Uncover aldehydes in biomass hydrolyzates: disproportionation of aldehydes in alkaline solution and subsequent measurement using an automated HPAEC-PAD method.

High-performance anion exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD) was used for developing a method for identifying and quantifying aldehydes in biomass hydrolyzates. This method was optimized to the requirements of HPAEC-PAD in order to allow for a simultaneous determination of aldehydes by respective Cannizzaro alcohols. To this end, sodium hydroxide concentration (0.1 to 5.0 mol/L), temperature (30 to 40 °C), and reaction time (0 to 24 h) were investigated for sufficient and reproducible disproportionation of the biomass-derived aldehydes. The optimized method for aldehyde disproportionation and subsequent measurement are 1 mol/L sodium hydroxide, 40 °C, and 1 h reaction time. The detection limits resulting from this method are lower than 68.55 mg/L and the sensitivity above 0.024 (nC min)/(mg/L) for 3,4-dimethoxybenzaldehyde. Linearity for aldehyde calibration always exceeded 0.98. Thus, HPAEC-PAD analysis allows for the quantification of biomass-derived compounds from all natural polymers and, therefore, it has exemplarily been used to quantify aldehyde concentration of beech wood, orange peel, and algae biomass hydrolyzates. Graphical abstract.

Efficient enzymatic saccharification of macroalgal biomass using a specific thermostable GH 12 endoglucanase from Aspergillus terreus JL1.

Stranded green macroalgae represents an important and renewable biomass that remains under valorized despite the numerous environmental problems generated by their accumulation in coastal regions. This work describes the isolation of a filamentous thermophile fungus identified as Aspergillus terreus JL1 that produces an efficient cellulolytic activity for green macroalgae saccharification. The characterization of the endoglucanase activity obtained after submerged fermentation showed a differential induction depending on the carbon source used with a unique isoform released when Ulva lactuca was used as inducer. The crude extract obtained hydrolyzed efficiently the untreated algal biomass (70.5%) compared to other cellulolytic extracts. The unique endoglucanase released was then purified to homogeneity (Yield: 49.6%; Specific activity: 30.1 U/mg; Purification fold: 4.36) and characterized biochemically. Its peptidic sequence was then determined and showed its belonging to the GH12. The described enzyme represents a promising biotechnological tool for algal biomass conversion.

Genotoxicity of three biofuel candidates compared to reference fuels.

Global demand for alternative energy sources increases due to concerns regarding energy security and greenhouse gas emissions. However, little is known regarding the impacts of biofuels to the environment and human health even though the identification of such impacts is important to avoid biofuels leading to undesired effects. In this study mutagenicity and genotoxicity of the three biofuel candidates ethyl levulinate (EL), 2-methyltetrahydrofuran (2-MTHF) and 2-methylfuran (2-MF) were investigated in comparison to two petroleum-derived fuels and a biodiesel. None of the samples induced mutagenicity in the Ames fluctuation test. However, the Micronucleus assay revealed significant effects in Chinese hamster (Cricetulus griseus) V79 cells caused by the potential biofuels. 2-MF revealed the highest toxic potential with significant induction of micronuclei below 20.0 mg/L. EL and 2-MTHF induced micronuclei only at very high concentrations (>1000.0 mg/L). In regard to the genotoxic potential of 2-MF, its usage as biofuel should be critically discussed.

Aquatic toxicity of biofuel candidates on Daphnia magna.

The increasing need for carbon-neutral, low-emission transportation sector has led to the development of advanced biofuels with tailor-made production and combustion processes. Even though the large-scale deployment of these advanced biofuels also increases the risk for their release into the environment, their toxic potency remains largely unknown. To identify hazardous biofuel candidates as early as possible, the fuel development process can be expanded by "Green Toxicology". To demonstrate such early Green Toxicology testing, this study investigates the aquatic toxicity for the two biofuel candidates 2-methyltetrahydrofuran (2-MTHF) and 2-methylfuran (2-MF) on Daphnia magna. We performed the prolonged acute immobilisation assay (96 h) and the D. magna reproduction test. 2-MF induced acute effects on D. magna that were two orders of magnitude stronger than those of 2-MTHF. Furthermore, both substances affected the growth and reproductive output of D. magna in a 21 d reproduction test, with 2-MF already inducing effects with concentrations one to two orders of magnitude lower than those of 2-MTHF. Thus, our assessment of the aquatic toxicity suggests that further biofuel development should focus on 2-MTHF. Furthermore, the acute immobilisation test with D. magna was identified as a promising tool for a rapid and sensitive "Green Toxicology" screening of further biofuel candidates.

Fast automated online xylanase activity assay using HPAEC-PAD.

In contrast to biochemical reactions, which are often carried out under automatic control and maintained overnight, the automation of chemical analysis is usually neglected. Samples are either analyzed in a rudimentary fashion using in situ techniques, or aliquots are withdrawn and stored to facilitate more precise offline measurements, which can result in sampling and storage errors. Therefore, in this study, we implemented automated reaction control, sampling, and analysis. As an example, the activities of xylanases on xylotetraose and soluble xylan were examined using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). The reaction was performed in HPLC vials inside a temperature-controlled Dionex™ AS-AP autosampler. It was started automatically when the autosampler pipetted substrate and enzyme solution into the reaction vial. Afterwards, samples from the reaction vial were injected repeatedly for 60 min onto a CarboPac™ PA100 column for analysis. Due to the rapidity of the reaction, the analytical method and the gradient elution of 200 mM sodium hydroxide solution and 100 mM sodium hydroxide with 500 mM sodium acetate were adapted to allow for an overall separation time of 13 min and a detection limit of 0.35-1.83 mg/L (depending on the xylooligomer). This analytical method was applied to measure the soluble short-chain products (xylose, xylobiose, xylotriose, xylotetraose, xylopentaose, and longer xylooligomers) that arise during enzymatic hydrolysis. Based on that, the activities of three endoxylanases (EX) were determined as 294 U/mg for EX from Aspergillus niger, 1.69 U/mg for EX from Bacillus stearothermophilus, and 0.36 U/mg for EX from Bacillus subtilis. Graphical abstract Xylanase activity assay automation.

Variations in Physicochemical Properties and Bioconversion Efficiency of Ulva lactuca Polysaccharides After Different Biomass Pretreatment Techniques.

Green macroalgae are an abundant and undervalued biomass with a specific cell wall structure. In this context, different pretreatments, namely ethanol organosolv (Org), alkaline, liquid hot water (LHW), and ionic liquid (IL) pretreatments, were applied to the green macroalgae Ulva lactuca biomass and then evaluated. Their effects on chemical composition, biomass crystallinity, enzymatic digestibility, and theoretical ethanol potential were studied. The chemical composition analysis showed that the Org and LHW pretreatments allowed the highest glucan recovery (80.8 ± 3.6 and 62.9 ± 4.4 g/100 g DM, respectively) with ulvan (80.0 and 99.1%) and hemicellulose (55.0 and 42.3%) removal. These findings were in agreement with both thermogravimetric analysis and scanning electron microscopy results that confirm significant structural changes of the pretreated biomasses. It was found that the employed pretreatments did not significantly affect the cellulose crystallinity; however, they both increased the whole crystallinity and the enzymatic digestibility. This later reached 97.5% in the case of LHW pretreatment. Our results showed high efficiency saccharification of Ulva lactuca biomass that will constitute the key step of the implementation of a biorefinery process.

Multi-step biocatalytic depolymerization of lignin.

Lignin is a biomass-derived aromatic polymer that has been identified as a potential renewable source of aromatic chemicals and other valuable compounds. The valorization of lignin, however, represents a great challenge due to its high inherent functionalization, what compromises the identification of chemical routes for its selective depolymerization. In this work, an in vitro biocatalytic depolymerization process is presented, that was applied to lignin samples obtained from beech wood through OrganoCat pretreatment, resulting in a mixture of lignin-derived aromatic monomers. The reported biocracking route comprises first a laccase-mediator system to specifically oxidize the Cα hydroxyl group in the ß-O-4 structure of lignin. Subsequently, selective ß-O-4 ether cleavage of the oxidized ß-O-4 linkages is achieved with ß-etherases and a glutathione lyase. The combined enzymatic approach yielded an oily fraction of low-molecular-mass aromatic compounds, comprising coniferylaldehyde and other guaiacyl and syringyl units, as well as some larger (soluble) fractions. Upon further optimization, the reported biocatalytic route may open a valuable approach for lignin processing and valorization under mild reaction conditions.

Automated chromatographic laccase-mediator-system activity assay.

To study the interaction of laccases, mediators, and substrates in laccase-mediator systems (LMS), an on-line measurement was developed using high performance anion exchange chromatography equipped with a CarboPac™ PA 100 column coupled to pulsed amperometric detection (HPAEC-PAD). The developed method was optimized for overall chromatographic run time (45 to 120 min) and automated sample drawing. As an example, the Trametes versicolor laccase induced oxidation of 1-(3,4-dimethoxyphenyl)-2-(2-methoxyphenoxy)-1,3-dihydroxypropane (adlerol) using 1-hydroxybenzotriazole (HBT) as mediator was measured and analyzed on-line. Since the Au electrode of the PAD detects only hydroxyl group containing substances with a limit of detection being in the milligram/liter range, not all products are measureable. Therefore, this method was applied for the quantification of adlerol, and-based on adlerol conversion-for the quantification of the LMS activity at a specific T. versicolor laccase/HBT ratio. The automated chromatographic activity assay allowed for a defined reaction start of all laccase-mediator-system reactions mixtures, and the LMS reaction progress was automatically monitored for 48 h. The automatization enabled an integrated monitoring overnight and over-weekend and minimized all manual errors such as pipetting of solutions accordingly. The activity of the LMS based on adlerol consumption was determined to 0.47 U/mg protein for a laccase/mediator ratio of 1.75 U laccase/g HBT. In the future, the automated method will allow for a fast screening of combinations of laccases, mediators, and substrates which are efficient for lignin modification. In particular, it allows for a fast and easy quantification of the oxidizing activity of an LMS on a lignin-related substrate which is not covered by typical colorimetric laccase assays. ᅟ.

Microscale In Vitro Assays for the Investigation of Neutral Red Retention and Ethoxyresorufin-O-Deethylase of Biofuels and Fossil Fuels.

Only few information on the potential toxic effectiveness of biofuels are available. Due to increasing worldwide demand for energy and fuels during the past decades, biofuels are considered as a promising alternative for fossil fuels in the transport sector. Hence, more information on their hazard potentials are required to understand the toxicological impact of biofuels on the environment. In the German Cluster of Excellence "Tailor-made Fuels from Biomass" design processes for economical, sustainable and environmentally friendly biofuels are investigated. In an unique and interdisciplinary approach, ecotoxicological methods are applied to gain information on potential adverse environmental effects of biofuels at an early phase of their development. In the present study, three potential biofuels, ethyl levulinate, 2-methyltetrahydrofuran and 2-methylfuran were tested. Furthermore, we investigated a fossil gasoline fuel, a fossil diesel fuel and an established biodiesel. Two in vitro bioassays, one for assessing cytotoxicity and one for aryl hydrocarbon receptor agonism, so called dioxin-like activity, as measured by Ethoxyresorufin-O-Deethylase, were applied using the permanent fish liver cell line RTL-W1 (Oncorhynchus mykiss). The special properties of these fuel samples required modifications of the test design. Points that had to be addressed were high substance volatility, material compatibility and low solubility. For testing of gasoline, diesel and biodiesel, water accommodated fractions and a passive dosing approach were tested to address the high hydrophobicity and low solubility of these complex mixtures. Further work has to focus on an improvement of the chemical analyses of the fuel samples to allow a better comparison of any effects of fossil fuels and biofuels.

Acute embryo toxicity and teratogenicity of three potential biofuels also used as flavor or solvent.

The demand for biofuels increases due to concerns regarding greenhouse gas emissions and depletion of fossil oil reserves. Many substances identified as potential biofuels are solvents or already used as flavors or fragrances. Although humans and the environment may be readily exposed little is known regarding their (eco)toxicological effects. In this study, the three potential biofuels ethyl levulinate (EL), 2-methyltetrahydrofuran (2-MTHF) and 2-methylfuran (2-MF) were investigated for their acute embryo toxicity and teratogenicity using the fish embryo toxicity (FET) test to identify unknown hazard potentials and to allow focusing further research on substances with low toxic potentials. In addition, two fossil fuels (diesel and gasoline) and an established biofuel (rapeseed oil methyl ester) were investigated as references. The FET test is widely accepted and used in (eco)toxicology. It was performed using the zebrafish Danio rerio, a model organism useful for the prediction of human teratogenicity. Testing revealed a higher acute toxicity for EL (LC50: 83mg/L) compared to 2-MTHF (LC50: 2980mg/L), 2-MF (LC50: 405mg/L) and water accommodated fractions of the reference fuels including gasoline (LC50: 244mg DOC/L). In addition, EL caused a statistically significant effect on head development resulting in elevated head lengths in zebrafish embryos. Results for EL reduce its likelihood of use as a biofuel since other substances with a lower toxic potential are available. The FET test applied at an early stage of development might be a useful tool to avoid further time and money requiring steps regarding research on unfavorable biofuels.

Multi-scale processes of beech wood disintegration and pretreatment with 1-ethyl-3-methylimidazolium acetate/water mixtures.

BACKGROUND: The valorization of biomass for chemicals and fuels requires efficient pretreatment. One effective strategy involves the pretreatment with ionic liquids which enables enzymatic saccharification of wood within a few hours under mild conditions. This pretreatment strategy is, however, limited by water and the ionic liquids are rather expensive. The scarce understanding of the involved effects, however, challenges the design of alternative pretreatment concepts. This work investigates the multi length-scale effects of pretreatment of wood in 1-ethyl-3-methylimidazolium acetate (EMIMAc) in mixtures with water using spectroscopy, X-ray and neutron scattering. RESULTS: The structure of beech wood is disintegrated in EMIMAc/water mixtures with a water content up to 8.6 wt%. Above 10.7 wt%, the pretreated wood is not disintegrated, but still much better digested enzymatically compared to native wood. In both regimes, component analysis of the solid after pretreatment shows an extraction of few percent of lignin and hemicellulose. In concentrated EMIMAc, xylan is extracted more efficiently and lignin is defunctionalized. Corresponding to the disintegration at macroscopic scale, SANS and XRD show isotropy and a loss of crystallinity in the pretreated wood, but without distinct reflections of type II cellulose. Hence, the microfibril assembly is decrystallized into rather amorphous cellulose within the cell wall. CONCLUSIONS: The molecular and structural changes elucidate the processes of wood pretreatment in EMIMAc/water mixtures. In the aqueous regime with >10.7 wt% water in EMIMAc, xyloglucan and lignin moieties are extracted, which leads to coalescence of fibrillary cellulose structures. Dilute EMIMAc/water mixtures thus resemble established aqueous pretreatment concepts. In concentrated EMIMAc, the swelling due to decrystallinization of cellulose, dissolution of cross-linking xylan, and defunctionalization of lignin releases the mechanical stress to result in macroscopic disintegration of cells. The remaining cell wall constituents of lignin and hemicellulose, however, limit a recrystallization of the solvated cellulose. These pretreatment mechanisms are beyond common pretreatment concepts and pave the way for a formulation of mechanistic requirements of pretreatment with simpler pretreatment liquors.

Benzoxazolinone detoxification by N-Glucosylation: The multi-compartment-network of Zea mays L.

The major detoxification product in maize roots after 24 h benzoxazolin-2(3H)-one (BOA) exposure was identified as glucoside carbamate resulting from rearrangement of BOA-N-glucoside, but the pathway of N-glucosylation, enzymes involved and the site of synthesis were previously unknown. Assaying whole cell proteins revealed the necessity of H2O2 and Fe(2+) ions for glucoside carbamate production. Peroxidase produced BOA radicals are apparently formed within the extraplastic space of the young maize root. Radicals seem to be the preferred substrate for N-glucosylation, either by direct reaction with glucose or, more likely, the N-glucoside is released by glucanase/glucosidase catalyzed hydrolysis from cell wall components harboring fixed BOA. The processes are accompanied by alterations of cell wall polymers. Glucoside carbamate accumulation could be suppressed by the oxireductase inhibitor 2-bromo-4´-nitroacetophenone and by peroxidase inhibitor 2,3-butanedione. Alternatively, activated BOA molecules with an open heterocycle may be produced by microorganisms (e.g., endophyte Fusarium verticillioides) and channeled for enzymatic N-glucosylation. Experiments with transgenic Arabidopsis lines indicate a role of maize glucosyltransferase BX9 in BOA-N-glycosylation. Western blots with BX9 antibody demonstrate the presence of BX9 in the extraplastic space. Proteomic analyses verified a high BOA responsiveness of multiple peroxidases in the apoplast/cell wall. BOA incubations led to shifting, altered abundances and identities of the apoplast and cell wall located peroxidases, glucanases, glucosidases and glutathione transferases (GSTs). GSTs could function as glucoside carbamate transporters. The highly complex, compartment spanning and redox-regulated glucoside carbamate pathway seems to be mainly realized in Poaceae. In maize, carbamate production is independent from benzoxazinone synthesis.