Marcos Henrique Luciano Silveira

Current Pretreatment Technologies for the Development of Cellulosic Ethanol and Biorefineries.

Lignocellulosic materials, such as forest, agriculture, and agroindustrial residues, are among the most important resources for biorefineries to provide fuels, chemicals, and materials in such a way to substitute for, at least in part, the role of petrochemistry in modern society. Most of these sustainable biorefinery products can be produced from plant polysaccharides (glucans, hemicelluloses, starch, and pectic materials) and lignin. In this scenario, cellulosic ethanol has been considered for decades as one of the most promising alternatives to mitigate fossil fuel dependence and carbon dioxide accumulation in the atmosphere. However, a pretreatment method is required to overcome the physical and chemical barriers that exist in the lignin-carbohydrate composite and to render most, if not all, of the plant cell wall components easily available for conversion into valuable products, including the fuel ethanol. Hence, pretreatment is a key step for an economically viable biorefinery. Successful pretreatment method must lead to partial or total separation of the lignocellulosic components, increasing the accessibility of holocellulose to enzymatic hydrolysis with the least inhibitory compounds being released for subsequent steps of enzymatic hydrolysis and fermentation. Each pretreatment technology has a different specificity against both carbohydrates and lignin and may or may not be efficient for different types of biomasses. Furthermore, it is also desirable to develop pretreatment methods with chemicals that are greener and effluent streams that have a lower impact on the environment. This paper provides an overview of the most important pretreatment methods available, including those that are based on the use of green solvents (supercritical fluids and ionic liquids).

Enzymatic hydrolysis of steam-exploded sugarcane bagasse using high total solids and low enzyme loadings

Hydrolysis of phosphoric acid-impregnated steam-treated sugarcane bagasse was pre-optimized using a face-centered central composite design in which the process variables were the substrate total solids (TS, %), agitation intensity (AI, rpm) and enzyme loading (EL, gg(-1)). Pretreatment was carried out at 180°C for 10min using cane bagasse with 50wt% moisture content containing 9.5mg of H3PO4 per gram of dry biomass. Hydrolyses were performed for 96h at 50°C using Cellic CTec2® and water-washed steam-treated substrates. The highest amount of fermentable sugars was obtained with 20wt% TS, producing 76.8gL(-1) of glucose equivalents, which corresponded to a total glucan conversion of 69.2wt% and to a theoretical net increase of 39% in ethanol production from the same sugarcane tonnage without considering the use of leaves, tops and the additional yields from C5 sugars.

Kinetics of enzyme-catalyzed hydrolysis of steam-exploded sugarcane bagasse.

This work presents the experimental kinetic data and the fractal modeling of sugarcane bagasse steam treatment and enzymatic hydrolysis. Sugarcane bagasse (50 wt% moisture) was pretreated by autohydrolysis at 210 °C for 4 min. Acid catalysis involved the use of 9.5mg g(-1) of H2SO4 or H3PO4 in relation to the substrate dry mass at these same pretreatment conditions. Unwashed, water-washed and alkali-washed substrates were hydrolyzed at 2.0 wt% using 8 and 15 FPU g(-1) (108.22 and 199.54 mg/g) total solids of a Celluclast 1.5 L and Novozym 188 mixture (Novozymes). The fractal kinetic modeling was used to describe the effect of pretreatment and both washing processes on substrate accessibility. Water and/or alkali washing was not strictly necessary to achieve high hydrolysis efficiencies. Also, the fractal model coefficients revealed that H3PO4 was a better pretreatment catalyst under the experimental conditions used in this study, resulting in the most susceptible substrates for enzymatic hydrolysis.

Supercritical carbon dioxide combined with 1-butyl-3-methylimidazolium acetate and ethanol for the pretreatment and enzymatic hydrolysis of sugarcane bagasse

The use of green solvents for the partial delignification of milled sugarcane bagasse (1mm particle size) and for the enhancement of its susceptibility to enzymatic hydrolysis was demonstrated. The experiments were carried out for 2h using 40 g of supercritical carbon dioxide combined with 1-butyl-3-methylimidazolium acetate and 15.8 g of ethanol. The effects of temperature (110-180 °C), pressure (195-250 bar) and IL-to-bagasse mass ratio (0:1-1:1) were investigated through a factorial design in which the response variables were the extent of delignification and both anhydroglucose and anhydroxylose contents in the pretreated materials. The highest delignification degree (41%) led to the best substrate for hydrolysis, giving a 70.7 wt% glucose yield after 12h using 5 wt% and Cellic CTec2® (Novozymes) at 10 mg g(-1) total solids. Hence, excellent substrates for hydrolysis were produced with a minimal IL requirement, which could be recovered by ethanol washing for its downstream processing and reuse.

A simple and fast method for the determination of endo- and exo-cellulase activity in cellulase preparations using filter paper.

This study describes a procedure for the selective determination of endo- (EG) and exo- (ExG) cellulase activities using filter paper as the sole substrate. The procedure is based on the enzymes mode of action whereby EG activity predominantly forms insoluble reducing sugars and ExG activity soluble reducing sugars. The procedure was developed using filter paper as substrate for hydrolysis with three cellulase preparations of Hypocrea jecorina containing either endoglucanase (EG), predominantly exoglucanase (ExG) or both endo- and exoglucanase activities. Hydrolysis experiments, which were followed assessing the formation of total, soluble and insoluble reducing sugars (RS), showed that up to 30 min of hydrolysis predominantly insoluble reducing sugars were formed, while after this initial hydrolysis stage soluble reducing sugar formation increased significantly, making it thus possible to measure separately EG and ExG activity. FPA activities obtained from the reaction products at different reaction times suggest that EG-activity (FPA(insol)) should be measured between 10 and 20 min of hydrolysis. The proposed procedure allows to evaluate the EG and ExG activity contribution to total cellulase activity and to calculate the endo/exo activity ratio of any cellulase preparation.

Influence of mechanical agitation on the pH profile of total, soluble and insoluble filter paper activity of Hypocrea jecorina cellulase preparations

The characterization of cellulases is complex and has gained increasing importance due to the application of cellulases in finishing of textile fibres and the conversion of lignocellulose into second generation ethanol. Usually two or three different substrates such as filter paper Whatman No. 1, carboxymethylcellulose and avicel are used to characterize total, endo (EG) and exoglucanase (ExG) activity, respectively, and assays are carried out without any mechanical agitation. Unfortunately, the different activities obtained are not directly comparable and do not reflect cellulase behaviour in industrial applications, mainly due to physic-chemical differences between the substrates and the adopted procedures. In this study the pH profiles of three different cellulase preparations from Hypocrea jecorina (former Trichoderma reesei) have been characterized with modified filter paper assays which permitted the distinction between EG and ExG activities using a single insoluble substrate. Furthermore, the influence of mechanical agitation was studied. With the modified procedures, it could be shown that the contribution of EG and ExG activity to total cellulase activity and the endo/exo activity ratio depends on the cellulase preparation and varies with pH. When the assay was carried out under orbital agitation, EG activity increased significantly and the endo/exo-activity ratio increased.

Pretreatment Processes for Cellulosic Ethanol Production: Processes Integration and Modeling for the Utilization of Lignocellulosics Such as Sugarcane Straw

Pretreatment is the key step for a viable and efficient cellulosic ethanol production process and, for this reason, it must be very selective in avoiding polysaccharide degradation and inhibitors formation. This work provides a brief overview of the leading pretreatment technologies available to date, with emphasis on those that are already closed to or eventually reached commercial scale such as steam explosion and/or dilute acid hydrolysis. Details are also given with regard to the fundamental effects of pretreatment on the chemical composition and organizational structure of the plant cell wall. Furthermore, the impact of steam explosion and enzymatic hydrolysis on the overall capital cost of cellulosic ethanol production has been determined in light of the following process integration approaches using sugarcane straw as the reference material: simultaneous saccharification and co-fermentation (SSCF), separated hydrolysis and co-fermentation (SHCF) and separated hydrolysis and fermentation (SHF). As a result, cellulosic ethanol produced from SSCF, SHCF, and SHF processes resulted in capital cost estimates of

Hydrolysis of sugarcane bagasse with enzyme preparations from Acrophialophora nainiana grown on different carbon sources

1.66,

Bioconversion of Hemicellulose Into Ethanol and Value-Added Products

1.75, and

Second Generation Ethanol Production

2.23 per liter of ethanol produced. The difference among these values is related to the easiness with which different unit operations are harmonized in a sustainable and fully operational biorefinery unit.