Martin Bertau

RecoPhos: Full-scale fertilizer production from sewage sludge ash

The substitution potential of sewage sludge for German primary phosphate imports has been estimated as 40%. Yet, a marketable option for the full scale recovery has been lacking. This study focuses on a full-scale process for the manufacture of a P-fertilizer from sewage sludge ash (SSA) adapted from the production of Triple Superphosphate. Given (i) conformity of the input with phosphate ores mined from sedimentary deposits, (ii) comparability of the product with a commercially available P-fertilizer regarding contaminant levels, P-fractionation and yield effects, and (iii) compliance of the output with the German Fertilizer Ordinance the RecoPhos P 38 fertilizer was discharged from the waste legislation regime. The fertilizer is currently being produced at a rate of 1000 tonnes per month and sold at a competitive price.

Valuable Metals-Recovery Processes, Current Trends, and Recycling Strategies

This Review provides an overview of valuable metals, the supply of which has been classified as critical for Europe. Starting with a description of the current state of the art, novel approaches for their recovery from primary resources are presented as well as recycling processes. The focus lies on developments since 2005. Chemistry strategies which are used in metal recovery are summarized on the basis of the individual types of deposit and mineral. In addition, the economic importance as well as utilization of the metals is outlined.

Engineering Cofactor Preference of Ketone Reducing Biocatalysts: A Mutagenesis Study on a γ-Diketone Reductase from the Yeast Saccharomyces cerevisiae Serving as an Example

The synthesis of pharmaceuticals and catalysts more and more relies on enantiopure chiral building blocks. These can be produced in an environmentally benign and efficient way via bioreduction of prochiral ketones catalyzed by dehydrogenases. A productive source of these biocatalysts is the yeast Saccharomyces cerevisiae, whose genome also encodes a reductase catalyzing the sequential reduction of the γ-diketone 2,5-hexanedione furnishing the diol (2S,5S)-hexanediol and the γ-hydroxyketone (5S)-hydroxy-2-hexanone in high enantio- as well as diastereoselectivity (ee and de >99.5%). This enzyme prefers NADPH as the hydrogen donating cofactor. As NADH is more stable and cheaper than NADPH it would be more effective if NADH could be used in cell-free bioreduction systems. To achieve this, the cofactor binding site of the dehydrogenase was altered by site-directed mutagenesis. The results show that the rational approach based on a homology model of the enzyme allowed us to generate a mutant enzyme having a relaxed cofactor preference and thus is able to use both NADPH and NADH. Results obtained from other mutants are discussed and point towards the limits of rationally designed mutants.

Headspace solid-phase microextraction―gas chromatography―mass spectrometry determination of the characteristic flavourings menthone, isomenthone, neomenthol and menthol in serum samples with and without enzymatic cleavage to validate post-offence alcohol drinking claims

A rapid HS-SPME-GC-MS (headspace solid-phase microextraction-gas chromatography-mass spectrometry) method has been developed for determination of menthone, isomenthone, neomenthol and menthol in serum samples with and without enzymatic cleavage. These flavour compounds are characteristic markers for consumption of peppermint liqueurs as well as certain digestif bitters, herbal and bitter liqueurs. This method enabled the detection of the four compounds with a limit of detection (LOD) of 2.1 ng mL(-1) (menthone and isomenthone), 2.8 ng mL(-1) (neomenthol) and 4.6 ng mL(-1) (menthol), and a limit of quantification (LOQ) of 3.1 ng mL(-1) (menthone and isomenthone), 4.2 ng mL(-1) (neomenthol) and 6.8 ng mL(-1) (menthol) in serum samples. The method shows good precision intraday (3.2-3.8%) and interday (5.8-6.9%) and a calibration curve determination coefficient (R(2)) of 0.990-0.996. Experiments were conducted with a volunteer, who consumed peppermint liqueur on three different days under controlled conditions. At defined intervals, blood samples were taken, and the concentration-time profiles for serum menthone, isomenthone, neomenthol and menthol, as free substances as well as glucuronides, were determined. Both menthol and neomenthol underwent a rapid phase II metabolism, but minor amounts of free substances were also detected. Menthone and isomenthone were rapidly metabolised and were found in lower concentrations and over a shorter time span than the other analytes. In blood samples taken from 100 drivers who claimed to have consumed peppermint liqueur prior to the blood sampling, menthone, isomenthone, neomenthol and menthol were detected in the serum as free substances in concentrations between 3.1 and 7.0 ng mL(-1) in eight cases (menthone), 3.1 and 11.3 ng mL(-1) in eight cases (isomenthone), 5.3 and 57.8 ng mL(-1) in nine cases (neomenthol) and 8.0 and 92.1 ng mL(-1) in nine cases (menthol). The sum values of free and conjugated substances ranged between 4.2 and 127.8 ng mL(-1) in 35 cases for neomenthol and 11.0 and 638.2 ng mL(-1) in 59 cases for menthol. Menthone and isomenthone were not conjugated. These test results confirmed that the analysis of characteristic beverage aroma compounds, such as menthone, isomenthone, neomenthol and menthol, can be used for specific verification of post-offence alcohol consumption claims.

Equilibrium-dependent hydration of ethyl 4,4,4-trifluoro-acetoacetate in aqueous solutions and consequences for the whole-cell biotransformation with Saccharomyces cerevisiae

Abstract The presence of a trifluoromethyl group has implications on the enantioselective biotransformation of ethyl 4,4,4-trifluoro-acetoacetate with Saccharomyces cerevisiae (Baker’s yeast). Reaction of the trifluoro-keto ester with the aqueous solvent leads to hydration of the carbonyl group and reduces bioavailability of the substrate. The hydrate forms an equilibrium with the enol and the keto substrate which was investigated in detail and was shown to be dependent on temperature and pH. Bioemulsifiers released in the reaction medium by the microorganism increase hydration rates substantially. Implications of hydration are most effective under fed-batch conditions. Batch processes remain unaffected, but suffer from xenobiotic stress of the substrate. Under fermentation conditions the enol fraction amounts to more than twice the concentration of the keto-form. Competing stereoselective reduction of the enol is discussed to possibly affect the overall enantioselectivity of the biotransformation.

Novel unusual microbial dehalogenation during enantioselective reduction of ethyl 4,4,4-trifluoro acetoacetate with baker's yeast

Abstract In the course of investigating microbial syntheses for chiral pharmaceutical intermediates, ethyl 4,4,4-trifluoro acetoacetate ( 1 ) was submitted to bakers yeast reduction. With the purpose of obtaining the d -carbinol in high enantiopurity, several additives were tested for l -reductase inhibitor activity. Allyl alcohol proved to be not only a suitable additive, but also an inducer for effective defluorination of the substrate.

Pulp properties resulting from different pretreatments of wheat straw and their influence on enzymatic hydrolysis rate

Wheat straw was subjected to three different processes prior to saccharification, namely alkaline pulping, natural pulping and autohydrolysis, in order to study their effect on the rate of enzymatic hydrolysis. Parameters like medium concentration, temperature and time have been varied in order to optimize each method. Milling the raw material to a length of 4mm beforehand showed the best cost-value-ratio compared to other grinding methods studied. Before saccharification the pulp can be stored in dried form, leading to a high yield of glucose. Furthermore the relation of pulp properties (i.e. intrinsic viscosity, Klason-lignin and hemicelluloses content, crystallinity, morphology) to cellulose hydrolysis is discussed.

Fungal Aerobic Reductive Dechlorination of Ethyl 2‐Chloroacetoacetate by Saccharomyces cerevisiae: Mechanism of a Novel Type of Microbial Dehalogenation

Saccharomyces cerevisiae reduces the β‐keto ester ethyl 2‐chloroacetoacetate to the respective chiral cis‐ and trans‐β‐hydroxy esters. In the course of chiral reduction, competing dehalogenation of the xenobiotic substrate to ethyl acetoacetate occurs, in a reaction mediated by cytosolic glutathione (GSH). Mechanistically, the dechlorination is a novel type of glutathione‐dependent dehalogenation catalysed by an as yet unidentified glutathione‐dependent dehalogenase. The first step consists of a nucleophilic replacement of the chloride substituent by glutathione. In the subsequent enzyme‐catalysed step, a second glutathione molecule liberates the dehalogenation product by thiolytic attack at the thioether bridge, resulting in a net transfer of two electrons to the substrate and in the formation of glutathione disulfide (GSSG). Being effective under aerobic conditions and catalysed by a fungus, this reductive dechlorination of an aliphatic substrate is an outstanding example of a novel, glutathione‐mediated microbial dehalogenation.

Predictive tools for the evaluation of microbial effects on drugs during gastrointestinal passage.

Importance of the field: Predicting drug metabolism after oral administration is highly complex, yet indispensable. Hitherto, drug metabolism mainly focuses on hepatic processes. In the intestine, drug molecules encounter the metabolic activity of microorganisms prior to absorption through the gut wall. Drug biotransformation through the gastrointestinal microflora has the potential to evoke serious problems because the metabolites formed may cause unexpected and undesired side effects in patients. Hence, in the course of drug development, the question has to be addressed if microbially formed metabolites are physiologically active, pharmaceutically active or even toxic. In order to provide answers to these questions and to keep the number of laboratory tests needed low, predictive tools – in vivo as well as in silico – are invaluable. Areas covered in this review: This review gives an outline of the current state of the art in the field of predicting the drug biotransformation through the gastrointestinal microflora on several levels of modelling. What the reader will gain: A comprehensive review of the literature with a thorough discussion on assets and drawbacks of the different modelling approaches. Take home message: The impact of the gastrointestinal drug biotransformation on patients’ health will grow with increasing complexity of drug entities. Predicting metabolic fates of drugs by combining in vitro and in silico models provides invaluable information which will be suitable to particularly reduce in vivo studies.

Biosimulation of drug metabolism--a yeast based model.

Computationally predicting the metabolic fates of drugs is a very complex task which is owed not only to the huge and diverse biochemical network in the living cell, but also to the majority of in vivo transformations that occur through the action of hepatocytes and gastro-intestinal micro-flora. Thus, xenobiotics are metabolised by more than a single cell type. However, the prediction of metabolic fates is definitely a problem worth solving since it would allow facilitate the development of drugs in a way less relying on animal testing. As a first step in this direction, PharmBiosim is being developed, a biosimulation tool which is based on substantial data reduction and on attributing metabolic fates of drug molecules to functional groups and substituents. This approach works with yeast as a model organism and is restricted to drugs that are mainly transformed by enzymes of the central metabolism, especially sugar metabolism. The reason for the latter is that the qualitative functioning of the involved biochemistry is very similar in diverse cell types involved in drug metabolism. Further it allows for using glycolytic oscillations as a tool to quantify interactions of a drug with this metabolic pathway.