Sugarcane bagasse as feedstock for cellulase production by Trichoderma harzianum in optimized culture medium

This work aimed at the production of cellulases from pretreated sugarcane bagasse by the filamentous fungus Trichoderma harzianum IOC 3844 and their application in the hydrolysis of this same substrate, for a future use in second-generation ethanol production. The production of cellulases was optimized, which resulted in high enzymatic activities after 42 hrs of process in an instrumented bioreactor (CMCase 27,017 U x L-1; FPase 1,225 U x L-1; and β-glucosidase 609 U x L-1). The enzymatic extract was concentrated by using a hollow fiber membrane filtration system. The concentrated extract was applied in the hydrolysis of pretreated sugarcane bagasse, after 28 hrs of enzymatic reaction, displaying a similar catalytic performance of that attained with a commercial enzymatic preparation (hydrolysis efficiency of roughly 50%). Finally, the enzymatic extract was partially characterized in terms of the molecular weights of the main activities of the enzymatic pool. Electrophoretic analysis identified eleven protein bands; six of them were related to CMCase activity and revealing molecular weights that varied from 48 to 78 kDa, and two bands were associated with β-glucosidase activity and having molecular weights of 75 and 85 kDa.

Cellulase production by Penicillium funiculosum and its application in the hydrolysis of sugar cane bagasse for second generation ethanol production by fed batch operation.

This study aimed to produce a cellulase blend and to evaluate its application in a simultaneous saccharification and fermentation (SSF) process for second generation ethanol production from sugar cane bagasse. The sugar cane bagasse was subjected to pretreatments (diluted acid and alkaline), as for disorganizing the ligocellulosic complex, and making the cellulose component more amenable to enzymatic hydrolysis. The residual solid fraction was named sugar cane bagasse partially delignified cellulignin (PDC), and was used for enzyme production and ethanol fermentation. The enzyme production was performed in a bioreactor with two inoculum concentrations (5 and 10% v/v). The fermentation inoculated with higher inoculum size reduced the time for maximum enzyme production (from 72 to 48). The enzyme extract was concentrated using tangential ultrafiltration in hollow fiber membranes, and the produced cellulase blend was evaluated for its stability at 37 °C, operation temperature of the simultaneous SSF process, and at 50 °C, optimum temperature of cellulase blend activity. The cellulolytic preparation was stable for at least 300 h at both 37 °C and 50 °C. The ethanol production was carried out by PDC fed-batch SSF process, using the onsite cellulase blend. The feeding strategy circumvented the classic problems of diffusion limitations by diminishing the presence of a high solid:liquid ratio at any time, resulting in high ethanol concentration at the end of the process (100 g/L), which corresponded to a fermentation efficiency of 78% of the maximum obtainable theoretically. The experimental results led to the ratio of 380 L of ethanol per ton of sugar cane bagasse PDC.

Purification, crystallization and preliminary crystallographic analysis of the catalytic domain of the extracellular cellulase CBHI from Trichoderma harzianum.

The filamentous fungus Trichoderma harzianum has a considerable cellulolytic activity that is mediated by a complex of enzymes which are essential for the hydrolysis of microcrystalline cellulose. These enzymes were produced by the induction of T. harzianum with microcrystalline cellulose (Avicel) under submerged fermentation in a bioreactor. The catalytic core domain (CCD) of cellobiohydrolase I (CBHI) was purified from the extracellular extracts and submitted to robotic crystallization. Diffraction-quality CBHI CCD crystals were grown and an X-ray diffraction data set was collected under cryogenic conditions using a synchrotron-radiation source.

Nitrogen source optimization for cellulase production by Penicillium funiculosum, using a sequential experimental design methodology and the desirability function.

The present study aimed at maximizing cellulase production by Penicillium funiculosum using sequential experimental design methodology for optimizing the concentrations of nitrogen sources. Three sequential experimental designs were performed. The first and the second series of experiments consisted of a 2(4) and a 2(3) factorial designs, respectively, and in the third one, a central composite rotational design was used for better visualizing the optimum conditions. The following nitrogen sources were evaluated: urea, ammonium sulfate, peptone, and yeast extract. Peptone and ammonium sulfate were removed from the medium optimization since they did not present significant statistical effect on cellulase production. The optimal concentrations of urea and yeast extract predicted by the model were 0.97 and 0.36 g/L, respectively, which were validated experimentally. By the use of the desirability function, it was possible to maximize the three main enzyme activities simultaneously, which resulted in values for FPase of 227 U/L, for CMCase of 6,917 U/L, and for beta-glucosidase of 1,375 U/L. These values corresponded to increases of 3.3-, 3.2-, and 6.7-folds, respectively, when compared to those obtained in the first experimental design. The results showed that the use of sequential experimental designs associated to the use of the desirability function can be used satisfactorily to maximize cellulase production by P. funiculosum.

Aproveitamento do camu-camu (Myrciaria dubia) para produção de bebida alcoólica fermentada / The use of camu-camu (Myrciaria dubia) for the production of a fermented alcoholic beverage

O elevado teor de ácido ascórbico no camu-camu (Myrciaria dubia McVaugh, Myrtaceae) desperta o interesse de extrativistas, agricultores e consumidores, e leva à necessidade de desenvolvimento de tecnologias adequadas para produção em terra firme e aproveitamento industrial do fruto. Este trabalho teve por objetivo verificar a adequação do camu-camu para a produção de bebida alcoólica fermentada, assim como o efeito do branqueamento do fruto e da incorporação da casca à polpa nas características nutricionais e sensoriais da bebida. Os frutos foram separados em quatro lotes, sendo dois branqueados (90 ºC por 7 min). Após a despolpa, as cascas de um lote de cada tratamento (com e sem branqueamento) foram incorporadas às respectivas polpas e avaliadas quanto à composição química (umidade, pH, acidez, sólidos solúveis, açúcares, ácido ascórbico, compostos fenólicos, antocianinas e flavonóides). Após a correção do mosto com açúcar, pasteurização, fermentação (25 dias), trasfega, pasteurização (70 ºC por 15 min), filtragem e clarificação, as bebidas foram avaliadas quanto a composição química, edulcoradas e submetidas à análise sensorial. O branqueamento reduziu a concentração de ácido ascórbico das polpas (33 por cento) e a agregação da casca aumentou os teores de matéria seca (39 por cento polpa), ácido ascórbico (33 por cento na polpa, 23 por cento no mosto e 50 por cento na bebida) e fenólicos (50 por cento bebida). O perfil sensorial e a aceitabilidade sugerem que o camu-camu é adequado para a produção de bebida alcoólica fermentada e que a agregação da casca à polpa contribuiu positivamente para a aceitabilidade (6,7 com casca e 6,2 sem casca, na escala de 9 pontos). As bebidas apresentaram flavor característico do fruto, limpidez, coloração vermelho-laranjada e sabor agradável.

The high levels of ascorbic acid in camu-camu (Myrciaria dubia McVaugh, Myrtaceae) have stimulated interest of extractivists, farmers and consumers. This has led to a need to develop adequate technology for it's production on non-flooded land and the industrial use of this fruit. This study had as its main objective to verify if camu-camu is adequate for the production of fermented alcoholic beverages, measuring the effect of blanching the fruit and the incorporation of the fruit peel with the fruit pulp on the nutritional and sensory characteristics of the drink. The fruits were separated into 4 groups, two being blanched (90 ºC for 7 minutes). After the pulp was removed, the peels of one group from each blanching treatment were incorporated into the respective pulps and their chemical composition evaluated. After sugar correction of the must, pasteurisation, fermentation (25 days), decanting, pasteurisation (70 ºC for 15 minutes), filtering and clarification, the beverages were evaluated as to their chemical composition, sweetened and submitted to sensory analysis. Blanching reduced the concentration of ascorbic acid in the pulps (33 percent) and the addition of the peel increased the amount of dry matter (39 percent in pulp), ascorbic acid (33 percent in pulp, 23 percent in must and 50 percent in drink) and phenolic compounds (50 percent in drink). The sensory profile and acceptability suggest that camu-camu is adequate for the production of fermented alcoholic beverages and that the addition of the peel to the pulp contributes positively to it's acceptability (6.7 with versus 6.2 without, of 9 points possible). The beverage had flavour characteristic of the fruit, a orangish-red color and agreeable taste.