Impact of process parameters on chitinase production by an alkalophilic marine Beauveria bassiana in solid state fermentation

A chitinolytic fungus, Beauveria bassiana was isolated from marine sediment and significant process parameters influencing chitinase production in solid state fermentation using wheat bran were optimised. The organism was strongly alkalophilic and produced maximum chitinase at pH 9·20. The NaCl and colloidal chitin requirements varied with the type of moistening medium used. Vegetative (mycelial) inoculum was more suitable than conidial inoculum for obtaining maximal enzyme yield. The addition of phosphate and yeast extract resulted in enhancement of chitinase yield. After optimisation, the maximum enzyme yield was 246·6 units g⁻¹ initial dry substrate (U gIDS⁻¹). This is the first report of the production of chitinase from a marine fungus.     

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.     

Enzymatic production of N-acetyl-D-glucosamine by solid state fermentation of chitinase by Penicillium ochrochloron MTCC 517 using agricultural residues

The optimization studies for production of chitinase were carried out by response surface methodology (RSM) based on statistics experimental design using three substrates, which were wheat, rice and red gram bran. 2⁴ full factorial central composite design was applied to evaluate optimal combinations of variables. These variables were chitin concentration, initial moisture content, inoculum level, and incubation time. The results of second order polynomial showed that all four variables had significant effect on chitinase production. Maximum chitinase activity was recorded for wheat bran (2443.23 U g⁻¹) than rice (1216.65 U g⁻¹) and red gram bran (961.32 U g⁻¹). An overall 3-fold increase in chitinase activity was achieved using optimized strategies of RSM. Growth of the fungus on all bran particles have been visualized by scanning electron microscopy. These results indicated the potential of Penicillium ochrochloron for economical production of chitinase using agricultural residues. TLC and HPLC analysis of colloidal chitin hydrolysate with partially purified chitinases revealed that the major reaction product was monomeric GlcNAc indicating the potential of these enzymes for efficient production of GlcNAc.     

Evaluation of alpha-galactosidase biosynthesis by Streptomyces griseoloalbus in solid-state fermentation using response surface methodology

Aim: The present study was aimed at evaluating the effects of the three crucial factors, galactose concentration, inoculum size and moisture content, on α‐galactosidase production by the filamentous actinobacterium Streptomyces griseoloalbus in solid‐state fermentation. Methods and Results: Central Composite design was adopted to derive a statistical model for the optimization of fermentation conditions. Maximum α‐galactosidase yield (117 U g^–1 of dry fermented substrate) was obtained when soya bean flour supplemented with 1·5% galactose and with initial moisture content of 40% was inoculated with 1·9 × 10⁶ CFU g^?1 initial dry substrate. Conclusions: The model was valid and could result in considerably enhanced enzyme yield. Significance and Impact of the Study: The results indicated a cost effective method for the production of α‐galactosidase using soya bean flour. This is the first report on exploitation of the potential of filamentous bacterium for the production of α‐galactosidase, an enzyme having versatile applications.     

Comparative profiles of α-amylase production in conventional tray reactor and GROWTEK bioreactor

GROWTEK bioreactor was used as modified solid-state fermentor to circumvent many of the problems associated with the conventional tray reactors for solid-state fermentation (SSF). Aspergillus oryzae IFO-30103 produced very high levels of α-amylase by modified solid-state fermentation (mSSF) compared to SSF carried out in enamel coated metallic trays utilizing wheat bran as substrate. High α-amylase yield of 15,833 U g⁻¹ dry solid in mSSF were obtained when the fungus were cultivated at an initial pH of 6.0 at 32°C for 54 h whereas α-amylase production in SSF reached its maxima (12,899 U g⁻¹ dry solid ) at 30°C after 66 h of incubation. With the supplementation of 1% NaNO₃, the maximum activity obtained was 19,665 U g⁻¹ dry solid (24% higher than control) in mSSF, whereas, in SSF maximum activity was 15,480 U g⁻¹ dry solid in presence of 0.1% Triton X-100 (20% higher than the control).     

Production of red pigments byMonascus purpureus in solid-state culture

To maximize and sustain the productivity ofMonascus pigments, various environmental and nutritional parameters, such as the initial moisture content, pH, inoculum size, sample size, and nutrient supplement, that influence pigment production were evaluated in solid-state cultures as follows: initial moisture content, 50%; pH, 6.0; inoculum size 1 x 10⁴ spore cells (grams of dry solid substrate)⁻¹; sample size, 300 g. All supplementary nutrients (carbon, nitrogen, and mineral sources) added has inhibitory effects on the cell growth and red pigment production. In open tray culture the maximum biomass yield and specific productivity of red pigments were 223 mg DCW (grams of initial dry substrate)⁻¹ and, 47.6 OD₅₀₀ (DCW grams)⁻¹ h⁻¹, respectively.     

Production and optimization of thermophilic alkaline protease in solid-state fermentation by <Emphasis Type="Italic">Streptomyces</Emphasis> sp. CN902

The purpose of the present research is to study the production of thermophilic alkaline protease by a local isolate, Streptomyces sp. CN902, under solid state fermentation (SSF). Optimum SSF parameters for enzyme production have been determined. Various locally available agro-industrial residues have been screened individually or as mixtures for alkaline protease production in SSF. The combination of wheat bran (WB) with chopped date stones (CDS) (5:5) proved to be an efficient mixture for protease production as it gave the highest enzyme activity (90.50 U g⁻¹) when compared to individual WB (74.50 U g⁻¹) or CDS (69.50 U g⁻¹) substrates. This mixed solid substrate was used for the production of protease from Streptomyces sp. CN902 under SSF. Maximal protease production (220.50 U g⁻¹) was obtained with an initial moisture content of 60%, an inoculum level of 1 × 10⁸ (spore g⁻¹ substrate) when incubated at 45°C for 5 days. Supplementation of WB and CDS mixtures with yeast extract as a nitrogen source further increased protease production to 245.50 U g⁻¹ under SSF. Our data demonstrated the usefulness of solid-state fermentation in the production of alkaline protease using WB and CDS mixtures as substrate. Moreover, this approach offered significant benefits due to abundant agro-industrial substrate availability and cheaper cost.     

Chitinase Production in Solid-State Fermentation from <Emphasis Type="Italic">Oerskovia xanthineolytica</Emphasis> NCIM 2839 and its Application in Fungal Protoplasts Formation

The present study reports the economic production of thermostable chitinase production from Oerskovia xanthineolytica NCIM 2839 by solid-state fermentation (SSF) technique and its application in fungal protoplasts formation. The Oerskovia xanthineolytica NCIM 2839 was found to produce thermostable chitinase 148 U g⁻¹ of solid substrate in SSF using wheat bran with colloidal chitin as base. Protoplasts of A. niger were formed by using crude chitinase produced in SSF and formed protoplasts were confirmed by using scanning electron microscopy. This is the simple and economical method for protoplast formation which makes it possible applications in strain improvement of various fungi by protoplasts fusion in Biotechnological industries.     

Enrichment of laccase production by Phoma herbarum isolate KU4 under solid-state fermentation by optimizing RSM coefficients using genetic algorithm

The process parameters were optimized to obtain enhanced enzyme activity from the fungus Phoma herbarum isolate KU4 using rice straw and saw dust as substrate under solid-state fermentation using Response surface methodology (RSM). Genetic algorithm was used to validate the RSM for maximum laccase production. Six variables, viz., pH of the media, initial moisture content, copper sulphate concentration, concentration of tannic acid, inoculum concentration and incubation time were found to be effective and optimized for enhanced production. Maximum laccase production was achieved by RSM at pH 5·0 and 86% of initial moisture content of the culture medium, 150 µmol l⁻¹ of CuSO₄, 1·5% tannic acid and 0·128 g inoculum g⁻¹ dry substrate inoculum size on the fourth day of fermentation. The highest laccase activity was observed as 79 008 U g⁻¹, which is approximately sixfold enhanced production compared to the unoptimized condition (12 085·26 U g⁻¹).     

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 an Antifungal Chitinase from Enterobacter sp. NRG4 and its Application in Protoplast Production

Summary In this study flake chitin, crab shell chitin, mushroom stalk, fungal cell wall, wheat bran and rice bran were used as substrate for chitinase production by Enterobacter sp. NRG4 under submerged and solid state fermentation (SSF) conditions. Enterobacter sp. NRG4 produced 72 and 49.7 U/ml of chitinase in presence of cell walls of Candida albicans and Fusarium moniliforme in submerged fermentation. Under SSF, maximum chitinase production was 965 U/g solid substrate with flake chitin and wheat bran (1:3 ratio) at 75% moisture level after 144 h. The purified chitinase inhibited hyphal extension of Fusarium moniliforme, Aspergillus niger, Mucor rouxi and Rhizopus nigricans. The chitinase was effective in release of protoplasts from Trichoderma ressei, Pleurotus florida, Agaricus bisporus and Aspergillus niger. Protoplasts yield was maximum with 60 mg of 24 h old fungal mycelium incubated with 60 U of chitinase and 60 U of cellulase.     

Thermoactive β-N-acetylhexosaminidase production by a soil isolate of Penicillium monoverticillium CFR 2 under solid state fermentation: parameter optimization and application for N-acetyl chitooligosaccharides preparation from chitin

Two fungal strains were evaluated for β-N-acetylhexosaminidase production by solid state fermentation using different agro-industrial residues such as commercial wheat bran (CWB) and shrimp shell chitin waste (SSCW), of which Penicillium monoverticillium CFR 2 a local soil isolate showed significantly (P ≤ 0.001) higher β-N-acetylhexosaminidase activity on CWB medium as compared with the activity of Fusarium oxysporum CFR 8. Fermentation parameters such as incubation temperature, incubation time, initial moisture content and inoculum concentration were optimized by statistically designed experiments, using 3**(4–1) fractional factorial design of Response Surface Methodology. The high R² (0.9512) observed during validation experiment showed the usefulness of the model. Highest level of enzyme activity (311.84 U/g IDS) was predicted at 75% (w/w) initial moisture content, 26 °C incubation temperature, 168 h incubation time and initial inoculum, at the highest concentration tested (2.95 ml spore suspension/5 g substrate). Statistical optimization yielded a 4.5 fold increase in β-N-acetylhexosaminidase activity. The crude β-N-acetylhexosaminidase showed optimum temperature of 57 ± 1 °C and pH of 3.6 and retained 50% activity after 1 h of incubation at 57 ± 1 °C. SDS–PAGE zymogram revealed crude enzyme was a monomer with an apparent molecular weight ~110 kDa. The crude enzyme formed 6.81 ± 0.03 mM/l of N-acetyl chitooligosaccharides from colloidal chitin in 24 h of incubation. HPLC analysis revealed hydrolysate contained 37.57% N-acetyl chitotriose and 62.43% N-acetyl chitohexose, indicating its potential for specific N-acetyl chitooligosaccharides production.     

Cloning,characterization and expression of the chitinase gene of <Emphasis Type="Italic">Enterobacter</Emphasis> sp. NRG4

A chitinase producing bacterium Enterobacter sp. NRG4, previously isolated in our laboratory, has been reported to have a wide range of applications such as anti-fungal activity, generation of fungal protoplasts and production of chitobiose and N-acetyl D-glucosamine from swollen chitin. In this paper, the gene coding for Enterobacter chitinase has been cloned and expressed in Escherichia coli BL21(DE3). The structural portion of the chitinase gene comprised of 1686 bp. The deduced amino acid sequence of chitinase has high degree of homology (99.0%) with chitinase from Serratia marcescens. The recombinant chitinase was purified to near homogeneity using His-Tag affinity chromatography. The purified recombinant chitinase had a specific activity of 2041.6 U mg⁻¹. It exhibited similar properties pH and temperature optima of 5.5 and 45°C respectively as that of native chitinase. Using swollen chitin as a substrate, the K_m, k_cat and catalytic efficiency (k_cat/K_m) values of recombinant chitinase were found to be 1.27 mg ml⁻¹, 0.69 s⁻¹ and 0.54 s⁻¹M⁻¹ respectively. Like native chitinase, the recombinant chitinase produced medicinally important N-acetyl D-glucosamine and chitobiose from swollen chitin and also inhibited the growth of many fungi.     

Solid state fermentation for production of α-amylase by a thermotolerant Bacillus subtilis from hot-spring water

The production of extracellular α-amylase by thermotolerant Bacillus subtilis was studied in solid state fermentation (SSF). The effect of wheat bran (WB) and rice husk (RH) was examined. The appropriate incubation period, moisture level, particle size and inoculum concentration was determined. Maximum yields of 159,520 and 21,760 U g⁻¹ were achieved by employing WB and RH as substrates in 0.1 M phosphate buffer at pH 7 with 30% initial moisture content at 24 and 48 h. Particle size and inoculum concentration were found to be 1000 μm, 20% and 500 μm, 15% for WB and RH, respectively. Enzyme yield was 7.3-fold higher with WB medium compared with RH.     

Bioreactor application on adventitious root culture of <Emphasis Type="Italic">Astragalus membranaceus</Emphasis>

Astragalus membranaceus is one of the most widely used traditional medicinal herbs in China, but the time required to generate a useful product in the field production is long. The growth of adventitious root cultures was compared between cultures grown in solid, liquid, or a 5-L balloon-type bubble bioreactor. The maximum growth ratio (final dry weight/initial dry weight) was determined for adventitious roots grown in the bioreactor. Studies carried out to optimize biomass production of adventitious roots compared adventitious root growth from various inoculum root lengths, inoculum densities, and aeration volume in the bioreactors. The maximum growth ratio occurred in treatments with a 1.5-cm inoculum root length, with 30 g (fresh weight) of inoculum per bioreactor or with an aeration volume of 0.1 vvm (air volume/culture medium volume per min). The polysaccharide, saponin, and flavonoid content of roots from bioreactor-grown cultures were compared to roots from field-grown plants grown for 1 and 3 yr. Total polysaccharide content of adventitious roots in the bioreactor (30.0 mg g⁻¹ dry weight (DW)) was higher than the roots of 1-yr-old (13.8 mg g⁻¹ DW) and 3-yr-old (21.1 mg g⁻¹ DW) plants in the field. Total saponin (3.4 mg g⁻¹ DW) and flavonoid (6.4 mg g⁻¹ DW) contents were nearly identical to 3-yr-old roots and higher than that of 1-yr-old roots under field cultivation.     

Chitinase production by Beauveria felinaRD 101: optimization of parameters under solid substrate fermentation conditions

Major parameters affecting the production of chitinase by Beauveria felinaRD 101 under solid substrate fermentation conditions have been optimized. Wheat bran moistened with 100 MS-HCl medium adjusted to pH 5.0, inoculated with 1 × 10¹⁰ conidia g^–1 initial dry bran and incubated at 28 °C for 6 days produced maximum chitinase activity of 6.34 U g^–1 initial dry substrate.     

Production of laccase and manganese peroxidase by<Emphasis Type="Italic"> Fomes sclerodermeus</Emphasis> grown on wheat bran

The aim of this work was to study the growth and production of ligninolytic enzymes by Fomes sclerodermeus using a natural medium based on wheat bran as the principal substrate in a solid-state fermentation. Growth was monitored by measuring the chitin content in the substrate. The maximum rate of growth was observed between days 7 and 18. A 38% total dry-weight loss of the substrate was measured after 28 days of cultivation. Differential hydrolysis of the substrate revealed that cellulose was more extensively degraded than lignin. In the 28-day incubation period, the losses of cellulose and lignin were 38 and 15%, respectively. No lignin peroxidase activity was found in any of the media tested. The maximum manganese-dependent peroxidase activity recorded was 6.3 U g⁻¹ at 14 days, while the maximum laccase activity was 270 U g⁻¹ at 28 days post-inoculation. Addition of commonly used inducers such as copper or manganese did not produce a further increase in the enzyme activities, nor did addition of glucose, asparagine, or malt extract. Electronic Publication     

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).     

Influence of the carbon source on the growth and lignocellulolytic enzyme production by Morchella esculenta strains

Growth and lignocellulolytic enzymes production by two Morchella esculenta strains (BAFC 1728 and BEL 124) growing in solid state fermentation using different lignocellulosic materials along 58 days was characterized. Both strains were able to grow on the three substrates: wheat bran, wheat bran plus corn starch, and rolled oat. The growth was characterized by measuring chitin content, reducing sugars, pH, dry weight loss, and extractable proteins, such parameters varied substantially with substrate and strain used. The maximum rate of growth in both strains was observed between 5 and 28 days. Regarding enzyme production, as a general trend strain BAFC 1728 produced the highest titres. The most evident difference was observed in laccase production by this strain on wheat bran, which exceeded that observed in strain BEL 124 by tenfold (7.45 U g⁻¹).     

Exoglucanase production by Aspergillus niger grown on wheat bran

β-Exoglucanase production on the lignocellulosic material, wheat bran, by Aspergillus niger under solid state fermentation (SSF) on a laboratory scale was investigated. Different fermentation parameters, such as moisture content, initial pH, temperature, depth of the substrate, and inoculum size on exoglucanase production were optimized. Moisture content of 40 %, pH of 7.0, substrate depth of 1.0 cm, inoculum size of 2?×?10⁶ spores/g of wheat bran, and temperature at 30 °C were optimal for maximum production of exoglucanase. Maximum yields of exoglucanase with 28.60 FPU/g of wheat bran were obtained within 3 days of incubation under optimal conditions.