Pré-tratamentos de baixo impacto ambiental das fibras lignocelulósicas : contribuições para a obtenção do etanol celulósico

Abstract

The lignocellulosic biomass is composed mainly of cellulose, hemicellulose and lignin. The latter has a recalcitrant structure that hampers the obtainment of carbohydrates. Processing of carbohydrates of biomasses may have different purposes, among them, the production of cellulosic ethanol, also known as second generation bioethanol. This ethanol is produced from wasted inexpensive material that results especially from agrobusiness operations. Some of the biomasses used to produce this biofuel are produced in large quantities and have potential for fuel production, as for example, Eucalyptus grandis sawdust and rice husk, biomasses that are seldom used for obtaining carbohydrates. In order to use the carbohydrates of these biomasses it is required to degrade the lignin and decrease the recalcitrance of cellulose. These two factors are priorities to be considered for using these biomasses with efficiency and low costs. Several processes are used for removing lignin and for decreasing the crystallinity of cellulose. They are known collectively as pretreatments. Most of the existing pretreatments are either very expensive or produce unwanted substances. Therefore, a pretreatment that minimizes these disadvantages is required. Additionally, any pretreatment, be it physical, chemical or biological, should allow to recover as much carbohydrates as possible. Taking into account these guidelines, the goal of this thesis was to propose pretreatments that allow a highly efficient carbohydrate recovery by reducing the cellulose degree of crystallinity or even by degrading lignin and that are, at the same time, environmentally friendly, i.e., pretreatments that do not produce large amounts of waste material. For this purpose, two pretreatments are proposed, a biological treatment with basidiomycetes fungi and a physico-chemical treatment with liquid nitrogen cryo crushing). The pretreatment with white rot fungi was used because these organisms possess an enzymatic apparatus capable of degrading lignin efficiently. The liquid nitrogen pretreatment was used due to the ultra-fast freezing that occurs, a phenomenon that may cause important breaks in the biomass structure. The enzymatic saccharification of the treated materials was measured in order to quantify the efficiency of the treatment. The time course of the enzymatic saccarification was analysed in terms of an equation that allows to determine the initial rate of hydrolysis and to discriminate the easily hydrolysable fraction from the fraction that can be less easily hydrolyzed. In addition, infrared spectrometry, scanning microscopy and other physical techniques were used in order to get an insight into the physico-chemical modifications caused by each treatment. For the biological pretreatments of E. grandis sawdust the following ligninolytic fungi were used:Ganoderma lucidum, Phanerochaete chrysosporium, Pleurotus ostreatus, Pleurotus pulmonarius and Trametes sp. Liquid nitrogen (cryo crushing) was used to treat E. grandis sawdust and rice hulls. For the biological treatment, the lignocellulosic biomass was dried, grinded and moistened with a mineral solution and utilized as a substrate for fungi cultures for 30 days. After this time, water was added to extract the soluble material and the insoluble fibers were dried to constant weight. For the pretreatment with liquid nitrogen the fibers in natura were treated with liquid nitrogen, followed by maceration with a pestle and mortar. The pre- and biologically post-treated fibers were subjected to saccharification using commercial cellulases of Trichoderma reesei ATCC 26921 (Sigma- Aldrich C 8546). Cellulase and β-glucosidase Cellic® CTec2, and hemicellulases Cellic HTec2 ® (Novozymes, Araucaria, PR, Brazil) were used in the saccharification of the liquid nitrogen-treated fibers.The mixtures were maintained in shaker cultures for 48 h at specific temperatures. After this time, the mixtures were vacuum filtered. The reducing sugars present in the filtrates were estimated by the 3,5-dinitrosalicylic acid method. Glucose was measured using a glucose oxidase-based assay in the case of the liquid nitrogen pre-treated fibers. The lignocellulosic fibers pre- and post-treatment were characterized using scanning electron microscopy and by Fourier Transform Infrared (FTIR) spectroscopy for characterizing modifications in the lignin, cellulose and hemicellulose absorption bands. X-ray diffraction was used for testing specifically the modifications caused by the liquid nitrogen pretreatment. Equation fitting to experimental data was done by an iterative nonlinear least-squares procedure using the Scientist software from Micro Matth Scientific Software (Salt Lake City, UT). The biological pretreatment of the E. grandis sawdust fiber was effective with all the fungi that were used. The best results, however, were obtained with P. ostreatus and P. pulmonarius. FTIR analysis showed decreases in the major absorption bands of lignin, namely 1515 cm-1 (aromatic vibrations of lignin) and 1427 cm-1 (condensed guaiacyl and syringyl). In the cellulose bands there were small decreases in 1098 cm-1 (crystalline cellulose), 1375 cm-1 (cellulose and hemicellulose) and 898 cm-1 (amorphous cellulose), besides crystalline cellulose transformation into amorphous cellulose, important factor for an efficient saccharification. Electron microscopy revealed the appearance of holes, indicating a change made by fungi in the fibers, increasing its digestibility. The results obtained from the kinetic analyses of the cellulase action indicated an easily hydrolyzable part of cellulose in addition to a second fraction more resistant to hydrolysis. Pleurotus ostreatus and P. pulmonarius were very effective in making the pulp from sawdust E. grandis more readily hydrolysable by T. reesei cellulase. In the pretreatment using liquid nitrogen both E. grandis sawdust fibers and rice hulls experienced several structural modifications suggesting a disruption or weakening of the interactions between lignin and hemicellulose as well as between lignin and cellulose. X-ray diffraction indicates an increase in cellulose crystallinity, an observation suggesting a detachment between cellulose and lignin. The lignin content, however, measured by the methods of Klason lignin and complete solubilization of lignin with acetyl bromide, was not significantly diminished. This suggests that changes in the cellulose structure and separation of lignin from cellulose may already turn the polysaccharide more easily hydrolysable. Good yields of reducing sugars and glucose were found in the saccharification of the treated fibers, with maximal values of 54% and 40% for the holocellulose of E. grandis sawdust and rice husk, respectively. The yields obtained with the non-treated fibers were approximately 3-5% for both E. grandis sawdust and rice hulls. In the biological pretreatment with E. grandis there was a selective degradation of lignin by the fungi P. ostreatus and P. pulmonarius, as revealed by electron microscopy and FTIR analysis, leading to an increased efficiency of the enzymatic saccharification of cellulose. The kinetics of saccharification of the treated biomasses resembles in many aspects that of pure cellulose. The pretreatment with liquid nitrogen was also very effective in increasing the yield of saccharification, mainly by the deconstruction of the lignocellulosic structure. The two forms of pretreatment are in conformity with a worldwide trend that postulates the use of lignocellulosic biomass pretreatments that are less harmful to the environment and that are at the same time highly efficient in the release of fermentable carbohydrates.