Nico Anders

Simultaneous determination of acid-soluble biomass-derived compounds using high performance anion exchange chromatography coupled with pulsed amperometric detection

A high performance anion exchange chromatography-pulsed amperometric detection (HPAEC-PAD) method using a CarboPac™ PA100 column for the simultaneous determination of 25 soluble compounds originating from all polymer classes (cellulose, hemicellulose, lignin and pectin) in biomass hydrolyzates was developed and validated. The method was optimized for resolution varying the column oven temperature (in the range of 30–50 °C) and the eluents (100 mM NaOH, 200 mM NaOH, 100 mM NaOH/500 mM NaOAc and water). The optimized method used gradient elution at 40 °C and had 70 min duration for one run. The detection limits ranged between 0.14 mg L−1 for both 2,6-dimethoxyphenol and 3,5-dimethoxy-4-hydroxybenzaldehyde and 21.9 mg L−1 for 4-methoxybenzyl alcohol, linearity was always >0.99, and sensitivity ranged between 0.0023 (nC min)/(mg L−1) for glucuronic acid and 8.4 (nC min)/(mg L−1) for 3,5-dimethoxy-4-hydroxybenzaldehyde. In contrast to the established photometric lignin measurement the HPAEC-PAD method allows for a distinction between several acid-soluble lignin compounds. The method was applied to 17 different biomass hydrolyzates and displayed the capability to quantify a wide range of soluble products covering very different hydrolyzate compositions.

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 β-O-4 structure of lignin. Subsequently, selective β-O-4 ether cleavage of the oxidized β-O-4 linkages is achieved with β-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.

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

ABSTRACT 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 Fe2+ 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.

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.

Exploring Plants Strategies for Allelochemical Detoxification

The success of allelopathic plants is characterized by the releases of allelochemicals that suppress the growth of receiver plants directly and indirectly, due to concomitant effects on their microbiome. Therefore, negative effects of allelochemicals on microorganisms can enhance repercussions on plants. On the other hand, plants exposed to allelochemicals develop strategies to eliminate, detoxify or degrade the compounds, whereby plants can take advantage of metabolic properties of microorganisms associated with the root surface and the rhizosphere. Peroxidases and glucosyltransferases have important functions in those processes. In this chapter, methods are presented which allow an estimation of the extent of microbiome damage due to allelochemicals (signature lipid biomarker analysis combined with next generation sequencing). Assays for the determination of glucosyltransferases and peroxidases, important enzyme classes in detoxification processes, are presented with emphasis on benzoxazolinone detoxification as an example for the involvement of microorganisms in reaction sequences. Finally, methods to study alterations in the composition of cell wall polymers are presented as cell wall polymers can be modified during detoxification reactions.

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.

Anion exchange chromatography in lignocellulosics analysis

C wiring into nanoscale holes with high aspect ratio by electrodeposition is an important problem for 3-D integration in integrated circuit technology toward miniaturization of electronic devices. However, void and pinhole found in Cu wiring for the integration can cause trouble for miniature device. Cu electroplating method without void and pinhole is needed. We have proposed novel electroplating methods with supercritical carbon dioxide (sc-CO2) emulsion (EP-SCE). The electrochemical reaction is carried out in an emulsion of sc-CO2 in electrolyte with surfactants. Sc-CO2 has low viscosity and compatibility of hydrogen. Thus, this method is applicable in fine Cu wiring. The aim of this report is to examine Cu electrodeposition by using sc-CO2 emulsified electrolyte into nano-scale Cu wiring on the viewpoints of dissolution of Cu seed layer, gap-filling capability into nano-scale holes and contamination in the plated Cu. Moreover a continuous-flow reaction system is proposed and examined for filling of Cu into holes with 60 nm in diameter and aspect ratio of 2 and 5 by EP-SCE on a round-type large-area hole test element group with diameter of 300 mm, which has an integrated structure of Cu seed layer on TiN barrier layer sputtered on Si substrates.T spectroscopy has gained popularity as a promising non-invasive investigation tool in recent decades. In studies of solid-state pharmaceuticals, its usefulness is enhanced, since it enables one to distinguish diff erent polymorphic and pseudo-polymorphic forms. Terahertz absorption spectra are additive, meaning that the resulting spectrum of two or more compounds in the sample is an algebraic sum, in the linear region of the Beer’s law. For this reason, the evolving-in-time linear mixture of unknown pure components can be resolved using the blind source separation approach, where both spectral sources and their concentrations are to be estimated. Such an evolution occurs naturally in temperature-induced (pseudo) polymorphic transitions, i.e. dehydration, where the terahertz spectrum is the hydrate and dehydrates’s response with timevarying proportions. Conventionally, to estimate the kinetics of a reaction–an important physical-chemical parameter–the area of the unique spectral peak is integrated and normalized to evaluate the abundance, but a diffi culty of strongly overlapping peaks unable its application. To overcome this problem, we show, that by employing the blind source separation procedure, we can resolve a complex, multi-compound spectral mixture with signifi cant cross-bands, where the peak area method failed. We evaluate the performance of our approach in studies on dehydration of a well-known polycrystalline hydrate–α-D-glucose monohydrate. Seeing that the polymorphic transitions can completely change the properties of a pharmaceutical, our approach can fi nd application in a drug development process, where their careful characterization is of utmost importance.