Optimizing some factors affecting protease production under solid state fermentation

Production of extracellular alkaline protease by a locally isolated fungal species, Rhizopus oryzae, under solid state fermentation was optimized. The maximum enzyme activity under the optimum conditions of temperature (32?°C), relative humidity (90%–95%), spore count (～2?×?10⁵/g wheat bran), moisture content of solid substrate (140%) adjusted suitably with salt solution (M-9) of pH?5.5 was 341 unit/g wheat bran.     

Production of thermostable hydrolases (cellulases and xylanase) from <Emphasis Type="Italic">Thermoascus aurantiacus</Emphasis> RCKK: a potential fungus

Thermophilic fungi are potential sources of thermostable enzymes and other value added products. Present study has focused on optimization of different physicochemical parameters for production of thermostable cellulases and xylanase by Thermoascus aurantiacus RCKK under SSF. Enzyme production was supported maximally on wheat bran fed with 20 % inoculum, at initial pH 5, temperature 45 °C and moisture ratio 1:3. The supplementation of wheat bran with yeast extract, Tween-80 and glycine further improved enzyme titres (CMCase 88 IU/g, FPase 15.8 IU/g, β-glucosidase 25.3 IU/g and xylanase 6,543 IU/g). The crude enzymes hydrolyzed phosphoric acid-swollen wheat straw, avicel and untreated xylan up to 74, 71 and 90 %, respectively. In addition, T. aurantiacus RCKK produced antioxidants as fermentation by-products with significant %DPPH^? scavenging, FRAP and in vivo antioxidant capacity against H₂O₂-treated Saccharomyces cerevisiae. These capabilities show that it holds potential to exploit crop by-products for providing various commodities.     

Cyclosporin-A production by Tolypocladium inflatum using solid state fermentation

Tolypocladium inflatum strains are known to produce Cyclosporin-A under submerged culture conditions. In the present study solid state fermentation was used to produce Cyclosporin-A. Tolypocladium inflatum strains when grown on moist wheat bran produced 310–459 mg of Cyclosporin-A kg of bran⁻¹. Tolypocladium inflatum ATCC 34921 which produced 459 mg of Cyclosporin-A kg of bran⁻¹ was improved to produce 1031±27 mg of Cyclosporin-A kg⁻¹ of bran, by subjecting the spores to different mutagenic treatments. The mutated strain, designated Tolypocladium inflatum DRCC 106, produced 4843 mg kg⁻¹ of bran under optimum fermentation conditions in 10 days when grown on wheat bran medium containing millet flour 20%, jowar flour 10%, zinc sulphate 0·15%, ferric chloride 0·25% and cobaltous chloride 0·05%. An inoculum of 60% initial moisture content 70%, initial bran pH 2·0 and incubation temperature 25°C were found to be optimal. Cyclosporin-A thus obtained was purified by solvent extraction, followed by column chromatography. The isolated product complies with the United States Pharmacopoeia specifications.     

Optimization of amylase production by Aspergillus niger in solid-state fermentation using sugarcane bagasse as solid support material

Synthesis of amylase by Aspergillus niger strain UO-01 under solid-state fermentation with sugarcane bagasse was optimized by using response surface methodology and empirical modelling. The process parameters tested were particle size of sugarcane bagasse, incubation temperature and pH, moisture level of solid support material and the concentrations of inoculum, total sugars, nitrogen and phosphorous. The optimum conditions for high amylase production (457.82 EU/g of dry support) were particle size of bagasse in the range of 6–8 mm, incubation temperature and pH: 30.2°C and 6.0, moisture content of bagasse: 75.3%, inoculum concentration: 1 × 10⁷ spores/g of dry support and concentrations of starch, yeast extract and KH₂PO₄: 70.5, 11.59 and 9.83 mg/g of dry support, respectively. After optimization, enzyme production was assayed at the optimized conditions. The results obtained corroborate the effectiveness and reliability of the empirical models obtained.     

Studies on the production of nigerloxin using agro-industrial residues by solid-state fermentation

Nigerloxin, a new and potent lipoxygenase inhibitor, was discovered in our laboratory through solid-state fermentation of wheat bran by Aspergillus niger V. Teigh (MTCC-5166). The aim of this study is to investigate the possibility of using different agro-industrial residues as nutritional supplements along with wheat bran to enhance the production of nigerloxin. Nigerloxin produced by SSF was quantified spectrophotometrically at 292 nm. The results indicate that the inhibitor production was influenced by the type of solid substrate supplemented, moisture content, pH and size of the inoculum. Individually optimized supplements were tested in different combinations to determine their effects on nigerloxin production. A twofold increase in the production of nigerloxin (4.9 ± 0.3 mg gds⁻¹) was achieved by supplementing wheat bran with 10% w/w sweet lemon peel and 5% v/w methanol at optimized process parameters, that is, an initial moisture content of 65% v/w and incubation period of 6 days with an initial inoculum size of 2 ml (8 × 10⁵ spores gds⁻¹). Nigerloxin production was stable between pH of 4 and 5.     

Production of Extracellular Alkaline α‐Amylase by Solid State Fermentation with a Newly Isolated Bacillus sp.

Abstract

Production of alkaline α‐amylase employing our laboratory isolate, Bacillus sp., under solid state fermentation, was optimized. The effect of wheat bran and lentil husk was examined. Lentil husk exhibited the highest enzyme production. The appropriate incubation time, inoculum size, moisture level, and buffer solution level were determined. Maximum yields of 216,000 and 172,800 U/g were achieved by employing lentil husk and wheat bran as substrates in 0.1 M carbonate/bicarbonate buffer at pH 10.0 with 30% initial moisture level at 24 h. Inoculum size and buffer solution level were found to be 20% and 1:0.5 for two solid substrates.     

Statistical Optimization of Culture Conditions for Milk-Clotting Enzyme Production by Bacillus Amyloliquefaciens Using Wheat Bran-An Agro-Industry Waste

In order to improve the production of the milk-clotting enzyme under submerged fermentation, two statistical methods were applied to optimize the culture conditions of Bacillus amyloliquefaciens D4 using wheat bran as nutrient source. First, initial pH, agitation speed, and fermentation time were shown to have significant effects on D4 enzyme production using the Plackett–Burman experimental design. Subsequently, optimal conditions were obtained using the Box–Behnken method, which were as follows: initial pH 7.57, agitation speed 241 rpm, fermentation time 53.3 h. Under these conditions, the milk-clotting enzyme production was remarkably enhanced. The milk-clotting enzyme activity reached 1996.9 SU/mL, which was 2.92-fold higher than that of the initial culture conditions, showing that the Plackett–Burman design and Box–Behnken response surface method are effective to optimize culture conditions. The research can provide a reference for full utilization of wheat bran and the production of milk-clotting enzyme by B. amyloliquefaciens D4 under submerged fermentation.     

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.     

Increased alkalotolerant and thermostable ribonuclease (RNase) production from alkaliphilic Streptomyces sp. M49-1 by optimizing the growth conditions using response surface methodology

Total of 171 alkaliphilic actinomycetes were evaluated for extracellular RNase production and Streptomyces sp. M49-1 was selected for further experiments. Fermentation optimization for RNase production was implemented in two steps using response surface methodology with central composite design. In the first step, the effect of independent fermentation variables including temperature, initial pH and process time were investigated. After identification of carbon and nitrogen sources affecting the production by one variable at a time method, concentrations of glucose and yeast extract and also inoculum size were chosen for the second central composite design. A maximum RNase activity was obtained under optimal conditions of 4.14 % glucose concentration, 4.63 % yeast extract concentration, 6.7 × 10⁶ spores as inoculum size for 50 ml medium, 42.9 °C, 91.2 h process time and medium initial pH 9.0. Optimum activity of the enzyme is achieved at pH 11 and temperature 60 °C. The enzyme is highly stable at pH range 9.0–12.0 and at 90 °C after 2 h. Statistical optimization experiments provide 2.25 fold increases in the activity of alkalotolerant and thermostable RNase and shortened the fermentation time compared to that of unoptimized condition. The members of Streptomyces can be promising qualified RNase producer for pharmaceutical industries.     

Tea stalks – a novel agro-residue for the production of tannase under solid state fermentation by Aspergillus niger JMU-TS528

A novel agro-residue, tea stalks, was tested for the production of tannase under solid-state fermentation (SSF) using Aspergillus niger JMU-TS528. Maximum yield of tannase was obtained when SSF was carried out at 28 °C, pH 6.0, liquid-to-solid ratio (v/w) 1.8, inoculum size 2 ml (1?×?10⁸ spores/ml), 5 % (w/v) ammonium chloride as nitrogen source and 5 % (w/v) lactose as additional carbon source. Under optimum conditions, tannase production reached 62 U/g dry substrate after 96 h of fermentation. Results from the study are promising for the economic utilization and value addition of tea stalks.     

Use of response surface methodology for optimizing process parameters for high inulinase production by the marine yeast <Emphasis Type="Italic">Cryptococcus aureus</Emphasis> G7a in solid-state fermentation and hydrolysis of inulin

The optimization of process parameters for high inulinase production by the marine yeast strain Cryptococcus aureus G7a in solid-state fermentation (SSF) was carried out using central composite design (CCD), one of the response surface methodologies (RSMs). We found that moisture, inoculation size, the amount ratio of wheat bran to rice husk, temperature and pH had great influence on inulinase production by strain G7a. Therefore, the CCD was used to evaluate the influence of the five factors on the inulinase production by strain G7a. Then, five levels of the five factors above were further optimized using the CCD. Finally, the optimal parameters obtained with the RSM were the initial moisture 61.5%, inoculum 2.75%, the amount ratio of wheat bran to rice husk 0.42, temperature 29 °C, pH 5.5. Under the optimized conditions, 420.9 U g⁻¹ of dry substrate of inulinase activity was reached in the solid-state fermentation culture of strain G7a within 120 h whereas the predicted maximum inulinase activity of 436.2 U g⁻¹ of inulinase activity of 436.2 U g⁻¹ of dry weight was derived from the RSM regression. This is the highest inulinase activity produced by the yeast strain reported so far. A large amount of monosaccharides and oligosaccharides were detected after inulin hydrolysis by the crude inulinase.     

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.     

Process optimization of xylanase production using cheap solid substrate by Trichoderma reesei SAF3 and study on the alteration of behavioral properties of enzyme obtained from SSF and SmF

This study aimed to assess the variability in respect of titer and properties of xylanase from Trichoderma reesei SAF3 under both solid-state and submerged fermentation. SSF was initially optimized with different agro-residues and among them wheat bran was found to be the best substrate that favored maximum xylanase production of 219 U (gws)^?1 at 96 h of incubation. The mycelial stage of the fungi and intracellular accumulation of Ca⁺⁺ and Mg⁺⁺ induced maximum enzyme synthesis. Inoculum level of 10 × 10⁶ spores 5 g^?1 of dry solid substrate and water activity of 0.6 were found to be optimum for xylanase production under SSF. Further optimization was made using a Box-Behnken design under response surface methodology. The optimal cultivation conditions predicted from canonical analysis of this model were incubation time (A) = 96–99 h, inoculum concentration (B) = 10 × 10⁶ spores 5 g^?1 of dry substrate, solid substrate concentration (C) = 10–12 g flask^?1, initial moisture level (D) = 10 mL flask^?1 (equivalent to a _w  = 0.55) and the level of xylanase was 299.7 U (gws)^?1. Subsequent verification of these levels agreed (97 % similar) with model predictions. Maximum amount of xylanase was recovered with water (6:1, v/w) and under shaking condition (125 rpm). Purified xylanase from SSF showed better stability in salt and pH, was catalytically and thermodynamically more efficient over enzyme from SmF, though molecular weight of both enzymes was identical (53.8 kDa).     

Production of mosquitocidal <Emphasis Type="Italic">Bacillus sphaericus</Emphasis> by solid state fermentation using agricultural wastes

In this study, Bacillus sphaericus NRC 69 was grown in culture media, in which 12 agricultural wastes were tested as the main carbon, nitrogen and energy sources under solid state fermentation. Of the 12 tested agricultural by-products, wheat bran was the most efficient substrate for the production of B. sphaericus mosquitocidal toxins against larvae of Culex pipiens (LC₅₀ 1.2 ppm). Mixtures of tested agricultural wastes separately with wheat bran enhanced the produced toxicity several folds and decreased LC₅₀ between 3.7- and 50-fold in comparison with that of agricultural wastes without mixing. The toxicity of B. sphaericus grown in wheat bran/rice hull at 8/2 (g/g) and wheat bran/barley straw at 1/4 (g/g) showed the same toxicity as that in wheat bran medium (LC₅₀ decreased 17- and 16-fold, in comparison with that in rice hull or barely straw media, respectively). In wheat bran medium, the maximum toxicity of the tested organism obtained at 50% moisture content, inoculum size 84 × 10⁶ CFU/g wheat bran and incubation for 6 days at 30°C. Addition of cheese whey permeate at 10% to wheat bran medium enhanced the toxicity of B. sphaericus NRC 69 about 46%.     

Optimization of Cultivating Conditions for Triterpenoids Production from Antrodia cinnmomea

The submerged cultivating conditions for triterpenoids production from Antrodia cinnamomea were optimized using uniform design method and the one-factor-at-a-time method was adopted to investigate the effect of plants oils and glucose supply on triterpenoids production and mycelia growth. Corn starch and culturing time were identified as more significant variables for triterpenoids production. The optimal conditions for triterpenoids production was 20.0 g/L corn starch, 20.0 g/L wheat bran, 1.85 g/L MgSO4, initial pH 3 and 16 days of cultivation. In addition, investigation of plant oils and glucose supply showed that 0.3 % (v/v) olive oil supply at the beginning of fermentation stimulated mycelia growth and significantly increased triterpenoids production; 0.2 % (w/v) glucose supplement at 10th day enhanced production of triterpenoids with slight effect on biomass, which is reported for the first time. The triterpenoids production experimentally obtained under the optimal conditions was 7.23 % (w/w). The uniform design method may be used to optimize many environmental and genetic factors such as temperature and agitation that can also affect the triterpenoids production from A. cinnamomea.     

Fungal utilization of a known and safe macroalga for pigment production using solid-state fermentation

Saccharina (Laminaria) japonica, a safe, cheap, and readily available macroalga can be used as a substrate for various microbial fermentations. This work investigated the feasibility of S. japonica as a substrate for production of pigments by the fungus Talaromyces amestolkiae GT11 in solid-state fermentation without additional salt and/or nitrogen sources. Under optimized conditions, the pigment exhibited maximum absorption spectrum at 410 (yellow) and 510 nm (red), and the pigment yield of 1,153.5 (yellow) and 506.2 (red) OD units g^?1 of dry fermented substrate were achieved with a particle size of 1.0 mm and pH 7, although visually the pigment was reddish in color. The optimum incubation period, pH, moisture, inoculum size, and temperature were observed to be at 192 h, pH 7.0, 80 % (w/w) moisture, 1.8?×?10⁶ spores mL^?1 of inoculum g^-1 of dry substrate and 28 °C. Hence, this study indicates the suitability of utilization of S. japonica as a substrate for natural pigment production by T. amestolkiae GT11 which can be used in food, cosmetics and pharmaceutical industries for various applications.     

Efficient mosquitocidal toxin production by Bacillus sphaericus using cheese whey permeate under both submerged and solid state fermentations

Whey permeate (WP) was used efficiently for production of mosquitocidal toxin by Bacillus sphaericus 2362 (B. sphaericus 2362) and the Egyptian isolate, B. sphaericus 14N1 (B. sphaericus 14N1) under both submerged and solid state fermentation conditions. Under submerged fermentation, high mosquitocidal activity was produced by B. sphaericus 2362 and B. sphaericus 14N1 at 50-100% and 25-70% WP, respectively. Initial pH of WP was a critical factor for toxin production by both tested organisms. The highest toxicity was obtained at initial pH 7. Egyptian isolate, B. sphaericus 14N1 was tested for growth and toxin production under solid state fermentation conditions (SSF) by using WP as moistening agent instead of distilled water. The optimum conditions for production of B. sphaericus 14N1 on wheat bran-WP medium were 10 g wheat bran/250 ml flask moistened with 10-70% WP at 50% moisture content, inoculum size ranged between 17.2 × 10⁷ and 34.4 × 10⁷ and 6 days incubation under static conditions at 30 °C. Preliminary pilot-scale production of B. sphaericus 14N1 under SSF conditions in trays proved that wheat bran-WP medium was efficient and economic for industrial production of mosquitocidal toxin by B. sphaericus.     

Endocellulase Production by <Emphasis Type="Italic">Cotylidia pannosa</Emphasis> and its Application in Saccharification of Wheat Bran to Bioethanol

For efficient bioconversion of lignocellulosic materials to bioethanol, the study screened 19 white-rot fungal strains for their endocellulolytic activity and saccharification potential. Preliminary qualitative and quantitative screening revealed Cotylidia pannosa to be the most efficient endocellulase producing fungal strain when compared to the standard strain of Trichoderma reesei MTCC 164. Ensuing initial screening, the production of endocellulase was further optimized using submerged fermentation to recognize process parameters such as temperature, time, agitation pH, and supplementation of salts in media required for achieving maximum production of endocellulase. The strain C. pannosa produced the maximum amount of endocellulase (8.48 U/mL) under submerged fermentation with wheat bran (2%) supplemented yeast extract peptone dextrose (YEPD) medium after an incubation time of 56 h at 30 °C and pH 5.0 at an agitation rate of 120 rpm with a saccharification value of 50.5%. The fermentation of wheat bran hydrolysate with Saccharomyces cerevisiae MTCC 174 produced 4.12 g/L of bioethanol after 56 h of incubation at 30 °C. The results obtained from the present investigation establish the potential of white-rot fungus C. pannosa for hydrolysis and saccharification of wheat bran to yield fermentable sugars for their subsequent conversion to bioethanol, suggesting its application in efficient bioprocessing of lignocellulosic wastes.     

Production of polysaccharide from Agaricus subrufescens Peck on solid-state fermentation

The interest upon products obtained from fungi has increased during the recent years. Among the most noticeable, nutraceuticals, enzymes, and natural drugs occupy a privileged position. Fungal biomass for the obtainment of those products can be produced either by solid-state fermentation (SSF) or submersed fermentation. SSF has been employed for the production of spawn on pretreated wheat grains with the objective of increasing the fungal polysaccharide (glucomannans) contents. Among the important factors for the production of spawn, time of cooking, time of resting after grain cooking, consequently grain moisture, substrate pH, temperature of incubation, and initial inoculum amount are among the most significant. For wheat grains, cooking time of 21 min followed by a 24-min resting time has been shown as optimal for the production of glucomannans by the fungus Agaricus subrufescens (=Agaricus brasiliensis). Amendments of CaSO₄ (up to 3 %) and CaCO₃ (up to 1 %) had an important influence on the substrate pH. In general, better results for glucomannan production were obtained when no supplement was added or when up to 0.25 % CaCO₃ (pH 6.6) has been added to the mix. Our results demonstrate that the inoculum amount necessary for the best polysaccharide levels is around 10.3 %, while the best temperature is around 27.2 °C. Besides using the spawn for its main purpose, it could potentially and alternatively be used as nutraceutical due to the high levels of glucomannan observed (6.89 %), a compound technically proven to be a potent immunostimulatory and antitumoral agent.     

Highly thermo–halo–alkali-stable β-1,4-endoxylanase from a novel polyextremophilic strain of Bacillus halodurans

A novel bacterial isolate, capable of producing extracellular highly thermostable, halo-alkali-stable and cellulase-free xylanase, was isolated from soil and identified as Bacillus halodurans TSPV1 by polyphasic approach. The Plackett–Burman design identified wheat bran, lactose, tryptone and NaCl as the factors that significantly affect xylanase production, and thus, these were optimized by response surface methodology. The data analysis suggested that optimum levels of wheat bran (15–20 g L^?1), lactose (1.0–1.5 g L^?1), tryptone (2–2.5 g L^?1) and NaCl (7.0–8.0 g L^?1) support 6.75-fold higher xylanase production than that in the un-optimized medium. The xylanase is optimally active at 90 °C and pH 10, and stable for 4 h at 90 °C (T _1/2 60 h) over a broad range of NaCl concentrations (0–29 %). This is the first report on the isolation of polyextremophilic B. halodurans strain that produces thermo-halo-alkali-stable xylanase in submerged fermentation. This enzyme efficiently saccharifies agro residues like wheat bran and corncobs. Fifty-six percent of hemicellulose of wheat bran could be hydrolyzed by xylanase (100 U g^?1 substrate) along with cellulase (22 U FPase and 50 U CMCase g^?1). The xylanase, being thermo-alkali stable and cellulase free, can find applications in pre-bleaching of paper pulps and hydrolysis of xylan in agricultural residues.