Response surface optimization of fermentation conditions for producing xylanase by <Emphasis Type="Italic">Aspergillus</Emphasis><Emphasis Type="Italic">niger</Emphasis> SL-05

Fermentation conditions were statistically optimized for producing extracellular xylanase by Aspergillus niger SL-05 using apple pomace and cotton seed meal. The primary study shows that culture medium with a 1:1 ratio of apple pomace and cotton seed meal (carbon and nitrogen sources) yielded maximal xylanase activity. Three significant factors influencing xylanase production were identified as urea, KH(2)PO(4), and initial moisture content using Plackett-Burman design study. The effects of these three factors were further investigated using a design of rotation-regression-orthogonal combination. The optimized conditions by response surface analysis were 2.5% Urea, 0.09% KH(2)PO(4), and 62% initial moisture content. The analysis of variance indicated that the established model was significant (P < 0.05), "while" or "and" the lack of fit was not significant. Under the optimized conditions, the model predicted 4,998 IU/g dry content, whereas validation experiments produced an enzymatic activity of xylanase at 5,662 IU/g dry content after 60 h fermentation. This study innovatively developed a fermentation medium and process to utilize inexpensive agro-industrial wastes to produce a high yield of xylanase.     

Cost-effective and concurrent production of industrially valuable xylano-pectinolytic enzymes by a bacterial isolate Bacillus pumilus AJK

Simultaneous production of xylanase and pectinase by Bacillus pumilus AJK under submerged fermentation was investigated in this study. Under optimized conditions, it produced 315?±?16 IU/mL acidic xylanase, 290?±?20 IU/mL alkaline xylanase, and 88?±?9 IU/mL pectinase. The production of xylano-pectinolytic enzymes was the highest after inoculating media (containing 2% each of wheat bran and Citrus limetta peel, 0.5% peptone, 10?mM MgSO₄, pH 7.0) with 2% of 21-hr-old culture and incubated at 37°C for 60?hr at 200?rpm. Xylanase retained 100% activity from pH 6.0 to10.0 after 3?hr of incubation, while pectinase showed 100% stability from pH 6.0 to 9.0 even after 6?hr of incubation. Cost-effective and concurrent production of xylanase and pectinase by a bacterial isolate in the same production media suggests its potential for various biotechnological applications. This is the first report of simultaneous production of industrially important extracellular xylano-pectinolytic enzymes by B. pumilus.     

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.     

Application of Plackett–Burman experimental design and Doehlert design to evaluate nutritional requirements for xylanase production by <Emphasis Type="Italic">Alternaria mali</Emphasis> ND-16

The objective of this study was to use statistically based experimental designs for the optimization of xylanase production from Alternaria mali ND-16. Ten components in the medium were screened for nutritional requirements. Three nutritional components, including NH₄Cl, urea, and MgSO₄, were identified to significantly affect the xylanase production by using the Plackett–Burman experimental design. These three major components were subsequently optimized using the Doehlert experimental design. By using response surface methodology and canonical analysis, the optimal concentrations for xylanase production were: NH₄Cl 11.34 g L⁻¹, urea 1.26 g L⁻¹, and MgSO₄ 0.98 g L⁻¹. Under these optimal conditions, the xylanase activity from A. mali ND-16 reached 30.35 U mL⁻¹. Verification of the optimization showed that xylanase production of 31.26 U mL⁻¹ was achieved.     

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.     

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     

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.     

Production and partial characterization of xylanase by Sporotrichum thermophile under solid-state fermentation

A number of factors affecting production of xylanase, by the thermophilic fungus Sporotrichum thermophile under solid state fermentation (SSF) were investigated. Initial moisture content and type of carbon source were consecutively optimized. Solid state fermentation in a laboratory horizontal bioreactor using the optimized medium allowed the production of 320 U g^–1 of carbon source which compared favourably with those reported for other microorganisms. Optimal xylanase activity was observed at pH 5 and 70 °C. Chromogenic (fluorogenic) 4-methylumbelliferyl -glycoside of xylobiose (MUX₂) was used to characterize the xylanase multienzyme component, after separation by isoelectric focusing and native PAGE electrophoresis. The zymograms indicated one major xylanase fraction exhibiting pI and molecular mass values 4 and 90–120 kDa, respectively.     

The improvement of cephalosporin C production by fed-batch culture ofCephalosporium acremonium M25 using rice oil

The objective of this study is to improve cephalosporin C (CPC) production by optimization of medium and culture conditions. A statistical method was introduced to optimize the main culture medium. The main medium for CPC production was optimized using a statistical method. Glucose and corn steep liquor (CSL) were found to be the most effective factors for CPC production. Glucose and CSL were optimized to 2.84 and 6.68%, respectively. CPC production was improved 50% by feeding of 5% rice oil at day 3rd and 5th day during the shake flask culture ofC. acremonium M25. The effect of agitation speeds on CPC production in a 2.5-L bioreactor was also investigated with fed-batch mode. The maximum cell mass (54.5 g/L) was obtained at 600 rpm. However, the maximum CPC production (0.98 g/L) was obtained at 500 rpm. At this condition, the maximum CPC production was improved about 132% compared to the result with batch flask culture.     

High activity xylanase production by Streptomyces olivaceoviridis E-86

The effects of different factors on xylanase production by Streptomyces olivaceoviridis E-86 were studied under shake flask conditions. The best initial pH value of growth medium for xylanase production was pH 6.0. Corn cob xylan and beef peptone were the best C source and N source, respectively. The enzyme activity was doubled by addition of 1.5% (v/v) Tween-80 in the medium. By the combination of the above variables, the highest xylanase activity obtained was 1653 U/ml which is the highest ever reported from Streptomyces sp.     

Improved xylanase production using apple pomace waste by Aspergillus niger in koji fermentation

Xylanase production by Aspergillus niger NRRL‐567 in solid‐state fermentation (koji fermentation) was optimized using 2⁴ factorial design and response surface methodology. The evaluated variables were the initial moisture level and concentration of inducers [veratryl alcohol (VA), copper sulphate (CS), and lactose (LAC)], leading to the response of xylanase production. Initial moisture level and LAC were found to be the most significant variable for xylanase production (p<0.05). The highest xylanase production was observed with 3578.8 ± 65.3 IU/gds (gram dry substrate) under optimal conditions using initial moisture of 85% (v/w), pH 5.0 and inducers VA (2 mM/kg), LAC 2% (w/w), and CS (1.5 mM/kg) after 48 h of incubation time. Higher xylanase activity of 3952 ± 78.3 IU/gds was attained during scale‐up of the process in solid‐state tray fermentation under optimum conditions after 72 h of incubation time. The present study demonstrates that A. niger NRRL‐567 can efficiently be used to achieve xylanase production with an economical and environmental benefit in solid‐state tray fermentation. The developed process can be used to develop an effective process for commercially feasible bioproduction of xylanases for speciality applications, such as conversion of lignocellulosic biomass to biofuels and other value‐added products.     

Enhanced Production and Partial Characterization of Thermostable α-galactosidase by Thermotolerant Absidia sp.WL511 in Solid-state Fermentation using Response Surface Methodology

Summary Response surface methodology was applied to optimize medium components for maximum production of a thermostable α-galactosidase by thermotolerant Absidia sp. WL511. First, the Plackett-Burman screening design was used to evaluate the effects of variables on enzyme production. Among these variables, MgSO₄ and soybean meal were identified as having the significant effects (with confidence level (90%). Subsequently, the concentrations of MgSO₄ and soybean meal were further optimized using central composite designs. The optimal parameters were determined by response surface and numerical analyses as 0.0503% (g/g) MgSO₄ and 0.406% (g/g) soybean meal and allowed α-galactosidase production to be increased from 4.4 IU g⁻¹ to 117.8 IU g⁻¹. The subsequent verification experiments confirmed the validity of the model. The optimum pH of enzymatic activity was 7.5 and the enzyme was stable at pH values ranging from 5.0 to 9.0. The optimum temperature was 73 °С. The enzyme was fairly stable at temperatures up to 60 °С and had 87% of its full activity at 65 °С after 2 h of incubation.     

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.     

Phytase production by fermentation of recombinant Pichia pastoris in monosodium glutamate wastewater

A novel method is proposed to produce both phytase and single-cell protein in recombinant Pichia pastoris fermentation using monosodium glutamate wastewater (MSGW) as the basal medium. Recombinant P. pastoris MR33 transformed with a phytase gene (AppA-m) from Escherichia coli was constructed and showed capability to utilize ammonium as the only nitrogen source. The fermentation medium was optimized in shake flasks by single-factor test and response surface methodology. A fed-batch system containing 30% MSGW, 50 g/l glucose, 1.58 g/l CaSO₄, 5.18 g/l MgSO₄ and 6.67 g/l KH₂PO₄ was developed in a 3.7-l bioreactor. The maximum phytase activity in the MSGW medium reached 3,380 U/ml, 84.2% of that in chemically defined medium, and the dry cell weight was 136 g/l. The single-cell protein (SCP; 46.66% dry cell weight) contains a variety of amino acids and is low in fat, which is ideal for utilization in animal feed. Thus, it is feasible to use MSGW medium for the production of enzymes that can be expressed in P. pastoris.     

Optimization of culture conditions for production of cellulases and xylanases by Scytalidium thermophilum using Response Surface Methodology

Summary Scytalidium thermophilum type culture Humicola insolens MTCC 4520 isolated from composting soil was optimized for production of cellulolytic and hemicellulolytic enzymes (endoglucanase, Avicel-adsorbable endoglucanase, FPase, β-glucosidase, xylanase and mannanase) by solid-state fermentation (SSF). Initial experiments showed that culture medium containing rice straw and wheat bran (1:3) as carbon source prepared in a synthetic basal medium supported maximal enzyme production at 45 °C. Further optimization of enzyme production was carried out using Box-Behnken design of experiments to study the influence of process variables (inoculum level, (NH₄)₂SO₄ and pH) on enzyme production. The response surface plots revealed the conditions for obtaining optimal enzyme levels. The models computed for R ² value ranged between 95% and 98.7% indicating they are appropriate and can be useful to predict the effect of inoculum level, (NH₄)₂SO₄ and pH on enzyme production. Under optimized conditions 62.5 ± 0.50, 23.0 ± 0.58, 3.0 ± 0.50, 151.00 ± 8.194, 196 ± 5.033 and 4.9 ± 0.32 (units/g substrate) of endoglucanase (EG), Avicel-adsorbable endoglucanase (AAEG), FPase, β-glucosidase, xylanase and mannanase were produced, respectively. Isoelectric focusing (IEF) of the crude extract showed that S. thermophilum produced six different EG isoforms, of which the EG corresponding to pI values of 8.4, 7.9 and 6.5 showed affinity for Avicel, thereby indicating the presence of a cellulose-binding domain (CBD). Furthermore, seven isoforms of β-glucosidase and ten multiple forms of xylanase distributed over a wide range of pI were also detected.     

Xylanase production under solid-state fermentation and its characterization by an isolated strain of <Emphasis Type="Italic">Aspergillus foetidus</Emphasis> in India

Summary A locally isolated strain of Aspergillus foetidus MTCC 4898 was studied for xylanase (EC 3.2.1.8) production using lignocellulosic substrates under solid state fermentation. Corncobs were found as the best substrates for high yield of xylanases with poor cellulase production. The influence of various parameters such as temperature, pH, moistening agents, moisture level, nitrogen sources and pretreatment of substrates were evaluated with respect to xylanase yield, specific activity and cellulase production. Influence of nitrogen sources on protease secretion was also examined. Maximum xylanase production (3065 U/g) was obtained on untreated corncobs moistened with modified Mandels and Strenberg medium, pH 5.0 at 1 5 moisture levels at 30 °C in 4 days of cultivation. Submerged fermentation under the same conditions gave higher yield (3300 U/g) in 5 days of cultivation, but productivity was less. Ammonium sulphate fractionation yielded 3.56-fold purified xylanase with 76% recovery. Optimum pH and temperature for xylanase activity were found to be 5.3 and 50 °C respectively. Kinetic parameters like K_m and V_max were found to be 3.58 mg/ml and 570 μmol/mg/min. Activity of the enzyme was found to be enhanced by cystiene hydrochloride, CoCl₂, xylose and Tween 80, while significantly inhibited by Hg⁺⁺, Cu⁺⁺ and glucose. The enzyme was found to be stable at 40 °C. The half life at 50 °C was 57.53 min. However thermostability was enhanced by glycerol, trehalose and Ca⁺⁺. The crude enzyme was stable during lyophilization and could be stored at less than 0 °C.     

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.     

Optimization of Medium Components for Production of Cellulases by Melanocarpus sp. MTCC 3922 under Solid-state Fermentation

Summary The medium components for the production of extracellular cellulases by Melanocarpus sp. MTCC 3922 were optimized using solid-state fermentation. Melanocarpus sp. cultured in optimized medium containing 1.5% urea, and 0.12% KH₂PO₄ along with a trace element solution and surfactant (Tween 20), produced endoglucanase (142.4 U/g of substrate), Avicel-adsorbable endoglucanase (27.0 U/g of substrate), Avicelase (0.65 U/ g of substrate), FPase (39.9 U/g of substrate) and β-glucosidase (109.0 U/g of substrate) activities. The presence of sulphate ions in traces stimulated endoglucanase yields. The IEF fractionation of the crude proteins from Melanocarpus sp. showed the expression of 3, 1 and 11 isoforms of endoglucanase, β-glucosidase and xylanase, respectively.     

Statistical screening and optimization of process variables for xylanase production utilizing alkali-pretreated rice husk

With the objective of the production of xylanase, local raw material (rice husk) and the indigenous isolate, Aspergillus niger ITCC 7678, were studied. Optimization of the cultivation system for enhancing xylanase production was studied via submerged fermentation. Statistical procedures were employed to study the effect of process variables, such as alkali-pretreated rice husk (as carbon source), NaNO₃ (as nitrogen source), KH₂PO₄, KCl, Tween 80 (as surfactant), MgSO₄, FeSO₄·7H₂O, pH, particle size, agitation, and temperature, on xylanase production by A. niger. The effect and significance of the variables was studied using Plackett–Burman (PBD) and central composite statistical design (CCD). It was found that alkali pretreated rice husk (weight/volume), pH, temperature, and NaNO₃ significantly influence xylanase production. So, these four factors were further optimized by CCD, and it was found that maximum xylanase activity of 10.9 IU/ml was observed at (6.5 % w/v) rice husk, pH (5.5), temperature (32.5 °C), and NaNO₃ (0.35 % w/v) concentration. Under optimum conditions, xylanase production was also studied at the bioreactor level and showed 12.8 % enhanced xylanase activity.     

Production of cellulase-free xylanase by a thermotolerant Streptomyces sp. grown on agricultural waste and media optimization using mixture design and Plackett–Burman experimental design methods

Cellulase-free xylanase was produced by Streptomyces sp. Ab106 on finely ground cane bagasse at 55 °C. The optimal medium composition was developed by applying the mixture design and linear mathematical program, and evaluated using the Plackett–Burman experimental design. The best composition of basal medium was found by using the mixture design method. The highest xylanase activity, 10.6 IU, was obtained after 6 days of fermentation in shaked flask at 100 rpm, 55 °C, pH 7. Both experimental designs showed that trace elements induced xylanase production. With fermentation in a 5-l fermenter, xylanase activity of 12.5 IU was achieved.