Evaluation of Saccharomyces cerevisiae modified via CRISPR/Cas9 as a cellulosic platform microorganism in simultaneously saccharification and fermentation processes.

The nonrenewable character and deleterious effects of fossil fuels foster the need for cleaner and more inexhaustible energy sources, such as bioethanol. Especially from lignocellulosic biomasses. However, the economic viability of this product in the market depends on process optimization and cost reduction. This research applied a sequential experimental project to investigate the process of enzymatic saccharification and simultaneous fermentation to produce ethanol with sugarcane bagasse. The differential of the work was the application of the strain of Saccharomyces cerevisiae AGY001 which was improved by evolutionary engineering to become thermotolerant and by a heterologous expression based on genomic integration by CRISPR/Cas9 to produce endoglucanase and ß-glucosidase (AsENDO-AsBGL). The maximum ethanol yield found was 89% of the maximum theoretical yield (released sugars), obtained at temperature concentrations, sugarcane bagasse and inoculum at 40 °C, 16.5%, and 4.0 g/L, respectively (12.5 FPU/g bagasse). The mathematical model obtained can predict approximately 83% of the data set with 95% confidence. Therefore, these findings demonstrated the potential of sugarcane bagasse and S. cerevisiae AGY001 strain (CRISPR/Cas9 modified) in bioethanol production without the need for impractical selection media on an industrial scale, in addition to providing useful insights for the development of SSF processes.

Application of the solid-state fermentation process and its variations in PHA production: a review.

Solid-state fermentation (SSF) is a type of fermentation process with potential to use agro-industrial by-products as a carbon source. Nonetheless, there are few studies evaluating SSF compared to submerged fermentation (SmF) to produce polyhydroxyalkanoates (PHAs). Different methodologies are available associating the two processes. In general, the studies employ a 1st step by SSF to hydrolyze the agro-industrial by-products used as a carbon source, and a 2nd step to produce PHA that can be carried out by SmF or SSF. This paper reviewed and compared the different methodologies described in the literature to assess their potential for use in PHA production. The studies evaluated showed that highest PHA yields (86.2% and 82.3%) were achieved by associating SSF and SmF by Cupriavidus necator. Meanwhile, in methodologies using only SSF, Bacillus produced the highest yields (62% and 56.8%). Since PHA (%) does not necessarily represent a higher production by biomass, the productivity parameter was also compared between studies. We observed that the highest productivity results did not necessarily represent the highest PHA (%). C. necator presented the highest PHA yields associating SSF and SmF, however, is not the most suitable microorganism for PHA production by SSF. Concomitant use of C. necator and Bacillus is suggested for future studies in SSF. Also, it discusses the lack of studies on the association of the two fermentation methodologies, and on the scaling of SSF process for PHA production. In addition to demonstrating the need for standardization of results, for comparison between different methodologies.

Clonostachys rosea: Production by Submerged Culture and Bioactivity Against Sclerotinia sclerotiorum and Bemisia tabaci.

Among the prospective biocontrol agents, the saprophytic filamentous fungus Clonostachys rosea is an excellent necrotrophic mycoparasite of numerous plant pathogenic fungi. However, its commercial development has been hampered by mass production difficulties during solid-state fermentation. Conversely, the submerged liquid fermentation shortens the cultivation time while increasing yields of fungal propagules. However, this method has been overlooked for C. rosea. In this work, we investigated the impact of liquid pre-culture inoculum on the spore production by the two-stage fermentation process using rice grains in comparison to the traditional solid-state fermentation. In parallel, we studied the submerged cultivation of C. rosea by manipulating carbon-to-nitrogen (C:N) ratio and nitrogen source, with the further optimization of spore production in a benchtop bioreactor. Additional bioassays included assessing the bioactivity of water-dispersible microgranules (that contained a submerged conidia) against the whitefly (Bemisia tabaci biotype B) and Sclerotinia sclerotiorum (causal agent of the white mold). Our results showed a maximum concentration of 1.1 × 109 conidia/g-dry-matter after 7 days of cultivation by two-stage fermentation process. The liquid fermentation yielded 1.4 × 109 submerged conidia/ml after 7 days using a medium with a 50:1 C:N ratio, and it also induced the production of microsclerotia (MS) up to 1.35 × 104/ml within 6 days with 10:1 C:N ratio; both media were supplemented with dextrose monohydrate and soybean meal. The fermentation batches carried out in a benchtop bioreactor with medium 50:1 C:N ratio and amended with soybean meal rendered a production peak on the fourth day, corresponding to 1.11 × 109 conidia/ml and 4.35 × 108 colony forming units (CFU)/ml. Following air-drying, the conidia production from air-dried microgranules of C. rosea biomass was estimated at 3.4 × 1010 conidia/g of formulated product upon re-hydration for 7 days. Both submerged conidia and MS of C. rosea inhibited 100% germination of S. sclerotiorum sclerotia by direct parasitism. The air-dried submerged conidia exhibited a suppressive activity on sclerotia (88% mycoparasitism) and early whitefly nymphs (76.2% mortality) that rendered LC50 values of 3.2 × 104 CFU/g soil and 1.5 × 107 CFU/ml, respectively. Therefore, the submerged liquid culture of C. rosea may offer a feasible and cost-effective method for its large-scale production, alleviating critical constraints to their commercial use while providing an additional tool for management of B. tabaci and S. sclerotiorum.

Comparison of Pigment Production by Filamentous Fungal Strains under Submerged (SmF) and Surface Adhesion Fermentation (SAF).

Although synthetic colorants are widely used in many industries due to their high stability at different conditions in industrial processes, evidence of its negative impact on health and the environment is undeniable. Filamentous fungi are well known for their use as alternative sources to produce natural pigments. However, an adequate comparison of the productivity parameters between the fermentation systems could be limited to their heterogeneous conditions. Even though Solid-State Fermentations (SSF) on natural substrates are widely used for pigments production, complex media, and non-controlled variables (T, pH, medium composition), these systems could not only hamper the finding of accurate productivity parameters, but also mathematical modeling and genomics-based optimization. In this context, the present study screened five pigment-producing fungi by comparing Submerged (SmF) and Surface Adhesion Fermentation [biofilm (BF) and Solid-State (SSF)] with defined media and controlled variables. For this purpose, we used the same defined media with sucrose as the carbon source for pigment production on SmF, BF, and SSF, and BF and SSF were carried out on inert supports. Five molecularly identified Penicillium and Talaromyces strains isolated from the Peruvian rainforest were selected for their ability to produce yellowish-orange colorants. Highest productivities were obtained from T. brunneus LMB-HP43 in SmF (0.18 AU/L/h) and SSF (0.17 AU/L/h), and P. mallochii LMB-HP37 in SSF (0.18 AU/L/h). Both strains also exhibited the highest yields (AU/g biomass) in the three fermentation systems, reaching values greater than 18-folds in SSF compared to the other strains. Conversely, T. wortmannii LMB-HP14 and P. maximae LMB-HP33 showed no ability to produce pigments in the SSF system. The performed experiments accurately compared the effect of the fermentation system on yield and productivity. From this, further genomics approaches can be considered for an extensive analysis of pigment synthesis pathways and a genomics-driven optimization in the best fermentation system.

ß-Mannanase Production Using Coffee Industry Waste for Application in Soluble Coffee Processing.

Soluble coffee offers the combined benefits of high added value and practicality for its consumers. The hydrolysis of coffee polysaccharides by the biochemical route, using enzymes, is an eco-friendly and sustainable way to improve the quality of this product, while contributing to the implementation of industrial processes that have lower energy requirements and can reduce environmental impacts. This work describes the production of hydrolytic enzymes by solid-state fermentation (SSF), cultivating filamentous fungi on waste from the coffee industry, followed by their application in the hydrolysis of waste coffee polysaccharides from soluble coffee processing. Different substrate compositions were studied, an ideal microorganism was selected, and the fermentation conditions were optimized. Cultivations for enzymes production were carried out in flasks and in a packed-bed bioreactor. Higher enzyme yield was achieved in the bioreactor, due to better aeration of the substrate. The best ß-mannanase production results were found for a substrate composed of a mixture of coffee waste and wheat bran (1:1 w/w), using Aspergillusniger F12. The enzymatic extract proved to be very stable for 24 h, at 50 °C, and was able to hydrolyze a considerable amount of the carbohydrates in the coffee. The addition of a commercial cellulase cocktail to the crude extract increased the hydrolysis yield by 56%. The production of ß-mannanase by SSF and its application in the hydrolysis of coffee polysaccharides showed promise for improving soluble coffee processing, offering an attractive way to assist in closing the loops in the coffee industry and creating a circular economy.

Evolution of World and Brazilian Markets for Enzymes Produced by Solid-state Fermentation: A Patent Analysis.

BACKGROUND: The use of enzymes in various industrial processes has become increasingly frequent. When added to productive processes, it can accelerate reactions and generate a number of new products. The solid state fermentation (SSF), among other applications, has been employed also to obtain enzymes. OBJECTIVE: The purpose of this prospection was to map registered patent documents about enzymes production by this type of fermentation in the world, identify the most obtained enzymes with patent documents and compilate information about the world and Brazilian enzyme markets. METHODS: The experimental design was carried out by the keyword-driven scope through the advanced search in the Espacenet database European Patent Office (EPO). The keywords selected were solid-state fermentation and the International Patent Classification code, C12N9 (enzymes; proenzymes), for prospecting of interest. RESULTS: In 2012, there was the higher number of registered patents (12). China holds 84% of deposited patents. Among the types of depositors, 54% of the selected patent documents were deposited by universities and institutes, and 44% by companies. 76.5% of the evaluated patents used fungi as enzyme producer. Analyzing the enzymes obtained in the registered patents, it is verified that the majority belongs to the group of carbohydrases with 43%, followed by proteases (25%), which are also the two classes of enzymes most commercialized in the market. CONCLUSION: China holds the majority of the registered patents but North America gets the largest global enzyme market revenue followed by Europe and Pacific Asia. Carbohydrases were the most commercialized enzymes and with the highest number of patents registered. Among the carbohydrases, cellulases, xylanases and amylases are the most frequent in patent registration while being fungi produced.

Avaliação de diferentes métodos de cozimento e de cisalhamento na determinação da maciez de lombos suínos de diferentes qualidades / Evaluation of different cooking and shear methods to assess tenderness of pork loins from different qualities

Objetivou-se avaliar a maciez de lombos suínos (M. Longissimus thoracis) de diferentes classes de qualidade (RFN, PSE e RSE) por dois métodos de cozimento (micro-ondas e grelha) e dois protocolos (SSF e WBsSF) de determinação de força de cisalhamento (FC). As classes de qualidade de carne suína não difeririam (P>0,05) quanto a FC, independentemente do método de cozimento ou protocolo de cisalhamento usado. Maior repetibilidade da FC foi observada quando do cozimento em micro-ondas (SSF = 0,77; WBsSF = 0,75), tendo alta correlação (r = 0,72) entre os dois protocolos. Concluiu-se que que as classes de qualidade da carne suína não diferem quanto à maciez instrumental e que o cozimento em micro-ondas associado ao protocolo SSF é altamente promissor para avaliação da maciez instrumental da carne suína.(AU)

Avaliação de diferentes métodos de cozimento e de cisalhamento na determinação da maciez de lombos suínos de diferentes qualidades / Evaluation of different cooking and shear methods to assess tenderness of pork loins from different qualities

Objetivou-se avaliar a maciez de lombos suínos (M. Longissimus thoracis) de diferentes classes de qualidade (RFN, PSE e RSE) por dois métodos de cozimento (micro-ondas e grelha) e dois protocolos (SSF e WBsSF) de determinação de força de cisalhamento (FC). As classes de qualidade de carne suína não difeririam (P>0,05) quanto a FC, independentemente do método de cozimento ou protocolo de cisalhamento usado. Maior repetibilidade da FC foi observada quando do cozimento em micro-ondas (SSF = 0,77; WBsSF = 0,75), tendo alta correlação (r = 0,72) entre os dois protocolos. Concluiu-se que que as classes de qualidade da carne suína não diferem quanto à maciez instrumental e que o cozimento em micro-ondas associado ao protocolo SSF é altamente promissor para avaliação da maciez instrumental da carne suína.

Ethanol production from sugarcane bagasse: Use of different fermentation strategies to enhance an environmental-friendly process.

Ethanol production by simultaneous saccharification and fermentation (SSF) using sugarcane bagasse as substrate was developed using batch and fed-batch mode. Acid, alkali, hydrothermal and hydrogen peroxide pretreatments to the sugarcane bagasse were tested. Experiments were carried out to optimize the enzyme load of cellulases and ß-glucosidase. Four strains, two of Saccharomyces cerevisiae and two of Kluyveromyces marxianus yeast species were evaluate using SSF to produce ethanol. A kinetic study in bioreactor was carried out to optimize the SSF. The batch process was optimized using 1.0 g/L of inoculum, 15.0 FPU/g cellulose of cellulases and 6.0% of initial cellulose reaching 92.0% of theoretical ethanol yield after 18 h using the bagasse pretreate by acid-alkali and S. cerevisiae PE-2. The fed-batch process with enzyme load three times lower than that was used in batch process, obtained 88% of theoretical ethanol yield in 40 h. Therefore, the use of the lignocellulosic biomass (sugarcane bagasse) for producing a biofuel (ethanol) reduces the need for oil and is an environmental-friendly process.

Integral process assessment of sugarcane agricultural crop residues conversion to ethanol.

This work focuses a whole process assessment on post-harvesting sugarcane residues for 2G ethanol production by different saccharification-fermentation conditions at high solids loading, performed after steam explosion, alkaline and acidic pretreatments. Carbohydrate recoveries and enzymatic digestibility results showed that alkali and steam explosion pretreatments were effective for the biomass assayed. Due to a significant improvement (60%) of the glucose released by combining hemicellulases and cellulases only after the NaOH pretreatment, the most favorable process settled comprised an alkali-based pretreatment followed by a pre-saccharification and simultaneous saccharification and fermentation (PSSF). The produced ethanol reached 4.8% (w/w) as a result of an 80% conversion of the glucose from the pretreated biomass. Finally, an ethanol concentration of 3.2% (w/w) was obtained by means of a steam explosion followed by PSSF, representing a suitable start point to further develop a low environmental impact alternative for ethanol production.

On-line identification of fermentation processes for ethanol production.

A strategy for monitoring fermentation processes, specifically, simultaneous saccharification and fermentation (SSF) of corn mash, was developed. The strategy covered the development and use of first principles, semimechanistic and unstructured process model based on major kinetic phenomena, along with mass and energy balances. The model was then used as a reference model within an identification procedure capable of running on-line. The on-line identification procedure consists on updating the reference model through the estimation of corrective parameters for certain reaction rates using the most recent process measurements. The strategy makes use of standard laboratory measurements for sugars quantification and in situ temperature and liquid level data. The model, along with the on-line identification procedure, has been tested against real industrial data and have been able to accurately predict the main variables of operational interest, i.e., state variables and its dynamics, and key process indicators. The results demonstrate that the strategy is capable of monitoring, in real time, this complex industrial biomass fermentation. This new tool provides a great support for decision-making and opens a new range of opportunities for industrial optimization.

Saccharomyces boulardii: Optimization of simultaneous saccharification and fermentation of cell production in organic and conventional apple substrate pulp.

A face-centered factorial design was used to study the influence of temperature, cellulase, and pectinase concentration on the production of Saccharomyces boulardii cells during simultaneous saccharification and fermentation of organic and conventional apple substrate pulp. The effects of the variables fermentation temperature (25-35 °C), pectinase concentration (5-25 µL/100 g), and cellulase concentration (4-8 µL/100 g) were analyzed by multiple regression and polynomial models of second order, providing the ideal conditions for yeast cultivation. Cellular production of apple substrates was expressed in log CFU/mL. The optimum condition for temperature was 27.5 °C, and 20 and 5 µL/100 g for pectinase and 8 and 7 µL/100 g for cellulase concentrations for organic and conventional apple pulp, respectively. The observed viability values were in agreement with the predicted values of 8.352 log CFU/mL (organic) and 8.317 log CFU/mL (conventional) apple pulps, thus proving the effectiveness of the models.

The functional properties of a xyloglucanase (GH12) of Aspergillus terreus expressed in Aspergillus nidulans may increase performance of biomass degradation.

Filamentous fungi are attractive hosts for heterologous protein expression due to their capacity to secrete large amounts of enzymes into the extracellular medium. Xyloglucanases, which specifically hydrolyze xyloglucan, have been recently applied in lignocellulosic biomass degradation and conversion in many other industrial processes. In this context, this work aimed to clone, express, and determine the functional properties of a recombinant xyloglucanase (AtXEG12) from Aspergillus terreus, and also its solid-state (SSF) and submerged (SmF) fermentation in bioreactors. The purified AtXEG12 showed optimum pH and temperature of 5.5 and 65 °C, respectively, demonstrating to be 90 % stable after 24 h of incubation at 50 °C. AtXEG12 activity increased in the presence of 2-mercaptoethanol (65 %) and Zn+2 (45 %), while Cu+2 and Ag+ ions drastically decreased its activity. A substrate assay showed, for the first time for this enzyme's family, xylanase activity. The enzyme exhibited high specificity for tamarind xyloglucan (K M 1.2 mg mL-1) and V max of 17.4 µmol min-1 mg-1 of protein. The capillary zone electrophoresis analysis revealed that AtXEG12 is an endo-xyloglucanase. The heterologous xyloglucanase secretion was greater than the production by wild-type A. terreus cultivated in SmF. On the other hand, AtXEG12 activity reached by SSF was sevenfold higher than values achieved by SmF, showing that the expression of recombinant enzymes can be significantly improved by cultivation under SSF.

By-products from the biodiesel chain as a substrate to citric acid production by solid-state fermentation.

In this study, we propose the use of tung cake for the production of organic acids, with an emphasis on citric acid by solid-state fermentation. We evaluated the conditions of production and the by-products from the biodiesel chain as raw materials involved in this bioprocess. First, we standardized the conditions of solid-state fermentation in tung cake with and without residual fat and with different concentrations of glycerine using the fungus Aspergillus niger The solid-state fermentation process was monitored for 7 days considering the biomass growth and pH level. Citric acid production was determined by high-performance liquid chromatography. Fungal development was better in the crude tung cake, consisting of 20% glycerine. The highest citric acid yield was 350 g kg(-1) of biomass. Therefore, the solid-state fermentation of the tung cake with glycerine led to citric acid production using the Aspergillus niger fungus.

“Comparación de técnicas de laboratorio para el diagnóstico de Giardia intestinalis” / “Comparison of Laboratory Techniques For the Diagnosis of Giardia Intestinalis”

Evaluar la sensibilidad y especificidad de tres técnicas de laboratorio para el diagnóstico de Giardia intestinalis. Materiales y Métodos: 31 muestras de heces provenientes de niños en edad preescolar, se procesaron a través del examen microscópico con SSF-lugol, método de concentración de Ritchie y método inmunológico “Giardia-Strip”. La Técnica de Ritchie fue considerada como “Gold Standard”. Resultados: Se identificaron quistes de Giardia intestinalis en 6 muestras (19,35%) a través de la técnica de Ritchie. La técnica del examen al fresco identificó en 5 de ellas quistes del protozoario (16,1%), mostrando una sensibilidad de 83% y especificidad del 100%. La técnica Giardia-Strip identificó quistes en 4 muestras (12,9%) con sensibilidad de 66,66% y especificidad del 100%. El valor predictivo positivo para el método de “Giardia-Strip” fue 14% y valor predictivo negativo de 93%. El examen al fresco mostró valor predictivo positivo del 100% y valor predictivo negativo de 96%. Conclusiones: El concentrado de Ritchie y el examen al fresco mostraron mayor sensibilidad y especificidad en el diagnóstico de Giardia intestinalis, cuando las muestras contienen solo quistes. Sin embargo, el método Giardia-Strip, demostró mayor rapidez en la obtención de los resultados.

Objective: To evaluate the sensitivity and specificity of three laboratory techniques for the diagnosis of Giardia intestinalis. Materials and Methods: 31 fecal samples from preschoolers were submitted to microscopic examination with SSF-Lugol, the Ritchie concentration method and the “Giardia-Strip” immunological method. The Ritchie technique was considered the “gold standard.“ Results: G. intestinalis cysts were identified in 6 samples (19.35%) using the Ritchie technique. The fresh test identified 5 cases of protozoan cysts (16.1%), showing a sensitivity of 83 % and specificity of 100 %. The Giardia-Strip identified cysts in 4 samples (12.9%) with 66.66 % sensitivity and 100% specificity. The positive predictive value for the “Giardia Strip” method was 14% and the negative predictive value was 93%. The fresh test showed a positive predictive value of 100% and a negative predictive value of 96 %. Conclusions: Ritchie concentrate and the fresh test showed higher sensitivity and specificity in the diagnosis of Giardia intestinalis, when samples contain only cysts. However, the Giardia-Strip method proved faster in obtaining results.

Evaluation of sweet potato for fuel bioethanol production: hydrolysis and fermentation.

The enzymatic starch hydrolysis and bioethanol production from a variety of sweet potato developed for bioenergy purposes (K 9807.1) on the basis of its high starch yields, was studied. Drying at 55°C and 95°C of sweet potato neither affected the sugar content nor the starch enzymatic hydrolysis efficiency. Simultaneous saccharification and ethanol fermentations for dry matter ratio of sweet potato to water from 1:8 to 1:2 (w/v) were studied. Fresh sweet potato and dried at 55°C (flour) were assayed. At ratios of 1:8, similar results for fresh sweet potato and flour in terms of ethanol concentration (38-45 g/L), fermentation time (16 h) and sugar conversion (~ 100%) were found. At higher dry matter content, faster full conversion were observed using flour. A higher ratio than that for fresh sweet potato (1:2.2) did not improve the final ethanol concentration (100 g/L) and yields. High ethanol yields were found for VHG (very high gravity) conditions. The sweet potato used is an attractive raw matter for fuel ethanol, since up to 4800 L ethanol per hectare can be obtained.

Selection of process alternatives for lignocellulosic bioethanol production using a MILP approach.

This work proposes a decision-making framework for the selection of processes and unit operations for lignocellulosic bioethanol production. Process alternatives are described by its capital and operating expenditures, its contribution to process yield and technological availability information. A case study in second generation ethanol production using Eucalyptus globulus as raw material is presented to test the developed process synthesis tool. Results indicate that production cost does not necessarily decrease when yield increases. Hence, optimal processes can be found at the inflexion point of total costs and yield. The developed process synthesis tool provides results with an affordable computational cost, existing optimization tools and an easy-to-upgrade description of the process alternatives. These features made this tool suitable for process screening when incomplete information regarding process alternatives is available.

Effect of a GFOD2 variant on responses in total and LDL cholesterol in Mexican subjects with hypercholesterolemia after soy protein and soluble fiber supplementation.

BACKGROUND: Although dietary treatments can successfully reduce blood lipids in hypercholesterolemic subjects, individual variation in that response has on occasion been linked to allelic differences. SNP rs12449157 has shown association with HDL-C concentrations in GWAS and falls in the glucose-fructose oxidoreductase domain containing 2 (GFOD2) locus. Of interest, previous data suggest that this SNP may be under environmentally driven selection. Thus, the aim of this study was to assess if rs12449157 may mediate the response of lipid traits to a dietary supplementation (DS) with soy protein and soluble fiber in a Mexican population with hypercholesterolemia. METHODS: Forty-one subjects with hypercholesterolemia were given a low saturated fat diet (LSFD) for 1 month, followed by a LSFD+DS that included 25 g of soy protein and 15 g of soluble fiber (S/SF) daily for 2 months. Anthropometric, clinical, biochemical and dietary variables were determined. We analyzed the gene-diet interaction between the GFOD2 genotype, with the minor allele frequency of 0.24, and the DS on total cholesterol (TC) and LDL-C concentrations. RESULTS: Hypercholesterolemic subjects with GFOD2 rs12449157 G allele had higher serum TC and LDL-C at the baseline and showed a greater response to the LSCD+S/SF (-83.9 and -57.5mg/dl, respectively) than those with GFOD2 AA genotype (-40.1 and -21.8 mg/dl, respectively) (P=0.006 for TC, 0.025 for LDL-C, respectively). CONCLUSION: The observed differences in allele-driven, diet-induced changes in blood lipids may be the result of a recent environmentally driven selection on the rs12449157 minor allele. Variation in the GFOD2 gene contributes to the genetic basis for a differential response to a cholesterol- or lipid-lowering diet.

Evaluación de las enzimas celulolíticas producidas por hongosnativos mediante fermentación en estado sólido (SSF) utilizando residuos de cosecha de caña de azúcar / Evaluation of cellulolytic enzymes produced by native fungi through solid state fermentation (SSF) using sugarcane harvesting residues

Los residuos agrícolas de cosecha de caña de azúcar (RAC), se constituyen en una materia prima alternativa para la producción de etanol carburante, dado su contenido de celulosa próximo al 40%. El aprovechamiento de la celulosa depende de la aplicación de tratamientos fisicoquímicos o bioquímicos, que permitan la liberación de la glucosa y su posterior utilización en procesos fermentativos. La hidrólisis enzimática de estos residuos requiere un complejo celulolítico producido por microorganismos, comprendido por tres actividades enzimáticas: Endoglucanasas, Exoglucanasas y β-Glucosidasas. En el presente estudio, se evaluaron las enzimas celulolíticas producidas por dos hongos nativos del género Aspergillus spp., CH 2016 y CH 2001, mediante procesos de fermentación en estado sólido utilizando como sustrato RAC pre-tratados con organosolventes (deslignificado) y sin este pre-tratamiento. La cepa CH 2016 presentó la mayor actividad endoglucanasa 11,0773 U/mL en el sustrato sin pre-tratar el día siete de fermentación; esta misma cepa, en el sustrato deslignificado presentó la mayor actividad exoglucanasa (0,042 U/mL) y celulasa total (0,287 UPF/mL) en el día cinco de fermentación. La cepa CH 2001 presentó la mayor actividad β-glucosidasa (0,1778 U/mL) en el sustrato sin pre-tratar el día cinco de fermentación. Se observó que las variables sustrato y tiempo de fermentación, inciden en la expresión de las enzimas celulolíticas obteniendo en este trabajo extractos enzimáticos que pueden llevar a cabo una acción hidrolítica sinérgica sobre la celulosa.

Sugarcane harvesting residues are considered as a raw material for fuel ethanol production due its high content of cellulose, around 40% DS. The use of cellulose depends of the application of physicochemical or biochemical treatments that allow the release of glucose and its subsequent uses in fermentation processes. The enzymatic hydrolysis of these residues requires a cellulolytic complex produced by microorganisms, including three enzymatic activities: Endoglucanases, β-Glucosidases and Exoglucanases. In the present study, cellulolytic enzymes produced by two native fungi Aspergillus spp., CH 2016 and CH 2001 was assessment, through of solid-state fermentation processes using as raw substrate RAC and pre-treated with organosolvents (delignified). Strain CH 2016 had the highest endoglucanase activity 11.0773 U/mL in the raw substrate on day seven of fermentation, the same strain, in the delignified substrate showed the highest activity exoglucanasa (0.042 U/mL ) and total cellulase (0.287 UPF/mL) on day five of fermentation. Strain CH 2001 got the highest β-glucosidase activity (0.1778 U/mL) in the substrate without pre-treatment on day 5 of fermentation. It was observed that the variables as substrate and fermentation time affected the expression of cellulolytic enzymes.

A biodegradation study of forest biomass by Aspergillus niger F7: correlation between enzymatic activity, hydrolytic percentage and biodegradation index

Aspergillus niger F7 isolated from soil was found to be the potent producer of cellulase and xylanase. The residue of forest species Toona ciliata, Celtris australis, Cedrus deodara and Pinus roxburghii was selected as substrate for biodegradation study due to its easy availability and wide use in industry. It was subjected to alkali (sodium hydroxide) treatment for enhancing its degradation. Biodegradation of forest waste by hydrolytic enzymes (cellulase and xylanase) secreted by A. niger under solid state fermentation (SSF) was explored. SSF of pretreated forest biomass was found to be superior over untreated forest biomass. Highest extracellular enzyme activity of 2201±23.91 U/g by A. niger was shown in pretreated C. australis wood resulting in 6.72±0.20 percent hydrolysis and 6.99±0.23 biodegradation index (BI). The lowest BI of 1.40±0.08 was observed in untreated saw dust of C. deodara having the least enzyme activity of 238±1.36 U/g of dry matter. Biodegradation of forest biomass under SSF was increased many folds when moistening agent i.e. tap water had been replaced with modified basal salt media (BSM). In BSM mediated degradation of forest waste with A. niger, extracellular enzyme activity was increased up to 4089±67.11 U/g of dry matter in turn resulting in higher BI of 15.4±0.41 and percent hydrolysis of 19.38±0.81 in pretreated C. australis wood. A. niger exhibited higher enzyme activity on pretreated biomass when moistened with modified BSM in this study. Statistically a positive correlation has been drawn between these three factors i.e. enzyme activity, BI and percent hydrolysis of forest biomass thus proving their direct relationship with each other.