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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.

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Targets and tools for optimizing lignocellulosic biomass quality of miscanthus
Weijde, R.T. van der - \ 2016
University. Promotor(en): Richard Visser, co-promotor(en): Luisa Trindade; Oene Dolstra. - Wageningen : Wageningen University - ISBN 9789462578388 - 231 p.
miscanthus - bioethanol - biomass - biofuels - lignocellulose - fuel crops - plant breeding - cell walls - cell wall components - genetic diversity - genetic variation - biomass conversion - biobased economy - biomassa - biobrandstoffen - brandstofgewassen - plantenveredeling - celwanden - celwandstoffen - genetische diversiteit - genetische variatie - biomassaconversie

Miscanthus is a perennial energy grass characterized by a high productivity and resource-use efficiency, making it an ideal biomass feedstock for the production of cellulosic biofuels and a wide range of other biobased value-chains. However, the large-scale commercialization of converting biomass into cellulosic biofuel is hindered by our inability to efficiently deconstruct the plant cell wall. The plant cell wall is a complex and dynamic structure and its components are extensively cross-linked into an unyielding matrix. The production of biofuel depends on the extraction, hydrolysis and fermentation of cell wall polysaccharides, which currently requires energetically and chemically intensive processing operations that negatively affect the economic viability and sustainability of the industry. To address this challenge it is envisioned that the bioenergy feedstocks can be compositionally tailored to increase the accessibility and extractability of cell wall polysaccharides, which would allow a more efficient conversion of biomass into biofuel under milder processing conditions.

Extensive phenotypic and genetic diversity in cell wall composition and conversion efficiency was observed in different miscanthus species, including M. sinensis, M. sacchariflorus and interspecific hybrids between these two species. In multiple experiments a twofold increase in the release of fermentable sugars was observed in ‘high quality’ accessions compared to ‘low quality’ accessions. The exhaustive characterization of eight highly diverse M. sinensis genotypes revealed novel and distinct breeding targets for different bioenergy conversion routes. The key traits that contributed favourably to the conversion efficiency of biomass into biofuel were a high content of hemicellulosic polysaccharides, extensive cross-linking of hemicellulosic polysaccharides (revealed by a high content of trans-ferulic acids and a high ratio of arabinose-to-xylose), a low lignin content and extensive incorporation of para-coumaric acid into the lignin polymer.

Lignin is widely recognized as one of the key factors conveying recalcitrance against enzymatic deconstruction of the cell wall. The incorporation of para-coumaric acid into the lignin polymer is hypothesized to make lignin more easily degradable during alkaline pretreatment, one of the most widely applied processing methods that is used to pretreat biomass prior to enzymatic hydrolysis. Previous studies have shown that reducing lignin content is often implicated in reduced resistance of plants to lodging. We hypothesize that extensively cross-linked hemicellulosic polysaccharides may fulfil a similar function in supporting cell wall structural rigidity and increasing the content of hemicellulosic polysaccharides may be a way to reduce lignin content without adversely affecting cell wall rigidity. This strategy can be used to improve biomass quality for biobased applications, as hemicellulosic polysaccharides are more easily degradable during industrial processing than lignin. Furthermore, hemicellulosic polysaccharides adhere to cellulose, which negatively affects the level of cellulose crystallinity. Crystalline cellulose is harder to degrade than its more amorphous form. Therefore the reduction of cellulose crystallinity is another mechanism through which increasing the content of hemicellulosic polysaccharides positively contributes to cell wall degradability. These results provided new insights into the traits that may be targeted to improve the quality of lignocellulose feedstocks.

However, evaluation of complex biochemical traits for selection purposes is hindered by the fact that their accurate quantification is a costly, lengthy and laborious procedure. To overcome these limitations an accurate and high-throughput method was developed based on near-infrared spectroscopy. Through extensive calibration we developed accurate prediction models for a wide range of biomass quality characteristics, which may be readily implemented as a phenotyping tool for selection purposes.

Additionally, progress through breeding may substantially be improved by marker-assisted selection, which will reduce the need for the evaluation of genotype performance in multi-year field trials. To this end, a biparental M. sinensis mapping population of 186 individuals was developed and genotyped using a genotyping-by-sequencing approach. A total of 564 short-sequence markers were used to construct a new M. sinensis genetic map. Cell wall composition and conversion efficiency were observed to be highly heritable and quantitatively inherited properties. This is the first genetic study in miscanthus to map quantitative trait loci (QTLs) for biomass quality properties and is a first step towards the application of marker-assisted selection for biomass quality properties.

Through the evaluation of a diverse set of miscanthus genotypes in multiple locations we demonstrated that in addition to genotypic variation, growing conditions may have a substantial influence on cell wall composition and conversion efficiency. While further research is needed to identify which specific environmental parameters are responsible for the observed effects, these results clearly indicate that the environmental influence on biomass quality needs to be taken into account in order to match genotype, location and end-use of miscanthus as a lignocellulose feedstock. Moreover, significant genotype-by-environment interaction effects were observed for cell wall composition and conversion efficiency, indicating variation in environmental sensitivity across genotypes. Although the magnitude of the genotypic differences was small in comparison to genotype and environmental main effects, this affected the ranking of accession across environments. Stability analysis indicated some stable accessions performed relatively across diverse locations.

In addition to trialing miscanthus in diverse locations, we also evaluated miscanthus biomass quality under drought conditions for a number of reasons: 1) drought stress is linked to a differential expression of cell wall biosynthesis genes, 2) incidence of drought events is increasing due to climate change, 3) irrigation is likely to be uneconomical during the cultivation of miscanthus and 4) miscanthus has many characteristics that make it a crop with a good potential for cultivation on marginal soils, where abiotic stresses such as drought may prevail. Drought stress was shown to result in a large reduction in cell wall and cellulose content and a substantial increase in hemicellulosic polysaccharides and cellulose conversion rates. We hypothesized that the reduction in cellulose content was due to an increase in the production of osmolytes, which are well-known for their role in plant protection against drought. The results indicated that drought stress had a positive effect on the cell wall degradability of miscanthus biomass.

Overall the compendium of knowledge generated within the framework of this thesis provided insights into the variation in biomass quality properties in miscanthus, increased our understanding of the molecular, genetic and environmental factors influencing its conversion efficiency into biofuel and provided tools to exploit these factors to expand the use of miscanthus as a lignocellulose feedstock.

Biorefinery of leafy biomass using green tea residue as a model material
Zhang, C. - \ 2016
University. Promotor(en): Johan Sanders, co-promotor(en): Marieke Bruins. - Wageningen : Wageningen University - ISBN 9789462576902 - 156 p.
biorefinery - biomass conversion - leaves - biomass - green tea - tea - alkaline pulping - pectins - lignocellulose - environmental impact - processes - plant protein - food - biobased economy - bioraffinage - biomassaconversie - bladeren - biomassa - groene thee - thee - alkalische pulpbereiding - pectinen - milieueffect - processen - plantaardig eiwit - voedsel


With the rapidly growing world population and improving living standards, food demand is increased with a simultaneous desire for less human impact on the environment, such that “Twice the food production at half the ecological footprint” could be the EU goal for 2050. In fact, a boost in food demand is mainly required in developing countries, where the farmlands are limited and/or they are of poor quality. Rather than improving crop-production yield, developing biorefinery technology with unused biomass, such as leaves, in developing countries may be the key to fulfil the food demand.

Four major components, protein, pectin, lignin, and (hemi-) cellulose, account for more than 70% of the materials in leaves in almost all species. Among these components, protein and pectin can be used in food and animal feed, and they are key components for supplementing food production. However, the production and application of leaf products is limited for four reasons: unstable raw materials, complex components, rigid plant cell walls, and underdeveloped leaf logistics and economics. The limitations cause low pectin and protein yields, and low cost-efficiency in current extraction technologies, including mechanical milling, chemical extraction (acid and alkaline), solvent extraction, and ammonia protein extraction. Development of an integrated process for multiple products might be a good option for leaf biorefinery, but the compatibilities of these processes were unknown.

The aim of this study was to develop new processes and applications that optimally utilize all components, particularly protein, of leafy biomass in the feed and/or food industry using green tea residues as a starting material. The method should also be applicable to other leafy biomass. The research started from the development of alkaline protein extraction technology as presented in Chapter 2. We found that in alkaline protein extraction, temperature, NaOH amount, and extraction time are the parameters determining protein yield, while pH and volume of extraction liquid are critical parameters for production cost. After optimization, more than 90% of leaf protein could be extracted at a cost of 102€/ton protein by single step alkaline extraction. The extracted protein nutritional value was comparable to soybean meal and this technique can be adapted to various leafy biomass. Main drawback of this technique is the overuse of alkali, generation of salts, and the destruction of key amino acids, such as lysine, during the extraction. We tried to overcome its drawbacks by developing integrated process with a recycle for chemicals.

Chapter 3, 4, 5, and 6 refer to the integrated biorefinery. For a better design, we investigated how the alkali aided protein extraction (Chapter 3), and proved that alkaline protein extraction was not facilitated by increased solubility or hydrolysis of protein, but positively correlated to leaf tissue disruption. HG pectin, RGII pectin, polyphenols, and organic acids can be extracted before protein. Protein extraction can then be followed by the extraction of cellulose and hemi-cellulose. RGI pectin and lignin yield were both linearly correlated to protein yield, which indicated that they are likely to be the key limitation to leaf protein extraction. Based on the above findings, an integrated biorefinery that combined protein extraction with a pre-treatment was proposed. In Chapter 4, ethanol, viscozyme, and H2O2 were selected for pre-treatments targeting on the removal of polyphenols and pigments, carbohydrates, and lignin accordingly. Ethanol and viscozyme could extract their targeting components efficiently while H2O2 could bleach GTR with no lignin extracted. The best pre-treatment was the combination of viscozyme and 50% ethanol extraction, which not only reduced the use of alkali by 50%, but also improved protein content and its nutritional value. As pectin can be applied for food or chemicals, enzyme and PBS buffer were investigated for pectin extraction (Chapter 5). Both enzyme and PBS buffer extraction could not only extract high yield HG pectin (predominated by galacturonic acid) with no protein extraction, but also reduced alkali usage in subsequent protein extraction. Pectin obtained using PBS buffer could be present in its native form, which can be precipitated by 40% ethanol. Buffer is suggested to extract pectins when pectins are to be used in food. Otherwise, hydrolyzed pectin that mainly contains galacturonic acid, can be converted to other useful chemicals. For this the enzymatic methods, such as using Viscozyme® L, are recommended.

Alkali usage was further optimized. It was found that by using potassium hydroxide, the protein extraction efficiency was similar to that using sodium hydroxide. The waste water, mainly containing potassium salts, can then be used as fertilizer. This technique is highly depending on the location of factories, which should be built close to the field. Alternatively, calcium hydroxide can be used. As calcium salts can be precipitated by CO2 and calcium hydroxide can be regenerated through burning of the precipitate, this scheme is sustainable and adaptable to most situations. However, as calcium also precipitated pectin, ployphenols, and even proteins, the protein yield is relatively low. Although a pre-treatment can improve extraction efficiency of calcium hydroxide, economic results suggested that a pre-treatment is not necessary unless the products obtained by pre-treatment have an attractive market value.

In Chapter 7, we extend our knowledge on leaf biorefinery with some additional experiments and literature. Simplified models of leaf tissues and cell walls were proposed and used to explain the mechanism of alkaline protein extraction. The models were also used to explain other mechanisms for protein extraction; mechanical milling, steam explosion, acid, and enzyme aided extraction. The possible improvements of leaf biorefinery economics were illustrated either by reducing production cost, by e.g. using counter current extraction or ultrafiltration, or by upgrading product value by applying protein and pectin in food. The processes recommended in this thesis show an excellent prospective, in which they are applicable to other leaf biomass and suitable for small-scale production.

Development of a lactic acid production process using lignocellulosic biomass as feedstock
Pol, E.C. van der - \ 2016
University. Promotor(en): Gerrit Eggink, co-promotor(en): Ruud Weusthuis. - Wageningen : s.n. - ISBN 9789462576735 - 167 p.
lignocellulose - biomass - biobased chemicals - sugarcane bagasse - lactic acid - sugars - byproducts - inhibitors - pretreatment - bacillus coagulans - furfural - saccharification - fermentation - quantitative techniques - production processes - biomassa - chemicaliën uit biologische grondstoffen - suikerrietbagasse - melkzuur - suikers - bijproducten - remmers - voorbehandeling - versuikering - fermentatie - kwantitatieve technieken - productieprocessen

The availability of crude oil is finite. Therefore, an alternative feedstock has to be found for the production of fuels and plastics. Lignocellulose is such an alternative feedstock. It is present in large quantities in agricultural waste material such as sugarcane bagasse.

In this PhD thesis, lignocellulose is chemically and enzymatically pretreated to depolymerise sugars present in this structure. The released sugar monomers are fermented by micro-organisms to lactic acid, which is a precursor for the bioplastic PLA. In this thesis, it is shown that it is possible to produce lactic acid from lignocellulosic biomass with high yields and high productivities.

Pretreatment of lignocellulose for biotechnological production of lactic acid
Harmsen, P.F.H. ; Lips, S.J.J. ; Bakker, R.R.C. - \ 2013
Wageningen UR FBR (Rapport / Wageningen UR Food & Biobased Research 1384, public version) - ISBN 9789461736079 - 104
voorbehandeling - biomassaconversie - lignocellulose - melkzuur - bioproceskunde - technologie - suikerriet - kosten - kostenanalyse - biobased economy - pretreatment - biomass conversion - lactic acid - bioprocess engineering - technology - sugarcane - costs - cost analysis
The breakdown of biomass in pretreatment facilitates enzymatic hydrolysis by disrupting cell wall structures, driving lignin into solution or modification of the lignin structure, and reducing cellulose crystallinity and chain length, while preventing hydrolysis of cellulose. In an ideal situation the pretreatment leads to high yields of fermentable sugars with a limited formation of degradation products that inhibit enzymatic hydrolysis and fermentation to lactic acid, while remaining cost effective. This review deals with these challenges by providing information on available pretreatment technologies in general (chapter 3), and more specific on pretreatment of the model feedstock sugarcane bagasse (chapter 4). Techno economic studies are described in chapter 5 with the NREL study from 2011 as benchmark. This review starts with characteristics of lignocellulosic biomass in relation to compostion and formation of inhibitors.
Lignin as a renewable aromatic resource for the chemical industry
Gosselink, R.J.A. - \ 2011
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 - 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.

Uit elkaar geplukt is stro goud waard
Harmsen, P.F.H. - \ 2010
Kennis Online 7 (2010)dec. - p. 9 - 9.
stro - maïs - lignocellulose - biobrandstoffen - biobased economy - bioethanol - biomassaconversie - straw - maize - biofuels - biomass conversion
Het onderzoek van Paulien Harmsen van het instituut Food & Biobased Research laat zien dat er meer mogelijk is met stro.
Bioraffinage : naar een optimale verwaarding van biomassa
Annevelink, E. ; Harmsen, P.F.H. - \ 2010
Wageningen : Wageningen UR - Food & Biobased Research (Groene Grondstoffen 10) - ISBN 9789085857617 - 42
chemische industrie - vervangbare hulpbronnen - nederland - biomassa - biomassa productie - lignocellulose - biobased economy - bioraffinage - productieprocessen - biomassaconversie - aquatische biomassa - chemical industry - renewable resources - netherlands - biomass - biomass production - biorefinery - production processes - biomass conversion - aquatic biomass
Dit boekje is geschreven met als doel bioraffinage meer bekendheid te geven. In hoofdstuk 1 is de Biobased Economy beschreven en de sleutelrol die bioraffinage daarin speelt, met een focus op Nederland. Bioraffinage is een zeer brede term en een classificatiesysteem is noodzakelijk; dit is beschreven in hoofdstuk 2. In de hoofdstukken 3 en 4 wordt vervolgens dieper ingegaan op de grondstof voor bioraffinage, de biomassa, en de verschillende conversiemethoden die kunnen worden toegepast voor de verwerking van biomassa tot waardevolle producten. Vervolgens wordt in de hoofdstukken 5 t/m 7 een aantal voorbeelden van bioraffinage beschreven. Dit boekje wordt afgesloten met een aantal aspecten die van belang zijn voor de verdere ontwikkeling van bioraffinage in de toekomst.
Literature review of physical and chemical pretreatment processes for lignocellulosic biomass
Harmsen, P.F.H. ; Huijgen, W. ; Bermudez, L. ; Bakker, R. - \ 2010
Wageningen : Wageningen UR - Food & Biobased Research (Report / Wageningen UR, Food & Biobased Research 1184) - ISBN 9789085857570 - 54
literatuuroverzichten - lignocellulose - biomassa - voorbehandeling - bio-energie - biomassaconversie - biobased economy - literature reviews - biomass - pretreatment - bioenergy - biomass conversion
Different pretreatment technologies published in public literature are described in terms of the mechanisms involved, advantages and disadvantages, and economic assessment. Pretreatment technologies for lignocellulosic biomass include biological, mechanical, chemical methods and various combinations thereof. The choice of the optimum pretreatment process depends very much on the objective of the biomass pretreatment, its economic assessment and environmental impact. Only a small number of pretreatment methods has been reported as being potentially cost-effective thus far. These include steam explosion, liquid hot water, concentrated acid hydrolysis and dilute acid pretreatments.
Biobutanol : butanol from cellulosic biomass
Bakker, R.R. ; Lopez Contreras, A.M. ; Claassen, P.A.M. ; Merck, K.B. ; Willemsen, J.H.A. ; Mozaffarian, H. ; Uil, H. den; Pels, J. ; Reith, H. - \ 2010
bio-energie - cellulose - fermentatie - butanol - clostridium - lignocellulose - biomassa - biobased economy - biobrandstoffen - bioenergy - fermentation - biomass - biofuels
Poster met onderzoeksinformatie over de vebetering van het ABE (aceton, butanol, ethanol) fermentatieproces. Het doel van dit onderzoek is om de productie van biobutanol uit biomassa dat cellulose bevat, rendabel te maken.
Industrial development : biofuels for transportation
Bakker, Rob - \ 2009
rice straw - lignocellulose - transport - diesel oil - biobased economy - biofuels - bioethanol - future
Lignocellulosic ethanol in Brazil : technical assessment of 1st and 2nd generation sugarcane ethanol in a Brazilian setting
Stojanovic, M. ; Bakker, R.R.C. - \ 2009
Wageningen : Agrotechnology and Food Innovations - 18
suikerriet - ethanol - lignocellulose - sucrose - fermentatie - technologie - brazilië - bioethanol - biobrandstoffen - sugarcane - fermentation - technology - brazil - biofuels
Brazil is currently the largest ethanol-biofuel producer worldwide. Ethanol is produced by fermenting the sucrose part of the sugarcane that contains only one third of the sugarcane energy. The rest of the plant is burned to produce energy to run the process and to generate electricity that is sold to the public grid, making the process a net energy producer. This paper evaluates current technology from an energy efficiency point of view and quantifies additional benefits from extra energy generated in during sugarcane processing.
Bioethanol uit lignocellulose
Bakker, R.R.C. ; Bos, H.L. - \ 2009
bio-energie - lignocellulose - bioethanol - biobrandstoffen - biobased economy - bioenergy - biofuels
Deze info sheet geeft een overzicht van ontwikkelingen op het gebied van productie van bioethanol, een hernieuwbare transportbrandstof, uit lignocellulose. Lignocellulose is een verzamelnaam voor alle vezelhoudende biomassa waaronder zowel houtgewassen, grassen als reststromen vallen.
Microbial conversion of lignocellulose-derived carbohydrates into bioethanol and lactic acid
Maas, R.H.W. - \ 2008
University. Promotor(en): Gerrit Eggink, co-promotor(en): Ruud Weusthuis. - [S.l.] : S.n. - ISBN 9789085048718 - 158
tarwestro - lignocellulose - lignocellulosehoudend afval - microbiële afbraak - conversie - melkzuur - fermentatie - hydrolyse - bio-energie - bioethanol - wheat straw - lignocellulosic wastes - microbial degradation - conversion - lactic acid - fermentation - hydrolysis - bioenergy
Houtachtige biomassa (rest)stromen kan één van de duurzame alternatieven gaan worden voor aardolie omdat het kan dienen als grondstof voor de productie van biobrandstoffen en bulkchemicaliën. Belangrijk voordeel van deze technologie is dat er geen gebruik hoeft te worden gemaakt van plantaardige producten die geschikt zijn voor voedseldoeleinden. Binnen het huidige onderzoek zijn we erin geslaagd om op grote schaal houtachtige biomassa zoals stro via verschillende fysisch-chemische behandelingen en een enzymatische hydrolyse af te breken tot enkelvoudige suikers. Deze suikers werden door verschillende soorten micro-organismen omgezet naar gewenste producten zoals ethanol voor biobrandstoftoepassing of melkzuur als bouwsteen voor de productie van biologisch afbreekbaar plastic. De delen van het stro welke niet door de micro-organismen konden worden omgezet werden gebruikt voor biogasproductie of fungeerden als brandstof voor het verkrijgen van warmte en elektriciteit. Een productieproces waarbij, via een combinatie van verschillende voorbehandelingen, stro door bacteriën efficiënt werd omgezet naar melkzuur werd gepatenteerd.
Bio-ethanol: nu en in de toekomst
Bakker, Rob - \ 2005
contemporary society - fuels - lignocellulose - biofuels - bioethanol - future - biobased economy
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 - maize - mutations - nutritive value - plant breeding - quality - ruminants
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
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 - lignin - sunflower oil - plant oils - palm kernel oil - sunflowers - oil palms - fermentation - food biotechnology - byproducts - carbohydrates - cell membranes - cell walls
<p>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.<p>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.<p>Sunflower ( <em>Helianthus annuus</em> L.) meal and palm-kernel ( <em>Elaeis guineensis</em> Jacq) meal, by-products from the production of edible oil and used as animal feed compounds, were chosen for our studies.<p>In <strong>chapter 1</strong> 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.<p>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 ( <strong>chapter 2</strong> ). 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).<p>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 ( <strong>chapter 3</strong> ). 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- <em>O</em> -methyl)-glucuronoxylans (3%), deriving from the endocarp fraction of the meal, were present in low amounts in this monocotyledenous material.<p>For a study of the enzymic hydrolysis of the cell wall materials ( <strong>chapter 4</strong> ), 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.<p>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 ( <strong>chapter 5</strong> ). 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.<p>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 ( <strong>chapter 6</strong> ). 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- <em>O</em> -methyl)-glucuronoxylans was studied <em>in-situ</em> 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.<p>In <strong>chapter 7</strong> , 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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