Chemical Characterization and Determination of the Anti-Oxidant Capacity of Two Brown Algae with Respect to Sampling Season and Morphological Structures Using Infrared Spectroscopy and Multivariate Analyses.

Brown algae biomass has been shown to be a highly important industrial source for the production of alginates and different nutraceutical products. The characterization of this biomass is necessary in order to allocate its use to specific applications according to the chemical and biological characteristics of this highly variable resource. The methods commonly used for algae characterization require a long time for the analysis and rigorous pretreatments of samples. In this work, nondestructive and fast analyses of different morphological structures from Lessonia spicata and Macrocystis pyrifera, which were collected during different seasons, were performed using Fourier transform infrared (FT-IR) techniques in combination with chemometric methods. Mid-infrared (IR) and near-infrared (NIR) spectral ranges were tested to evaluate the spectral differences between the species, seasons, and morphological structures of algae using a principal component analysis (PCA). Quantitative analyses of the polyphenol and alginate contents and the anti-oxidant capacity of the samples were performed using partial least squares (PLS) with both spectral ranges in order to build a predictive model for the rapid quantification of these parameters with industrial purposes. The PCA mainly showed differences in the samples based on seasonal sampling, where changes were observed in the bands corresponding to polysaccharides, proteins, and lipids. The obtained PLS models had high correlation coefficients (r) for the polyphenol content and anti-oxidant capacity (r > 0.9) and lower values for the alginate determination (0.7 < r < 0.8). Fourier transform infrared-based techniques were suitable tools for the rapid characterization of algae biomass, in which high variability in the samples was incorporated for the qualitative and quantitative analyses, and have the potential to be used on an industrial scale.

Condensed lignin structures and re-localization achieved at high severities in autohydrolysis of Eucalyptus globulus wood and their relationship with cellulose accessibility.

Eucalyptus globulus wood was subjected to autohydrolysis pretreatment at different severity factors. The pretreated materials were enzymatically saccharified at a substrate load of 10% (w/v) using a cellulase enzyme complex. Around 82-95% of original glucans were retained in the pretreated material, and the enzymatic hydrolysis yields ranged from 58% to 90%. The chemical and structural changes in the pretreated materials were investigated by microscopic (SEM, LSCM) and spectroscopic (2D-HSQC NMR and FT-IR) techniques. 2D-NMR results showed a reduction in the amounts of ß-O-4 aryl-ether linkages and suggested the presence of newly condensed structures of lignin in the biomass pretreated at the more severe conditions. Furthermore, the microscopic analysis showed that lignin migrates out of the cell wall and re-deposits in certain regions of the fibers at the more severe conditions to form droplet-like structures and expose the cellulose surface. These changes improved the glucose yield up to 69%, on dry wood basis.

Fourier transform infrared imaging and microscopy studies of Pinus radiata pulps regarding the simultaneous saccharification and fermentation process.

The distribution and chemical patterns of lignocellulosic components at microscopic scale and their effect on the simultaneous saccharification and fermentation process (SSF) in the production of bioethanol from Pinus radiata pulps were analyzed by the application of diverse microscopical techniques, including scanning electronic microscopy (SEM), confocal laser scanning microscopy (CLSM) and attenuated total reflectance (ATR) - Fourier transform infrared microspectroscopy. This last technique was accompanied with multivariate methods, including principal component analysis (PCA) and multivariate curve resolution with alternating least squares (MCR-ALS) to evaluate the distribution patterns and to generate pure spectra of the lignocellulosic components of fibers. The results indicate that the information obtained by the techniques is complementary (ultrastructure, confocality and chemical characterization) and that the distribution of components affects the SSF yield, identifying lignin coalescence droplets as a characteristic factor to increase the SSF yield. Therefore, multivariate analysis of the infrared spectra enabled the in situ identification of the cellulose, lignin and lignin-carbohydrates arrangements. These techniques could be used to investigate the lignocellulosic components distribution and consequently their recalcitrance in many applications where minimal sample manipulation and microscale chemical information is required.

Reactivity of catecholamine-driven Fenton reaction and its relationships with iron(III) speciation.

INTRODUCTION: Fenton reaction is the main source of free radicals in biological systems. The reactivity of this reaction can be modified by several factors, among these iron ligands are important. Catecholamine (dopamine, epinephrine, and norepinephrine) are able to form Fe(III) complexes whose extension in the coordination number depends upon the pH. Fe(III)-catecholamine complexes have been related with the development of several pathologies. METHODS: In this work, the ability of catecholamines to enhance the oxidative degradation of an organic substrate (veratryl alcohol, VA) through Fenton and Fenton-like reactions was studied. The initial VA degradation rate at different pH values and its relationship to the different iron species present in solution were determined. Furthermore, the oxidative degradation of VA after 24 hours of reaction and its main oxidation products were also determined. RESULTS: The catecholamine-driven Fenton and Fenton-like systems showed higher VA degradation compared to unmodified Fenton or Fenton-like systems, which also showed an increase in the oxidation state of the VA degradation product. All of this oxidative degradation takes place at pH values lower than 5.50, where the primarily responsible species would be the Fe(III) mono-complex. CONCLUSION: The presence of Fe(III) mono-complex is essential in the ability of catecholamines to increase the oxidative capacity of Fenton systems.

Infrared spectroscopy as alternative to wet chemical analysis to characterize Eucalyptus globulus pulps and predict their ethanol yield for a simultaneous saccharification and fermentation process.

Bioethanol can be obtained from wood by simultaneous enzymatic saccharification and fermentation step (SSF). However, for enzymatic process to be effective, a pretreatment is needed to break the wood structure and to remove lignin to expose the carbohydrates components. Evaluation of these processes requires characterization of the materials generated in the different stages. The traditional analytical methods of wood, pretreated materials (pulps), monosaccharides in the hydrolyzated pulps, and ethanol involve laborious and destructive methodologies. This, together with the high cost of enzymes and the possibility to obtain low ethanol yields from some pulps, makes it suitable to have rapid, nondestructive, less expensive, and quantitative methods to monitoring the processes to obtain ethanol from wood. In this work, infrared spectroscopy (IR) accompanied with multivariate analysis is used to characterize chemically organosolv pretreated Eucalyptus globulus pulps (glucans, lignin, and hemicellulosic sugars), as well as to predict the ethanol yield after a SSF process. Mid (4,000-400 cm(-1)) and near-infrared (12,500-4,000 cm(-1)) spectra of pulps were used in order to obtain calibration models through of partial least squares regression (PLS). The obtained multivariate models were validated by cross validation and by external validation. Mid-infrared (mid-IR)/NIR PLS models to quantify ethanol concentration were also compared with a mathematical approach to predict ethanol yield estimated from the chemical composition of the pulps determined by wet chemical methods (discrete chemical data). Results show the high ability of the infrared spectra in both regions, mid-IR and NIR, to calibrate and predict the ethanol yield and the chemical components of pulps, with low values of standard calibration and validation errors (root mean square error of calibration, root mean square error of validation (RMSEV), and root mean square error of prediction), high correlation between predicted and measured by the reference methods values (R (2) between 0.789 and 0.997), and adequate values of the ratio between the standard deviation of the reference methods and the standard errors of infrared PLS models relative performance determinant (RPD) (greater than 3 for majority of the models). Use of IR for ethanol quantification showed similar and even better results to the obtained with the discrete chemical data, especially in the case of mid-IR models, where ethanol concentration can be estimated with a RMSEV equal to 1.9 g L(-1). These results could facilitate the analysis of high number of samples required in the evaluation and optimization of the processes.

Structural change in wood by brown rot fungi and effect on enzymatic hydrolysis.

The effects of biological pretreatment on Pinus radiata and Eucalyptus globulus, were evaluated after exposure to two brown rot fungi Gloephylum trabeum and Laetoporeus sulphureus. Changes in chemical composition, structural modification, and susceptibility to enzymatic hydrolysis in the degraded wood were analyzed. After eight weeks of biodegradation, the greatest loss of weight and hemicellulose were 13% and 31%, respectively, for P. radiata with G. trabeum. The content of glucan decreased slightly, being the highest loss of 20% for E. globulus with G. trabeum. Consistent with degradation mechanism of these fungi, lignin was essentially undegraded by both brown rot fungi. Both brown rot fungi cause a sharp reduction in the cellulose degree of polymerization (DP) in the range between 58% and 79%. G. trabeum depolymerized cellulose in both wood faster than L. sulphureus. Also, structural characteristic of crystalline cellulose were measured by using two different techniques - X-ray diffraction (XRD) and infrared spectroscopy (FT-IR). The biological pretreatments showed an effect on cellulose crystallinity structure, a decrease between 6% and 21% was obtained in the crystallinity index (CrI) calculated by IR, no changes were observed in the XRD. Material digestibility was evaluated by enzymatic hydrolysis, the conversion of cellulose to glucose increased with the biotreatment time. The highest enzymatic hydrolysis yields were obtained when saccharification was performed on wood biopretreated with G. trabeum (14% P. radiata and 13% E. globulus). Decreasing in DP and CrI, and hemicellulose removal result in an increase of enzymatic hydrolysis performance. Digestibility was better related to DP than with other properties. G. trabeum can be considered as a potential fungus for biological pretreatment, since it provides an effective process in breaking the wood structure, making it potentially useful in the development of combined pretreatments (biological-chemical). A viable alternative to pretreatment process that can be used is a bio-mimetic system, similar to low-molecular complexes generated by fungi such as G. trabeum combined pretreatments (biological-chemical).

Bioethanol production from tension and opposite wood of Eucalyptus globulus using organosolv pretreatment and simultaneous saccharification and fermentation.

During tree growth, hardwoods can initiate the formation of tension wood, which is a strongly stressed wood on the upper side of the stem and branches. In Eucalyptus globulus, tension wood presents wider and thicker cell walls with low lignin, similar glucan and high xylan content, as compared to opposite wood. In this work, tension and opposite wood of E. globulus trees were separated and evaluated for the production of bioethanol using ethanol/water delignification as pretreatment followed by simultaneous saccharification and fermentation (SSF). Low residual lignin and high glucan retention was obtained in organosolv pulps of tension wood as compared to pulps from opposite wood at the same H-factor of reaction. The faster delignification was associated with the low lignin content in tension wood, which was 15% lower than in opposite wood. Organosolv pulps obtained at low and high H-factor (3,900 and 12,500, respectively) were saccharified by cellulases resulting in glucan-to-glucose yields up to 69 and 77%, respectively. SSF of the pulps resulted in bioethanol yields up to 35 g/l that corresponded to 85-95% of the maximum theoretical yield on wood basis, considering 51% the yield of glucose to ethanol conversion in fermentation, which could be considered a very satisfactory result compared to previous studies on the conversion of organosolv pulps from hardwoods to bioethanol. Both tension and opposite wood of E. globulus were suitable raw materials for organosolv pretreatment and bioethanol production with high conversion yields.

Evaluation of a combined brown rot decay-chemical delignification process as a pretreatment for bioethanol production from Pinus radiata wood chips.

Wood chips of Pinus radiata softwood were biotreated with the brown rot fungus (BRF) Gloeophyllum trabeum for periods from 4 and 12 weeks. Biodegradation by BRF leads to an increase in cellulose depolymerization with increasing incubation time. As a result, the intrinsic viscosity of holocellulose decreased from 1,487 cm(3)/g in control samples to 783 and 600 cm(3)/g in 4- and 12-week decayed wood chips, respectively. Wood weight and glucan losses varied from 6 to 14% and 9 to 21%, respectively. Undecayed and 4-week decayed wood chips were delignified by alkaline (NaOH solution) or organosolv (ethanol/water) processes to produced cellulosic pulps. For both process, pulp yield was 5-10% lower for decayed samples than for control pulps. However, organosolv bio-pulps presented low residual lignin amount and high glucan retention. Chemical pulps and milled wood from undecayed and 4-week decayed wood chips were pre-saccharified with cellulases for 24 h at 50 degrees C followed by simultaneous saccharification and fermentation (SSF) with the yeast Saccharomyces cerevisiae IR2-9a at 40 degrees C for 96 h for bioethanol production. Considering glucan losses during wood decay and conversion yields from chemical pulping and SSF processes, no gains in ethanol production were obtained from the combination of BRF with alkaline delignification; however, the combination of BRF and organosolv processes resulted in a calculated production of 210 mL ethanol/kg wood or 72% of the maximum theoretically possible from that pretreatment, which was the best result obtained in the present study.

Evaluation of the white-rot fungi Ganoderma australe and Ceriporiopsis subvermispora in biotechnological applications.

Ganoderma australe is a white-rot fungus that causes a selective wood biodelignification in some hardwoods found in the Chilean rainforest. Ceriporiopsis subvermispora is also a lignin-degrading fungus used in several biopulping studies. The enzymatic system responsible for lignin degradation in wood can also be used to degrade recalcitrant organic pollutants in liquid effluents. In this work, two strains of G. australe and one strain of C. subvermipora were comparatively evaluated in the biodegradation of ABTS and the dye Poly R-478 in liquid medium, and in the pretreatment of Eucalyptus globulus wood chips for further kraft biopulping. Laccase was detected in liquid and wood cultures with G. australe. Ceriporiopsis subvermispora produce laccase and manganese peroxidase when grown in liquid medium and only manganese peroxidase was detected during wood decay. ABTS was totally depleted by all strains after 8 days of incubation while Poly R-478 was degraded up to 40% with G. australe strains and up to 62% by C. subvermispora after 22 days of incubation. Eucalyptus globulus wood chips decayed for 15 days presented 1-6% of lignin loss and less than 2% of glucan loss. Kraft pulps with kappa number 15 were produced from biotreated wood chips with 2% less active alkali, with up to 3% increase in pulp yield and up to 20% less hexenuronic acids than pulps from undecayed control. Results showed that G. australe strains evaluated were not as efficient as C. subvermispora for dye and wood biodegradation, but could be used as a feasible alternative in biotechnological processes such as bioremediation and biopulping.

Determination of arylglycerol-beta-aryl ether linkages in enzymatic mild acidolysis lignins (EMAL): comparison of DFRC/(31)P NMR with thioacidolysis.

Enzymatic mild acidolysis lignins (EMAL) isolated from different species of softwood and Eucalyptus globulus were submitted to comparative analysis that included thioacidolysis, derivatization followed by reductive cleavage (DFRC), and DFRC followed by quantitative (31)P NMR (DFRC/(31)P NMR). While gas chromatography (GC) was used to determine the monomer yields from both thioacidolysis and DFRC, (31)P NMR studies quantified the various phenolic hydroxy groups released by DFRC. The monomer yields from thioacidolysis and DFRC were substantially different, with thioacidolysis resulting in higher yields. In contrast, an excellent agreement was obtained in the total number of beta-aryl ether structures determined by thioacidolysis and DFRC/(31)P NMR, indicating that the combination of DFRC with quantitative (31)P NMR overcomes, at least in part, the limitations presented by the DFRC method. Both thioacidolysis and DFRC/(31)P NMR were further used to better understand the lignin isolation process from wood. The results show that mild rotary ball milling minimizes, but does not prevent, the degradation of beta-O-4 structures during the early stages of wood pulverization. The extent of such degradation was found to be higher for E. globulus than for a variety of softwoods examined. Furthermore, the structures of the EMALs isolated at yields ranging from 20% to 62% were very similar, indicating structural homogeneity in the lignin biopolymer within the secondary wall.

Enhanced hydroxyl radical production by dihydroxybenzene-driven Fenton reactions: implications for wood biodegradation.

Brown rot fungi degrade wood, in initial stages, mainly through hydroxyl radicals (.OH) produced by Fenton reactions. These Fenton reactions can be promoted by dihydroxybenzenes (DHBs), which can chelate and reduce Fe(III), increasing the reactivity for different substrates. This mechanism allows the extensive degradation of carbohydrates and the oxidation of lignin during wood biodegradation by brown rot fungi. To understand the enhanced reactivity in these systems, kinetics experiments were carried out, measuring .OH formation by the spin-trapping technique of electron paramagnetic resonance spectroscopy. As models of the fungal DHBs, 1,2-dihydroxybenzene (catechol), 2,3-dihydroxybenzoic acid and 3,4-dihydroxybenzoic acid were utilized as well as 1,2-dihydroxy-3,5-benzenedisulfonate as a non-Fe(III)-reducing substance for comparison. Higher amounts and maintained concentrations of .OH were observed in the driven Fenton reactions versus the unmodified Fenton process. A linear correlation between the logarithms of complex stability constants and the .OH production was observed, suggesting participation of such complexes in the radical production.

An evaluation of British Columbian beetle-killed hybrid spruce for bioethanol production.

The development of bioconversion technologies for production of fuels, chemicals, and power from renewable resources is currently a high priority for developed nations such as the United States, Canada, and the European Union as a way to improve national energy security and reduce greenhouse gas emissions. The widespread implementation of such technologies will require a sustainable supply of biomass from forestry and agriculture. Forests are a major source of feedstocks for biofuels production in Canada. Woody biomass includes residues from logging and forest thinning, and from wood processing and pulp production. More recently, damaged wood caused by beetle infestations has become available on a large scale in Western Canada. This study evaluates beetle-killed British Columbian hybrid spruce (HS) (Picea glauca x P. engelmannii) as a feedstock for the production of bioethanol. In the past 30 yr, attack by the beetle Dendroctonus rufipennis and associated fungi has resulted in estimated losses of more than three billion board feet in British Columbia alone. Here we describe the chemical and some physical characteristics of both healthy (HHS) and beetle-killed (BKHS) British Columbian HS and evaluate the technical feasibility of using these feedstocks as a source of biomass for bioethanol production. Untreated HHS and BKHS did not differ significantly in chemical composition except for the moisture content, which was significantly lower in BKHS (approx 10%) compared with HHS (approx 18%). However, the yields of carbohydrates in hydrolyzable and fermentable forms were higher at mild pretreatment conditions (H-Factor <1000) for BKHS compared with HHS. At medium (H-Factor 1000-2000) and severe (H-Factor >2000) pretreatment conditions HHS and BKHS behaved similarly. Organosolv pretreated HHS and BKHS demonstrated good ethanol theoretical yields, approx 70 and 80%, respectively.

Thiobarbituric acid reactive substances, Fe3+ reduction and enzymatic activities in cultures of Ganoderma australe growing on Drimys winteri wood.

Ganoderma australe is a basidiomycete responsible for a natural process of selective and extensive lignin degradation. Fatty acids, thiobarbituric acid reactive substances (TBARS), Fe3+-reduction and enzymatic activities were monitored in cultures of G. australe growing on Drimys winteri wood chips. Linoleic acid was de novo synthesized, and steadily increased during 12 weeks of cultivation. Part of the unsaturated fatty acids underwent peroxidation as TBARS accumulated with biodegradation time. TBARS accumulation was proportional to the wood weight and component losses. Manganese-dependent peroxidase and lignin peroxidase were not detected in the culture extracts, whereas laccase-induced oxidation of syringaldazine peaked after 2 weeks (104+/-9 micromol oxidized min(-1) kg(-1) of dry wood), subsequently decreasing. On the other hand, nonenzymatic Fe3+-reducing activity increased as a function of cultivation time and could be involved in the initiation of lipid peroxidation.

Evidence of chemical reactions between di- and poly-glycidyl ether resins and tannins isolated from Pinus radiata D. Don bark.

This work evaluates the feasibility of reacting tannins isolated from Pinus radiata D. Don bark with epoxide resins of the diglycidyl and polyglycidyl ether type. To this end, gel times of aqueous tannin dispersions (40% w/w) with every one of nine selected resins (5% w/w), at previously established pH values (initial equal to 3.3, 4, 7 and 10), have been determined. Products of these reactions were analyzed by FT-IR spectroscopy, and the results were compared with those obtained from tannin-p-formaldehyde and (+)-catechin-p-formaldehyde systems, at the same pH values. Their mechanical properties were evaluated, by dynamic mechanical analysis, at two pH values (3.3 and 10). In general, it was concluded that tannin-epoxide resin systems behave similarly to tannin-paraformaldehyde systems, especially at basic pH values.

Removal of metal ions by modified Pinus radiata bark and tannins from water solutions.

Pinus radiata bark and tannins, chemically modified with an acidified formaldehyde solution were used for removing metal ions from aqueous solutions and copper mine acidic residual waters. The adsorption ability to different metal ions [V(V), Re(VII), Mo(VI), Ge(IV), As(V), Cd(II), Hg(II), Al(III), Pb(II), Fe(II), Fe(III), Cu(II)] and the factors affecting their removal from solutions were investigated. Effect of pH on the adsorption, desorption, maximum adsorption capacity of the adsorbents, and selectivity experiments with metal ion solutions and waste waters from copper mine were carried out. The adsorbents considerably varied in the adsorption ability to each metal ion. The adsorption depends largely upon the pH of the solution. Modified tannins showed lower adsorption values than the modified bark. For the same adsorbent, the maximum capacity at pH 3 for the different ions were very different, ranging for modified bark from 6.8 meqg(-1) for V to 0.93 meqg(-1) for Hg. Waste waters were extracted with modified bark as adsorbent and at pH 2. The ions Cu(II) (35.2 mgL(-1)), Fe(III) (198 mgL(-1)), Al(III) (83.5 mgL(-1)) and Cd(II) (0.15 mgL(-1)) were removed in 15.6%, 46.9%, 83.7% and 3.3%, respectively, by using 1g of adsorbent/10 mL of waste water. In general, a continuous adsorption on a packed column gave higher adsorbed values than those observed in the batchwise experiment.

A screening method for detecting iron reducing wood-rot fungi.

A plate assay using the Fe(II) selective dye, ferrozine, for detecting wood-rot fungi with Fe(III) reductive abilities, was developed. The assay is fast, simple and, in most cases, more sensitive than the corresponding liquid medium test. The brown rot fungi, Gloeophyllum trabeum and Laetiporeus sulphureus, displayed higher iron reductive capabilities than white rot fungi, Trametes versicolor, Ganoderma australe and Ceriporiopsis subvermispora.

Lignocellulosic materials: a binational project in single cell protein production

En este trabajo se analizan las proporciones de algunos aminoácidos de importancia nutricional en fuentes de proteínas convencionales, con las obtenidas a partir de materiales lignocelulósicos. También se comparan las reacciones de aminoácidos individuales con los aminoácidos esenciales totales (a/e) y la relación entre los aminoácidos esenciales totales y los totales recuperados como nitrógeno (e/t). Estos resultados junto a los valores químicos de losaminoácidos esenciales nos permitieron comparar las cualidades de estas proteínas obtenidas de las diferentes fuentes de carbono. Este tipo de análisis permitióseleccionar las proteínas más adecuadas para una función definida, ya sea como para una ración animal o como posible alimento humano