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

BACKGROUND: The 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. RESULTS: Cross polarization by multiple contact periods (Multi-CP) method was used to obtain quantitative (13)C 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 (13)C 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. CONCLUSIONS: The 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.

Nuclear magnetic resonance investigation of water accessibility in cellulose of pretreated sugarcane bagasse.

BACKGROUND: Enzymatic hydrolysis is a crucial step of biomass conversion into biofuels and different pretreatments have been proposed to improve the process efficiency. Amongst the various factors affecting hydrolysis yields of biomass samples, porosity and water accessibility stand out due to their intimate relation with enzymes accessibility to the cellulose and hemicellulose fractions of the biomass. In this work, sugarcane bagasse was subjected to acid and alkali pretreatments. The changes in the total surface area, hydrophilicity, porosity and water accessibility of cellulose were investigated by scanning electron microscopy (SEM) and nuclear magnetic resonance (NMR). RESULTS: Changes in chemical and physical properties of the samples, caused by the partial removal of hemicellulose and lignin, led to the increase in porosity of the cell walls and unwinding of the cellulose bundles, as observed by SEM. (1)H NMR relaxation data revealed the existence of water molecules occupying the cores of wide and narrow vessels as well as the cell wall internal structure. Upon drying, the water molecules associated with the structure of the cell wall did not undergo significant dynamical and partial moisture changes, while those located in the cores of wide and narrow vessels kept continuously evaporating until reaching approximately 20% of relative humidity. This indicates that water is first removed from the cores of lumens and, in the dry sample, the only remaining water molecules are those bound to the cell walls. The stronger interaction of water with pretreated bagasse is consistent with better enzymes accessibility to cellulose and higher efficiency of the enzymatic hydrolysis. CONCLUSIONS: We were able to identify that sugarcane bagasse modification under acid and basic pretreatments change the water accessibility to different sites of the sample, associated with both bagasse structure (lumens and cell walls) and hydrophilicity (lignin removal). Furthermore, we show that the substrates with increased water accessibility correspond to those with higher hydrolysis yields and that there is a correlation between experimentally NMR-measured transverse relaxation times and the efficiency of enzymatic hydrolysis. This might allow for semiquantitative estimates of the enzymatic hydrolysis efficiency of biomass samples using inexpensive and non-destructive low-field (1)H NMR relaxometry methods.

Chemical and morphological characterization of sugarcane bagasse submitted to a delignification process for enhanced enzymatic digestibility.

BACKGROUND: In recent years, biorefining of lignocellulosic biomass to produce multi-products such as ethanol and other biomaterials has become a dynamic research area. Pretreatment technologies that fractionate sugarcane bagasse are essential for the successful use of this feedstock in ethanol production. In this paper, we investigate modifications in the morphology and chemical composition of sugarcane bagasse submitted to a two-step treatment, using diluted acid followed by a delignification process with increasing sodium hydroxide concentrations. Detailed chemical and morphological characterization of the samples after each pretreatment condition, studied by high performance liquid chromatography, solid-state nuclear magnetic resonance, diffuse reflectance Fourier transformed infrared spectroscopy and scanning electron microscopy, is reported, together with sample crystallinity and enzymatic digestibility. RESULTS: Chemical composition analysis performed on samples obtained after different pretreatment conditions showed that up to 96% and 85% of hemicellulose and lignin fractions, respectively, were removed by this two-step method when sodium hydroxide concentrations of 1% (m/v) or higher were used. The efficient lignin removal resulted in an enhanced hydrolysis yield reaching values around 100%. Considering the cellulose loss due to the pretreatment (maximum of 30%, depending on the process), the total cellulose conversion increases significantly from 22.0% (value for the untreated bagasse) to 72.4%. The delignification process, with consequent increase in the cellulose to lignin ratio, is also clearly observed by nuclear magnetic resonance and diffuse reflectance Fourier transformed infrared spectroscopy experiments. We also demonstrated that the morphological changes contributing to this remarkable improvement occur as a consequence of lignin removal from the sample. Bagasse unstructuring is favored by the loss of cohesion between neighboring cell walls, as well as by changes in the inner cell wall structure, such as damaging, hole formation and loss of mechanical resistance, facilitating liquid and enzyme access to crystalline cellulose. CONCLUSIONS: The results presented herewith show the efficiency of the proposed method for improving the enzymatic digestibility of sugarcane bagasse and provide understanding of the pretreatment action mechanism. Combining the different techniques applied in this work warranted thorough information about the undergoing morphological and chemical changes and was an efficient approach to understand the morphological effects resulting from sample delignification and its influence on the enhanced hydrolysis results.

Biological activities of the fermentation extract of the endophytic fungus alternaria alternata isolated from coffea arabica L / Atividades biológicas do extrato da fermentação do fungo endofítico alternaria alternata isoladas de coffea arabica L

A total of 22 endophytic fungi isolated from coffee (Coffea arabica L.) were cultivated in vitro and their crude extracts tested. The screening was carried out using the agar diffusion method against Staphylococcus aureus, Escherichia coli and Candida albicans. The most effective isolate was Alternaria alternata, and subsequently, its extract was assayed. The total phenolic content was 3.44 μg GAE/mg of the crude extract. For the antibacterial and antifungal activity assays, minimum inhibitory concentrations (MIC) and minimum bactericidal and fungicidal concentrations (MBC and MFC) were determined. The ranges of MIC values were 50-100 μg/mL for S. aureus and 400-800 μg/mL for E. coli. The extract did not show activity in the tested concentrations for C. albicans. The fungal crude extract was assayed for antioxidant activities. Its ability to scavenge DPPH radicals and antioxidant activity by β-carotene/linoleic acid system oxidation was not significant. In addition, antitumor activity was studied using the MTT assay. At a dilution of 400 μg/mL, the extract displayed a cytotoxic activity of approximately 50 percent towards HeLa cells in vitro. The results indicate that endophytic fungi could be a promising source of bioactive compounds and warrant further study.

Total de 22 fungos endofíticos isolados de café (Coffea arabica L.) foi cultivado in vitro e seus extratos testados. A triagem foi conduzida pelo método de difusão em agar contra bactérias Gram-positiva, Gram-negativa e uma levedura. O isolado mais efetivo foi Alternaria alternata e, subsequentemente, seu extrato foi analisado. O conteúdo de fenólicos totais do extrato bruto foi de 3,44 μg EAG/mg de extrato. Para os testes de atividade antimicrobiana, a concentração inibitória mínima (CIM) e concentração bactericida e fungicida mínima (CBM e CFM) contra Staphylococcus aureus, Escherichia coli e Candida albicans foram determinadas. Resultados da CIM variaram entre 50-100 μg/mL para S. aureus e 400-800 μg/mL para E. coli. O extrato bruto não apresentou atividade nas concentrações testadas para C. albicans. Foram analisadas as atividades antioxidantes do extrato bruto. Sua habilidade para seqüestrar radicais DPPH e a atividade antioxidante pela oxidação do sistema β-caroteno/ácido linoléico não foram significativas. Além disso, a atividade antitumoral foi estudada pelo teste do MTT. À diluição de 400 μg/mL, o extrato apresentou atividade de aproximadamente 50 por cento sobre as células HeLa in vitro. Os resultados indicam que fungos endófitos poderiam ser uma fonte promissora de compostos bioativos necessitando de estudos futuros.