Use of a new membrane-reactor saccharification assay to evaluate the performance of celluloses under simulated ssf conditions

A new saccharification assay has been devised, in which a continuously buffer-swept membrane reactor is used to remove the solubilized saccharification products, thus allowing high extents of substrate conversion without significant inhibitory effects from the buildup of either cellobiose or glucose. This diafiltration saccharification assay (DSA) can, therefore, be used to obtain direct measurements of the performance of combinations of cellulase and substrate under simulated SSF conditions, without the saccharification results being complicated by factors that may influence the subsequent fermentation step. This assay has been used to compare the effectiveness of commercial and special in-house-producedTrichoderma reeSci. cellulase preparations in the saccharification of a standardized microcrystalline (Sigmacell) substrate and a dilute-acid pretreated lignocellulosic substrate. Initial results strongly suggest that enzyme preparations produced in the presence of the targeted lignocellulosic substrate will saccharify that substrate more effectively. These results call into question the widespread use of the “filter paper assay” as a reliable predictor of enzyme performance in the extensive hydrolysis of substrates that are quite different from filter paper in both physical properties and chemical composition.

     

Use of analytical microscopy to analyze the speciation of copper and chromium ions onto a low-cost biomaterial

In this paper, we present our study of the speciation of copper and hexavalent chromium sorbed onto a lignocellulosic substrate, using analytical microscopy. The lignocellulosic substrate constitutes a low-cost biomaterial that can be used in wastewater treatment. Transmission electron microscopy (TEM) coupled with energy-dispersive X-ray spectroscopy (EDXS) was used to determine the speciation of the two metal ions on the constitutive moieties of the lignocellulosic substrate. The use of a staining agent sensitive to carbon unsaturation allowed us to differentiate between the microstructures rich in lignin entities and those rich in cellulose entities. The EDX analysis showed that metal ions are preferentially sorbed onto microstructures rich in lignin moieties. The energy electron loss spectroscopy (EELS) was used to determine the oxidation states of chromium in association with lignocellulosic moieties. We showed that the sorption process of hexavalent chromium requires the reduction of Cr(VI) into Cr(III) and the probable oxidation of lignin moieties.     

Metal ions binding onto a lignocellulosic substrate extracted from wheat bran: a NICA-Donnan approach

In this study ion binding to solid organic matter was investigated. We used the NICA-Donnan model to describe the interaction between Cu(II), Pb(II), and Cd(II) ions and a lignocellulosic substrate extracted from wheat bran. Such a material represents a very cheap and flexible substrate, which can be used as a natural filter to remove toxic metal ions from industrial effluents or applied in the decontamination and rehabilitation of abandoned industrial sites. Moreover, such a material also represents a very simple model of natural organic matter derived from lignin and cellulose, for which there is now a lack of thermodynamic data as far as their interaction with toxic metal ions is concerned. Experimental results were obtained in various conditions of pH and ionic strength. In our modeling, we used the acidity constants and the concentration of sites that were determined in a previous work. The parameters deduced from the NICA-Donnan model were also compared to thermodynamic data available for other cases of natural organic matter, mainly humic substances.     

Prospecting fungal ligninases using corncob lignocellulosic fractions

Microorganisms play an important role in the bioconversion of organic residues and have therefore become promising for obtaining value-added enzymes. In an attempt to take advantage of the by-products and residues of bioconversion, this work sought to use lignocellulosic fractions extracted from corncob as fermentation substrate for ligninase induction by Pleurotus sajor-caju. To obtain the corncob lignocellulosic fractions, biomass was submitted to treatment by alkaline extraction (NaOH 0.75 mol L^?1, 55 °C for 2 h) and organosolv (40% ethanol/water, 185 °C for 20 min). The in natura biomass and lignocellulosic fractions were used as substrates in the subsequent fermentation processes: 2% in natura corncob; 2% cellulose–lignin complex fraction; 2% lignin-enriched fraction; 1% lignin-enriched fraction; and synthetic medium fungal (SMF) as standard. Chemical and physical–chemical analyses indicated the effectiveness of the lignocellulosic extraction process. According to the results, the developed system promoted the induction of ligninases by P. sajor-caju. The enzymatic analysis showed laccase production (768 U L^?1) using the 1% lignin-enriched fraction as substrate. Manganese peroxidase production was 1050 U L^?1 with the use of the 2% lignin-enriched fraction. The presence of lignocellulosic fractions extracted from corncob’s lignin-enriched fraction in the culture medium favored the induction of ligninases in comparison to the use of residue alone.     

Metal Ions Binding to Natural Organic Matter Extracted from Wheat Bran: Application of the Surface Complexation Model

Ions binding to solid organic matter was investigated in this study. A simple surface complexation model, the diffuse double-layer model, was used to describe Pb(II), Cd(II), Zn(II), and Ni(II) binding to a lignocellulosic substrate extracted from wheat bran. The lignocellulosic substrate was represented by two acid sites: a low-pH ("carboxylic") site and a high-pH ("phenolic") site, the phenolic-type sites contributing significantly to the binding behavior, even at relatively low pH. By using the previously determined concentrations of sites and acidity constants, the surface complexation model was applied to Pb, Cd, Zn, and Ni binding as a function of pH in a 0.1 M NaNO(3) medium. The model fits were good over a wide range of pH (2相似文献   

Use of Multi-parameter Flow Cytometry as Tool to Monitor the Impact of Formic Acid on Saccharomyces carlsbergensis Batch Ethanol Fermentations

The use of lignocellulosic materials as substrate for bioethanol production is considered a cost-effective approach to make the biofuel production process economically sustainable. However, lignocellulosic hydrolysis releases toxic compounds such as weak acids which inhibit microorganism growth and ethanol production. In order to understand the physiological response of Saccharomyces carlsbergensis when fermenting glucose in the presence of formic acid (HF), the yeast growth was monitored by multi-parameter flow cytometry. Cytoplasmic membrane potential decreased as the HF concentration increased and as the yeast culture reached the stationary phase. However, the proportion of cells with permeabilized membrane did not increase with the HF concentration increase. The accumulation of reactive oxygen species was also monitored. Control and fermentations at low HF concentrations (<1 g/L) resulted in a high proportion of highly oxidized cells at the stationary phase. The multi-parameter flow cytometry approach proved to be a useful tool to monitor the physiological stress response of S. carlsbergensis growth and ethanol production in the presence of HF, an inhibitor present in lignocellulosic hydrolysates. The information here obtained at near real time can be used to enhance second-generation bioethanol production process efficiency.     

Determination of some steroidal bases in non-aqueous media by potentiometric titrations at a small constant current

In this work the volumetric and coulometric titrations of some newly synthesized androstene steroidal compounds with pyridine ring are carried out. The titrations were followed potentiometrically at a small constant current using pairs of identical platinum or glassy carbon electrodes, both in presence of quinhydrone. As the titration reagent, in volumetric titrations served 0.01 mol dm^?3 perchloric acid, and in coulometric titrations hydrogen ions were anodically generated in situ from quinhydrone at the platinum electrode. Sodium perchlorate served as the supporting electrolyte.Amounts of 1.18-5.35 mg of the investigated steroidal compounds were determined with a relative standard deviation less than 1.4% at volumetric and 1.2% at coulometric determinations. A negative influence of the supporting electrolyte on magnitude of the signal change in potentiometric determinations was observed. Glassy carbon electrodes are more advantageous over platinum ones.The achieved results are in good agreement with those of catalytic thermometric titrations.     

Simultaneous saccharification and extractive fermentation of lignocellulosic materials into lactic acid in a two-zone fermentor-extractor system

Simultaneous saccharification and extractive fermentation of lignocellulosic materials into lactic acid was investigated using a two-zone bioreactor. The system is composed of an immobilized cell reactor, a separate column reactor containing the lignocellulosic substrate and a hollow-fiber membrane. It is operated by recirculating the cell free enzyme (cellulase) solution from the immobilized cell reactor to the column reactor through the membrane. The enzyme and microbial reactions thus occur at separate locations, yet simultaneously. This design provides flexibility in reactor operation as it allows easy separation of the solid substrate from the microorganism, in situ removal of the product and, if desired, different temperatures in the two reactor sections. This reactor system was tested using pretreated switchgrass as the substrate. It was operated under a fed-batch mode with continuous removal of lactic acid by solvent extraction. The overall lactic acid yield obtainable from this bioreactor system is 77% of the theoretical.     

Wetting behaviour,moisture up-take and electrokinetic properties of lignocellulosic fibres

The wetting and moisture up-take behaviour, as well as the electrokinetic properties of various lignocellulosic fibres were characterised. Knowledge of surface and water uptake properties of this kind of materials will help to tailor their potential use in different end user applications. The surface tension of the fibres was determined from wetting measurements using the capillary rise technique. The wetting data were used to determine the surface tension of the fibres. Our results show that the surface tension of the lignocellulosic fibres is a linear function of their cellulose content. Zeta-potential measurements were exploited to characterise the surface chemistry of the fibres. Measuring the zeta-potential as function of time enables the rapid assessment of the water up-take, i.e. the swelling behaviour of the fibres. The results obtained by the zeta potential measurements correlate, with the exception of flax, in a linear manner with the results obtained from conventional moisture uptake measurements. Even though all lignocellulosic fibres are very hydrophilic due to the presence of polar oxygen containing groups they have different grades of hydrophilicity, which is also reflected in the different water uptake capabilities measured. The wetting, moisture uptake and electrokinetic properties of the lignocellulosic fibres are determined by the availability of the surface functional groups present, which is usually consequence of the processes used to separate, and extract the fibres from the plant (retting), as well as any further processing used to improve the fibre quality.     

Characterization of Mercury – Humic Acids Interaction by Potentiometric Titration with a Modified Carbon Paste Mercury Sensor

Stability constant for mercury binding by commercial and natural humic acids (HA) were determined using a new potentiometric mercury(II) sensor based on dithiosalicylic acid modified carbon paste electrode. The sensor present a high selective and sensitive response to mercury(II) ions, and a low detection limit of 1.8×10^?8 M. The potentiometric titrations curves of humic acids against mercury(II) ions were modeled. For 1.00×10^?7 to 3.00×10^?4 M mercury(II) ion concentration levels the results are consistent with the presence of two different binding sites in the humic acid macromolecule. The strongest binding sites (log K₁ ranging from 10.1 to 6.8) are probably due to interaction with carboxylic acid and amine groups in the molecule, whereas weakest binding sites (log K₂ ranging from 8.8 to 4.5) can be associated to phenolic groups.     

Ultramicro photometric titrations using a long-path photometer

The long-path photometer previously described was adapted for photometric microtitrations. The instrument was tested employing several types of titrations. Turbidimetric titrations of silver with chloride and of barium with sulfate did not yield good results. However, the following titrations gave excellent results down to the levels indicated in parentheses: iron(II) with permanganate (0.4 ppm), zinc with EDTA in the presence of excess Zincon (6 ppm), copper with EDTA in the presence of excess SNAZOXS (9 ppb !), and calcium with EDTA in the presence of excess arsenazo III (100 ppb). The last of these titrations gave good results although a blank of about 25% of the actual volume of titrant required was encountered.     

New methods of heterogeneous catalysis for lignocellulosic biomass conversion to chemicals

This review deals with the use of solid catalysts for the enhancement of the efficiency and the development of a new generation of environmentally friendly, energy and resource efficient processes for the deep processing of lignocellulosic biomass to desired chemicals. The oxidative delignification of wood with hydrogen peroxide in the presence of the suspended TiO₂ catalyst, the oxidation of wood with molecular oxygen in the presence of copper catalysts, the acidcatalyzed conversion of cellulose to glucose and levulinic acid, and the thermal conversion of lignin to fuel additives on solid acid catalysts are analyzed. New integrated processes based on the heterogeneous catalytic oxidation are suitable for the complex processing of lignocellulosic biomass to produce valuable chemicals and engine fuel components without the use of toxic and corrosion-active reagents.     

Enhancing the enzymatic hydrolysis of cellulosic materials using simultaneous ball milling

One of the limiting factors restricting the effective and efficient bioconversion of softwood-derived lignocellulosic residues is the recalcitrance of the substrate following pretreatment. Consequently, the ensuing enzymatic process requires relatively high enzyme loadings to produce monomeric carbohydrates that are readily fermentable by ethanologenic microorganisms. In an attempt to circumvent the need for larger enzyme loadings, a simultaneous physical and enzymatic hydrolysis treatment was evaluated. A ball-mill reactor was used as the digestion vessel, and the extent and rate of hydrolysis were monitored. Concurrently, enzyme adsorption profiles and the rate of conversion during the course of hydrolysis were monitored. α-Cellulose, employed as a model substrate, and SO₂-impregnated steam-exploded Douglas-fir wood chips were assessed as the cellulosic substrates. The softwood-derived substrate was further posttreated with water and hot alkaline hydrogen peroxide to remove >90% of the original lignin. Experiments at different reaction conditions were evaluated, including substrate concentration, enzyme loading, reaction volumes, and number of ball beads employed during mechanical milling. It was apparent that the best conditions for the enzymatic hydrolysis of α-cellulose were attained using a higher number of beads, while the presence of air-liquid interface did not seem to affect the rate of saccharification. Similarly, when employing the lignocellulosic substrate, up to 100% hydrolysis could be achieved with a minimum enzyme loading (10 filter paper units/g of cellulose), at lower substrate concentrations and with a greater number of reaction beads during milling. It was apparent that the combined strategy of simultaneous ball milling and enzymatic hydrolysis could improve the rate of saccharification and/or reduce the enzyme loading required to attain total hydrolysis of the carbohydrate moieties.     

Investigating adsorption of bovine serum albumin on cellulosic substrates using magnetic resonance imaging

Cellulosic biomass is recalcitrant to enzymatic hydrolysis which greatly reduces the efficiency of biofuels production. Specifically, the lignin component of biomass is thought to provide non-productive binding sites for glycosyl hydrolases, effectively disabling the enzymes from completing further digestion. A thorough understanding of the adsorption rates of protein molecules on celluloses—especially lignocelluloses—is crucial to improving the cyclic steps of adsorption, diffusion, and reaction. We use magnetic resonance imaging (MRI) to detect concentrations of bovine serum albumin (BSA) in equilibrium with various cellulose substrates, including delignified and acid-treated lignocellulosic substrates. BSA is believed to be an effective adsorption blocker during enzymatic hydrolysis of lignocellulosics, and has been correlated with an increase in reaction yield. We found BSA to have little adsorption onto the chosen cellulose substrates in the low concentration range studied. Ultraviolet (UV) absorption measurements of reaction supernatants at 280 nm were used to confirm the MRI results for each of the substrate types. The advantages of the MRI technique are compared with that of the traditional UV measurement.     

Anion binding to a tetracopper resorcinarene-based complex

UV–vis and electron paramagnetic resonance (EPR) spectroscopic studies have been carried out on the multinuclear copper(II) complex Cu₄BpaRes. The copper atoms are in a tetragonal distorted geometry with nitrogens from bispicolylamine (Bpa) coordinating in the equatorial plane and water molecules or anions completing four coordination sites. The interaction of anions with this polynuclear copper complex in aqueous solution supports the formation of different complex species, which depend on the type and the concentration of the anions. In the presence of excess anions, frozen solution EPR parameters show the formation of species in which the in-plane coordination is characterised by the presence of three nitrogen atoms coming from the ligand and a donor atom from the specific anion. For the bidentate anion ligands and especially for malonate, UV–vis titrations indicate the formation of a 1:4 (Cu₄BpaRes:anion) species. EPR experiments support the formation of such a species and indicate that the four copper centres are equivalent and reach penta-coordination via the coordination of both oxygens from the bidentate ligand.     

Adsorption of aspartic acid on kaolinite

The interaction of aspartic acid with kaolinite was studied by potentiometric titrations and by adsorption measurements both at constant aspartate concentration (but varying pH) and at a constant pH of 5.5. The temperature was 25 degrees C, and the ionic medium 5 mM KNO3. Aspartic acid dissociation constants estimated from titrations agreed with those from the literature. The adsorption of aspartic acid to kaolinite was weak and varied only slightly with pH; 10-18% of 100 microM aspartic acid adsorbed to kaolinite at 100 m(2)L(-1) between pH 3 and 10. Data from the titrations and adsorption experiments were fitted closely by an extended constant-capacitance surface complexation model, in which monodentate outer-sphere complexes formed between deprotonated aspartic acid molecules and protonated sites on the variable-charge edges of the kaolinite crystals. There appeared to be no adsorption to the permanently charged crystal faces.     

Comparison of a single-use pH test strip and a glass electrode for potentiometric titration

The potentiometric titration of a carbonate mixture or an acetate solution is a common experiment in analytical laboratories. Typically, a glass electrode combined with a calomel or Ag/AgCl reference electrode is used to locate the equivalence points in neutralization titrations. The dissociation constants of weak acids and bases can be calculated from the pH at the half-neutralization point. Recently, a new commercial product for measuring pH has been developed. This novel acid–base detection strip is a single-use sensor that requires neither storage in a preservation liquid nor calibration prior to use. This study examined its suitability for the continuous monitoring of pH changes in potentiometric titrations of carbonate mixtures, acetate solutions, or ammonia solutions. There were no significant differences in the concentrations of solutions tested using a glass electrode and a pH test strip. The pK_a, pK_b, and pH values determined using the two systems differed by less than 5%. The results confirmed that the pH strips are suitable for continuously monitoring pH changes during neutralization titrations. However, the strips can only be used once.     

Weak lignin-binding enzymes

Economic barriers preventing commercialization of lignocellulose-to-ethanol bioconversion processes include the high cost of hydrolytic enzymes. One strategy for cost reduction is to improve the specific activities of cellulases by genetic engineering. However, screening for improved activity typically uses “ideal” cellulosic substrates, and results are not necessarily applicable to more realistic substrates such as pretreated hardwoods and softwoods. For lignocellulosic substrates, nonproductive binding and inactivation of enzymes by the lignin component appear to be important factors limiting catalytic efficiency. A better understanding of these factors could allow engineering of cellulases with improved activity based on reduced enzyme-lignin interaction (“weak lignin-binding cellulases”). To prove this concept, we have shown that naturally occurring cellulases with similar catalytic activity on a model cellulosic substrate can differ significantly in their affinities for lignin. Moreover, although cellulose-binding domains (CBDs) are hydrophobic and probably participate in lignin binding, we show that cellulases lacking CBDs also have a high affinity for lignin, indicating the presence of lignin-binding sites on the catalytic domain.     

Spectroscopy and reactivity of the type 1 copper site in Fet3p from Saccharomyces cerevisiae: correlation of structure with reactivity in the multicopper oxidases

Fet3p is a multicopper oxidase recently isolated from the yeast, Saccharomyces cerevisiae. Fet3p is functionally homologous to ceruloplasmin (Cp) in that both are ferroxidases. However, by sequence homology Fet3p is more similar to fungal laccase, and both contain a type 1 Cu site that lacks the axial methionine ligand present in the functional type 1 sites of Cp. To determine the contribution of the electronic structure of the type 1 Cu site of Fet3p to the ferroxidase mechanism, we have examined the absorption, circular dichroism, magnetic circular dichroism, electron paramagnetic resonance, and resonance Raman spectra of wild-type Fet3p and type 1 and type 2 Cu-depleted mutants. The spectroscopic features of the type 1 Cu site of Fet3p are nearly identical to those of fungal laccase, indicating a very similar three-coordinate geometry. We have also examined the reactivity of the type 1 Cu site by means of redox titrations and stopped-flow kinetics. From poised potential redox titrations, the E degrees of the type 1 Cu site is 427 mV, which is low for a three-coordinate type 1 Cu site. The kinetics of reduction of the type 1 Cu sites of four different multicopper oxidases with two different substrates were compared. The type 1 site of a plant laccase (Rhus vernicifera) is reduced moderately slowly by both Fe(II) and a bulky organic substrate, 1,4-hydroquinone (with 6 equiv of substrate, k(obs) = 0.029 and 0.013 s(-)(1), respectively). On the other hand, the type 1 site of a fungal laccase (Coprinus cinereus) is reduced very rapidly by both substrates (k(obs) > 23 s(-)(1)). In contrast, both Fet3p and Cp are rapidly reduced by Fe(II) (k(obs) > 23 s(-)(1)), but only very slowly by 1,4-hydroquinone (10- and 100-fold more slowly than plant laccase, respectively). Semiclassical theory is used to analyze the origin of these differences in reactivity in terms of type 1 Cu site accessibility to specific substrates.     

Amperometric complex-formation titrations with a dropping bismuth amalgam electrode in halide medium

The use of the dropping bismuth amalgam electrode has been investigated for the selective determination of metal ions in the presence of large concentrations of halides by means of amperometric complex-formation titrations, using normal pulse polarography. Concentrations of metal ions down to 3 x 10(-7)M have been determined with adequate accuracy in the presence of about 0.1M chloride or 0.01M bromide. Calculated and experimental current-voltage curves have been compared and found to be in reasonable agreement.