• Energy Research

Le développement de méthodes efficaces de production de carburants renouvelables à partir de la biomasse lignocellulosique est nécessaire pour maximiser les rendements et réduire les coûts d'exploitation. L'un des principaux défis de l'application industrielle du processus de conversion lignocellulosique est le coût élevé des enzymes cellulolytiques. Le recyclage des enzymes peut présenter une solution potentielle pour atténuer ce problème. Dans la présente étude, les enzymes associées à la fraction insoluble ont été recyclées après l'hydrolyse enzymatique de la bagasse de canne à sucre prétraitée, en utilisant différentes conditions de traitement, charges enzymatiques et charges solides. Il a été constaté que le mélange d'enzymes de Chrysoporthe cubensis et Penicillium pinophilum était efficace pour l'hydrolyse enzymatique et qu'une partie importante de l'activité enzymatique pouvait être récupérée lors du recyclage de la fraction insoluble. Les valeurs de productivité des enzymes (g de glucose/mg de protéine enzymatique) sur toutes les périodes de recyclage étaient de 2,4 et 3,7 pour l'application de 15 et 30 FPU/g de glucane, ce qui représente une augmentation de plus de dix fois celle obtenue dans un processus discontinu avec le même mélange d'enzymes et une augmentation encore plus importante par rapport aux enzymes cellulases commerciales. Contrairement à ce qui peut être attendu, l'augmentation des concentrations de lignine tout au long de la période de recyclage n'a pas eu d'influence négative sur l'efficacité de l'hydrolyse, mais les rendements de conversion se sont améliorés continuellement. Le recyclage de la totalité de la fraction solide insoluble était suffisant pour le recyclage des enzymes adhérées avec la biomasse, indiquant une méthode efficace pour augmenter la productivité enzymatique. El desarrollo de métodos eficientes para la producción de combustibles renovables a partir de biomasa lignocelulósica es necesario para maximizar los rendimientos y reducir los costos operativos. Uno de los principales retos para la aplicación industrial del proceso de conversión lignocelulósica son los altos costes de las enzimas celulolíticas. El reciclaje de enzimas puede presentar una posible solución para aliviar este problema. En el presente estudio, las enzimas asociadas con la fracción insoluble se reciclaron después de la hidrólisis enzimática del bagazo de caña de azúcar pretratado, utilizando diferentes condiciones de procesamiento, cargas enzimáticas y cargas sólidas. Se encontró que la mezcla enzimática de Chrysoporthe cubensis y Penicillium pinophilum fue eficiente para la hidrólisis enzimática y que una parte significativa de la actividad enzimática se pudo recuperar al reciclar la fracción insoluble. Los valores de productividad enzimática (g de glucosa/mg de proteína enzimática) durante todos los períodos de reciclaje fueron 2.4 y 3.7 para la aplicación de 15 y 30 FPU/g de glucano, lo que representa un aumento superior a diez veces el obtenido en un proceso por lotes con la misma mezcla enzimática y un aumento aún mayor en comparación con las enzimas celulasa comerciales. Contrariamente a lo que se puede esperar, el aumento de las concentraciones de lignina a lo largo del período de reciclaje no influyó negativamente en la eficiencia de la hidrólisis, pero las eficiencias de conversión mejoraron continuamente. El reciclaje de toda la fracción de sólidos insolubles fue suficiente para el reciclaje de las enzimas adheridas junto con la biomasa, lo que indica un método eficaz para aumentar la productividad enzimática. Development of efficient methods for production of renewable fuels from lignocellulosic biomass is necessary to maximize yields and reduce operating costs. One of the main challenges to industrial application of the lignocellulosic conversion process is the high costs of cellulolytic enzymes. Recycling of enzymes may present a potential solution to alleviate this problem. In the present study enzymes associated with the insoluble fraction were recycled after enzymatic hydrolysis of pretreated sugarcane bagasse, utilizing different processing conditions, enzyme loadings, and solid loadings.It was found that the enzyme blend from Chrysoporthe cubensis and Penicillium pinophilum was efficient for enzymatic hydrolysis and that a significant portion of enzyme activity could be recovered upon recycling of the insoluble fraction. Enzyme productivity values (g glucose/mg enzyme protein) over all recycle periods were 2.4 and 3.7 for application of 15 and 30 FPU/g of glucan, representing an increase in excess of ten times that obtained in a batch process with the same enzyme blend and an even greater increase compared to commercial cellulase enzymes.Contrary to what may be expected, increasing lignin concentrations throughout the recycle period did not negatively influence hydrolysis efficiency, but conversion efficiencies continuously improved. Recycling of the entire insoluble solids fraction was sufficient for recycling of adhered enzymes together with biomass, indicative of an effective method to increase enzyme productivity. يعد تطوير طرق فعالة لإنتاج الوقود المتجدد من الكتلة الحيوية اللجنية السليولوزية أمرًا ضروريًا لزيادة الغلة وتقليل تكاليف التشغيل. يتمثل أحد التحديات الرئيسية التي تواجه التطبيق الصناعي لعملية التحويل الليجنوسلوزية في ارتفاع تكاليف الإنزيمات المحللة للسليلوز. قد تقدم إعادة تدوير الإنزيمات حلاً محتملاً للتخفيف من هذه المشكلة. في هذه الدراسة، تم إعادة تدوير الإنزيمات المرتبطة بالجزء غير القابل للذوبان بعد التحلل المائي الإنزيمي لقصب قصب السكر المعالج مسبقًا، باستخدام ظروف المعالجة المختلفة، وتحميل الإنزيمات، والتحميلات الصلبة. وجد أن مزيج الإنزيم من Chrysoporthe cubensis و Penicillium pinophilum كان فعالًا في التحلل المائي الإنزيمي وأنه يمكن استرداد جزء كبير من نشاط الإنزيم عند إعادة تدوير الجزء غير القابل للذوبان. كانت قيم إنتاجية الإنزيم (جرام من بروتين إنزيم الجلوكوز/ملجم) خلال جميع فترات إعادة التدوير 2.4 و 3.7 لتطبيق 15 و 30 وحدة حماية من الحرائق/جم من الجلوكان، مما يمثل زيادة تزيد عن عشرة أضعاف تلك التي تم الحصول عليها في عملية دفعية مع نفس مزيج الإنزيم وزيادة أكبر مقارنة بإنزيمات السليلوز التجارية. على عكس ما يمكن توقعه، فإن زيادة تركيزات اللجنين طوال فترة إعادة التدوير لم تؤثر سلبًا على كفاءة التحلل المائي، ولكن كفاءة التحويل تحسنت باستمرار. كانت إعادة تدوير جزء المواد الصلبة غير القابلة للذوبان بالكامل كافية لإعادة تدوير الإنزيمات الملتصقة مع الكتلة الحيوية، مما يدل على وجود طريقة فعالة لزيادة إنتاجية الإنزيم.

The aim of this work was to evaluate the biochemical features of the white-rot fungi Pycnoporus sanguineus cellulolytic complex and its utilization to sugarcane bagasse hydrolysis. When cultivated under submerged fermentation using corn cobs as carbon source, P. sanguineus produced high FPase, endoglucanase, β-glucosidase, xylanase, mannanase, α-galactosidase, α-arabinofuranosidase, and polygalacturonase activities. Cellulase activities were characterized in relation to pH and temperature. β-Glucosidase and FPase activities were higher at 55 °C, pH 4.5, and endoglucanase activity was higher at 60 °C, in a pH range of 3.5-4.0. All cellulase activities were highly stable at 40 and 50 °C through 48 h of pre-incubation. Crude enzymatic extract from P. sanguineus was applied in a saccharification experiment using acid-treated and alkali-treated sugarcane bagasse as substrate, and the hydrolysis yields were compared to that obtained by a commercial cellulase preparation. Reducing sugar yields of 60.4% and 64.0% were reached when alkali-treated bagasse was hydrolyzed by P. sanguineus extract and commercial cellulase, respectively. Considering the glucose production, it was observed that P. sanguineus extract and commercial cellulase ensured yields of 22.6% and 36.5%, respectively. The saccharification of acid-treated bagasse was lower than that of alkali-treated bagasse regardless of the cellulolytic extract. The present work showed that P. sanguineus has a great potential as an enzyme producer for biomass saccharification.

Enzymatic hydrolysis is an important but expensive step in the production of ethanol from biomass. Thus, the production of efficient enzymatic cocktails is of great interest for this biotechnological application. The production of endoglucanase and xylanase activites from F. verticillioides were optimized in a factorial design (2(5)) followed by a CCDR design. Endoglucanase and xylanase activities increased from 2.8 to 8.0 U/mL and from 13.4 to 114 U/mL, respectively. The optimal pH and temperature were determined for endoglucanase (5.6, 80 °C), cellobiase (5.6, 60 °C), FPase (6.0, 55 °C) and xylanase (7.0, 50 °C). The optimized crude extract was applied in saccharification and fermentation of sugarcane bagasse from which 9.7 g/L of ethanol was produced at an ethanol/biomass yield of 0.19.

The plant pathogenic fungus Chrysoporthe cubensis was cultivated under solid state employing different substrates and the highest endoglucanase (33.84Ug(-1)), FPase (2.52Ug(-1)), β-glucosidase (21.55Ug(-1)) and xylanase (362.38Ug(-1)) activities were obtained using wheat bran as carbon source. Cellulases and xylanase produced by C. cubensis showed maximal hydrolysis rate at pH 4.0 and in a temperature range of 50-60°C. All enzymatic activities were highly stable at 40 and 50°C through 48h of pre-incubation. Saccharification of alkaline pretreated sugarcane bagasse by crude enzyme extract from C. cubensis resulted in release of 320.8mg/g and 288.7mg/g of glucose and xylose, respectively. On another hand, a similar assay employing commercial cellulase preparation resulted in release of 250.6mg/g and 62.1mg/g of glucose and xylose, respectively. Cellulolytic extract from C. cubensis showed a great potential to be used in biomass saccharification processes.

HIGHLIGHTS Brachiaria brizantha proved to be a promising biomass for ethanol production. Fermentation was not impaired by the inhibitors furfural and hydroxymethylfurfural.

Blending of the enzyme extracts produced by different fungi can result in favorable synergetic enhancement of the enzyme blend with regards to the main cellulase activities, as well as the inclusion of accessory enzymes that may not be as abundant in enzyme extracts produced by predominantly cellulase producing fungi. The Chrysoporthe cubensis:Penicillium pinophilum 50:50 (v/v) blend produced herein presented good synergy, especially for FPase and endoglucanase activities which were 76% and 48% greater than theoretical, respectively. This enzyme blend was applied to sugarcane bagasse previously submitted to a simple alkali pretreatment. Glucan hydrolysis efficiency reached an excess of 60% and xylan conversion exceeded 90%. Increasing the hydrolysis temperature from 45 to 50°C also resulted in a 16-20% increase in conversion of both glucan and xylan fractions. The blended enzyme extract obtained therefore showed great potential for application in the lignocellulose hydrolysis process.

Production of ethanol with two corn endophytic fungi, Fusarium verticillioides and Acremonium zeae, was studied. The yield of ethanol from glucose, xylose and a mixture of both sugars were 0.47, 0.46 and 0.50g/g ethanol/sugar for F. verticillioides and 0.37, 0.39 and 0.48g/g ethanol/sugar for A. zeae. Both fungi were able to co-ferment glucose and xylose. Ethanol production from 40g/L of pre-treated sugarcane bagasse was 4.6 and 3.9g/L for F. verticillioides and A. zeae, respectively, yielding 0.31g/g of ethanol per consumed sugar. Both fungi studied were capable of co-fermenting glucose and xylose at high yields. Moreover, they were able to produce ethanol directly from lignocellulosic biomass, demonstrating to be suitable microorganisms for consolidated bioprocessing.