Ayla Sant’Ana da Silva

Major improvement in the rate and yield of enzymatic saccharification of sugarcane bagasse via pretreatment with the ionic liquid 1-ethyl-3-methylimidazolium acetate ([Emim] [Ac]).

In this study, sugarcane bagasse was pretreated by six ionic liquids (ILs) using a bagasse/IL ratio of 1:20 (wt%). The solubilization of bagasse in the ILs was followed by water precipitation. On using 1-ethyl-3-methylimidazolium acetate [Emim] [Ac] at 120 °C for 120 min, 20.7% of the bagasse components remained dissolved and enzymatic saccharification experiments resulted on 80% glucose yield within 6h, which evolved to over 90% within 24 h. Moreover, FE-SEM analysis of the precipitated material indicated a drastic lignin extraction and the exposure of nanoscopic cellulose microfibrils with widths of less than 100 nm. The specific surface area (SSA) of the pretreated bagasse (131.84 m2/g) was found to be 100 times that of untreated bagasse. The ability of [Emim] [Ac] to simultaneously increase the SSA and to decrease the biomass crystallinity is responsible for the improved bagasse enzymatic saccharification rates and yields obtained in this work.

Amino acids interference on the quantification of reducing sugars by the 3,5-dinitrosalicylic acid assay mislead carbohydrase activity measurements

This study evaluated the interference of the amino acids tryptophan, cysteine, histidine, tyrosine, hydroxyproline, leucine, proline, serine, glycine, valine, glutamic acid, phenylalanine, and methionine on the measurement of reducing sugars using a phenol-free 3,5-dinitrosalicylic acid (DNS) reagent. It was found that in reaction mixtures containing 20mM of either tryptophan, cysteine, histidine, tyrosine, or hydroxyproline the measurement of 3.7 mM glucose was overestimated by 76%, 50%, 35%, 18%, and 10%, respectively. The amino acids valine, glutamic acid, and phenylalanine did not affect the DNS reaction, while methionine decreased the color development by 5%. The measurement of glucose, xylose, arabinose, and cellobiose at the 3.7-12.4 mM range in the presence of 20 mM cysteine resulted in an overestimated concentration of 34.8-50%. Enzymatic assays for measuring xylanolytic and filter paper activity (FPAse) were conducted in the presence of 20-60 mM cysteine, and compared to cysteine-free assays. In the presence of cysteine, the measured xylanase activity increased threefold and the FPAse activity increased twofold due to the overestimation of the reducing sugar concentrations in the assays. The interference from cysteine was reduced to a maximum of 8.6% when a DNS reagent containing phenol was used.

Continuous pretreatment of sugarcane bagasse at high loading in an ionic liquid using a twin-screw extruder

Ionic liquids (ILs) are innovative and effective solvents for pretreating lignocellulose biomasses because they bring about a noticeable increase in the enzymatic saccharification of these materials. However, the reported solid loadings in ILs of approximately 5.0 wt% reflect their large consumption per gram of biomass and hinder their use in biomass pretreatment. In the present study, a twin-screw extruder with high shearing force was used as a pretreatment reactor to process sugarcane bagasse at high loadings in the IL 1-ethyl-3-methylimidazolium acetate. This procedure allowed effective pretreatment of biomasses at loadings as high as 25 wt% for 8 min at 140 °C, resulting in glucose yields of more than 90% after 24 h of enzymatic saccharification of the pretreated material. This glucose yield was comparable to that of a 4.8 wt% bagasse loading that was pretreated for 120 min at 120 °C in a stirring reactor and enzymatically hydrolyzed under the same conditions. A higher bagasse loading, 50 wt%, also afforded a high glucose yield of 76.4%. Characterization of the pretreated materials by examining the surface morphology at the nanoscopic scale, measuring the specific surface area (SSA), and analyzing the degree of crystallinity showed that the use of the extruder as a pretreatment reactor significantly decreased the crystallinity and increased the SSA by more than 100-fold. In addition, the extrusion process can be conducted continuously and is appropriate for industrial-scale biomass processing, allowing higher reactant concentrations and throughputs, higher mixing rates, and more uniform products compared to batch processes.

Efficient production of lignocellulolytic enzymes xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by the mutant strain Aspergillus awamori 2B.361 U2/1.

The production of xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by Aspergillus awamori 2B.361 U2/1, a hyper producer of glucoamylase and pectinase, was evaluated using selected conditions regarding nitrogen nutrition. Submerged cultivations were carried out at 30 °C and 200 rpm in growth media containing 30 g wheat bran/L as main carbon source and either yeast extract, ammonium sulfate, sodium nitrate or urea, as nitrogen sources; in all cases it was used a fixed molar carbon to molar nitrogen concentration of 10.3. The use of poor nitrogen sources favored the accumulation of xylanase, β-xylosidase and ferulic acid esterase to a peak concentrations of 44,880; 640 and 118 U/L, respectively, for sodium nitrate and of 34,580, 685 and 170 U/L, respectively, for urea. However, the highest β-glucosidase accumulation of 10,470 U/L was observed when the rich organic nitrogen source yeast extract was used. The maxima accumulation of filter paper activity, xylanase, β-xylosidase, ferulic acid esterase and β-glucosidase by A. awamori 2B.361 U2/1 was compared to that produced by Trichoderma reesei Rut-C30. The level of β-glucosidase was over 17-fold higher for the Aspergillus strain, whereas the levels of xylanase and β-xylosidase were over 2-fold higher. This strain also produced ferulic acid esterase (170 U/L), which was not detected in the T. reesei culture.

Combining biomass wet disk milling and endoglucanase/β-glucosidase hydrolysis for the production of cellulose nanocrystals.

Cellulose nanocrystals (CNCs), a biomaterial with high added value, were obtained from pure cellulose, Eucalyptus holocellulose, unbleached Kraft pulp, and sugarcane bagasse, by fibrillating these biomass substrates using wet disk milling (WDM) followed by enzymatic hydrolysis using endoglucanase/β-glucosidase. The hydrolysis experiments were conducted using the commercial enzyme OptimashBG or a blend of Pyrococcus horikoshii endoglucanase and Pyrococcus furiosus β-glucosidase. The fibrillated materials and CNCs were analyzed by X-ray diffraction, atomic force microscopy, scanning electron microscopy, and the specific surface area (SSA) was measured. WDM resulted in the formation of long and twisted microfibers of 1000-5000 nm in length and 4-35 nm in diameter, which were hydrolyzed into shorter and straighter CNCs of 500-1500 nm in length and 4-12 nm in diameter, with high cellulose crystallinity. Therefore, the CNCs aspect ratio was successfully adjusted by endoglucanases under mild reaction conditions, relative to the reported acidic hydrolysis method.

Use of cellobiohydrolase-free cellulase blends for the hydrolysis of microcrystalline cellulose and sugarcane bagasse pretreated by either ball milling or ionic liquid [Emim][Ac]

This study investigated the requirement of cellobiohydrolases (CBH) for saccharification of microcrystalline cellulose and sugarcane bagasse pretreated either by ball milling (BM) or by ionic liquid (IL) [Emim][Ac]. Hydrolysis was done using CBH-free blends of Pyrococcus horikoshii endoglucanase (EG) plus Pyrococcus furiosus β-glucosidase (EGPh/BGPf) or Optimash™ BG while Acremonium Cellulase was used as control. IL-pretreated substrates were hydrolyzed more effectively by CBH-free enzymes than were the BM-pretreated substrates. IL-treatment decreased the crystallinity and increased the specific surface area (SSA), whereas BM-treatment decreased the crystallinity without increasing the SSA. The hydrolysis of IL-treated cellulose by EGPh/BGPf showed a saccharification rate of 3.92 g/Lh and a glucose yield of 81% within 9h. These results indicate the efficiency of CBH-free enzymes for the hydrolysis of IL-treated substrates.

Biomass pretreatment: a critical choice for biomass utilization via biotechnological routes

The necessary biomass pretreatment step, to render the material accessible to the relevant enzyme pool, has been under thorough investigation as the production of biomass syrups, via enzymatic hydrolysis, with high sugars concentrations and yields and low inhibitors concentrations requires the pretreatment to be both efficient and low cost. A good choice for biomass pretreatment should be made by considering: (i) the possibility to use high biomass concentration; (ii) a highly digestible pretreated solid by either increasing the biomass superficial area or decrease in crystallinity or both; (iii) no significant sugar degradation into toxic compounds; (iv) yeast and bacterial fermentation compatibility of the derived sugar syrups; (v) lignin recovery; (vi) operation in reasonably sized and moderately priced reactors and (vii) minimum heat and power requirements [1]. Considering the most known pretreatments, such as diluted acid, hydrothermal processes, steam explosion, milling, extrusion, and ionic liquids, different pretreatment methods produce different effects on the biomass in terms of its structure and composition [2]. For example, the hydrothermal, steam explosion and acidic pretreatments conceptually remove mainly the biomass hemicellulose fraction whereas alkaline pretreatments remove lignin. On the other hand the product of a milling-based pretreatment retains the biomass initial composition. Furthermore, cellulose crystallinity is not significantly reduced by pretreatments based on steam, or hydrothermal, or acidic procedures, whereas ionic liquid-based techniques can shift crystalline cellulose into amorphous cellulose, substantially increasing the enzymatic hydrolysis rates and yields. As such, the choice of pretreatment and its operational conditions as well as the composition of the enzyme blend used in the hydrolysis step, determines the hexose and pentose sugars composition, the concentration and toxicity of the resulting biomass syrups. The activity profile of the enzyme blend and the enzyme load for an effective saccharification may also vary according to the pretreatment. Indeed, a low hemicellulase load can be used for a xylan-free biomass and a lower cellulase load will be needed for the hydrolysis of a low crystalline and highly amorphous pretreated biomass material. As the pretreatment choice will also be affected by the type of biomass, the envisaged biorefinery model will need to consider the main types of biomass that will be used for the biorefinery operation so as to select an appropriate, and versatile pretreatment method [3]. Considering the biorrefinery concept which broadens the biomass derived products, the C6 sugars could be fermented into ethanol, while the C5 stream could be used for the production, via biotechnological routes, of a wide range of chemicals with higher added value. To date, sugarcane and woody biomass, depending on the geographic location, are strong candidates as the main renewable resources to be fed into a biorefinery. However, due to major differences regarding their physical properties and chemical composition, the relevant pretreatments to be used in each case are expected to be selective and customized. Moreover, a necessary conditioning step for wood size reduction, prior to the pretreatment, may not be necessary for sugarcane bagasse, affecting the pretreatment energy consumption and costs. Moreover, the choice of pretreatment should take into account the foreseen utilization of the main biomass molecular components (cellulose, hemicelluloses and lignin). It is important to point out that lignin can be used as a valuable solid fuel or as a source of aromatic structures for the chemical industry. Sugarcane is one of the major agricultural crops when considering ethanol production, especially in tropical countries. In Brazil, sugarcane occupies 8.4 million hectares, which corresponds to 2.4% of farmable lands in Brazil. The gross revenue of this sector is about US

Milling pretreatment of sugarcane bagasse and straw for enzymatic hydrolysis and ethanol fermentation.

20 billion (54% as ethanol, 44% as sugar, and 2% as bioelectricity) [4]. In addition, up to 50% of all vehicles in Brazil are flex fuel cars, which corresponds to approximately 15 million cars [5]. Given the above, Brazil is an important player in this scenario, and, consequently, sugarcane bagasse and straw are promising feed stocks for biomass ethanol. Brazil produced, in 2008, 415 million tons of sugar cane residues, 195 million tons of sugarcane bagasse, and 220 million tons of sugarcane straw, whereas the forecast for the 2011 sugarcane production is 590 million tons, which would correspond to 178 million tons of bagasse, and 200 million tons of straw [6]. Currently, in Brazil, R&D on the use of biomass via biotechnological routes has been focused mainly on agricultural residues such as sugarcane residual biomass.