Improving enzyme production by solid-state cultivation in packed-bed bioreactors by changing bed porosity and airflow distribution.

Enzymes production by solid-state cultivation in packed-bed bioreactor needs to be improved by mathematical modeling and also by experimentation. In this work, a mixture of sugarcane bagasse and wheat bran was used for the growth of the fungus Myceliophthora thermophila I-1D3b, able to secrete endoglucanase and xylanase, enzymes of interest in the second-generation ethanol production. Bench and pilot-scale bioreactors were used for the experiments, while critical parameters as bed porosity and airflow distribution were evaluated. Results showed enzymes with higher activities for the most porous medium, even though the less substrate amount to be cultivated. For the pilot-scale bioreactor, only the most porous medium was evaluated using different airflow distribution techniques. Using an inner tube for air supply resulted in more homogeneous enzyme production, with higher activities. The results here presented will be helpful for the scale-up of this class of bioreactor into industrial apparatuses.

Growth kinetics of Myceliophthora thermophila M.7·7 in solid-state cultivation.

AIMS: This work aimed to estimate the growth of Myceliophthora thermophila M.7·7 in solid-state cultivation (SSC) through quantification of N-acetyl-d-glucosamine (NAG) and enzyme activity. METHODS AND RESULTS: The fungus was cultivated in sugarcane bagasse and wheat bran. A consistent statistical analysis was done to assess the reliability of experimental data. Logistic model equation was fitted to experimental data and growth parameters were estimated. The results showed strong influence of the sample size on NAG and a minimum recommended sample size was identified. Scanning electron microscopy (SEM) was used to identify the strategy of substrate colonization. Wheat bran was attacked firstly, while sugarcane bagasse was consumed after wheat bran depletion. The biomass growth was poorly estimated by secretion kinetics of α-amylase, endoglucanase, protease and xylanase, but enzyme kinetics were important for understanding substrate colonization. CONCLUSIONS: In conclusion, the NAG concentration was strongly affected by the sample size and sampling procedure. The strategy of fungal colonization on the substrates was well characterized through SEM analysis. The colonization strategy has direct influence on the kinetic parameters of the logistic model. Myceliophthora thermophila has a well-defined dynamic of enzyme secretion to degrade the substrate, although the kinetics of enzyme secretion has shown not adequate to characterize the kinetics of fungal growth. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper provides reliable growth kinetic parameters in the SSC of the cellulase producer fungus M. thermophila M.7·7, as well as a robust analysis on three indirect methods (NAG, enzymes and SEM) for estimation of fungal development.

The DNA-methyltransferase inhibitor 5-aza-2-deoxycytidine affects Humicola grisea enzyme activities and the glucose-mediated gene repression.

Humicola grisea var. thermoidea (Hgvt) is a thermophilic ascomycete that produces lignocellulolytic enzymes and it is proposed for the conversion of agricultural residues into useful byproducts. Drugs that inhibit the DNA methyltransferases (DNMTs) activity are employed in epigenetic studies but nothing is known about a possible effect on the production of fungal enzymes. We evaluated the effect of 5-aza-2'-deoxycytidine (5-Aza; a chemical inhibitor of DNMTs activity) on the secreted enzyme activity and on the transcription of cellulase and xylanase genes from Hgvt grown in agricultural residues and in glucose. Upon cultivation on wheat bran (WB), the drug provoked an increase in the xylanase activity at 96 h. When Hgvt was grown in glucose (GLU), a repressor of Hgvt glycosyl hydrolase genes, 5-Aza led to increased transcript accumulation for the cellobiohydrolases and for the xyn2 xylanase genes. In WB, 5-Aza enhanced the expression of the transcription factor CreA gene. Growth on WB or GLU, in presence of 5-Aza, led to a significant increase in transcripts of the pH-response regulator PacC gene. To our knowledge, this is the first report on the effect of a DNMT inhibitor in the production of fungal plant cell wall degradation enzymes.

Box-Behnken analysis and storage of spray-dried collagenolytic proteases from Myceliophthora thermophila submerged bioprocess.

Enzymes do not have long-term storage stability in soluble forms, thus drying methods could minimize the loss of enzymatic activity, the spray dryer removes water under high temperatures and little time. The aims of this study were to improve the stability of enzymatic extract from Myceliophthora thermophila for potential applications in industry and to evaluate the best conditions to remove the water by spray drying technique. The parameters were tested according to Box-Behnken and evaluated by analysis of variance (ANOVA), all the parameters measured were found to influence the final enzyme activity and spray drying process yield ranged from 38.65 to 63.75%. Enzyme powders showed increased storage stability than extract and maintained about 100% of collagenolytic activity after 180 days of storage at 30°C. The results showed that the microbial enzymes maintained activity during the spray drying process and were stable during long-term storage; these are promising characteristics for industrial applications.

Filamentous fungi and media for cellulase production in solid state cultures.

Cellulase production was evaluated in two reference strains (T. reesei Rut-C30 and T. reesei QM9414), two strains isolated from a sugarcane cultivation area (Trichoderma sp. IPT778 and T. harzianum rifai IPT821) and one strain isolated in a program for biodiversity preservation in São Paulo state (Myceliophthora thermophila M77). Solid state cultures were performed using sugarcane bagasse (C), wheat bran (W) and/or soybean bran (S). The highest FPA was 10.6 U/gdm for M77 in SC (10:90) at 80% moisture, which was 4.4 times higher than production in pure W. C was a strong inducer of cellulase production, given that the production level of 6.1 U/gdm in WC (40:60) was 2.5 times higher than in pure W for strain M77; T. reesei Rut-C30 did not respond as strongly with about 1.6-fold surplus production. S advantageously replaced W, as the surplus production on SC (20:80) was 2.3 times relative to WC (20:80) for M77.

Filamentous fungi and media for cellulase production in solid state cultures

Cellulase production was evaluated in two reference strains (T. reesei Rut-C30 and T. reesei QM9414), two strains isolated from a sugarcane cultivation area (Trichoderma sp. IPT778 and T. harzianum rifai IPT821) and one strain isolated in a program for biodiversity preservation in São Paulo state (Myceliophthora thermophila M77). Solid state cultures were performed using sugarcane bagasse (C), wheat bran (W) and/or soybean bran (S). The highest FPA was 10.6 U/gdm for M77 in SC (10:90) at 80% moisture, which was 4.4 times higher than production in pure W. C was a strong inducer of cellulase production, given that the production level of 6.1 U/gdm in WC (40:60) was 2.5 times higher than in pure W for strain M77; T. reesei Rut-C30 did not respond as strongly with about 1.6-fold surplus production. S advantageously replaced W, as the surplus production on SC (20:80) was 2.3 times relative to WC (20:80) for M77.(AU)

Filamentous fungi and media for cellulase production in solid state cultures

Cellulase production was evaluated in two reference strains (T. reesei Rut-C30 and T. reesei QM9414), two strains isolated from a sugarcane cultivation area (Trichoderma sp. IPT778 and T. harzianum rifai IPT821) and one strain isolated in a program for biodiversity preservation in São Paulo state (Myceliophthora thermophila M77). Solid state cultures were performed using sugarcane bagasse (C), wheat bran (W) and/or soybean bran (S). The highest FPA was 10.6 U/gdm for M77 in SC (10:90) at 80% moisture, which was 4.4 times higher than production in pure W. C was a strong inducer of cellulase production, given that the production level of 6.1 U/gdm in WC (40:60) was 2.5 times higher than in pure W for strain M77; T. reesei Rut-C30 did not respond as strongly with about 1.6-fold surplus production. S advantageously replaced W, as the surplus production on SC (20:80) was 2.3 times relative to WC (20:80) for M77.