At present, the United States generates biofuels (ethanol) from corn grain. Unfortunately, low crop yields and limited growth regions result in limited availability. Furthermore, the use of staple food crops for ethanol production has generated a highly controversial food vs. fuel debate. Because of its high abundance and relatively low cost, lignocellulosic biomass is a promising alternative feedstock for biofuel production; however, structural features of lignocellulose limit accessibility of enzymes or microorganisms. These structural barriers include high lignin content, acetyl groups on hemicellulose, high cellulose crystallinity, cellulose degree of polymerization, and small pore volume. To overcome these barriers, a variety of pretreatment processes (chemical and mechanical) have been developed. Oxidative-lime pretreatment (OLP) is highly effective at reducing lignin content and removing acetyl groups from hemicellulose. Combining OLP with a mechanical treatment process greatly enhances the enzymatic digestibility of lignocellulose. Recommended OLP conditions were determined for Dacotah (120 °C, 6.89-bar O2, 240 min) and Alamo (110 °C, 6-89-bar O2, 240 min) switchgrass. Using recommended conditions, 72-h glucan digestibilities (g glucan hydrolyzed/100 g glucan in raw biomass; 15 filter paper units/g raw glucan) of 85.2 and 88.5 were achieved for Dacotah and Alamo, respectively. Adding ball milling to OLP further enhanced glucan digestibility to 91.1 (Dacotah) and 90.0 (Alamo). In previous studies, shock treatment achieved promising results, but was often inconsistent. This work refined shock treatment with a focus on using consistent procedures and performance analysis. The combination of OLP and shock treatment enhanced the 72-h glucan digestibility of several promising biomass feedstocks: bagasse (74.0), corn stover (92.0), poplar wood (94.0), sorghum (71.8), and switchgrass (89.0). Highly digestible lignocellulose can also be used as ruminant animal feed. Shock treatment plus OLP increased the total digestible nutrients (TDNN; g nutrients digested/100 g organic matter) of corn stover from 51.9 (untreated) to 72.6. Adding in pre-washed corn stover solubles to produce a combined feed (17.8 percent corn stover solubles and 82.2 percent shock OLP corn stover) increased TDNN to 74.9. Mixing in enough solubilized protein to match the crude protein content of corn grain further improved TDNN to 75.5, only 12.6 less than corn grain.

Reproduction de : Thèse de doctorat : Sciences des agroressources : Toulouse, INPT : 1995. / Titre provenant de l'écran-titre. Bibliogr. 334 réf.

Energy utilization is a determining factor for the standards of living around the world, and the current primary source of energy is fossil fuels. A potential source of liquid fuels that could ease the strain caused by diminishing petroleum resources is bioethanol. Effective exploitation of biomass materials requires a pretreatment to disrupt the lignin and cellulose matrix. The pretreatment utilized for this research was oxygen delignification, which is a standard process stage in the production of bleached chemical pulp. The model substrate utilized as a feedstock for bioethanol was recovered fibre. An analysis of the substrates digestibility resulted in a hexose yield of approximately 23%, which justified the need for an effective pretreatment. An experimental design was performed to optimize the delignification conditions by performing experiments over a range of temperature, caustic loadings, and reaction times. Equations were developed that outline the dependence of various response parameters on the experimental variables. An empirical model that can predict sugar concentrations from enzymatic hydrolysis based on the Kappa number, enzyme loading, and initial fibre concentration was also developed. A study of hydrolysis feeding regimes for untreated recovered fibre (87 Kappa), pretreated recovered fibre (17 Kappa), and bleached pulp (6 Kappa) showed that the batch feeding regime offers reduced complexity and high sugar yields for lower Kappa substrates. In order to evaluate the possibility of lignin recovery, the pH of delignification liquor was reduced by the addition of CO₂ and H₂SO₄, resulting in up to 25% lignin yield. An experiment that looked at effect of post-delignification fibre washing on downstream hydrolysis found that a washing efficiency of approximately 90% is required in order to achieve a hexose sugar yield of 85%.

Pretreatment of recalcitrant lignocellulosic biomass to release sugars for bioconversion into ethanol produces fermentation inhibitors. Increasing yeast inhibitor tolerance should reduce production time and cost. UV mutagenesis followed by genome shuffling using cross mating was performed on Scheffersomyces stipitis strain GS301, a genome shuffled strain with increased tolerance to spent sulphite liquor (SSL). The main fermentation inhibitors in SSL are acetic acid, hydroxymethylfurfural (HMF), and various phenolics. UV mutagenesis resulted in acetic acid tolerant mutants, but they were phenotypically unstable. However, two rounds of UV mutagenesis followed by five rounds of genome shuffling resulted in strains EVB105, EVB205 and EVB505 with increased SSL tolerance and improved acetic acid and HMF tolerance. When fermenting undiluted SSL at pH 5.5, the three strains utilized sugars faster producing higher maximum ethanol than GS301. This study demonstrates that UV mutagenesis with genome shuffling can significantly improve inhibitor tolerance and fermentation performance of yeast. / NSERC Bioconversion Network

<p>To ensure the sustainability of bioethanol production, major attention has been directed to develop feedstocks which provide an alternative to food-crop biomass. Lignocellulosic (LC) biomass, which is chiefly composed of industrial plant residues, is a carbon-rich reservoir that is presently attracting much attention. However LC material is highly recalcitrant to bioprocessing and requires a mixture of physical and enzymatic pretreatment in order to liberate fermentable sugars. Thermostable enzymes are extremely desirable for use in thermophilic fermentations due to their inherent stability. Hemicellulose, a core constituent of LC, requires a cascade of hemicellulases to stimulate the depolymerisation of its xylan backbone. &alpha / -L-arabinofuranosidase (AFase) increases the rate of lignocellulose biodegradation by cleaving arabinofuranosyl residues from xylan thereby increasing the accessibility of other hemicellulases. Twenty thermophilic Actinomycete isolates were screened for AFase activity using pnp-arabinofuranoside as the substrate. Three strains (ORS #1, NDS #4 and WBDS #9) displayed significant AFase activity and were identified as Streptomyces species with 16S rRNA gene sequence analysis. Genomic DNA was isolated from these strains and a cosmid library constructed in the shuttle vector pDF666. Subsequent functional and PCR-based screening revealed no positive clones.</p>

Energy utilization is a determining factor for the standards of living around the world, and the current primary source of energy is fossil fuels. A potential source of liquid fuels that could ease the strain caused by diminishing petroleum resources is bioethanol. Effective exploitation of biomass materials requires a pretreatment to disrupt the lignin and cellulose matrix. The pretreatment utilized for this research was oxygen delignification, which is a standard process stage in the production of bleached chemical pulp. The model substrate utilized as a feedstock for bioethanol was recovered fibre. An analysis of the substrates digestibility resulted in a hexose yield of approximately 23%, which justified the need for an effective pretreatment. An experimental design was performed to optimize the delignification conditions by performing experiments over a range of temperature, caustic loadings, and reaction times. Equations were developed that outline the dependence of various response parameters on the experimental variables. An empirical model that can predict sugar concentrations from enzymatic hydrolysis based on the Kappa number, enzyme loading, and initial fibre concentration was also developed. A study of hydrolysis feeding regimes for untreated recovered fibre (87 Kappa), pretreated recovered fibre (17 Kappa), and bleached pulp (6 Kappa) showed that the batch feeding regime offers reduced complexity and high sugar yields for lower Kappa substrates. In order to evaluate the possibility of lignin recovery, the pH of delignification liquor was reduced by the addition of CO₂ and H₂SO₄, resulting in up to 25% lignin yield. An experiment that looked at effect of post-delignification fibre washing on downstream hydrolysis found that a washing efficiency of approximately 90% is required in order to achieve a hexose sugar yield of 85%.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 128-133). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri-Columbia, 1998. / Typescript. Vita. Includes bibliographical references (leaves 128-133). Also available on the Internet.

Thesis (MSc)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Lignocellulosic material is composed of three major macromolecule components i.e., cellulose, hemicelluloses and lignin. These components are chemically associated and directly linked to each other through covalent bonding which is scientifically denoted as lignin-carbohydrate complexes (LCCs) and their interaction is fundamentally important as to understand wood formation and reactivity during chemical and biological processing e.g. pulping and enzymatic hydrolysis. The association of lignin with polysaccharides (covalent linkages) has been surrounded by contradictions and controversy in several wood chemistry studies. These linkages exist in lignocellulosic materials from wood to herbaceous plants. In woody plants, they consist of ester and ether linkages through sugar hydroxyl to α-carbonyl of phenyl-propane unit on lignin. However, in herbaceous plants ferulic and p-coumaric acids are esterified to hemicelluloses and lignin respectively. In recent studies, the existence of the bonds has been shown by applying indirect analysis strategies which resulted to low yields and contaminations. The general aim of the current study was to isolate and fractionate LCCs from raw lignocellulosic materials (E. grandis and sugarcane bagasse) and corresponding processed materials (chemical pulps and water-insoluble residues (WIS)) in order to determine the chemical structure of the residual lignin associated with polysaccharides and how they affected industrial processing. The objective of the study is to compile a document that when the development of pulping and bio-ethanol bio-refinery will greatly depends on the detailed wood chemistry on how the components interact with each before and after hemicelluloses pre-extraction prior to pulping and steam explosion pre-treatment prior to enzymatic hydrolysis. The current study was focusing on understanding the effect LCCs isolated from two different industrial processing methods, i.e. pulping and enzymatic hydrolysis (EH). There were two lignocelluloses feedstocks used for pulping, i.e. Eucalyptus grandis and sugarcane bagasse whereas sugarcane bagasse was the only feedstock used for enzymatic hydrolysis. Hemicelluloses pre-extracted (mild alkali or dilute acid and autohydrolysis for sugarcane bagasse) pulps of Kraft or soda AQ from E. grandis and sugarcane bagasse were used to understand the effect of xylan pre-extraction prior to pulping on lignin-carbohydrate complexes has not been reported to the best knowledge of the primary author. Also prior to EH the material was subjected to two different treatment methods, i.e. steam explosion and ionic liquid fractionation in varying conditions. The study illustrated the types of extracted and fractionated LCCs from hemicelluloses pre-extracted pulps and WIS in comparison to the non-extracted pulps and reports from the literature. Lignin-carbohydrate complexes (LCCs) were isolated and fractionated by an inorganic method which yielded reasonable quantification quantities and no contamination and low yields for the hardwood compared to reports of using an enzymatic method. To the best knowledge of the authors, no work has been done on WIS material. The lignocelluloses were subjected to ball milling which was followed by a sequence of inorganic solvents swelling and dissolution into 2 fractions i.e. glucan-lignin and xylan-lignin-glucan. Characterisation of the isolated LCCs was made using a variety of analytical tools such as FTIR-PCA, HPLC, GPC and GC-MS. LCCs were evident when FTIR and HPLC studies were conducted. Residual lignin isolated from the lignocelluloses was assumed to be chemically bonded to carbohydrates and mostly to xylan. Approximately 60% and 30% of the lignin was linked to xylan while for the second and first fractions respectively. It is reported that lignin associated with xylan is more resistant and reduce the delignification process than when linked to glucan that is easily hydrolysable. With the FTIR and GPC analyses of LCC fractions, it was evident that the ester bonds of LCCs were destroyed through pre-extraction and pre-treatment, where this resulted to more cellulose being more accessible to alkaline pulping and enzymatic hydrolysis respectively. The linkages were either partially broken down or completely destroyed leading to significant changes of chemical structures. The polydispersity of the LCCs assisted in determining the structure of lignin, either existing as monolignols on the surfaces of fibres or a as complex two or three-dimensional structure that is linked to carbohydrates as the Mw increased or decreased. In general, these findings may have an important implication for the overall efficiency on bio-refinery. The molecular weights (Mw) of the extracted LCCs were measured by gel permeation chromatography. From the chromatograms, it was observed that the materials that were subjected to pre-processing prior to further processing, the Mw shifted to lower Mws regions. It was found that LCCs isolated from mild alkali pre-extracted pulps had high lignin syringyl to guaiacyl lignin contents than LCCs isolated from dilute acid pre-extracted pulps. High syringyl/guaiacyl ratio (S/G ratio) was an indication of low lignin content as a result of processing which will result to high product yields after downstream processing. The 5 average S/G ratio for the pulps from E. grandis and sugarcane bagasse was ranging between 1.1 to 19.01 and 1.4 to 18.16 respectively, while for the WIS-material generated from ionic liquid fractionated and steam exploded materials ranged from 3.29 to 9.27 and 3.5 to 13.3 respectively. The S/G ratios of the LCCs extracted from E. grandis and sugarcane bagasse pulps ranged from 0.42 to 2.39 and 0.041 to 0.31 was respectively while for the LCCs extracted from water-insoluble-solids (WIS) material generated from steam exploded material was from 4.87 to 10.40. The determination of S/G ratio is recommended for the LCC extraction and characterisation study as an evaluation of residual lignin in processed materials such as pulps and WIS. The obtained saccharifications were low, possibly due to the severity of the steam explosion pre-treatment and ionic liquid fractionation conditions which resulted on high accumulation of acetic acid and increased in cellulose crystallinity respectively. From quantitative analysis of the LCCs perspective it could be concluded that free lignin was present in mild alkali pre-extracted pulps than for the dilute acid pre-extracted pulps. / AFRIKAANSE OPSOMMING: Cellulose materiaal is saamgestel uit drie groot makromolekule komponente naamlik, sellulose, hemisellulose en lignien. Hierdie komponente is chemies verwante en direk met mekaar verbind deur kovalente binding wat wetenskaplik aangedui as lignien-koolhidraat komplekse (LCCs) en hul interaksie is fundamenteel belangrik as hout vorming en reaktiwiteit tydens chemiese en biologiese verwerking bv om te verstaan verpulping en ensiematiese hidrolise. Die vereniging van lignien met polisakkariede (kovalente verbindings) is omring deur teenstrydighede en omstredenheid in verskeie hout chemie studies. Hierdie skakeling bestaan in cellulose materiaal uit hout te kruidagtige plante. In houtagtige plante, hulle bestaan uit ester en eter bindings deur suiker hidroksiel te α-karboniel van feniel-propaan eenheid op lignien. Maar in kruidagtige plante ferulic en p-coumaric sure veresterd te hemisellulose en lignien onderskeidelik. In onlangse studies, het die bestaan van die bande is getoon deur die toepassing van indirekte analise strategieë wat gelei tot lae opbrengste en kontaminasie. Die algemene doel van die huidige studie was om te isoleer en fraksioneer LCCs van rou cellulose materiaal (E. grandis en suikerriet bagasse) en die ooreenstemmende verwerkte materiaal (chemiese pulp en water-oplosbare residue (WIS)) ten einde die chemiese struktuur van die te bepaal oorblywende lignien wat verband hou met polisakkariede en hoe hulle geaffekteerde industriële verwerking. Die doel van die studie is 'n dokument op te stel dat wanneer die ontwikkeling van verpulping en bio-etanol bio-raffinadery sal grootliks afhang van die gedetailleerde hout chemie oor hoe om die komponente met mekaar voor en na hemisellulose pre-onttrekking voor verpulping en stoom ontploffing pre-behandeling voor ensiematiese hidrolise. Die huidige studie was die fokus op die begrip van die effek LCCs geïsoleerd van twee verskillende industriële verwerking, maw verpulping en ensiematiese hidrolise (EH). Daar was twee lignocelluloses voerstowwe gebruik vir verpulping, dws Eucalyptus grandis en suikerriet bagasse terwyl suikerriet bagasse was die enigste grondstof gebruik vir ensiematiese hidrolise. Hemisellulose pre-onttrek (ligte alkali of verdunde suur en autohydrolysis vir suikerriet bagasse) pulp van Kraft of soda AQ van E. grandis en suikerriet bagasse is gebruik om die effek van Xylan pre-onttrekking te voor verstaan verpulping op lignien-koolhidraat komplekse het nie aan die berig is beste kennis van die primêre outeur. Ook voor EH die materiaal is onderworpe aan twee verskillende behandeling metodes, naamlik stoom ontploffing en ioniese vloeistof fraksionering in wisselende toestande. Die studie geïllustreer die tipes onttrek en gefractioneerd LCCs van hemisellulose pre-onttrek pulp en WIS in vergelyking met die nie-onttrek pulp en verslae van die literatuur. Lignien-koolhidraat komplekse (LCCs) is geïsoleer en gefraksioneer deur 'n anorganiese metode wat redelike kwantifisering hoeveelhede en geen besoedeling en lae opbrengste opgelewer vir die hardehout vergelyking met verslae van die gebruik van 'n ensiematiese metode. Na die beste kennis van die skrywers, het geen werk op WIS materiaal gedoen. Die lignocelluloses is onderworpe aan die bal maal wat gevolg is deur 'n reeks van anorganiese oplosmiddels swelling en ontbinding in 2 breuke dws glucan-lignien en Xylan-lignien-glucan. Karakterisering van die geïsoleerde LCCs is gemaak met behulp van 'n verskeidenheid van analitiese gereedskap soos FTIR-PCA, HPLC, GPC en GC-MS. LCCs was duidelik wanneer FTIR en HPLC studies is uitgevoer. Residuele lignien geïsoleerd van die lignocelluloses is aanvaar moet word chemies gebind aan koolhidrate en meestal te xylan. Ongeveer 60% en 30% van die lignien is gekoppel aan xylan terwyl dit vir die tweede en eerste breuke onderskeidelik. Dit is gerapporteer dat lignien wat verband hou met Xylan is meer bestand en die delignification proses as wanneer gekoppel aan glucane wat maklik hidroliseerbare verminder. Met die FTIR en GPC ontledings van LCC breuke, was dit duidelik dat die ester bande van LCCs is deur pre-ontginning en pre-behandeling, waar dit gelei tot meer sellulose om meer toeganklik te alkaliese verpulping en ensiematiese hidrolise onderskeidelik vernietig. Die skakeling is óf gedeeltelik afgebreek of heeltemal vernietig lei tot beduidende veranderinge van chemiese strukture. Die polydispersity van die LCCs bygestaan in die bepaling van die struktuur van lignien, hetsy bestaande as monolignols op die oppervlak van die vesel of 'n as komplekse twee of drie-dimensionele struktuur wat gekoppel is aan koolhidrate as die Mw vermeerder of verminder. In die algemeen, kan hierdie bevindinge het 'n belangrike implikasie vir die algehele doeltreffendheid op bio-raffinadery. Die molekulêre gewigte (Mw) die onttrek LCCs gemeet deur gelpermeasie- chromatografie. Van die chromatograms, was dit opgemerk dat die materiaal wat blootgestel is aan die pre-verwerking voor verdere verwerking, die Mw verskuif MWS streke te verlaag. Daar is gevind dat LCCs geïsoleerd van ligte alkali pre-onttrek pulp het hoë lignien syringyl lignien inhoud as LCCs geïsoleerd van verdunde suur vooraf onttrek pulp te guaiacyl. Hoë syringyl / guaiacyl verhouding (S/G-verhouding) was 'n aanduiding van 'n lae lignien inhoud as 'n resultaat van verwerking wat sal lei tot 'n hoë produk opbrengste ná stroomaf verwerking. Die gemiddelde S/G-verhouding vir die pulp van E. grandis en suikerriet bagasse was wat wissel tussen 1,1-19,01 en 1,4-18,16 onderskeidelik, terwyl dit vir die WIS-materiaal gegenereer uit ioniese vloeistof gefraksioneer en stoom ontplof materiaal het gewissel 3,29-9,27 en 3.5 13,3 onderskeidelik. Die S/G verhoudings van die LCCs onttrek uit E. grandis en suikerriet bagasse pulp gewissel 0,42-2,39 en ,041-,31 was onderskeidelik terwyl dit vir die LCCs onttrek uit water-oplosbare-vastestowwe (WIS) materiaal gegenereer uit stoom ontplof materiaal was van 4,87-10,40. Die bepaling van S/G-verhouding word aanbeveel vir die LCC ontginning en karakterisering studie as 'n evaluering van die oorblywende lignien in verwerkte materiaal soos pulp en WIS. Die verkry saccharifications was laag, moontlik as gevolg van die erns van die stoom ontploffing pre-behandeling en ioniese vloeistof fraksionering voorwaardes wat gelei op 'n hoë opeenhoping van asynsuur en vermeerder in sellulose kristalliniteit.

Scaling biological pretreatment from the bench scale to the production scale may be more economical if unsterilized feedstock are used, however these allow for microbial competition from contaminates. An accurate and rapid method for identifying the desired biological pretreatment organism is necessary to confirm the presence of the desired organism when contaminates are morphologically similar to the target organism. Traditional methods, such as visual identification, sequencing, and selective plating can be time consuming and are sometimes still inconclusive. Based on methods described in the literature, plasmid DNA containing the marker genes gus (�-glucuronidase), LacZ, and gfp (green fluorescence protein) incorporated into the lignin-degrading basidiomycete Phanerochaete chrysosporium would result in a rapid genetic test for the desired organism. The presence of these genes can be confirmed either through an X-Gluc (cyclohexylammonia salt), X-Gal histochemical assay or observing the gfp’s fluorescence by a specially equipped confocal microscope. Each reporter systems will allow for rapid, reliable identification of the target species. This study will report on the success of the transformation methods in creating a transformed fungus to be used in the context of a large-scale fermentation operation. Additionally, a novel in-harvest lignocellulose feedstock biological pretreatment inoculation trial was performed comparing lignolytic performance between fungal inoculum application techniques. Optimization of carbohydrate availability for enhanced saccharification was determined by analyzing glucose release by treated and non-treated unsterilized switchgrass. This study also focused on identifying parameters to enhance saccharification efficacy at the farm-scale.