Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

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

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    Assessing methane emission from dairy cows : modeling and experimental approaches on rumen microbial metabolism
    Lingen, Henk J. - \ 2017
    Wageningen University. Promotor(en): W.H. Hendriks, co-promotor(en): J. Dijkstra; A. Bannink; C.M. Plugge. - Wageningen : Wageningen University - ISBN 9789463431590 - 207
    dairy cows - methane - emission - microbial degradation - rumen metabolism - rumen fermentation - models - fatty acids - biochemical pathways - animal nutrition - nutrition physiology - melkkoeien - methaan - emissie - microbiële afbraak - pensmetabolisme - pensfermentatie - modellen - vetzuren - biochemische omzettingen - diervoeding - voedingsfysiologie

    Methane (CH4) is a greenhouse gas (GHG) with a global warming potential of 28 CO2 equivalents. The livestock sector was estimated to emit 7.1 gigatonnes of CO2 equivalents, which is approximately 14.5% of total global anthropogenic GHG emissions. Enteric CH4 production is the main source of GHG emissions from dairy cattle, representing 46% of the global GHG emissions in dairy supply chains. Dairy production has great value in view of the ability of ruminants to effectively turn human inedible biomass into human edible food and to produce food from non-arable land. Consequently, there is an urgent need to develop strategies to decrease dairy cattle enteric CH4 emission. Evaluation of these strategies requires meticulous quantification and increased understanding of anaerobic fermentation and methanogenesis in the rumen ecosystem. The overall aim of this PhD research was, therefore, to quantitatively evaluate enteric CH4 emission from dairy cows as affected by feeding and rumen microbial metabolism.

    A meta-analysis was performed to quantify relationships between enteric CH4 yield (per unit of feed and unit of milk) and milk FA profile in dairy cattle and to develop equations to predict CH4 yield based on milk FA profile of cows fed a wide variety of diets. Various milk FA concentrations were significantly or tended to be positively or negatively related to CH4 yield per unit of feed or milk. Mixed model multiple regression resulted in various milk FA included in optimal equations to predict CH4 yield per unit of feed and per unit of milk. These regression equations indicated a moderate potential for using milk FA profile to predict CH4 yield.

    For the development of a mechanistic model of CH4 production in the rumen, the thermodynamic control of pH2 on reaction rates of specific fermentation pathways, NADH oxidation and methanogenesis was theoretically explored. This control was determined using the thermodynamic potential factor (FT), which is a dimensionless factor that corrects a predicted kinetic reaction rate for the thermodynamic control exerted. The thermodynamic feasibility of these microbial conversions showed that the control of pH2 on individual VFA produced and associated yield of H2 and CH4 cannot be explained without considering NADH oxidation, with a considerable effect of pH.

    For obtaining experimental support of the conclusions drawn from the theoretical exploration, diurnal patterns of gaseous and dissolved metabolite concentrations in the bovine rumen, H2 and CH4 emitted, and the rumen microbiota were monitored. In addition, the effect of dietary inclusion of linseed oil on these patterns was assessed. An in vivo experiment with rumen cannulated dairy cows was performed to study the anaerobic metabolism and the microbiota composition in the rumen. A 100-fold increase in pH2 in the rumen headspace was observed at 0.5 h after feeding, followed by a decline. Qualitatively similar patterns after feeding were observed for H2 and CH4 emission, ethanol and lactate concentrations, and propionate molar proportion, whereas an opposite pattern was seen for acetate molar proportion. Associated with these patterns, a temporal biphasic change in the microbial composition was observed as based on 16S ribosomal RNA with certain taxa specifically associated with each phase. Bacterial concentrations were affected by time and increased by linseed oil supplementation. Archaeal concentrations tended to be affected by time and were not affected by diet, despite linseed oil supplementation tending to decrease the partial pressure and emission of CH4 and tending to increase propionate molar proportion. The various diurnal profiles that were monitored support the key role of the NAD+ to NADH ratio in rumen fermentation and the importance of diurnal dynamics when understanding VFA, H2 and CH4 production.

    A dynamic mechanistic model was developed, in which the thermodynamic control of pH2 on VFA fermentation pathways, and methanogenesis in the bovine rumen are incorporated. The model represents substrate degradation, microbial fermentation and methanogenesis in the rumen, with the type of VFA formed to be controlled by the NAD+ to NADH ratio, which in turn is controlled by pH2. Feed composition and feed intake rate representing a twice daily feeding regime were used as model input. The model predicted a marked peak in pH2 after feeding that rapidly declined in time. This peak in pH2 caused a decrease in NAD+ to NADH ratio followed by an increased propionate molar proportion at the expense of acetate molar proportion. In response to feeding, the model predicted a sudden increase and a steady decrease in CH4 production in time. The pattern of CH4 emission rate followed the patterns of pH2 and H2 emission rate, but its magnitude of increase in response to feeding was less pronounced. A global sensitivity analysis indicated the parameter that determines the NADH oxidation rate to explain the most substantial part of the variation of predicted daily CH4 emission. The modeling effort provides the integration of more detailed knowledge than accomplished in previous rumen fermentation models and enables assessment of diurnal dynamics of rumen metabolic pathways yielding VFA, H2 and CH4.

    For assessing the general value of the research reported in this thesis, the potential for predicting enteric CH4 emission from dairy cattle based on milk FA profile was discussed in the light of published studies and compared with empirical modeling of enteric CH4 based on feed input. Moreover, the concept of NAD-controlled fermentation was considered in a more general perspective by comparing the rumen ecosystem with bioreactor systems. Furthermore, the feasibility of the developed models as an alternative for IPCC tiered approaches was explored. In conclusion, the research reported in this thesis contributes to an increased understanding of rumen fermentation and microbial metabolism, and has provides a basis to further improve prediction models of enteric CH4 emissions from dairy cattle.

    Tospovirus : induction and suppression of RNA silencing
    Hedil, Marcio - \ 2016
    Wageningen University. Promotor(en): Just Vlak, co-promotor(en): Richard Kormelink. - Wageningen : Wageningen University - ISBN 9789462577848 - 137
    tospovirus - rna - plants - immunity - gene silencing - biochemical pathways - rna interference - viral proteins - plant viruses - tospovirus - rna - planten - immuniteit - uitschakelen van genexpressie - biochemische omzettingen - rna-interferentie - viruseiwitten - plantenvirussen

    While infecting their hosts, viruses must deal with host immunity. In plants the antiviral RNA silencing pathway is an important part of plant innate immunity. Tospoviruses are segmented negative-stranded RNA viruses of plants. To counteract the antiviral RNA silencing response in plants, tospoviruses have evolved a silencing suppressor function via its NSs protein. This viral protein has previously been shown to bind dsRNA that likely arises from secondary RNA folding structures in viral RNAs. The aim of the present research was to further investigate the interaction between tospoviruses and the plant antiviral RNA silencing response, including the target sequences in the viral RNA and the further role of the NSs protein as part of the tospovirus counterdefence strategy.

    In order to identify the target and inducer for RNA silencing against tospoviruses, small RNAs purified from plants infected with three tospoviral species, tomato spotted wilt virus (TSWV), groundnut ringspot virus (GRSV) and tomato yellow ring virus (TYRV), were probed against the viral RNA segments of these three different tospoviruses (Chapter 3). Virus-derived siRNAs (vsiRNAs) were found to be derived from all three genomic RNA segments but predominantly the ambisense M and S RNAs. Further profiling on the S RNA sequence revealed that vsiRNAs were found from almost the entire S RNA sequence, except the predicted AU-rich hairpin (HP) structure encoded by the intergenic region (IGR) from where hardly any vsiRNAs were found. Similar profiles were observed with the closely related GRSV as well as the distantly related TYRV. Dicer cleavage assays using Drosophila melanogaster embryo extracts showed that synthetic transcripts of the IGR-HP region were recognized as substrate for Dicer. Transient agroinfiltration assays of a GFP-sensor construct containing the IGR-HP sequence at its 3′-UTR did not show more rapid/strong silencing, and profiling of the corresponding siRNAs generated outside the context of a viral infection still revealed relatively low levels of IGR-HP-derived siRNAs. These data support the idea that the IGR-HP region/structure is a weak inducer of RNA silencing and plays a minor role in the amplification of a strong antiviral RNA silencing response.

    Next, a biochemical analysis was performed using E. coli-expressed and purified NSs from GRSV and TYRV. The binding of both purified NSs proteins to small and long dsRNA indicated that this is likely a generic feature of all tospoviral NSs proteins (Chapter 4). Binding of siRNAs to NSs furthermore revealed two shifts on polyacrylamide gels i.e. a first shift at low NSs concentrations followed by a second larger one at higher concentrations. When the NSs protein of TSWV resistant breaker (RB) isolates (of Tsw-gene based resistance), which lack RSS activity when transiently expressed, were analyzed using extracts from infected plants still a major (second) shift of siRNAs was observed, similar to the case with extracts containing TSWV resistant inducer (RI) isolates. In contrast, plant extracts containing transiently expressed NSs proteins alone (no infection) showed only the smaller, first shift for NSsRI but no shift for NSsRB.

    The ability of NSs to suppress systemic silencing is demonstrated for the NSs proteins of TSWV, GRSV and TYRV, and their relative strengths to suppress local and systemic silencing were compared (Chapter 5). A system was developed to quantify suppression via GFP silencing constructs, allowing comparison of relative RNA silencing suppressor strength. In this case NSs proteins of all three tospoviruses are suppressors of local and systemic silencing. Unexpectedly, suppression of systemic RNA silencing by NSsTYRV was just as strong as those by NSsTSWV and NSsGRSV, even though NSsTYRV was expressed in lower amounts. Moreover, a set of selected NSsTSWV gene constructs mutated in predicted RNA binding domains, as well as NSs from TSWV isolates 160 and 171 (resistance breakers of the Tsw resistance gene), were analyzed for their ability to suppress systemic GFP silencing. The results indicate another mode of RNA silencing suppression by NSs that acts further downstream of the biogenesis of siRNAs and their sequestration.

    In summary, evidence is presented showing that sequences from all three genomic segments from tospovirus are targeted by the plant RNA silencing machinery. The predicted hairpin sequence in the IGR is poorly targeted. Biochemical experiments with purified NSs proteins further support the view that binding to small and long dsRNA is a characteristic common to all tospovirus NSs proteins. Furthermore, tospovirus NSs proteins suppress systemic silencing and there are indications that local and systemic silencing suppression can be uncoupled in NSs. Collectively, these results add to our current understanding of the tospovirus-plant interaction involving antiviral RNA silencing and the viral counter-defence (NSs protein). Lastly, the results of the research presented in this thesis are discussed in light of the current knowledge on RNA silencing and to present some future perspectives and questions that remain open and/or resulted from this thesis (Chapter 6).

    Ecophysiology of novel intestinal butyrate-producing bacteria
    Bui, Thi Phuong Nam - \ 2016
    Wageningen University. Promotor(en): Willem de Vos, co-promotor(en): Caroline Plugge. - Wageningen : Wageningen University - ISBN 9789462577015 - 202
    butyrates - butyric acid bacteria - intestines - microbial interactions - faecal examination - mice - man - infants - genomics - intestinal physiology - microbial physiology - biochemical pathways - lysine - sugar - butyraten - boterzuurbacteriën - darmen - microbiële interacties - fecesonderzoek - muizen - mens - zuigelingen - genomica - darmfysiologie - microbiële fysiologie - biochemische omzettingen - lysine - suiker

    The human intestinal tract harbours a trillion on microbial cells, predominantly anaerobes. The activity and physiology of these anaerobes is strongly associated with health and disease. This association has been investigated for a long time.However, this has not been fully understood. One of the reasons is the limited availability of cultured representatives. It is estimated that there may be more than 3000 species colonised in the gut of healthy individuals, however, only a bit over 1000 species have been isolated and characterised. Among the intestinal microbes, butyrate-producing bacteria are of special interest as the butyrate produced, is crucial to maintain a healthy gut. In addition, butyrate-producing bacteria have shown a reverse correlation with several intestinal diseases. In Chapter 2 we described a novel species Anaerostipes rhamnosivorans 1y2T isolated from an infant stool. This strain belonged to genus Anaerostipes within Clostridium cluster XIVa. A. rhamnosivorans had a capability of converting rhamnose into butyrate that is unique within intestinal butyrate-producing bacteria. The genomic analysis also revealed the entire rhamnose fermentation pathway as well as the acetyl-CoA pathway for butyrate production. This bacterium is able to produce butyrate from a wide range of sugars as well as lactate plus acetate. In Chapter 3, we described the microbial interactions between A. rhamnosivorans and Bacteriodes thetaiotaomicron in dietary pectins; Blautia hydrogenotrophica in lactate and small amount of acetate; Methanobrevibacter smithii in glucose. We observed that A. rhamnosivorans was able to benefit from its partners in all cocultures for butyrate production. This is likely due to its high metabolic flexibility. While the interaction between A. rhamnosivorans and B. thetaiotaomicron appeared as syntrophy, the interaction between A. rhamnosivorans and hydrogenotrohic microbes were cross-feeding type where hydrogen was transferred between two species. The latter resulted in an increase in butyrate level. In Chapter 4 we described a novel species Intestinimonas butyriciproducens SRB521T representing a novel genus Intestinimonas from a mouse caecum within Clostridium cluster IV. This bacterium produced butyrate and acetate as end products from Wilkins-Chalgren-Anaerobe broth.

    Butyrate production is assumed to derive from carbohydrate employing acetyl-CoA pathway. No gut bacterium is known to convert proteins or amino acids to butyrate although butyrogenic pathways from amino acid degradation have been detected in the human gut using metagenomic approach. In Chapter 5 we discovered a novel butyrate synthesis pathway from the amino acid lysine and the Amadori product fructoselysine in Intestinimonas butyriciproducens AF211 that was isolated from human stool. This strain appeared to grow much better in lysine as compared to sugars although lysine and acetyl-CoA pathways were both detected in its complete genome. Moreover, the strain AF211 was able to metabolise efficiently fructoselysine into butyrate, and acetate was found to affect the fructoselysine fermentation, representing the impact of the environmental conditions where acetate is abundant in the gut. While the lysine pathway was found in the gut of many individuals, the fructoselysine pathway was present in only half of those samples. The finding that strain I. butyriciproducens AF211 is capable of the butyrogenic conversion of amino acid lysine and fructoselysine, an Amadori product formed in heated foods via the Maillard reaction, indicated a missing link that coupling protein metabolism and butyrate formation. As this Amadori product has been implicated to play a role in aging process, the use of strain AF211 as fructoselysine clearance in the gut needs further investigation. In Chapter 6 we performed genomic and physiological comparison between the I. butyriciproducens strain AF211 (human isolate) and SRB521T (mouse isolate). I. butyriciproducens was the most abundant species within the Intestinimonas genus and highly prevalent in humans based on metadata analysis on 16S amplicons. We confirmed that the butyrogenesis from lysine was a shared characteristic between the two I. butyriciproducens strains. We also observed the host specific features including tolerance to bile, cellular fatty acid composition, more efficient capability of converting sugars into butyrate, especially galactose and arabinose, in the human strain AF211. In addition, genomic rearrangements as well as variations in bacteriophages differed among strains.

    Transformation by photolysis in water in the pesticide model TOXSWA : implementation report
    Beltman, W.H.J. ; Mulder, H.M. ; Horst, M.M.S. ter; Wipfler, E.L. - \ 2015
    Wageningen : Alterra (Alterra report 2649) - 47
    waterverontreiniging - pesticiden - fotolyse - biochemische omzettingen - waterbodems - adsorptie - ecotoxicologie - modellen - water pollution - pesticides - photolysis - biochemical pathways - water bottoms - adsorption - ecotoxicology - models
    The TOXSWA model has been extended with the functionality to simulate photolysis in water. TOXSWA simulates the fate of substances in water bodies to calculate exposure concentrations for aquatic organisms or sediment-dwelling organisms as part of the risk assessment of plant protection products (PPP). Photolysis is modelled as a first-order process, where transformation occurs in the water phase only. The transformation rate is considered to be linearly proportional to global radiation. Studies in outdoor surface water systems can in principle be used to derive the PPP transformation rates due to photolysis.
    The formation of endosymbiotic membrane compartments: membrane identity markers and the regulation of vesicle trafficking
    Ivanov, S. - \ 2012
    Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Elena Fedorova; Erik Limpens. - S.l. : s.n. - ISBN 9789461733436 - 121
    planten - rhizobium - stikstof - stikstoffixatie - medicago - endosymbiose - celmembranen - blaasjes - biochemische omzettingen - moleculaire biologie - wortels - mycorrhizae - plants - rhizobium - nitrogen - nitrogen fixation - medicago - endosymbiosis - cell membranes - vesicles - biochemical pathways - molecular biology - roots - mycorrhizas

    In symbiosis of plants and arbuscular mycorrhizal fungi as well as in rhizobium-legume symbiosis the microbes are hosted intracellularly, inside specialized membrane compartments of the host. These membrane compartments are morphologically different but similar in function, since they control the exchange of compounds between host and its microsymbiont thus forming a highly specialized symbiotic interface. These are the arbuscules, containing highly branched fungal hyphae, and organelle-like symbiosomes containing rhizobium bacteria. Recent studies have markedly extended our insight in the evolution of the signaling mechanism underlying the formation of these symbiotic interfaces. These studies strongly suggest that rhizobium co-opted the complete signaling mechanism (including lipo-oligosaccharides signal molecules) from the more ancient AM fungi symbiosis. Further, in plant species (Parasponia) where rhizobium nodulation evolved rather recent and independent from legumes, the same lipo-oligosaccharide receptor is essential for the formation of the rhizobium symbiotic interface as well as arbuscules. Therefore it seems likely that rhizobium also co-opted the cellular mechanism controlling arbuscule formation to form a rhizobium symbiotic interface. This would imply that even after co-evolution in legumes the key regulators involved in the formation of these interfaces are similar or even identical.
    In this thesis I have shown that rhizobium symbiosis shares with AM symbiosis molecular and cell biological mechanisms that control symbiotic interface formation. I identified a plant exocytotic pathway marked by two highly homologous vesicle associated membrane proteins (VAMP) that control the formation of the symbiotic interface in both symbioses. RNAi of these two Medicago VAMP genes did not affect non-symbiotic plant development nor nodule formation. However, it hampered the formation of cell wall free regions at infection threads, and therefore blocks symbiosome formation. Further arbuscule formation was blocked, whereas root colonization was not affected. By identifying these VAMPs as common symbiotic regulators in secretory vesicle trafficking, I postulated that during evolution of rhizobium symbiosis pre-existing cellular mechanisms of the AM fungal symbiosis have been co-opted. These findings also revealed a primary role of exocytosis in symbiosome formation and allowed to postulate the apoplastic nature of symbiosome. Using identity markers of endocytotic compartments of plant cell (early endosome and late endosome) such as small GTPases belonging to the Rab family and SNARE (soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins, I have shown that they never occur on symbiosome membranes at any stage of symbiosome formation and development. This makes untenable long-standing hypothesis that symbiosomes originate from endocytosis-like process and represent endocytic (vacuolar) compartments. Instead symbiosomes have an apoplastic nature. Although symbiosomes have an apoplastic nature they acquire the vacuolar marker MtRab7 when they reach an elongated stage. However, vacuolar SNAREs which execute fusion of membranes are not present on functional symbiosomes, but they do appear on symbiosome membranes at the onset of senescence when symbiosomes are turned into a lytic compartment. Therefore I postulate that the acquisition of Rab7 primes the symbiosomes for degradation by the host. By this the host has full control over its microsymbiont.
    The finding that rhizobium symbiosis has co-opted the signaling mechanism as well as cellular mechanism from AM fungi symbiosis to facilitate an intracellular life style, has major implications for strategies to transfer the nodule symbiosis to non-legume crops. This is a “dream” that is already about a century old. The AM fungal symbiosis is far more ancient than the rhizobial symbiosis. It is also wide spread in the plant kingdom and almost 80% of plant species can establish an AM symbiosis. This implies that plants which are able to interact with AM fungi contain in principle the genes that are necessary for the intracellular accommodation of rhizobium. So the question is no longer why the rhizobium-legume symbiosis is specific for legumes, but why non-legumes are not yet able to establish this symbiosis?

    Post-genomic characterization of metabolic pathways in Sulfolobus solfataricus
    Walther, J. - \ 2012
    Wageningen University. Promotor(en): John van der Oost; Willem de Vos. - S.l. : s.n. - ISBN 9789461732033 - 162
    sulfolobus solfataricus - biochemische omzettingen - koolstofpathways - transcriptomica - bioreactoren - sulfolobus solfataricus - biochemical pathways - carbon pathways - transcriptomics - bioreactors

    The physiological functions and mode of actions of different biomolecules are of continuous interest and a prerequisite to fully understand and appreciate the potential of Archaea and their molecules. We chose to study Sulfolobus solfataricus for its stable (heat-resistant) enzymes and specific metabolic potential, the ease of cultivation of this organism, and the relative large amount of knowledge about this heat-loving acidophilic organism. We selected a systems approach to study the behaviour of this organism trying to make steps forward into the unknown, whenever possible trying to link exploration to exploitation. The cultivation of S.solfataricus is an essential element in all systems approaches that link genotype to phenotype. Hence, specific attention is given to the advanced culturing systems for this extremophile that have been used in all experimental studies described here (Chapters 3-6).

    Systems analysis includes the integration of all available omics data and is increasingly used in the analysis of Archaea (Chapters 3 and 4). However, most attention has been given to archaeal transcriptome analysis and hence the most important literature on heat-loving Archaea is summarized (Chapter 2).

    In the experimental chapters (Chapters 3-6) various systems approaches are applied to gain understanding of metabolic pathways in Sulfolobus. Chapter 3 describes the study of the central carbon pathways, consisting of the (non-) phosphorilated Entner-Douderoff (ED) pathway and the citric acid cycle. Different functional genomic approaches were applied on the model organism Sulfolobus solfataricus to study the response of growth on different carbon sources, D-Glucose vs. Tryptone and Yeast Extract. The complete transcriptome was studied using PCR-based microarrays. In addition the proteome was studied using 2D-electrophoresis map in combination with 13N- labelling technique to determine protein fluctuations. Despite the large difference in medium, very few significant differences on protein or RNA level were observed for the two conditions. Therefore regulation of these pathways does in all probability not occur through changes in protein abundance but presumably rather by direct changes in enzyme activity. This is unlike two thermophilic Euryarchaea: Thermococcus kodaaraensis (Kanai, Akerboom et al. 2007)and Pyrococcus furiosus (Schut, Brehm et al. 2003)where extensive regulation of glycolytic genes was observed in a similar situation.

    Chapter 4describes the study of the degradation of D-arabinose through a similar approach as was described in chapter 3. S. solfataricus was grown on either D-arabinose or D-glucose and a comprehensive transcriptome and proteome study was carried out. The result of these studies was not only elucidation of the D-arabinose degradation route, but also a general prokaryotic pentose, hexaric acids and hydroxyproline degradation route, which supports the theory of metabolic pathway genesis by enzyme recruitment. Also this study predicted a cis-regulatory element to induce the arabinose degrading pathway when needed. The enzymes involved in the proposed pathway were cloned, expressed and their function was biochemically measured. This showed that using these enzymes, D-arabinose can be degraded to 2-oxogluterate, one of the metabolites that are part of the citric acid cycle.

    Chapter 5reports on the effects of different oxygen concentrations on the behaviour of Sulfolobus solfataricus. The oxygen amount can be controlled relatively easily in a bioreactor, which is crucial for rapid and reproducible growth. Based on growth experiments in microcosms, different types of behaviour could be seen. At 35% (v/v gas phase) the cultures did not grow, indicating that S. solfa-taricus experiences a lethal dose of oxygen. At 26-32% growth was impaired, suggesting a moderate toxicity compared to the reference (21%). In the ranges 16-24% of oxygen, standard growth was observed, suggesting that S. solfataricus is comfortable in these oxygen ranges. For the lower amounts of oxygen (1.5-15%), the growth was comparable to the reference, but the respiratoryefficiency was increased. To get some more insight into this behaviour, we looked at the transcriptome. It showed differential expression of several genes, including genes encoding terminal oxidases, indicating that the organism adapts to lower oxygen concentrations by adapting its respiratory machinery.

    Chapter 6 describes the zeaxanthin pathway in the Sulfolobus species. Zeaxanthin is a colorant and of vital importance for the function of the human eye. In this chapter the genes responsible for zeaxanthin production are presented. For this, DNA microarrays, bioinformatics as well as molecular genetics techniques were used. A crtx-like gene is operational in most of the known Sulfolobus species that is able to attach sugar-like molecules to zeaxanthin, which improves its solubility in water, which is very important in many food uses. We have cloned this crtx-like gene of S. solfataricus, S. shibatae, and S. acidocaldarius in a zeaxanthin overproducing E. coli strain. It has been demonstrated that the gene products of S. shibatae and S. acidocaldarius were responsible for attaching sugar-like molecules to zeaxanthin. The ctrx-like gene of S. solfataricus was not operating in E. coli. This is probably due to the fact that the gene is truncated. This chapter has further improved the understanding of archaeal carotenoid pathways and it has shown that the Sulfolobus species are able to modify zeaxanthin, although each species produces different zeaxanthin modifications.

    Autogenerative high pressure digestion: anaerobic digestion and biogas upgrading in a single step reactor system
    Lindeboom, R.E.F. ; Fermoso, F.G. ; Weijma, J. ; Zagt, K. ; Lier, J.B. van - \ 2011
    Water Science and Technology 64 (2011)3. - ISSN 0273-1223 - p. 647 - 653.
    biogas - gasproductie - biochemische omzettingen - methaan - anaërobe afbraak - drukbehandeling - kooldioxide - oplosbaarheid - anaërobe behandeling - afvalwaterbehandeling - biobased economy - biogas - gas production - biochemical pathways - methane - anaerobic digestion - pressure treatment - carbon dioxide - solubility - anaerobic treatment - waste water treatment - biobased economy
    Conventional anaerobic digestion is a widely applied technology to produce biogas from organic wastes and residues. The biogas calorific value depends on the CH4 content which generally ranges between 55 and 65%. Biogas upgrading to so-called ‘green gas’, with natural gas quality, generally proceeds with add-on technologies, applicable only for biogas flows >100 m3/h. In the concept of autogenerative high pressure digestion (AHPD), methanogenic biomass builds up pressure inside the reactor. Since CO2 has a higher solubility than CH4, it will proportion more to the liquid phase at higher pressures. Therefore, AHPD biogas is characterised by a high CH4 content, reaching equilibrium values between 90 and 95% at a pressure of 3–90 bar. In addition, also H2S and NH3 are theoretically more soluble in the bulk liquid than CO2. Moreover, the water content of the already compressed biogas is calculated to have a dew point
    Chasing organohalide respirers: ecogenomics approaches to assess the bioremediation capacity of soils
    Maphosa, F. - \ 2010
    Wageningen University. Promotor(en): Willem de Vos, co-promotor(en): Hauke Smidt. - [S.l. : S.n. - ISBN 9789085856566 - 172
    microbiële afbraak - biochemische omzettingen - gehalogeneerde koolwaterstoffen - organische halogeenverbindingen - bioremediëring - bodemverontreiniging - bodembiologie - microbial degradation - biochemical pathways - halogenated hydrocarbons - organic halogen compounds - bioremediation - soil pollution - soil biology
    Het opsporen van organohalogeen-reducerende bacteriën: ecogenomics benaderingen om de bioremediatie-capaciteit van de bodem te beoordelen. Organohalogeen-reducerende bacteriën (OHRB) zijn efficiënte afbrekers van organische chloorverbindingen, zoals gechloreerde ethenen, chloorfenolen en andere gehalogeneerde alifatische en aromatische koolwaterstoffen. Desondanks, lijken deze organische chloorverbindingen te volharden op verschillende locaties. De reden voor dit gebrek aan afbraak kan worden toegeschreven aan het ontbreken van OHRB in voldoende aantallen of aan verkeerde fysisch-chemische omstandigheden voor hun groei en activiteit. Derhalve is er een dringende behoefte aan snelle, robuuste en gevoelige methoden die het voorspellen van en het toezicht houden op het bioremediatie potentieel en de activiteit van OHRB mogelijk maken. Moleculaire monitoring en modelsimulaties werden toegepast om de in-situ afbraak prestaties van een on-site dechlorerende bioreactor te bepalen en zijn invloed op de vervuilingsspluim. De toepasbaarheid van dit systeem werd getest in verschillende verontreinigde bodems.
    Bayesian networks for omics data analysis
    Gavai, A.K. - \ 2009
    Wageningen University. Promotor(en): Jack Leunissen; Michael Muller, co-promotor(en): Guido Hooiveld; P.J.F. Lucas. - [S.l.] : S.n. - ISBN 9789085853909 - 98
    bio-informatica - waarschijnlijkheidsmodellen - bayesiaanse theorie - netwerkanalyse - genexpressie - roken - vluchtige verbindingen - biochemische omzettingen - voedingsonderzoek bij de mens - genexpressieanalyse - microarrays - netwerken - nutrigenomica - bioinformatics - probabilistic models - bayesian theory - network analysis - gene expression - smoking - volatile compounds - biochemical pathways - human nutrition research - genomics - microarrays - networks - nutrigenomics
    This thesis focuses on two aspects of high throughput technologies, i.e. data storage and data analysis, in particular in transcriptomics and metabolomics. Both technologies are part of a research field that is generally called ‘omics’ (or ‘-omics’, with a leading hyphen), which refers to genomics, transcriptomics, proteomics, or metabolomics. Although these techniques study different entities (genes, gene expression, proteins, or metabolites), they all have in common that they use high-throughput technologies such as microarrays and mass spectrometry, and thus generate huge amounts of data. Experiments conducted using these technologies allow one to compare different states of a living cell, for example a healthy cell versus a cancer cell or the effect of food on cell condition, and at different levels.
    The tools needed to apply omics technologies, in particular microarrays, are often manufactured by different vendors and require separate storage and analysis software for the data generated by them. Moreover experiments conducted using different technologies cannot be analyzed simultaneously to answer a biological question. Chapter 3 presents MADMAX, our software system which supports storage and analysis of data from multiple microarray platforms. It consists of a vendor-independent database which is tightly coupled with vendor-specific analysis tools. Upcoming technologies like metabolomics, proteomics and high-throughput sequencing can easily be incorporated in this system.
    Once the data are stored in this system, one obviously wants to deduce a biological relevant meaning from these data and here statistical and machine learning techniques play a key role. The aim of such analysis is to search for relationships between entities of interest, such as genes, metabolites or proteins. One of the major goals of these techniques is to search for causal relationships rather than mere correlations. It is often emphasized in the literature that "correlation is not causation" because people tend to jump to conclusions by making inferences about causal relationships when they actually only see correlations. Statistics are often good in finding these correlations; techniques called linear regression and analysis of variance form the core of applied multivariate statistics. However, these techniques cannot find causal relationships, neither are they able to incorporate prior knowledge of the biological domain. Graphical models, a machine learning technique, on the other hand do not suffer from these limitations.
    Graphical models, a combination of graph theory, statistics and information science, are one of the most exciting things happening today in the field of machine learning applied to biological problems (see chapter 2 for a general introduction). This thesis deals with a special type of graphical models known as probabilistic graphical models, belief networks or Bayesian networks. The advantage of Bayesian networks over classical statistical techniques is that they allow the incorporation of background knowledge from a biological domain, and that analysis of data is intuitive as it is represented in the form of graphs (nodes and edges). Standard statistical techniques are good in describing the data but are not able to find non-linear relations whereas Bayesian networks allow future prediction and discovering nonlinear relations. Moreover, Bayesian networks allow hierarchical representation of data, which makes them particularly useful for representing biological data, since most biological processes are hierarchical by nature. Once we have such a causal graph made either by a computer program or constructed manually we can predict the effects of a certain entity by manipulating the state of other entities, or make backward inferences from effects to causes. Of course, if the graph is big, doing the necessary calculations can be very difficult and CPU-expensive, and in such cases approximate methods are used.
    Chapter 4 demonstrates the use of Bayesian networks to determine the metabolic state of feeding and fasting mice to determine the effect of a high fat diet on gene expression. This chapter also shows how selection of genes based on key biological processes generates more informative results than standard statistical tests. In chapter 5 the use of Bayesian networks is shown on the combination of gene expression data and clinical parameters, to determine the effect of smoking on gene expression and which genes are responsible for the DNA damage and the raise in plasma cotinine levels of blood of a smoking population. This study was conducted at Maastricht University where 22 twin smokers were profiled. Chapter 6 presents the reconstruction of a key metabolic pathway which plays an important role in ripening of tomatoes, thus showing the versatility of the use of Bayesian networks in metabolomics data analysis.
    The general trend in research shows a flood of data emerging from sequencing and metabolomics experiments. This means that to perform data mining on these data one requires intelligent techniques that are computationally feasible and able to take the knowledge of experts into account to generate relevant results. Graphical models fit this paradigm well and we expect them to play a key role in mining the data generated from omics experiments.
    Assessment of the pectinolytic network of Aspergillus niger by functional genomics : insights from the transcriptome
    Martens-Uzunova, E.S. - \ 2008
    Wageningen University. Promotor(en): Willem de Vos; C.A.M.J.J. van den Hondel, co-promotor(en): Peter Schaap. - Wageningen : Wageningen University - ISBN 9789085048879 - 208
    aspergillus niger - polygalacturonase - galacturonzuur - metabolisme - biochemische omzettingen - functionele genomica - transcriptomics - aspergillus niger - polygalacturonase - galacturonic acid - metabolism - biochemical pathways - functional genomics - transcriptomics
    More than a century ago, in 1889, A. Fernbach presented a detailed report about the invertase of Aspergillus niger in the third edition of “Annales De L'institut Pasteur”. Since then, many of the enzymes secreted by A. niger have found a broad range of applications, and today they are produced on an industrial scale. This filamentous fungus is also used as a primary source for the production of organic acids and other economically important metabolites. Although, many of these fermentation processes are well established, the underlying genetics are still not well understood. The recent determination of the genome sequence of A. niger illustrated the versatile metabolic capacities of this fungus and created the opportunity for challenging research aiming to unravel the fine tuned metabolic network and the full enzymatic potential of A. niger.
    The work described in this thesis is focused on the genome mining and transcriptional profiling of the enzymatic network of A. niger involved in pectin degradation and utilization. Pectin was chosen because of the broad application of it and its derivates in food, feed, medicines, and cosmetics and the economical importance of this polymer in several technological processes. This study addresses several issues concerning pectin degradation by A. niger:
    i) the complete inventory of the known and potential pectinolytic activities encoded in the genome of this fungus; ii) the understanding of dynamics of their gene-expression on (complex) carbon sources in order to unravel underling metabolic networks; iii) the evolutionary aspects of the pectinolytic system of A. niger and other filamentous fungi.
    Chapter 1 of this thesis discusses the importance of fungi and A. niger for the biotech industry, and gives a brief introduction to the structural elements of pectin, the types of enzymatic activities involved in its degradation, and the present knowledge of the metabolism of its major constituent – galacturonic acid. The current advances in fungal ‘omics research are pointed out. Chapter 2 presents the construction of pectin specific cDNA libraries from A. niger, and the annotation of more than 200 of the obtained expressed sequence tags. Chapter 3 focuses on the roles of and the interactions between the 21 genes within glycoside hydrolase family 28 - the largest group of A. niger pectinases. A special emphasis is given to a new, previously unanticipated, group of exo-acting enzymes. Chapter 4 describes the survey for all genes encoding pectinolytic activities in A. niger and their transcriptional profiles. In this chapter, a hypothetical model of the pectinolytic network of A. niger is proposed. Chapter 5 focuses on the identification of the genes involved in galacturonic acid catabolism in A. niger, the verification of some of the enzymatic activities encoded by them, and elaborates on the presence of an evolutionary conserved galacturonic acid pathway operating only in filamentous fungi capable of plant cell wall degradation. In Chapter 6, the evolutionary conservation of transcriptional response of the pectinolytic system of A. niger is compared to that of the model organism A. nidulans. This chapter further describes the identification of the evolutionary conserved, regulatory core of functional orthologs involved in galacturonic acid utilization and metabolism. In Chapter 7, the results of the work described in this thesis are summarized and discussed.
    Inhibition factors of hydrogen production by the extreme thermophile Caldicellulosiruptor saccharolyticus
    Niel, E.W.J. van; Claassen, P.A.M. ; Stams, A.J.M. - \ 2007
    thermofiele bacteriën - waterstof - sucrose - biochemische omzettingen - biobased economy - biobrandstoffen - thermophilic bacteria - hydrogen - sucrose - biochemical pathways - biobased economy - biofuels
    Poster met onderzoeksinformatie over de productie van waterstof uit sucrose door de thermofiele bacterie Caldicellulosiruptor saccharolyticus.
    Feasibility of Biological Hydrogen Production from Biomass for utilization in fuel cells
    Claassen, P.A.M. ; Groenestijn, J.W. van; Janssen, A.J.H. ; Niel, E.W.J. van; Wijffels, R.H. - \ 2002
    waterstof - biomassa - haalbaarheidsstudies - fermentatie - biochemische omzettingen - biobased economy - biobrandstoffen - hydrogen - biomass - feasibility studies - fermentation - biochemical pathways - biobased economy - biofuels
    Poster met onderzoeksinformatie over de haalbaarheid van biologische waterstofproductie uit biomassa.
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