Improved Cellulase Production by <Emphasis Type="Italic">Trichoderma reesei </Emphasis>RUT C30 under SSF Through Process Optimization

The major constraint in the enzymatic saccharification of biomass for ethanol production is the cost of cellulase enzymes. Production cost of cellulases may be brought down by multifaceted approaches which includes the use of cheap lignocellulosic substrates for fermentation production of the enzyme, and the use of cost efficient fermentation strategies like solid state fermentation (SSF). The current study investigated the production of cellulase by Trichoderma reesei RUT C30 on wheat bran under SSF. Process parameters important in cellulase production were identified by a Plackett and Burman design and the parameters with significant effects on enzyme production were optimized for maximal yield using a central composite rotary design (CCD). Higher initial moisture content of the medium had a negative effect on production whereas incubation temperature influenced cellulase production positively in the tested range. Optimization of the levels of incubation temperature and initial moisture content of the medium resulted in a 6.2 fold increase in production from 0.605 to 3.8 U/gds of cellulase. The optimal combination of moisture and temperature was found to be 37.56% and 30 °C, respectively, for maximal cellulase production by the fungus on wheat bran.     

Phytase Production by Aspergillus oryzae in Solid-State Fermentation and its Applicability in Dephytinization of Wheat Ban

Aspergillus oryzae SBS50 secreted a high titre of phytase in solid-state fermentation (SSF) using wheat bran at 30 °C after 96 h at the initial substrate to moisture ratio of 1:2 and a water activity of 0.95. The production of phytase increased when wheat bran was supplemented with sucrose and beef extract. Further enhancement in enzyme production was recorded when the substrate was supplemented with the surfactant Triton X-100 (145 U/g of DMB). An overall 29-fold improvement in phytase production was achieved owing to optimization. Under optimized conditions, the mould secreted 9.3-fold higher phytase in SSF as compared to submerged fermentation (SmF). The mesophilic mould also secreted amylase, cellulase (CMCase), pectinase and xylanase along with phytase in SSF. Scanning electron microscopy revealed luxuriant growth of A. oryzae on wheat bran with abundant spores. The enzyme dephytinized wheat bran with concomitant liberation of inorganic phosphate.     

Xylanase production by the thermophilic mold Humicola lanuginosa in solid-state fermentation

Among the lignocellulosic substrates tested, wheat bran supported a high xylanase (EC 3.2.1.8) secretion by Humicola lanuginosa in solid-state fermentation (SSF). Enzyme production reached a peak in 72 h followed by a decline thereafter. Enzyme production was very high (7832 U/g of dry moldy bran) when wheat bran was moistened with tap water at a substrate-to-moistening agent ratio of 1:2.5 (w/v) and an inoculum level of 3 × 10⁶ spores/10 g of wheat bran at a water activity (a _w ) of 0.95. Cultivation of the mold in large enamel trays yielded a xylanase titer comparable with that in flasks. Parametric optimization resulted in a 31% increase in enzyme production in SSF. Xylanase production was approx 23-fold higher in SSF than in submerged fermentation (SmF). A threshold constitutive level of xylanase was secreted by H. lanuginosa in a medium containing glucose as the sole carbon source. The enzyme was induced by xylose and xylan. Enzyme synthesis was repressed beyond 1.0% (w/v) xylose in SmF, whereas it was unaffected up to 3.0% (w/w) in SSF, suggesting a minimization of catabolite repression in SSF.     

Utilization of Horticultural Waste for Laccase Production by <Emphasis Type="Italic">Trametes versicolor</Emphasis> Under Solid-State Fermentation

Horticultural waste collected from a landscape company in Singapore was utilized as the substrate for the production of laccase under solid-state fermentation by Trametes versicolor. The effects of substrate particle size, types of inducers, incubation temperature and time, initial medium pH value, and moisture content on laccase production were investigated. The optimum productivity of laccase (8.6 U/g substrate) was achieved by employing horticultural waste of particle size greater than 500 μm and using veratryl alcohol as the inducer. The culture was at 30 °C for 7 days at moisture content of solid substrate of 85% and initial pH 7.0. The decolorization was also investigated in order to assess the degrading capability of the ligninolytic laccase obtained in the above-mentioned cultures. The decolorization degree of a model dye, phenol red, was around 41.79% in 72 h of incubation. By far, this is the first report on the optimization of laccase production by T. versicolor under solid-state fermentation using horticultural waste as the substrate.     

Amylase Production by Saccharomycopsis fibuligera A11 in Solid-State Fermentation for Hydrolysis of Cassava Starch

The optimization of process parameters for high amylase production by Saccharomycopsis fibuligera A11 in solid-state fermentation was carried out using central composite design. Finally, the optimal parameters obtained with the response surface methodology (RSM) were moisture 610.0 ml/kg, inoculum 30.0 ml (OD_600 nm = 20.0)/kg, the amount ratio of wheat bran to rice husk 0.42, cassava starch concentration 20.0 g/kg, temperature 28 °C, and natural pH. Under the optimized conditions, 4,296 U/g of dry substrate of amylase activity was reached in the solid-state fermentation culture of the yeast strain A11 within 160 h, whereas the predicted maximum amylase activity of 4,222 U/g of dry substrate of amylase activity was derived from the RSM regression. It was found that cassava starch can be actively converted into monosaccharides and oligosaccharides by the crude amylase.     

Production of ligninolytic enzymes for dye decolorization by cocultivation of white-rot fungi Pleurotus ostreatus and Phanerochaete chrysosporium under solid-state fermentation

Lignocellulosic wastes such as neem hull, wheat bran, and sugarcane bagasse, available in abundance, are excellent substrates for the production of ligninolytic enzymes under solid-state fermentation by white-rot fungi. A ligninolytic enzyme system with high activity showing enhanced decomposition was obtained by cocultivation of Pleurotus ostreatus and Phanerochaete chrysosporium on combinations of lignocellulosic waste. Among the various substrate combinations examined, neem hull and wheat bran wastes gave the highest ligninolytic activity. A maximum production of laccase of 772 U/g and manganese peroxidase of 982 U/g was obtained on d 20 and lignin peroxidase of 656 U/g on d 25 at 28±1 °C under solid-state fermentation. All three enzymes thus obtained were partially purified by acetone fractionation and were exploited for decolorizing different types of acid and reactive dyes.     

High-yield bacillus subtilis protease production by solid-state fermentation

A Bacillus subtilis isolate was shown to be able to produce extracellular protease in solid-state fermentations (SSF) using soy cake as culture medium. A significant effect of inoculum concentration and physiological age on protease production was observed. Maximum activities were obtained for inocula consisting of exponentially growing cells at inoculum concentrations in the range of 0.7–2.0 mg g⁻¹. A comparative study on the influence of cultivation temperature and initial medium pH on protease production in SSF and in submerged fermentation (SF) revealed that in SSF a broader pH range (5–10), but the same optimum temperature (37°C), is obtained when compared to SF. A kinetic study showed that enzyme production is associated with bacterial growth and that enzyme inactivation begins before biomass reaches a maximum level for both SF and SSF. Maximum protease activity and productivity were 960 U g⁻¹ and 15.4 U g⁻¹ h⁻¹ for SSF, and 12 U mL⁻¹ and 1.3 U mL⁻¹ h⁻¹ for SF. When SSF protease activity was expressed by volume of enzyme extract, the enzyme level was 10-fold higher and the enzyme productivity 45% higher than in SF. These results indicate that this bacterial strain shows a high biotechnological potential for protease production in solid-state fermentation.     

Production of l-asparaginase, an anticancer agent, from Aspergillus niger using agricultural waste in solid state fermentation

This article reports the production of high levels of l-asparaginase from a new isolate of Aspergillus niger in solid state fermentation (SSF) using agrowastes from three leguminous crops (bran of Cajanus cajan, Phaseolus mungo, and Glycine max). When used as the sole source for growth in SSF, bran of G. max showed maximum enzyme production followed by that of P. mungo and C. cajan. A 96-h fermentation time under aerobic condition with moisture content of 70%, 30 min of cooking time and 1205–1405 μ range of particle size in SSF appeared optimal for enzyme production. Enzyme yield was maximum (40.9±3.35 U/g of dry substrate) at pH 6.5 and temperature 30±2°C. The optimum temperature and pH for enzyme activity were 40°C and 6.5, respectively. The study suggests that choosing an appropriate substrate when coupled with process level optimization improves enzyme production markedly. Developing an asparaginase production process based on bran of G. max as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries.     

Enhancement of L-Asparaginase Production by Isolated <Emphasis Type="Italic">Bacillus circulans</Emphasis> (MTCC 8574) Using Response Surface Methodology

L-asparaginase production was optimized using isolated Bacillus circulans (MTCC 8574) under solid-state fermentation (SSF) using locally available agricultural waste materials. Among different agricultural materials (red gram husk, bengal gram husk, coconut, and groundnut cake), red gram husk gave the maximum enzyme production. A wide range of SSF parameters were optimized for maximize the production of L-asparaginase. Preliminary studies revealed that incubation temperature, moisture content, inoculum level, glucose, and L-asparagine play a vital role in enzyme yield. The interactive behavior of each of these parameters along with their significance on enzyme yield was analyzed using fractional factorial central composite design (FFCCD). The observed correlation coefficient (R ²) was 0.9714. Only L-asparagine and incubation temperature, were significant in linear and quadratic terms. L-asparaginase yield improved from 780 to 2,322 U/gds which is more than 300% using FFCCD as a means of optimizing conditions.     

Fungi allergens produced by solid-state fermentation process

Allergenic extracts were produced from Drechslera (Helminthosporium) monoceras biomass cultured by solid-state fermentation using wheat bran as the substrate. The main fermentation variables were selected by statistical design, and the optimized biomass yield (1.43 mg/[g of dry substrate · d]) was obtained at pH 9.5 and 45.8% moisture. The allergenic extracts were produced from crude extract by protein precipitation and polyphenol removal. Proteins in the range of 16–160 kDa were identified in the extracts. Their reactions in patients were characterized by in vivo cutaneous tests (positive in 40% of the atopic patients) and by dot-blotting assays.     

Optimization of Spore and Antifungal Lipopeptide Production During the Solid-state Fermentation of <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Bacillus subtilis strain TrigoCor 1448 was grown on wheat middlings in 0.5-l solid-state fermentation (SSF) bioreactors for the production of an antifungal biological control agent. Total antifungal activity was quantified using a 96-well microplate bioassay against the plant pathogen Fusarium oxysporum f. sp. melonis. The experimental design for process optimization consisted of a 2⁶⁻¹ fractional factorial design followed by a central composite face-centered design. Initial SSF parameters included in the optimization were aeration, fermentation length, pH buffering, peptone addition, nitrate addition, and incubator temperature. Central composite face-centered design parameters included incubator temperature, aeration rate, and initial moisture content (MC). Optimized fermentation conditions were determined with response surface models fitted for both spore concentration and activity of biological control product extracts. Models showed that activity measurements and spore production were most sensitive to substrate MC with highest levels of each response variable occurring at maximum moisture levels. Whereas maximum antifungal activity was seen in a limited area of the design space, spore production was fairly robust with near maximum levels occurring over a wider range of fermentation conditions. Optimization resulted in a 55% increase in inhibition and a 40% increase in spore production over nonoptimized conditions.     

Remediation of Textile Dye Waste Water Using a White-Rot Fungus Bjerkandera adusta Through Solid-state Fermentation (SSF)

A strict screening strategy for microorganism selection was followed employing a number of white-rot fungi for the bioremediation of textile effluent, which was generated from one Ireland-based American textile industry. Finally, one fungus Bjerkandera adusta has been investigated in depth for its ability to simultaneously degrade and enrich the nutritional quality of highly coloured textile effluent-adsorbed barley husks through solid-state fermentation (SSF). Certain important parameters such as media requirements, moisture content, protein/biomass production and enzyme activities were examined in detail. A previously optimised method of dye desorption was employed to measure the extent of dye remediation through effluent decolorisation achieved as a result of fungal activity in SSF. B. adusta was capable of decolourising a considerable concentration of the synthetic dye effluent (up to 53%) with a moisture content of 80-85%. Protein enrichment of the fermented mass was achieved to the extent of 229 g/kg dry weight initial substrate used. Lignin peroxidase and laccase were found to be the two main enzymes produced during SSF of the dye-adsorbed lignocellulosic waste residue.     

Production of lovastatin by wild strains of Aspergillus terreus

A wild fungal strain of Aspergillus terreus, labeled as PM3, was isolated by using the Candida albicans bioassay and confirmed by 18S r DNA analyses. Lovastatin was produced by submerged and solid state fermentations. Of the 30 isolated fungal strains, 11 showed lovastatin production with Aspergillus terreus PM3 being the best with a yield of 240 mg/L at the 10th day of submerged fermentation. Carboxymethylcellulose had a stimulatory effect on lovastatin production. It restricted uncontrolled filamentous growth, induced pellet formation and, thereby, improved lovastatin yield. In solid state fermentation (SSF), of the agro wastes from five crops (bran of wheat and rice, husks of red gram and soybean, and green gram straw), wheat bran showed maximum lovastatin production (12.5 mg/g of dry substrate) at pH 7.1 and a temperature of 30 +/- 2 degrees C. Development of a lovastatin production process based on wheat bran as a substrate in SSF is economically attractive as it is a cheap and readily available raw material in agriculture-based countries.     

Optimization of Influential Parameters for Extracellular Keratinase Production by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> (MTCC9102) in Solid State Fermentation Using Horn Meal—A Biowaste Management

A Bacillus subtilis (MTCC9102) isolate was shown to produce significant amount of keratinase under optimized conditions in solid-state fermentation using Horn meal as a substrate. Optimized value for moisture, inoculum, and aeration were found to be 100% (v/w), 50% (v/w), and 150% (w/w), respectively, and the optimum nitrogen source was peptone and carbon source was dextrose. Maximum keratinolytic activity was observed at 48 h after incubation, and the optimum age (24 h) of inoculum was significant. The influence of cultivation temperature and initial pH of the medium on keratinase production revealed the optimum values for the temperature and pH as 37 °C and 7, respectively. Maximum keratinase activity of the crude extract was 15,972 U/mg/ml. These results indicate that this bacterial strain shows a high biotechnological potential for keratinase production in solid-state fermentation, and use of the horn meal as the substrate can be implemented for keratinous solid wastes management.     

Xylanase Production by <Emphasis Type="Italic">Penicillium canescens</Emphasis> on Soya Oil Cake in Solid-State Fermentation

There is an increasing interest for the organic residues from various sectors of agriculture and industries over the past few decades. Their application in the field of fermentation technology has resulted in the production of bulk chemicals and value-added products such as amino acid, enzymes, mushroom, organic acids, single-cell protein, biologically active secondary metabolites, etc. (Ramachandran et al., Bioresource Technology 98:2000–2009, 2007). In this work, the production of extracellular xylanase by the fungus Penicillium canescens was investigated in solid-state fermentation using five agro-industrial substrates (soya oil cake, soya meal, wheat bran, whole wheat bran, and pulp beet). The best substrate was the soya oil cake. In order to optimize the production, the most effective cultivation conditions were investigated in Erlenmeyer flasks and in plastic bags with 5 and 100 g of soya oil cake, respectively. The initial moisture content, initial pH, and temperature of the culture affected the xylanase synthesis. The optimal fermentation medium was composed by soya oil cake crushed to 5 mm supplemented with 3% and 4% (w/w) of casein peptone and Na₂HPO₄.2H₂O. After 7 days of incubation at 30 °C and under 80% of initial moisture, a xylanase production level of 18,895 ± 778 U/g (Erlenmeyer flasks) and 9,300 ± 589 U/g (plastic bags) was reached. The partially purified enzyme recovered by ammonium sulfate fractionation was completely stable at freezing and refrigeration temperatures up to 6 months and reasonably stable at room temperature for more than 3 months.     

Intraspecific Diversity within <Emphasis Type="Italic">Ganoderma lucidum</Emphasis> in the Production of Laccase and Mn-Oxidizing Peroxidases During Plant Residues Fermentation

Comparison of the potential for laccase and Mn-oxidizing peroxidases synthesis by ten strains of Ganoderma lucidum, originating from different worldwide areas, during solid-state fermentation of selected plant raw materials was the aim of this study. The great intraspecific variability in the production of analyzed enzymes as well as the dependence of the enzyme activity on plant raw materials were reported. The strain HAI 957 was the best laccase producer in the presence of corn stem, as a unique carbon source (129.46 U/L). The highest level of Mn-dependent peroxidase activity was noted after wheat straw fermentation by G. lucidum HAI 246 (78.64 U/L), while the maximal versatile peroxidase production (59.72 U/L) was observed in strain HAI 957 in the medium with oak sawdust.     

Efficient production of arachidonic acid of <Emphasis Type="Italic">Mortierella</Emphasis> sp. by solid-state fermentation using combinatorial medium with spent mushroom substrate

Solid-state fermentation (SSF) is a bioconversion process for turning cheap agro-industrial materials to added-value products. For enrichment of agro-industrial materials with arachidonic acid (ARA; C20:4 n-6), SSF process of Mortierella sp. was developed by optimizing cultivation medium and parameters. The results showed that the fungal cultivation on the medium with optimal ratio of selected agricultural materials provided the fermented mass containing high ARA proportion of total fatty acid. Inclusion of the optimal medium with suitable amount of spent mushroom substrate, which was used as an internal support, significantly promoted the ARA production yield. Using the predicted quadratic model generated by Box–Behnken design, the maximal ARA production yield was achieved, thereby the fermentation parameter set for ARA production was experimentally validated using the developed medium formula. Of variables studied, the culture temperature and initial moisture content were important for the ARA production. The developed SSF process would provide a prospect for larger scale production of ARA by this fungal strain.     

Improvement on Citric Acid Production in Solid-state Fermentation by <Emphasis Type="Italic">Aspergillus niger</Emphasis> LPB BC Mutant Using Citric Pulp

Citric acid (CA) production has been conducted through a careful strain selection, physical–chemical optimization and mutation. The aim of this work was to optimize the physical–chemical conditions of CA production by solid-state fermentation (SSF) using the Aspergillus niger LPB BC strain, which was isolated in our laboratory. The parental and mutant strain showed a good production of CA using citric pulp (CP) as a substrate. The physical–chemical parameters were optimized and the best production was reached at 65% moisture, 30 °C and pH 5.5. The influence of the addition of commercial and alternative sugars, nitrogen sources, salts, and alcohols was also studied. The best results (445.4 g of CA/kg of CP) were obtained with sugarcane molasses and 4% methanol (v/w). The mutagenesis induction of LPB BC was performed with UV irradiation. Eleven mutant strains were tested in SSF where two mutants showed a higher CA production when compared to the parental strain. A. niger LPB B3 produced 537.6 g of CA/kg of CP on the sixth day of fermentation, while A. niger LPB B6 produced 616.5 g of CA/kg of CP on the fourth day of fermentation, representing a 19.5% and 37% gain, respectively.     

Keratinase Production by Endophytic <Emphasis Type="Italic">Penicillium</Emphasis> spp. Morsy1 Under Solid-State Fermentation Using Rice Straw

Among all endophytic keratinolytic fungal isolates recovered from marine soft coral Dendronephthya hemprichii, Penicillium spp. Morsy1 was selected as the hyperactive keratinolytic strain under solid substrate fermentation of different agriculture and poultry wastes. The optimization of extraction process, physicochemical parameters affecting the keratinase production in solid-state fermentation, and the purified keratinase parameters were studied. Maximum keratinase activity (1,600 U g⁻¹, initial dry substrate) was recovered from moldy bran with 0.1% Tween 80. The optimized production conditions were rice straw as carbon source, pH of medium 6, growth temperature 26 °C, initial moisture content of 80% (v/w), inoculum size of 10⁵ spores ml⁻¹, and an average particle size of the substrate 0.6 mm (3,560 U g⁻¹, initial dry substrate after 5 days of fermentation). Two types of keratinase (Ahm1 and Ahm2) were purified from the culture supernatant through ammonium sulfate precipitation, DEAE-Sepharose, and gel filtration chromatography. Enzyme molecular weights were 19 kDa (Ahm1) and 40 kDa (Ahm2). The kinetic parameters of purified keratinases were optimized for the hydrolysis of azokeratin by Ahm1 (pH 7.0–8.0, stable in pH range of 6.0 to 8.0 at 50 °C) and Ahm2 enzymes (pH 10.0–11.0, stable in pH range of 6.0 to 11.0 at 60–65 °C). Whereas inhibitors of serine (phenylmethylsulfonyl fluoride) and cysteine (iodoacetamide) proteases had minor effects on both Ahm1 and Ahm2 activity, both keratinases were strongly inhibited by chelating agents EDTA and EGTA. These findings suggest that serine and cysteine residues are not involved in the catalytic mechanisms, and they are metalloproteases.     

High-Yield Hypocrellin A Production in Solid-State Fermentation by <Emphasis Type="Italic">Shiraia</Emphasis> sp. SUPER-H168

Hypocrellin A production by Shiraia sp. SUPER-H168 was studied under solid-state fermentation. Corn was found to be the best substrate after evaluating eight kinds of agro-industrial crops and residues. The optimized solid-state fermentation conditions were as follows: inoculum size 3 × 10⁶ spores, substrate particle size 0.8–1 mm, initial moisture content 50%, and temperature 30 °C. Six kinds of external carbon source and seven kinds of external nitrogen source were evaluated, respectively, for HA production. Glucose and NaNO₃ were the best. The combination of them was optimized by the response surface method. The optimum compositions of the supplementary glucose and NaNO₃ were 1.65 g/100 g and 0.43 g/L, respectively. Hypocrellin A production reached 4.7 mg/g.