Master of Science / Department of Grain Science and Industry / Yonghui Li / Antioxidants are widely used in food industries to delay lipid oxidation and prevent oxidative deterioration. In recent years, growing interests in developing safe and efficient antioxidants from natural sources due to the health-related risks associated with synthetic antioxidants. Recently, peptide antioxidants have drawn growing interests as since proteins are a macronutrient with various functionalities and high consumer acceptability. A lot of dietary proteins have been validated for their antioxidant potentials especially those obtained from animal proteins, nuts and pulses. Relatively less information is available on characterizing the antioxidant profile of cereal protein, and even less for sorghum protein. Sorghum is the fifth largest crop worldwide and is the third in United States. U.S. is leading in global sorghum production and distribution, and the state of Kansas is producing nearly half of U.S. sorghum. Currently, about one third of the U.S. sorghum is being used for ethanol production, resulting in more than 450 kilotons of by-products (e.g., DDGS) annually, which were often discarded or underutilized. DDGS is a premium protein source (~ 30% protein) that could be potentially modified into value-added products such as peptide antioxidants. In this study, relevant literatures detailing the extraction of cereal proteins, enzymatic hydrolysis of proteins, purification and characterization of hydrolysates, and evaluation of antioxidant profiles were extensively reviewed in Chapter 1. As preliminary experiments, sorghum kafirin protein was extracted from defatted sorghum white flour and hydrolyzed by 10 different types of enzymes from microbial, plant and animal sources. Hydrolysates prepared with Neutrase, Alcalase, and Papain displayed the most promising antioxidant activities as well as total protein recovery were primarily selected and investigated in depth described in Chapter 2, Chapter 3, and Chapter 4. The reaction conditions including substrate content, enzyme-to-substrate ratio, and hydrolysis time are critical parameters in producing peptides with desired activity and consistency, were therefore examined and optimized for each case of kafirin hydrolysates. The antioxidant capacity of the resulting hydrolysates was measured for antioxidant capacity through in vitro assays (DPPH, ABTS, ORAC, reducing power, and metal chelating) and then demonstrated in model systems (oil-in-water emulsion and ground meat). The fractions of hydrolysates possessing strongest activities were further fractionated by gel filtration and HPLC. Peaks representing the largest areas from HPLC were identified for major sequences by MALDI-TOF-MS. The experiment results indicated that all the three selected fractions of kafirin hydrolysates revealed excellent inhibition effects against oil and fat oxidations, which could be employed as tools to predict their performances in real food products. In addition, the structure studies showed that medium-sized hydrolysates of Neutrase (3 – 10 kDa) and Alcalase (5 – 10 kDa), and small-sized hydrolysates of Papain (1 – 3 kDa) exhibited relatively stronger activities. This study provided a workable processing method and critical reaction parameters for the production of peptide antioxidants from sorghum protein. The experiment results revealed that the sorghum peptide antioxidant could act through multiple mechanisms including free radical scavenging, metal ion chelation, hydrogen donating, and forming physical barriers to minimize the contact of oxidative agents to targets. These antioxidative peptides are a promising ingredient that can be potentially incorporated to food and feed products as alternatives to synthetic antioxidants or synergetic elements to nonpeptic antioxidants for protection of susceptible food ingredients. This study also made a positive impact to sorghum ethanol industry by guiding the conversion of sorghum protein-rich by-products into value-added antioxidant products as an additional revenue stream.

AN ABSTRACT OF THE THESIS OF Thara G. Siddaramu, for the Master of Science degree in Civil and Environmental Engineering, presented on February 5, 2011, at Southern Illinois University Carbondale. TITLE: EVALUATION OF DIFFERENT PRETREATMENT APPROACHES FOR DISRUPTING LIGNOCELLULOSIC STRUCTURES MAJOR PROFESSOR: Dr. Yanna Liang There are two major steps in biofuel production- pretreatment of lignocellulosic materials and enzymatic hydrolysis. The present study investigated the ability of two pretreatment methods, namely traditional oven and microwave oven treatments for disrupting lignocellulosic structures. The substrates tested were Jatropha seed cake and sweet sorghum bagasse. In recent years, Jatropha curcas also known as physic nut or purging nut has attracted extensive attention due to its several unique characteristics. Similarly, sweet sorghum has the potential to provide great value to energy sectors and food industries being that the entire plant is rich in various sugars and nutrients. Both crops can adapt to various climates, and can withstand extended drought conditions compared to other crops. Additionally, both Jatropha seed cakes and sweet sorghum bagasse are good sources of lignin and carbohydrates, which could be used for production of biofuels only if the sugars can be unlocked. Several treatment methods such as mechanical, physical, chemical and biological treatments have been reported to breakdown the cellulosic structure of biomass. However, other low cost and quicker methods, such as ovenpretreatment and microwave irradiation have not been evaluated for Jatropha seed cake and Sweet Sorghum Bagasse (SSB), respectively. Composition change of Jatropha seed cake samples was evaluated upon lime pretreatment at 100 oC with different parameters. With a lime dose of 0.2 g and a water content of 10 ml per gram of cake and a treatment period of 1 h, 38.2 ± 0.6% of lignin was removed. However, 65 ± 16% of hemicellulose was also lost under this condition. For all the treatments tested, cellulose content was not affected by lime supplementation. Through further examining total reducing sugar (TRS) release by enzymatic hydrolysis after lime pretreatment, results indicated that 0.1 g of lime and 9 ml of water per gram of cake and 3 h pretreatment produced the maximal 68.9% conversion of cellulose. Without lime pretreatment, the highest cellulose conversion was 33.3%. Finally, this study shows that Jatropha seed cake samples could be hydrolyzed by enzymes. Even though the cellulose content was not high for this Jatropha cake sample, the fractionation by lime presented in this study opened the door for other applications, such as removal of lignin and toxicity for use as animal feed and fertilizer. The microwave radiation pretreatment of SSB was evaluated with or without lime (0.1 g/g bagasse) at 10 ml water/g bagasse for 4 min. TRS release over 72-h enzymatic hydrolysis was different for samples treated differently and at different solid loadings. The TRS concentration was increased by 2 and 5-fold from 0 to 24 hours in non lime-pretreated and lime-pretreated samples, respectively. Further incubation of samples for 48 and 72 h did not result in increased TRS. Comparing different solid loadings of samples treated with or without lime, 1% solid content resulted in 1.4 times higher TRS increase than that of 5% solid concentration. Therefore, lime was effective in disintegrating lignocellulosic structures and making cellulose more accessible for saccharification. Higher solid loadings which can lead to higher sugar concentrations are desired for downstream biofuel production. But, as shown in this study, higher concentration of bagasse samples decreased rate of cellulose hydrolysis due to poorer mixing efficiency and hindrance to interactions between enzymes and solid materials. Thus, an optimal solid content needs to be determined for maximal cellulose hydrolysis and for preparing the hydrolysates for downstream processes, either bioethanol or lipid production.

Made available in DSpace on 2014-06-11T19:28:03Z (GMT). No. of bitstreams: 0 Previous issue date: 2012-02-02Bitstream added on 2014-06-13T19:57:22Z : No. of bitstreams: 1 bassan_jc_me_arafcf_parcial.pdf: 63703 bytes, checksum: 8b9bc74a9baab69ea2ec520e84ab90dc (MD5) Bitstreams deleted on 2015-03-12T12:02:45Z: bassan_jc_me_arafcf_parcial.pdf,Bitstream added on 2015-03-12T12:03:14Z : No. of bitstreams: 1 000682632.pdf: 939229 bytes, checksum: 4f9b453ce3606382c9d71aaba154fcb5 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / Universidade Estadual Paulista (UNESP) / O leite de búfala representa 12% da produção mundial de leite, com maiores teores de proteínas e gorduras que o de vaca. O soro de leite é o co-produto da indústria queijeira que vem sendo, muitas vezes, descartado no meio ambiente, causando grande impacto ambiental em função de sua constituição rica em proteínas e lactose. Hidrólise enzimática é um avanço tecnológico importante por melhorar propriedades físicas, químicas e funcionais dessas proteínas. O objetivo do trabalho foi produzir hidrolisados protéicos a partir das proteínas presentes no soro do leite de búfala e simular a digestão in vitro pelo método da dialisabilidade. O soro lácteo bubalino foi dialisado para retirada de lactose e pequenos peptídeos e aminoácidos, e tratado com caulim para adsorção da gordura. Os produtos de médio e alto grau de hidrólise foram obtidos por ação da pepsina, tripsina, quimotripsina e carboxipeptidase A em pHs e temperaturas específicos, adicionados ao soro conjuntamente e em separado, em diferentes tempos de hidrólise. A determinação quantitativa de proteínas, aminoácidos, lactose e gordura foram realizadas segundo os métodos Bradford (1976), cromatografia líquida de alta eficiência, Miller (1959) e Gerber, respectivamente. A caracterização qualitativa das proteínas e produtos de hidrólise fez-se por eletroforese não-desnaturante (PAGE) e desnaturante (SDS-PAGE). Os ensaios de biodisponibilidade dos produtos de hidrólise foram realizados pelo método in vitro da dialisabilidade segundo metodologia descrita por Luten et al. 1996. Os resultados encontrados para o soro deslactosado e desengordurado foram 6,53g prot.L -1 , redução de 99% de lactose e < 0,10 % de gordura. Bandas protéicas... / Buffalo milk represents 12% of world production of milk, with high protein and fat content of the cow. The whey is the co-product of cheese industry that has been often discarded into the environment, causing great environmental impact due to its rich protein and lactose constitution. Enzymatic hydrolysis is an important technological advance to improve the physical, chemical and functional properties of these proteins. The objective of this research was to produce protein hydrolysates from whey proteins present in buffalo milk and to simulate the digestion in vitro by the method of dialyzability. The whey was dialyzed to remove lactose and small peptides and amino acids, and treated with kaolin for fat adsorption. The partial and total hydrolysates were obtained by the action of pepsin, trypsin, chymotrypsin and carboxypeptidase A in specific pH and temperature, added to the whey together or separately at different times of hydrolysis. Quantitation of proteins, amino acids, lactose and fat were performed according to Bradford (1976), HPLC, Miller (1959) and Gerber respectively. The qualitative characterization of proteins and hydrolysis products was made by PAGE and SDS-PAGE. Tests of absorption of hydrolysis products were performed in vitro by dialyzability. The results for dialyzed and defatted whey was 6.53 g prot L -1 , reduction of 99% lactose and <0.10% fat. Protein bands... (Complete abstract click electronic access below)

Orientador: Rubens Maciel Filho / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Química / Made available in DSpace on 2018-08-26T08:54:48Z (GMT). No. of bitstreams: 1 Oliveira_JohnattAllanRochade_D.pdf: 5982686 bytes, checksum: 807df203b0fc6c390cc3dd62aeca05fc (MD5) Previous issue date: 2014 / Resumo: O aproveitamento de resíduos lignocelulósicos para a produção de etanol combustível tem se tornado um foco de pesquisas em diversas partes do mundo. Cada região do mundo focou em um resíduo de sua indústria local o que é típico da industria de biocombustíveis. Entretanto o Brasil é um país de imensa produção agroindustrial e com diferentes resíduos que podem ser explorados para cada região, como é o caso do caroço de açaí na região norte do Brasil. Dentro deste contexto o objetivo desse trabalho é propor e investigar as etapas de processos para a obtenção de etanol a partir do caroço de açaí (Euterpe oleracea). Para tanto foi realizado um planejamento experimental central composto rotacional 23 para a avaliação do pré-tratamento do caroço de açaí com H2SO4 diluído, e as condições do estudo foram: concentração de H2SO4 (0,5 - 1,5% m/v), carga de sólidos (5-15% m/v) e o tempo (30-90 min). Em seguida a deslignificação do caroço de açaí pré-tratado foi avaliada através de um planejamento experimental do tipo 22 nas seguintes condições: 1% de NaOH (m/v), carga de sólidos (10-40% (m/v), temperatura (50-100 °C) e tempo de 60 minutos. Ambos os materiais foram hidrolisados enzimaticamente (carga enzimática de 15FPU/g de caroço e 25CBU/g de caroço) e caracterizados quimicamente via HPLC (cromatografia líquida de alta eficiência). As melhores condições obtidas para o pré-tratamento e deslignificação foram utilizadas para avaliar o efeito da carga de sólidos (5-15%) e agitação (100-200 rpm) na hidrólise enzimática (em reator) através de um planejamento experimental 32. A fermentação dos hidrolisados obtidos foi realizada e os hidrolisados e fermentados foram caracterizados via HPLC. As condições consideradas ótimas para o pré-tratamento com H2SO4 diluído foi de 1% de ácido, 10% de sólidos durante 60°C, enquanto que a melhor condição para a deslignificação sequencial foi de 25% de sólidos a 75°C. Para a hidrólise enzimática, a carga de sólidos de 3% foi a que permitiu maiores valores de conversão (78,97%). Entretanto, os maiores valores de glicose (g/L) foram obtidos para 10 e 15% de sólidos em reator a 200rpm (melhor condição de agitação). A fermentação do hidrolisado obtido a partir do material pré-tratado com H2SO4 diluído e sequencialmente deslignificado foi aquela que produziu o maior índice de etanol (21,65 g/L). Foi possível concluir que as etapas de pré-tratamento e deslignificação do caroço de açaí permitiram a liberação de índices aceitáveis de açúcares e posterior produção de etanol / Abstract: The use of lignocellulosic wastes to ethanol production has become a focus of research in several parts of the world. Each region of the world focused in a specific waste from your local industry. However the Brazil is a country of immense agro-industrial production and with different residues that can be explored within each region one of its regions. Such as açaí seeds in northern of Brazil. A central composite experimental design rotational 23 for evaluation of pretreatment with H2SO4 diluted of açaí seeds was carried outj and study conditions were : concentration of H2SO4 (0.5 - 1.5% w/v), solids loading (5-15% w/v) and time (30-90 min). Then delignification of pretreated seeds açaí was evaluated through an experimental design 22 under the following conditions: 1% NaOH (w/v), solids loading (10-40% w/v), temperature (50-100 °C) and time 60 minutes. Both materials were hydrolysed enzymatically (enzyme load of 15FPU / g seed and 25CBU /g of seed) chemically and characterized via HPLC (high performance liquid chromatography). The best conditions achieved for the pretreatment and delignification were used to evaluate the effect of loading solids (5-15% w/v) and agitation (100-200 rpm) on the enzymatic hydrolysis (in reactor) through an experimental design 32. The optimum conditions for pre-treatment dilute sulfuric acid was 1% acid, 10% solids at 60°C. While the optimum conditions for delignification sequential was 25% solids at 75°C. For the enzymatic hydrolysis, the solids loading of 3% allowed for conversion the highest values (78.97%). However the highest values of g glucose (g/L) were obtained for 10 and 15% solids in the reactor 200rpm (best condition of agitation). The fermentation of the hydrolyzate from the material pretreated with dilute sulfuric acid and sequentially deslignificated was one which produced the highest yield of ethanol (21,65g/L). It was concluded that the steps of pretreatment and delignification of seeds of acai allowed the release of good levels of sugars and subsequent ethanol production / Doutorado / Desenvolvimento de Processos Químicos / Doutor em Engenharia Química

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%. / Applied Science, Faculty of / Chemical and Biological Engineering, Department of / Graduate

This master’s thesis is aimed at process of enzymatic hydrolysis of lignocellulosic material – waste paper as a source of raw material for production of liquid biofuels. In the theoretical part of this work are summarized previously used methods of hydrolysis and lignocellulosic materials used for the process of hydrolysis as a source of fermentable sugars for fermentation technology. The different types of waste paper are evaluated from the composition and usability with consideration to the papermaking process in order to select the appropriate type of waste paper for the enzymatic hydrolysis process. In the next part of this work are suggested technological premises and procedures for the preparation of raw materials and the subsequent enzymatic hydrolysis of these pre–treated materials. In the experimental part were optimized parameters of enzymatic hydrolysis using the Novozymes company enzyme package. Enzymatic degradation of cellulose to reducing sugars was observed using Somogyi – Nelson method. For the verification of hydrolysis conditions were used model materials with high cellulose content – pulp and filter paper. Conditions, which seems to be the best after testing on the model materials, were verified on specific waste paper materials – offset cardboard, recycled paper, matte MYsol paper and for comparison again on model materials – pulp and filter paper. The highest yields was achieved with the use of cardboard, which was further tested using various combinations of pretreatment to material for purpose of increase the yields of hydrolysis.

This master’s thesis discusses the useof enzymatic hydrolysis process of waste cardboard using simultaneous saccharification and fermentation (SSF) as a source of raw materials for production of liquid biofuels. This thesis is based on theses written by Ing. Brummer and Ing.Lepař.Thus, results gained in these works have been used and also further developed. The theoretical part summarizes the reasons for further development of SSF method and discusses, as well, the achievements reached in the processing of lignocellulosic waste materials by the SSF method so far.This section also discusses the general characteristics of lignocellulosic materials and also of the cellulolytic enzymes. It focusses also on individual pretreatment methods of lignocellulosic material and options of increasing the yield of the whole process. The experimental part verifies the particular results reached in previous theses and at the same time a further optimization of the method has been carried out because of the transfer of the whole process into a fermenter. Cardboard was set as the substrate for the experiments as it was evaluated by Ing. Brummer as the best one for enzymatic hydrolysis which was carried out by enzymes from Novozymes®. Parameters such as temperature, pH and kind of used buffer, the loading concentration of substrate and enzymes, were set according to the thesis of Ing. Lepař, which was aimed to their optimization. The SSF process done in fermenter of 2.0 l volume confirmed the previous results and furthermore it has been more effective through optimization of the added inoculum volume. It has been confirmed that the best substrate is cardboard finely grinded by vibrating mill. Also experiments with added nutrients had been done as an effort to increase the ethanol concentration, but these haven’t resulted insatisfying results. The maximal concentration of ethanol was 23,49 g/l, which was achieved after further optimization of various conditions. This result equals to experimental yield of 84,79 %.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: Maceration enzymes for the wine industry are preparations containing mainly pectinases, cellulases and hemicellulases, used during wine making to degrade the berry cell walls and release polyphenolic and aroma molecules to increase wine quality. These types of enzymes are also used for the harvesting of revenue-generating molecules from pomace (skins, pulp and seeds from grape processing waste), or as processing aids when used in the production of bioethanol. Grape berry cell walls are recalcitrant towards degradation, therefore knowledge about their structures and compositions, as well as how the application of enzymes modify these structures is essential in order to optimise these processes. The aim of this study was to extend current knowledge by using a mixture of existing and novel methodologies to study grape berry cell walls by focusing on the profiles of polymers present in the walls. Cell wall profiling techniques used in this study include the Comprehensive Microarray Polymer Profiling (CoMPP) method that employs monoclonal antibodies and Carbohydrate Binding Modules (CBM) which specifically recognise the polymers in the plant cell wall. With this method we measured the abundance of specific polymers and traced the fluctuation in their levels of abundance as influenced by external factors such as enzyme hydrolysis. The CoMPP method was coupled with monosaccharide profile analysis by GC-MS to determine the building blocks of the cell wall polymers, as well as with Infrared Spectroscopy to monitor the changes in the bulk chemistry profile. Data sets generated by the cell wall profiling methods were analysed with uni- and multivariate statistical methods to detect the major patterns in the data. This study highlighted the cell wall differences on the polymer level, in the berry skin cells of Pinotage grapes at different ripeness levels and how it changes during a standard wine fermentation, leading to the release of homogalacturonans and the exposing of arabinogalactan proteins. When maceration enzymes were added, further depectination was evident and the enzymes unravelled the cell wall of the ripe grapes. In overripe grapes no additional degradation could be observed due to maceration enzyme actions, presumably indicating that the endogenous grape enzymes already caused extensive degradation. When purified enzymes were incubated under buffered conditions with isolated skin cell walls from Pinotage grapes or with Chardonnay grape pomace, different levels of enzymatic hydrolysis were observed and defined. The sequence in which cell wall polymers were extracted, and the influence of specific enzymes in facilitating the extraction process, provided important information on the accessibility of specific cell wall polymers. Synergistic action between, for example an endo-polygalacturonase (EPG) and an endo-glucanase (EG) was demonstrated with CoMPP. This EPG and EG synergism was also demonstrated with a yeast strain (a Saccharomyces paradoxus x S. cerevisiae hybrid) fermented in a buffered pomace suspension. This yeast strain has a native EPG and was engineered to also express a recombinant EG from a genome integrated cassette. The cell walls isolated from the pomace after fermentation were unravelled and depectination took place, as evident from CoMPP data. The cell wall profiling techniques used in this study were proven to be fast and sensitive. It provided insights into the structure of grape cell walls and was used to evaluate the changes due to ripening, fermentation, enzymatic hydrolysis and a heat pre-processing treatment. In addition to the knowledge gained, we also demonstrated that these techniques can be used to evaluate different enzymes and enzyme combinations as well as the potential of microorganisms to degrade grape tissue. / AFRIKAANSE OPSOMMING: Maserasie ensieme vir die wynindustrie is ensiem mengsels wat hoofsaaklik pektinases, sellulases en hemisellulases bevat en word tydens wynbereiding gebruik om die druifkorrel se selwand af te breek, die polifenole en aroma molekules vry te stel en sodoende die wyn kwaliteit te verbeter. Hierdie soort ensieme word ook gebruik om inkomste-genererende molekules vanuit druiweprosesserings afval (doppe, pulp en pitte) te isoleer, en ook as prosesserings hulpmiddels in die produksie van bioetanol. Druifkorrel selwande is weerstandig teen ensiem afbraak en daarom is kennis oor die struktuur en samestelling van die selwand, asook hoe die selwand strukture deur die toediening van ensieme verander word noodsaaklik om sodoende hierdie prosesse te optimaliseer. Die doel van hierdie studie was om die huidige kennis uit te brei deur bestaande asook nuwe metodes te gebruik om die druifkorrel selwand te bestudeer met die fokus op die polimeerprofiel van die selwande. Selwand karakteriserings tegnieke wat in hierdie studie gebruik is sluit in die Comprehensive Microarray Polymer Profiling (CoMPP) metode wat monoklonale teenliggaampies en koolhidraat bindende modules (Carbohydrate binding modules, CBMs) wat spesifiek die selwandpolimere van die plant selwand herken, gebruik. Met hierdie metode het ons die vlakke van spesifieke polimere gemeet asook die skommeling in hulle vlakke soos dit beïnvloed is deur eksterne faktore soos ensiem hidroliese. Die CoMPP metode is tesame met monosakkaried profiel analise, met behulp van GC-MS, wat die boublokke van die selwand polimere bepaal, asook infrarooi spektroskopie om die veranderinge in die oorhoofse chemiese profiel te bepaal, gebruik. Datastelle wat met die selwand karakteriserings tegnieke gegenereer is, is ontleed met een- en multiveranderlike statistiese metodes om die hoof tendense in die data op te spoor. Hierdie studie het die selwand verskille, op die polimeervlak, van Pinotage druiwe uitgelig. Verskillende rypheidsgrade asook hoe dit verander tydens ‘n standaard wynfermentasie is gevolg. Laasgenoemde het die vrystelling van homogalakturonaan en die ontbloting van arabinogalaktoproteïene tot gevolg gehad. Met die byvoeging van maserasie ensieme was dit duidelik dat addisionele pektienverwydering plaasgevind het en dat die ensieme die selwand van die ryp druiwe ontrafel het. In oorryp druiwe was daar geen addisionele selwand afbreking sigbaar as gevolg van die aksie van maserasie ensieme nie, wat moontlik aandui dat die inherente druif ensieme reeds uitgebreide selwand afbraak versoorsaak het. Wanneer gesuiwerde ensieme met geïsoleerde selwande van Pinotage druiwedoppe en met Chardonnay druiweprosesserings afval geïnkubeer is onder gebufferde kondisies, is verskillende vlakke van ensiematiese hidroliese waargeneem en geklassifiseer. Die volgorde waarin die selwand polimere geëkstraheer is, asook die invloed van spesifieke ensieme in die bevordering van die ekstraksie proses, het belangrike inligting verskaf oor die toeganglikheid van spesifieke selwand polimere. Sinergistiese aksie tussen, byvoorbeeld ‘n endo-poligalakturonase (EPG) en ‘n endo-glukanase (EG) is geidentifiseer met behulp van die CoMPP data. Hierdie EPG en EG sinergisme is ook geïllustreer met ‘n gisras (‘n Saccharomyces paradoxus x S. cerevisiae hibried) wat in ‘n gebufferde druifprosesserings afval suspensie gefermenteer het. Hierdie gisras het ‘n endogene EPG en is ontwerp om ook ‘n rekombinante EG uit te druk vanaf ‘n genoom geïntegreerde kasset. Die selwande van die druiweprosesserings afval wat na die fermentasie geïsoleer is, was ontrafel en pektienverwydering het plaasgevind, soos bevestig met CoMPP data. In hierdie studie is bewys dat die selwand karakteriserings tegnieke vinnig en sensitief is. Dit het insigte verskaf oor die struktuur van die druifselwand en is gebruik om die veranderinge as gevolg van rypheidsverskille, wynfermentasie, ensiem hidroliese en hitte prosessering te evalueer. Buiten die bydraes tot kennis oor hierdie onderwerpe, is die bruikbaarheid van hierdie tegnieke ook aangetoon, veral in die evaluasie van verskillende ensieme en ensiemkombinasies, asook mikroörganismes vir die afbraak van druifweefsel.

The work of the present thesis focused on conversion of the cellulosic part of waste textiles into biogas and ethanol, and its challenges. In 2009, the global annual fiber consumption exceeded 70 Mt, of which around 40% consisted of cellulosic material. This huge amount of fibers is processed into apparel, home textiles, and industrial products, ending up as waste after a certain time delay. Regretfully, current management of waste textiles mainly comprises incineration and landfilling, in spite of the potential of cellulosic material being used in the production of ethanol or methane. The volume of cellulose mentioned above would be sufficient for producing around 20 billion liters of ethanol or 11.6 billion Nm3 of methane per year. Nevertheless, waste textiles are not yet accepted as a suitable substrate for biofuel production, since their processing to biofuel presents certain challenges, e.g. high crystallinity of cotton cellulose, presence of dyes, reagents and other materials, and being textiles as a mixture of natural and synthetic fibers. High crystallinity of cotton cellulose curbs high efficient conversion by enzymatic or bacterial hydrolysis, and the presence of non-cellulosic fibers may create several processing problems. The work of the present thesis centered on these challenges. Cotton linter and blue jeans waste textiles, all practically pure cellulose, were converted to ethanol by SSSF, using S. cerevisiae, with a yield of about 0.14 g ethanol/g textile, only 25% of the theoretical yield. To improve the yield, a pretreatment process was required and thus, several methods were examined. Alkaline pretreatments significantly improved the yield of hydrolysis and subsequent ethanol production, the most effective condition being treatment with a 12% NaOH-solution at 0 °C, increasing the yield to 0.48 g ethanol/g textile (85% of the theoretical yield). Waste textile streams, however, are mixtures of different fibers, and a separation of the cellulosic fibers from synthetic fibers is thus necessary. The separation was not achieved using an alkaline pretreatment, and hence another approach was investigated, viz. pretreatment with N-methyl-morpholine-N-oxide (NMMO), an industrially available and environment friendly cellulose solvent. The dissolution process was performed under different conditions in terms of solvent concentration, temperature, and duration. Pretreatment with 85% NMMO at 120 °C under atmospheric pressure for 2.5 hours, improved the ethanol yield by 150%, compared to the yield of untreated cellulose. This pretreatment proved to be of major advantage, as it provided a method for dissolving and then recovering the cellulose. Using this method as a foundation, a novel process was developed, refined and verified, by testing polyester/cellulose-blended textiles, which predominate waste textiles. The polyesters were purified as fibers after the NMMO treatments, and up to 95% of the cellulose content was regenerated. The solvent was then recovered, recycled, and reused. Furthermore, investigating the effect of this treatment on anaerobic digestion of cellulose disclosed a remarkable enhancement of the microbial solubilization; the rate in pretreated textiles was twice the rate in untreated material. The overall yield of methane was, however, not significantly affected. The process developed in the present thesis appears promising for transformation of waste textiles into a suitable raw material, to subsequently be used for biological conversion to ethanol and biogas. / <p>Thesis to be defended in public on Friday, May 27, 2011 at 13.00 at KC-salen, Kemigården 4, Göteborg, for the degree of Doctor of Philosophy.</p>

Ce travail de thèse s'inscrit dans le cadre de la valorisation des co-produits issus des industries de transformation et de conserve de thon. Il porte sur la mise en oeuvre de l'hydrolyse enzymatique et des procédés de séparation membranaires en vue d'obtenir des composés d'intérêt comme les peptides et les acides aminés. Les techniques utilisées dans ce travail font partie des « technologie propre », visant une dépense énergétique et un investissement modérés. L'hydrolyse enzymatique a été menée pour identifier l'efficacité des enzymes sur la matrice afin de déterminer les conditions optimales d'hydrolyse en utilisant la méthodologie des plans d'expériences avec pour objectif l'enrichissement de la phase soluble en peptides de petite taille dotés d'activités biologiques intéressantes. Le fractionnement par la taille par ultrafiltration et Nanofiltration a été utilisé comme un procédé pour agir sur la distribution de la taille moléculaire de la population peptidique et sur les propriétés d'hydrolysat produit. Tout d'abord, un fractionnement à petite échelle a été réalisé avec des membranes de différents seuils de coupure et de différentes natures (organique et inorganique) afin de sélectionner la meilleure membrane répondant à notre objectif.Ensuite une étude d'optimisation des conditions opératoire a été réalisée pour les deux étapes de fractionnement UF et NF de manière à déterminer les meilleures conditions séparatives en utilisant un hydrolysat commercial. La validation du procédé de fractionnement en utilisant l'hydrolysat des co-produits de thon a été effectuée. Durant cette étude, différents modes de fractionnement utilisant différentes combinaisons d'UF et NFont été testés pour déterminer le meilleur procédé permettant la récupération du maximum de fractions peptidiques intéressantes. L'originalité de ce travail de thèse réside d'une part, dans l'enrichissement de l'hydrolysat des co-produit de thon en composés valorisables comme les acides aminés essentiels ainsi que les peptides dotés de valeur ajoutée et d'autre part, la mise en place d'un procédé de fractionnement pour la récupération des différentes fractions afin de mettre en évidence la conception d'un bioréacteurs enzymatique couplé à la technique membranaire. / This work is performed in the framework of up-grading of tuna by-products generated from processing and conditioning industries. The enzymatic hydrolysis combined with membrane separation processes in order to obtain the fraction of interest peptides and amino acids was studied. The optimal conditions during enzymatic hydrolysis were determined using the methodology of design of experiments in order to enrich the soluble phase in small peptides with interesting biological activities. The fractionation by Ultrafiltration and Nanofiltration following a suitable combination was studied. For this, firstly, a small-scale fractionation was performed with membranes of different cut-off and different natures (organic and inorganic) to select the best membrane processes combination and to optimize the used conditions. Then, a validation study of the fractionation using the hydrolysate of tuna by-products produced during was performed. In this study, different modes of fractionation combination of concentration and diafiltration steps were tested to determine the best method for the recovery of large quantities of interesting peptide fractions. The originality of this PhD work is the enrichment of the tuna by-products hydrolysate with valuable compounds such as essential amino acids and peptides with a high biological activity.