Oigres D. Bernardinelli

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

BackgroundIn 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).ResultsCompositional 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.ConclusionOur 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.

Multi-scale structural and chemical analysis of sugarcane bagasse in the process of sequential acid-base pretreatment and ethanol production by Scheffersomyces shehatae and Saccharomyces cerevisiae

BackgroundHeavy usage of gasoline, burgeoning fuel prices, and environmental issues have paved the way for the exploration of cellulosic ethanol. Cellulosic ethanol production technologies are emerging and require continued technological advancements. One of the most challenging issues is the pretreatment of lignocellulosic biomass for the desired sugars yields after enzymatic hydrolysis. We hypothesized that consecutive dilute sulfuric acid-dilute sodium hydroxide pretreatment would overcome the native recalcitrance of sugarcane bagasse (SB) by enhancing cellulase accessibility of the embedded cellulosic microfibrils.ResultsSB hemicellulosic hydrolysate after concentration by vacuum evaporation and detoxification showed 30.89 g/l xylose along with other products (0.32 g/l glucose, 2.31 g/l arabinose, and 1.26 g/l acetic acid). The recovered cellulignin was subsequently delignified by sodium hydroxide mediated pretreatment. The acid–base pretreated material released 48.50 g/l total reducing sugars (0.91 g sugars/g cellulose amount in SB) after enzymatic hydrolysis. Ultra-structural mapping of acid–base pretreated and enzyme hydrolyzed SB by microscopic analysis (scanning electron microcopy (SEM), transmitted light microscopy (TLM), and spectroscopic analysis (X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, Fourier transform near-infrared (FT-NIR) spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy) elucidated the molecular changes in hemicellulose, cellulose, and lignin components of bagasse. The detoxified hemicellulosic hydrolysate was fermented by Scheffersomyces shehatae (syn. Candida shehatae UFMG HM 52.2) and resulted in 9.11 g/l ethanol production (yield 0.38 g/g) after 48 hours of fermentation. Enzymatic hydrolysate when fermented by Saccharomyces cerevisiae 174 revealed 8.13 g/l ethanol (yield 0.22 g/g) after 72 hours of fermentation.ConclusionsMulti-scale structural studies of SB after sequential acid–base pretreatment and enzymatic hydrolysis showed marked changes in hemicellulose and lignin removal at molecular level. The cellulosic material showed high saccharification efficiency after enzymatic hydrolysis. Hemicellulosic and cellulosic hydrolysates revealed moderate ethanol production by S. shehatae and S. cerevisiae under batch fermentation conditions.

Folding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR

Exploitation of plant lignocellulosic biomass is hampered by our ignorance of the molecular basis for its properties such as strength and digestibility. Xylan, the most prevalent non-cellulosic polysaccharide, binds to cellulose microfibrils. The nature of this interaction remains unclear, despite its importance. Here we show that the majority of xylan, which forms a threefold helical screw in solution, flattens into a twofold helical screw ribbon to bind intimately to cellulose microfibrils in the cell wall. 13C solid-state magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy, supported by in silico predictions of chemical shifts, shows both two- and threefold screw xylan conformations are present in fresh Arabidopsis stems. The twofold screw xylan is spatially close to cellulose, and has similar rigidity to the cellulose microfibrils, but reverts to the threefold screw conformation in the cellulose-deficient irx3 mutant. The discovery that induced polysaccharide conformation underlies cell wall assembly provides new principles to understand biomass properties.

Evaluating the composition and processing potential of novel sources of Brazilian biomass for sustainable biorenewables production

BackgroundThe search for promising and renewable sources of carbohydrates for the production of biofuels and other biorenewables has been stimulated by an increase in global energy demand in the face of growing concern over greenhouse gas emissions and fuel security. In particular, interest has focused on non-food lignocellulosic biomass as a potential source of abundant and sustainable feedstock for biorefineries. Here we investigate the potential of three Brazilian grasses (Panicum maximum, Pennisetum purpureum and Brachiaria brizantha), as well as bark residues from the harvesting of two commercial Eucalyptus clones (E. grandis and E. grandis x urophylla) for biofuel production, and compare these to sugarcane bagasse. The effects of hot water, acid, alkaline and sulfite pretreatments (at increasing temperatures) on the chemical composition, morphology and saccharification yields of these different biomass types were evaluated.ResultsThe average yield (per hectare), availability and general composition of all five biomasses were compared. Compositional analyses indicate a high level of hemicellulose and lignin removal in all grass varieties (including sugarcane bagasse) after acid and alkaline pretreatment with increasing temperatures, whilst the biomasses pretreated with hot water or sulfite showed little variation from the control. For all biomasses, higher cellulose enrichment resulted from treatment with sodium hydroxide at 130°C. At 180°C, a decrease in cellulose content was observed, which is associated with high amorphous cellulose removal and 5-hydroxymethyl-furaldehyde production. Morphological analysis showed the effects of different pretreatments on the biomass surface, revealing a high production of microfibrillated cellulose on grass surfaces, after treatment with 1% sodium hydroxide at 130°C for 30 minutes. This may explain the higher hydrolysis yields resulting from these pretreatments, since these cellulosic nanoparticles can be easily accessed and cleaved by cellulases.ConclusionOur results show the potential of three Brazilian grasses with high productivity yields as valuable sources of carbohydrates for ethanol production and other biomaterials. Sodium hydroxide at 130°C was found to be the most effective pretreatment for enhanced saccharification yields. It was also efficient in the production of microfibrillated cellulose on grass surfaces, thereby revealing their potential as a source of natural fillers used for bionanocomposites production.

Quantitative 13C MultiCP solid-state NMR as a tool for evaluation of cellulose crystallinity index measured directly inside sugarcane biomass

BackgroundThe crystallinity index (CI) is often associated with changes in cellulose structure after biological and physicochemical pretreatments. While some results obtained with lignocellulosic biomass demonstrate a progressive increase in the CI as a function of pretreatments, it is also shown that the CI can significantly vary depending on the choice of the measurement method. Besides, the influence of the CI on the recalcitrance of biomass has been controversial for a long time, but the most recent results tend to point out that the efficiency of pretreatments in reducing the recalcitrance is not clearly correlated with the decrease of the CI. Much of this controversy is somewhat associated with the inability to distinguish between the CI of the cellulose inside the biomass and the CI of the full biomass, which contains other amorphous components such as lignin and hemicellulose.ResultsCross polarization by multiple contact periods (Multi-CP) method was used to obtain quantitative 13C solid-state nuclear magnetic resonance (ssNMR) spectra of sugarcane bagasse biomass submitted to two-step pretreatments and/or enzymatic hydrolysis. By comparing the dipolar filtered Multi-CP 13C NMR spectra of untreated bagasse samples with those of samples submitted to acid pretreatment, we show that a 1% H2SO4-assisted pretreatment was very effective in removing practically all the hemicellulose signals. This led us to propose a spectral editing procedure based on the subtraction of MultiCP spectra of acid-treated biomass from that of the extracted lignin, to obtain a virtually pure cellulose spectrum. Based on this idea, we were able to evaluate the CI of the native cellulose inside the sugarcane bagasse biomass.ConclusionsThe results show the validity of the proposed method as a tool for evaluating the variations in the CI of the cellulose inside biomasses of similar kinds. Despite a clear increase in the CI of biomass as measured by X-ray diffraction, no significant variations were observed in the CI of the cellulose inside the biomass after a particular 1% H2SO4/0.25–4% NaOH chemical-assisted pretreatments. The CI of cellulose inside the biomass solid fraction that remained after the enzymatic hydrolysis was also evaluated. The results show a slight increase in crystallinity.

Correlation between molecular conformation, packing, and dynamics in oligofluorenes: a theoretical/experimental study.

Fluorene-based systems have shown great potential as components in organic electronics and optoelectronics (organic photovoltaics, OPVs, organic light emitting diodes, OLEDs, and organic transistors, OTFTs). These systems have drawn attention primarily because they exhibit strong blue emission associated with relatively good thermal stability. It is well-known that the electronic properties of polymers are directly related to the molecular conformations and chain packing of polymers. Here, we used three oligofluorenes (trimer, pentamer, and heptamer) as model systems to theoretically investigate the conformational properties of fluorene molecules, starting with the identification of preferred conformations. The hybrid exchange-correlation functional, OPBE, and ZINDO/S-CI showed that each oligomer exhibits a tendency to adopt a specific chain arrangement, which could be distinguished by comparing their UV/vis electronic absorption and (13)C NMR spectra. This feature was used to identify the preferred conformation of the oligomer chains in chloroform-cast films by comparing experimental and theoretical UV/vis and (13)C NMR spectra. Moreover, the oligomer chain packing and dynamics in the films were studied by DSC and several solid-state NMR techniques, which indicated that the phase behavior of the films may be influenced by the tendency that each oligomeric chain has to adopt a given conformation.

Research data supporting “Folding of xylan onto cellulose fibrils in plant cell walls revealed by solid-state NMR”

Solid state NMR data files. Files for the DIPSHIFT calculations are listed below. The original Origin.c code may not be widely available therefore Matlab versions are also attached 1) the original Origin.c code - dipshift.c (inside are the codes for CH and CH2 groups, which can be called directly from the origin command window) 2) A .m file code for calculating DIPSHIFT curves for CH groups - DIPSHIFT_CH.m. 3) A .m file code for calculating DIPSHIFT curves for CH2 groups DIPSHIFT_CH2.m. 4) a set of angles and weight for powder average based on the repulsion method - powder_angles.dat (this can be used for both Matlab or Origin.c, but in the latter case it should be loaded into an Origin worksheet before running).

Analyses of Biomass Products by Nuclear Magnetic Resonance Spectroscopy

Nuclear magnetic resonance spectroscopy has been one of the most important analytical techniques to analyze biomass materials and their transformation products. 1H, 13C, 15N, and 31P NMR techniques have been used to analyze carbohydrates, proteins, lipids, and polycondensed compounds in raw and processed biomass, from plants, animals, fungi, algae, and other living beings. NMR has been used in qualitative and quantitative analyses of biomass materials, determination of the chemical composition, and structure and dynamics of monomers, oligomers, and polymeric materials. One of the major NMR advantages is its nondestructive nature that maintains sample integrity and the analyzed samples can be analyzed by other methods. This chapter provides basic information about NMR measurements and spectroscopic parameters, analysis in solid state, liquid state and in heterogeneous samples.