Effects of pretreatment on morphology, chemical composition and enzymatic digestibility of eucalyptus bark: a potentially valuable source of fermentable sugars for biofuel production – part 1

Abstract Background In recent years, the growing demand for biofuels has encouraged the search for different sources of underutilized lignocellulosic feedstocks that are available in sufficient abundance to be used for sustainable biofuel production. Much attention has been focused on biomass from grass. However, large amounts of timber residues such as eucalyptus bark are available and represent a potential source for conversion to bioethanol. In the present paper, we investigate the effects of a delignification process with increasing sodium hydroxide concentrations, preceded or not by diluted acid, on the bark of two eucalyptus clones: Eucalyptus grandis (EG) and the hybrid, E. grandis x urophylla (HGU). The enzymatic digestibility and total cellulose conversion were measured, along with the effect on the composition of the solid and the liquor fractions. Barks were also assessed using Fourier-transform infrared spectroscopy (FTIR), solid-state nuclear magnetic resonance (NMR), X-Ray diffraction, and scanning electron microscopy (SEM). Results Compositional analysis revealed an increase in the cellulose content, reaching around 81% and 76% of glucose for HGU and EG, respectively, using a two-step treatment with HCl 1%, followed by 4% NaOH. Lignin removal was 84% (HGU) and 79% (EG), while the hemicellulose removal was 95% and 97% for HGU and EG, respectively. However, when we applied a one-step treatment, with 4% NaOH, higher hydrolysis efficiencies were found after 48 h for both clones, reaching almost 100% for HGU and 80% for EG, in spite of the lower lignin and hemicellulose removal. Total cellulose conversion increased from 5% and 7% to around 65% for HGU and 59% for EG. NMR and FTIR provided important insight into the lignin and hemicellulose removal and SEM studies shed light on the cell-wall unstructuring after pretreatment and lignin migration and precipitation on the fibers surface, which explain the different hydrolysis rates found for the clones. Conclusion Our results show that the single step alkaline pretreatment improves the enzymatic digestibility of Eucalyptus bark. Furthermore, the chemical and physical methods combined in this study provide a better comprehension of the pretreatment effects on cell-wall and the factors that influence enzymatic digestibility of this forest residue.

Physico-chemical, spectroscopical and thermal characterization of biodiesel obtained by enzymatic route as a tool to select the most efficient immobilized lipase

Two microbial lipases from Burkholderia cepacia and Pseudomonas fluorescens were evaluated as catalysts for the enzymatic transesterification of beef tallow with ethanol and the most efficient lipase source was selected by taking into account the properties of the product to be used as fuel. Both lipases were immobilized on an epoxy silica-polyvinyl alcohol composite by covalent immobilization and used to perform the reactions under the following operational conditions: beef tallow-to-ethanol molar ratio of 1:9, 45ºC and 400 units of enzymatic activity per gram of fat. Products, characterized using Fourier Transform Infrared spectroscopy (FTIR), viscosimetry, thermogravimetry and ¹H NMR spectroscopy, suggested that the biodiesel sample obtained in the reaction catalyzed by Burkholderia cepacia lipase has the best set of properties for fuel usage.

Production and action of an Aspergillus phoenicis enzymatic pool using different carbon sources

Aspergillus phoenicis is an interesting heat tolerant fungus that can synthesize enzymes with several applications in the food industry due to its great hydrolytic potential. In this work, the fungus produced high enzymatic levels when cultivated on inexpensive culture media consisting of flakes from different origins such as cassava flour, wheat fibre, crushed soybean, agro-industrial wastes, starch, glucose or maltose. Several enzymatic systems were produced from these carbon sources, but amylase was the most evident, followed by pectinase and xylanase. Traces of CMCases, avicelase, lipase, β-xylosidase, β-glucosidase and α-glucosidase activities were also detected. Amylases were produced on rye flakes, starch, oat flakes, corn flakes, cassava flour and wheat fibre. Significant amylolytic levels were produced in the culture medium with glucose or when this sugar was exhausted, suggesting an enzyme in the constitutive form. Cassava flour, rye, oats, barley and corn flakes were also used as substrates in the hydrolytic reactions, aiming to verify the liberation potential of reducing sugars. Corn flakes induced greater liberation of reducing sugars as compared to the others. Thin layer chromatography of the reaction end products showed that the hydrolysis of cassava flour liberated maltooligosaccharides, but cassava flour and corn, rye, oats and barley flakes were hydrolyzed to glucose. These results suggested the presence of glucoamylase and α-amylase as part of the enzymatic pool of A. phoencis.