How lignin sticks to cellulose-insights from atomic force microscopy enhanced by machine-learning analysis and molecular dynamics simulations.

Elucidating cellulose-lignin interactions at the molecular and nanometric scales is an important research topic with impacts on several pathways of biomass valorization. Here, the interaction forces between a cellulosic substrate and lignin are investigated. Atomic force microscopy with lignin-coated tips is employed to probe the site-specific adhesion to a cellulose film in liquid water. Over seven thousand force-curves are analyzed by a machine-learning approach to cluster the experimental data into types of cellulose-tip interactions. The molecular mechanisms for distinct types of cellulose-lignin interactions are revealed by molecular dynamics simulations of lignin globules interacting with different cellulose Iß crystal facets. This unique combination of experimental force-curves, data-driven analysis, and molecular simulations opens a new approach of investigation and updates the understanding of cellulose-lignin interactions at the nanoscale.

Metabolomic profiling of Spathaspora passalidarum fermentations reveals mechanisms that overcome hemicellulose hydrolysate inhibitors.

Understanding the mechanisms involved in tolerance to inhibitors is the first step in developing robust yeasts for industrial second-generation ethanol (E2G) production. Here, we used ultra-high-performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) and MetaboAnalyst 4.0 for analysis of MS data to examine the changes in the metabolic profile of the yeast Spathaspora passalidarum during early fermentation of hemicellulosic hydrolysates containing high or low levels of inhibitors (referred to as control hydrolysate or CH and strategy hydrolysate or SH, respectively). During fermentation of SH, the maximum ethanol production was 16 g L-1 with a yield of 0.28 g g-1 and productivity of 0.22 g L-1 h-1, whereas maximum ethanol production in CH fermentation was 1.74 g L-1 with a yield of 0.11 g g-1 and productivity of 0.01 g L-1 h-1. The high level of inhibitors in CH induced complex physiological and biochemical responses related to stress tolerance in S. passalidarum. This yeast converted compounds with aldehyde groups (hydroxymethylfurfural, furfural, 4-hydroxybenzaldehyde, syringaldehyde, and vanillin) into less toxic compounds, and inhibitors were found to reduce cell viability and ethanol production. Intracellularly, high levels of inhibitors altered the energy homeostasis and redox balance, resulting in lower levels of ATP and NADPH, while that of glycolytic, pentose phosphate, and tricarboxylic acid (TCA) cycle pathways were the most affected, being the catabolism of glucogenic amino acids, the main cellular response to inhibitor-induced stress. This metabolomic investigation reveals interesting targets for metabolic engineering of ethanologenic yeast strains tolerant against multiple inhibitors for E2G production. KEY POINTS: • Inhibitors in the hydrolysates affected the yeast's redox balance and energy status. • Inhibitors altered the glycolytic, pentose phosphate, TCA cycle and amino acid pathways. • S. passalidarum converted aldehyde groups into less toxic compounds.

Engineering aspects of hydrothermal pretreatment: From batch to continuous operation, scale-up and pilot reactor under biorefinery concept.

Different pretreatments strategies have been developed over the years mainly to enhance enzymatic cellulose degradation. In the new biorefinery era, a more holistic view on pretreatment is required to secure optimal use of the whole biomass. Hydrothermal pretreatment technology is regarded as very promising for lignocellulose biomass fractionation biorefinery and to be implemented at the industrial scale for biorefineries of second generation and circular bioeconomy, since it does not require no chemical inputs other than liquid water or steam and heat. This review focuses on the fundamentals of hydrothermal pretreatment, structure changes of biomass during this pretreatment, multiproduct strategies in terms of biorefinery, reactor technology and engineering aspects from batch to continuous operation. The treatise includes a case study of hydrothermal biomass pretreatment at pilot plant scale and integrated process design.

Development of hemicellulolytic enzyme mixtures for plant biomass deconstruction on target biotechnological applications.

An essential step in the conversion of lignocellulosic biomass to ethanol and other biorefinery products is conversion of cell wall polysaccharides into fermentable sugars by enzymatic hydrolysis. The objective of the present study was to understand the mode of action of hemicellulolytic enzyme mixtures for pretreated sugarcane bagasse (PSB) deconstruction and wheat arabinoxylan (WA) hydrolysis on target biotechnological applications. In this study, five hemicellulolytic enzymes-two endo-1,4-xylanases (GH10 and GH11), two α-L-arabinofuranosidases (GH51 and GH54), and one ß-xylosidase (GH43)-were submitted to combinatorial assays using the experimental design strategy, in order to analyze synergistic and antagonistic effects of enzyme interactions on biomass degradation. The xylooligosaccharides (XOSs) released from hydrolysis were analyzed by capillary electrophoresis and quantified by high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Based on this analysis, it was possible to define which enzymatic combinations favor xylose (X1) or XOS production and thus enable the development of target biotechnological applications. Our results demonstrate that if the objective is X1 production from WA, the best enzymatic combination is GH11 + GH54 + GH43, and for xylobiose (X2) production from WA, it is best to combine GH11 + GH51. However, if the goal is to produce XOS, the five enzymes used in WA hydrolysis are important, but for PSB hydrolysis, only GH11 is sufficient. If the final objective is bioethanol production, GH11 is responsible for hydrolyzing 64.3 % of hemicellulose from PSB. This work provides a basis for further studies on enzymatic mechanisms for XOS production, and the development of more efficient and less expensive enzymatic mixtures, targeting commercially viable lignocellulosic ethanol production and other biorefinery products.

Industrial-scale steam explosion pretreatment of sugarcane straw for enzymatic hydrolysis of cellulose for production of second generation ethanol and value-added products.

Steam explosion at 180, 190 and 200°C for 15min was applied to sugarcane straw in an industrial sugar/ethanol reactor (2.5m(3)). The pretreated straw was delignificated by sodium hydroxide and hydrolyzed with cellulases, or submitted directly to enzymatic hydrolysis after the pretreatment. The pretreatments led to remarkable hemicellulose solubilization, with the maximum (92.7%) for pretreatment performed at 200°C. Alkaline treatment of the pretreated materials led to lignin solubilization of 86.7% at 180°C, and only to 81.3% in the material pretreated at 200°C. All pretreatment conditions led to high hydrolysis conversion of cellulose, with the maximum (80.0%) achieved at 200°C. Delignification increase the enzymatic conversion (from 58.8% in the cellulignin to 85.1% in the delignificated pulp) of the material pretreated at 180°C, but for the material pretreated at 190°C, the improvement was less remarkable, while for the pretreated at 200°C the hydrolysis conversion decreased after the alkaline treatment.

Mass balance of pilot-scale pretreatment of sugarcane bagasse by steam explosion followed by alkaline delignification.

Five pilot-scale steam explosion pretreatments of sugarcane bagasse followed by alkaline delignification were explored. The solubilised lignin was precipitated with 98% sulphuric acid. Most of the pentosan (82.6%), and the acetyl group fractions were solubilised during pretreatment, while 90.2% of cellulose and 87.0% lignin were recovered in the solid fraction. Approximately 91% of the lignin and 72.5% of the pentosans contained in the steam-exploded solids were solubilised by delignification, resulting in a pulp with almost 90% of cellulose. The acidification of the black liquors allowed recovery of 48.3% of the lignin contained in the raw material. Around 14% of lignin, 22% of cellulose and 26% of pentosans were lost during the process. In order to increase material recovery, major changes, such as introduction of efficient condensers and the reduction in the number of washing steps, should be done in the process setup.

Second generation ethanol in Brazil: can it compete with electricity production?

Much of the controversy surrounding second generation ethanol production arises from the assumed competition with first generation ethanol production; however, in Brazil, where bioethanol is produced from sugarcane, sugarcane bagasse and trash will be used as feedstock for second generation ethanol production. Thus, second generation ethanol production may be primarily in competition with electricity production from the lignocellulosic fraction of sugarcane. A preliminary technical and economic analysis of the integrated production of first and second generation ethanol from sugarcane in Brazil is presented and different technological scenarios are evaluated. The analysis showed the importance of the integrated use of sugarcane including the biomass represented by surplus bagasse and trash that can be taken from the field. Second generation ethanol may favorably compete with bioelectricity production when sugarcane trash is used and when low cost enzyme and improved technologies become commercially available.

A study on the pretreatment of a sugarcane bagasse sample with dilute sulfuric acid.

Experiments based on a 2(3) central composite full factorial design were carried out in 200-ml stainless-steel containers to study the pretreatment, with dilute sulfuric acid, of a sugarcane bagasse sample obtained from a local sugar-alcohol mill. The independent variables selected for study were temperature, varied from 112.5°C to 157.5°C, residence time, varied from 5.0 to 35.0 min, and sulfuric acid concentration, varied from 0.0% to 3.0% (w/v). Bagasse loading of 15% (w/w) was used in all experiments. Statistical analysis of the experimental results showed that all three independent variables significantly influenced the response variables, namely the bagasse solubilization, efficiency of xylose recovery in the hemicellulosic hydrolysate, efficiency of cellulose enzymatic saccharification, and percentages of cellulose, hemicellulose, and lignin in the pretreated solids. Temperature was the factor that influenced the response variables the most, followed by acid concentration and residence time, in that order. Although harsher pretreatment conditions promoted almost complete removal of the hemicellulosic fraction, the amount of xylose recovered in the hemicellulosic hydrolysate did not exceed 61.8% of the maximum theoretical value. Cellulose enzymatic saccharification was favored by more efficient removal of hemicellulose during the pretreatment. However, detoxification of the hemicellulosic hydrolysate was necessary for better bioconversion of the sugars to ethanol.

Fermentation of cellulosic hydrolysates obtained by enzymatic saccharification of sugarcane bagasse pretreated by hydrothermal processing.

This work aims to evaluate the fermentability of cellulosic hydrolysates obtained by enzymatic saccharification of sugarcane bagasse pretreated by hydrothermal processing using Candida guilliermondii FTI 20037 yeast. The inoculum was obtained from yeast culture in a medium containing glucose as a carbon source supplemented with rice bran extract, CaCl(2)·2H(2)O and (NH(4))(2)SO(4) in 50 mL Erlenmeyer flasks, containing 20 mL of medium, initial 5.5 pH under agitation of an orbital shaker (200 rpm) at 30°C for 24 h. The cellulosic hydrolysates, prior to being used as a fermentation medium, were autoclaved for 15 min at 0.5 atm and supplemented with the same nutrients employed for the inoculum, except the glucose, using the same conditions for the inoculum, but with a period of 48 h. Preliminary results showed the highest consumption of glucose (97%) for all the hydrolysates, at 28 h of fermentation. The highest concentration of ethanol (20.5 g/L) was found in the procedure of sugarcane bagasse pretreated by hydrothermal processing (195°C/10 min in 20 L reactor) and delignificated with NaOH 1.0% (w/v), 100°C, 1 h in 500 mL stainless steel ampoules immersed in an oil bath.

Scale-up of diluted sulfuric acid hydrolysis for producing sugarcane bagasse hemicellulosic hydrolysate (SBHH).

Sugarcane bagasse was pretreated with diluted sulfuric acid to obtain sugarcane bagasse hemicellulosic hydrolysate (SBHH). Experiments were conducted in laboratory and semi-pilot reactors to optimize the xylose recovery and to reduce the generation of sugar degradation products, as furfural and 5-hydroxymethylfurfural (HMF). The hydrolysis scale-up procedure was based on the H-Factor, that combines temperature and residence time and employs the Arrhenius equation to model the sulfuric acid concentration (100 mg(acid)/g(dm)) and activation energy (109 kJ/mol). This procedure allowed the mathematical estimation of the results through simulation of the conditions prevailing in the reactors with different designs. The SBHH obtained from different reactors but under the same H-Factor of 5.45+/-0.15 reached similar xylose yield (approximately 74%) and low concentration of sugar degradation products, as furfural (0.082 g/L) and HMF (0.0071 g/L). Also, the highest lignin degradation products (phenolic compounds) were rho-coumarilic acid (0.15 g/L) followed by ferulic acid (0.12 g/L) and gallic acid (0.035 g/L). The highest concentration of ions referred to S (3433.6 mg/L), Fe (554.4 mg/L), K (103.9 mg/L). The H-Factor could be used without dramatically altering the xylose and HMF/furfural levels. Therefore, we could assume that H-Factor was directly useful in the scale-up of the hemicellulosic hydrolysate production.

Production, characterization and application of activated carbon from brewer's spent grain lignin.

Different types of activated carbon were prepared by chemical activation of brewer's spent grain (BSG) lignin using H(3)PO(4) at various acid/lignin ratios (1, 2, or 3g/g) and carbonization temperatures (300, 450, or 600 degrees C), according to a 2(2) full-factorial design. The resulting materials were characterized with regard to their surface area, pore volume, and pore size distribution, and used for detoxification of BSG hemicellulosic hydrolysate (a mixture of sugars, phenolic compounds, metallic ions, among other compounds). BSG carbons presented BET surface areas between 33 and 692 m(2)/g, and micro- and mesopores with volumes between 0.058 and 0.453 cm(3)/g. The carbons showed high capacity for adsorption of metallic ions, mainly nickel, iron, chromium, and silicon. The concentration of phenolic compounds and color were also reduced by these sorbents. These results suggest that activated carbons with characteristics similar to those commercially found and high adsorption capacity can be produced from BSG lignin.