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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    De toekomst van hout in de biobased economy
    Annevelink, E. ; Harmsen, P.F.H. ; Spijker, J.H. - \ 2018
    Vakblad Natuur Bos Landschap 15 (2018)141. - ISSN 1572-7610 - p. 7 - 11.
    biobased economy - hout - biobrandstoffen - biomassa - hernieuwbare energie - materialen uit biologische grondstoffen - cellulose - lignine - vezels - biobased economy - wood - biofuels - biomass - renewable energy - biobased materials - cellulose - lignin - fibres
    De laatste jaren is de biobased economy sterk gegroeid door allerlei activiteiten, variërend van fundamenteel onderzoek naar nieuwe biobased toepassingen, tot het op commerciële schaal vervaardigen van biobased producten. Welke kansen biedt dit voor hout en houtige biomassa en wat zijn de verwachte effecten op de houtmarkt?
    Samenstelling van blad, stengel en rhizomen in relatie tot optimaal oogst-tijdstip van Miscanthus x giganteus
    Kasper, G.J. ; Kolk, J.C. van der; Putten, J.C. van der - \ 2017
    Wageningen : Wageningen Livestock Research (Wageningen Livestock Research rapport 1022) - 27
    brandstofgewassen - biobased economy - miscanthus - grassen - oogsttijdstip - gewasopbrengst - akkerbouw - plantensamenstelling - suikergehalte - lignine - pectinen - droge stof - koolhydraten - stengels - wortelstokken - fuel crops - biobased economy - miscanthus - grasses - harvesting date - crop yield - arable farming - plant composition - sugar content - lignin - pectins - dry matter - carbohydrates - stems - rhizomes
    A plurality of components (such as sugars, lignin, pectin) of Miscanthus x giganteus has been studied in stem, leaf, and rhizomes for the harvest times July and January in view of the optimal harvest time. Additional literature search shows that the end of October is the optimum time for harvesting on the basis of the maximum above-ground dry matter yield and sugar yield, and dry matter yield in the next year. It will have to be investigated whether the optimal harvest time also applies to long-term research.
    Lignine : groene grondstof voor chemicaliën en materialen
    Dam, Jan van; Harmsen, Paulien ; Bos, Harriëtte ; Gosselink, Richard - \ 2016
    Wageningen : Wageningen Food & Biobased Research (Groene grondstoffen ) - ISBN 9789463430197 - 54
    lignine - chemicaliën uit biologische grondstoffen - biobased economy - toepassingen - bioraffinage - productieprocessen - lignin - biobased chemicals - biobased economy - applications - biorefinery - production processes
    De mogelijkheden voor het gebruik van lignine worden in dit boekje toegelicht. Deze uitgave heeft als doel meer informatie te geven over de eigenschappen van lignine en de vele mogelijkheden die lignine biedt als grondstof voor chemicaliën en materialen voor de biobased economy.
    Alkaline pretreatments of lignin-rich by-products and their implications for enzymatic degradation
    Murciano-Martinez, P. - \ 2016
    Wageningen University. Promotor(en): Harry Gruppen, co-promotor(en): Mirjam Kabel. - Wageningen : Wageningen University - ISBN 9789462576629 - 156
    degradation - enzymes - pretreatment - byproducts - lignin - food chemistry - delignification - sugarcane bagasse - degradatie - enzymen - voorbehandeling - bijproducten - lignine - voedselchemie - delignificatie - suikerrietbagasse

    The increasing interest in plant biomass based biofuels and chemicals arouses mainly from the increased awareness of a possible finiteness of fuels. The current main challenge to produce such biofuels and biochemicals is economic efficiency, but also knowledge concerning type and effectiveness of both thermally assisted chemical and enzymatic treatments, needed to generate fermentable sugars, is lacking. The subject of this thesis is to gain understanding of the effect of both sulphuric acid and NaOH catalysed pretreatments of sugar cane bagasse and oil palm empty fruit bunches on subsequent enzymatic saccharification, with a focus on the NaOH catalysed pretreatments. The fate of the main polymers present, lignin, cellulose and xylan, was studied and also single-activity xylan degrading enzymes, all from Rasamsonia emersonii, were studied for their mode-of-action.

    Evaluation of king oyster mushroom strains (Pleurotus eryngii) on selective lignin degradation in wheat straw: An update
    Sonnenberg, A.S.M. ; Visser, M.H.M. ; Lavrijssen, B. ; Cone, J.W. ; Hendrickx, P.M. - \ 2016
    Wageningen : Wageningen UR (PPO/PRI report 2016-4) - 21
    mushrooms - edible fungi - lignin - postharvest quality - agaricus bisporus - composts - nutrient use efficiency - pleurotus eryngii - lentinula edodes - wheat straw - miscanthus - straw - paddestoelen - eetbare paddestoelen - lignine - kwaliteit na de oogst - agaricus bisporus - compost - nutriëntengebruiksefficiëntie - pleurotus eryngii - lentinula edodes - tarwestro - miscanthus - stro
    The collection of Plant Breeding contains a large number of strains of Pleurotus eryngii and Lentinula edodes, two fungi producing edible mushrooms and also known by their selective degradation of lignin in lignocellulosic materials.
    De lignineraffinaderij
    Didde, R. ; Gosselink, R.J.A. ; Haveren, J. van - \ 2014
    WageningenWorld 2014 (2014)2. - ISSN 2210-7908 - p. 32 - 39.
    lignine - chemie op basis van biologische grondstoffen - biobased economy - chemicaliën uit biologische grondstoffen - bioraffinage - biomassaconversie - onderzoek - lignin - biobased chemistry - biobased economy - biobased chemicals - biorefinery - biomass conversion - research
    Lignine geeft planten stevigheid, maar deze verbinding is ook een schatkist vol waardevolle basischemicaliën. Wageningse onderzoekers maken die stap voor stap open. Een duurzaam alternatief voor aardolie is daarmee binnen handbereik.
    Doodzonde om op te stoken. Lignine de grondstof van de toekomst
    Zundert, M. ; Gosselink, R.J.A. - \ 2014
    Chemie Magazine 2014 (2014)3. - ISSN 1572-2996 - p. 24 - 27.
    biopolymeren - lignine - toepassingen - bioraffinage - innovaties - chemie op basis van biologische grondstoffen - biobased economy - aromatische koolwaterstoffen - biopolymers - lignin - applications - biorefinery - innovations - biobased chemistry - biobased economy - aromatic hydrocarbons
    Onhandelbaar, wee barstig en recalcitrant. Zo staat het houtpolymeer lignine bekend. Maar de stof vervangt steeds vaker fenol. En onderzoekers zijn hoopvol over de ontwikkeling van bio-BTX en koolstofvezels uit lignine.
    Availability of lignocellulosic feedstocks for lactic acid production - Feedstock availability, lactic acid production potential and selection criteria
    Bakker, R.R.C. - \ 2013
    Wageningen : Wageningen UR - Food & Biobased Research - 60
    melkzuur - lignine - biomassa - oogstresten - beschikbaarheid - inventarisaties - biomassa productie - biobased economy - lactic acid - lignin - biomass - crop residues - availability - inventories - biomass production - biobased economy
    The overall objective of this study is to assess the worldwide availability and suitability of agricultural residues for lactic acid production, based on fermentation of carbohydrates. The study focuses on lignocellulosic biomass that is produced as a by-product of agricultural production. The results of the study can be used to rank different biomass types on their lactic acid or fermentable sugar production potential. For each residue, both total production (ton of fermentable sugars per year) and productivity (fermentable sugars produced per ha of agricultural land) are considered. Furthermore, the production of non-fermentable residues (e.g. lignin) is included as well in the study. The study is concluded by series of recommendations on what factors to consider when choosing a suitable lignocellulosic feedstock for production of lactic acid, or for other fermentation processes. The results of this study can be used to further evaluate suitability, cost and sustainability of using agricultural residues as feedstock for fermentative production of lactic acid production, or other biochemical conversion processes.
    Lignin as a renewable aromatic resource for the chemical industry
    Gosselink, R.J.A. - \ 2011
    Wageningen University. Promotor(en): Johan Sanders, co-promotor(en): G. Gellerstedt; Jan van Dam. - [S.l.] : S.n. - ISBN 9789461731005 - 191
    vervangbare hulpbronnen - lignine - lignocellulose - lignocellulosehoudend afval - pulp- en papierwarenindustrie - chemicaliën uit biologische grondstoffen - chemie op basis van biologische grondstoffen - renewable resources - lignin - lignocellulose - lignocellulosic wastes - pulp and paper industry - biobased chemicals - biobased chemistry

    Valorization of lignin plays a key role in the further development of lignocellulosic biorefinery processes for biofuels and biobased materials production. Today’s increased demand for alternatives to fossil carbon-based products expands the interest and the need to create added value to the unconverted lignin fraction. The aim of the research was to study the potential of lignin to become a renewable aromatic resource for the chemical industry. Lignin can be considered as an abundantly available and cheap raw material for the manufacturing of an array of products. Development of applications needs to go hand in hand with the anticipated increased production of technical lignins derived from the pulp and paper industry and the emerging lignocellulosic biorefinery industry. Two promising lignin applications are studied in this thesis:

    1) the use of lignin in wood adhesives

    2) the use of lignin for the production of aromatic chemicals

    PCA modeling was performed aimed at the prediction of the application potential of different technical lignins for wood adhesive production. The lignins and their fractions could be classified in different clusters based on their structure dependent properties. Lignins exhibiting sufficient reactive sites, medium molar mass and low level of impurities are most promising candidates for the development of lignin based wood adhesives. Both lignin reactivity and formaldehyde-free crosslinking agents are needed to develop emission-free adhesives. Periodate oxidation was studied as method to improve the lignin reactivity. Alternatives to formaldehyde- based glues are under investigation and a combination of lignin and furans might be an interesting concept to develop renewable adhesives.

    In this research a reliable SEC methodology was developed for the analysis of the molar mass distribution of a wide range of different lignins. The major drawback of this method is that the molar masses are calculated on a relative basis to sulfonated polystyrenes. Using MALDI-TOF-MS and prior fractionation of lignin did not solve all problems associated with the determination of the absolute molar mass of lignin.

    Supercritical depolymerisation of lignin using a carbon dioxide/acetone/water fluid resulted in a depolymerised lignin oil. In this oil some monomeric compounds are present in relatively high amounts up to 3.6% (based on dry lignin). These products maybe further isolated by downstream processing to obtain purified fine chemicals. For continuous operation of this supercritical process, the formation of char should be further limited.

    The results presented in this thesis are expected to contribute - together with the many on-going activities worldwide - to the increased commercial utilisation of lignin in the future. Moreover, the obtained results contribute to the increasing knowledge on lignin analysis, chemistry and reactivity.

    Chemical composition of lamina and sheath of Lolium perenne as affected by herbage management
    Hoekstra, N.J. ; Struik, P.C. ; Lantinga, E.A. ; Schulte, R.P.O. - \ 2007
    NJAS Wageningen Journal of Life Sciences 55 (2007)1. - ISSN 1573-5214 - p. 55 - 73.
    afsnijhoogte - grasbestand - voedergewassen - lolium perenne - hergroei - rotaties - chemische samenstelling - graslandbeheer - voedingswaarde - lignine - totale hoeveelheid droge stof - koolhydraten - stikstof - vezelgehalte - bloeiwijzen - cutting height - herbage - fodder crops - lolium perenne - regrowth - rotations - chemical composition - grassland management - nutritive value - lignin - total solids - carbohydrates - nitrogen - fibre content - inflorescences - water-soluble carbohydrate - neutral detergent fiber - dairy-cows - nitrogen application - nutritive-value - ryegrass varieties - animal nutrition - milk-production - rumen function - l. cultivars
    The quality of grass in terms of form and relative amounts of energy and protein affects both animal production per unit of intake and nitrogen (N) utilization. Quality can be manipulated by herbage management and choice of cultivar. The effects of N application rate (0, 90 or 390 kg N ha¿1 year¿1), duration of regrowth period (2¿3, 4¿5, or 6¿7 weeks), and cutting height (8 or 12 cm) on the mass fractions of nitrogen (N), water-soluble carbohydrates (WSC), neutral detergent fibre (NDF), acid detergent fibre (ADF), lignin and ash in lamina and sheath material of a high-sugar (Aberdart) and a low-sugar (Respect) perennial ryegrass (Lolium perenne) cultivar, were studied in a factorial field experiment during four seasons in 2002 and 2003. Expressing NDF and ADF mass fractions in g per kg WSC-free dry matter (DM) increased the consistency of treatment effects. The high-sugar cultivar had generally higher WSC mass fractions than the low-sugar cultivar, especially during the late season. Moreover, the relative difference in WSC mass fraction between the two cultivars tended to be higher for the lamina material than for the sheath material, which suggests that the high-sugar trait may be more important under grazing conditions, when lamina forms the bulk of the intake, than under mowing regimes. Longer regrowth periods and lower N application rates increased WSC mass fractions and decreased N mass fractions; interactions between regrowth period and N application rate were highly significant. The mass fractions of NDF and ADF were much less influenced. The NDF mass fraction in terms of g per kg WSC-free DM tended to be higher at lower N application rates and at longer regrowth periods. The effect of cutting height on herbage chemical composition was unclear. In conclusion, high-sugar cultivars, N application rate and length of the regrowth period are important tools for manipulating herbage quality.
    Managing organic resources for soil amendment
    Giller, K.E. ; Misiko, M. ; Tittonell, P.A. - \ 2006
    LEISA : ILEIA newsletter for low-external-input and sustainable agriculture 22 (2006)4. - ISSN 1569-8424 - p. 16 - 17.
    organisch afval - agrarische afvalstoffen - plantenresten - organisch bodemmateriaal - mulches - voedingsstoffen - lignine - polyfenolen - stikstof - agrarische bedrijfsvoering - grondbeheer - agrarische bedrijfsplanning - organic wastes - agricultural wastes - plant residues - soil organic matter - mulches - nutrients - lignin - polyphenols - nitrogen - farm management - land management - farm planning
    Soil fertility management is a key issue for sustaining agricultural production in the tropics. Organic resources are important for short-term nutrient availability, as well as for longer-term maintenance of soil organic matter. For smallholder farmers, organic materials are an important source of nutrients, and necessary to manage soil fertility. However, the amount of organic material available on-farm is often limited in supply, and differs widely in quality. This is why the little that is available needs to be used as efficiently as possible.
    Coir based building and packaging materials : final report of project CFC/FIGHF/11
    Snijder, M.H.B. ; Keijsers, E.R.P. ; Oever, M.J.A. van den; Dam, J.E.G. van - \ 2006
    Wageningen : Agrotechnology & Food Innovations (Report / Wageningen UR, Agrotechnology & Food Innovations 592) - ISBN 9789067549943 - 121
    kokosvezel - plantenvezels - bouwmaterialen - verpakkingsmaterialen - chemische samenstelling - lignine - fabricage - proefprojecten - haalbaarheidsstudies - ontwikkelingslanden - biobased economy - coir - plant fibres - building materials - packaging materials - chemical composition - lignin - manufacture - pilot projects - feasibility studies - developing countries - biobased economy
    Kick-off meeting EUROLIGNIN : September 19-20 2002, Evian-les-Bains, France
    Gosselink, R.J.A. ; Dam, J.E.G. van; Jong, E. de - \ 2002
    toepassingen - applications - research - kleurstoffen (dyes) - stralingsbeschermingsmiddelen - radiation protection agents - eigenschappen - lignine - properties - onderzoek - hechtmiddelen - Non-food Products (General) - coatings - adhesives - dyes - lignin - afdeklagen - biobased economy - Non-food producten (algemeen) - toepassingen - applications - research - kleurstoffen (dyes) - stralingsbeschermingsmiddelen - radiation protection agents - eigenschappen - lignine - properties - onderzoek - hechtmiddelen - Non-food Products (General) - coatings - adhesives - dyes - lignin - afdeklagen - biobased economy - Non-food producten (algemeen)
    Presentatie aan het begin van het project EUROLIGNIN. Er wordt onderzoek gedaan naar verschillende toepassingen van lignine.
    Binderless board and moulded products produced from whole coconut husks
    Dam, J.E.G. van; Snijder, M.H.B. - \ 2002
    Wageningen : Wageningen UR, Agrotechnology & Food Innovations - 4
    bouwmaterialen - kokosvezel - bouwplaten - kokosnootproducten - lignine - economische haalbaarheid - biobased economy - productieprocessen - building materials - coir - wallboard - coconut products - lignin - economic viability - biobased economy - production processes
    A simple and efficient technology has been developed to produce high strength - high density board materials from whole coconut husks, without the addition of chemical binders. The board material has been shown to exhibit excellent properties, which are comparable with or even superior to commercial wood based panels.
    Hoog ligninegehalte in compost lijkt nadelig voor productie
    Straatsma, G. - \ 2001
    Groenten en Fruit. Vakdeel paddestoelen 2001 (2001)12. - ISSN 0925-9716 - p. 16 - 17.
    paddestoelen - compost - groeimedia - compostering - opbrengsten - oogsttoename - oogstverliezen - lignine - steriliseren - mushrooms - composts - composting - growing media - yields - yield increases - yield losses - lignin - sterilizing
    Effect van pasteurisatie, conditonering tijdens compostering en het lignine-gehalte op de champignonopbrengst
    Lignin peroxidase mediated biotransformations useful in the biocatalytic production of vanillin
    Have, R. ten - \ 2000
    Agricultural University. Promotor(en): J.A.M. de Bont; J.A. Field. - S.l. : S.n. - ISBN 9789058081698 - 90
    vanilline - lignine - peroxidase - vanillin - lignin - peroxidase

    This research concentrates on lignin peroxidase (LiP) mediated biotrans-formations that are useful in producing vanillin.

    In order to obtain this extracellular enzyme, the white-rot fungus Bjerkandera sp. strain BOS55 was cultivated on nitrogen rich medium. This procedure resulted in a successful LiP production of 600 U/L. Peptone in the culture medium was shown to interfere with the standard LiP assay in which the formation of veratraldehyde (VAD) from veratryl alcohol (VA) is monitored. Removal of peptone by (NH 4 ) 2 SO 4 precipitation minimized the interference.

    BOS55 excreted at least seven LiP isozymes of which two were purified and characterized. The predominant LiP isozyme (LiP-2) oxidized VA to VAD in the pH range from 2.5 to 6.5. The VA oxidizing activity was optimal at the lowest pH. The K m for H 2 O 2 was strongly depended on the pH. At pH 5.0, a physiological pH, the Km for H 2 O 2 was similar to the extracellular H 2 O 2 concentration measured in cultures of BOS55.

    A model based on calculated ionisation potentials (IP) was developed to predict which potential vanillin precursor would be oxidized by LiP. By testing a series of non-phenolic aromatic compounds, of which the IP was calculated, an IP-threshold value of 9.0 eV was determined. This value was used to select compounds with a lower IP like O -acetyl coniferyl alcohol, and O -acetyl isoeugenol (isoeugenyl acetate, IEA). Indeed, these compounds were consumed and in part converted into vanillyl acetate, the acetyl ester of vanillin.

    IEA was studied to elucidate mechanisms of its oxidation andCalpha - Cbetacleavage of the propenyl side chain in IEA into vanillyl acetate. It was found that IEA was consumed via redox mediation. IEA was only oxidized in the presence of the redox mediator VA. The latter was first oxidized by LiP to the radical cation (VA · +) which in its turn oxidized IEA to its corresponding radical cation IEA · +. The followingCalpha - Cbetacleavage reaction was the result of O 2 -dependent chemical reactions which resulted in vanillyl acetate and ethanal.

    The isoeugenol methyl ether (DMPP) was also used to investigate the consumption and theCalpha - Cbetacleavage mechanism. DMPP was not only consumed enzymatically, but also by O 2 -dependent self-propagating reactions. Mn 2+inhibited this chemical consumption. Since Mn 2+also inhibited the molar yield of the predominantCalpha - Cbetacleavage product, VAD, it was concluded that this product was formed also during these chemical reactions. An other VAD producing route was discovered by incubating a DMPP oxidation side product, 1-(3',4'-dimethoxyphenyl)-2-propanone (DMPA) with LiP. Interestingly, VAD was only formed in the presence of O 2 . Without O 2 , solely DMPA dimers were detected by GC-MS.

    Role of manganese and veratryl alcohol in the ligninolytic system of Bjerkandera sp. strain BOS55
    Mester, T. - \ 1998
    Agricultural University. Promotor(en): J.A.M. de Bont; J.A. Field. - S.l. : Mester - ISBN 9789054859048 - 116
    verhouting - lignine - mangaan - lignification - lignin - manganese

    Introduction
    Lignin is a three dimensional hydrophobic plant polymer derived from the random coupling of phenylpropanoid precursors. The chemical and physical characteristics of lignin require a nonspecific, extracellular oxidative process for biodegradation. White rot basidiomycetes are the only group of organisms having an efficient extracellular ligninolytic system. These fungi produce peroxidases and laccases that are involved in the initial attack of lignin. The peroxidases work with H 2 O 2 which is also enzymatically produced by the fungi by different H 2 O 2 -producing oxidases. For their proper operation, peroxidases require appropriate cofactors which are the best substrates of the enzymes. Lignin peroxidase (LiP) uses the de novo produced secondary metabolite, veratryl alcohol (3,4-dimethoxybenzyl alcohol) as cofactor; whereas, the cofactor of manganese peroxidase (MnP) is the metal Mn(II) which occurs naturally in wood. The in vitro oxidation of lignin preparations by LiP or MnP is only feasible in the presence of veratryl alcohol or manganese, respectively. Additionally, simple aliphatic organic acid metabolites such as oxalate are involved in lignin degradation. On the one hand, oxalate is a good chelator of Mn(III) generated by MnP. This complex is relatively stable forming a low molecular weight diffusible oxidant capable of oxidizing phenolic lignin. Oxalate is also oxidized by ligninolytic enzymes in the presence of their cofactors generating reactive oxygen species that possibly participate in the oxidation of lignin.

    The low molecular weight cofactors also influence the physiological regulation of white rot fungi. Veratryl alcohol is known to increase LiP activities in white rot fungal cultures, although, veratryl alcohol itself does not induce lip gene transcription. Manganese is essential for the induction of mnp gene expression and MnP activity in various white rot fungi. In contrast, manganese lowers LiP production so that the highest LiP activities are measured under manganese deficiency. Nonetheless manganese has no direct repressive effect on lip gene transcription.

    Despite the great research efforts conducted so far on various white rot fungi, the true mechanism of lignin degradation is still not fully understood. However, it is becoming clear that low molecular weight cofactors are important catalytic and physiological regulating components of the ligninolytic system.

    This thesis was dedicated to study the interrelationship between low molecular weight cofactors and ligninolytic enzymes. Bjerkandera sp. strain BOS55 was used as the fungus of study, since it was found to be an outstanding producer of ligninolytic enzymes and a great variety of secondary aryl metabolites. Moreover, this strain was observed to be a good degrader of polycyclic aromatic hydrocarbons (PAH) and biobleacher of kraft pulp in various screenings. The main objective of the thesis was to gain insight into the role of veratryl alcohol and manganese in regulating the physiology and participating in the ligninolytic system of Bjerkandera sp. strain BOS55.

    Chapter 2
    In Chapter 2, it was demonstrated for the first time that manganese inhibits the biosynthesis of veratryl alcohol in white rot fungi. This explains at least in part the general observation that LiP production is lowered in the presence of manganese although manganese itself does not repress the transcription of lip genes. The ten-fold increase in veratryl alcohol biosynthesis caused by manganese deficiency stimulated the LiP titres by protecting the enzyme from inactivation by physiological levels of H 2 O 2 . Adding veratryl alcohol to manganese containing cultures of the fungus sustained high LiP titres similar to that found under manganese deficient conditions. Moreover, a good correlation was observed between the LiP titres and veratryl alcohol concentrations irrespective of whether veratryl alcohol was produced by the fungus or added exogenously.

    Chapter 3
    In Chapter 3, the mechanism resulting in manganese inhibition of veratryl alcohol biosynthesis was studied. Potential biosynthetic precursors of veratryl alcohol were added to manganese deficient and sufficient cultures of Bjerkandera sp. strain BOS55 in order to bypass the inhibited step. The addition of fully methylated precursors (veratrate, veratraldehyde) equally increased the production of veratryl alcohol irrespective of the manganese concentration. This observation indicated that the reduction of the benzylic acid to benzyl alcohol group is not inhibited by manganese. All the other known precursors such as phenylalanine, cinnamate, benzoate/benzaldehyde as well as the partially hydroxylated benzylic compounds (e.g. 3-hydroxybenzoate, 4-hydroxybenzoate, protocatechuate, vanillate, isovanillate) did increase the veratryl alcohol production in the presence of manganese but never as much as that under manganese deficiency. From these observations it was concluded that no single step along the biosynthetic pathway was inhibited by the presence of manganese. Instead, the availability of phenolic precursors is limited when manganese was added. From this study, we also learned that there are several alternative precursors resulting in increased veratryl alcohol production which can potentially originate from lignin degradation. In addition, it was demonstrated that many of the veratryl alcohol precursors (phenylalanine, cinnamate, benzoate, 4-hydroxybenzoate) enhanced the production of anisyl and chloroanisyl metabolites indicating the existence of common precursors in these biosynthetic pathways. Deuterium labelled benzoate and 4-hydroxybenzoate were converted to a broad spectrum of labelled aryl metabolites.

    Chapter 4
    In Chapter 4, the interrelationship between cofactors and peroxidases in cultures grown on natural substrates (beech wood and hemp stem wood sawdust) was studied. Beech wood and hemp stem wood substrates, which contain 6 and 25 mg kg -1dry wood of soluble manganese, respectively were favourable for MnP production. Many studies in the past have failed to demonstrate the presence of LiP on natural substrates even in fungi having lip genes. Surprisingly, Bjerkandera sp. strain BOS55 produced very high LiP titres on the wood substrates. The high LiP activity observed suggested that this enzyme may have an important role during wood decay. The significant LiP production in spite of the presence of manganese can be explained by the very high production of veratryl alcohol throughout the incubation. The fact that veratryl alcohol was produced in high amounts although soluble manganese was always present apparently contradicts the findings presented in Chapter 2 . However, the high veratryl alcohol production can be explained by the presence of lignin degradation products such as 4-hydroxybenzoate, protocatechuate, vanillate, isovanillate entering the biosynthetic pathway of veratryl alcohol as was suggested in Chapter 3.

    Chapter 5
    Bjerkandera sp. strain BOS55 is a good MnP producer. In Chapter 5, the conditions for optimal MnP production were examined. The highest production was observed in nitrogen rich medium with 0.2 to 1 mM manganese at a pH value of 5.2 and at a temperature of 30 oC. Two interesting phenomena were discovered while studying the physiology of MnP production. Firstly, significant MnP production was also observed in the absence of manganese which previously only has been shown to be the case for Pleurotus spp. However, unlike Pleurotus spp., MnP production in cultures of Bjerkandera sp. strain BOS55 was enhanced in response to increasing Mn levels. Secondly, it was demonstrated that the addition of various organic acid metabolites significantly increased the MnP titres under manganese sufficient conditions. The best results were obtained with glycolate.

    Chapter 6
    In Chapter 6, the study on the MnP production was continued in order to better understand why MnP is produced in the absence of manganese and to elucidate the induction mechanism under manganese deficient conditions. In the absence of manganese, oxalate and related organic acids, such as glycolate or glyoxylate were found to induce MnP production. The stimulatory effect of organic acids on MnP production was demonstrated to be due to the increased production of MnP proteins. Additionally, it was shown that the acids induced mnp gene transcription (unpublished data).

    The major MnP isozyme produced in the absence of Mn and in the presence of glycolate was purified and characterized. Like other MnP isozymes, this enzyme was able to efficiently oxidize Mn. However, unlike other MnP isozymes, it was also able to directly oxidize veratryl alcohol and 1,4-dimethoxybenzene with a very high affinity in the absence of manganese. These nonphenolic substrates are typical substrates of LiP. Methoxyphenols and aromatic amines could also be oxidized in the absence of manganese. The optimal pH for the manganese independent oxidation of all the substrates tested was 3.0 similar to that observed for LiP isozymes. On the other hand, the oxidation of Mn(II) and consequently the manganese dependent oxidation of phenolic substrate reached the highest rate at pH 4.5 as described for many MnP isozymes. The kinetic values in terms of turnover number and affinity for Mn(II) and veratryl alcohol oxidation were similar to those found for other MnP and LiP isozymes. Therefore, the Bjerkandera MnP could be best described as a hybrid enzyme between MnP and LiP, having a binding site for Mn(II) as well as for methoxy aromatics/phenols. This conclusion is supported by the finding that Mn(II) at concentrations greater than 0.1 mM severely inhibited veratryl alcohol oxidation by the enzyme; whereas Mn(II) has no effect on LiP. The fact that this enzyme can oxidize Mn(II) as well as directly oxidize veratryl alcohol and other aromatic amines and phenols clarifies the physiological relevance of the occurrence of this MnP under Mn deficient and sufficient conditions.

    Conclusions
    In conclusion, this thesis has resulted in new insights into the key regulatory role of manganese in lignin degradation such as the repressive effect of manganese on the production of aryl metabolites. The addition of manganese at concentrations as low as 33μM completely changed the pattern of peroxidases and aryl metabolites production compared to that under manganese deficiency. Additionally, it was observed that MnP is purposefully produced and regulated by organic acids in the total absence of manganese. Under these conditions, a novel type of MnP-LiP hybrid isozyme was isolated which was functional under any manganese regime using either veratryl alcohol or Mn(II) as a cofactor. Under natural conditions, such an enzyme would have considerable physiological significance since soluble manganese is known to be leached out or become oxidized to insoluble MnO 2 during fungal attack, possibly resulting in manganese deficient areas in wood. In manganese deficient areas, veratryl alcohol biosynthesis is stimulated and the enzyme can use this secondary metabolite as an alternative to manganese.

    Future research should elucidate the newly discovered role of organic acids in regulating MnP in white rot fungi. The physiological significance of this regulation may be due to the role of organic acids as an important source of reduced oxygen radicals. The oxidative stress resulting from radicals may be the signal for MnP gene expression as has been shown to be the case with H 2 O 2 . The radicals are also required for extensive degradation of lignin. Additionally, the presence of organic acids may serve as an early warning for the upcoming presence of bioavailable manganese. The fungal organic acid metabolites are well known for their ability to solubilize insoluble MnO 2 deposits in fungal attacked wood.

    Effect bm-genen op verteerbaarheid onderzocht.
    Dolstra, O. ; Putten, P.E.L. van der; Loo, E.N. van; Marvin, H. - \ 1994
    Prophyta 48 (1994)3. - ISSN 0921-5506 - p. 32 - 34.
    verteerbaarheid - voer - lignine - lignocellulose - maïs - mutaties - voedingswaarde - plantenveredeling - kwaliteit - herkauwers - zea mays - digestibility - feeds - lignin - lignocellulose - maize - mutations - nutritive value - plant breeding - quality - ruminants - zea mays
    De snijmaisveredeling is gericht op het ontwikkelen van rassen met een hoge voederwaarde. Recessieve mutanten met een laag ligninegehalte worden gekenmerkt door bladeren met opvallend bruine pigmentatie van de middennerven en worden daarom aangeduid met de afkorting bm voor 'brown midrib'. Vergelijkende proeven met herkauwers hebben duidelijk positieve effecten van bm-genen op voeropname, smakelijkheid, in vivo verteerbaarheid en dierprestatie aangetoond. Onderzoek is gedaan naar het effect van 1 dosis van een bm-gen, dat wil zeggen heterozygotie, op expressie van deze eigenschappen
    Physiological roles and metabolism of fungal aryl alcohols
    Jong, E. de - \ 1993
    Agricultural University. Promotor(en): J.A.M. de Bont, co-promotor(en): M.M.G.R. Bol; J.A. Field. - S.l. : De Jong - ISBN 9789054851943 - 224
    enzymen - bosbouw - pulpbereiding - biodegradatie - oxidoreductasen - peroxidasen - katalase - basidiomycotina - scheurvorming - decompositie - degradatie - lignine - chemische verbindingen - enzymes - forestry - pulping - biodegradation - oxidoreductases - peroxidases - catalase - basidiomycotina - cracking - decomposition - degradation - lignin - chemical compounds

    The major structural elements of wood and other vascular tissues are cellulose, hemicellulose and generally 20-30% lignin. Lignin gives the plant strength, it serves as a barrier against microbial attack and it acts as a water impermeable seal across cell walls of the xylem tissue. However, the presence of lignin has practical drawbacks for some of the applications of lignocellulosic materials. First, lignin has to be removed for the production of high quality pulps. Second, lignin reduces the digestibility of lignocellulosic materials. High quality pulps can be produced with chemical methods, however the abundant use of chemicals and energy, and the formation of an enormous waste stream has led scientists to investigate the possibilities of biodelignification. White-rot fungi give the most rapid and extensive degradation and have become subject of intensive research. Results obtained with the model organism Phanerochaete chrysosporium and other strains have revealed that lignin biodegradation is an extracellular, oxidative and non-specific process. This unique biodegradative potential has been considered for broader applications such as waste water treatment and the degradation of xenobiotic compounds. The research described in this thesis concentrates on the function of aryl alcohols in fungal physiology.

    Aryl alcohols In the physiology of white-rot fungi. White-rot fungi have a versatile machinery of enzymes, including peroxidases and oxidases, which work in harmony with secondary aryl alcohol metabolites to degrade the recalcitrant, aromatic biopolymer lignin. In chapter 2 literature concerning the important physiological roles of aryl (veratryl, anisyl and chlorinated anisyl) alcohols in the ligninolytic enzyme system has been reviewed. Their functions include stabilization of lignin peroxidase, charge-transfer reactions and as substrate for oxidases generating extracellular H 2 O 2 .

    The experimental research described in this thesis was initiated to evaluate the possibilities of white-rot fungi in the biopulping of hemp stem wood. Sixty-seven basidiomycetes were isolated and screened for high peroxidative activity (chapter 3). Several of the new isolates were promising manganese peroxidase-producing white-rot fungi. Enzyme assays indicated that for the production of H 2 O 2 either extracellular glyoxal or aryl alcohol oxidase were present. In contrast, lignin peroxidase was only detected in P. chrysosporium , despite attempts to induce this enzyme in other strains with oxygen and oxygen/veratryl alcohol additions. A highly significant correlation was found between two ligninolytic indicators: ethene formation from α-keto-γ- methylthiolbutyric acid and the decolorization of a polymeric dye, Poly R-478. Three of the new isolates had significantly higher Poly R decolorizing activities compared to P. chrysosporium .

    One of the best Poly R decolorizing strains, Bjerkandera sp. BOS55 was selected for further characterization. A novel enzyme activity (manganese independent peroxidase) was detected in the extracellular fluid of Bjerkandera sp. BOS55 (chapter 4). The purified enzyme could oxidize several compounds like phenol red, 2,6-dimethoxyphenol (DMP), Poly R-478, 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and guaiacol with H 2 O 2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H 2 O 2 . Bjerkandera sp. BOS55 also produced de novo several aromatic metabolites. Besides veratryl alcohol and veratraldehyde, compounds which are known to be involved in the ligninolytic system of several other white-rot fungi (chapter 2), other metabolites were formed. These included anisaldehyde, 3-chloro-anisaldehyde, 3,5-dichloro-anisaldehyde and small amounts of the corresponding anisyl, 3-chloro-anisyl and 3,5-dichloro-anisyl alcohol (chapters 5 and 6). This was the first report of de novo biosynthesis of simple chlorinated aromatic compounds by a white-rot fungus. These unexpected findings led us investigate the physiological role(s) of the denovo biosynthesized chlorinated anisyl alcohols (chapter 6). All metabolites were produced simultaneously with the extracellular ligninolytic enzymes. The monoand dichlorinated anisyl alcohols appeared to be excellent substrates for the extracellular aryl alcohol oxidases. The formed aldehydes were readily recycled via reduction by washed fungal mycelium, thus creating an extracellular H 2 O 2 production system regulated by intracellular enzymes. Lignin peroxidase does not oxidize the chlorinated anisyl alcohols both in the absence and in the presence of veratryl alcohol. It was therefore concluded that the chlorinated anisyl alcohols are well protected against the fungus's own aggressive ligninolytic enzymes. The relative amounts of veratryl alcohol and the chlorinated anisyl alcohols differ significantly depending on the growth conditions, indicating that the production of veratryl alcohol and the (chlorinated) anisyl metabolites are independently regulated.

    It was concluded that the chlorinated anisyl metabolites, biosynthesized by the white-rot fungus Bjerkandera sp. BOS55, are purposeful for ecologically significant processes such as lignin degradation. These results made us speculate if a significant biogenesis of chlorinated aromatics by fungi occurs in natural environments (chapter 7). Many common wood- and forest litter-degrading fungi were indeed detected that produced chlorinated anisyl metabolites (CAM). These compounds, which are structurally related to xenobiotic chloroaromatics, were present in the environment and occur at high concentrations of approximately 75 mg CAM kg -1wood or litter. The ubiquity among common fungi to produce large amounts of chlorinated aromatic compounds in the environment leads to the conclusion that these kind of compounds can no longer be considered to originate from anthropogenic sources only.

    Degradation of aryl alcohols by fungi. In chapter 2 the anabolic and catabolic routes of aryl alcohols by white-rot fungi has been reviewed. These fungi can not use veratryl alcohol as sole source of carbon and energy. However, several bacteria, yeasts and fungi were selectively isolated from paper mill waste water that grew on veratryl alcohol (chapter 8). Penicilliumsimplicissimum was selected for the characterization of the veratryl alcohol degradation route. P. simplicissimum oxidized veratryl alcohol via a NAD(P) +-dependent veratryl alcohol dehydrogenase to veratraldehyde which was further oxidized to veratric acid in a NAD(P) +-dependent reaction. Veratric acid-grown cells contained NAD(P)H-dependent O -demethylase activity for veratrate, vanillate and isovanillate. Ring-cleavage of protocatechuate was by a protocatechuate 3,4-dioxygenase. An interesting aspect of P. simplicissimum is the production of vanillyl alcohol oxidase with covalently bound FAD (chapter 9). The intracellular enzyme was purified 32-fold. SDS-PAGE of the purified enzyme revealed a single fluorescent band of 65 Kda. Gel filtration and sedimentation-velocity experiments indicated that the purified enzyme exists in solution as an octamer, containing 1 molecule flavin/subunit. The covalently bound prosthetic group of the enzyme was identified as 8α-(N 3-histidyl)FAD from pH dependent fluorescence quenching (p Ka = 4.85) and no decrease in fluorescence upon reduction with sodium borohydride. The enzyme showed a narrow and rather peculiar substrate specificity. In addition to vanillyl alcohol and 4-hydroxybenzyl alcohol, eugenol and chavicol are substrates for the enzyme (chapter 10). The formed products, coniferyl and coumaryl alcohol are the natural precursors of lignin in plants. This reaction has a potential application to produce coniferyl alcohol and subsequent synthetic lignin (DHP) from the inexpensive precursor eugenol.

    Characterisation and enzymic degradation of non-starch polysccharides in lignocellulosic by-products : a study on sunflower meal and palm-kernel meal
    Duesterhoeft, E.M. - \ 1993
    Agricultural University. Promotor(en): A.G.J. Voragen, co-promotor(en): W. Pilnik. - S.l. : Duesterhoeft - ISBN 9789054850762 - 134
    lignocellulose - lignine - zonnebloemolie - asteraceae - plantaardige oliën - palmpitolie - helianthus annuus - zonnebloemen - elaeis guineensis - oliepalmen - fermentatie - voedselbiotechnologie - bijproducten - koolhydraten - cellulose - celmembranen - celwanden - lignocellulose - lignin - sunflower oil - asteraceae - plant oils - palm kernel oil - helianthus annuus - sunflowers - elaeis guineensis - oil palms - fermentation - food biotechnology - byproducts - carbohydrates - cellulose - cell membranes - cell walls

    Non-starch polysaccharides (NSP) constitute a potentially valuable part of plant by- products deriving from the food and agricultural industries. Their use for various applications (fuel, feed, food) requires the degradation and modification of the complex plant materials. This can be achieved by enzymatic processes which, in comparison with chemical or physical methods, are regarded as energy-saving and non-polluting. However, a major disadvantage of enzymic processes often is their low effectivity and consequently high costs.

    The investigations described in this thesis were conducted to understand the reasons for the low susceptibility to enzymic hydrolysis of such by-products and, in particular, of their non-starch polysaccharides, and to find out whether and how the efficacy of enzymic treatment could be enhanced. The studies should provide information necessary for the development of polysaccharidase-preparations, tailored for the use in different applications.

    Sunflower ( Helianthus annuus L.) meal and palm-kernel ( Elaeis guineensis Jacq) meal, by-products from the production of edible oil and used as animal feed compounds, were chosen for our studies.

    In chapter 1 an introduction is given to the macroscopic and microscopic structure of the raw materials, to plant cell walls and their constituent polymers. The biodegradation of cell walls and its limitations are briefly reviewed and major non-starch polysaccharide degrading enzymes are summarised. Chapter 1 closes with an outline of the thesis.

    For a detailed study of type and structure of the non-starch polysaccharides, cell wall materials (CWM) were prepared from the meals by enzymatic digestion of protein and removal of small amounts of buffer-extractable material ( chapter 2 ). The resulting CWM's were enriched in NSP (55% of sunflower CWM and 75% of palm-kernel CWM) and had a high lignin content. Two different chemical methods, sequential extraction with alkali and sodium chlorite and treatment with 4-methylmorpholine-N-oxide (MMNO) were tested to extract all constituent polysaccharides. Almost complete dissolution could be achieved by a combination of both methods, but the recovery of sugars, especially during MMNO treatment, was low. From the sugar composition of polysaccharide fractions, obtained by sequential chemical extraction, a tentative identification of major polysaccharides was achieved. Their distribution in different botanical fractions of the meals could be deduced by comparison with data from literature (sunflower) or by own experiments (palm-kernel).

    The polysaccharide extracts of different purity were further fractionated by graded precipitation with ethanol, size-exclusion or adsorption-chromatography. By determination of the sugar- and glycosidic linkage composition of extracts, (partially) purified subfractions and intact cell wall materials, the identification, partial characterisation and quantification of major non-starch polysaccharides were achieved ( chapter 3 ). In sunflower meal, cellulose (42% of NSP), pectic polysaccharides (24%) and (4-O-methyl)-glucuronoxylans (24%) with about 10% glucuronosyl-substitution were major constituents. Minor amounts of (gluco)mannans (5%) and fucoxyloglucans (4.5%) were also identified. Major polysaccharides in palm-kernel meal were mannans (78% of NSP) with very low degrees of galactose-substitution and of apparently small molecular size (DP 12 to 14), and cellulose (12%). Arabinoxylans (3%) and (4- O -methyl)-glucuronoxylans (3%), deriving from the endocarp fraction of the meal, were present in low amounts in this monocotyledenous material.

    For a study of the enzymic hydrolysis of the cell wall materials ( chapter 4 ), three multi-component enzyme preparations were chosen. Solubilisation occurred as a bi-phasic process with high reaction velocities in the first stage of the incubation and only slow progress during extended incubation up to 72h. The solubilisation could markedly be improved by reduction in particle size; partial delignification or increasing enzyme concentration, however, had almost no effect. Maximally 30% of NSP in sunflower meal and 50% in palm-kernel meal could be solubilised from the finely milled CWM's. Although the composition of the enzyme preparations was found to influence the type of reaction products, the extent of their release and, as observed by transmission electron microscopy, the site of enzymic attack in different cell wall layers, our results suggested that substrate accessibility was the major factor limiting enzymic hydrolysis.

    A detailed study of the reaction products obtained by incubation with the crude enzyme preparations or fractions thereof (prepared by anion-exchange chromatography) revealed, that pectic compounds and mannose-containing polysaccharides in sunflower CWM were readily degradable ( chapter 5 ). The hydrolysis of mannans in palm-kernel CWM varied from 20% to 50%. In both CWM's, xylans and cellulose were most resistant to hydrolysis. The results indicate the preferential degradation of parenchyma and endosperm tissues and the resistance of hull and endocarp fractions to enzymic hydrolysis. The reaction products formed during all stages of the treatment were of small oligomeric and monomeric size.

    The contribution of different enzyme activities to the total solubilisation achieved by the heterogeneous enzyme preparations was studied with (partially) purified subfractions which were prepared by various chromatographic techniques from the crude preparations, and with highly purified enzymes from other microbial sources ( chapter 6 ). In general, the effect of these purified enzyme fractions was low (solubilisation of NSP: 1 % to 5 %). Supplementation of main enzyme fractions with pectolytic, cellulolytic or mannanolytic subfractions did only slightly enhance the total solubilisation. Synergistic action was observed between glucanases and mannanases in palm-kernel incubations and between arabinanases, polygalacturonases and rhamnogalacturonan-degrading enzyme fractions in the hydrolysis of pectic polysaccharides in sunflower CWM. The enzymic hydrolysis of (4- O -methyl)-glucuronoxylans was studied in-situ and with the isolated polysaccharide. The results indicated that the resistance of the xylans to enzymic degradation is not only due to their interlinkage with other polymers and location in the cell wall but also to their primary structure.

    In chapter 7 , important aspects concerning the approach and the methodology used are discussed. Implications arising for different fields of application are shown and suggestions for the formulation of enzyme preparations, which merit further research, are made.

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