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.     

Precursor Supply Strategy for Tetramethylpyrazine Production by Bacillus Subtilis on Solid-State Fermentation of Wheat Bran

Tetramethylpyrazine (TTMP) is a widely used flavoring additive with a nutty and roasted taste. Solid-state fermentation (SSF) of wheat bran for producing TTMP was studied with Bacillus subtilis CCTCC M208157, which was an exogenous precursor-independent TTMP-producing strain. Factors influencing endogenous precursor supply and TTMP formation in this strain were investigated. According to the findings, glucose and diammonium phosphate contributed to TTMP production but excess salts caused an inhibition on cell growth and TTMP formation. Then a two-step supply strategy was applied: 10 % glucose was added at the beginning of the process to allow acetoin formation, which was the precursor of TTMP, while 3 % diammonium phosphate was added only after acetoin accumulation reached its maximum. By applying this strategy, acetoin increased from 5.44 to 13.2 g/kg dry substrate (kgds), and consequently the yield of TTMP increased by 6.8 folds from 0.44 to 3.01 g/kgds. This was the first report of using a two-step supply strategy for TTMP production by SSF, which proved to be conducive to TTMP production in this strain.     

Lipase Production in Solid-State Fermentation Monitoring Biomass Growth of Aspergillus niger Using Digital Image Processing

The aim of this study was to monitor the biomass growth of Aspergillus niger in solid-state fermentation (SSF) for lipase production using digital image processing technique. The strain A. niger 11T53A14 was cultivated in SSF using wheat bran as support, which was enriched with 0.91% (m/v) of ammonium sulfate. The addition of several vegetable oils (castor, soybean, olive, corn, and palm oils) was investigated to enhance lipase production. The maximum lipase activity was obtained using 2% (m/m) castor oil. In these conditions, the growth was evaluated each 24 h for 5 days by the glycosamine content analysis and digital image processing. Lipase activity was also determined. The results indicated that the digital image process technique can be used to monitor biomass growth in a SSF process and to correlate biomass growth and enzyme activity. In addition, the immobilized esterification lipase activity was determined for the butyl oleate synthesis, with and without 50% v/v hexane, resulting in 650 and 120 U/g, respectively. The enzyme was also used for transesterification of soybean oil and ethanol with maximum yield of 2.4%, after 30 min of reaction.     

Microwave Pretreatment of Substrates for Cellulase Production by Solid-State Fermentation

The agricultural residues, wheat bran and rice hulls, were used as substrates for cellulase production with Trichoderma sp 3.2942 by solid-state fermentation. Microwave irradiation was employed to pretreat the substrates in order to increase the susceptibility. Although the highest cellulase yield was obtained by the substrates pretreated by 450 W microwave for 3 min, pretreatment time and microwave power had no significant effect on cellulase production. The initial reducing sugar content (RSC) of substrates was decreased by microwave irradiation, but more reducing sugars were produced in later fermentation. Alkali pretreatment combined with microwave pretreatment (APCMP) of rice hulls could significantly increase cellulase yields and reducing sugar. The maximum filter paper activity, carboximethylcellulase (CMC)ase, and RSC were increased by 35.2%, 21.4%, and 13%, respectively, compared with those of untreated rice hulls. The fermented residues could produce more cellulase and reducing sugars than fresh rice hulls after they were treated by APCMP. The increased accessibility of the substrates by microwave pretreatment was mainly achieved by rupture of the rigid structure of rice hulls. However, for alkali pretreatment and APCMP, delignification and removal of ash played very important roles for increasing the acceptability of substrates.     

Production, Purification, and Characterization of Exoglucanase by Aspergillus fumigatus

Fungi are considered good producers of industrially valuable enzymes with higher enzymatic activities. Among these cellulases are group of extracellular enzymes commonly employed in many industries for the hydrolysis of cellulolytic material. Aspergillus fumigatus produced exoglucanase having high enzymatic activity (83 U/gds) during the solid-state fermentation of wheat straw under optimum physical and nutritional conditions. Maximum production was obtained after 72 h of fermentation, at 55 °C temperature, pH 5.5, 80 % moisture level, and 2 mL fungal inoculum. Production was further increased by the addition of fructose (0.3 %) as additional carbon source, peptone (0.4 %) as nitrogen source, Tween-80 (0.3 %) as surfactant, and ammonium sulfate (0.2 %) in media. Exoglucanase was 2.30-folds purified by adding 40 % ammonium sulfate with volumetric activity 95.4 U/gds and specific activity 14.74 U/mg. Further, it was 5.18-folds purified by gel filtration chromatography with volumetric activity 115.2 U/gds and specific activity 33.10 U/mg. Purified exoglucanase has maximum activity at 55 °C and pH 4.8 using 1 % Avicel aqueous solution as substrate. The K _m and V _max were 4.34 mM and 7.29 μM/min, respectively. Calcium, magnesium, and zinc ions have positive effect on exoglucanase activity.     

Enzymatic Hydrolysis and Ethanol Fermentation of High Dry Matter Wet-Exploded Wheat Straw at Low Enzyme Loading

Wheat straw was pretreated by wet explosion using three different oxidizing agents (H₂O₂, O₂, and air). The effect of the pretreatment was evaluated based on glucose and xylose liberated during enzymatic hydrolysis. The results showed that pretreatment with the use of O₂ as oxidizing agent was the most efficient in enhancing overall convertibility of the raw material to sugars and minimizing generation of furfural as a by-product. For scale-up of the process, high dry matter (DM) concentrations of 15–20% will be necessary. However, high DM hydrolysis and fermentation are limited by high viscosity of the material, higher inhibition of the enzymes, and fermenting microorganism. The wet-explosion pretreatment method enabled relatively high yields from both enzymatic hydrolysis and simultaneous saccharification and fermentation (SSF) to be obtained when performed on unwashed slurry with 14% DM and a low enzyme loading of 10 FPU/g cellulose in an industrial acceptable time frame of 96 h. Cellulose and hemicellulose conversion from enzymatic hydrolysis were 70 and 68%, respectively, and an overall ethanol yield from SSF was 68%.     

Examining the Potential of Plasma-Assisted Pretreated Wheat Straw for Enzyme Production by <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

Plasma-assisted pretreated wheat straw was investigated for cellulase and xylanase production by Trichoderma reesei fermentation. Fermentations were conducted with media containing washed and unwashed plasma-assisted pretreated wheat straw as carbon source which was sterilized by autoclavation. To account for any effects of autoclavation, a comparison was made with unsterilized media containing antibiotics. It was found that unsterilized washed plasma-assisted pretreated wheat straw (which contained antibiotics) was best suited for the production of xylanases (110 IU ml⁻¹) and cellulases (0.5 filter paper units (FPU) ml⁻¹). Addition of Avicel boosted enzyme titers with the highest cellulase titers (1.5 FPU ml⁻¹) found with addition of 50 % w/w Avicel and with the highest xylanase production (350 IU ml⁻¹) reached in the presence of 10 % w/w Avicel. Comparison with enzyme titers from other nonrefined feedstocks suggests that plasma pretreated wheat straw is a promising and suitable substrate for cellulase and hemicellulase production.     

Cellulase Production by Aspergillus japonicus URM5620 Using Waste from Castor Bean (Ricinus communis L.) Under Solid-State Fermentation

The activity of β-glucosidase (βG), total cellulase (FPase) and endoglucanase (CMCase), produced by Aspergillus japonicus URM5620, was studied on solid-state fermentation using castor bean meal as substrate. The effect of the substrate amount, initial moisture, pH, and temperature on cellulase production was studied using a full factorial design (2(4)). The maximum βG, FPase, and CMCase activity was 88.3, 953.4, and 191.6 U/g dry substrate, respectively. The best enzyme activities for all three enzymes were obtained at the same conditions with 5.0 g of substrate, initial moisture 15% at 25 °C and pH 6.0 with 120 h of fermentation. The optimum activity for FPase and CMCase was found at pH 3.0 at an optimum temperature of 50 °C for FPase and of 55 °C for CMCase. The cellulases were stable in the range of pH 3.0-10.0 at 50 °C temperature. The enzyme production optimization demonstrated clearly the impact of the process parameters on the yield of the cellulolytic enzymes.     

Ethanol Production from Wet-Exploded Wheat Straw Hydrolysate by Thermophilic Anaerobic Bacterium Thermoanaerobacter BG1L1 in a Continuous Immobilized Reactor

Thermophilic ethanol fermentation of wet-exploded wheat straw hydrolysate was investigated in a continuous immobilized reactor system. The experiments were carried out in a lab-scale fluidized bed reactor (FBR) at 70°C. Undetoxified wheat straw hydrolysate was used (3–12% dry matter), corresponding to sugar mixtures of glucose and xylose ranging from 12 to 41 g/l. The organism, thermophilic anaerobic bacterium Thermoanaerobacter BG1L1, exhibited significant resistance to high levels of acetic acid (up to 10 g/l) and other metabolic inhibitors present in the hydrolysate. Although the hydrolysate was not detoxified, ethanol yield in a range of 0.39–0.42 g/g was obtained. Overall, sugar efficiency to ethanol was 68–76%. The reactor was operated continuously for approximately 143 days, and no contamination was seen without the use of any agent for preventing bacterial infections. The tested microorganism has considerable potential to be a novel candidate for lignocellulose bioconversion into ethanol. The work reported here also demonstrates that the use of FBR configuration might be a viable approach for thermophilic anaerobic ethanol fermentation.     

Potential of fluorescence polarization immunoassay for the detection of Aspergillus fumigatus galactomannan

Russian Chemical Bulletin - Galactomannan (GM) is a specific polysaccharide antigen of opportunistic mold fungi of the genus Aspergillus. The detection of GM in patients’ biological fluids is...     

Banana Peel: A Potential Substrate for Laccase Production by <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> VkJ2.4.5 in Solid-State Fermentation

In solid-state fermentation, among various solid supports evaluated, banana peel was found to be an ideal support and resulted into higher levels of laccase (6281.4 ± 63.60 U l⁻¹) along with notable levels of manganese peroxidase production (1339.0 ± 131.23 U l⁻¹) by Aspergillus fumigatus VkJ2.4.5. Maximum levels of laccase was achieved under derived conditions consisting of 80% of moisture level, 6 days of incubation period, 6% inoculum level, and an aeration level of 2.5 l min⁻¹. A column-tray bioreactor was designed to scale up and economize the enzyme production in three successive cycles of fermentation using the same fungal biomass. Thermal and pH stability profiles revealed that enzyme was stable up to 50°C and at varying pH range from 5–9 for up to 2 h. The apparent molecular weight of laccase was found to be 34 ± 1 kDa. MALDI-TOF/TOF analysis of the protein showed significant homology with maximum identity of 67% to other laccases reported in database.     

Fermentation of Biologically Pretreated Wheat Straw for Ethanol Production: Comparison of Fermentative Microorganisms and Process Configurations

The pretreatment of lignocellulosic biomass with white-rot fungi to produce bioethanol is an environmentally friendly alternative to the commonly used physico-chemical processes. After biological pretreatment, a solid substrate composed of cellulose, hemicellulose and lignin, the two latter with a composition lower than that of the initial substrate, is obtained. In this study, six microorganisms and four process configurations were utilised to ferment a hydrolysate obtained from wheat straw pretreated with the white-rot fungus Irpex lacteus. To enhance total sugars utilisation, five of these microorganisms are able to metabolise, in addition to glucose, most of the pentoses obtained after the hydrolysis of wheat straw by the application of a mixture of hemicellulolytic and cellulolytic enzymes. The highest overall ethanol yield was obtained with the yeast Pachysolen tannophilus. Its application in combination with the best process configuration yielded 163 mg ethanol per gram of raw wheat straw, which was between 23 and 35 % greater than the yields typically obtained with a conventional bioethanol process, in which wheat straw is pretreated using steam explosion and fermented with the yeast Saccharomyces cerevisiae.     

Plasma-Assisted Pretreatment of Wheat Straw for Ethanol Production

The potential of wheat straw for ethanol production after pretreatment with O(3) generated in a plasma at atmospheric pressure and room temperature followed by fermentation was investigated. We found that cellulose and hemicellulose remained unaltered after ozonisation and a subsequent washing step, while lignin was degraded up to 95% by O(3). The loss of biomass after washing could be explained by the amount of lignin degraded. The washing water of pretreated samples (0-7 h) was analyzed for potential fermentation inhibitors. Approximately 30 lignin degradation products and a number of simple carboxylic acids and phenolic compounds were found, e.g., vanillic acid, acetic acid, and formic acid. Some components had the highest concentration at the beginning of the ozonisation process (0.5, 1 h), e.g., 4-hydroxybenzladehyde, while the concentration of others increased during the entire pretreatment (0-7 h), e.g., oxalic acid and acetovanillon. Interestingly, washing had no effect on the ethanol production with pretreatment times up to 1 h. Washing improved the glucose availability with pretreatment times of more than 2 h. One hour of ozonisation was found to be optimal for the use of washed and unwashed wheat straw for ethanol production (maximum ethanol yield, 52%). O(3) cost estimations were made for the production of ethanol at standard conditions.     

Optimization of β-1,4-Endoxylanase Production by an Aspergillus niger Strain Growing on Wheat Straw and Application in Xylooligosaccharides Production

Plant biomass constitutes the main source of renewable carbon on the planet. Its valorization has traditionally been focused on the use of cellulose, although hemicellulose is the second most abundant group of polysaccharides on Earth. The main enzymes involved in plant biomass degradation are glycosyl hydrolases, and filamentous fungi are good producers of these enzymes. In this study, a new strain of Aspergillus niger was used for hemicellulase production under solid-state fermentation using wheat straw as single-carbon source. Physicochemical parameters for the production of an endoxylanase were optimized by using a One-Factor-at-a-Time (OFAT) approach and response surface methodology (RSM). Maximum xylanase yield after RSM optimization was increased 3-fold, and 1.41- fold purification was achieved after ultrafiltration and ion-exchange chromatography, with about 6.2% yield. The highest activity of the purified xylanase was observed at 50 °C and pH 6. The enzyme displayed high thermal and pH stability, with more than 90% residual activity between pH 3.0–9.0 and between 30–40 °C, after 24 h of incubation, with half-lives of 30 min at 50 and 60 °C. The enzyme was mostly active against wheat arabinoxylan, and its kinetic parameters were analyzed (K_m = 26.06 mg·mL⁻¹ and V_max = 5.647 U·mg⁻¹). Wheat straw xylan hydrolysis with the purified β-1,4 endoxylanase showed that it was able to release xylooligosaccharides, making it suitable for different applications in food technology.     

Production of Alkaline Protease by Bacillus altitudinis GVC11 using Castor Husk in Solid-State Fermentation

Castor (Ricinus communis L.) is an important oil seed crop having its main cultivated area in India, China, and Brazil in dry land farming. Castor husk is generated as waste in castor oil production. Use of castor husk waste as substrate is studied for alkaline protease production by Bacillus altitudinis GVC11 in solid-state fermentation. Various parameters like moisture content, incubation period, particle size, effect of carbon and nitrogen sources are studied and optimized for enzyme production. Highest enzyme production of 419,293 units per gram husk is obtained. Cost of enzyme production can be reduced by using castor husk as substrate.     

Effect of Biosurfactants on Laccase Production and Phenol Biodegradation in Solid-State Fermentation

The effects of two biosurfactants, tea saponin (TS) and rhamnolipid (RL), on the production of laccase and the degradation of phenol by P. simplicissimum were investigated in solid-state fermentation consisting of rice straw, rice bran, and sawdust. Firstly, the effects of phenol on the fermentation process were studied in the absence of surfactants. Then, a phenol concentration of 3 mg/g in the fermentation was selected for detailed research with the addition of biosurfactants. The results showed that TS and RL at different concentrations had stimulative effects on the enzyme activity of laccase. The highest laccase activities during the fermentation were enhanced by 163.7%, 68.2%, and 23.3% by TS at concentrations of 0.02%, 0.06%, and 0.10%, respectively. As a result of the enhanced laccase activity, the efficiency of phenol degradation was also improved by both biosurfactants. RL caused a significant increase of fungal biomass in the early stage of the fermentation, while TS had an inhibitory effect in the whole process. These results indicated that RL could mitigate the negative effects of phenol on fungal growth and consequently improve laccase production and phenol degradation. TS was potentially applicable to phenol-polluted solid-state fermentation.     

Production of Thermostable Lipase by Thermomyces lanuginosus on Solid-State Fermentation: Selective Hydrolysis of Sardine Oil

A naturally immobilized biocatalyst with lipase activity was produced by Thermomyces lanuginosus on solid-state fermentation with perlite as inert support. Maxima lipase activities (22 and 120 U/g of dry matter, using p-nitrophenyl octanoate and trioctanoine, respectively, as substrates) were obtained after 72 h of solid culture, remaining nearly constant up to 120 h. Maxima lipase activity was found at 60 to 85 °C and pH 10. The biocatalyst was stable at 60 °C for at least 4 h of incubation and a pH from 7 to 10. Energy values of activation and deactivation of lipase were of 26 and 6.7 kJ/mol, respectively. The biocatalyst shows high selectivity for the release of the omega-3 polyunsaturated fatty acids, eicosapentaenoic (EPA) and docosahexaenoic acids (DHA), during the hydrolysis of sardine oil. The EPA/DHA ratio (16:6) released by this biocatalyst was superior to that obtained with the commercial preparations of T. lanuginosus.     

Optimization of Xylanase Production by Filamentous Fungi in Solid-State Fermentation and Scale-up to Horizontal Tube Bioreactor

Five microorganisms, namely Aspergillus niger CECT 2700, A. niger CECT 2915, A. niger CECT 2088, Aspergillus terreus CECT 2808, and Rhizopus stolonifer CECT 2344, were grown on corncob to produce cell wall polysaccharide-degrading enzymes, mainly xylanases, by solid-state fermentation (SSF). A. niger CECT 2700 produced the highest amount of xylanases of 504?±?7 U/g dry corncob (dcc) after 3 days of fermentation. The optimization of the culture broth (5.0 g/L NaNO₃, 1.3 g/L (NH₄)₂SO₄, 4.5 g/L KH₂PO₄, and 3 g/L yeast extract) and operational conditions (5 g of bed loading, using an initial substrate to moistening medium of 1:3.6 (w/v)) allowed increasing the predicted maximal xylanase activity up to 2,452.7 U/g dcc. However, different pretreatments of materials, including destarching, autoclaving, microwave, and alkaline treatments, were detrimental. Finally, the process was successfully established in a laboratory-scale horizontal tube bioreactor, achieving the highest xylanase activity (2,926 U/g dcc) at a flow rate of 0.2 L/min. The result showed an overall 5.8-fold increase in xylanase activity after optimization of culture media, operational conditions, and scale-up.     

Enhanced Saccharification of Biologically Pretreated Wheat Straw for Ethanol Production

The biological pretreatment of lignocellulosic biomass with white-rot fungi for the production of bioethanol is an alternative to the most used physico-chemical processes. After biological treatment, a solid composed of cellulose, hemicellulose, and lignin—this latter is with a composition lower than that found in the initial substrate—is obtained. On the contrary, after applying physico-chemical methods, most of the hemicellulose fraction is solubilized, while cellulose and lignin fractions remain in the solid. The optimization of the combination of cellulases and hemicellulases required to saccharify wheat straw pretreated with the white-rot fungus Irpex lacteus was carried out in this work. The application of the optimal dosage made possible the increase of the sugar yield from 33 to 54 %, and at the same time the reduction of the quantity of enzymatic mixture in 40 %, with respect to the initial dosage. The application of a pre-hydrolysis step with xylanases was also studied.