Biobased industrial chemicals from glutamic acid
Lammens, T.M. - \ 2011
Wageningen University. Promotor(en): Johan Sanders, co-promotor(en): Maurice Franssen; Elinor Scott. - [S.l.] : S.n. - ISBN 9789461730237
glutaminezuur - derivaten - chemicaliën uit biologische grondstoffen - synthese - biobased economy - glutamic acid - derivatives - biobased chemicals - synthesis - biobased economy
In dit onderzoek is op zoek gegaan naar routes om van glutaminezuur vier producten te maken die van waarde zijn voor de industrie, die nu uit olie gemaakt worden. Dat zijn grondstoffen voor allerlei soorten kunststof, zoals nylon en rubbers. Het onderzoek laat zien dat alle vier die producten inderdaad gemaakt kunnen worden uit glutaminezuur. Vervolgens is niet alleen gekeken naar de manier waarop dat zou kunnen, maar ook naar de praktische uitvoerbaarheid, de economische haalbaarheid en de impact op het milieu. Daarbij bleek dat er voor twee van de vier producten nog verbeteringen nodig zijn om te kunnen concurreren met de productie uit olie, maar dat de andere twee industrieel haalbaar kunnen zijn. Productie daarvan uit glutaminezuur vermindert onze afhankelijkheid van fossiele brandstoffen en is beter voor het milieu.
Metabolic engineering toward 1-butanol derivatives in solvent producing clostridia
Siemerink, M.A.J. - \ 2010
Wageningen University. Promotor(en): John van der Oost, co-promotor(en): Servé Kengen. - [S.l. : S.n. - ISBN 9789085857976 - 202
industriële microbiologie - clostridium - butanol - derivaten - metabolisme - industrial microbiology - clostridium - butanol - derivatives - metabolism
Chapter 1 of this thesis gives an overview about the history of the acetone, butanol and ethanol (ABE) fermentation. The responsible solventogenic clostridia with their central metabolism are briefly discussed. Despite the fact that scientific research on the key organisms of the ABE process has continued over the past decades and even increased in recent years, still numerous aspects remain unclear.
Economically, the biggest challenge within the ABE fermentation field, remains the 1-butanol toxicity. Due to its toxicity the yield of 1-butanol does not exceed 1-2% (w/v), and only little progress has been made over the past years. Nevertheless, the ABE fermentation process became interesting again, because of the global interest in biofuels or biofuel additives.
Nowadays, in an attempt to reach higher yields, other microorganisms are also being explored as hosts for the production of solvents. E. coli is often chosen as a promising host organism for the microbial production of biofuels. Nevertheless, clostridial hosts remain interesting, due to several reasons, like i) availability of a genetic system, ii) natural production of solvents and iii) relatively high tolerance towards solvents. A disadvantage of the usage of these solventogenic organisms is the inability to use cellulose and hemi-cellulose as substrate.
With respect to the production of potentially interesting 1-butanol derivatives, we focussed on 2,3-butanediol. This industrially valuable compound is already produced in nature by several bacteria, but not by C. acetobutylicum. In this thesis, the production of 2,3-butanediol by Clostridium acetobutylicum is investigated. In Chapter 1 the 2,3-butanediol biosynthesis pathway is extensively described.
The two industrially most important and sequenced solventogenic clostridia are C. acetobutylicum ATCC 824 and C. beijerinckii NCIMB 8052. A lot of biochemical information is known about the various metabolic steps of the central catabolic pathway of C. acetobutylicum ATCC 824. In Chapter 2, comparisons are made between the pathways of both species. With the genome sequence of C. beijerinckii NCIMB 8052 also available, likely candidates for the 34 involved enzymatic conversions within the central catabolic pathway of C. beijerinckii, could be predicted. The enzymatic conversions involved in glucose uptake, glycolysis, gluconeogenesis, pyruvate conversion and acetyl-CoA conversion towards the different end products are being discussed.
Chapter 3 describes and investigates a novel approach in solving the problem of the 1-butanol toxicity towards C. acetobutylicum. Increasing of the tolerance of 1-butanol has been tried more often in other research groups. In our approach, we chose for the introduction of new biosynthesis pathways that enable the production of less toxic 1-butanol derivatives. The toxicity of compounds is expected to correlate with its lipophilicity, e.g. the tendency to accumulate in cell membranes. It can be expressed as the logarithm of the partition coefficient with octanol and water (log Kow value). Chapter 3 describes the growth experiments that were performed to access the toxicity of the various derivatives that were selected, viz. iso-butanol, 2-butanol, tert-butanol, 2,3-butanediol, iso-amyl alcohol, butyl acetate, butyl butyrate and butyl lactate. Iso-butanol, 2-butanol and 2,3-butanediol emerged as likely alternatives to 1-butanol, based on their log Kow value and their behaviour in the toxicity test.
2,3-Butanediol appeared to be a potential candidate, which is less toxic to C. acetobutylicum than 1-butanol. The precursor of 2,3-butanediol, acetoin, which is already produced by C. acetobutylicum in small amounts, is formed by the decarboxylation of acetolactate. The conversion is catalyzed by acetolactate decarboxylase. In Chapter 4 the identification, heterologous production, purification and biochemical characterization of a acetolactate decarboxylase from C. acetobutylicum ATCC 824 is described. Ca-ALD encoded by CAC2967 was proven to exhibit acetolactate decarboxylase activity. Size exclusion chromatography revealed that the native enzyme mainly exists as dimer of 27 kDa subunits. Optimal activity was found around 40 °C, and at pH 5.2. The enzyme is dependent on the presence of bivalent metal ions, like Zn2+ or Co2+. The half life is estimated as 25 hours at 37 °C. The Ca-ALD binds acetolactate cooperatively with a Hill coefficient of 1.49. Also, a K1/2 of 16.8 mM and a Vmax of 51.9 U/mg was determined. Furthermore, a shuttle vector was constructed to express Ca-ald under control of the strong adc promoter in C. acetobutylicum ATCC 824. However, despite successful transformation, no significant increase in acetoin production was observed in the Ca-ALD overexpressing strain.
Only one additional enzyme is needed to complete the 2,3-butanediol biosynthesis pathway in C. acetobutylicum. This enzyme, catalyzing the reduction of acetoin to 2,3-butandiol is called an acetoin reductase. Chapter 5 describes the identification, heterologous production, purification and biochemical characterization of an acetoin reductase from C. beijerinckii. A bioinformatic screening within the genome of C. beijerinckii , revealed eight putative acetoin reductases. Out of six successfully cloned genes, one (CBEI_1464) showed substantial acetoin reductase activity after heterologous expression in E. coli. This purified enzyme (Cb-ACR) was found to exist predominantly as homodimer of 37 kDa subunits. The enzyme has a preference for NADPH (Km = 0.32 μM) as electron donor, with a specific activity amounting to 76 U. mg-1. Optimal activity was found around 68 °C, for both reactions and at pH 6.5 and 9.5, for the reduction and oxidation reaction, respectively. ICP-AES analysis revealed the presence of ~2 Zn2+ atoms and ~1 Ca2+ atom per monomer. To gain insight into the reaction mechanism, but also into the substrate- and cofactor-specificity, a structural model was constructed with a ketose reductase (sorbitol dehydrogenase) from Bemisia argentifolii (silverleaf whitefly) as template. The catalytic zinc atom is likely coordinated by Cys37, His70, Glu71 in Cb-ACR, while the structural zinc site is probably composed of Cys100, Cys103, Cys106, and Cys114.
The acetoin reductase (Cb-ACR) found in C. beijerinckii is used for in vivo experiments in C. acetobutylicum. In Chapter 6 the production of D-2,3-butanediol production in C. acetobutylicum ATCC 824 by heterologous expression of Cb-ACR is described. Under certain conditions Clostridium acetobutylicum ATCC 824 (and derived strains) generates both D and L stereoisomers of acetoin, but due to the lack of an ACR enzyme, does not produce 2,3 butanediol. A gene encoding ACR from Clostridium beijerinckii NCIMB 8052 has been functionally expressed in C. acetobutylicum under control of two strong promoters, i.e. the constitutive thl promoter and the late exponential adc promoter. Both ACR-overproducing strains have been grown in batch cultures, during which 89 90% of the natively produced acetoin has been converted to 20 22 mM D 2,3 butanediol. Addition of a racemic mixture of acetoin did lead to the production of both, D 2,3 butanediol and meso 2,3 butanediol. A metabolic network is proposed that is in agreement with the experimental data. Native 2,3 butanediol production is a first step towards a potential homo fermentative 2 butanol producing strain of C. acetobutylicum as will be discussed in this thesis.
Hydrogen bonding in the recovery of phenols and methyl-t-butyl ether : molecular modeling and calorimetry
Cuypers, R. - \ 2010
Wageningen University. Promotor(en): Han Zuilhof; Ernst Sudhölter. - [S.l. : S.n. - ISBN 9789085857754 - 189
afvalwaterbehandeling - verwijdering - fenol - ethers - derivaten - harsen - extractie - waste water treatment - removal - phenol - ethers - derivatives - resins - extraction
The purification of waste water is very important, for clean potable water is a common good and a necessity. Surface water purification is nowadays carried out on a massive industrial scale, and clean water is at our disposal virtually everywhere and always. However, cleaning industrial waste water can be a difficult task. Although apolar and slightly polar compounds can be removed from water relatively easily e.g. by extraction to an apolar phase, more polar pollutants like phenol and methyl-tert-butyl ether (MTBE), the two main compounds that this thesis deals with, cannot be removed as easily. A more effective method is therefore needed to clean water that is contaminated with either phenol or MTBE.
Solvent-Impregnated Resins (SIRs) are porous polymer beads containing apolar organic extractant liquids. They are used as three-phase separation systems. When brought into contact with a SIR, a solute will preferentially partition from the aqueous phase into the impregnated solvent phase. A drawback to the use of solely the organic extraction liquid in SIRs is the limited solubility of more polar compounds like phenols and ethers in such a medium. In order to enhance extraction, complex-forming extractants can be added to the organic solvent. By means of complex formation inside the organic solvent, the overall equilibrium distribution can be shifted towards the SIR, with a concomitant enhancement of the extraction efficiency. A tight binding of the pollutant molecules to the extractant will eventually ensure high distribution coefficients. However, a moderate binding strength would enable a relatively easy regeneration of the complexing agent after a binding event, enabling multiple uses of the same compound.
In this thesis hydrogen-bond (H-bond) complexation, a specific and strong yet reversible way of binding is investigated for phenol recovery and MTBE recovery from aqueous environments, involving the use of organic complexing agents that can be used inside the SIR to enhance the extraction. Potentially interesting compounds were investigated on a molecular scale by quantum chemical modeling methods and subsequent synthesis and physical characterization by primarily calorimetric means. H-bond complex formation has been evaluated and the important parameters determining the binding process have been described.
After a general introduction in Chapter 1, Chapters 2 through 4 describe phenol complexation by several different classes of complexing agents. In Chapter 2, the binding of phenol and thiophenol by phosphine oxides, phosphates, and their thio-analogs was investigated. Modeling experiments, isothermal titration calorimetry (ITC) measurements, and liquid-liquid extraction experiments showed that, in principle, the binding affinity of the oxide compounds for phenol is high, whereas the sulfide compounds show only low affinity. In particular, the binding behavior of tri-n-octylphosphine oxide towards phenol and a series of electron-withdrawing group (EWG)-substituted phenols was studied, both in the presence and absence of water in the system. It was found that the presence of water in the system – as can be expected in industrial applications – yields lower binding affinities by as much as 60 %, but the binding stoichiometry remained specific and 1 : 1 complexes were still found. Electronic and steric effects were shown to play an important role in phenol binding in the investigated environment.
In Chapter 3, these investigations were extended to the modeling of the full homologous series of mono-, di- and tri-substituted phosphine oxides and phosphates and their thio-analogs. Different modeling methods were used to investigate both structural and electronic elements. Dimethylphosphate was found to form the strongest complexes to the investigated phenols, but because this compound forms very strong homo-dimers in solution it cannot be used as an effective extractant. The SCS-MP2 method, that was relatively unexplored for H-bonding until now, was found to yield very accurate energy predictions, whereas the CBS-Q method was found to predict false binding affinities. Solvent effects are shown to immensely influence the binding behavior.
Another, even stronger group of H-bond acceptors, amine-N-oxides, was investigated as described in Chapter 4. The binding properties for phenol and thiophenol with three different amine-N-oxides yielded very high binding affinities (up to 30 times higher than for the phosphine oxide compounds). Introduction of EWGs in the amine-N-oxides was shown to yield markedly lower binding affinities towards phenols, providing a handle to fine-tune the interaction and facilitating easier regeneration of the complexing agent in future SIR applications. Solvent effects and the influence of water in the system were investigated, and it was shown that they both influence the phenol binding strength. The results in Chapters 2 through 4 show that phosphates, phosphine oxides, and amine-N-oxides could all be used in future SIR extraction systems, and the choice between these classes of compounds can be made based on more detailed considerations.
MTBE binding by several complexing agents was described in Chapter 5. A detailed modeling study of a number of different substituted phenols for MTBE binding was carried out, and the influence of solvents on the binding behavior was investigated, using a.o. the recently developed M06-2X functional and SMD solvent model. The investigated complexing agents were found to show moderate binding affinities to MTBE with binding strengths being closely linked to the acidity of the extractant. Steric effects and a proper consideration of entropic effects are also found to be important to yield successful binding of MTBE. In combination with the existing MTBE distribution coefficient for apolar phases, these moderate binding affinities were found to be able to enhance extraction, in principle, up to the point where it becomes industrially relevant to use such extractants in SIR-based extractions.
Finally, in Chapter 6 the performed research is reviewed, and conclusions, recommendations and a wider perspective for future scientific challenges are given.
Enzymatic production of hyaluronan oligo- and polysaccharides
Kooy, F.K. - \ 2010
Wageningen University. Promotor(en): Gerrit Eggink; Hans Tramper, co-promotor(en): Carmen Boeriu. - [S.l. : S.n. - ISBN 9789085856481 - 174
hyaluronzuur - derivaten - oligosacchariden - polysacchariden - industriële microbiologie - industriële enzymen - hyaluronic acid - derivatives - oligosaccharides - polysaccharides - industrial microbiology - industrial enzymes
Hyaluronan oligo- and polysaccharides are abundant in the human body. Depending on the chain length, hyaluronan is an important structural component or is involved in influencing cell responses during embryonic development, healing processes, inflammation and cancer. Due to these diverse roles of hyaluronan, there are multiple applications already in use or in development, such as supplementation of fluid in eyes and joints, cosmetic tissue augmentation, enhancing wound healing, tissue engineering, cancer treatment, controlled drug release and targeted drug delivery. State-of-the-art hyaluronan production techniques include bacterial fermentation to produce long hyaluronan polymers with a small chain length distribution and in vitro enzymatic systems to produce hyaluronan oligosaccharides of one chain length. Both production strategies make use of hyaluronan synthase (HAS), an enzyme that elongates UDP-glucuronic acid (UDP-GlcUA) and UDP-N-acetylglucosamine (UDP-GlcNAc) into hyaluronan.
The main question in hyaluronan production today is how the chain length of the products can be controlled. Since most production processes use hyaluronan synthases, the aim of this thesis was to elucidate the polymerization mechanism of Pasteurella multocida hyaluronan synthase (PmHAS) from a biochemical point of view. In addition, the acquired knowledge is used for improving the control on hyaluronan chain length in polymerization reactions using PmHAS. Valuable information important for production processes on the intrinsic properties of the enzyme, such as substrate affinity, can be obtained by kinetic studies using single-step elongations. Kinetic studies also provide insights on how polymerization is achieved and, combined with structural studies, the identification of amino acid residues that are important for polymerization. This knowledge can be used for improving the hyaluronan synthesis performance of the enzyme.
Kinetic studies require purified substrates in quantities of mg-scale. Hyaluronan (HA) oligosaccharides were obtained through stepwise hyaluronan cleavage using hyaluronidase and consecutive separation of the reaction mixture by flash-chromatography (Chapter 2). The enzymatic hydrolysis was optimized by experimental design studies with pH, enzyme concentration and reaction time as parameters. Empirical models were developed for the yield of each individual target HA oligosaccharide using the results from a central composite design. Selective production of short HA oligomers (HA ≤ 10) or longer oligosaccharides (HA > 10) was made possible through implementation of the reaction conditions indicated by the empirical models. Separated HA oligomers were characterized by a combination of anion exchange chromatography and matrix-assisted laser desorption/ionization mass spectrometry with time-off-flight analysis. Using these techniques, the desired quantities of purified target HA oligosaccharides (n = 4, 6, 8 and 10) were obtained and used in further studies.
Besides the single-step elongations assessed in kinetic studies, full polymerization studies with both UDP-sugars available were used to investigate the influence of substrate concentrations on the chain length distribution of the hyaluronan products. In order to quantify all oligosaccharides formed during PmHAS polymerization in μl-scale reactions, HA templates consisting of a fluorophore-labeled HA tetrasaccharide (HA4) were generated (Chapter 3). A fast, simple and sensitive assay was developed based on fluorophore-assisted carbohydrate electrophoresis (FACE) that was used for quantification and characterization of PmHAS polymerization products.
The individual β1,3-glucuronyl-transferase (UA-transferase) and β1,4-N-acetylglucosamine-transferase (NAc-transferase) activities of PmHAS were investigated separately using kinetic studies, where the reaction of an HA oligosaccharide was followed with, respectively, UDP-GlcUA or UDP-GlcNAc in single-step elongations. In Chapter 4, the influence of HA oligosaccharide length (n = 4, 5, 6, 7, 8 and 9) on the polymerization reaction was investigated by one-substrate kinetics, varying only the HA oligosaccharide concentration at saturating UDP-sugar concentration. These reactions followed Michaelis Menten kinetics, although HA oligosaccharides may become inhibiting at elevated concentrations above 6 mM. The observed kcat values increased with increasing HA oligosaccharide length to a constant value at HA6 and HA7. The specificity constant kcat/Km values for HA oligosaccharides in the UA-transferase domain increased at increasing oligosaccharide length, whereas in the NAc-transferase domain kcat/Km values were constant at a low value. This indicates that there are two separate oligosaccharide binding sites of different lengths, one in each transferase domain of PmHAS. In Chapter 4, it was demonstrated that the chain-lenght distribution in PmHAS polymerization reactions can be decreased, and thus improved, by using saturating concentrations of both HA oligosaccharides and UDP-sugars.
Chapter 5 describes two-substrate kinetic studies, where in single-step elongations both HA oligosaccharide and one of the UDP-sugars were varied, to investigate the polymerization mechanism of each individual transferase domain in PmHAS. Dead-end inhibition studies and goodness-of-fit parameters were used to distinguish between two-substrate models. From this analysis follows that both transferase domains elongate the UDP-sugar through a sequential mechanism, which is most likely an ordered one. In this proposed mechanism, the UDP-sugar is first bound followed by binding of the HA oligosaccharide, after which first the elongated HA oligosaccharide and then UDP is released. Large differences between Km values for UDP-GlcNAc and UDP-GlcUA, also found in Class I HAS enzymes, suggest that UDP-GlcNAc concentration is involved in the regulation of HAS activity and thus the chain length of hyaluronan products.
Structural studies were used to evaluate the results obtained with kinetic studies. In Chapter 4, a structural homology model of PmHAS was built based on crystal structure K4CP chondroitin polymerase in E. coli, which has a high sequence identity of 62% and high sequence homology of 78% with PmHAS. The active sites of PmHAS are structurally related to other glycosyltransferases and this provided information on where the oligosaccharide binding sites could be located. These putative oligosaccharide binding sites differ in size, as was predicted by kinetic studies (Chapter 4). Furthermore, structural similarities between PmHAS, α1,3-galactosyltransferase (α3GT) and β1,4-galactosyltransferase (β4Gal-T1) demonstrated that PmHAS contains in each transferase domain one flexible loop that forms a bridge over the active site. In crystal structures of α3GT and β4Gal-T1, these flexible loops have been shown to change conformation upon binding the UDP-sugar. Based on similarities in kinetic mechanisms and structures between PmHAS, α3GT and β4Gal-T1, it is likely that the flexible loops in PmHAS follow a similar conformational change, which makes the proposed ordered mechanism the only possible mechanism (Chapter 5).
In Chapter 6, the knowledge on the PmHAS polymerization mechanism gained in earlier chapters is reviewed and used to create new insights in the polymerization mechanism of Class I HAS enzymes. Both Class I HASs and PmHAS are used in hyaluronan production, and, therefore, the differences and similarities are discussed in Chapter 6. During hyaluronan production, there are many different aspects, such as intrinsic properties of the enzyme, cell metabolism and fermentation reaction conditions, that influence hyaluronan chain length and yield (Chapter 6). Moreover, hyaluronan production systems that are able to produce hyaluronan of desired length are discussed in Chapter 6 and a personal view of how these systems can be improved is presented.
Synthetic ganglioside analogues for sensitive biosensing : improved probes for antibodies and bacterial toxins
Pukin, A.V. - \ 2010
Wageningen University. Promotor(en): Han Zuilhof. - [S.l. : S.n. - ISBN 9789085856054 - 131
gangliosiden - derivaten - biosensoren - synthetische materialen - gangliosides - derivatives - biosensors - synthetic materials
This thesis describes the synthesis of analogues of human gangliosides and applications thereof for the detection and inhibition of bacterial toxins and antibodies. An efficient glycosylation method was developed for the synthesis of ω-functionalized alkyl lactosides (Chapter 2). These lactosides were further used as starting compounds in chemo-enzymatic syntheses of analogues of human gangliosides GM3, GM2, GM1, GD1a and GalNAc-GD1a (Chapters 3 and 4). In addition, divalent, tetravalent and octavalent GM2 and GM1 gangliosides were obtained (Chapter 5). A complete NMR characterization of the synthesized compounds was performed (Chapter 3).
The potential of these ganglioside mimics to detect toxins and antibodies was shown in a variety of diagnostic tests:
- in inhibition studies of the synthesized oligosaccharide-linked dendrimers with the cholera toxin B-subunit (Chapter 5) unprecedentedly large multivalency effects were observed: the tetra- and octa- GM1-substituted dendrimers are, respectively, 80 000 and 380 000 times stronger than a monovalent GM1 derivative as binding ligands for the toxin;
- in various modified ELISAs the synthetic ganglioside analogues were used to modify the plates (Chapters 3, 4 and 6) and thus tested for their performance in the detection of the cholera toxin B-subunit and the B subunit of E. coli heat-labile enterotoxin;
- for the detection of IgG and IgM antibodies in serum samples from neuropathy patients by the covalently attached ganglioside analogues a proof of principle was demonstrated (Chapter 6).
Multifunctional starch derivatives: synthesis, characterization and properties
Huijbrechts, A.M.L. - \ 2008
Wageningen University. Promotor(en): Ernst Sudhölter, co-promotor(en): Carmen Boeriu; Maurice Franssen. - [S.l.] : S.n. - ISBN 9789085852506 - 135
maïs - zetmeel - derivaten - synthese - fysicochemische eigenschappen - biodegradatie - maize - starch - derivatives - synthesis - physicochemical properties - biodegradation
The number of food poisoning cases caused by enteropathogens has increased in recent years. A significant part of the outbreaks associated with the consumption of raw vegetables has been attributed to Escherichia coli O157:H7 and Salmonella enterica subsp. enterica serovar Typhimurium. Bovine manure and slurry are the main environmental sources of these pathogens. Thus, reduction of the multiplication of E. coli O157:H7 and Salmonella serovar Typhimurium in cattle and their survival in manure and slurry are important tasks to minimize the risks of contamination of plant products and outbreaks of food-borne diseases. This thesis describes the influence of various environmental factors on survival of E. coli O157:H7 and Salmonella serovar Typhimurium in manure, slurry and soil amended with manure or slurry. Manure or slurry were inoculated with green fluorescent protein transformed strains of both enteropathogens at 106 - 107 cells g-1 dry weight, and their survival was studied in these substrates and in soil amended with inoculated manure or slurry. Population densities of the pathogens and autochthonous microbial communities were determined by dilution plating. The obtained survival data were fitted to non-linear models such as modified logistic or Weibull models, and estimated survival times in various substrates were compared. Analysis of the estimated parameter values showed that the pathogens survived longer at relatively low temperatures under anaerobic conditions especially at high concentrations of easily available substrate. Salmonella serovar Typhimurium was more resistant to environmental stresses than E. coli O157:H7. Survival of both pathogens significantly declined with increasing temperature amplitudes of daily temperature oscillations. Variations in fluctuations of E. coli O157:H7 populations around the decline curve were evaluated by the Approximate Entropy (ApEn) procedure. The instability of E. coli O157:H7 populations around the decline curve was greater in conventional than in organic and in loamy than in sandy soils, even though the mean survival periods did not differ. Multiple regression analysis of instability of E. coli O157:H7 survival on various soil characteristics showed a positive relation with the ratio of copiotrophic / oligotrophic bacteria, suggesting greater instability at higher available substrate concentrations. Percolation experiments with soil columns showed that surface application of solid manure decreased the risk of contamination of ground water and lettuce roots compared to injection of slurry, as more pathogen cells percolated to greater depths after slurry than after manure application. Detection of E. coli O157:H7 could be improved by incubation of Petri plates in anaerobic conditions, as this resulted in significantly higher numbers of recovered cells in comparison with the common aerobic plating procedure. Finally, a simulation model was developed based on our experimental data. The relative effects of temperature and substrate content were more important than that of oxygen concentration. The interaction with substrate resulted in oscillatory behavior of E. coli O157:H7 populations in manure and manure amended soil. Competition for substrate was the most important factor affecting the final survival time. The model was used to evaluate the effects of various manure and soil management scenarios on the survival of E. coli O157:H7. This simulation model provides a new approach to investigate dynamic changes of invasive microorganisms in natural substrates. The results presented in this thesis can be used for risk assessment of E. coli O157:H7 and Salmonella serovar Typhimurium in dairy farming systems and will help to identify and evaluate potential control strategies to minimize the chance of pathogen spread in the vegetable production chain.
Customised commodity derivates; the case of natural gas in the Dutch horticulture sector
Horsager, K. ; Baltussen, W.H.M. ; Backus, G.B.C. - \ 2006
Den Haag : LEI (Report / LEI : Domain 2, Business development and competitive position ) - ISBN 9789086150885 - 43
agrarische economie - banken - industrie - financiële instellingen - landbouwprijzen - risico - risicovermindering - financiën - derivaten - nederland - agricultural economics - banks - industry - financial institutions - agricultural prices - risk - risk reduction - finance - derivatives - netherlands
Financial banks in the Netherlands are developing customised derivatives for the agriculture industry to decrease the volatility of input or output prices. These derivatives can also be attractive for Dutch agriculture producers because a big part of the business risk in agriculture is caused by fluctuating commodity input and output prices. This report provides insight on how customised derivatives can be constructed and what the advantage is for a farmer. This insight is given by a simulation of the natural gas market for horticulture producers in the Netherlands
Bioavailability of genistein and its glycoside genistin
Steensma, A. - \ 2006
Wageningen University. Promotor(en): Ivonne Rietjens, co-promotor(en): H.P.J.M. Noteborn. - [S.l.] : S.n. - ISBN 9789085044031 - 161
genisteïne - derivaten - glycosiden - biologische beschikbaarheid - darmslijmvlies - cellen - ratten - genistein - derivatives - glycosides - bioavailability - intestinal mucosa - cells - rats
Genistein belongs to the class of isoflavones. The main sources of isofiavones in food are soybeans and soy-based products. Most isoflavones in plants are bound to sugars such as the glycosides genistin and daidzin. Understanding the various factors that influence absorption and metabolism of isoflavones and especially of their naturally occurring glycosides, is essential to fully elucidate and understand the physiological activity of these dietary ingredients in the human body. It is also essential to fully explore the possible beneficial health effects of the isoflavones, which are considered to be promising as healthy constituents in enriched (novel) foods or supplements. The aim of this thesis is to obtain data on the transport (uptake) and metabolism of the isoflavones genistein and daidzein and their glycosides genistin and diadzin. Special focus is directed to the possible role of SGLTl in the mechanism involved in the transport and absorption of the isoflavonic glycoside genistin. Another objective is to explore, develop, validate and optimise in vitro, in situ and in vivo gastrointestinal model systems for studying the bioavailability and metabolism of genistein, daidzein and their glycosides.
This thesis reveals that in all intestinal models studied genistein is more bioavailable than its glycoside genistin. The major route of absorption of genistin appeared to be deglycosylation of genistin to genistein and the subsequent intestinal metabolism of genistein to genistein glucuronides and sulfates. Mechanistic studies show that the bioavailability of genistin could be improved by the naturally occurring compound phloridzin, present in apples, because phloridzin inhibits the efflux-transporters located at the brush border (apical) membrane of the enterocyte. On the basis the results described in this thesis, it is concluded that the best gastrointestinal model system appears to be freely moving unanaesthetized cannulated rats. However, animal experiments are expensive and time-consuming and should be reduced, refined or replaced whenever feasible also for ethical reasons. The Caco-2 cells provide a good alternative for studying the mechanism of isoflavones absorption, although the hydrolase activity in this cell line should be improved to mimic the human situation to a better extent.
All together the results presented in this thesis provides an insight in the mechanisms underlying the bioavailability of this important class of health beneficial soy based food ingredients and even point at inhibition of apical transporters as a way to improve the bioavailability of genistin and genistein.
Groene cellulose : eindrapportage project 0351-04-03-08-004
Molenveld, K. ; Giezen, F.E. ; Litjens, M.J.J. ; Teunissen, W. ; Bezemer, R.C. - \ 2005
onbekend : Agrotechnology & Food Sciences Group (Rapport Agrotechnology & Food Innovations 404) - ISBN 9789067549110 - 44
cellulose - derivaten - verwerking - kooldioxide - oplosmiddelen - weekmakers - fysicochemische eigenschappen - markten - cellulose - derivatives - processing - carbon dioxide - solvents - plasticizers - physicochemical properties - markets
Groene cellulose : tussenrapportage project 0351-04-03-08-004
Molenveld, K. ; Giezen, F.E. ; Litjens, M.J.J. ; Teunissen, W. - \ 2004
onbekend : Agrotechnology & Food Sciences Group (Rapport / Wageningen UR, Agrotechnology & Food Innovations 264) - ISBN 9789067548328 - 23
cellulose - derivaten - verwerking - kooldioxide - oplosmiddelen - weekmakers - fysicochemische eigenschappen - markten - biobased economy - cellulose - derivatives - processing - carbon dioxide - solvents - plasticizers - physicochemical properties - markets - biobased economy
This intermediate report describes the progress in the Green cellulose project. The literature studies are in progress and a start has been made with the market study. Furthermore, two experimental trails have been performed. The literature report contains general information on cellulose derivatives, supercritical carbon dioxide processing using supercritical carbon dioxide. The market overview gives insight in market volume, the most important products and producers of cellulose derivatives. So far, the experiments have not been successful since no change in crystallinity was observed and severe cellulose degradation occurred. However, this could indicate that using supercritical carbon dioxide might be used to modify solely the amorphous regions. In future experiments reaction times will be lowered and co-solvents will be added.
Survey of existing measured concentration data for 1,1-dichloroethene, 2,4-dichlorophenol and monochloromethane in water, sediment and aquatic biota
Leonards, P.E.G. ; Leslie, H.A. - \ 2004
onbekend : RIVO Milieu en Voedselveiligheid (Report / RIVO-Netherlands Institute for Fisheries Research C087/04) - 9
ethyleendichloride - 2,4-dichloorfenol - methaan - derivaten - aquatisch milieu - risicoschatting - analytische scheikunde - ethylene dichloride - 2,4-dichlorophenol - methane - derivatives - aquatic environment - risk assessment - analytical chemistry
Adducten van nitrofuranen: metabolisme, uitscheidingskinetiek en analytiek
Zuidema, T. - \ 2003
Wageningen : RIKILT (Rapport / RIKILT 2003.022) - 56
nitrofuranen - derivaten - vleeskuikens - blootstelling - metabolisme - furazolidon - in vitro - heterocyclische verbindingen - analytische scheikunde - nitrofurans - derivatives - broilers - exposure - metabolism - furazolidone - in vitro - heterocyclic compounds - analytical chemistry
Zorg over nieuw type contaminant: vlamvertragers in Nederlandse voeding
Boer, J. de; Leonards, P.E.G. ; Baumann, B. - \ 2003
Voeding Nu 5 (2003)10. - ISSN 1389-7608 - p. 21 - 23.
elektronica - elektrische apparatuur - kunststoffen - vuurvertragingsmiddelen - derivaten - biodegradatie - milieueffect - voedselketens - voedingsmiddelen - toxische stoffen - volksgezondheid - electronics - electrical equipment - plastics - fire retardants - derivatives - biodegradation - environmental impact - food chains - foods - toxic substances - public health
Gebromeerde vlamvertragers vinden toepassing in elektronische apparatuur zoals computers en teevee's en in tal van andere materialen. De productie van decabroomdifenylether (deca BDE) en hexabroomcyclododecaan (HBCD) neemt in Europa toe. Deze stoffen worden aangetroffen in een aantal Nederlandse voedingsmiddelen. De effecten op de volksgezondheid moeten nader worden vastgesteld. De moeilijke afbreekbaaarheid maakt het wenselijk om alternatieve vlamvertragers te ontwikkelen
Chemoenzymatic synthesis of enantiopure 1,4-dihydropyridine derivatives
Sobolev, A. - \ 2003
Wageningen University. Promotor(en): Aede de Groot, co-promotor(en): Maurice Franssen. - [S.I.] : S.n. - ISBN 9789058088222 - 160
synthese - enzymactiviteit - enantiomeren - pyridines - derivaten - synthesis - enzyme activity - enantiomers - pyridines - derivatives
Chirality is important for the activity of many biologically active compounds, since differences in biological properties of stereoisomers occur frequently. The exact stereochemical composition of each new chiral compound as well as toxicological and pharmacological data for racemic and enantiomerically pure compounds are required for their approval as new chiral medicines in the EU.
The research described in this thesis deals with the chemoenzymatic synthesis of 1,4-dihydropyridine derivatives (1,4-DHPs) in enantiopure form as the key intermediates for chiral analogues of symmetrical biologically active compounds. The use of enzymes is an advantageous alternative to classical chemical methods, as enzymes are efficient catalysts with high chemo-, regio- and stereoselectivity under mild conditions.
In Chapter 1, a literature review is given about the synthesis of 1,4-dihydropyridine derivatives, and their biological activities. Synthesis of 1,4-DHPs by cyclocondensation reactions and reduction of pyridines are described whereby special attention is paid to stereoselective chemical and biotechnological methods for the synthesis of enantiopure 1,4-DHPs.
Derivatives of bis(ethoxycarbonylmethyl) 1,4-dihydropyridine-3,5-dicarboxylates have shown antimetastatic activities as well as activities against the Herpes simplex virus. The first objective of the current research is the enzyme-catalysed hydrolysis of these compounds as described in Chapter 2.
Prochiral bis(ethoxycarbonylmethyl) substituted 4-aryl-1,4-dihydropyridine-3,5-dicarboxylates 1a-f are hydrolysed enantioselectively by Candida antarctica lipase B (Novozym 435 ®) (Scheme 1). The enantiomeric excesses range from 68 to 97%, depending on the substituent at position 4. In some cases, the e.e. can be significantly increased by changing the solvent system.
Cerebrocrast (2,6-dimethyl-3,5-bis[2-(propoxy)ethoxycarbonyl]-4-[2-(difluoromethoxy)phenyl]-1,4-dihydropyridine) is a highly active neuroprotector. This compound has been found active in the treatment of diabetes and various inflammatory disorders. Chapter 3 is devoted to the synthesis of chiral analogues of cerebrocrast in enantiopure form via enzyme-catalysed kinetic resolution of 2,6-dimethyl-4-[2-(difluoromethoxy)phenyl]-1,4-dihydropyridine 3,5-diesters.
Alkyl esters at the 3- and 5-positions of 2,6-dimethyl-4-aryl-1,4-dihydropyridine-3,5-dicarboxylates are not hydrolysed by commercially available hydrolases, and 4-substituted bis(ethoxycarbonylmethyl) 1,4-dihydropyridine-3,5-dicarboxylates can be cleaved by lipases only at the 'outer' ester group. Therefore, derivatives have been prepared which contain a spacer that spontaneously detaches after enzymatic hydrolysis of the 'outer' ester group (Chapter 3). Seven acyloxymethyl esters of 5-methyl- and 5-(2-propoxyethyl) 4-[2-(difluoromethoxy)phenyl]-2,6-dimethyl-1,4-dihydro-3,5-pyridinedicarboxylate 3 have been synthesised and subjected to Candida rugosa lipase (CRL) catalysed hydrolysis in wet diisopropyl ether (Scheme 2). A methyl ester at the 5-position and a long or branched acyl chain at C(3) give the highest enantiomeric ratio ( E value). The most stereoselective reaction ( E =21) is obtained with 3-[(isobutyryloxy)methyl] 5-methyl 4-(2-difluoromethoxyphenyl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate 3 , and this compound is used to prepare both enantiomers of 3-methyl 5-(2-propoxyethyl) 4-[2-(difluoromethoxy)phenyl]-2,6-dimethyl-1,4-dihydro-3,5-pyridinedicarboxylate 5 .
The absolute configuration of the enzymatically-produced carboxylic acid has been established to be 4 R by X-ray crystallographic analysis of its 1-( R )-phenylethyl amide.
In Chapter 4, an efficient chemoenzymatic synthesis of (—)-( R )- 5 is described (Scheme 3). The key step in this approach is the asymmetrisation of a symmetrical bifunctional substrate. The enantioselectivity of Candida rugosa lipase-mediated asymmetrisation of the prochiral bis[(isobutyryloxy)methyl] 4-[2-(difluoromethoxy)phenyl]-1,4-dihydro-2,6-dimethyl-3,5-pyridinedicarboxylate is excellent (≥99%).
The disodium salt of 2-(2,6-dimethyl-3,5-diethoxycarbonyl-1,4-dihydropyridine-4-carboxamido)-glutaric acid (glutapyrone) possesses an unusually broad spectrum of biological activities at low concentrations such as neuromodulatory and neuroregulatory action. It is an anticonvulsant, stress-protective, antiarrhythmic, cognition and memory enhancing compound.
3,4,5-Trialkyl 2,6-dimethyl-1,4-dihydro-3,4,5-pyridinetricarboxylates like glutapyrone are inert to the attack of hydrolytic enzymes. The exchange of at least one alkyl group to an enzymatically labile moiety ( e.g. acyloxymethyl or ethoxycarbonylmethyl) turns these 1,4-dihydroisonicotinic acid derivatives into substrates for hydrolytic enzymes. Since acyloxymethyl and ethoxycarbonylmethyl derivatives of 2,6-dimethyl-4-aryl-1,4-dihydropyridine-3,5-dicarboxylates have been recognised as being susceptible to lipases (see Chapters 2-4), the corresponding derivatives of 1,4-dihydroisonicotinic acid 8 and 10 have been prepared (see Scheme 4). The lipase-catalysed kinetic resolution of five derivatives of 4-[(acyloxy)methyl] and 4-ethoxycarbonylmethyl 3-methyl 5-propyl 2,6-dimethyl-1,4-dihydro-3,4,5-pyridinetricarboxylates 8 and 10 has been investigated, and the results are described in Chapter 5. Whereas the enantioselectivity of lipases towards the acyloxymethyl derivatives 8 is rather low, the Candida antarctica lipase B (Novozym 435 ®) catalysed hydrolysis of the ethoxycarbonylmethyl ester of 1,4-dihydroisonicotinic acid 10 is enantioselective. In water-saturated diisopropyl ether at 45°C the enantioselectivity of Novozym 435 ®toward the ethoxycarbonylmethyl ester 10 is rather moderate ( E =13.8), but it is enhanced at rt and +4°C ( E =21.5 and E =28.9, respectively). A high enantiomeric ratio ( E =45.3) is reached at subzero temperatures, although at the expense of the reaction rate.
Polycyclic 1,4-DHPs in enantiopure form are desired for extended pharmacological studies, since racemic 1,4-dihydrobenzothieno[3,2- b ]pyridine-5,5-dioxides and 5-oxo-4,5-dihydro-1,4-indeno[1,2- b ]pyridines possess various biological activities such as coronary dilating and anticancer; they have also been found active as glutathione S-transferase inhibitors.
The lipase-catalysed kinetic resolution of enzymatically labile 3-(isobutyryloxy)methyl 4-[2-(difluoromethoxy)phenyl]-2-methyl-5,5-dioxo-1,4-dihydrobenzothieno[3,2- b ]pyridine-3-carboxylate rac -12 is described in Chapter 6 (Scheme 5). The most enantioselective reaction ( E =28) is a CRL-mediated transesterification with n -butanol in water-saturated toluene, at 45°C.
The main results of this study are evaluated in Chapter 7. The approaches to overcome inactivity of hydrolytic enzymes toward simple esters of 1,4-DHPs, with their advantages, disadvantages and limitations, are discussed. The perspectives of the applications of chemoenzymatic approaches to the synthesis of enantiopure pharmacologically important novel dihydropyridine derivatives are also outlined.
It can be concluded that the use of hydrolytic enzymes, acting on hydrolysable groups on spacers, is a useful and widely applicable method for the enantioselective synthesis of hydrogenated pyridines.
Aggregation behavior of cholic acid derivatives in organic solvents and in water
Willemen, H.M. - \ 2002
Wageningen University. Promotor(en): E.J.R. Sudhölter; A.T.M. Marcelis. - S.l. : S.n. - ISBN 9789058087140 - 112
cholinezuur - derivaten - oplosmiddelen - gelering - micellen - fysische toestand - cholic acid - derivatives - solvents - gelation - micelles - physical state
In this thesis various cholic acid derivatives are reported that display aggregation in water or in organic solvents. Spontaneous aggregation of single molecules into larger, ordered structures occurs at the borderline of solubility. Amphiphilic compounds, or surfactants, which possess a hydrophobic as well as a hydrophilic part, have a high tendency to form aggregates to minimize unfavorable polar-apolar interactions with the surrounding solvent. There are different types of aggregates in water as well as in organic solvents. Examples of aggregates in water are micelles and vesicles. The apolar tails of the surfactants are located in the interior of these aggregates, shielded from water, and the polar head groups are located on the surface. The shape of the surfactant determines for a large part the shape of the aggregate. An example of an aggregate in organic solvents is the organogel. Fibers can be formed by means of various interactions between the single organogelator molecules and these fibers then build a network. A small amount of organogelator is sufficient to increase the viscosity of the solvent.
Cholic acid is a main bile acid, a special class of micelle-forming biosurfactants. It has a carboxylate group as an ionic head group attached to a steroid unit via a small spacer. The steroid unit is facial amphiphilic due to the presence of three hydroxyl groups, which are all located on one side. Cholic acid can be easily derivatized via the carboxylate group and hydroxyl groups. In this thesis numerous newly synthesized cholic acid derivatives are reported of which the aggregation behavior in solution has been studied. Special emphasis is on the variation in molecular structure and its influence on aggregation properties.
A large number of cholic acid derivatives with either an amino acid ester group or a long alkyl group attached to the carboxylic acid group via an amide bond display organogel formation in various organic solvents, mostly aromatic solvents. Both types of compounds form thermoreversible gels that are completely transparent and stable for prolonged periods of time. The sensitivity of the gels toward water suggests the construction of a hydrogen bonded gel network. Molecular variation and infrared spectroscopy confirm this and show that both the amide bond and the hydroxyl groups are involved. Electron microscopy shows the presence of thin fibers. From SANS measurements the diameter of the fibers was established to be around 20 to 40 Å, so the fibers are monomolecular in thickness. Further details about the fibrous shape and network structure were also gained by SANS. Derivatives with an alkyl tail or an amino acid butyl ester form flat fibers that are connected via loose entanglements. Derivatives with an amino acid methyl ester group form round fibers that are connected via crystalline knots of about 100 to 200 Å. This last type of gel network breaks down stepwise upon heating, which could be monitored with DSC.
Small variations in the molecular structure of these organogelators could induce a change in the gelation behavior. Introduction of an aromatic unit in the alkyl tail limits the number of solvents, suited for gelation. Reversing the amide bond increases the melting point of the gels. The stereochemistry of the amino acid unit seems to play a role in some compounds. After substitution of the amide group by a urea group a longer alkyl tail is necessary for gelation, whereas after substitution by an ester group no gelation occurs in aromatic solvents. These alkyl ester derivatives, however, are capable of forming a two-component gel in hexane and octane, in the presence of isomannide or isosorbide. The optimal ratio for the two components is 1:1.
Hydrolysis of the alkyl ester group of several cholyl amino acid ester derivatives creates a series of compounds analogous to the natural bile salt glycocholate. These water-soluble compounds form small micelles of about 5 nm. Only a small variation in the cmc values was found for the various compounds, although the amino acid side groups range from a single hydrogen atom to an aromatic unit or an ionic group. The cmc value of the tyrosine derivative undergoes a small increase at high pH.
Another series of micelle forming cholic acid derivatives was prepared in which the amphiphilicity of cholic acid was completely altered. A long alkyl tail was attached to the carboxylic acid group and ionic groups were attached to the hydroxyl groups via alkyl spacers, resulting in compounds with three ionic head groups. These are the first facial amphiphiles based on cholic acid with three permanent ionic groups. These compounds form micelles in water and the size of the micelles is dependent of the alkyl tail length. Increasing the length of the spacer or of the alkyl tail causes a decrease in cmc, although the decrease is not as large as found for other surfactants. The thermodynamics of micellization was studied using ITC and the temperature-dependent behavior of these compounds is comparable to other surfactants. Some of these compounds have a strong inhibitory effect on the growth of gram-positive and gram-negative bacteria. In their antimicobial activity they are comparable to some other facial amphiphiles with nitrogen-containing groups.
Mestsamenstelling: berekenen of bemonsteren?
Backus, G. ; Adams, J. - \ 1998
Praktijkonderzoek varkenshouderij 12 (1998)3. - ISSN 1382-0346 - p. 21 - 21.
mineralen - boekhouding - varkens - dierlijke meststoffen - drijfmest - stikstof - fosfaten - fosforpentoxide - derivaten - kunstmeststoffen - samenstelling - onderzoek - proeven - minerals - accounting - pigs - animal manures - slurries - nitrogen - phosphates - phosphorus pentoxide - derivatives - fertilizers - composition - research - trials
De afvoer van mineralen in de mest (N en P) kan worden berekend of worden gemeten. In MINAS gaat men alleen uit van gemeten mineralengehaltes in de mest. Het Praktijkonderzoek onderzocht de verschillen tussen berekenen en meten voor de zeugenhouderij
Hoog ureumgehalte soms moeilijk te vermijden
Teenstra, E. ; Boxem, Tj. - \ 1998
Praktijkonderzoek Rundvee, Schapen en Paarden. Praktijkonderzoek 11 (1998)4. - ISSN 1386-8470 - p. 9 - 11.
dierlijke producten - mineraalgehalte - melkvee - melkveehouderij - melkopbrengst - melkkwaliteit - derivaten - ureum - analyse - testen - controle - animal products - mineral content - dairy cattle - dairy farming - milk yield - milk quality - derivatives - urea - analysis - testing - control
Het ureumgehalte is een indicator voor de stikstofbenutting van het melkvee. Dit artikel beschrijft kort wat het ureumgehalte is, wat het zegt en wat je er als veehouder mee kunt.
Fosfaatverliesnormen: nieuw graslandonderzoek van start
Schils, R. ; Middelkoop, J.C. - \ 1998
Praktijkonderzoek Rundvee, Schapen en Paarden. Praktijkonderzoek 11 (1998)3. - ISSN 1386-8470 - p. 35 - 37.
bodem - fosfor - graslanden - uitspoelen - fosfaten - fosforpentoxide - derivaten - soil - phosphorus - grasslands - leaching - phosphates - phosphorus pentoxide - derivatives
In 1997 is door het PR, in samenwerking met het Nutriënten Management Instituut (NMI), het Staring Centrum (SC-DLO) en het Instituut voor Agrobiologisch en Bodemvruchtbaarheidsonderzoek (AB-DLO), onderzoek gestart naar de gevolgen van verschillende fosfaatverliesnormen op graslandproductie en fosfaatverliezen. Dit artikel beschrijft de opzet van het onderzoek en behandelt enkele resultaten na een jaar onderzoek
Leaching of nitrogen and phosphorus from rural areas to surface waters in the Netherlands
Boogaard, H.L. ; Kroes, J.G. - \ 1998
Nutrient Cycling in Agroecosystems 50 (1998)1/3. - ISSN 1385-1314 - p. 321 - 324.
bodem - uitspoelen - nitraten - fosfaten - fosforpentoxide - derivaten - rivieren - waterlopen - kanalen - water - oppervlaktewater - waterverontreiniging - waterkwaliteit - modellen - onderzoek - soil - leaching - nitrates - phosphates - phosphorus pentoxide - derivatives - rivers - streams - canals - water - surface water - water pollution - water quality - models - research
|Agricultural nutrient losses to surface water in the Netherlands: impact, strategies, and perspecties
Molen, D.T. van der; Breeuwsma, A. ; Boers, P.C.M. - \ 1998
Journal of Environmental Quality 27 (1998)1. - ISSN 0047-2425 - p. 4 - 11.
bodem - uitspoelen - fosfaten - fosforpentoxide - derivaten - dierlijke meststoffen - nederland - soil - leaching - phosphates - phosphorus pentoxide - derivatives - animal manures - netherlands