Production of highly antioxidant lignin nanoparticles from a hardwood technical lignin.

Eucalypt kraft lignin isolated in a LignoBoost™ pilot plant was characterized by GC-MS, ICP-OES, DSC, HPSEC, 31P NMR, and HSQC 2D-NMR to be used without any further processing to produce lignin nanoparticles (LNPs) by nanoprecipitation. Tetrahydrofuran (THF) was used as a solvent, and water as a non-solvent. Microscopic analysis (TEM) showed that LNPs were regularly spherical with some hollow particles dispersed in-between, and sizes were tunable by changing the solvent dripping rate onto the non-solvent. LNP particle sizes had a bimodal distribution, with the largest population having an average apparent hydrodynamic diameter ranging from 105.6 to 75.6 nm. Colloidal dispersions of LNPs in water presented good stability in different dilutions without significant size changes upon storage at pH close to neutral for as long as 45 days. Zeta potentials around -40 mV were obtained for LNP suspensions at pH ranging from 7 to 9. The high carbohydrate content (circa 10 % on a dry basis, mostly xylans) of the lignin precursor did not interfere in LNP formation, whose antioxidant activity was expressive as demonstrated by the ABTS assay at pH 7.4, with an EC50 of 4.04 µg mL-1. Also, the Trolox® equivalent antioxidant capacity (TEAC) of LNPs reached 1.90 after 40 min reaction time.

Ethanol organosolv pretreatment of sugarcane bagasse assisted by organic acids and supercritical carbon dioxide.

The scCO2-assisted organosolv pretreatment of sugarcane bagasse was carried out using aqueous ethanol and organic acid catalysts. Variables involved were temperature (150-190 °C), time (0-60 min), type of catalyst (acetic, citric, and oxalic acids), amount of CO2 (25-50 g), ethanol titer in water (0-80 vol%), and catalyst concentration (0.5 to 1.5 % w·v-1). The best delignification (86 wt%) and glucan retention (89 wt%) were achieved at 170 °C for 15 min using 60 vol% ethanol in water, 1 wt% oxalic acid, and 25 g CO2. Organic acid esterification was a limitation for pretreatment operations using ethanol titers above 60 vol%. Enzymatic hydrolysis of pretreated materials at 1 % (w·v-1) glucans released 74.3 ± 0.2 % glucose in 96 h using Cellic CTec3 (Novozymes) at 9.89 FPU·gglucans-1. The concentrated pretreatment liquor allowed lignin recovery by water precipitation in high yields, while the aqueous supernatant contained low levels of fermentation inhibitors.

The boosting effect of recombinant hemicellulases on the enzymatic hydrolysis of steam-treated sugarcane bagasse.

To increase the efficiency of enzyme cocktails in deconstructing cellulose and hemicelluloses present in the plant cell wall, a combination of enzymes with complementary activities is required. Xylan is the main hemicellulose component of energy crops and for its complete hydrolysis a system consisting of several enzymes acting cooperatively, including endoxylanases (XYN), ß-xylosidases (XYL) and α-l-arabinofuranosidases (ABF) is necessary. The current work aimed at evaluating the effect of recombinant hemicellulolytic enzymes on the enzymatic hydrolysis of steam-exploded sugarcane bagasse (SEB). One recombinant endoxylanase (HXYN2) and one recombinant ß-xylosidase (HXYLA) from Humicola grisea var thermoidea, together with an α-l-arabinofuranosidase (AFB3) from Penicillium pupurogenum, all produced in Pichia pastoris, were used to formulate an efficient enzyme mixture for SEB hydrolysis using a 23 Central Composite Rotatable Design (CCRD). The most potent enzyme for SEB hydrolysis was ABF3. Subsequently, the optimal enzyme mixture was used in combination with commercial cellulases (Accellerase 1500), either simultaneously or in sequential experiments. The supplementation of Accellerase 1500 with hemicellulases enhanced the glucose yield from SEB hydrolysis by 14.6%, but this effect could be raised to 50% when hemicellulases were added prior to hydrolysis with commercial cellulases. These results were supported by scanning electron microscopy, which revealed the effect of enzymatic hydrolysis on SEB fibers. Our results show the potential of complementary enzyme activities to improve enzymatic hydrolysis of SEB, thus improving the efficiency of the hydrolytic process.

Integrated biomarker response index to assess toxic effects of environmentally relevant concentrations of paracetamol in a neotropical catfish (Rhamdia quelen).

The nonsteroidal anti-inflammatory drugs (NSAIDs) are amongst the most commonly detected classes of pharmaceuticals in freshwater environments, with paracetamol being the most abundant. The aim of this study was to evaluate the possible toxic effects of environmentally relevant concentrations (0.25, 2.5 and 25 µg.L-1) of paracetamol in Rhamdia quelen fish exposed for 14 days using different biomarkers. The total count of leukocytes and thrombocytes was reduced at the highest concentration. In the gills, all concentrations of paracetamol reduced the glutathione S-transferase (GST) activity and the reduced glutathione (GSH) levels compared to the control group. The activity of catalase (CAT) was not altered and glutathione peroxidase (GPx) activity increased at the highest concentrations. The superoxide dismutase (SOD) activity decreased at 25 µg.L-1 and the LPO levels increased at 2.5 µg.L-1 when compared to the control group. The concentration of ROS was not different among the groups. In the posterior kidney the activities of GST (2.5 µg.L-1), CAT (2.5 µg.L-1 and at 25 µg. L-1) and GPx and GSH levels increased at all concentrations when compared to the control group. The SOD activity and LPO levels did not change. Paracetamol caused genotoxicity in the blood and gills at concentrations of 2.5 µg.L-1 and in the posterior kidney at 2.5 and 25 µg.L-1. An osmoregulatory imbalance in plasma ions and a reduction in the carbonic anhydrase activity in the gills at 0.25 µg.L-1 were observed. Histopathological alterations occurred in the gills of fish exposed to 25 µg.L-1 and in the posterior kidney at 0.25 and 25 µg.L-1 of paracetamol. The integrated biomarker index showed that the stress caused by the concentration of 25 µg.L-1 was the highest one. These results demonstrated toxic effects of paracetamol on the gills and posterior kidneys of fish, compromising their physiological functions and evidencing the need for monitoring the residues of pharmaceuticals released into aquatic environment.

Metadata Analysis Approaches for Understanding and Improving the Functional Involvement of Rumen Microbial Consortium in Digestion and Metabolism of Plant Biomass.

Rumen is one of the most complex gastro-intestinal system in ruminating animals. With bountiful of microorganisms supporting in breakdown and consumption of minerals and nutrients from the complex plant biomass. It is predicted that a table spoon of ruminal fluid can reside up to 150 billion microorganisms including various species of bacteria, fungi and protozoa. Several studies in the past have extensively explained about the structural and functional physiology of the rumen. Studies based on rumen and its microbiota has increased significantly in the last decade to understand and reveal applications of the rumen microbiota in food processing, pharmaceutical, biofuel and biorefining industries. Recent high-throughput meta-genomic and proteomic studies have revealed humongous information on rumen microbial diversity. In this study, we have extensively reviewed and reported present-day's progress in understanding the rumen microbial diversity. As of today, NCBI resides about 821,870 records based on rumen with approximately 889 genome sequencing studies. We have retrieved all the rumen-based records from NCBI and extensively catalogued the rumen microbial diversity and the corresponding genomic and proteomic studies respectively. Also, we have provided a brief inventory of metadata analysis software packages and reviewed the metadata analysis approaches for understanding the functional involvement of these microorganisms. Knowing and understanding the present progress on rumen microbiota and performing metadata analysis studies will significantly benefit the researchers in identifying the molecular mechanisms involved in plant biomass degradation. These studies are also necessary for developing highly efficient microorganisms and enzyme mixtures for enhancing the benefits of cattle-feedstock and biofuel industries.

Effects of low concentrations of ibuprofen on freshwater fish Rhamdia quelen.

Ibuprofen is a pharmaceutical drug widely used by the global population and it has been found in aquatic ecosystems in several countries. This study evaluated the effects of ibuprofen in environmental concentrations (0, 0.1, 1 and 10 µg/L) on the freshwaterspecies Rhamdia quelen exposed for 14 days. In the posterior kidney, ibuprofen increased glutathione-S-transferase activity in all groups exposed. Furthermore, increased glutathione peroxidase activity and the levels of reduced glutathione in the group exposed to 10 µg/L. Ibuprofen decreased the carbonic anhydrase activity in the posterior kidney in all exposed groups, and increased the activity in the gills in group exposed to 0.1 µg/L. The levels of plasma magnesium increased in groups exposed to 0.1 and 1 µg/L. In the blood, ibuprofen decreased the white blood cell count in groups exposed to 0.1 e 1.0 µg/L. Therefore, these results indicated that ibuprofen caused nephrotoxicity and demonstrated immunosuppressive effect in Rhamdia quelen.

Evaluation of Castor Oil Cake Starch and Recovered Glycerol and Development of "Green" Composites Based on Those with Plant Fibers.

Continuous efforts are being made in some countries for the recovery of crude glycerin (RG/CG) and castor oil cake (COC), the two byproducts of biodiesel production. These are expected to help, not only in addressing environmental safety, but also in adding value to those byproducts, which otherwise may go to waste. Finding ways to utilize those byproducts underlines the main objective of this study. This paper presents the evaluation of (i) COC, glycerin and banana and sugarcane fibers for moisture content; (ii) COC for structural and thermal properties; and (iii) CG for its chemical characteristics. The possibility of using COC and CG with the selected fibers as reinforcement in the development of bio-composites is attempted through thermo-molding. Results revealed enhanced mechanical properties for these composites. The obtained results are discussed in terms of the observed morphology.

Steam explosion pretreatment of oil palm empty fruit bunches (EFB) using autocatalytic hydrolysis: A biorefinery approach.

The oil palm empty fruit bunches (EFB) are an attractive source of carbon for the production of biochemical products, therefore, the aim of this work is to analyze the effect of the steam explosion (SE) pretreatment under autocatalytic conditions on EFB using a full experimental design. Temperature and reaction time were the operational variables studied. The EFB treated at 195°C for 6 min showed an increase of 34.69% in glycan (mostly cellulose), and a reduction of 68.12% in hemicelluloses, with increased enzymatic digestibility to 33% producing 4.2 g L(-1) of glucose. Scanning electron micrographs of the steam treated EFB exhibited surface erosion and an increased fiber porosity. Fourier transform infrared spectroscopy showed the solubilization of hemicellulose and modification of cellulose in treated EFB.

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.

Ethanol production from sugars obtained during enzymatic hydrolysis of elephant grass (Pennisetum purpureum, Schum.) pretreated by steam explosion.

In this work, steam explosion was used a pretreatment method to improve the conversion of elephant grass (Pennisetum purpureum) to cellulosic ethanol. This way, enzymatic hydrolysis of vaccum-drained and water-washed steam-treated substrates was carried out with Penicillium echinulatum enzymes while Saccharomyces cerevisiae CAT-1 was used for fermentation. After 48 h of hydrolysis, the highest yield of reducing sugars was obtained from vaccum-drained steam-treated substrates that were produced after 10 min at 200 °C (863.42 ± 62.52 mg/g). However, the highest glucose yield was derived from water-washed steam-treated substrates that were produced after 10 min at 190 °C (248.34 ± 6.27 mg/g) and 200 °C (246.00 ± 9.60 mg/g). Nevertheless, the highest ethanol production was obtained from water-washed steam-treated substrates that were produced after 6 min at 200 °C. These data revealed that water washing is a critical step for ethanol production from steam-treated elephant grass and that pretreatment generates a great deal of water soluble inhibitory compounds for hydrolysis and fermentation, which were partly characterized as part of this study.

Production of cellulosic ethanol from cotton processing residues after pretreatment with dilute sodium hydroxide and enzymatic hydrolysis.

In this study, production of cellulosic ethanol from two cotton processing residues was investigated after pretreatment with dilute sodium hydroxide. Pretreatment performance was investigated using a 2(2) factorial design and the highest glucan conversion was achieved at the most severe alkaline conditions (0.4g NaOH g(-1) of dry biomass and 120°C), reaching 51.6% and 38.8% for cotton gin waste (CGW) and cotton gin dust (CGD), respectively. The susceptibility of pretreated substrates to enzymatic hydrolysis was also investigated and the best condition was achieved at the lowest total solids (5wt%) and the highest enzyme loading (85mg of Cellic CTec2 g(-1) of dry substrate). However, the highest concentration of fermentable sugars - 47.8 and 42.5gL(-1) for CGD and CGW, respectively - was obtained at 15wt% total solids using this same enzyme loading. Substrate hydrolysates had no inhibitory effects on the fermenting microorganism.

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.

Sugarcane biomass for biorefineries: comparative composition of carbohydrate and non-carbohydrate components of bagasse and straw.

Two fractions of sugarcane, namely bagasse and straw (or trash), were characterized in relation to their chemical composition. Bagasse presented values of glucans, hemicelluloses, lignin and ash of 37.74, 27.23, 20.57 and 6.53%, respectively, while straw had 33.77, 27.38, 21.28 and 6.23% of these same components. Ash content was relatively high in both cane biomass fractions. Bagasse showed higher levels of contaminating oxides while straw had a higher content of alkaline and alkaline-earth oxides. A comparison between the polysaccharide chemical compositions of these lignocellulosic materials suggests that similar amounts of fermentable sugars are expected to arise from their optimal pretreatment and enzymatic hydrolysis. Details about the chemical properties of cane biomass holocellulose, hemicelluloses A and B and α-cellulose are provided, and these may offer a good opportunity for designing more efficient enzyme cocktails for substrate saccharification.

Assessment of the enzymatic hydrolysis profile of cellulosic substrates based on reducing sugar release.

The activity profile of a 1:0.30 mixture of Celluclast 1.5L FG and Novozym 188 (Novozymes) was investigated using Whatman #1 filter paper (W1FP) as a single substrate for hydrolysis. The procedure was based on the ability of the enzymes to release total (RS(Tot)), insoluble (RS(Insol)) and soluble (RS(Sol)) reducing sugars from W1FP. RS(Insol) was used to estimate endoglucanase (EnG) activity whereas exoglucanases (ExG) were assessed by measuring RSSol in the presence of δ-gluconolactone. Finally, the ß-glucosidase (ßG) activity was derived from the difference between RS(Sol) measurements in the presence and absence of δ-gluconolactone. When this analytical procedure was applied to W1FP using 9.64 mg mL(-1) of the enzyme mixture, the relative contributions of EnG, ExG and ßG to the total cellulase activity were 63.28%, 12.02% and 24.70%, respectively. Also, this ratio changed with changes in the enzyme loading, giving a new insight into the synergy that exists among the enzymes.

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.

Acid activated montmorillonite as catalysts in methyl esterification reactions of lauric acid.

The catalytic activity of acid activated montmorillonite in the esterification of free fatty acids (FFA) is reported. Standard Montmorillonite (MMT) type STx-1 provided by the Clay Mineral Society repository was activated using phosphoric, nitric and sulphuric acids under different conditions and the resulting materials were characterized and evaluated as catalysts in the methyl esterification of lauric acid. Blank reactions carried out in the absence of any added catalyst presented conversions of 32.64, 69.79 and 79.23%, for alcohol:lauric acid molar ratios of 60:1, 12:1 and 6:1, respectively. In the presence of the untreated clay and using molar ratios of 12:1 and 6:1 with 12% of catalyst, conversions of 70.92 and 82.30% were obtained, respectively. For the acid activated clays, conversions up to 93.08% of lauric acid to methyl laurate were obtained, much higher than those observed for the thermal conversion or using untreated montmorillonite. Relative good correlations were observed between the catalytic activity and the development of acid sites and textural properties of the resulting materials. Therefore, a simple acid activation was able to improve the catalytic activity and produce clay catalysts that are environmental friendly, cost effective, noncorrosive and reusable.

Bioethanol from lignocelluloses: Status and perspectives in Brazil.

The National Alcohol Program--PróAlcool, created by the government of Brazil in 1975 resulted less dependency on fossil fuels. The addition of 25% ethanol to gasoline reduced the import of 550 million barrels oil and also reduced the emission CO(2) by 110 million tons. Today, 44% of the Brazilian energy matrix is renewable and 13.5% is derived from sugarcane. Brazil has a land area of 851 million hectares, of which 54% are preserved, including the Amazon forest (350 million hectares). From the land available for agriculture (340 million hectares), only 0.9% is occupied by sugarcane as energy crop, showing a great expansion potential. Studies have shown that in the coming years, ethanol yield per hectare of sugarcane, which presently is 6000 L/ha, could reach 10,000 L/ha, if 50% of the produced bagasse would be converted to ethanol. This article describes the efforts of different Brazilian institutions and research groups on second generation bioethanol production, especially from sugarcane bagasse.

New multifunctional materials obtained by the intercalation of anionic dyes into layered zinc hydroxide nitrate followed by dispersion into poly(vinyl alcohol) (PVA).

Different anionic blue and orange dyes have been immobilized on a zinc hydroxide nitrate (Zn(5)(OH)(8)(NO(3))(2)nH(2)O--Zn-OH-NO(3)) by anion exchange with interlayer and/or outer surface nitrate ions of the layered matrix. Orange G (OG) was totally intercalated, orange II (OII) was partially intercalated, while Niagara blue 3B (NB) and Evans blue (EV) were only adsorbed at the outer surface. Several composite films of poly(vinyl alcohol)--PVA were prepared by casting through the dispersion of the hybrid material (Zn-OH-OG) into a PVA aqueous solution and evaporation of water in a vacuum oven. The obtained composite films were transparent, colored, and capable of absorbing UV radiation. Improved mechanical properties were also obtained in relation to the nonfilled PVA films. These results demonstrate the onset of a new range of potential applications for layered hydroxide salts in the preparation of polymer composite multifunctional materials.

Optimization of the ethanolysis of Raphanus sativus (L. Var.) crude oil applying the response surface methodology.

Raphanus sativus (L. Var) is a perennial plant of the Brassicaceae (or Cruciferae) family whose oil has not been investigated in detail for biodiesel production, particularly when ethanol is used as the alcoholysis agent. In this work, response surface methodology (RSM) was used to determine the optimum condition for the ethanolysis of R. sativus crude oil. Three process variables were evaluated at two levels (2(3) experimental design): the ethanol:oil molar ratio (6:1 and 12:1), the catalyst concentration in relation to oil mass (0.4 and 0.8 wt% NaOH) and the alcoholysis temperature (45 and 65 degrees C). When the experimental results were tentatively adjusted by linear regression, only 58.15% of its total variance was explained. Therefore, a quadratic model was investigated to improve the poor predictability of the linear model. To apply the quadratic model, the 2(3) experimental design had to be expanded to a circumscribed central composite design. This allowed the development of a response surface that was able to predict 97.75% of the total variance of the system. Validation was obtained by performing one ethanolysis experiment at the conditions predicted by the model (38 degrees C, ethanol:oil molar ratio of 11.7:1 and 0.6 wt% NaOH). The resulting ester yield (104.10 wt% or 99.10% of the theoretical yield of 105.04 wt%) was shown to be the highest among all conditions tested in this study. The second ethanolysis stage of the best RSM product required 50% less ethanol and 90% less catalyst consumption. The amount of ethyl esters obtained after this procedure reached 94.5% of the theoretical yield. The resulting ethyl esters were shown to comply with most of the Brazilian biodiesel specification parameters except for oxidation stability. Addition of 500 ppm of BHT to the esters, however, complied with the specification target of 6h. The application of 2 wt% Magnesol after the second ethanolysis stage eliminated the need for water washing and helped generate a final product with less unreacted glycerides.

Comparison of Penicillium echinulatum and Trichoderma reesei cellulases in relation to their activity against various cellulosic substrates.

Penicillium echinulatum has been identified as a potential cellulase producer for bioconversion processes but its cellulase system has never been investigated in detail. In this work, the volumetric activities of P. echinulatum cellulases were determined against filter paper (0.27 U/mL), carboxymethylcellulose (1.53 U/mL), hydroxyethylcellulose (4.68 U/mL), birchwood xylan (3.16 U/mL), oat spelt xylan (3.29 U/mL), Sigmacell type 50 (0.10 U/mL), cellobiose (0.19 U/mL), and p-nitrophenyl-glucopiranoside (0.31 U/mL). These values were then expressed in relation to the amount of protein and compared those of Trichoderma reesei cellulases (Celluclast 1.5L FG, Novozymes). Both enzyme complexes were shown to have similar total cellulase and xylanase activities. Analysis of substrate hydrolysates demonstrated that P. echinulatum enzymes have higher beta-glucosidase activity than Celluclast 1.5L FG, while the latter appears to have greater cellobiohydrolase activity. Unlike Celluclast 1.5L FG, P. echinulatum cellulases had enough beta-glucosidase activity to remove most of the cellobiose produced in hydrolysis experiments. However, Celluclast 1.5L FG became more powerful than P. echinulatum cellulases when supplemented with exogenous beta-glucosidase activity (Novozym 188). Both cellulase complexes displayed the same influence over the degree of polymerization of cellulose, revealing that hydrolyzes were carried out under the typical endo-exo synergism of fungal enzymes.