long duration, along with high-level production, are particularly suited to the paper and pulp industry.     

Statistical optimization of medium components for the production of xylanase byAspergillus niger KK2 in submerged cultivation

Medium composition was optimized for the production of xylanase byAspergillus niger KK2 using statistical experimental designs. Corn steep liquor (CSL) and industrial yeast extract (IYE) were the most important factors affecting xylanase activity. The medium that produced the optimum conditions for the production of xylanase contained 3% rice straw, 1% wheat bran, 6.3% CSL, 0.15% IYE, and 0.5% KH₂PO₄. After 4 days of cultivation under optimized conditions in a 2.5-L stirred tank reactor the activity and productivity of xylanase were 620 IU/mL and 6,458 IU/L.h, respectively. The highest xylanase activity obtained using the optimized medium was 80% greater than the activity obtained using basal medium. The xylanase activity predicted by a polynomial model was 670 IU/ml.     

Biocatalytic potential of protease-resistant phytase of Aspergillus oryzae SBS50 in ameliorating food nutrition

Production of protease-resistant phytase by Aspergillus oryzae SBS50 was optimized in solid state fermentation using wheat bran as substrate. An integrated statistical optimization approach involving the Placket–Burman design followed by response surface methodology was employed. Among all the variables tested, incubation period, triton X-100, moisture ratio, and magnesium sulphate were identified as significant and further optimized using response surface methodology that resulted in 3.35-fold improvement in phytase production from 55.43 to 185.75 U/g dry mouldy bran (DMB). Optimal conditions for maximum phytase production (185.75 U/g DMB) included wheat bran 10 g per 250 ml flask moistened with 35 ml distilled water supplemented with 3.0% triton X-100, 0.04% magnesium sulphate, 1.0% sucrose and 0.5% yeast extract incubated at 30?°C for an incubation time of 48 h. Phytase titers were sustainable (179.55 to 185.75 U/g DMB), when the mould was grown in shake flasks of varied volumes and enamel-coated metallic trays under optimized conditions. Fermentation time was reduced to half from 96 h to 48 h after optimization resulting in a 6.7-fold enhancement in the phytase productivity from 577.39 to 3868.75 U/Kg/h and thus, reducing the cost of enzyme production. Phytase released inorganic phosphate, reducing sugars and soluble proteins from different food samples in a time dependent manner as a result of phytate hydrolysis.     

Thermostable, alkalophilic and cellulase free xylanase production by Thermoactinomyces thalophilus subgroup C

Thermoactinomyces thalophilus produced cellulase free extracellular endo-1,4-beta-xylanase (EC 3.2.1.8) at 50 degrees C and pH 8.5. Maximum xylanase production was achieved in fermentation medium using birchwood xylan as substrate after 96 h of growth at 50 degrees C. Other agricultural substrates such as wheat bran, wheat straw, sugarcane bagasse and cornstover produced less xylanase. The crude enzyme preparation from mutant T. thalophilus P2 grown under optimised fermentation conditions showed no cellulase contamination and maximum xylanase activity of 42 U/ml at 65%deg;C and pH 8.5-9.0. This enzyme with initial xylanase activity of 42 U/ml was found thermostable up to 65 degrees C and retaining 50% of its activity after its incubation for 125 min at 65 degrees C.     

Enhanced endoxylanase production by Myceliophthora thermophila using rice straw and its synergism with phytase in improving nutrition

The goal of the present investigation was to attain the enhanced production of endoxylanase in submerged fermentation using different approaches followed by its utility in improving nutrition of wheat and rice flours along with phytase. Myceliophthora thermophila BJTLRMDU3 produced 51.70 U/mL of xylanase using rice straw as a substrate after optimization with ‘one variable at a time’ approach. After Plackett-Burman design study, sodium nitrate, K₂HPO₄ and Tween 20 were selected as critical factors and further optimized by response surface methodology. Increased xylanase production (80.15 U/mL) was attained with 2.5 % (w/v) sodium nitrate, 1.25 % (w/v) K₂HPO₄, and 2 % (v/v) Tween 20 at 40 °C. An overall 1.5-fold increase in xylanase production was achieved after statistical optimization. Applicability of M. thermophila xylanase (200 U/g flour) alone and in combination with phytase (15 U/g flour) from Aspergillus oryzae SBS50 in wheat and rice flours showed enhancement in nutritional qualities of both flours. About 45.67 %, 29.73 %, and 107.91 % increase in reducing sugars, soluble proteins and inorganic phosphate, respectively in wheat flour, while 94.16 %, 134.52 %, and 473.33 % increase in reducing sugars, soluble proteins and inorganic phosphate, respectively in rice flour was achieved at 60 °C and pH 5.0 by synergistic action of xylanase and phytase as compared to control having only xylanase.     

Alkaline xylanases from Bacillus mojavensis A21: Production and generation of xylooligosaccharides

Medium composition and culture conditions for the xylanases production by Bacillus mojavensis A21 were optimized using two statistical methods: Plackett-Burman design applied to find the key ingredients and conditions for the best yield of enzyme production and Box-Behnken design used to optimize the value of the four significant variables: barley bran, NaCl, agitation, and cultivation time. The optimal conditions for higher production of xylanases were barley bran 18.66g/l, NaCl 1.04g/l, speed of agitation 176rpm and cultivation time 34.08h. Under these conditions, the xylanase experimental yield (7.45U/ml) closely matched the yield predicted by the statistical model (7.23U/ml) with R(2)=0.98. The medium optimization resulted in a 6.83-fold increase in xylanase production compared to that of the initial medium. Best xylanase activity was observed at the temperature of 50°C and at pH 8.0. The enzyme retained more 96% of its activity after 24h at pH ranges from 7.0 to 90.0. The enzyme preserved more 80% of its initial activity after 60min of pre-incubation from 30°C to 60°C. The main hydrolysis products yielded from corncob extracted xylan were xylobiose and xylotriose, suggesting the good potential of strain A21 in xylooligosaccharides production.     

Utilization of agro-industrial waste for xylanase production by Aspergillus foetidus MTCC 4898 under solid state fermentation and its application in saccharification

Xylanase production by Aspergillus foetidus MTCC 4898 was carried out under solid state fermentation using wheat bran and anaerobically treated distillery spent wash. Response surface methodology involving Box–Behnken design was employed for optimizing xylanase production. The interactions among various fermentation parameters viz. moisture to substrate ratio, inoculum size, initial pH, effluent concentration and incubation time were investigated and modeled. The predicted xylanase activity under optimized parameters was 8200–8400 U/g and validated xylanase activity was 8450 U/g with very poor cellulase activity. Crude xylanase was used for enzymatic saccharification of agroresidues like wheat straw, rice straw and corncobs. Dilute NaOH and ammonia pretreatments were found to be beneficial for the efficient enzymatic hydrolysis of all the three substrates. Dilute NaOH pretreated wheat straw, rice straw and corncobs yielded 4, 4.2, 4.6 g/l reducing sugars, respectively whereas ammonia treated wheat straw, rice straw and corncobs yielded 4.9, 4.7, 4.6 g/l reducing sugars, respectively. The hydrolyzates were analysed by HPTLC. Xylose was found to be the major end product with traces of glucose in the enzymatic hydrolyzates of all the substrates.     

Production of Aspergillus xylanase by lignocellulosic waste fermentation and its application

Strains of Aspergillus terreus and A. niger, known to produce xylanase with undetectable amounts of cellulase, were studied for xylanase (EC 3.2.1.8) production on various lignocellulosic substrates using solid state fermentation. Of the lignocellulosic substrates used, wheat bran was the best for xylanase production. The effects of various parameters, such as moistening agent, level of initial moisture content, temperature of incubation, inoculum size and incubation time, on xylanase production were studied. The best medium for A. terreus was wheat bran moistened with 1:5 Mandels and Strenberg mineral solution containing 0.1% tryptone, at 35 degrees C, and at inoculum concentration 2x107-2x108 spores 5 g-1 substrate; for A. niger, the best medium was wheat bran moistened with 1:5 Mandels and Strenberg mineral solution containing 0.1% yeast extract, at 35 degrees C, and at an inoculum concentration of 2x107-2x108 spores 5 g-1 substrate. Under these conditions, A. terreus produced 68.9 IU ml-1 of xylanase, and A. niger, 74.5 IU ml-1, after 4 d of incubation. A crude culture filtrate of the two Aspergillus strains was used for the hydrolysis of various lignocellulosic materials. Xylanase preparations from the two strains selectively removed the hemicellulose fraction from all lignocellulosic materials tested.     

Xylanase production in solid state fermentation by Aspergillus niger mutant using statistical experimental designs

The initial moisture content, cultivation time, inoculum size and concentration of basal medium were optimized in solid state fermentation (SSF) for the production of xylanase by an Aspergillus niger mutant using statistical experimental designs. The cultivation time and concentration of basal medium were the most important factors affecting xylanase activity. An inoculum size of 5 x 10(5) spores/g, initial moisture content of 65%, cultivation time of 5 days and 10 times concentration of basal medium containing 50 times concentration of corn steep liquor were optimum for xylanase production in SSF. Under the optimized conditions, the activity and productivity of xylanase obtained after 5 days of fermentation were 5,071 IU/g of rice straw and 14,790 IU l(-1) h(-1), respectively. The xylanase activity predicted by a polynomial model was 5,484 IU/g of rice straw.