Rosangela Donizete Perpetua Buzon Pirota

Improving the extraction conditions of endoglucanase produced by Aspergillus niger under solid-state fermentation

Production of cellulases under solid-state fermentation (SSF) is a promising technique that can help to reduce costs. Besides optimizing the production process, it is also important to consider enzyme recovery during the extraction step. Here, an experimental design methodology was used to investigate the effects of the operational parameters solid to liquid ratio (1:3, 1:6 and 1:9), stirring rate (80, 120 and 160 rpm), and temperature (10, 22 and 35 °C) on the recovery of endoglucanases produced by Aspergillus niger cultivated under SSF. Statistical analysis revealed that only the solid to liquid ratio had a significant influence on endoglucanase extraction. The highest endoglucanase recovery (35.7 U/g) was achieved using 0.2 mol/L acetate buffer at pH 4.8, together with a solid to liquid ratio of 1:9 and an agitation time of 10 minutes. In sequential extraction experiments, it was shown that most of the enzyme was recovered during the first extraction. The procedure adopted increased the efficiency of endoglucanase extraction by 70%, emphasizing the importance of selection of suitable operational conditions during SSF processes.

Saccharification of biomass using whole solid‐state fermentation medium to avoid additional separation steps

The enzymatic hydrolysis of steam‐exploded sugarcane bagasse (SESB) was investigated using enzymatic extracts (EE) and whole fermentation media (WM), produced in‐house, from Aspergillus niger 3T5B8 and Trichoderma reesei Rut‐C30 cultivated on wheat bran under solid‐state fermentation (SSF). A detailed and quantitative comparison of the different hydrolysis conditions tested was carried out using the Chrastil approach for modeling enzymatic reactions by fitting the experimental data of total reducing sugar (TRS) released according to hydrolysis time. Conversion of SESB using A. niger enzymatic complex were up to 3.2‐fold higher (in terms of TRS) than T. reesei at similar enzyme loadings, which could be correlated to the higher β‐glucosidase levels (up to 35‐fold higher) of A. niger enzymatic complex. Conversion yields after 72 h exceeded 40% in terms of TRS when the WM was supplemented with a low dosage of a commercial enzyme preparation. When the combination of WM (from either T. reesei or A. niger) and commercial cellulase was used, the dosage of the commercial enzyme could be reduced by half, while still providing a hydrolysis that was up to 36% more efficient. Furthermore, SESB hydrolysis using either EE or WM resulted in similar yields, indicating that the enzyme extraction/filtration steps could be eliminated from the overall process. This procedure is highly advantageous in terms of reduced enzyme and process costs, and also avoids the generation of unnecessary effluent streams. Thus, the enzymatic conversion of SESB using the WM from SSF is cost‐effective and compatible with the biorefinery concept.

Isolation and characterization of a β-galactosidase from a new Amazon forest strain of Aspergillus niger as a potential accessory enzyme for biomass conversion

Abstract The selection of enzyme-producing fungi is useful to obtain enzymes required to hydrolyze lignocellulosic material and thereby contribute to biomass conversion into fuels and chemicals. Besides cellulases, the presence of accessory enzymes in enzyme cocktails is necessary to enhance hydrolysis efficiency. This study evaluates the production, purification, and biochemical-kinetic characterization of β-galactosidase produced by a new strain of Aspergillus niger (P47C3) isolated from the Amazon Forest. The A. niger (P47C3) was cultured under SmF conditions and β-galactosidase was purified in a three-step purification, using an ultrafiltration membrane, ion exchange (TSK-SP), and gel filtration (Sephacryl S-200). The calculated molecular weight of the purified enzyme was 125 kDa. Optimum pH (4.0) and temperature (55°C) of β-galactosidase activity were determined. The values of the kinetic parameters obtained from p-nitrophenyl-β-D-galactopyranoside (PNPG) hydrolysis were 2.2 mM and 0.285 mM/min for Km and Vmax, respectively. The inhibition of PNPG hydrolysis by β-galactosidase in the presence of the inhibitor galactose gave a Ki value of 5.01 mM. As a precursor to elucidating the tertiary structure using X-ray diffraction, the β-galactosidase was crystallized using 0.2 M Tris–HCl buffer, with 12% PEG 4000 as the precipitation agent; the largest crystals were formed at pH 8.6. These results provide the basis for further structural and functional studies of this accessory enzyme to evaluate its potential biotechnological applications.

Addendum to Issue 1 - ENZITEC 2012Simultaneous biosynthesis of biomass-degrading enzymes using co-cultivation of Aspergillus niger and Trichoderma reesei

Abstract The biotransformation of lignocellulosic materials into biofuels and chemicals requires the simultaneous action of multiple enzymes. Since the cost of producing an efficient enzyme system maybe high, mixed cultures of microorganisms maybe an alternative to increase enzymatic production and consequently reduce costs. This study investigated the effects of different inoculum ratios and inoculation delays on the biosynthesis of cellulases and xylanases during co-cultivation of Aspergillus niger and Trichoderma reesei under solid-state fermentation (SSF). While the monoculture of T. reesei was more efficient for CMCase production than the co-cultivation of A. niger and T. reesei, a significant increase in β-glucosidase and xylanase production was achieved by co-cultivation of both species. The maximum CMCase activity of 153.91 IU/g was obtained with T. reesei after 48 h of cultivation, while the highest β-glucosidase activity of 119.71 IU/g (after 120 h) was obtained by co-cultivation of A. niger and T. reesei with a 3:1 inoculum ratio (A. niger: T. reesei). The greatest xylanase activity observed was 589.39 IU/g after 72 h of mixed culturing of A. niger and T. Reesei with a 1:1 inoculum ratio. This is the first study where the effects of inoculum ratio and inoculation delay in mixed culture of T. reesei and A. niger under SSF have been systematically assessed, and it indicates co-cultivation as a feasible alternative to increase enzymatic production.