Enzymatic modification of the functional properties of xylan from lignocellulose feedstocks

Gomes, Katiana Raquel da Gama (2012-12)

Thesis (MScEng)--Stellenbosch University, 2012.

Thesis

ENGLISH ABSTRACT: In the past decades, sustainable alternatives to petroleum-derived products have been explored. While fossil derived products are still the main source of energy and chemicals worldwide, they are the major contributors to the increased emission of green house gasses (GHG’s), responsible for the climate change. Lignocellulose represents a more sustainable alternative since it is biodegradable, renewable and does not contribute to GHG emissions to the same extent as fossil-based resources. Hemicelluloses are the second most abundant class of polysaccharide biopolymers on earth after cellulose, based on widespread availability in nature of the lignocellulosic plant biomass in which they occur. Xylan represents between 15 and 30% of lignocellulose in hardwood plant species, between 25-35% in grasses and in lower proportions in softwood (between 7 and 12%). In pulp and paper industries, xylan is dissolved and separated from cellulose along with the lignin under harsh pulping conditions, and subsequently burned as black liquor for energy generation. This process represents an under-utilisation of hemicellulose feedstocks, due to its low specific heating value (13.6 MJ/kg) compared with lignin (27 MJ/kg). However, the xylans that can be extracted from lignocellulose, either during pulping or in dedicated processes, have limited applications due to low functionality as a biopolymer, mainly high solubility in water. The objective of this study was to investigate the enzymatic hydrolysis of xylans, extracted from different lignocellulosic feedstocks available in South Africa and to find optimum conditions for modifying chemical and functional properties of the xylans for industrial applications. The glucuronoxylan from Eucalyptus grandis was extracted using protocols adopted from Höije et al. (Höije et al., 2005) and Pinto et al. (Pinto et al., 2005). The arabinoglucuronoxylan from sugarcane bagasse was extracted following the protocol adopted from Höije et al. (Höije et al., 2005). Beechwood xylan (Sigma) was used as the model xylan. The xylan was extracted from E. grandis and sugarcane bagasse using the Höije protocol with yields of 20 and 71% and uronic acid contents of 21 and 7.05%, respectively. The molecular weight distribution showed that the major fraction had a degree of polymerization of 287 in E. grandis xylan (Höije). The xylan was extracted from E. grandis by the Pinto protocol with a yield of 89%. However, the degree of polymerization was 133 due to polysaccharide degradation. The model xylan from beechwood had the highest purity levels in terms of xylose content but the lowest degree of polymerization, corresponding to 77. Selective removal of arabinose and 4-O-methyl glucuronic acid by the enzymes α-L-arabinofuranosidase and α-D-glucuronidase, respectively, caused formation of water-insoluble xylan particles. Partially purified α-D-glucuronidase at concentrations between 416 and 462 mg/L were used in the selective enzymatic hydrolysis of 4-O-methyl glucuronic acid side-groups in xylans extracted from different lignocellulosic feedstocks. The minimum time required for selective hydrolysis of the glucuronoxylans at concentrations ranging from 1.1 to 5.0% (w/v) in study was 24 hours. Consequently, the minimum degree of substitution required for the precipitation of beechwood xylan, E. grandis xylan (Höije) and E. grandis xylan (Pinto) was 1:40, 1:6 and 1:25, respectively. The highest release of 4-O-methyl glucuronic acid was found at α-D-glucuronidase dosage of 6.4 mg/g combined with 1.87% (w/v) substrate in beechwood xylan, 3.08% (w/v) in E. grandis xylan (Hoije) and 5.03% (w/v) in E. grandis xylan (Pinto). Optimum conditions for increase in viscosity were found at an enzyme dosage of 6.4 mg/g combined with 4.98% (w/v) of substrate in beechwood xylan, 4.3% (w/v) in E. grandis xylan (Höije) and 4.5% (w/v) in E. grandis xylan (Pinto). The most significant factor for the release of 4-O-MeGlcA was the α-D-glucuronidase dosage (p-value < 0.03), whereas the substrate concentration (p < 0.006) was the most significant factor to maximise viscosity. Enzymatic hydrolysis with both α-D-glucuronidase and α-L-arabinofuranosidase for release of 4-O-methyl glucuronic acid and arabinose from sugarcane bagasse xylan resulted in precipitation. A positive synergy was verified in the release of both side-chains, which was dependent on the dosage ratio between α-L-arabinofuranosidase and α-D-glucuronidase. Optimum conditions for precipitation were found at a dosage of 6.4 mg/g of α-D-glucuronidase and 150 nkat/g of α-L-arabinofuranosidase, with 25% precipitation. On the other hand, the highest release of side-chains was verified at a dosage of 6.4 mg/g of α-D-glucuronidase and 350 nkat/g of α-L-arabinofuranosidase, with 69.5% release of arabinose and 24% release of 4-O-MeGlcA. Morphological analysis of the modified xylans indicated that enzymatic treatment improved both the gelling and plasticising properties. Removal of 4-O-methyl glucuronic acid from glucuronoxylans resulted in increased viscosity, and formation of hydrogels. Arabinose and 4-O-methyl glucuronic acid removal from arabinoglucuronoxylan also resulted in formation of insoluble xylan particles that settled with gravity. The highest viscosity rheological properties was observed with E. grandis xylan (Höije), followed by the model xylan from beechwood. Particle agglomeration was more evident on less substituted glucuronoxylans with a minimum degree of substitution of 4-O-MeGlcA of 3%. The micro particles were found to increase in size with an increase in the xylan concentration, from a minimum concentration of 4.45% (w/v), during enzymatic hydrolysis. The glucuronoxylan hydrogels and arabinoglucuronoxylan insoluble particles formed at optimum conditions had particles sizes ranging from 0.4 to 1.97 μm. It was concluded that the enzymatic modification is a mild and selective chemical process that can add functionality to beechwood, E. grandis and sugarcane bagasse xylans, by reducing their solubility in water and can be further applied as pulp additives, paper coatings, packaging films and gel encapsulation matrices.

AFRIKAANSE OPSOMMING: Gedurende die laaste paar dekades is ondersoek ingestel na die gebruik van volhoubare alternatiewe vir petroleum gebasseerde produkte. Alhoewel produkte afkomstig van fossielbrandstof steeds die hoof bron van energie en chemikalieë wêreldwyd is, lewer hierdie produkte ook die grootste bydrae tot die verhoogde vrystelling van kweekhuisgasse (KHG’e) wat verantwoordelik is vir klimaatsverandering. Lignosellulose is ‘n meer volhoubare alternatief omdat dit biodegradeerbaar en hernubaar is en ook minder bydra tot KHG emissies as fossielbrandstof gebasseerde hulpbronne. Hemisellulose is die tweede volopste klas van polisakkaried biopolimere op aarde na sellulose, weens die wydverspreide voorkoms van lignosellulose plantaardige biomassa waarin dit in die natuur voorkom. Xilaan verteenwoordig tussen 15 en 30% van lignosellulose in hardehout plantspesies, tussen 25 en 35% in grasse en laer proporsies in sagtehout (tussen 7 en 12%). In die pulp en papier industrieë word xilaan opgelos en geskei van sellulose en lignien deur strawwe verpulpingskondisies, en word daarna verbrand as “swart vloeistof” vir energieproduksie. Hierdie proses verteenwoordig ‘n nie-optimale gebruik van hemisellulose toevoermateriale weens die lae spesifieke verhittingswaarde (13.6 MJ/kg) daarvan in vergelyking met dié van lignien (27 MJ/kg). Die xilaan wat wel geëkstraheer kan word uit lignosellulose deur verpulping of toegewyde xilaan ekstraksie prosesse, het beperkte toepassings. Die rede hiervoor is die lae funksionaliteit daarvan as biopolimeer weens die hoë oplosbaarheid daarvan in water. Die doelwit van hierdie studie was om die ensiematiese hidrolise van xilane (geëkstraheer uit verskillende lignosellulose toevoermateriale beskikbaar in Suid-Afrika) te ondersoek. Verder ook om die optimale kondisies te bepaal vir die ensiematiese wysiging van die chemiese en funksionele eienskappe van xilane vir gebruik in industriële toepassings. Die glukuronoxilaan van Eucalyptus grandis is geëkstraheer deur gebruik te maak van metodes aangepas uit Höije et al. (2005) en Pinto et al. (2005). Die arabinoglukuronoxilaan afkomstig van suikerriet bagasse is geëkstraheer volgens die protokol aangepas uit Höije et al. (2005). Boekenhout xilaan (Sigma) is gebruik as ‘n xilaan model. Die xilaan is geëkstraheer uit E. grandis en suikerriet bagasse volgens die Höije protokol en het ‘n geëkstraheerde xilaan opbrengs van 20 en 71% en ‘n uroonsuurinhoud van 21 en 7.05% onderskeidelik gelewer. Die molekulêre gewigsverspreiding het getoon dat die hooffraksie in E. grandis xilaan ‘n graad van polimerisasie van 287 het (volgens die Höije protokol). Die xilaan is geëkstraheer uit E. grandis volgens die Pinto protokol met ‘n opbrengs van 89%, maar die graad van polimerisasie was 133 weens polisakkaried degradasie. Die model xilaan uit boekenhout het die hoogste suiwerheidsvlak gehad in terme van xilose inhoud maar ook die laagste graad van polimerisasie, naamlik 77. Die selektiewe verwydering van syketting arabinose en 4-O-metielglukuroonsuur groepe deur die ensieme α-L-arabinofuranosidase en α-D-glukuronidase respektiewelik, het gelei tot die vorming van water-onoplosbare xilaan partikels. Gedeeltelik gesuiwerde α-glukuronidase in konsentrasies tussen 416 en 462 mg/L is gebruik vir die selektiewe hidrolise van 4-O-metielglukuroonsuur sykettings in xilane geëkstraheer uit verskillende lignosellulose toevoermateriale. Hierdie studie het vasgestel dat 24 uur die minimum tyd is wat benodig word vir die selektiewe hidrolise van die glukuronoxilane by substraatkonsentrasies wat varieer van 1.1 tot 5.0%. Gevolglik was die minimum graad van substitusie vereis vir die presipitasie van boekenhout xilaan, E. grandis xilaan (volgens die Höije protokol) en E. grandis xilaan (volgens die Pinto protokol) 1:40, 1:6 en 1:25 onderskeidelik. Die hoogste vrystelling van 4-O-metielglukuroonsuur sykettinggroepe het voorgekom by ‘n ensiemdosering van 6.4 mg/g in kombinasie met 1.87% (w/v) substraat in boekenhout xilaan, 3.08% (w/v) in E. grandis xilaan (volgens die Höije protokol) en 5.03% (w/v) in E. grandis xilaan (volgens die Pinto protokol). Optimale kondisies vir ‘n toename in viskositeit is verkry by ‘n ensiemdosering van 6.4 mg/g in kombinasie met 4.98% (w/v) substraat in boekenhout xilaan, 4.3% (w/v) in E. grandis xilaan (volgens die Höije protokol) en 4.5% (w/v) in E. grandis xilaan (volgens die Pinto protokol). Die mees noemenswaardige faktor vir die vrystelling van 4-O-metielglukuroonsuur was die α-D-glukuronidase dosering (p-waarde < 0.03), en vir die maksimalisering van die reologiese viskositeitseienskappe was dit die substraatkonsentrasie (p < 0.0006). Ensiematiese hidrolise met beide α-D-glukuronidase en α-L-arabinofuranosidase vir die vrystelling van 4-O-metielglukuroonsuur en arabinose uit suikerriet bagasse xilaan het gelei tot presipitasie. ‘n Positiewe sinergie in die vrystelling van beide sykettings is aangetoon. Hierdie sinergie was afhanklik van die doseringsverhouding van α-L-arabinofuranosidase tot α-D-glukuronidase. Optimale kondisies vir ‘n toename in viskositeit is gevind by ‘n dosering van 6.4 mg/g α-glukuronidase en 150 nkat/g α-arabinofuranosidase met 25% presipitasie. Die hoogste vrystelling van sykettings is waargeneem by ‘n dosering van 6.4 mg/g α-glukuronidase en 350 nkat/g α-arabinofuranosidase , met 69.5% vrystelling van arabinose en 24% vrystelling van 4-O-metielglukuroonsuur. Morfologiese analise van die gewysigde xilane het aangetoon dat ensiematiese behandeling beide die gelvormings- en plastisiseringseienskappe daarvan verbeter het. Die verwydering van 4-O-metielglukuroonsuur sykettings vanaf die glukuronoxilane het tot ‘n verhoogde viskositeit gelei asook die vorming van hidrojels. Arabinose en 4-O-metielglukuroonsuur verwydering uit arabinoglukuronoxilaan het gelei tot die vorming van onoplosbare xilaan partikels wat onder swaartekrag uitgesak het. Die hoogste reologiese viskositeitseienskappe is waargeneem by E. grandis xilaan (geëkstraheer volgens Höije), gevolg deur die model xilaan uit boekenhout. Partikel agglomerasie was meer duidelik by die minder gesubstitueerde glukuronoxilane met ‘n minimum graad van 4-O-metielglukuroonsuur substitusie van 3%. Daar is gevind dat die mikro-partikels toegeneem het in grootte met ‘n toename in xilaan konsentrasie, vanaf ‘n minimum konsentrasie van 4.45% (w/v) gedurende ensiematiese hidrolise. Die glukuronoxilaan hidrojels en arabinoglukuronoxilaan onoplosbare partikels gevorm by die optimale kondisies het partikelgroottes gehad wat varieer van 0.4 tot 1.97 μm. Die ensiematiese modifikasie van xilaan is dus ‘n matige en selektiewe proses wat die funksionaliteit van boekenhout, E. grandis en suikerriet bagasse xilane kan verbeter deur hul oplosbaarheid in water te verlaag. Sodoende kan hierdie gemodifiseerde xilane verder toegepas word as verpulpingstoevoegings, papierbedekkings, verpakkingsfilms en jel inkapselingsmatrikse.

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