# Staff Publications

## Staff Publications

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'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

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 Photosynthetic efficiency in microalgal lipid productionRemmers, Ilse M. - \ 2017Wageningen University. Promotor(en): R.H. Wijffels, co-promotor(en): P.P. Lamers. - Wageningen : Wageningen University - ISBN 9789463434607 - 200 algae - biofuels - light - triacylglycerols - lipids - metabolism - algae culture - cultural methods - algen - biobrandstoffen - licht - triacylglycerolen - lipiden - metabolisme - algenteelt - cultuurmethoden Microalgae can contain large amounts of lipids which make them a promising feedstock for sustainable production of food, feed, fuels and chemicals. Various studies, including pilot-scale, have been performed and the knowledge on microalgal processes has advanced quickly. Unfortunately, current production costs for cultivation are still too high for bulk lipid production from microalgae. One of the major causes for the high costs of bulk lipid production is the reduced solar-to-lipid conversion efficiency. Current research, however, does not provide sufficient insight to identify optimization targets. Therefore, in this thesis we have studied the lipid production in microalgae in depth. Different TAG-accumulation strategies were investigated from a process engineering and metabolic point of view. The combination of all findings were used in the general discussion to thoroughly evaluate the microalgal lipid accumulation strategies. Current phototrophic microalgal lipid yields are still 10 times lower than the theoretical maximum. There is, however, still an enormous potential for further improvements. Future research should focus on (genetically) improved strains and advanced cultivation strategies, including adaptation to fluctuating outdoor weather conditions. This thesis was performed within the EU FP7 FUEL4ME project under grand agreement No 308938. Objective of this program is to develop a sustainable and scalable process for biofuels from microalgae and to valorize the by-products. In vivo 1H NMR methods to study dynamics of chloroplast water and thylakoid membrane lipids in leaves and in photosynthetic microorganismsPagadala, Shanthi - \ 2017Wageningen University. Promotor(en): H. van Amerongen, co-promotor(en): H. van As. - Wageningen : Wageningen University - ISBN 9789463431569 - 130 cell membranes - membranes - chloroplasts - thylakoids - photosynthesis - in vivo experimentation - stress conditions - stress - proteins - lipids - mobility - dynamics - celmembranen - membranen - chloroplasten - thylakoïden - fotosynthese - in vivo experimenten - stress omstandigheden - stress - eiwitten - lipiden - mobiliteit - dynamica Dynamics of thylakoid membranes and mobility of pigment-protein complexes therein are essential for survival of photosynthetic organisms under changing environmental conditions. The published approaches to probe mobility of the thylakoid membrane lipids and protein complexes are either dependent on the use of external labels or are used only for in vitro studies. Here, we present non-invasive 1H NMR methods (DOSY and DRCOSY) to study dynamics of water in chloroplasts, lipids in oil bodies and in thylakoid membranes and pigment-protein complexes under complete in vivo conditions in leaf disks of F. benjamina and A. platanoides and in suspensions of the green alga Chlamydomonas reinhardtii and blue-green alga Synechocystissp.PCC 6803. In leaf disks of Ficus benjamina and Acer platanoides, water in chloroplasts could be clearly discriminated from other pools. Both water in chloroplasts, and water in vacuoles of palisade and spongy cells showed resonances in the high field part of the spectra (with respect to pure water), in contrast to what has been reported in literature. Subepidermal cells (present only in F. benjamina but not in A. platanoides) may act as a water storage, buffer pool during drought. This pool prevented the fast loss of water from the chloroplasts. Nutrient stress and excess salt stress resulted in accumulated lipid bodies and in striking differences in the dynamics and spectra/composition of the different components. T2 values of the different components are compared with those observed in suspensions of Synechocystissp.PCC 6803. The differences in membrane composition (ratio of the different membrane lipids) were clearly observed in the DANS of the oil bodies and the (thylakoid) membranes, but the diffusion coefficients were quite comparable. Also the DANS of the component that is assigned to the pigment-protein complexes are quite different, reflecting the differed composition. The diffusion coefficients of this component in isolated spinach thylakoids and in C. reinhardtii are very comparable, but about a factor of 10 lower with respect to that of Synechocystis at short diffusion times. The dynamics of these complexes in these systems are thus quite different. The fatter the better : selecting microalgae cells for outdoor lipid productionDominguez Teles, I. - \ 2016Wageningen University. Promotor(en): Rene Wijffels, co-promotor(en): Maria Barbosa; Dorinde Kleinegris. - Wageningen : Wageningen University - ISBN 9789462578821 - 164 algae - chlorococcum - lipids - lipogenesis - fat - production - phenotypes - inoculum - diameter - cells - sorting - algen - chlorococcum - lipiden - lipogenese - vet - productie - fenotypen - entstof - diameter - cellen - sorteren In chapter 1 we introduce microalgae, photosynthetic microorganisms with potential to replace commodities (such as food, feed, chemicals and fuels). Production costs are still high, reason why microalgae are still only economically feasible for niche markets. We suggest to borrow the concept of plant domestication to select industrial microalgae cells. Two approaches can be successfully used to domesticate microalgae: adaptive laboratory evolution (ALE) and fluorescence assisted cell sorting (FACS). ALE takes advantage of the natural adaptability of microorganisms to different environments, while FACS actually select cells with specific phenotypes. This thesis aimed to select cells of Chlorococcum littorale with improved phenotypes, assuming that these cells could establish new populations with increased industrial performance. In Chapter 2 we wanted to know what happened during time to biomass and lipid productivities of Chlorococcum littorale repeatedly subjected to N-starvation. We tested 2 different cycles of N-starvation, short (6 days) and long (12 days). Short cycles didn’t affect lipid productivity, highlighting the potential of C. littorale to be produced in semi-continuous cultivation. Repeated cycles of N-starvation could have caused adaptations of the strain. Hence, we also discussed the implications of using repeated N-starvation for adaptive laboratory evolution (ALE) experiments. Chapter 3 introduces a method to detect and to select microalgae cells with increased lipid content. The method requires only the fluorescence dye Bodipy505/515 dissolved in ethanol, and the method was designed to maintain cellular viability so the cells could be used to produce new inoculum. In chapter 4 we evaluated a question that emerged while deciding which criteria to use to sort lipid-rich cells: does cellular size affects lipid productivity of C. littorale? We hypothesized that cells with different diameters have different division rates, which could affect lipid productivity. Therefore, we assessed the influence of cell diameter, as a sorting parameter, on both biomass and lipid productivity of Chlorococcum littorale (comparing populations before and after sorting, based on different diameters). Results showed that the size of vegetative cells doesn’t affect the lipid productivity of C. littorale. In chapter 5 we present a strategy to sort cells of C. littorale with increased TAG productivity using the method developed at chapter 3. Both the original and the sorted population with the highest lipid productivity (namely, S5) were compared under simulated Dutch summer conditions. The results confirmed our data from experiments done under continuous light: S5 showed a double TAG productivity. Our results showed also that the selected phenotype was stable (1.5 year after sorting) and with potential to be used under industrial conditions. In chapter 6 we extrapolated our results (indoor and outdoor) to other climate conditions. We ran simulations changing the light conditions to four different locations worldwide (the Netherlands, Norway, Brazil and Spain) to estimate both biomass and TAG productivities. Results indicated that biomass yields were reduced at locations with higher light intensities (Brazil/Spain) when compared with locations with lower light intensities (Norway/Netherlands). Hence, the choice of location should not be based on light intensity, but on how stable irradiation is. Chapter 7 is the general discussion of the thesis, demonstrating that both ALE and FACS are effective approaches to select industrial microalgae cells. We also present our view on how ALE and FACS could further improve microalgae strains for industry. Lipid bilayer stability in relation to oxide nanoparticlesPera, H. - \ 2015Wageningen University. Promotor(en): Frans Leermakers, co-promotor(en): Mieke Kleijn. - Wageningen : Wageningen University - ISBN 9789462574670 - 144 lipids - membranes - stability - nanotechnology - particles - analytical methods - models - modeling - lipiden - membranen - stabiliteit - nanotechnologie - deeltjes - analytische methoden - modellen - modelleren Lipid bilayer stability in relation to oxide nanoparticles All living organisms are composed of cells that are filled with a thick molecular soup. These molecules constitute a complex machinery that brings these cells to life. To contain these molecules, and to protect them from the hostile outer environment, a phospholipid bilayer envelopes the cell. It is essential that this lipid bilayer, also known as the cell membrane, should remain intact and form a perfect barrier at all times. Industrially manufactured nanoparticles are suspect to be able to penetrate this barrier, and thus endanger living organisms in the environment. This thesis deals with some aspects of the structural integrity of lipid bilayers, and especially how this integrity is affected by the interaction with nanoparticles. Experiments were performed with silica and titanium dioxide nanoparticles, interacting with lipid bilayers, using a variety of experimental techniques. In addition, a theoretical model was applied that is based on the Scheutjens-Fleer Self Consistent Field (SCF) theory. This model delivered detailed structural and thermodynamic information about the lipid bilayer. The modelling work helped us to improve our understanding of lipid bilayer stability, and showed the effect of the interaction with the nanoparticles on the phospholipid bilayer. These latter results could be related directly to our experiments. Let us first experimentally regard the interaction of lipid bilayers with synthetic oxide nanoparticles. We developed a protocol for high-throughput screening of the nanoparticle-bilayer interaction using a fluorescence technique. Results from this method were combined with reflectometry measurements and atomic force microscopy (AFM). The combination of these methods allowed us to relate lipid bilayer integrity to its interaction with nanoparticles and their adsorption onto the bilayer. In addition, the AFM results yielded detailed structural information at the nano-scale. We found that the interaction strongly depends on both lipid bilayer and nanoparticle charge. However, the specific interaction that depends on the nanoparticle type, starts to play a role when the charges are low. When the total interaction strength is regarded, a regime was found at which interaction is strong enough for the nanoparticles to adsorb onto the bilayer, but too weak to disrupt the bilayer. If, however, the bilayer is disrupted by the nanoparticles, the particle may steal away some lipid molecules from the bilayer, and leave again to disrupt the bilayer elsewhere. Let us now go into more detail on the SCF modelling. Bilayers are composed of phospholipids, which consist of a hydrophilic head group, and a hydrophobic tail. These bilayers were modelled using a single lipid molecule type, of which the head group structure and lipid tail length could be varied. We thus obtained bilayers that varied in their thickness, and the space that a single lipid takes within the bilayer. Changes in bilayer composition affect the bilayer mechanical properties, such as those constants that describe bilayer stretching or bending. This thesis shows how vesicles, which are bilayers in a globular shape, may become unstable if the bilayer lipid composition is changed. Under certain conditions, a vesicle would prefer to fall apart into many smaller vesicles, which is when highly charged head groups start to repel each other. Or the bilayer may form continuous cubic phases, which might occur if lipids with uncharged head groups but with very long tails are used to form the bilayer. Under very specific and finely tuned conditions, a lipid bilayer may become unstable to form stable pores in the membrane, or to fall apart into tiny lipid discs. Effect of dietary protein on lipid and glucose metabolism: implications for metabolic healthRietman, A. - \ 2015Wageningen University. Promotor(en): Frans Kok; D. Tomé, co-promotor(en): Marco Mensink. - Wageningen : Wageningen University - ISBN 9789462573482 - 160 voeding en gezondheid - stofwisselingsstoornissen - eiwitinname - dieet - metabolisme - lipiden - glucose - macronutriënten - nutrition and health - metabolic disorders - protein intake - diet - metabolism - lipids - glucose - macronutrients Abstract Background: Diet is an important factor in the development of the Metabolic Syndrome (Mets) and type 2 Diabetes Mellitus. Accumulation of intra hepatic lipid (IHL) can result in non-alcoholic fatty liver disease (NAFLD), which is sometimes considered the hepatic manifestation of Mets. Manipulation of the dietary macronutrient composition – altering either fat or simple carbohydrates – has the potential to change lipid storage in the liver. Protein also has this ability, however human data is scarce. Moreover, high dietary protein intake is linked with an increased type 2 Diabetes risk. Therefore, it is essential to study the metabolic consequences of changes in macronutrient composition focussing on altering dietary protein quantity. Objective: In this thesis the effects of dietary protein on metabolic health focusing on lipid and glucose metabolism were investigated in both observational studies as well as in a human dietary intervention trial. Methods: In an observational study (n=1283), Fatty Liver Index (FLI) was calculated and related to macronutrient consumption from dietary assessment data. In a controlled dietary intervention, participants (n=27) were assigned to either a control-diet for 4 weeks, or a high-fat, hypercaloric diet, with either a high-protein or a normal-protein content for two weeks, and vice versa. Measurements of IHL (1H-MRS) and blood plasma glucose and lipid concentrations were performed, both in the fasting state and following a meal. Results: In the observational study, the prevalence of fatty liver as indicated by an FLI>60, was 22.0%. Compared to persons with a normal FLI score of <30, protein intake was positively related with high FLI score >60 (OR: 1.26 per 1 en%, 95%CI 1.16-1.37). This was in particular the case for protein intake from animal sources. In the dietary intervention study, the high-protein diet compared to the normal-protein diet resulted in lower IHL and plasma TG concentrations (IHL: 0.35 ± 0.04 % vs. 0.51 ± 0.08 %; p=0.08; TG: 0.65 ± 0.03 vs. 0.77 ± 0.05 mmol/L; p=0.07). Furthermore, after the meal challenge the free fatty acids (FFA) response was significant different between all three intervention diets (p=0.03). Moreover, the postprandial glucose response was significantly lower after adaptation to NP compared with HP (p=0.03), without differences in the postprandial insulin responses (p=0.37). Conclusions: From data of the intervention study and observational studies reported in this thesis, it can be concluded that dietary protein intake is associated with alterations in metabolic profile, with both favourable and potential unfavorable health outcomes. On the short term increasing dietary protein in healthy subjects improved lipid metabolism, as seen by lower TG and IHL levels, but not glucose metabolism. On the long term, however, a high-protein intake was related to a fatty liver, and associated to insulin resistance. Lipoxygenase : a game-changing enzymeHeshof, R. - \ 2015Wageningen University. Promotor(en): Willem de Vos; Vitor Martins dos Santos, co-promotor(en): Leo de Graaff. - Wageningen : Wageningen University - ISBN 9789462571761 - 160 lipoxygenase - aminozuursequenties - lipiden - schimmels - bio-informatica - kaliumjodide - industriële toepassingen - biobased economy - lipoxygenase - amino acid sequences - lipids - fungi - bioinformatics - potassium iodide - industrial applications - biobased economy Many challenges lie ahead in using LOXs as tools in industrial oleochemistry. One of these challenges is the supply of PUFAs. Although we are moving towards a biobased economy where second and third generation biomass is taking a leading role, it is still faster and cheaper to use first generation biomass. Industrialization of microbial oils is a good alternative to supply the demand of PUFAs. Another challenge is the production of heterologous LOX in sufficient quantities. Since the last decade this problem is being tackled and more research is being done in heterologous expression of LOXs. The LOX with the highest potential so far is the secreted Pseudomonas aeruginosa LOX produced in Escherichia coli. During this thesis research different lox genes were tried for heterologous production of LOX using different Aspergillus niger and Aspergillus nidulans strains as expression hosts. These LOXs were identified as discussed in Chapter 3 and Chapter 6. Unfortunately, heterologous production in sufficient quantities was unsuccessful using these expression hosts as discussed in Chapter 5 and Chapter 6. Since production of Gaeumannomyces graminis LOX was successful in Trichoderma reesei, as discussed in Chapter 4, the production of polymers used for bioplastics could be demonstrated in this ERA-NOEL project anyway. Therefore this thesis shifted its focus on resolving the question of the difficulties in the heterologous expression of LOX in different Aspergillus species. Chapter 5 is the result of a systematic approach to analyze different aspects of G. graminis LOX expression in A. nidulans. Chapter 2 shows that heterologous expression of extracellular fungal LOX can be performed using T. reesei and Pichia pastoris as production hosts, and E. coli can be used for the production of intracellular LOXs of plant, mammal, bacterial, and fungal origin. As shown in Chapter 2, E. coli is not very efficient in the production of heterologous LOX due to the formation of inclusion bodies and low induction temperature necessary for production. The use of Aspergillus oryzae can be exploited further in the heterologous production of LOXs. Due to the choice of using A. niger and A. nidulans as expression hosts, this expression host was not exploited for its potential. The last challenge is to synthetically engineer LOX to broaden its use in industry. In this way more building blocks for chemicals can be synthetically produced and more products based on LOX origin can be made. Therefore, LOX can be a world-wide game-changing enzyme in a biobased economy as its use can decrease the demand for petroleum-based products. Production of fungal lipids : kinetic modeling and process designMeeuwse, P. - \ 2011Wageningen University. Promotor(en): Hans Tramper, co-promotor(en): Arjen Rinzema. - [S.l.] : S.n. - ISBN 9789461730930 - 239 mortierella - lipiden - bioproceskunde - afvalhergebruik - biodiesel - mortierella - lipids - bioprocess engineering - waste utilization - biodiesel Finding alternatives for fossil fuels is currently urgent. One of the new processes in this field is the production of biodiesel from lipids accumulated by microorganisms. Some yeasts and fungi accumulate lipids when a component needed for growth, usually the N-source, is limiting while the C-source is in excess. These oleaginous yeasts and fungi were previously mainly used for unsaturated fatty acid production, but now also come into view for production of lipids as a source of biodiesel. This thesis takes the first steps in the development of a new process to produce lipids with an oleaginous fungus in solid-state fermentation on agro-industrial waste. Solid-state fermentation is the cultivation on solid substrate particles without (free) flowing water, and has several advantages over submerged fermentation such as less waste water production, less energy use for oxygen transfer and lower production costs. In this thesis, we focused on growth and lipid production kinetics in submerged as well as solid-state fermentation. The models developed for these systems provide insight in the lipid production mechanism, needed to develop the new process based on solid-state fermentation. The thesis starts with the selection of a model strain (Chapter 2). With this strain, the kinetics of growth and lipid accumulation were studied and modeled. We started with a steady-state model (Chapter 3 and 4) in submerged chemostat culture, and extended this to a dynamic model for submerged batch culture (Chapter 5). As the next step towards solid-state fermentation, we developed a model for growth and lipid accumulation on κ-carrageenan plates with monomers (Chapter 6). These three models were finally used to calculate potential lipid yield and energy use in a biodiesel production system (Chapter 7). For the system we want to develop, we need a fungus that can utilize different substrates and can produce lipids. For this purpose, we tested two oleaginous fungi: Mortierella alpina and Umbelopsis isabellina, which is described in Chapter 2. We cultivated both fungi on agar plates containing glucose, xylose, starch, cellulose or pectin, and on sugar beet pulp in a packed bed. M. alpina did not utilize xylose, cellulose and pectin, utilized starch much slower than glucose and only consumed approximately 40% of the sugar beet pulp in 20 days. This shows that M. alpina is not a suitable organism for our production system. U. isabellina utilized pectin and xylose with the same rate as glucose, but used starch slower and (crystalline) cellulose not at all. It consumed approximately 75% of the sugar beet pulp after 8 days and approximately 100% after 20 days. Also, it accumulated some lipids (3% of remaining dry mass) in the culture on sugar beet pulp; optimization of this process by addition of enzymes increased the lipid content to 9% of remaining dry mass. This shows that U. isabellina is a promising strain for lipid production from agro-industrial waste, and is therefore a good strain to use in our research. The lipid concentrations found in SSF culture were quite low; we therefore decided to look in more depth into the kinetics of lipid production in different model systems. The first model system was a submerged chemostat culture, because the substrate supply rates can be varied in this system by varying the dilution rate as well as the concentrations in the feed. Chapter 3 describes the development of a mathematical model that includes growth, lipid accumulation and substrate consumption of oleaginous fungi in submerged chemostat cultures. Key points of the model are: (1) If the C-source supply rate is limited, maintenance has a higher priority than growth, which has a higher priority than lipid production; (2) the maximum specific lipid production rate of the fungus is independent of the actual specific growth rate. This model was validated with chemostat cultures of U. isabellina grown on mineral media with glucose and NH4+. Because of practical problems at low dilution rates, the model could only be validated for D>0.04 h‑1. For further validation, published data sets for chemostat cultures of oleaginous yeasts and a published data set for a poly-hydroxyalkanoate accumulating bacterial species were used, which is described in Chapter 4. All data sets could be described well by the model. Analysis of all data showed that the maximum specific lipid production rate is in most cases very close to the specific production rate of membrane and other functional lipids for cells growing at their maximum specific growth rate. The limiting factor suggested by Ykema et al.(1986, Antonie van Leeuwenhoek 52: 491-506), i.e. the maximum glucose uptake rate, did not give good predictions of the maximum lipid production rate. The model shows that both the C/N-ratio of the feed as well as the dilution rate has a large influence on the lipid production rate. When these data are translated to SSF, it means that a low substrate supply rate can prevent lipid production, even when the C/N-ratio of the substrate is high. The next step towards understanding lipid accumulation was a model that also describes changes in time. Therefore, we developed a model for growth, lipid production and lipid turnover in submerged batch fermentation, which is shown in Chapter 5. This model describes three subsequent phases: exponential growth when both a C-source and an N-source are available, carbohydrate and lipid production when the N-source is exhausted, and turnover of accumulated lipids when the C-source is exhausted. The model was validated with submerged batch cultures of U. isabellina with two different initial C/N-ratios. In batch culture, the specific lipid production rate was almost four times higher than in chemostat cultures and it decreased exponentially in time. This indicates that different mechanisms for lipid production are active in batch and chemostat cultures. The model could also describe several data sets from literature very well. Furthermore, the model shows that local limitation of C-source in SSF can cause lipid turnover before the average C-source concentration in the substrate is zero. The next step towards an SSF system is the inclusion of diffusion in the batch model. We did this by developing a model that describes growth, lipid production and lipid turnover in a culture on κ-carrageenan plates containing the monomers glucose and alanine as C-source and N-source, respectively. This is described in Chapter 6. The model includes reaction kinetics and diffusion of glucose, alanine and oxygen. It was validated with U. isabellina and describes the different phases of the culture very well: exponential growth, linear growth because of oxygen limitation, accumulation of lipids and carbohydrates after local N-depletion and turnover of lipids after local C-depletion. Extending the model with an unidentified extracellular product improved the fit of the model to the data. The model shows that oxygen limitation is extremely important in solid-state cultures using monomers. Together with the low specific lipid production rate found in SSF, it explains the difference in production rate with submerged cultures. In Chapter 7, we used the models from Chapter 3, 5 and 6 together with basic engineering principles to calculate lipid yield and energy use in the modeled systems. We evaluated a process including pretreatment, cultivation and down-stream processing with sugar beet pulp and wheat straw as substrate, described different reactor types, and considered both a yeast and a fungus as microorganisms. According to the models, lipid yields on substrate were between 5% w/w and 19% w/w, depending on the culture system. With the same models, improvement of the yield to 25-30% w/w was shown to be possible, for example by genetic modification of the microorganism. The net energy ratio of the non-optimized systems varied between 0.8 and 2.5 MJ produced per MJ used; energy use for pretreatment and for oxygen transfer were most important. For the optimized systems, the net energy ratio increased to 2.9 – 5.5 MJ produced per MJ used, which can compete very well with other biofuels such as bioethanol or algal biodiesel. So although there is still quite some work to be done, microbial lipids have the potential to be tomorrow’s source of biodiesel. Mitofood conference : bioactive food components, energy metabolism and human health, 13-15 April 2011, Wageningen, The Netherlands : celebrating 90 years of human and animal physiology at Wageningen University : final conference of COST Action FA0602: bioactive food components, mitochondrial function and healthKeijer, J. - \ 2011[Wageningen] : Wageningen UR - 87 voedselsamenstelling - bioactieve verbindingen - voedingsstoffen - energiemetabolisme - gezondheid - polyfenolen - lipiden - vitaminen - hormonen - verouderen - bio-energetica - mitochondria - voeding - food composition - bioactive compounds - nutrients - energy metabolism - health - polyphenols - lipids - vitamins - hormones - aging - bioenergetics - nutrition more info ... The role of lipids in the global organization of thylakoid membranes of higher plantsKrumova, S.K.B. - \ 2009Wageningen University. Promotor(en): Herbert van Amerongen, co-promotor(en): G. Garab. - [S.l.] : S.n. - ISBN 9789085852926 - 132 planten - thylakoïden - membranen - lipiden - chloroplasten - fotosynthese - plants - thylakoids - membranes - lipids - chloroplasts - photosynthesis The first steps of photosynthesis (capturing of light and conversion of light energy into chemical energy) occur in the thylakoid membrane of the chloroplasts. It consists for 75% of membrane proteins and 25% of lipid molecules. In this thesis the global properties of the lipids in the thylakoid membrane are studied with a variety of spectroscopic techniques. Special attention is paid to the lipid packing, phase behavior and membrane permeability, as well as the role of the lipids in structural rearrangements and the overall organization of the membrane. The presented results reveal heterogeneity in the packing of the bulk lipid molecules and the participation of the lipids in both bilayer and non-bilayer structures. It is demonstrated that the lipid mixture has an active role in the lateral arrangement of the protein complexes, the formation of macrodomains and their thermal stability and indirectly in the excitation energy trapping by the photosynthetic complexes. This work provides a basis for further future investigations of the properties of the thylakoid lipid matrix and of the lipid-protein interactions in thylakoid membranes and their relevance for different functions of the membrane. Probing the role of PPAR alpha in the small intestine : a functional nutrigenomics approachBünger, M. - \ 2008Wageningen University. Promotor(en): Michael Muller, co-promotor(en): Guido Hooiveld. - [S.l.] : S.n. - ISBN 9789085049739 - 166 dunne darm - voedingsfysiologie - transcriptiefactoren - lipiden - lipidenabsorptie - nutrigenomica - small intestine - nutrition physiology - transcription factors - lipids - lipid absorption - nutrigenomics Background The peroxisome proliferator-activated receptor alpha (PPARα) is a ligand-activated transcription factor known for its control of metabolism in response to diet. Although functionally best characterized in liver, PPARα is also abundantly expressed in small intestine, the organ by which nutrients, including lipids, enter the body. Dietary fatty acids, formed during the digestion of triacylglycerols, are able to profoundly influence gene expression by activating PPARα. Since the average Western diet contains a high amount of PPARα ligands, knowledge on the regulatory and physiological role of PPARα in the small intestine is of particular interest. Aim In this thesis the function of PPARα in the small intestine was studied using a combination of functional genomics experiments, advanced bioinformatics tools, and dietary intervention studies. Results Detailed analyses on the expression of PPARα in small intestine showed that PPARα is most prominently expressed in villus cells of the jejunum, coinciding with the main anatomical location where fatty acids are digested and absorbed. Genome-wide transcriptome analysis in combination with feeding studies using the synthetic agonist WY14643 and several nutritional PPARα agonists revealed that PPARα controls processes ranging from fatty acid oxidation and cholesterol-, glucose- and bile acid metabolism to apoptosis and cell cycle. In addition, we connected PPARα with the intestinal immune system. In a more focussed study we showed that PPARα controls the barrier function of the intestine. By comparing the intestinal and hepatic PPARα transcriptome we found that PPARα controls in these two organs the expression of two distinct, but overlapping sets of genes. Finally, by performing a range of functional studies deduced from the transcriptome analysis, we demonstrated that PPARα controls intestinal lipid absorption. Conclusion By maximally utilizing the unique possibilities offered in the post-genome era, the studies described in this thesis reported on the function of PPARα in small intestine. We conclude that intestinal PPARα plays an important role, is relevant for nutrition, and its effects are distinguishable from the hepatic PPARα response. Our results provide a better understanding of normal intestinal physiology, and may be of particular importance for the development of fortified foods, and prevention and therapies for treating obesity and inflammatory bowel diseases. Glucogeen rantsoen helpt koe : verlagen melkvet via glucogeen rantsoen zorgt voor verbetering energiebalansKnegsel, A.T.M. van; Dijkstra, J. ; Brand, H. van den - \ 2007Veeteelt 24 (2007)11. - ISSN 0168-7565 - p. 12 - 14. melkkoeien - melkproductie - melkkwaliteit - rundveevoeding - lactatie - voedingsrantsoenen - energiebalans - energetische waarde - zetmeelwaarde - lipiden - zetmeelvertering - dairy cows - milk production - milk quality - cattle feeding - lactation - feed rations - energy balance - energy value - starch equivalent - lipids - starch digestion Elke koe kampt aan het begin van de lactatie met een negatieve energiebalans. Aandacht voor de juiste vorm van energie in het rantsoen is geen overbodige luxe, blijkt uit onderzoek. Een glucogeen rantsoen heeft de voorkeur boven een lipogeen rantsoen Lipid bilayers and interfacesKik, R.A. - \ 2007Wageningen University. Promotor(en): Martien Cohen Stuart; Frans Leermakers, co-promotor(en): Mieke Kleijn. - [S.l.] : S.n. - ISBN 9789085048374 - 169 elektrische dubbellaag - lipiden - grensvlak - oppervlakte-eiwitten - ionensterkte-effecten - electrical double layer - lipids - interface - surface proteins - ion strength effects In biological systems lipid bilayers are subject to many different interactions with other entities. These can range from proteins that are attached to the hydrophilic region of the bilayer or transmembrane proteins that interact with the hydrophobic region of the lipid bilayer. Interaction between two membranes is also very common. To gain more insight into the thermodynamic, structural and mechanical consequences we experimentally and theoretically investigated the interactions of a lipid bilayer with various types of interfaces. More specifically, we have analysed the transmembrane protein-lipid interaction by a computational self-consistent field method and have studied the adhesion of vesicles onto gold experimentally. Some aspects of the latter problem were also analysed theoretically. There exists a computationally inexpensive, yet qualitatively accurate and realistic method to molecularly model the bilayer membrane in the presence of surfaces, namely the self-consistent field theory. This approach makes use of a large number of approximations. Important ones are: the discretisation of space by using a lattice, the non-self-avoidance of chains implying freely jointed chains and the replacement of binary interactions by an external potential leading to the (local) mean field ansatz. When a transmembrane hydrophobic inclusion is present in the lipid membrane the bilayer around it is disturbed. The structural perturbation of the lipid bilayer around these inclusions have an exponentially decaying wave-like appearance. There are many factors that influence this. The most important ones are the shape of the inclusion, the hydrophobic length of the inclusion, the local interaction between the inclusion and the bilayer, the hydrophobic bilayer thickness and the mechanical characteristics of the lipid bilayer. At distances larger than the bilayer thickness the wavelength and the decaylength of this exponentially decaying wave are exclusively determined by the mechanical and structural properties of the bilayer. This means that the wavelength and the decaylength can be described by the thickness, the bending modulus and the area compression-expansion modulus of the bilayer. The amplitude and the offset of the perturbation are on the other hand set by the properties of the inclusion. Indeed, the hydrophobic length mismatch, i.e., the difference between the hydrophobic length of the inclusion and the hydrophobic thickness of the lipid bilayer, and the contact interaction between the inclusion and the lipid bilayer are the key variables. The free energy of insertion is mainly determined by the contact interaction energy between the inclusion and the lipid bilayer and it shows a parabolic dependence on the hydrophobic length mismatch. The free energy of insertion is minimal at a hydrophobic length mismatch where the bilayer perturbations are minimal. We argued that there is a subtle interplay between the entropy loss of the lipid tails adjacent to the surface and the contact interaction between the inclusion and the lipid tails. Important for the biological performance, we found that overlap of the perturbed regions of the bilayer around two or more inclusions can cause attractive or repulsive interaction between such inclusions depending on the distance between them. This non-monotonic interaction force with the distance between inclusions, is directely linked to the non-monotonic structural perturbations mentioned already. This lipid mediated free energy of interaction between the inclusions can be divided into three different regimes each with their own length scale. These are the short-range segmental, the intermediate-range conformational and the long-range elastic contributions. The short-range contribution is only present when one or two lipids are in between the inclusions. This interaction depends strongly on the Flory-Huggins interaction between the inclusion and the lipid tails. The intermediate-range contribution is present at separations on the length scale of approximately the bilayer thickness. This interaction shows an exponentially decaying dependence on the separation between these inclusions and is a consequence of the confinement of the lipid tails in between these inclusions. The long-range contribution is determined by the elastic properties of the bilayer and has an exponentially decaying wave-like appearance, with a wavelength that is the same as the perturbation wave length of the bilayer. Our SCF analysis complements available simulations on the one hand and mesoscopic models on the other. Moreover, they may help to analyse experiments and explain observations in biomembranes. In the second part of this thesis we examined the adhesion of negatively charged DOPG vesicles and zwitterionic DOPC vesicles to a gold surface using quartz crystal microbalance and surface plasmon resonance techniques. Gold has a hydrophilic surface where lipid vesicles adsorb intact. When the vesicle radius was above approximately $40$ nm the DOPC vesicles completely cover the surface, whereas below this radius the surface coverage decreases with decreasing vesicles size. When spherical vesicles adsorb onto a surface they deform. The shape deformation of the adsorbed vesicles increases with increasing vesicles size. The diminished deformation for the smaller vesicles results in a relatively small interaction area between the vesicles and the gold surface resulting in less lipid-surface interactions. Self-consistent field model calculations on a single vesicle are in line with these experimental results. The calculations showed that the relative deformation of the vesicles has a linear dependence on the vesicles radius. They furthermore showed that below a certain minimal vesicle radius the deformation is completely absent resulting in a lipid-surface interaction energy that vanishes. Self-consistent field calculations further indicate that the lipid-surface interaction can be divided into three different regimes. In the weak interaction regime the adhesion of the vesicles is not accompanied by drastic changes in the bilayer structure and the vesicle is deformed elastically. In this case the adhesion of the vesicles is energetically favourable over the adhesion of an equally sized bilayer patch. The adhesion of lipid vesicles to the gold surface can most likely be categorised in this regime. In the second intermediate interaction regime the adsorbed vesicles are energetically unfavourable compared to equally sized bilayer patches. The deformation of these vesicles remain in the elastic regime and therefore they do not transform into an adsorbed lipid bilayer patch. In the strong interaction regime the adsorption of the vesicles is strongly energetically unfavourable compared to equally sized bilayer patches and this interaction is so strong that local molecular rearrangements take place to increase the bilayer curvature. This results in adsorbed vesicles that are very susceptible to fusion and/or rupture. An interesting prediction is that the adsorption energy of a vesicle does not depend on the bilayer rigidity. This means that the adsorption energy is a constant, and fixed by the interaction energy between the lipid molecules and the surface. At the same time the deformations of the vesicles increase with diminishing rigidity, which means that although the interaction is the same, the vesicles with different rigidity can be present in different interaction regimes. As already mentioned, lipid vesicles adsorb intact onto a gold surface. However, on many other surfaces lipid vesicles transform after adsorption into a supported lipid bilayer. We studied the importance of electrostatic interactions for the adhesion strength of DOPC and DOPG vesicles to a gold surface. This was done by varying the pH, the ionic strength and an externally applied electrostatic potential. Varying the pH of the solution has an effect on the protonation of the oxide groups present at the gold surface. As a consequence the surface charge ranges from a positive charge below pH$=5$ to negative charge above pH$=5$. In the case of negatively charged DOPG vesicles, we showed that there is a relation between the adsorbed amount and the pH. The adsorbed amount was larger at low pH compared to high pH and remained approximately constant in the pH range $6-10$. There is still some adsorption in this pH range, from which it can be concluded that besides the electrostatic interaction also other interactions, such as the van der Waals or other chemical interactions, play a role. The ionic strength has a rather strong influence on the adhesion of DOPG vesicles, while the adsorbed amount of DOPC vesicles remains approximately constant. Both experiments and self-consistent field modelling showed that the adsorbed amount decreases with decreasing ionic strength. This relation can be attributed to the fact that the headgroup density of the DOPG vesicle decreases with decreasing ionic strength, which results in less favourable non-electrostatic lipid-surface interactions. The externally applied potential had no effect on the adsorption DOPG vesicles. This can be attributed to the fact that externally applied potential can only be varied over a limited range, because otherwise redox reactions reaction at the gold surface start to play a role. This means that the surface potential range is too small to influence the interaction energy of the DOPG and the DOPC bilayer. With self-consistent field modelling it was shown that if redox reaction did not occur and the externally applied potential could be varied over a larger range, the interaction energy between the lipid bilayer and the gold surface could be divided into four different regimes. These regimes vary from weakly attractive to strongly attractive. It can be concluded that the adhesion of DOPG vesicles onto gold is parly determined by electrostatic interactions. Because the vesicles are weakly bound to the gold surface, the electrostatic interaction can influence the adsorption of intact vesicles. However they are never strong enough to induce transition of the adsorbed vesicles to a flat supported bilayer. In the case of DOPC vesicles the electrostatic interactions have a negligible effect The organisation of proteins in lipid membranes is identified as one of the central issues in molecular biology. We have tried to unravel the role of the lipid matrix in the protein insertion problem. Our results may be important, for example in the case of transmembrane proteins with multiple transmembrane $\\alpha$-helices, because the short-range lipid-mediated interactions of these transmembrane helices can directly influence the quaternary structure of these proteins. Besides generic issues discussed in the present thesis there are numerous molecular specific aspects. These problems will undoubtelly attract many scientific activities in the years to come. Lipid vesicles at surfaces attracted a lot of attention in the last ten years. Vesicles adhesion is used frequently to generate supported lipid bilayers. Such interfacial layers gives the opportunity to study the properties and interactions of these lipid bilayers and use these layers in biotechnological applications. We tried to unravel some details of the interactions of lipid layers with a gold surface. Our results may be used to understand why in some cases supported bilayers are formed while in other vesicles stay intact at the surface. Understanding this will give us the opportunity to control the fusion of lipid vesicles on a surface. Fusion of vesicles in a plane is also an issue in biological processes such as the formation of the cell plate in plant cell division. Fecundity in relation to lipid content in North Sea herringSchouten, M. - \ 2007IJmuiden : IMARES (Report / IMARES 07.012) - 37 p. haringen - vruchtbaarheid - eierproductie - lipidenmetabolisme - lipiden - periode van kuitschieten - vissen - noordzee - herrings - fertility - egg production - lipid metabolism - lipids - spawning season - fishes - north sea Energy partitioning in dairy cows : effects of lipogenic and glucogenic diets on energy balance, metabolites and reproduction variables in early lactationKnegsel, A.T.M. van - \ 2007Wageningen University. Promotor(en): Bas Kemp; Seerp Tamminga, co-promotor(en): Henry van den Brand; Jan Dijkstra. - [S.l.] : S.n. - ISBN 9789085047346 - 166 melkkoeien - energiebalans - diëten - voer - voedingsstoffen - lipiden - glucose - stofwisselingsstoornissen - voortplanting - immuunsysteem - lactatie - dairy cows - energy balance - diets - feeds - nutrients - lipids - glucose - metabolic disorders - reproduction - immune system - lactation Keywords: dairy cows; dietary energy source; glucogenic nutrients; lipogenic nutrients; negative energy balance; metabolic disorders; reproduction, immune system Dairy cows experience a negative energy balance (NEB) in early lactation which results from high energy requirements for milk production accompanied by a limited energy intake. Nutrition has been indicated as an important factor in the incidence and severity of NEB and NEB–related metabolic and reproductive disorders, like ketosis and delayed resumption of ovarian activity. A metabolic effect of a NEB in dairy cows is suggested to be an unbalanced availability from glucogenic and lipogenic nutrients. The objective of this thesis was to study the effect of lipogenic and glucogenic diets on the energy balance (EB) and risk of metabolic and reproductive disorders in dairy cows in early lactation. The first study, a literature survey, illustrated that feeding extra glucogenic nutrients relative to lipogenic nutrients, decreased milk fat and seemed to decrease plasma non-esterified fatty acid (NEFA) and β-hydroxybutyrate (BHBA) concentrations. Since studies were scarce and mostly included a confounding effect of dietary energy source with energy intake, it was difficult to draw conclusions on the energy source effects on EB and fertility. Therefore, in the second study, 16 dairy cows were either fed a glucogenic or a lipogenic diet. Diets were isocaloric and equal in intestional digestible protein. Energy balance was determined in climate-respiration chambers from week 2 until week 9 of lactation. The glucogenic diet decreased milk fat yield and milk energy and tended to decrease body fat mobilisation compared with the lipogenic diet. The objective of the third study was to study the effect of dietary energy source on EB, metabolites and some reproduction variables. Dairy cows (n=111) were fed glucogenic, lipogenic or mixed diet from week -3 until week 9 relative to calving. Multiparous cows fed the glucogenic diet had lower milk fat yield, higher calculated EB, and lower plasma NEFA, BHBA and liver tri-acyl glyceride concentration and tended to have less days postpartum till first ovulation. Additionally, in the third study, the presence of natural antibodies (NAb) in plasma and milk of individual dairy cows was determined. Relations were detected between NAb and EB and plasma metabolites, suggesting that alterations in immune competence peripartum are reflected in the humoral part of the innate immune system. In conclusion, increasing the glucogenic nutrient availability improved the EB and had potential to reduce the risk of metabolic disorders and to improve reproductive performance in dairy cows. Seafood research from fish to dish : quality, safety and processing of wild and farmed fishLuten, J.B. - \ 2006Wageningen : Wageningen Academic Publishers - ISBN 9789086860050 - 567 zeevruchten - visproducten - visverwerking - voedselkwaliteit - voedselveiligheid - lipiden - voedselbewaring - houding van consumenten - methodologie - seafoods - fish products - fish processing - food quality - food safety - lipids - food preservation - consumer attitudes - methodology In this book, scientists from various disciplines address the advances in seafood research with respect to quality, safety, consumer’s demands and processing of wild and farmed fish. The nutritional properties of marine lipids and lipid oxidation from model systems to seafood are presented. Several contributions on the effects of natural anti-oxidants to prevent oxidation are also included. Effects of dietary factors on muscle tissue quality, pre-rigor processing and brining of farmed cod are covered. The development of rigor mortis and the quality of muscle in relation to commercial and experimental slaughter techniques are also discussed. Consumer’s knowledge, perception and need for information about seafood are discussed. Topics such as shelf life and microbial quality of seafood are covered in a range of contributions. Inactivation of micro organisms or biopreservation of seafood are included. Attention is paid to the development of the Quality Index Method for the evaluation of the quality of fresh fish and products. The characterisation and the quality of processed by-products are also presented. The presence of trace elements and organic contaminants in variety of seafood products is highlighted. Finally, several contributions regarding advanced methodologies to determine the quality of seafood are presented. Proceedings of the Frontis workshop on fresh herbage for dairy cattle : the key to a sustainable food chainElgersma, A. ; Dijkstra, J. ; Tamminga, S. - \ 2006Dordrecht : Springer (Wageningen UR Frontis series vol. 18) - ISBN 9781402054518 melkvee - grasbestand - begrazing - soortenrijkdom - lolium perenne - voeropname - voedingsstoffen - voedersupplementen - graslandbeheer - stikstofmetabolisme - lipiden - melkkwaliteit - diervoeding - duurzaamheid (sustainability) - dairy cattle - herbage - grazing - species richness - lolium perenne - feed intake - nutrients - feed supplements - grassland management - nitrogen metabolism - lipids - milk quality - animal nutrition - sustainability Product quality and a sustainable food chain of ruminant products are largely determined by animal nutrition, in which forage is the major feed source. Forages and grasslands play a unique role in agriculture because they contribute through animals to our food supply and to the abatement of environmental problems. Interest in grassland management and grass utilization for dairy production in temperate and subtropical regions has recently led to considerable research efforts. In the past this research often emphasized on plant and animal aspects separately. However, the interrelationship between pasture and the grazing ruminant is a dynamic, two-way process. Many people are not aware of these complex relations, and scientists often focus on either soil-plant interactions, plant production, animal nutrition, animal production or product quality issues. This book contains the contributions of researchers and experts in the field of grassland management and grass utilization. Chapters are arranged in an order that allows progressive development within the food chain, moving from the big picture to basic principles of grassland management and effects on herbage quality, intake and nutrient flows. Elucidating the mechanism behind the lipid-raising effect of cafestolBoekschoten, M.V. - \ 2004Wageningen University. Promotor(en): M.B. Katan, co-promotor(en): Guido Hooiveld. - [S.I.] : S.n. - ISBN 9789085041139 cafestol - koffie - cholesterolmetabolisme - lipiden - hart- en vaatstoornissen - gezondheid - cafestol - coffee - cholesterol metabolism - lipids - cardiovascular disorders - health The objective of this thesis was to identify genes that control the response of serum lipid levels to diet. To this end we used cafestol as model substance for a food component that affects serum lipids and therefore health. Cafestol is a cholesterol‑raising diterpene present in coffee beans and unfiltered coffee types.A possible explanation for the cholesterol-raising effect of cafestol is inhibition of bile acid synthesis. This is observed in APOE3Leiden mice upon treatment with cafestol. The nuclear receptors FXR and PXR are key regulators of genes involved in lipid and bile acid metabolism and detoxification.Both these nuclear receptorscan mediate inhibition of cholesterol 7a‑hydroxylase, the rate-limiting enzyme in bile acid synthesis. Therefore, we hypothesized that cafestol is able to activate FXR and/or PXR.We used promoter-reporter gene assays to show that cafestol interacts with FXR and PXR in vitro .This suggests that cafestol can regulate gene expression via these receptors. Indeed cafestol regulated several mRNA levels of target genes of FXR and PXR in livers of APOE3Leiden mice. For a number of target genes these effects were absent in livers of FXR and PXR knockout mice. This confirms that FXR and PXR are involved in the regulation of gene expression by cafestol. However, we could not confirm suppression of bile acid synthesis in humans. We measured plasma levels of7a‑hydroxy-4-cholesten-3-one, a marker for activity of cholesterol 7a‑hydroxylase, in volunteers that consumed coffee oil. Surprisingly, we observed an increase rather than a decrease in the level of 7a‑hydroxy‑4‑cholesten‑3‑one upon coffee oil treatment. In conclusion, it is likely that the interaction with FXR and PXR is at least partly responsible for the effect of cafestol on serum lipids in humans. However, the exact mechanism by which cafestol raises serum cholesterol remains to be elucidated. Elucidation of this mechanism will provide insight into how dietary components can affect serum lipid levels. Vetoxidatie en het voorspellen van houdbaarheidDekker, M. - \ 2002Voedingsmiddelentechnologie 35 (2002)1-2. - ISSN 0042-7934 - p. 37 - 39. vetten - lipiden - geoxideerde vetten - oxidatie - chemische reacties - houdbaarheid (kwaliteit) - kwaliteit - bewaartijd - opslagkwaliteit - voedingsmiddelen - conferenties - fats - lipids - oxidized fats - oxidation - chemical reactions - keeping quality - quality - storage life - storage quality - foods - conferences Verslag van een symposium als afsluiting van een onderzoeksproject Biological ensilage of fish : optimization of stability, safety and functionalityEnes Dapkevicius, M.L.N. - \ 2002Wageningen University. Promotor(en): F.M. Rombouts; M.J.R. Nout; J.H. Houben. - S.l. : [s.n.] - ISBN 9789058085702 - 170 voedselmicrobiologie - kuilvoerbereiding - micromestistius poutassou - trachurus - scomber scombrus - biogene aminen - lipiden - eiwitafbraak - food microbiology - silage making - micromestistius poutassou - trachurus - scomber scombrus - biogenic amines - lipids - protein degradation This thesis deals with stability, safety, and functionality aspects of biological fish silage (BFS) obtained by lactic acid fermentation. BFS may provide an economically viable, environment friendly way of upgrading fish waste.BFS has been found advantageous when compared to the so-called acid process, since it yielded lower levels of peroxides in the silage oil and led to lower non-protein nitrogen values. Fermentation by lactic acid bacteria (LAB) was shown to efficiently inhibit pathogens such as L. monocytogenes and E. coli O157:H7 as well as some spoilage microorganisms. Histamine degradation by some of the most promising available LAB strains was also tested, as an innovative means of ensuring low levels of this amine in the fermented product. Several potential starter strains were found to degrade histamine as single strain cultures and could find application in fish silage and other fish products in which histamine accumulation might pose a certain risk.Temperatures of 35 - 37°C were regarded as most suitable from the point of view of starter growth and acidification. Strains with a short acidification lag time, such as Lb. plantarum 009, or with very fast growth and good antibacterial properties, such as Lb. curvatus 15.35, are most suitable.Sodium chloride inhibited growth of biogenic amine producing bacteria and additionally decreased non-protein nitrogen and total volatile basic nitrogen values in BFS. Potassium sorbate was shown to inhibit growth and/or acidification by potential starter lactic acid bacteria. The role of sludge retention time in the hydrolysis and acidification of lipids, carbohydrates and proteins during digestion of primary sludge in CSTR systemsMiron, Y. ; Zeeman, G. ; Lier, J.B. van; Lettinga, G. - \ 2000Water Research 34 (2000)5. - ISSN 0043-1354 - p. 1705 - 1713. afvalwaterbehandeling - rioolslib - anaërobe behandeling - koolhydraten - lipiden - eiwitten - verzuring - hydrolyse - waste water treatment - sewage sludge - anaerobic treatment - carbohydrates - lipids - proteins - acidification - hydrolysis CSTR systems: completely stirred tank reactor systems
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