Thesis (MScEng)--Stellenbosch University, 2012 / ENGLISH ABSTRACT: Through many years of research, a number of production schemes have been developed for converting lignocellulosic biomass into transport fuels. These technologies have been assessed through a number of techno-economic studies for application in a particular context in terms of the technical and economic feasibility. However, previous studies using these methods have tended to lack vigour in various aspects. Either the energy efficiency of the processes were not maximised through adequate heat integration, or a competing technology which existed was not considered. From an economic perspective, the financial models would often lack the vigour to account for the risk and uncertainty that is inherent in the market prices of the commodities. This phenomenon is especially relevant for the biofuel industry that faces the full fledge of uncertainties experienced by the agricultural sector and the energy sector. Furthermore, from an environmental perspective, the techno-economic studies had often ignored the environmental impacts that are associated with biofuel production. Thus, a comparative study could have favoured an option due to its economic feasibility, while it could have had serious environmental consequences. The aim of this study was to address these issues in a South African context, where biofuels could be produced from sugarcane bagasse. The first step would be to modify an existing simulation model for a bioethanol scenario that operates with a Separate Hydrolysis and Fermentation (SHF process) configuration into a second processing scenario that operates with a Simultaneous Saccharification and Fermentation (SSF process) configuration using reliable experimental data. The second step was to ensure that the maximum energy efficiency of each scenario was realised by carrying out pinch point analysis as a heat integration step. In contrast to these biological models is the thermochemical model that converts bagasse to gasoline and diesel via gasification, Fischer-Tropsch synthesis and refining (GFT process). While there were no significant advances in technology concerning this type of process, the energy efficiency was to be maximised with pinch point analysis. The GFT process obtained the highest energy efficiency of 50.6%. Without the affects of pinch point technology, the efficiency dropped to 46%, which thus emphasises the importance of heat integration. The SSF had an efficiency of 42.8%, which was superior to that of the SHF at 39.3%. This resulted from a higher conversion of biomass to ethanol in the SSF scenario. Comparing the SHF model to an identical model found in literature that did not have pinch point retrofits, this study showed lower efficiency. This arose because the previous study did not account for the energy demands of the cold utility systems such as the cooling tower operation, which has been shown in this study to account for 40% of the electrical energy needs. The economic viability of all three processes was assessed with Monte Carlo Simulations to account for the risks that the fluctuations in commodity prices and financial indices pose. This was accomplished by projecting the fluctuations of these parameters from samples of a historical database that has been transformed into a probability distribution function. The consequences were measured in terms of the Net Present Value (NPV) and Internal Rate of Return (IRR) for a large number of simulations. The results of these variables were aggregated and were then assessed by testing the probability that the NPV<0, and that the IRR recedes below the interest rate of 12.64%. The investment was thus deemed unfeasible if these probabilities were greater than 20%. Both biological models were deemed profitable in terms of this standard. The probabilities were 13% for the SSF and 14% for the SHF. The GFT process however was deemed completely unfeasible because the probability that the NPV<0 was 78%. Given that the GFT process had the highest energy efficiency, this result arises mainly because the capital investment of 140,000USD/MWHHV of biomass energy input is to enormous for any payback to be expected. The environmental footprint of each process was measured using Life Cycle Assessments (LCAs). LCAs are a scientifically intricate way of quantifying and qualifying the effects of a product or process within a specified boundary. The impacts are assessed on a range of environmental issues, such as Global Warming, Acidification, Eutrophication and Human toxicity. Furthermore, if the project under concern has multiple output products, then the impacts are distributed between the output products in proportion to the revenue that each generates. The impacts were either relative to the flow of feedstock, which was 600MW of bagasse, or to the functional unit, which was the amount of fuel required to power a standard vehicle for a distance of 1 kilometre. In either case, the GFT scenario was the least burdening on the environmental. This was expected because the GFT process had the highest energy efficiency and the process itself lacked the use of processing chemicals. Relative to the feedstock flow, the SSF was the most environmentally burdening scenario due to the intensive use of processing chemicals. Relative to the functional unit, the SHF was the most severe due to its low energy efficiency. Thus, the following conclusions were drawn from the study:  The GFT is the most energy and environmentally efficient process, but it showed no sign of economic feasibility. iv  There is no significant difference in the economic and environmental evaluation of the SSF and SHF process, even though the SSF is considered to be a newer and more efficient process. The major cause of this is because the setup of the SSF model was not optimised. / AFRIKAANSE OPSOMMING: Deur baie jare van navorsing is ‘n aantal produksie-skemas vir die omskakeling van lignosellulose biomassa na vloeibarebrandstof ontwikkel. Hierdie tegnologië is geassesseer ten opsigte van die tegniese en ekonomiese haalbaarheid deur middel van tegno-ekonomiese studies in bepaalde tekste. Tog het hierdie vorige studies besliste beperkings gehad. Of die energie-doeltreffendheid van die proses is nie gemaksimeer deur voldoende hitte-integrasie nie, of 'n mededingende tegnologie wat bestaan is nie oorweeg nie. Vanuit 'n ekonomiese perspektief, was die finansiële modelle dikwels nie die omvattend genoeg om rekening te hou met die risiko en onsekerheid wat inherent is in die markpryse van die kommoditeite nie. Hierdie verskynsel is veral relevant vir die biobrandstof bedryf wat die volle omvang van onsekerhede ervaar waaraan die landbousektor en die energiesektoronderhewig is. Verder het die tegno-ekonomiese studies dikwels die omgewingsimpakte wat verband hou met biobrandstofproduksie geïgnoreer. Dus kon ‘n opsie deur die ekonomiese haalbaarheid bevoordeel word, ten spyte van die ernstige omgewingsimpakte wat dit kon inhou. Die doel van hierdie studie was om hierdie kwessies aan te spreek in 'n Suid-Afrikaanse konteks, waar biobrandstof uit suikerriet bagasse geproduseer kan word. Die eerste stap was om 'n bestaande simulasiemodel vir 'n bio-scenario wat met Afsonderlike Hidroliese en Fermentasie (SHF proses) stappe werk, te modifiseer vir 'n tweede verwerking scenario wat met 'n gelyktydige Versuikering en Fermentasie (SSF proses) konfigurasie werk. Die verandering is gedoen deur die gebruik van betroubare eksperimentele data. Die tweede stap was om te verseker dat elke scenario die maksimum energie-doeltreffendheid het, deur 'n hitte-integrasie stap, wat gebruik maak van “pinch-point” analise. In teenstelling met hierdie biologiese modelle, is daar die thermochemiese roete waar petrol en diesel van bagasse vervaardig word via vergassing, Fischer-Tropsch-sintese en rafinering (GFT proses). Daar was geen betekenisvolle vooruitgang in tegnologie vir hierdie proses nie, maar die energie-doeltreffendheid is gemaksimeer word deur energie-integrasie. Die GFT proses toon die hoogste energie-doeltreffendheid van 50,6%. Sonder die invloed van energie-integrasie het die doeltreffendheid gedaal tot 46%, wat dus die belangrikheid van hitte-integrasie beklemtoon. Die SSF het 'n effektiwiteit van 42,8% gehad, wat beter was as dié 39,3% van die SHF opsie. Hierdie hoër effektiwiteit wasas gevolg van die hoër omskakeling van biomassa na etanol in die SSF scenario. Die energie doeltreffendheid vir die SHF-model was laer as met 'n identiese model (sonder energie-integrasie) wat in die literatuur gevind wat is. Dit het ontstaan omdat die vorige studie nie 'n volledig voorsiening gemaak het met die energie-eise van die verkillingstelselsnie, wat tot 40% van die elektriese energie behoeftes kan uitmaak. Die ekonomiese lewensvatbaarheid van al drie prosesse is bepaal met Monte Carlo simulasies om die risiko's wat die fluktuasies in kommoditeitspryse en finansiële indekse inhou, in berekening te bring. Hierdie is bereik deur die projeksie van die fluktuasies van hierdie parameters aan die hand van 'n historiese databasis wat omskep is in 'n waarskynlikheid verspreiding funksie. Die gevolge is gemeet in terme van die netto huidige waarde (NHW) en Interne Opbrengskoers (IOK) vir 'n groot aantal simulasies. Die resultate van hierdie veranderlikes is saamgevoeg en daarna, deur die toets van die waarskynlikheid dat die NPV <0, en dat die IRR laer as die rentekoers van 12,64% daal, beoordeel. Die belegging is dus nie realiseerbaar geag as die waarskynlikhede meer as 20% was nie. Beide biologieseprosesse kan as winsgewend beskou word in terme van bostaande norme. Die waarskynlikhede was 13% vir die SSF en 14% vir die SHF. Aangesien die NHW van die GFT-proses onder 0 met ‘n waarskynlikheid van 78% is, is die opsie as nie-winsgewend beskou. Gegewe dat die GFT-proses die hoogste energie-doeltreffendheid het, is die resultaat hoofsaaklik omdat die kapitale belegging van 140,000 USD / MWHHV-biomassa energie-inset te groot is, om enige terugbetaling te verwag. Die omgewingsvoetspoor van elke proses is bepaal deur die gebruik van Lewens Siklus Analises (“Life Cycle Assessments”) (LCAS). LCAS is 'n wetenskaplike metodeom die effek van ‘n produk of proses binne bepaalde grense beide kwalitatief en kwantitatief te bepaal. Die impakte word beoordeel vir 'n verskeidenheid van omgewingskwessies, soos aardverwarming, versuring, eutrofikasie en menslike toksisiteit. Voorts, indien die projek onder die saak verskeie afvoer produkte het, word die impakte tussen die afvoer produkte verdeel, in verhouding tot die inkomste wat elkeen genereer. Die impak was met of relatief tot die vloei van roumateriaal (600MW van bagasse), of tot die funksionele eenheid, wat die hoeveelheid van brandstof is om 'n standaard voertuig aan te dryf oor 'n afstand van 1 kilometer. In al die gevalle het die GFT scenario die laagste belading op die omgewing geplaas. Hierdie is te verwagte omdat die GFT proses die hoogste energie-doeltreffendheid het en die proses self nie enige addisionele chemikalieë vereis nie. Relatief tot die roumateriaal vloei, het die SSF die grootse belading op die omgewing geplaas as gevolg van die intensiewe gebruik van verwerkte chemikalieë. Relatief tot die funksionele eenheid, was die SHF die swakste as gevolg van sy lae energie-doeltreffendheid.

Made available in DSpace on 2017-05-12T14:47:05Z (GMT). No. of bitstreams: 1 JULIANA_ MOCO CORREA (2).pdf: 1734110 bytes, checksum: 24beceb790c634d766ff59c9de448991 (MD5) Previous issue date: 2014-11-27 / PAPER 1 Depletion of the xynB2 gene upregulates &#946;-Xylosidase expression in C. crescentus. Caulobacter crescentus is able to express several enzymes involved in the utilization of lignocellulosic biomasses. Five genes, xynB1-5, that encode &#946;-xylosidases are present in the genome of this bacterium. In this study, the xynB2 gene, which encodes &#61538;- xylosidase II (CCNA_02442), was cloned under the control of the PxylX promoter to generate the O-xynB2 strain, which overexpresses the enzyme in the presence of xylose. In addition, a null mutant strain, &#61508;-xynB2, was created by two homologous recombination events where the chromosomal xynB2 gene was replaced by a copy that was disrupted by the spectinomycin-resistant cassette. It was demonstrated that C. crescentus cells lacking &#61538;-xylosidase II up-regulates the xynB genes inducing &#946;- xylosidase activity. Transcriptional analysis revealed that xynB1 (RT-PCR analysis) and xynB2 (lacZ transcription fusion) gene expression was induced in the &#61508;-xynB2 cells, and high &#61538;-xylosidase activity was observed in the presence of different agroindustrial residues in the null mutant strain, a characteristic that can be explored and applied in biotechnological processes. In contrast, overexpression of the xynB2 gene caused down-regulation of the expression and activity of the &#61538;-xylosidase. For example, the &#946;-xylosidase activity that was obtained in the presence of sugar cane bagasse was 7-fold and 16-fold higher than the activity measured in the C. crescentus parental and O-xynB2 cells, respectively. Our results suggest that &#61538;-xylosidase II may have a role in controlling the expression of the xynB1 and xynB2 genes in C.crescentus. PAPER 2 - OPTIMIZATION OF THE PRODUCTION &#946;-XYLOSIDASE: A NEW Thermomyces lanuginosus ISOLATED FROM ATLANTIC FOREST BIOME. The successful production of enzymes for the deconstruction of plant biomass depends not only on the isolation and identification of new microorganism producers of hemicellulases, but also on the implementation and improvement of experimental strategies that lead to maximal induction of enzymatic activities. In this work, a new strain of Thermomyces lanuginosus (T. lanuginosus) was isolated from the Atlantic Forest biome in Brazil, and its &#946;-xylosidase activity in response to agro-industrial residues was tested. Using the (CCRD) statistical approach as a strategy for optimization, the induction of &#946;-xylosidase activity was evaluated in residual corn straw, which was used as a carbon source, and improved so that the optimum condition achieved high &#946;-xylosidase activity (1,003 U ml -1; specific activity = 1.683 U mg-1) with 214 U ml -1. The optimal conditions for the crude enzyme extract were pH 5.5 and 60° C showing better thermostability at 55° C. The saccharification ability of &#946;-xylosidase in the presence of hemicellulose obtained from corn straw and xylan from beechwood substrates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%, respectively, at 50° C. These data suggest that &#946;-xylosidase from T. lanuginosus isolated from the Atlantic Forest can be used for the saccharification of hemicellulose derived from corn straw, an abundant residue in the American continents, thus providing an interesting alternative for future tests for energy production that relies on the conversion of plant biomass. / RESUMO GERAL As Beta-D-Xilosidases (1,4-&#946;-D-xilano xilohidrolase; EC 3.2.1.37) são glicosídeo hidrolases que tem papel crucial em catalizar a liberação de unidades de xilose a partir de xilo-oligossacarídeos derivados da degradação do xilano. A completa degradação do xilano é um passo chave do ciclo do carbono na natureza e é um processo também realizado por microrganismos. A bioconversão de materiais lignocelulósicos é vantajosa não somente do ponto de vista ambiental mais também econômico o que é recebido nos setores produtivos com um considerável interesse, pois esses materiais representam vasta fonte de carbono, que podem ser empregados no desenvolvimento de bioprocessos que resultam em produtos de alto valor agregado; entre os quais estão os açúcares fermentáveis, combustíveis, fármacos, enzimas e substâncias de interesse industrial, além de fazer uma gestão integrada do efluente que por não haver um desenvolvimento biotecnológico adequado é descartado e acumulado na natureza. Em face disso, o presente trabalho teve por objetivos estudar a regulação gênica do gene xynB2 da bactéria Caulobacter crescentus que codifica para a Beta-xilosidase II através de abordagens moleculares e otimizar a produção enzimática de Betaxilosidases de Thermomyces lanuginosus na presença de diferentes resíduos de biomassa vegetal por delineamento experimental. No primeiro artigo exploramos a bactéria aquática Caulobacter crescentus por possuir várias enzimas envolvidas na utilização de biomassas lignocelulósicas; contendo em seu genoma cinco genes que codificam &#946;-xilosidases. A partir do gene xynB2, que codifica para enzima &#61538;-xylosidase II (CCNA_02442), desenvolvemos duas linhagens mutantes denominadas O-xynB2, que super-expressa a enzima na presença de xilose e &#61508;-xynB2 que tem o gene xynB2 interrompido, o que possibilitou avaliar que a ausência da enzima &#61538;-xylosidase II em células de C. crescentus regula positivamente os genes xynB, induzindo a atividade global de &#946;-xilosidases, revelando um papel regulatório para a mesma. No segundo trabalho um fungo da linhagem Thermomyces lanuginosus isolado de bioma de Mata Atlântica foi identificado e analisado quanto à capacidade de produzir Beta-xilosidases na presença de diferentes resíduos vegetais; em decorrência disso foi otimizado a produção enzimática com delineamento experimental DCCR, o que permitiu alcançar altos níveis de atividade enzimática beta-xilosidásica na presença de palha de milho.

Lignocellulosic biomass is composed of cellulose, hemicellulose, lignin and extraneous compounds (waxes, fats, gums, starches, alkaloids, resins, tannins, essential oils, silica, carbonates, oxalates, etc). The sugars within the complex carbohydrates (cellulose and hemicellulose) can be accessed for cellulosic bioethanol production through ethanologenic microorganisms. However, the composite nature of lignocellulosic biomass, particularly the lignin portion, presents resistance and recalcitrance to biological and chemical degradation during enzymatic hydrolysis/saccharification and the subsequent fermentation process. This leads to a very low conversion rate, which makes the process uneconomically feasible. Thus, biomass structure requires initial breakdown of the lignocellulosic matrix. In this study, two types of biomass pretreatment were applied on barley straw grind: radio-frequency (RF)-based dielectric heating technique using alkaline (NaOH) solution as a catalyst and steam explosion pretreatment at low severity factor. The pretreatment was applied on barley straw which was ground in hammer mill with a screen size of 1.6 mm, so as to enhance its accessibility and digestibility by enzymatic reaction during hydrolysis. Three levels of temperature (70, 80, and 90oC), five levels of ratio of biomass to 1% NaOH solution (1:4, 1:5, 1:6, 1:7, & 1:8), 1 h soaking time, and 20 min residence time were used for the radio frequency pretreatment. The following process and material variables were used for the steam explosion pretreatment: temperature (140-180oC), retention time (5-10 min), and 8-50% moisture content (w.b). The effect of both pretreatments was assessed through chemical composition analysis and densification of the pretreated and non-pretreated biomass samples. Results of this investigation show that lignocellulosic biomass absorbed more NaOH than water, because of the hydrophobic nature of lignin, which acts as an external crosslink binder on the biomass matrix and shields the hydrophilic structural carbohydrates (cellulose and hemicellulose). It was observed in the RF pretreatment that the use of NaOH solution and the ratio of biomass to NaOH solution played a major role, while temperature played a lesser role in the breakdown of the lignified matrix, as well as in the production of pellets with good physical quality. The heat provided by the RF is required to assist the alkaline solution in the deconstruction and disaggregation of lignocellulosic biomass matrix. The disruption and deconstruction of the lignified matrix is also associated with the dipole interaction, flip flop rotation, and friction generated between the electromagnetic charges from the RF and the ions and molecules from the NaOH solution and the biomass. The preserved cellulose from the raw sample (non-treated) was higher than that from the RF alkaline pretreated samples because of the initial degradation of the sugars during the pretreatment process. The same observation applies to hemicellulose. This implies that there is a trade-off between the breakdown of the biomass matrix/creating pores in the lignin and enhancing the accessibility and digestibility of the cellulose and hemicellulose. The use of dilute NaOH solution in biomass pretreatment showed that the higher the NaOH concentration, the lower was the acid insoluble lignin and the higher was the solubilized lignin moieties. The ratio of 1:6 at the four temperatures studied was determined to be the optimal. Based on the obtained data, it is predicted that this pretreatment will decrease the required amount and cost of enzymes by up to 64% compared to using non-treated biomass. However, the use of NaOH led to an increase in the ash content of biomass. The ash content increased with the decreasing ratio of biomass to NaOH solution. This problem of increased ash content can be addressed by washing the pretreated samples. RF assisted-alkaline pretreatment technique represents an easy to set-up and potentially affordable route for the bio-fuel industry, but this requires further energy analysis and economic validation, so as to investigate the significant high energy consumption during the RF-assisted alkaline pretreatment heating process. Data showed that in the steam explosion (SE) pretreatment, considerable thermal degradation of the energy potentials (cellulose and hemicellulose) with increasing acid soluble and insoluble lignin content occurred. The high degradation of the hemicellulose can be accounted for by its amorphous nature which is easily disrupted by external influences unlike the well-arranged crystalline cellulose. It is predicted that this pretreatment will decrease the required amount and cost of enzymes by up to 33% compared to using non-treated biomass.The carbon content of the solid SE product increased at higher temperature and longer residence time, while the hydrogen and oxygen content decreased. The RF alkaline and SE treatment combinations that resulted to optimum yield of cellulose and hemicellulose were selected and then enzymatically digested with a combined mixture of cellulase and β-glucosidase enzymes at 50oC for 96 h on a shaking incubator at 250 rev/min. The glucose in the hydrolyzed samples was subsequently quantified. The results obtained confirmed the effectiveness of the pretreatment processes. The average available percentage glucose yield that was released during the enzymatic hydrolysis for bioethanol production ranged from 78-96% for RF-alkaline pretreated and 30-50% for the SE pretreated barley straw depending on the treatment combination. While the non-treated sample has available average percentage glucose yield of just below 12%. The effects of both pretreatment methods (RF and SE) were further evaluated by pelletizing the pretreated and non-pretreated barley straw samples in a single pelleting unit. The physical characteristics (pellet density, tensile strength, durability rating, and dimensional stability) of the pellets were determined. The lower was the biomass:NaOH solution ratio, the better was the quality of the produced pellets. Washing of the RF-alkaline pretreated samples resulted in pellets with low quality. A biomass:NaOH solution ratio of 1:8 at the three levels of temperature (70, 80, and 90oC) studied are the RF optimum pretreatment conditions. The higher heating value (HHV) and the physical characteristics of the produced pellets increased with increasing temperature and residence time. The steam exploded samples pretreated at higher temperatures (180ºC) and retention time of 10 min resulted into pellets with good physical qualities. Fourier transform infrared-photoacoustic spectroscopy (FTIR-PAS) was further applied on the RF alkaline and SE samples in light of the need for rapid and easy quantification of biomass chemical components (cellulose, hemicellulose, and lignin). The results obtained show that the FTIR-PAS spectra can be rapidly used for the analysis and identification of the chemical composition of biofuel feedstock. Predictive models were developed for each of the biomass components in estimating their respective percentage chemical compositions.

Made available in DSpace on 2014-12-17T15:01:34Z (GMT). No. of bitstreams: 1 PedroFSF_DISSERT.pdf: 2707475 bytes, checksum: 2a6ef9226a3d6d607615f74305bb9e07 (MD5) Previous issue date: 2014-05-09 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The need for new sources of energy and the concern about the environment have pushed the search for renewable energy sources such as ethanol. The use of lignocellulosic biomass as substrate appears as an important alternative because of the abundance of this raw material and for it does not compete with food production. However, the process still meets difficulties of implementation, including the cost for production of enzymes that degrade cellulose to fermentable sugars. The aim of this study was to evaluate the behavior of the species of cactus pear Opuntia ficus indica and Nopalea cochenillifera, commonly found in northeastern Brazil, as raw materials for the production of: 1) cellulosic ethanol by simultaneous saccharification and fermentation (SSF) process, using two different strains of Saccharomyces cerevisiae (PE-2 and LNF CA-11), and 2) cellulolytic enzymes by semi-solid state fermentation (SSSF) using the filamentous fungus Penicillium chrysogenum. Before alcoholic fermentation process, the material was conditioned and pretreated by three different strategies: alkaline hydrogen peroxide, alkaline using NaOH and acid using H2SO4 followed by alkaline delignification with NaOH. Analysis of composition, crystallinity and enzymatic digestibility were carried out with the material before and after pretreatment. In addition, scanning electron microscopy images were used to compare qualitatively the material and observe the effects of pretreatments. An experimental design 2? with triplicate at the central point was used to evaluate the influence of temperature (30, 40 and 45 ?C) and the initial charge of substrate (3, 4 and 5% cellulose) in the SSF process using the material obtained through the best condition and testing both strains of S. cerevisiae, one of them flocculent (LNF CA-11). For cellulase production, the filamentous fungus P. chrysogenum was tested with N. cochenillifera in the raw condition (without pretreatment) and pretrated hydrothermically, varying the pH of the fermentative medium (3, 5 and 7). The characterization of cactus pear resulted in 31.55% cellulose, 17.12% hemicellulose and 10.25% lignin for N. cochenillifera and 34.86% cellulose, 19.97% hemicellulose and 15.72% lignin for O. ficus indica. It has also been determined, to N. cochenillifera and O. ficus indica, the content of pectin (5.44% and 5.55% of calcium pectate, respectively), extractives (26.90% and 9.69%, respectively) and ashes (5.40% and 5.95%). Pretreatment using alkaline hydrogen peroxide resulted in the best cellulose recovery results (86.16% for N. cochenillifera and 93.59% for O. ficus indica) and delignification (48.79% and 23.84% for N. cochenillifera and O. ficus indica, respectively). This pretreatment was also the only one which did not increase the crystallinity index of the samples, in the case of O. ficus indica. However, when analyzing the enzymatic digestibility of cellulose, alkali pretreatment was the one which showed the best yields and therefore it was chosen for the tests in SSF. The experiments showed higher yield of conversion of cellulose to ethanol by PE-2 strain using the pretreated N. cochenillifera (93.81%) at 40 ?C using 4% initial charge of cellulose. N. cochenillifera gave better yields than O. ficus indica and PE-2 strain showed better performance than CA-11. N. cochenillifera proved to be a substrate that can be used in the SSSF for enzymes production, reaching values of 1.00 U/g of CMCase and 0.85 FPU/g. The pretreatment was not effective to increase the enzymatic activity values / A necessidade de novas fontes de energia e a preocupa??o com o meio-ambiente t?m impulsionado a pesquisa por fontes renov?veis de energia, como o etanol. O uso de biomassa lignocelul?sica como substrato aparece como uma importante alternativa devido ? abund?ncia desta mat?ria-prima e por n?o concorrer com a produ??o de alimentos. Entretanto, o processo ainda encontra dificuldades de implementa??o, entre elas o custo para produ??o das enzimas que degradam a celulose em a??cares fermentesc?veis. O objetivo deste trabalho foi avaliar o comportamento das esp?cies de palma forrageira Opuntia ficus indica (gigante) e Nopalea cochenillifera (mi?da), comumente encontradas na regi?o Nordeste do Brasil, como mat?rias-primas para produ??o de: 1) etanol celul?sico pelo processo de sacarifica??o e fermenta??o simult?neas (SFS) usando duas cepas diferentes de Saccharomyces cerevisiae (PE-2 e LNF CA-11) e 2) enzimas celulol?ticas atrav?s da fermenta??o em estado semiss?lido (FES) usando o fungo filamentoso Penicillium chrysogenum. Antes do processo de fermenta??o alco?lica, o material foi condicionado e pr?-tratado por tr?s diferentes estrat?gias: per?xido de hidrog?nio alcalino, alcalino usando NaOH e ?cido usando H2SO4 seguido de deslignifica??o alcalina com NaOH. An?lises de composi??o, cristalinidade e digestibilidade enzim?tica foram feitas com o material antes e depois do pr?-tratamento. Adicionalmente, imagens de microscopia eletr?nica de varredura foram usadas para comparar qualitativamente o material e observar os efeitos dos pr?-tratamentos. Um planejamento fatorial 2? com triplicata no ponto central foi utilizado para avaliar a influ?ncia da temperatura (30, 40 e 45 ?C) e da carga inicial de substrato (3, 4 e 5% de celulose) no processo SFS, usando o material obtido nas melhores condi??es de pr?-tratamento e testando duas cepas de S. cerevisiae, sendo uma delas floculante (LNF CA-11). Para a produ??o de celulase, o fungo filamentoso P. chrysogenum foi testado com a esp?cie de palma N. cochenillifera no estado in-natura (sem pr?-tratamento) e submetida a um pr?-tratamento hidrot?rmico, variando-se o pH do meio fermentativo (3, 5 e 7). A caracteriza??o das palmas forrageiras resultou em 31,55% de celulose, 17,12% de hemicelulose e 10,25% de lignina para a esp?cie N. cochenillifera e 34,86% de celulose, 19,97% de hemicelulose e 15,72% de lignina para a esp?cie O. ficus indica. Analisou-se ainda, para as palmas mi?da e gigante, o teor de pectina (5,44% e 5,55% de pectato de c?lcio, respectivamente), extrativos (26,90% e 9,69%, respectivamente) e cinzas (5,40% e 5,95%). O pr?-tratamento usando per?xido de hidrog?nio alcalino apresentou os melhores resultados de recupera??o de celulose (86,16% para a palma mi?da e 93,59% para a palma gigante) e de deslignifica??o (48,79% e 23,84% para as palmas mi?da e gigante, respectivamente). Este pr?-tratamento foi tamb?m o ?nico a n?o elevar o ?ndice de cristalinidade das amostras, no caso da palma gigante. Entretanto, quando analisada a digestibilidade enzim?tica da celulose, o pr?-tratamento alcalino foi o que proporcionou os melhores rendimentos e, portanto, este foi o escolhido para os testes de SFS. Os experimentos demonstraram maior rendimento da convers?o de celulose em etanol pela cepa PE-2 usando a palma mi?da pr?-tratada (93,81%) a 40 ?C e usando 4% de carga inicial de celulose. A palma mi?da demonstrou melhores rendimentos que a gigante e a cepa PE-2 resultou melhor desempenho que a CA-11. A palma mi?da se mostrou um substrato poss?vel de ser usado na FES para produ??o de enzimas, alcan?ando valores de 1,00 U/g de CMCase e 0,85 FPU/g. O pr?-tratamento n?o se mostrou eficaz para aumentar os valores de atividade enzim?tica

AgÃncia Nacional do PetrÃleo / CoordenaÃÃo de AperfeÃoamento de Pessoal de NÃvel Superior / In this work, the ethanol production from cashew bagasse was studied after acid followed by alkali pretreatment (CAB-OH) using the Separate Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF) processes. In SHF process, the hydrolysate obtained from enzymatic hydrolysis of CAB-OH was used as carbon source for fermentation with different strains of Saccharomyces (S. cerevisiae CCA008, S. cerevisiae 01, S. cerevisiae 02 and Saccharomyces sp. 1238), Kluyveromyces (K. marxianus CCA510, CE025 and ATCC36907) and Hanseniaspora sp. GPBio03. The bioprocess was conducted at 30 ÂC and 50 g.L-1 initial glucose concentration. The K. marxianus ATCC36907 achieved ethanol concentration of 20 g.L-1 with consumption of all glucose in the hydrolysate. Similar results were obtained with Saccharomyces strains and higher ethanol concentration (23.43 g.L-1) was obtained by Saccharomyces sp. 1238. The maximum ethanol concentration of 24.54 g.L-1 was achieved by Hanseniaspora sp. GPBio03. Focused on further studies using SSF process, it was evaluated the temperature influence of thermotolerant yeast K. marxianus ATCC36907 in glucose and enzymatic hydrolysate from CAB-OH. The results showed that the temperature (30, 35, 40, 45 and 50 ÂC) did not affect the values of YE/G (0.45 to 0.46 gethanol/gglucose) using glucose as substrate. Moreover, the ethanol yields obtained with enzymatic hydrolysate were slightly influenced by temperature, 0.39 and 0.43 gethanol/gglucose were obtained at 30 and 40 ÂC, respectively. Based on this, the SSF of CAB-OH and K. marxianus ATCC36907 was conducted at 40 ÂC with cellulases from Celluclast 1.5L at 15 FPU/gcellulose. The highest ethanol concentration (24.90  0.89 g.L-1) was obtained with 76h of fermentation with 0.33 g.L-1.h-1, 0.34 gethanol/gglucose and 66.3% of productivity, Y&#884;E/G and of ethanol efficiency, respectively. In enzymatic hydrolysis studies, the cellulase NS 22074 at 30 FPU/gcellulose without cellobiases supplementation resulted in glucose yield of 93.77  2.72% which is promising for studies of SSF with this enzyme complex. The temperature (40, 42 , 45 and 50 ÂC) influence in SSF process using microcrystalline cellulose, in contrast with SHF results, higher ethanol concentration, 19.86  0.32 g.L-1, was obtained at 40 ÂC. The SSF using CAB-OH, 30 FPU/gcellulose cellulases NS 22074 at 40 ÂC showed higher ethanol concentration of 37.35  0.64 g.L-1 at 80h, with productivity of 0.46 g.L-1.h-1. In this condition, there was an increase of Y&#884;E/G from 0.34 to 0.49 gethanol/gglucose and the ethanol efficiency from 66.3% to 95.59% when compared to results obtained with SSF using Celluclast 1.5L. Based on the results of efficiency and ethanol yield (Y&#884;E/G), the cashew apple bagasse showed as lignocelulose feedstock promising material for second generation ethanol production by SSF process using the yeast K. marxianus ATCC36907 and NS 22074 cellulases complex. / Nesse trabalho, estudou-se a produÃÃo de etanol de bagaÃo de caju apÃs prÃ-tratamento Ãcido seguido de Ãlcali (CAB-OH) atravÃs dos processos de FermentaÃÃo e HidrÃlise Separadas (SHF) e FermentaÃÃo e HidrÃlise SimultÃneas (SSF). No processo SHF, o hidrolisado obtido da hidrÃlise enzimÃtica de CAB-OH foi submetido à etapa de fermentaÃÃo com diferentes linhagens de Saccharomyces (S. cerevisiae CCA008, Saccharomyces sp. 1238, S. cerevisiae 01, S. cerevisiae 02), Kluyveromyces (K. marxianus CCA510, CE025 e ATCC36907) e Hanseniaspora sp. GPBio03. A fermentaÃÃo do hidrolisado foi conduzida a 30 ÂC com concentraÃÃo inicial de glicose de 50 g.L-1. ApÃs o screening de leveduras, a linhagem de K. marxianus ATCC36907 destacou-se com maior concentraÃÃo de etanol de 20 g.L-1 com consumo de toda glicose no hidrolisado. Resultados similares foram obtidos com Saccharomyces sp. 1238 e com a levedura isolada do caju (Hanseniaspora sp. GPBio03) com maiores concentraÃÃes de etanol de 22,41 g.L-1 e 24,54 g.L-1, respectivamente. Com o propÃsito de estudos posteriores de SSF, avaliou-se a influÃncia da temperatura da levedura termotolerante K. marxianus ATCC36907 em glicose PA e hidrolisado enzimÃtico de CAB-OH. Os resultados mostraram que para a glicose PA, a variaÃÃo da temperatura (30, 35, 40, 45 e 50 ÂC) nÃo influenciou nos valores de conversÃo de glicose em etanol (YE/G) obtendo-se valores na faixa de 0,45-0,46 getanol/gglicose. Por outro lado, os resultados de YE/G em hidrolisado enzimÃtico foram ligeiramente influenciados pela temperatura, obtendo-se 0,39 getanol/gglicose a 30ÂC e 0,43 getanol/gglicose a 40 ÂC. Em seguida, realizou-se a SSF de CAB-OH com K. marxianus ATCC36907 a 40 ÂC e celulases de Celluclast 1.5L a 15 FPU/gcelulose. A maior concentraÃÃo de etanol (24,90  0,89 g.L-1) foi obtida em 76h de fermentaÃÃo com produtividade de 0,33 g.L-1.h-1, conversÃo de glicose em etanol (Y&#884;E/G) de 0,34 e eficiÃncia de produÃÃo de etanol de 66,3%. Contudo, visando aumentar a produÃÃo de etanol em estudos posteriores de SSF, realizou-se o estudo de hidrÃlise enzimÃtica com outros complexos de celulases (NS 22074) e celobiases (NS 50010). Os resultados de hidrÃlise enzimÃtica mostraram que a atividade de celulases NS 22074 a 30 FPU/gcelulose sem suplementaÃÃo de celobiase resultou no rendimento de glicose de 93,77  2,72% sendo resultado promissor para estudos de SSF com esse complexo enzimÃtico. Nos ensaios de SSF com celulases do complexo NS 22074, inicialmente realizou-se o estudo da temperatura (40, 42, 45 e 50 ÂC) com K. marxianus ATCC36907 utilizando celulose microcristalina; e, em contrapartida com os resultados SHF, na temperatura de 40 ÂC foi obtida a maior concentraÃÃo de etanol de 19,86  0,32 g.L-1, em 72h de fermentaÃÃo. Diante desses resultados, realizou-se o processo de SSF de CAB-OH nas seguintes condiÃÃes: 40 ÂC de temperatura e 30 FPU/gcelulose do complexo de celulases NS 22074. A maior concentraÃÃo de etanol (37,35  0,64 g.L-1) foi obtida em 80h de fermentaÃÃo, com produtividade de 0,46 g.L-1.h-1. Diante desses resultados, observa-se que a mudanÃa do complexo enzimÃtico de Celluclast 1.5L para NS 22074 proporcionou o aumento no valor de Y&#884;E/G de 0,34 getanol/gglicose para 0,49 getanol/gglicose e no rendimento de etanol de 66,3% para 95,59%, o que torna o bagaÃo de caju prÃ-tratado promissor como matÃria-prima para produÃÃo de etanol de segunda geraÃÃo por processo SSF utilizando a levedura K. marxianus ATCC36907.

Made available in DSpace on 2017-07-10T19:23:52Z (GMT). No. of bitstreams: 1 JULIANA_ MOCO CORREA (2).pdf: 1734110 bytes, checksum: 24beceb790c634d766ff59c9de448991 (MD5) Previous issue date: 2014-11-27 / PAPER 1 Depletion of the xynB2 gene upregulates &#946;-Xylosidase expression in C. crescentus. Caulobacter crescentus is able to express several enzymes involved in the utilization of lignocellulosic biomasses. Five genes, xynB1-5, that encode &#946;-xylosidases are present in the genome of this bacterium. In this study, the xynB2 gene, which encodes &#61538;- xylosidase II (CCNA_02442), was cloned under the control of the PxylX promoter to generate the O-xynB2 strain, which overexpresses the enzyme in the presence of xylose. In addition, a null mutant strain, &#61508;-xynB2, was created by two homologous recombination events where the chromosomal xynB2 gene was replaced by a copy that was disrupted by the spectinomycin-resistant cassette. It was demonstrated that C. crescentus cells lacking &#61538;-xylosidase II up-regulates the xynB genes inducing &#946;- xylosidase activity. Transcriptional analysis revealed that xynB1 (RT-PCR analysis) and xynB2 (lacZ transcription fusion) gene expression was induced in the &#61508;-xynB2 cells, and high &#61538;-xylosidase activity was observed in the presence of different agroindustrial residues in the null mutant strain, a characteristic that can be explored and applied in biotechnological processes. In contrast, overexpression of the xynB2 gene caused down-regulation of the expression and activity of the &#61538;-xylosidase. For example, the &#946;-xylosidase activity that was obtained in the presence of sugar cane bagasse was 7-fold and 16-fold higher than the activity measured in the C. crescentus parental and O-xynB2 cells, respectively. Our results suggest that &#61538;-xylosidase II may have a role in controlling the expression of the xynB1 and xynB2 genes in C.crescentus. PAPER 2 - OPTIMIZATION OF THE PRODUCTION &#946;-XYLOSIDASE: A NEW Thermomyces lanuginosus ISOLATED FROM ATLANTIC FOREST BIOME. The successful production of enzymes for the deconstruction of plant biomass depends not only on the isolation and identification of new microorganism producers of hemicellulases, but also on the implementation and improvement of experimental strategies that lead to maximal induction of enzymatic activities. In this work, a new strain of Thermomyces lanuginosus (T. lanuginosus) was isolated from the Atlantic Forest biome in Brazil, and its &#946;-xylosidase activity in response to agro-industrial residues was tested. Using the (CCRD) statistical approach as a strategy for optimization, the induction of &#946;-xylosidase activity was evaluated in residual corn straw, which was used as a carbon source, and improved so that the optimum condition achieved high &#946;-xylosidase activity (1,003 U ml -1; specific activity = 1.683 U mg-1) with 214 U ml -1. The optimal conditions for the crude enzyme extract were pH 5.5 and 60° C showing better thermostability at 55° C. The saccharification ability of &#946;-xylosidase in the presence of hemicellulose obtained from corn straw and xylan from beechwood substrates showed a xylo-oligosaccharide to xylose conversion yield of 80 and 50%, respectively, at 50° C. These data suggest that &#946;-xylosidase from T. lanuginosus isolated from the Atlantic Forest can be used for the saccharification of hemicellulose derived from corn straw, an abundant residue in the American continents, thus providing an interesting alternative for future tests for energy production that relies on the conversion of plant biomass. / RESUMO GERAL As Beta-D-Xilosidases (1,4-&#946;-D-xilano xilohidrolase; EC 3.2.1.37) são glicosídeo hidrolases que tem papel crucial em catalizar a liberação de unidades de xilose a partir de xilo-oligossacarídeos derivados da degradação do xilano. A completa degradação do xilano é um passo chave do ciclo do carbono na natureza e é um processo também realizado por microrganismos. A bioconversão de materiais lignocelulósicos é vantajosa não somente do ponto de vista ambiental mais também econômico o que é recebido nos setores produtivos com um considerável interesse, pois esses materiais representam vasta fonte de carbono, que podem ser empregados no desenvolvimento de bioprocessos que resultam em produtos de alto valor agregado; entre os quais estão os açúcares fermentáveis, combustíveis, fármacos, enzimas e substâncias de interesse industrial, além de fazer uma gestão integrada do efluente que por não haver um desenvolvimento biotecnológico adequado é descartado e acumulado na natureza. Em face disso, o presente trabalho teve por objetivos estudar a regulação gênica do gene xynB2 da bactéria Caulobacter crescentus que codifica para a Beta-xilosidase II através de abordagens moleculares e otimizar a produção enzimática de Betaxilosidases de Thermomyces lanuginosus na presença de diferentes resíduos de biomassa vegetal por delineamento experimental. No primeiro artigo exploramos a bactéria aquática Caulobacter crescentus por possuir várias enzimas envolvidas na utilização de biomassas lignocelulósicas; contendo em seu genoma cinco genes que codificam &#946;-xilosidases. A partir do gene xynB2, que codifica para enzima &#61538;-xylosidase II (CCNA_02442), desenvolvemos duas linhagens mutantes denominadas O-xynB2, que super-expressa a enzima na presença de xilose e &#61508;-xynB2 que tem o gene xynB2 interrompido, o que possibilitou avaliar que a ausência da enzima &#61538;-xylosidase II em células de C. crescentus regula positivamente os genes xynB, induzindo a atividade global de &#946;-xilosidases, revelando um papel regulatório para a mesma. No segundo trabalho um fungo da linhagem Thermomyces lanuginosus isolado de bioma de Mata Atlântica foi identificado e analisado quanto à capacidade de produzir Beta-xilosidases na presença de diferentes resíduos vegetais; em decorrência disso foi otimizado a produção enzimática com delineamento experimental DCCR, o que permitiu alcançar altos níveis de atividade enzimática beta-xilosidásica na presença de palha de milho.

Orientador: Ljubica Tasic / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-22T23:34:19Z (GMT). No. of bitstreams: 1 Awan_AlmasTaj_D.pdf: 4796874 bytes, checksum: 185a66c389aae68c266f385689030ef0 (MD5) Previous issue date: 2013 / Resumo: Os biocombustíveis de segunda geração surgiram como fontes energéticas promissoras, podendo ser obtidos a partir de vários tipos de biomassa que não seja utilizada para alimentos. Um tipo de biomassa que apresenta baixo custo além de apresentar níveis elevados de carboidratos, é a biomassa obtida após o processamento da laranja (Citrus processing waste from oranges, CPWO). Há um grande interesse na exploração desta biomassa em termos da produção do bioetanol (etanol da 2G). Nosso trabalho visa melhorar os processos de hidrólise do CPWO comparando o rendimento do processo clássico de hidrólise ácida com aplicação de enzimas comerciais ou provenientes do microrganismo Xanthomonas axonopodis pv. citri, cepa 306 (um fitopatógeno). Os resultados obtidos com a presente investigação evidenciam que ocorreu a conversão bem-sucedida do CPWO em uma mistura de açúcares. A posteriori, os açúcares redutores que foram obtidos foram convertidos em bioetanol por meio da fermentação em mono- e co-cultura. Para tanto, foi empregada a espécie Saccharomyces cerevisiae e duas cepas de Candida parapsilosis IFM 48375 e NRRL Y-12969, sendo que as duas últimas foram isoladas a partir do bagaço da laranja. Os rendimentos em termos de bioetanol obtido nas fermentações aplicando co-culturas estavam ao redor de 50 a 62%, constituindo valores muito maiores comparados com os obtidos por cepas usadas individualmente. Além disso, os açúcares foram consumidos mais rapidamente (6 h), tornando tais processos atraentes em termos de custo e aplicações comerciais / Abstract: Second generation biofuels from renewable resources have come forth as a result of energy security coupled with diminishing fossil fuel resources. Lignocellulosic biomass is a renewable resource, which can be converted in to liquid transportation fuels. Utilization of agro-industrial waste for the generation of biofuels makes it a cleaner production (Green Chemistry). Brazil is the world¿s largest producer of oranges. The current project deals with Citrus Processing Waste from Oranges (CPWO), and obtaining valuable products such as bioethanol, hesperidin, and essential oil. The process of hydrolyzing CPWO was improved and the classical way of biomass saccharification, i.e. acid hydrolysis, was compared with the enzyme hydrolysis. In enzyme hydrolysis, apart from applying commercial enzymes, saccharification was also investigated with protein extracts of Xanthomonas axonopodis pv. citri strain 306 (Xac 306), a potent pathogen that causes Citrus canker disease. Later, the obtained reducing sugars were converted into bioethanol by submerged mono- and co-culture fermentations that involved three yeast strains: Saccharomyces cerevisiae, Candida parapsilosis IFM 48375 and NRRL Y-12969, the last two being isolated from bagasse. Results demonstrated successful hydrolyses by Xac enzymes that released high levels of fermentable sugars. Also during co-culture fermentation processes, it was noticed that ethanol yield was improved from 50% to 62% w/w (calculated on the basis of total dry matter contents) and sugars were consumed faster. Thus by employing co-culture fermentation strategy, apart from getting better bioethanol yields, fermentation time is also reduced that makes it a cost effective technique / Doutorado / Quimica Organica / Doutora em Ciências

Xylanases have an important role as biocatalysts in different agroindustrial processes, such as saccharification of plant residues for the production of ethanol and bleaching of wood pulp for the production of pulp. On an industrial scale, these processes may require extreme pH or temperature, which require enzymes compatible with these conditions. Most enzymes available or showing commercial potential is synthesized by fungi or bacteria belonging to a few genera. Evaluation of rare isolation bacteria is an important strategy to expand the diversity of xylanases and their potential in terms of activity, stability and technological application. The aim of this study was to evaluate a collection of common and rare isolation soil bacteria regarding to xylanase activity and characterize the stability conditions of temperature and pH. The analyzed collection consists of 120 isolates with representatives from six phyla that were subjected to screening for xylanase activity in pure cultures and in the extracellular proteic extract (EPE). The ratio between the halos diameters of xylan hydrolysis and in the colonies on solid medium (ratio H:C), incubated at 30 ° C for up to 14 days, was used for the evaluation of cultures as a selection criteria. The effects of different sources of variation in the bacteria isolation stage (original soil, culture medium, solidifying agent, inoculum dilution and plating method and incubation time to colony appearance) and the group of bacterial isolation (rare or common) on the frequency of isolates with high xylanase activity were evaluated based on the ratio H:C. EPEs were obtained in liquid media containing xylan inoculated with the eleven isolates with highest ratios H:C. The extracts were evaluated for the specific xylanase activity at 50 °C for 1 h. Extracts of the three isolates with the highest potential for activity under this condition were evaluated for optimum activity, stability, activity at 60 oC and at pH 4.0, 5.5 and 8.0. Twenty-two isolates (25%), including eight from rare isolation, showed xylanase activity under the conditions evaluated, and found a high variability (> 230%) between these isolates. No isolation factor or rare or common isolation condition were associated with efficiency of xylanase activity in solid medium. High variability in specific xylanase activity in EPEs was also found among isolates (1500%), highlighting two rare isolates (TC119 and TC21), from Alfaproteobacteria class, and one common isolate (TC99), from Ralstoniaceae family. Extracellular xylanases from TC21 and TC119 showed high relative activity at temperatures up to 70 oC and were insensitive to pH in the range 4.0 to 8.0; however, TC99 isolate showed optimum temperature at 40 °C and low stability to temperature and pH. Extracellular xylanases from TC119 showed no cellulolytic activity. Rare isolation soil bacteria show high potential as a source of extracellular xylanases adapted to extreme pH and temperature conditions, which are required in agroindustrial processes. / As xilanases apresentam papel relevante como biocatalisadores de diferentes processos agroindustriais, como a sacarificação de resíduos vegetais para a produção de etanol e o clareamento de polpas de madeira para a produção de celulose. Em escala industrial, estes processos podem requerer pH e, ou, temperatura extremos, os quais demandam enzimas compatíveis com estas condições. A maioria das enzimas disponíveis ou com potencial comercial é sintetizada de fungos ou de bactérias pertencentes a poucos gêneros. A avaliação de bactérias de isolamento raro constitui-se em estratégia importante para ampliar a diversidade de xilanases e suas potencialidades em termos de atividade, estabilidade e aplicação tecnológica. O objetivo deste trabalho foi avaliar uma coleção de bactérias do solo de isolamento comum e raro quanto à atividade de xilanases e caracterizar estas enzimas quanto à estabilidade a condições contrastantes de temperatura e pH. A coleção analisada é composta de 120 isolados com representantes de seis filos e foi submetida à seleção quanto à atividade de xilanases em culturas puras e no extrato proteico extracelular (EPE). Para a avaliação das culturas, utilizou-se como critério de seleção a relação entre os diâmetros de halos de hidrólise de xilana e das colônias em meio sólido (relação H:C), incubado a 30 oC por até 14 dias. Os efeitos de diferentes fontes de variação da fase de isolamento destas bactérias (solo de origem, meio de cultura, agente solidificante, diluição do inóculo e método de plaqueamento e tempo de incubação até surgimento de colônias) e do grupo de isolamento bacteriano (raro ou comum) sobre a frequência de isolados com alta atividade de xilanase foram avaliados com base na relação H:C. EPEs foram obtidos em meios líquidos contendo xilana inoculados com os onze isolados com maiores relações H:C. Os extratos foram avaliados quanto à atividade específica de xilanases a 50 oC, por 1 h. Os extratos dos três isolados com maior potencial de atividade sob esta condição foram avaliados quanto à temperatura ótima de atividade, estabilidade de atividade a 60 oC e a valores de pH 4,0, 5,5 e 8,0. Vinte e dois isolados (25%), incluindo oito de isolamento raro, apresentaram atividade de xilanase nas condições avaliadas, sendo encontrada uma alta variabilidade (>230%) entre estes isolados. Nenhum fator de isolamento ou a condição de isolado raro ou comum foram associadas à eficiência de atividade de xilanases em meio sólido. Alta variabilidade de atividade específica de xilanases nos EPEs também foi encontrada entre os isolados (1500%), com destaque para dois de cultivo raro (TC119 e TC21), da classe Alfaproteobacteria, e um de cultivo comum (TC99), da família Ralstoniaceae. As xilanases extracelulares de TC119 e TC21 apresentaram elevada atividade relativa em temperaturas de até 70 oC e foram pouco sensíveis ao pH na faixa de 4,0 a 8,0; entretanto, as de TC99 apresentaram temperatura ótima de 40 oC, baixa estabilidade a temperatura e pH. Xilanases extracelulares de TC119 não apresentaram atividade celulolítica. Bactérias do solo de isolamento raro apresentam alto potencial como fonte de xilanases extracelulares adaptadas a condições extremas de pH e temperatura requeridas em processos agroindustriais.

Abstract Lignocellulosic biomass is the most abundant renewable raw material on the earth and it is so far the most suitable and promising resource for the production of biofuels to replace long-term use of fossil oil. This research aims to convert lignocellulose-based industrial residuals, fibre sludge (FS) from a pulp mill and pine sawdust (PSD) from a sawmill, into platform sugars by two different bifunctionalised pretreatments of lignocellulosic biomass. The bifunctionalised pretreatment combines the ordinary pretreatment (deconstruction) of lignocellulosic biomass with lignocellulosic polysaccharides saccharification. The outcome from both pretreatments can be further transformed into biofuels and chemicals. PSD and FS were converted into platform sugars by acid-catalysed mechanical depolymerisation in a planetary ball mill in the first part of this research. The efficiency of the conversion was mainly affected by the transferred energy caused by collisions, the total milling time, acid concentration and moisture content in the reaction. Approximately 30 wt% of the sugars was yielded from PSD and FS both in the short milling process with a low acid/substrate (A/S) concentration without any prior treatment. The second part of this research focuses upon the conversion of FS into platform sugars using hydroxyalkylimidazolium hydrogen sulphate ionic liquids (ILs). Around 29 wt% of the sugars was produced from FS using an IL/water mixture. The added water acted as a co-solvent and played a critical role in the utilisation of these ILs. The blended water reduced the viscosity of the ILs and enhanced the mass transfer between solvent and solute. In addition, the anions of the ILs provided their acidic property in an aqueous solution and offered an acidic environment for hydrolysis simultaneously. / Tiivistelmä Lignosellulossapohjainen biomassa on runsaimmin saatavilla oleva ja yksi lupaavimmista raaka-aineista biopolttoaineiden valmistukseen korvaamaan fossiilisia polttoaineita. Väitöskirjassa tutkitaan teollisuuden lignoselluloosapohjaisten sivutuotteiden, selluteollisuuden kuitulietteen ja sahateollisuuden sahanpurun (mäntypuru), muuntamista sokereiksi kahdella erilaisella ns. bifunktionaalisella esikäsittelyllä, joissa yhdistyvät lignoselluloosabiomassan perinteinen esikäsittely (hajotus) ja polysakkaridien sokeroituminen. Muodostuneet sokerit voidaan edelleen muuntaa biopolttoaineiksi ja -kemikaaleiksi. Tutkimuksen ensimmäisessä vaiheessa sahanpuru ja kuituliete muunnettiin sokereiksi happokatalysoidussa mekaanisessa käsittelyssä, joka tehtiin kuulamyllyssä. Reaktiossa katalyyttisen käsittelyn tehokkuuteen vaikuttivat erityisesti jauhatuksen kineettinen energia, jauhatusaika, happokonsentraatio ja reaktioseoksen kosteus. Tulosten perusteella todettiin, että ilman lähtöaineen esikäsittelyä sekä sahanpurun että kuitulietteen sokerisaanto oli noin 30 massa% lyhyen, matalassa happokonsentraatiossa tehdyn jauhatuksen jälkeen. Tutkimuksen toisessa vaiheessa kuituliete muutettiin sokereiksi käyttämällä ionista liuotinta (IL), hydroksialkyyli-imidatsoliumvetysulfaattia. Sokerisaanto kuitulietteestä oli noin 29 massa% IL-vesiseoksessa. Vesi toimi reaktiossa apuliuottimena ja sen rooli on keskeinen ionisten liuottimien käytössä. Sekoittunut vesi laski ionisen liuottimen viskositeettia sekä edisti aineensiirtoa liuottimen ja liukenevan aineen välillä. IL:n anionit lisäsivät happamuutta vesiliuoksessa ja mahdollistivat happamat olosuhteet samanaikaiselle hydrolyysille. / Abstract Biomasse aus Lignocellulose ist der am häufigsten vorkommende nachwachsende Rohstoff der Erde und wird aktuell als eine der besten Alternativen für die Produktion von Biokraftstoffen gesehen. Diese sollen langfristig die fossilen Öl-basierten Produkte ersetzen. Diese Forschungsarbeit untersucht die Herstellung von Zucker aus Lignocellulose basierten Abfällen. Faserschlamm aus der Zellstoffindustrie und Kiefern-Sägemehl aus der Holzverarbeitung wurden durch zwei unterschiedliche Bifunktionelle Vorbehandlungen aufgespalten. Diese Bifunktionelle Vorbehandlung kombiniert zwei Schritte in einem Prozess; die gewöhnliche Dekonstruktion der Biomasse und die Verzuckerung von Polysacchariden aus der Lignocellulose. Das so erzeugte Produkt dient als Ausgangsstoff für die weitere Herstellung von Biokraftstoffen und Chemikalien. Im ersten Teil dieser Forschungsarbeit wurden Kiefern-Sägemehl und Faserschlamm in einer Planeten-Kugelmühle zermahlen und gleichzeitig durch eine Säure depolymerisiert. Der Wirkungsgrad dieser säurekatalysierten mechanischen Depolymerisation wurde hauptsächlich durch die Übertragung der Reibungsenergie, der Mahldauer der Zerkleinerung, der Konzentration der Säure und der Feuchtegehalt der Proben beeinflusst. Etwa 30 wt% Zucker wurde so durch den kurzen Zermahlungsprozess aus Kiefern-Sägemehl und Faserschlamm gewonnen. Dabei wurden die Proben nicht vorbehandelt und enthielten eine geringe Säure/Probe Konzentration. Der zweite Teil der Forschungsarbeit untersucht die Umwandlung von Faserschlamm in Zucker mittels der Ionischen Flüssigkeit (ILs) Hydroxyalkyl Imidazolium Hydrogensulfat. Aus den Faserschlamm Proben konnte 29 wt% Zucker durch eine Mischung von ILs und Wasser gewonnen werden. Das zugesetzte Wasser spielte als Co-Lösemittel eine wichtige Rolle in der Nutzung der Ionischen Flüssigkeit, dessen Viskosität so reduziert wurde. Dies führte zu einem erhöhten Stoffübergang zwischen dem Lösemittel und dem Solvat. Zusätzlich sorgten die Anionen der Ionischen Flüssigkeit für ein saures Milieu in der wässrigen Lösung und ermöglichten so eine gleichzeitige Hydrolyse.

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