Thèses sur le sujet « Lignocellulosic inhibitor »

Lignocellulosic biomass has attracted considerable attention as an alternative feedstock for the production of fuels, energy and chemicals, due to its renewability, abundance and reduced cost. Pretreatment and hydrolysation of lignocellulose releases sugars that are subsequently converted by fermentation. However, by-products such as aliphatic acids and furans could be generated during the upstream processes, which could inhibit enzymes and fermenting microorganisms. In addition, fermentation of low-concentrated sugars would lead to low products concentration and, consequently, to higher recovery and purification costs. Therefore, it is beneficial to reduce the concentration of the inhibitors and to concentrate the fermentable sugars in the liquor, prior to the fermentation process. This study was focused on the identification of the operating conditions to effectively remove aliphatic acids and furans from birch pretreatment liquor by nanofiltration. Two commercial NF membranes (NF90 and TS40) were employed. Effects of main operating parameters such as pH, feed concentration, temperature, pressure and tangential velocity on the separation performances were investigated, in both dead-end and cross-flow modes. The membrane performances were compared in terms of retention of sugars and inhibitors, permeate flux and permeability loss. It was found that NF90 membrane was more suitable than TS40 for simultaneous sugars concentration and beneficial removal of the inhibitors. Better separation performances were achieved at pH 1.5, 8 bar, room temperature and 3000 rpm. Dilution of 5 times of the liquor promoted the rejection of the sugars and kept low the retention of the inhibitory compounds. Water permeability loss was recovered by flushing with NaOH and water. The protocol and the operating conditions proposed in this study are suitable to perform the filtration process not only on laboratory scale, but also on industrial scale.

The fossil energy resources decrease and climate changes, caused by carbon dioxide (CO2) emissions, have led most industrialized countries to undertake policies aimed at the development and use of renewable energy sources. Among the renewable energies, vegetal biomasses play a key role because widely available and potentially able to cover up to 200% of the global energy demand. Vegetal biomasses can be used mainly as raw materials for the production of chemicals, biofuels and energy, in the increasingly important green economy concept based on biorefineries creation. Although the vegetal biomasses result widely available, rising costs of food raw material such as wheat, corn and sugar beet have raised a serious ethical problem using these resources. To avoid the use of such raw materials, the exploitation of lignocellulosic biomasses plays a fundamental role in the industry. However, for an efficient utilization of lignocellulosic biomasses, new technologies are required in order to transform the starting biomass into simple molecules, such as pentoses and hexoses sugars, more easily to use by the microrganism, which will have the task of producing both fine chemicals and bulk chemicals in an economically and environmentally sustainable processes. In this regard, industrial biotechnologies should be able to develop new microrganisms capable to face the harsh environmental conditions that occur during an industrial production process. For many of these productions the yeast Saccharomyces cerevisiae is largely used, not only because of its naturally ability to produce large ethanol amount, but also is widely known both at genetic and metabolic level, outlining a good starting point for the development of producers strains with high tolerance against different stresses occur during an industrial process. This is the view adopted by NEMO project (Novel high performance Enzymes and Microrganisms for conversion of lignocellulosic biomass to ethanol), belonging to the European Union seventh framework program, where it become of primary importance the development of microrganisms, especially S.cerevisiae, for the second generation ethanol production. Microrganisms must be, on the one hand able to efficiently utilize all the sugars released from lignocellulosic biomass pre-treatment, on the other hand should be more tolerant against process conditons, such as inhibitory compounds and environmental stresses. A point of relevant importance is the ability to utilize pentose sugars, like D-xylose, released in large amount after lignocellulose pre-treatment. Currently, worldwide researches are focused on the development of yeast strains engineered with xylose degradation pathways involving the pentose phosphate pathway. In fact the fungal pathway exploits xylose reductase and the xylitol dehydrogenase while the bacterial pathway exploits xylose isomerase; both pathways degrade D-xylose into D-xylulose, which will enter into pentose phosphate pathway. In addition to these two pathways studied since the ‘80s of the last century, there also two other poorly known metabolisms, described for the first time in the ‘70s, which produce alpha-ketoglutarate or pyruvate and glycolaldehyde through an oxidative xylose degradation. These pathways are composed of 5 enzymatic reactions by the Weimberg’s pathway and of 4 enzymatic reactions by the Dahms’ pathway, however they share the first 3 enzymatic reactions. After bioinformatics we were identified the presence of Weimberg’s pathway into Burkholderia xenovorans, while the reaction that characterizes the Dahms’ pathway has been identified in Escherichia coli. The encoding genes for these enzymatic activities were expressed in S.cerevisiae, and the capacity to grow on D-xylose as carbon source are evaluated. The reconstruction of these two pathways showed a poorly growth capacity on xylose. Such growth limitation seems to be related to several factors: the presence of bottlenecks associated to enzymes functionality, like D-xylonate dehydratase activity; the yeast ability to internalize xylose efficiently; the involved genes optimization. Another important aspect is the yeast ability to face and overcome environmental stresses encountered during an industrial process. The cytoplasmic membrane plays a key role in cellular homeostasis, being at the interface between the cell and the external environment, and reacting at environmental changes. The plant membrane protein TIL gives particular strength to the yeast cells when these are subjected to environmental stresses of industrial relevance, such as the presence of oxidative agents or during temperature changes. However, when TIL is expressed in an industrial and/or in an engineered laboratory strains, for industrial use, the protective effect against prolonged stress exposure and process conditions disappear. Finally, a further important aspect during an industrial process is the S.cerevisiae ability to tolerate the growth inhibitory compounds presence into pre-treated lignocellulose. In fact has been largely described how chemical compounds like aldehydes, organic acids and phenolic compounds, released during lignocellulose pre-treatment process, are toxic at certain concentration, inhibiting S.cerevisiae growth or causing yeast death. The growth performance of different wild type or engineered yeast strains are evaluated on spruce and giant cane lignocellulose pre-treated: in addition the same strains were tested on minimal formulated medium according to the spruce pre-treated composition. The results showed that the combination of low pH and the presence of organic acids, especially acetic acid and formic acid, are dramatically harmful for growth of both industrial strain, naturally more tolerant, and engineered strain, for the production and recycle of L-ascorbic acid. However, the behavior of engineered strain for production and recycle of L-ascorbic acid is interesting at low pH, because showed higher tolerance than other strains in terms of growth rate and ethanol production and productivity. Despite the positive results obtained by engineering microrganisms, especially S.cerevisiae, in laboratory, their industrial uses still remain limited. Therefore, appears extremely important the construction of more robustness strains, able to withstand different environmental conditions along an entire industrial process, with consequent influence on yields, production and productivity. For these reasons, the research is aimed to combine these aspects to provide the best microrganism possible to industry productions.

The rapid depletion of fossil fuel reserves and concurrent increase in global temperatures has resulted in global demand for the production of alternative environmentally friendly fuels. First-generation biofuels that utilise cash crops for the extraction of fermentable sugars currently exist, but are highly controversial due to socioeconomic and environmental reasons such as diverting food production or deforestation. Therefore, second-generation biofuels that utilise lignocellulosic waste materials are a more attractive prospect. In Europe, lignocellulosic biomass wastes such as wheat straw, display great potential for the production of alternative energy sources such as bioethanol for transportation. Conversion to this biofuel requires microorganisms that will effectively utilise the constituent sugars to produce a high yield of product. Saccharomyces cerevisiae (S. cerevisiae) strains possess the most desirable phenotypes for this objective. However, the components of wheat straw are difficult to break down, therefore pretreatment is required. Pretreatment methods vary but often utilise various chemicals that produce compounds that are inhibitory to yeast. This affects the efficiency of fermentations. The focus of this work is on formic acid and a synthetic media containing the main inhibitor compounds released during pre-treatment of steam exploded wheat straw. Six pair-wise F1 crosses between four distinct parental S. cerevisiae clean lineage populations have been generated previously by Cubillos et al., 2009. The 96 F1 progeny from each cross have been assayed for tolerance phenotypes in order to determine QTLs (Quantitative Trait Loci), which will enable us to map genes contributing to the multi-genic trait of inhibitor tolerance. Overall, three QTLs were identified for formic acid and five QTLs were identified from the synthetic inhibitor mix. Candidate genes were selected from the QTL analysis and were tested by performing reciprocal hemizygosity assays to determine which genes are responsible for inhibitor resistance to enable the development of yeast strains suitable for second-generation biofuel production.

Mestrado em Biotecnologia - Biotecnologia Industrial e Ambiental
The present work aimed to tackle two of the major challenges in bioethanol production from lignocellulosic feedstocks: (i) high tolerance of microorganisms to lignocellulosic inhibitors, and (ii) microbial contamination avoidance. Lignocellulosic inhibitors are an important fraction of spent sulphite liquor (SSL), a by-product of the pulp and paper industries. Hardwood SSL (HSSL) is rich in pentose sugars, mainly xylose, which can be converted to ethanol by the yeast Scheffersomyces stipitis. In this work, a population of S. stipitis previously adapted to 60 % (v/v) of HSSL was used, and its stability on the absence of inhibitors during ten sequential transfers was investigated at single-clone level. During the screening trials, all the isolated clones showed higher xylose and acetate uptake rates and lower ethanol productivities than the parental strain. The clone exhibiting higher xylose uptake rate (0.558 g L-1 h-1) was named isolate C4. The effect of short-term adaptation on isolate C4 fermentation performance was evaluated by pre-cultivating the clone in the presence or absence of 60 % (v/v) of HSSL. The uptake rates of glucose and xylose were similar under both conditions, but a higher acetate consumption rate (0.101 g L-1 h-1) and maximum ethanol concentration (4.51 g L-1) were achieved without pre-adaptation step, suggesting the robustness of isolate C4. The industrial bioethanol production is mostly carried out under non-sterile conditions, which favours microbial contamination. In this work, the mechanism that triggers Lactobacillus pentosus contamination in SSL plants was investigated. A simulated synthetic hydrolysate mimicking the average composition of sugars and inhibitors of softwood SSL (SSSL) was used and the impact of different factors in bacterial and Saccharomyces cerevisiae viability was analysed. The presence of yeast extract led to an increase in lactate production (9-fold higher) and L. pentosus viability when only bacteria was inoculated. Using different inoculation ratios of yeast/bacteria, the ethanol production rates were not affected after 48 h, and L. pentosus failed to overtake S. cerevisiae. The presence of inhibitors delayed yeast growth, but the bacteria did not outcompete S. cerevisiae. When the pH was optimal to L. pentosus in co-culture experiments, the bacterial cell viability decreased slower. The results indicate that L. pentosus was unable to overtake S. cerevisiae. The presence of yeast extract and favourable pH to bacteria are important factors that can play a role in the mechanism that triggers the bacterial contamination in ethanol plants.
A presente dissertação tem como objetivo abordar dois dos maiores desafios na produção de bioetanol a partir de biomassa lenhocelulósica: (i) elevada tolerância de microrganismos a inibidores, e (ii) prevenção de contaminação microbiana. Os inibidores lenhocelulósicos são uma fração relevante do licor de cozimento ao sulfito ácido (SSL), um subproduto das indústrias do papel e pastas. O SSL de folhosas (HSSL) é rico em pentoses, principalmente xilose, que podem ser fermentadas em etanol pela levedura Scheffersomyces stipitis. Neste estudo, utilizou-se uma população de S. stipitis previamente adaptada a 60 % (v/v) HSSL, e avaliou-se a sua estabilidade na ausência de inibidores durante dez transferências sequenciais. Comparando com a estirpe original, todos os clones isolados exibiram taxas de consumo de xilose e ácido acético superiores e produtividades em etanol inferiores. O clone que demonstrou a maior taxa de consumo de xilose (0,558 g L-1 h-1) foi designado isolado C4, e o efeito de adaptação de curta duração no seu desempenho fermentativo foi investigado através do seu pré-cultivo na presença ou ausência de 60 % (v/v) HSSL. Nas duas condições, as taxas de consumo de glucose e xilose foram idênticas, contudo, atingiu-se maior taxa de consumo de ácido acético (0,101 g L-1 h-1) e maior concentração máxima de etanol (4,51 g L-1) foram atingidas na ausência do processo de adaptação de curta duração. Tais resultados demonstram a robustez do isolado C4. A maioria dos processos de produção industrial de bioetanol é realizada na ausência de esterilidade, favorencendo a contaminação por microrganismos. Neste estudo, investigou-se o mecanismo responsável pela contaminação com Lactobacillus pentosus na indústria de SSL. Para tal, utilizou-se um hidrolisado sintético mimetizando a composição média de açúcares e inibidores de SSL de resinosas (SSSL) e averiguou-se o impacto de vários fatores na viabilidade de L. pentosus e S. cerevisiae. A presença de extrato de levedura foi responsável pelo aumento da produção de ácido lático (9 vezes) e da viabilidade bacteriana quando L. pentosus foi cultivado na ausência de levedura. Diferentes proporções de inóculo de levedura/bactéria não afetaram a produção de etanol após 48 h de fermentação, e L. pentosus foi incapaz de ser a estirpe dominante durante os ensaios de co-cultura. A presença de inibidores retardou o crescimento da levedura, mas a bactéria foi de novo incapaz de se a espécie dominante. Ajustando o valor de pH para o ótimo de L. pentosus nos ensaios de co-cultura, a viabilidade celular da bactéria diminuiu mais lentamente. Os resultados demonstram que L. pentosus não foi a espécie dominante nos ensaios de co-cultura. A presença de extrato de levedura e de valores de pH favoráveis a L. pentosus podem desempenhar um papel importante no mecanismo responsável pela contaminação bacteriana nas indústrias de produção de bioetanol.

Le développement de technologies enzymatiques constitue un enjeu majeur pour le fractionnement maîtrisé et la valorisation des ressources lignocellulosiques (biocarburants, biopolymères, synthons…). L’efficacité de ces biocatalyseurs est cependant limitée par de multiples facteurs liés à la fois à leurs caractéristiques structurales et fonctionnelles, mais également à la nature complexe de la biomasse lignocellulosique (riche en parois secondaires lignifiées). Dans le but d’identifier les paramètres clés pour une conversion efficace des hémicelluloses, constituants majeurs des lignocelluloses, nous avons centré notre étude sur l’endoxylanase (Tx-Xyl) de Thermobacillus xylanilyticus appartenant à la famille 11 des glycoside- hydrolases (GH11). Dans une approche biomimétique, nous avons étudié, l’action de la xylanase (Tx-Xyl) sur des substrats différents et de complexité croissante (hétéroxylanes isolés, assemblages de copolymères reconstitués in vitro. . . ). L’emploi de nano-composites hétéroxylanes extraits - lignines de synthèse (DHPs) nous a permis de souligner l’impact de l’agencement tridimensionnel des complexes covalents (LCC) sur la limitation de l’accessibilité des hétéroxylanes à l’enzyme. Une corrélation directe a pu, également, être établie entre l’augmentation du contenu en lignines des nano-composites et la baisse de l’activité de l’enzyme, suggérant des interactions non spécifiques directes enzyme-lignines. Par ailleurs, l’étude des interactions de Tx-Xyl avec divers acides hydroxycinamiques (p-coumarique, férulique, cafféique…) a permis de mettre en évidence un phénomène d’inhibition non compétitif de l’enzyme par ces composés phénoliques. Moyennant des outils de biologie moléculaire, nous avons développé une stratégie qui vise à modifier l’architecture protéique et/ou la spécificité de fixation de Tx-Xyl en la fusionnant via des séquences "linker" à des modules protéiques différents: le CBM1 de la cellulase Cel7A de Trichoderma reesei fixant spécifiquement la cellulose cristalline et la GFP (Green Fluoerescent Protein). Les protéines chimériques Tx-Xyl-CBM1 et Tx-Xyl-GFP obtenues sont moins efficaces sur les xylanes solubles (faible kcat) comparées à Tx-Xyl. Cependant, leurs modes d’action sur des substrats lignocellulosiques (tels que les coproduits du blé : paille et son de blé) semblent différents. En effet, des rendements d’hydrolyse légèrement augmentés sont obtenus dans le cas de Tx-Xyl-CBM1, suggérant un impact positif du CBM1 sur l’action de l’enzyme in situ, contrairement à Tx-Xyl-GFP dont la taille serait un facteur limitant sa diffusion/pénétration au sein des parois végétales
The development of enzymatic technologies offers an alternative, environmentally-friendly interesting strategy for controlled fractionation and upgrading of lignocellulosic biomass (biofuels, biopolymers, industrially-relevant chemicals. . . ). The effectiveness of these biocatalysts is, nevertheless, limited by multiple factors related to their structural and functional characteristics, but also to the complex nature of the lignocellulosic biomass (rich in lignified secondary cell walls). In order to identify the key parameters for an effective bioconversion of hemicelluloses, the major components of lignocelluloses, we have focused our study on the endoxylanase (Tx-Xyl) of Thermobacillus xylanilyticus, a family 11 glycoside- hydrolase (GH11). In a biomimetic approach, we have studied the action pattern of Tx-Xyl on different substrates displaying increasing complexity (isolated heteroxylans, in vitro reconstituted copolymer assemblies. . . ). The use of nano-composites of heteroxylans - lignins (DHPs) synthesized in vitro has enabled us to reveal that supramolecular organization of the covalent complexes (LCC) would severely hamper the enzyme's access to carbohydrates. Otherwise, a direct correlation has been established between the increase in the lignin content of the nano-composites and the decrease of the enzyme activity suggesting direct nonspecific lignins-enzyme interactions. In addition, the study of the interactions of Tx-Xyl with various hydroxycinamic acids (p-coumaric, ferulic, caffeic acids. . . ) has revealed a non-competitive inhibition of the enzyme by these phenolic compounds. Using protein engineering, we have developed a strategy which aims at modifying the Tx-Xyl architecture and/or specificity by grafting, through "linker" sequences, different protein modules: the CBM1 of the cellulase Cel7A from Trichoderma reesei binding specifically crystalline cellulose and the GFP (Green Fluoerescent Protein). The chimeric fusion proteins Tx-Xyl-CBM1 and Tx-Xyl-GFP obtained have been less effective on soluble xylans (low kcat) than Tx-Xyl. However, their efficiency on lignocellulosic substrates (such as wheat by products; straw and bran) was different. Indeed, modestly enhanced hydrolysis rates were obtained in the case of Tx-Xyl-CBM1, suggesting that the CBM1 may potentiate in situ action of the enzyme, contrary to Tx-Xyl-GFP whose size would be a factor limiting its diffusion/action within the cell wall network

Lignocellulosic biomass is a renewable resource that can be utilized for the production of biofuels, chemicals, and bio-based materials. Biochemical conversion of lignocellulose to advanced biofuels, such as cellulosic ethanol, is generally performed through microbial fermentation of sugars generated by thermochemical pretreatment of the biomass followed by an enzymatic hydrolysis of the cellulose. The aims of the research presented in this thesis were to address problems associated with pretreatment by-products that inhibit microbial and enzymatic biocatalysts, and to investigate the potential of utilizing residual streams from pulp mills and biorefineries to produce hydrolytic enzymes. A novel method to detoxify lignocellulosic hydrolysates to improve the fermentability was investigated in experiments with the yeast Saccharomyces cerevisiae. The method is based on treatment of lignocellulosic slurries and hydrolysates with reducing agents, such as sodium dithionite and sodium sulfite. The effects of treatment with sodium borohydride were also investigated. Treatment of a hydrolysate of Norway spruce by addition of 10 mM dithionite resulted in an increase of the balanced ethanol yield from 0.03 to 0.35 g/g. Similarly, the balanced ethanol yield of a hydrolysate of sugarcane bagasse increased from 0.06 to 0.28 g/g after treatment with 10 mM dithionite. In another study with a hydrolysate of Norway spruce, addition of 34 mM borohydride increased the balanced ethanol yield from 0.02 to 0.30 g/g, while the ethanol productivity increased from 0.05 to 0.57 g/(L×h). While treatment with sulfur oxyanions had a positive effect on microbial fermentation and enzymatic hydrolysis, treatment with borohydride resulted in an improvement only for the microbial fermentation. The chemical effects of treatments of hydrolysates with sodium dithionite, sodium sulfite, and sodium borohydride were investigated using liquid chromatography-mass spectrometry (LC-MS). Treatments with dithionite and sulfite were found to rapidly sulfonate inhibitors already at room temperature and at a pH that is compatible with enzymatic hydrolysis and microbial fermentation. Treatment with borohydride reduced inhibitory compounds, but the products were less hydrophilic than the products obtained in the reactions with the sulfur oxyanions. The potential of on-site enzyme production using low-value residual streams, such as stillage, was investigated utilizing recombinant Aspergillus niger producing xylanase and cellulase. A xylanase activity of 8,400 nkat/ml and a cellulase activity of 2,700 nkat/ml were reached using stillages from processes based on waste fiber sludge. The fungus consumed a large part of the xylose, the acetic acid, and the oligosaccharides that were left in the stillages after fermentation with S. cerevisiae. In another study, the capability of two filamentous fungi (A. niger and Trichoderma reesei) and three yeasts (S. cerevisiae, Pichia pastoris, and Yarrowia lipolytica) to grow on inhibitory lignocellulosic media were compared. The results indicate that the two filamentous fungi had the best capability to utilize different nutrients in the media, while the S. cerevisiae strain exhibited the best tolerance against the inhibitors. Utilization of different nutrients would be especially important in enzyme production using residual streams, while tolerance against inhibitors is desirable in a consolidated bio-process in which the fermenting microorganism also contributes by producing enzymes.

Orientador: Francisco Maugeri Filho
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia de Alimentos
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Resumo: Na fermentação de produtos como etanol, utilizando biomassa lignocelulósica como matéria-prima, existem dois fatores principais que limitam a produtividade e eficiência do processo: inibição pelo produto e inibição por substâncias no caldo hidrolítico provenientes da hidrólise. Neste trabalho, é proposta a remoção simultânea de ambos os fatores para eliminar seus efeitos negativos na fermentação alcoólica. Produtos de fermentação prejudicam muitas vezes a integridade da membrana celular do micro-organismo utilizado como fermento. Portanto, a toxidez do produto não permite que a fermentação ocorra de forma ilimitada, uma vez que o produto está presente no meio em certa concentração. O crescimento do micro-organismo, a produtividade e o rendimento são prejudicados pela presença do mesmo. Compostos como furfural, hidroximetil furfural, compostos fenólicos e ácidos, que são produzidos durante o pré-tratamento ou hidrólise da biomassa lignocelulósica, introduzem outros efeitos inibidores, como a extensão da fase lag da levedura, prejudicam o crescimento e a produção. Esta tese propõe empregar um solvente orgânico na dorna do biorreator, com o fim de extrair o produto inibidor e todos os componentes inibidores existentes no substrato, de tal forma que o processo de fermentação não seja prejudicado. Com esse objetivo, primeiramente foi definida a relação entre o tamanho molecular de agentes extrativos, bio-compatibilidade e propriedades extrativas dos mesmos. Em seguida, um solvente foi escolhido, sendo o biodiesel à base de óleo de mamona, através de características como biocompatibilidade, coeficientes de partição, seletividade, alta disponibilidade e reutilização. Foram feitas fermentações em regime batelada em fermentadores de bancada, utilizando o biodiesel como agente extrativo, demonstrando os efeitos positivos no desempenho da fermentação de um licor hidrolítico. Adicionalmente, o comportamento de uma cepa de levedura industrial foi estudado na presença de inibidores e foi construído um modelo matemático que descreve as taxas de conversão dos principais inibidores e as condições em que a levedura, ao invés de manter uma fase lag, inicia a produção de biomassa e etanol. Finalmente, foi elaborado, como exemplo da utilização da tecnologia proposta, um modelo do sistema contínuo de fermentação alcoólica com a extração líquido-líquido, incluindo a recuperação do produto e resfriamento do meio de fermentação pelo próprio solvente orgânico. Por meio desta modelagem e uma série de simulações, foram determinadas as faixas ideais das principais variáveis na produção de etanol pelo sistema bifásico, sendo elas a fração de licor hidrolítico no mosto, concentração de substrato, temperatura de fermentação, e taxa de diluição do solvente. Assim, o trabalho demonstra as vantagens, efeitos positivos e os limites da utilização de extração líquido-líquido na fermentação de substrato da segunda geração. Entre as vantagens se destacam: maior tolerância de caldo hidrolítico no mosto, elevada produtividade, maior rendimento e maior custo-benefício do substrato
Abstract: There are two main factors that limit fermentation productivity and eficiency during the production of chemicals like ethanol when using lignocelulosic biomass as raw material: product inhibition and inhibition by substances in hydrolitic liquor generated during hydrolyzis. In this work, the simultaneous removal of both factors is proposed to eliminate their negative effects on ethanol fermentation. Fermentation products often damage the cellular wall of the micro-organism that is used as ferment. As a result, the toxicicity of the product does not permit that the fermentation continues unhindered once the product concentration has reached a certain level; growth of the micro-organism, productivity and yield are effected. Substances like furfural, hydroximetil furfural, phenolic compounds and organic acids, that are produced or released during pre-treatment or hydrolyzis of ligno-celulosic biomass, introduce other inhibiting effects, like the extension of the lag phase of the ferment or decreasing growth and production. This thesis proposes the use of an organic solvent as a second liquid phase in the bioreactor, to extract both the inhibiting product and all inhibiting compounds present in the substrate, such that the fermentation process remains unhindered. With this objective, first the relation between the molecular size of an extractive agent and its biocompatibility and extractive properties was determined. Next, a solvent was chosen, being biodiesel based on castor oil, by prioritizing characteristics as biocompatibility, partition coeficients, selectivity, availability and possibilities for recycling and reuse. Batch fermentations were executed in bench-scale, using biodiesel as extractive agent, demonstrating the improvements of fermentation of hydrolytic liquor. Aditionaly, the performance of an industrial yeast strain was studied in the presence of inhibitors and a mathematical model was constructed that descibes the conversion rates of the main inhibitors and conditions at which the yeast, instead of maintaining a lag phase, starts production of biomass and ethanol. Finally, as a practical example of the proposed technology, simulations were performed for an integrated process including continuous ethanol fermentation with liquid-liquid extraction, product recovery and cooling of the fermentation broth by the extractive agent itself. The simulation results reveiled the optimal ranges for the most important variables of the two-phase ethanol production process, i.e. fraction of hydrolitic liquor in the must, substrate concentration, fermentation temperature and dilution rate of the solvent. In all, the work shows the advantages, positive effects of and limits to the use of liquid-liquid extraction in fermentation of second-generation substrate. Advantages are, among others, higher tolerance of hydrolyzate in the must, higher yield, higher productivity and higher return on investment of raw-material
Doutorado
Engenharia de Alimentos
Doutor em Engenharia de Alimentos

O hidrogênio é uma fonte de energia limpa, pois sua combustão gera apenas água. Porém, ainda há a necessidade de se encontrar soluções tecnologicamente eficientes, econômicas e seguras para sua geração e uso. A produção do H2 por vias biológicas, conhecido como biohidrogênio, vem ganhando grande destaque nos últimos anos, pois possibilita o uso de materiais renováveis como matéria-prima. Materiais lignocelulósicos são potenciais substratos para a produção de H2 por fermentação, no entanto se faz necessário dispor de métodos de hidrólise que disponibilizem os componentes destes materiais para a fermentação. A maior parte dos métodos disponíveis para hidrolisar materiais lignocelulósicos resulta em produtos de degradação de carboidratos, que são reconhecidamente inibidores de fermentação. Este estudo, primeiramente, avaliou o efeito de 3 diferentes grupos de inibidores sobre a produção de H2 por fermentação: (1) ácido orgânico, como o ácido acético; (2) derivados de furano, tais como o furfural e o 5-hidroximetilfurfural (5-HMF); (3) monômeros fenólicos derivados da lignina, tais como o siringaldeído, vanilina e ácido 4-hidroxibenzóico (AHB). Ensaios de fermentação para a produção de H2 em batelada utilizaram como inóculo uma cultura mista (lodo) e foram realizados na presença de glicose e diferentes concentrações dos mencionados inibidores. O modelo de Gompertz modificado foi utilizado para estimar os parâmetros cinéticos dos ensaios de fermentação, como o volume máximo de H2 (P), velocidade máxima de produção de H2 (Rm) e o tempo necessário para o início da produção de H2 (). A partir destes ensaios foi verificado como a adição de diferentes concentrações de inibidores afetou tais parâmetros cinéticos em relação a um controle (apenas contendo glicose). Desta forma foi possível estimar as concentrações dos inibidores que reduzem em 50% as velocidades máximas de produção de H2 a concentração inibitória 50 (CI 50). Em termos de CI 50, o AHB proporcionou a maior inibição (0,38 g.L-1), seguido do 5-HMF e o furfural, com valores de CI 50 de 0,48 e 0,62 g.L-1, respectivamente. A vanilina, o siringaldeído e o ácido acético apresentaram os menores efeitos inibitórios sobre a produção de H2 dentre os inibidores testados, com CI 50 de 0,71; 1,05; e 5,14 g L-1, respectivamente. Numa segunda etapa do trabalho foi avaliado o efeito inibitório da associação de 3 inibidores, representantes de cada uma das classes de inibidores, o ácido acético, o 5-HMF e o siringaldeído. Foi observado um efeito aditivo da inibição quando o ácido acético foi adicionado juntamente com o 5-HMF, porém em ensaios contendo siringaldeído o efeito inibitório tornou-se sinérgico. Por fim, foi utilizado um hidrolisado de bagaço de cana de açúcar como substrato na produção de H2 por fermentação. A produção de H2 a partir deste substrato só foi possível após o tratamento do hidrolisado com carvão ativado. Portanto, concluiu-se que os compostos inibitórios presentes em hidrolisados de materiais lignocelulósicos condicionam a viabilidade da produção de H2 com estes materiais. Este estudo permitiu concluir que os compostos estudados, exceto os monossacarídeos, resultantes da hidrólise de materiais lignocelulósicos, inibem a produção de H2 pela cultura mista utilizada em diferentes graus, sendo o AHB o mais inibidor. A combinação de compostos inibidores potencializa ainda mais o efeito inibitório sobre a produção de H2. O ácido acético, que pode se originar dos hidrolisados, mas que também é um metabólito da produção de H2 por fermentação aumentou ainda mais a inibição do siringaldeído. Assim, sugere-se que a hidrólise de materiais lignocelulósicos deve ser conduzida de forma a minimizar a presença dos inibidores nos hidrolisados, a fim de maximizar o aproveitamento da biomassa lignocelulósica como matéria-prima no processo fermentativo.
Hydrogen is a clean energy source because its combustion produces only water. However, there is still the need to find technologically efficient, economic and safe solutions for their generation and use. The production of H2 by biological pathways, known as biohydrogen, has gained great prominence in recent years because it enables the use of renewable materials as raw material. Lignocellulosic materials are potential substrates for H2 production by fermentation, however it is necessary to have methods that provide hydrolysis of the components of these materials for fermentation. Most methods are available for hydrolyzing lignocellulosic materials results in carbohydrate degradation products are fermentation inhibitors known. This study was primarily to evaluate the effect of 3 different groups inhibitors of the H2 production by fermentation: (1) organic acid such as acetic acid; (2) furan derivatives such as furfural and 5-hydroxymethylfurfural (5-HMF); (3) phenolic derivatives of lignin monomers, such as syringaldehyde, vanillin and 4-hydroxybenzoic acid (HBA). Fermentation tests for H2 production batch used as a mixed culture inoculum (sludge) and were carried out in the presence of glucose and different concentrations of the inhibitors mentioned. The modified Gompertz model was used to estimate the kinetic parameters of the fermentation test, the maximum volume of H2 (H), maximum rate of H2 production (Rm) and the time required for the commencement of production of H2 () . From these tests it was observed how the addition of different concentrations of inhibitors affect these kinetic parameters relative to a control (containing only glucose). Thus it was possible to estimate the concentrations of inhibitors that reduce by 50% the maximum production speeds H2 - The inhibitory concentration 50 (IC 50). In terms of IC 50, the AHB provided the greatest inhibition (0.38 g L-1), followed by 5-HMF and furfural, with IC 50 values of 0.48 and 0.62 g L-1, respectively. Vanillin, syringaldehyde and the acetic acid had minor inhibitory effects on H2 production from the tested inhibitors with IC50 of 0.71; 1.05; and 5.14 g L-1, respectively. In a second stage of work, the inhibitory effect of 3 inhibitors association representatives of each class inhibitors, acetic acid, and 5-HMF syringaldehyde. An additive effect of inhibition when acetic acid was added along with 5-HMF was observed in assays containing syringaldehyde but the inhibitory effect became synergistic. Finally, we used a hydrolyzate of sugarcane bagasse as substrate in H2 production by fermentation. The production of H2 from this substrate was only possible after the hydrolyzate treatment with activated carbon. Therefore, it was concluded that the inhibitory compounds present in hydrolyzed lignocellulosic materials affect the viability of H2 production with these materials. This study concluded that the studied compounds, other monosaccharides resulting from the hydrolysis of lignocellulosic materials, inhibit the production of H2 by mixed culture used in varying degrees, being most AHB inhibitor. The combination of compounds further enhances the inhibitory effect of inhibitors on the production of H2. Acetic acid, which can originate the hydrolysates, but is also a metabolite of H2 production by fermentation further increased inhibition of syringaldehyde. Thus, it is suggested that the hydrolysis of lignocellulosic materials should be conducted to minimize the presence of inhibitors of the hydrolysates, in order to maximize the utilization of lignocellulosic biomass as a raw material in the fermentation process.

Efforts are made to change from 1st to 2nd generation bioethanol production, using lignocellulosics as raw materials rather than using raw materials that alternatively can be used as food sources. An issue with lignocellulosics is that a harsh pretreatment step is required in the process of converting them into fermentable sugars. In this step, inhibitory compounds such as furan aldehydes and carboxylic acids are formed, leading to suboptimal fermentation rates. Another issue is that lignocellulosics may contain a large portion of pentoses, which cannot be fermented simultaneously with glucose by Saccharomyces cerevisiae. In this thesis, high local cell density has been investigated as a means of overcoming these two issues. Encapsulation of yeast in semi-permeable alginate-chitosan capsules increased the tolerance towards furan aldehydes, but not towards carboxylic acids. The selective tolerance can be explained by differences in the concentration of compounds radially through the cell pellet inside the capsule. For inhibitors, gradients will only be formed if the compounds are readily convertible, like the furan aldehydes. Conversion of inhibitors by cells close to the membrane leads to decreased concentrations radially through the cell pellet. Thus, cells closer to the core experience subinhibitory levels of inhibitors and can ferment sugars. Carbohydrate gradients also give rise to nutrient limitations, which in turn trigger a stress response in the yeast, as was observed on mRNA and protein level. The stress response is believed to increase the robustness of the yeast and lead to improved tolerance towards additional stress. Glucose and xylose co-consumption by a recombinant strain, CEN.PK XXX, was also improved by encapsulation. Differences in affinity of the sugar transporters normally result in that glucose is taken up preferentially to xylose. However, when encapsulated, cells in different parts of the capsule experienced high and low glucose concentrations simultaneously. Xylose and glucose could thus be taken up concurrently. This improved the co-utilisation of the sugars by the system and led to 50% higher xylose consumption and 15% higher final ethanol titres. A protective effect by the capsule membrane itself could not be shown. Hence, the interest in flocculation was triggered, as a more convenient way to keep the cells together. To investigate whether flocculation increases the tolerance, like encapsulation, recombinant flocculating yeast strains were constructed and compared with the non-flocculating parental strain. Experiments showed that strong flocculation did not increase the tolerance towards carboxylic acids. However, the tolerance towards a spruce hydrolysate and especially against furfural was indeed increased. The results of this thesis show that high local cell density yeast cultures have the potential to aid against two of the major problems for 2nd generation bioethanol production: inhibitors and simultaneous hexose and pentose utilisation.

Akademisk avhandling som för avläggande av teknologie doktorsexamen vid Chalmers tekniska högskola försvaras vid offentlig disputation den 19 februari 2014,klockan 13.30 i KA-salen, Kemigården 4, Göteborg.

Bioethanol from lignocellulose is expected to be the most likely fuel alternative in the near future. SEKAB E-Technology in Örnsköldsvik, Sweden develops the technology of the 2nd generation ethanol production; to produce ethanol from lignocellulosic raw material. The objective of this master’s thesis was to achieve a better knowledge of the potential and limitations of separate hydrolysis and fermentation (SHF) as a process concept for the 2nd generation ethanol production. The effects of enzyme concentration, temperature and pH on the glucose concentration in the enzymatic hydrolysis were investigated for pretreated spruce at 10% DM using a multiple factor design. Enzyme concentration and temperature showed significant effects on the glucose concentration, while pH had no significant effect on the concentration in the tested interval of pH 4.5-5.5. To obtain the maximum glucose concentration (46.4 g/l) for a residence time of 48 h, the optimal settings within the studied parameter window are a temperature of 45.7⁰C and enzyme concentration of 15 FPU/g substrate. However, a higher enzyme concentration would probably further increase the glucose concentration. If enzymatic hydrolysis should be performed for very short residence times, e.g. 6 h, the temperature should be 48.1⁰C to obtain maximum glucose concentration. The efficiency of the enzymes was inhibited when additional glucose was supplied to the slurry prior to enzymatic hydrolysis. It could be concluded that end product inhibition by glucose occurs and results in a distinct decrease in glucose conversion. No clear conclusions could be drawn according to different techniques for slurry and enzymes, i.e. batch and fed-batch, in the enzymatic hydrolysis process. Investigations of the fermentability of the hydrolysate revealed that the fermentation step in SHF is problematic. Inhibition of the yeast decrease the fermentation efficiency and it is therefore difficult to achieve the 4% ethanol limit. Residence time for enzymatic hydrolysis (48 h) and fermentation (24 h) need to be prolonged to achieve a sufficient SHF process. However, short processing times are a key parameter to an economically viable industrial process and to prolong the residence times should therefore not be seen as a desirable alternative. SHF as a process alternative in an industrial bioethanol plant has both potential and limitations. The main advantage is the possibility to separately optimize the process steps, especially to be able to run the enzymatic hydrolysis at an optimal temperature. Although, it is important to include all the process steps in the optimization work. The fermentation difficulties together with the end product inhibition are two limitations of the SHF process that have to be improved before SHF is a preferable alternative in a large scale bioethanol plant.

Dans le contexte énergétique et climatique mondial, le coût élevé des enzymes Cellulolytiques (cellulases) freine le développement des bioraffineries lignocellulosiques, pour produire des biocarburants et composés chimiques à partir d'une matière première végétale renouvelable. L'objectif de ce travail est de caractériser et de modéliser le métabolisme du micro-organisme Trichoderma reesei, afin d'optimiser le protocole industriel de production de cellulases. Cette étude a été réalisée sur des milieux modèles représentatifs de ceux attendus à l'échelle industrielle. Tout d'abord, la stoechiométrie des réactions de croissance et de production a été établie, puis une étude cinétique a été menée pour mesurer précisément le comportement du micro-organisme à forte induction de la production de cellulases. Le modèle résultant a été utilisé pour optimiser le protocole industriel de production. Ensuite, l'intégration de cette étape dans une bioraffinerie lignocellulosique a été étudiée, avec l'effet sur le métabolisme i) des mélanges de sucres disponibles, ii) des composés inhibiteurs issus de la dégradation de la lignocellulose, et iii) du changement d'échelle. Ces travaux ont fait progresser de façon substantielle les connaissances du métabolisme de T. reesei en ce qui concerne la production de cellulases, et les modèles développés sont des outils d'aide rationnelle à la définition d'un procédé de production de cellulases intégré dans une bioraffinerie lignocellulosique
In the global energetic and climatic context, the high cost of the cellulolytic enzymes (cellulases) postpones the development of lignocellulosic biorefineries, dedicated to produce biofuels and chemical compounds from renewable vegetable feedstocks. The aim of this work was to measure and model the metabolism of the micro-organism Trichoderma reesei, in order to optimize the industrial protocol for the production of cellulase. This study was carried out using synthetic media representative of industrial ones. First, the stoichiometries of growth and protein production reactions were determined. Then, a kinetic study was conducted to precisely measure the specific rates of T. reesei at high induction of cellulase production. The resulting model was used to optimize the industrial production protocol. Finally the integration of this step in a lignocellulosic biorefinery was studied by determining the impacts on the metabolism of i) available sugar mixtures, ii) inhibitory compounds from lignocellulosic biomass degradation, and iii) scale-up. These results significantly contributed to improve the knowledge of T. reesei metabolism on cellulase production. The developed models are rational tools for the optimization of a cellulase production protocol suited to lignocellulosic biorefineries

Global climate change and volatility of petroleum price have driven the necessity to reduce fossil fuel utilization and replace it by renewable energy. Bioethanol production in the United States and Brazil from cornstarch and sugarcane, respectively, is already established. However, the bioethanol industry appears unsustainable in view of the potential stress that its production places on food commodities. In contrast, second-generation biofuels produced from cheap and abundant lignocellulosic biomass, has been viewed as one plausible solution to this \"food versus fuel\" problem. Sugarcane bagasse is an abundant source of lignocellulosic biomass in Brazil and is generally recognized as a very promising feedstock for lignocellulosic ethanol production. Nevertheless, inhibitors such as furfural, 5-hydroxymethyl furfural (HMF) and carboxylic acids are formed during an acid thermochemical pretreatment of lignocellulosic biomass, which has a negative effect on the fermentative microorganisms - Saccharomyces cerevisiae. Second-generation (2G) ethanol in Brazil has the possibility to use a novel substrate, prepared as a blend of sugarcane bagasse hydrolysate and cane molasses. Molasses supplements the nutritional deficiencies of bagasse hydrolysate, contributing with minerals, amino acids and vitamins. However, molasses also contains additional inhibitors, such as HMF, sulfite, and toxic concentration of some minerals (K, Ca), which affect S. cerevisiae fermentation performance. The goal of this work was to generate tolerant derivatives of S. cerevisiae industrial strains that are able to cope with inhibitors present in bagasse hydrolysate and molasses, by means of sexual hybridization and adaptive evolution, which can be used for 2G-ethanol production. The industrial strains PE-2, CAT-1 and SA-1 were sporulated, and haploids were irradiated by ultraviolet (UV) light in order to increase genetic and phenotypic diversity. After direct mating and screening in molasses and hydrolysate media, 234 hybrid strains were selected for further study. In parallel, mass matings (intra and interlines) of PE-2, CAT-1 and SA-1 from non-irradiated haploids were performed and the generated strains were subjected to adaptive evolution for about 100 generations. The 120 strains derived from mass mating and adaptive evolution were then screened for growth in molasses-hydrolysate media. Six isolates showed good fermentation properties compared to the reference strains, showing that hybridization and adaptive evolution of Brazilian industrial yeast strains was a good strategy to develop new tolerant strains for 2G-ethanol production. To better utilize all the sugars present in bagasse hydrolysate, a cassette containing the three genes responsible for xylose fermentation (xylose reductase, xylitol dehydrogenase and xylulose kinase) was integrated into the genome of a haploid derivative (272-1a) of one of the six selected hybrids (272), which had the highest tolerance to Miscanthus x giganteus hydrolysate. Fermentation studies demonstrated that this engineered strain was able to metabolize xylose into ethanol. Finally, the haploid 272-1a was analyzed by quantitative trait loci (QTL) mapping to identify the genetic basis of hydrolysate tolerance. Although the causative gene(s) were not identified in this work, a number of QTL peaks were identified that will serve as the starting point for future fine-mapping studies.
Mudança climática global e a volatilidade do preço do petróleo tem impulsionado a necessidade de redução e substituição de combustíveis fósseis por energias renováveis. A produção de bioetanol nos Estados Unidos e no Brasil a partir de milho e cana-de-açúcar, respectivamente, está estabelecida. Todavia, a produção de bioetanol mostra-se insustentável, pelo fato da utilização de produtos alimentares para tal produção. Em contrapartida, biocombustíveis produzidos a partir de resíduos lignocelulósicos têm sido vistos como uma solução plausível para o problema \"alimento versus combustível\". No Brasil, o bagaço de cana é uma fonte disponível de biomassa lignocelulósica. No entanto, inibidores como furfural, 5-hidroximetil-furfural (HMF) e ácidos carboxílicos formados durante o prétratamento ácido da biomassa lignocelulósica, têm efeito negativo sobre os microorganismos fermentadores - Saccharomyces cerevisiae. No Brasil, o etanol de segunda-geração (2G) tem possibilidade de utilizar um novo substrato, preparado a partir da mistura de melaço e hidrolisado de bagaço. O melaço será um adjuvante para suprir a deficiência nutricional do hidrolisado, contribuindo com minerais, aminoácidos e vitaminas. Por outro lado, o melaço apresenta alguns inibidores, como HMF, sulfito, e concentração tóxica de alguns minerais, como potássio (K) e cálcio (Ca), que afetam o crescimento e desempenho fermentativo de S. cerevisiae. O objetivo deste trabalho foi gerar descendentes tolerantes de linhagens industriais de S. cerevisiae, capazes de lidar com inibidores presentes no melaço e no hidrolisado de bagaço, por meio de hibridação e evolução adaptativa, para produção do etanol 2G. As linhagens industriais PE-2, CAT-1 e SA-1 foram esporuladas, seus haplóides foram irradiados por luz ultravioleta (UV), objetivando o aumento da diversidade genética e fenotípica das linhagens. Após cruzamento direcionado, 234 híbridos foram selecionados pelo crescimento (DO570nm) em meios de melaço e hidrolisado. Em paralelo, cruzamentos massais (intra e interlinhagens) de haplóides não-irradiados de PE-2, CAT-1 e SA-1 foram realizados e submetidos a evolução adaptativa por cerca de 100 gerações. As 120 estirpes de cruzamentos massais seguidos de evolução adaptativa foram selecionadas pelo crescimento em meios de melaço e hidrolisado. Seis isolados apresentaram boas características fermentativas em comparação às cepas referências, mostrando que hibridação e evolução adaptativa de linhagens de leveduras industriais brasileiras são boas estratégias para desenvolver novas linhagens para produção do etanol-2G. Para uma melhor utilização dos açúcares do hidrolisado, a cassete contendo os três genes responsáveis pela fermentação de xilose (xilose redutase, xilitol desidrogenase e xiluloquinase) foi integrada no genoma do haplóide segregante (272-1a) de uma das seis estirpes selecionadas (272), que apresentou a maior tolerância em hidrolisado de Miscanthus x giganteus. Estudos de fermentação mostraram que a estirpe foi capaz de metabolizar a xilose em etanol. Por fim, o haploide 272-1a foi analisado por quantitative trait loci (QTL) afim de identificar a base genética da tolerância ao hidrolisado. Apesar, do(s) gene(s) causativos não terem sido identificados nesse trabalho, os picos do mapa de QTL identificados servirão como ponto de partida para futuro mapeamento.

The sugarcane bagasse has a high content of lignocellulosic material, which enables the study for the production of second-generation ethanol, requiring the application of a pretreatment that promotes the rupture of the fiber, to make accessible sugars for fermentation. There are several pretreatments aimed at the break and in the search for the most productive one, it is applied severe conditions of temperature and pressure. This promotes the formation of undesirable products in the bioethanol production process, requiring detoxification step for removal of inhibitors. In this study, we used the detoxifying step for two pretreatments, hydrothermal and acid. The methodology raised the pH of the hydrolysates resulting from the acid pretreatment to 7.0 with calcium oxide and then decay to pH 4.0 with phosphoric acid. The hydrolysates of the hydrothermal pretreatment had its pH reduced to 4.0 by addition of phosphoric acid, both pretreated were subjected to adsorption on activated carbon (1% w /v , 100 rpm , 30 minutes at 50 ° C), conditions chosen after design 22 after triplicate with the center point. The evaluation of the efficacy of these procedures was made as to the removal of toxic compounds depending on the fermentation yield with the yeast Saccharomyces cerevisiae, hydrolysates with and without detoxification, assessing the amount of released sugars for conversion into second-generation ethanol. According to the results , the change of pH combined with activated carbon adsorption led to higher fermentation yields in both pretreated 38.51% acid and hydrothermal 44.85% hydrolyzed , when compared to the yield of samples not detoxified , these results may be associated with interference of lignin in the pulp, which can form condensation products able to interfere with the detoxification. However the best results were found in the hydrolysate hydrothermally pretreated with 87.94% efficiency and fermentation alcohol content of 7.41%, compared to the pre-treated hydrolysate with acid and 5.11% 75.05% respectively.
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O bagaço de cana-de-açúcar possui alto teor de material lignocelulósico, o que viabiliza o estudo para a produção do etanol de segunda geração, sendo necessária a aplicação de um pré tratamento que promova a ruptura da fração fibrosa, para tornar os açucares acessíveis para fermentação. Existem vários pré-tratamentos que visam essa quebra, e na busca pelo mais produtivo são aplicadas condições severas de temperatura e pressão. Isso propicia a formação de produtos indesejáveis ao processo de produção do bioetanol, sendo necessária a etapa de destoxificação para remoção os inibidores. Nesse trabalho, foi empregado a etapa de destoxificação para dois pré-tratados acido e hidrotérmico, na metodologia utilizada elevou-se o pH dos hidrolisados provenientes do pré-tratamento acido para 7,0 com oxido de cálcio e em seguida o decaimento ate pH 4,0 com acido fosfórico, os hidrolisados do pré-tratamento hidrotérmico tiveram seu pH reduzidos para 4,0 com a adição do acido fosfórico, ambos os pré-tratados foram submetidos a adsorção em carvão ativado (1% m/v, 100rpm, 30 minutos a 50°C), condições escolhidas apos planejamento 22 com triplicata no ponto central. Avaliação da eficácia destes procedimentos foi feita quanto a remoção dos compostos tóxicos em função do rendimento fermentativo com a levedura Saccharomyces cerevisiae, de hidrolisados com e sem destoxificação, avaliando a quantidade de açúcares liberados para conversão em etanol de segunda geração. De acordo com os resultados, a alteração de pH combinada a adsorção com carvão ativo propiciou maiores rendimentos fermentativos em ambos os hidrolisados pré-tratados acido 38,51% e hidrotérmico 44,85%, quando comparados ao rendimento de amostras não destoxificadas, a esses resultados pode estar associado a interferência da lignina no bagaço, que pode formar produtos de condensação capazes de interferir na destoxificação. No entanto os melhores resultados foram encontrados no hidrolisado pré-tratado hidrotémicamente com 87,94% de eficiência de fermentação e teor alcoólico de 7,41%, quando comparado ao hidrolisado pré-tratado com acido de 75,50% e 5,11%, respectivamente.

A busca por soluções sustentáveis, levando a um processo energético mais eficiente induziu a novas tecnologias e esforços estão sendo realizados para viabilizar o etanol de segunda geração, com o aproveitamento da biomassa celulolítica como substrato para a fermentação alcoólica. Contudo, durante a hidrólise do bagaço, muitos compostos tóxicos à levedura são formados como o furfural, hidroximetilfurfural, ácido acético, e compostos fenólicos com efeitos depressivos sobre a fermentação. A adição de melaço no hidrolisado de bagaço poderia permitir uma fermentação com maior teor alcoólico, contribuindo para um balanço energético favorável da destilação, além de propiciar nutrientes minerais e orgânicos para a levedura. Tais nutrientes poderiam permitir um processo fermentativo com reciclo de células de leveduras aproveitando assim uma estrutura e conhecimentos já existentes na destilaria de etanol de primeira geração. O reciclo de células permitiria fermentações rápidas, porém impõe repetidas condições estressantes, o que torna um grande desafio a obtenção de linhagens com o perfil de tolerância desejado. Assim este trabalho se propôs a selecionar linhagens de Saccharomyces cerevisiae com múltiplas tolerâncias em relação aos inibidores tanto presentes no hidrolisado como no melaço. Para tal, foram impostas condições estressantes sobre culturas da linhagem SA-1 e de leveduras isoladas de destilarias brasileiras durante cerca de 62 gerações, forçando uma evolução adaptativa ou mesmo um enriquecimento/seleção de indivíduos mais tolerantes. Paralelamente a biodiversidade de linhagens isoladas de destilarias foi avaliada quanto aos atributos de tolerância aos compostos tóxicos presentes no hidrolisado do bagaço. As linhagens com maiores desempenhos foram avaliadas em fermentações com reuso de células empregando-se substrato constituído de hidrolisado e melaço, sendo que 4 linhagens se mostraram superiores às linhagens referenciais. Destas, dois isolados (242 e 408) foram esporulados e os conjuntos dos haploides foram empregados em cruzamentos massais. Simultaneamente, 273 haploides isolados das linhagens 242 e 408 foram avaliados quanto ao crescimento (DO600nm) em substrato constituído por hidrolisado e melaço, sendo que 32 foram selecionados. Após a tipificação segundo o \"mating type\" os mesmos foram utilizados em cruzamentos direcionados mediante micromanipulação, resultando em 35 cruzamentos. Cinco híbridos de cada cruzamento direcionado foram resgatados (155 isolados), que juntamente com 80 isolados oriundos do cruzamento massal, foram novamente avaliados quanto ao crescimento (DO600nm) e a seguir em fermentações com reciclo de células. Cinco linhagens se destacaram como superiores aos parentais, demonstrando que mediante o protocolo empregado foi possível incrementar o perfil de tolerância de Saccharomyces cerevisiae para suportar os estresses impostos por um substrato para produção do etanol de segunda geração.
The search for sustainable solutions to improve process efficiency has promoted the development of new technologies, and the use of cellulolytic biomass as the substrate for fermentation has emerged as a promising second-generation ethanol production strategy. However, the hydrolysis of this material results in the formation of toxic compounds to yeast such as furfural, hydroxymethylfurfural, acetic acid and phenolic compounds, with deleterious effects on fermentation. Addition of molasses in the bagasse hydrolysate could allow fermentation with higher alcohol content contributing to a favorable energy balance in the distillation, as well as providing minerals and organic nutrients for the yeast. These nutrients could allow a fermentative process with yeast cell recycle, utilizing the structure and knowledge already existing in first generation process. The cell recycle enables a rapid fermentation, but imposes repeated stress conditions, making it challenging to obtain strains with the desired tolerance profile. The purpose of this study was to select Saccharomyces cerevisiae strains with multiple tolerances to inhibitors present in the hydrolysate and molasses. Stressful conditions were imposed on cultures of SA-1 strain and indigenous strains from Brazilian distilleries for around 62 generations, forcing an adaptive evolution or even an enrichment / selection of more tolerant individuals. In parallel, the biodiversity of the strains from Brazilian distilleries were evaluated with respect to their tolerance to the toxic compounds present in bagasse hydrolysate. The strains that showed higher performance were assessed in fermentations with cell reuse employing substrate composed by hydrolyzate and molasses. Four of the analyzed strains exhibited better performance than the reference strain. Of these, two isolates (242 and 408) were sporulated and the haploids were subjected to mass mating. Simultaneously, 273 haploids rescued from the strains 242 and 408 were evaluated for growth (OD 600 nm) in the substrate consisting of hydrolysate and molasses, and among them 32 were selected. After the characterization according to the \"mating type\", the haploids were utilized in direct mating induced by micromanipulation, totaling 35 crossings. Five hybrids from each direct mating were rescued (totaling 155 isolates), which together with 80 isolated from the mass mating, were evaluated for growth (OD 600 nm) and then in fermentation with cell recycle. 5 strains have excelled as superiors to the reference strain showing that by the protocol employed was possible to increase profile of tolerance of Saccharomyces cerevisiae to resist pressures imposed by a substrate for second-generation ethanol production.

O desenvolvimento de alternativas aos combustíveis fósseis como fonte de energia é uma prioridade global. A biomassa celulósica representa uma alternativa para satisfazer a procura de bicombustíveis renováveis. O bagaço de cana-de-açúcar é um abundante subproduto proveniente da produção atual de etanol no Brasil. Tal subproduto pode ser hidrolisado a fim de se obter açúcares fermentáveis para a produção do etanol de segunda-geração. Porém, no processo de pré-tratamento são gerados diversos inibidores como ácido acético, furfural e hidroximetilfurfural que causam efeitos adversos para a levedura no processo de fermentação alcoólica. A adição de melaço ao hidrolisado é uma forma de diminuir os efeitos dos inibidores no metabolismo das leveduras e também permite uma fermentação com maior teor alcoólico contribuindo para um balanço energético favorável da destilação, contribui também com o fornecimento de nutrientes minerais e orgânicos necessários a levedura para um processo empregando reciclo de células. Assim, objetivou-se selecionar linhagens de Saccharomyces cerevisiae com melhores características de multitolerância ao hidrolisado a partir de cruzamento direcionado e cruzamento massal seguido de evolução adaptativa. Para tal, linhagens S. cerevisiae industriais CAT-1, BG-1, PE-2 e SA-1 foram esporuladas e mediante micromanipulação foram obtidos 604 haploides, que foram avaliados quanto ao crescimento (DO570nm) em substrato constituído por hidrolisado e melaço. Os haploides selecionados (25) tiveram o \"mating type\" determinado permitindo a realização de 51 cruzamentos direcionados, gerando 398 zigotos, que foram igualmente avaliados para o crescimento no meio seletivo. Paralelamente, foram realizados cruzamentos massais, resultando em 7 diferentes populações, as quais foram submetidas à evolução adaptativa por 25 gerações, sendo que os isolados selecionados de cada cruzamento foram avaliados em fermentação com reciclo de células. Quatro linhagens se destacaram como superiores aos parentais, evidenciando que a estratégia utilizada permitiu a obtenção de linhagens de S. cerevisiae com maior tolerância aos estresses impostos por um substrato para produção do etanol de segunda geração.
The development of alternatives to fossil fuels as a source of energy is a global priority. Cellulosic biomass is an alternative to meet the demand for renewable biofuels. The sugarcane bagasse, an abundant byproduct generated from ethanol production in Brazil, can be hydrolysed to obtain fermentable sugars to produce second-generation ethanol. However, inhibitors produced in the pre-treatment process such as acetic acid, furfural and hydroxymethylfurfural, cause adverse effects to the yeast in the fermentation process. Addition of molasses in the bagasse hydrolyzate is one way to reduce the effects of inhibitors in the metabolism of yeast and also could allow fermentation with higher alcohol content contributing to a favorable energy balance in the distillation, as well as providing minerals and organic nutrients for the yeast. The main goal of this study was to select strains of Saccharomyces cerevisiae with better features of multi-tolerance to the bagasse hydrolyzate by directed crossing and mass mating followed by adaptive evolution. For that S. cerevisiae lineages CAT-1, BG-1, PE-2 e SA-1 were sporulated and 604 haploid cultures were obtained by micromanipulation and evaluated for growth (OD 570nm) in the substrate consisting of hydrolyzate and molasses. Selected haploids (25) were identified regarding their \"mating type\" (a and α) and used in. 51 directed crossings generating 398 zygotes, which were rescued by micromanipulation and also evaluated for growth in the same selective medium. Mass mating were performed with 7 different haploid populations from the parental strains, followed by an adaptive evolution for 25 generations. The selected zygotes were then subjected to fermentation trails with cell recycling, resulting in 4 strains with superior traits when compared with the parentals, allowing to conclude that the used strategy was successful in obtaining hybrids of Saccharomyces cerevisiae with increased profile of tolerance towards a substrate for second-generation ethanol production.

Orientador: Mario de Oliveira Neto
Resumo: A biomassa lignocelulósica pode ser usada para a produção de energia ou de novos bioprodutos potenciais substitutos de químicos convencionais. Porém a conversão dos polissacarídeos estruturais presentes na parede celular vegetal das células que compõe a biomassa não é simples. Isto se deve principalmente pela presença da lignina, que juntamente com a hemicelulose, formam uma estrutura coesa de microfibrilas que entrelaçam a celulose. Compostos que inibem as enzimas celulolíticas, incluindo fenólicos solúveis (derivados da lignina), açúcares solúveis, aldeídos de furano e ácidos fracos são gerados durante os diversos pré-tratamentos utilizados atualmente. Neste estudo, observamos como os fenólicos solúveis interagem com -glicosidases. Para isso, combinamos simulações de ensaio enzimático, docking molecular e dinâmica molecular para descrever o processo de ligação. Notavelmente, o ácido tânico, um dos fenólicos solúveis estudados, foi a molécula com maior poder inibitório em comparação com todos os demais fenólicos. Possivelmente devido ao seu comprimento e suas substituições de grupos químicos. A alta presença de anéis aromáticos e grupos hidroxilas no ácido tânico, leva a maior interação entre as moléculas e consequente inibição/desativação das β-glicosidases bacterianas, enquanto os grupos carboxílicos presentes nos demais fenólicos alteram os efeitos físico-químicos aumentando a hidrofobicidade; criando cargas eletrostáticas e aumentando a ligação de hidrogênio, afetando a... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Lignocellulosic biomass can be transformed to chemicals or energy products. However converting polysaccharides present on the cell wall can be limitated due to the high recalcitrance caused by the presence of lignin. Compounds that inhibit enzymes, including lignin-derived phenolics, soluble sugars, furan aldehydes, and weak acids, are generated during the various pre-treatments currently used. In this study was observed how the soluble phenolics generated significantly impede the enzymatic hydrolysis of cellulose. For this were combine enzymatic assay, molecular docking and molecular dynamics simulations to describe the binding process between soluble phenolics and bacterial β-glycosidases. Notably, tannic acid, one of the soluble phenolics generated, was the strongest inhibitory molecule in comparison with all phenolics studied. Possibly because of its length and its substitutions of chemical groups. The high presence of aromatic rings and hydroxyl groups in tannic acid leads to greater interaction between the molecules and consequent inhibition / deactivation of bacterial β-glycosidases. Taken together, our studies of the interaction suggest that there is a high correlation between exposed hydrophobic surface areas and the number of binding sites on the inhibition of βglucosidases. These data may provide a useful basis for future biotechnological applications of microbial β-glucosidases, especially in the field of biofuel production.
Doutor

Im Laborversuch konnte der positive Einfluss einer thermobarischen Vorbehandlung auf die Hydrolysier- und Vergärbarkeit von Rinderfestmist und Rindergülle nachgewiesen werden. Die Laborergebnisse wurden innerhalb eines theoretischen Modells in den Praxismaßstab übertragen, um den Einfluss auf Treibhausgasemissionen, Energiebilanz und Ökonomie zu bewerten. Die Vorbehandlungstemperaturen im Labor lagen zwischen 140 und 220°C in Schritten von 20 K und einer Vorbehandlungszeit von jeweils 5 Minuten. Die höchste Methanmehr¬ausbeute von 58 % konnte bei einer Temperatur von 180°C ermittelt werden. Das Auftreten von Inhibitoren und nicht vergärbaren Bestandteilen führte bei einer Aufbereitungstemperatur von 220°C zu Methanausbeuten, die geringer waren als die des unaufbereiteten Einsatzstoffes. In einer erweiterten Analyse konnte ein funktioneller Zusammenhang zwischen der Methanausbeute nach 30 Tagen und der Methanbildungsrate und -ausbeute während der Beschleunigungsphase gezeigt werden. Mittels einer Regressionsanalyse der so ermittelten Werte wurde nachgewiesen, dass die optimale Aufbereitungstemperatur 164°C ist und die minimale größer als 115°C zu sein hat. Treibhausgasemissionen und Energiebilanz wurden im Rahmen einer Ökobilanz nach ISO 14044 (2006) ermittelt, sowie eine Kosten-Nutzen-Analyse durchgeführt. Dazu wurde eine Anlage zur thermobarischen Vorbehandlung entwickelt und innerhalb eines Modells in eine Biogasanlage integriert. Weiterhin wurde in diesem Modell Maissilage durch Rinderfestmist und / oder Rindergülle als Einsatzstoff ersetzt. Rinderfestmist, ein Einsatzstoff mit hohem organischen Trockenmassegehalt, der ohne Vorbehandlung nicht einsetzbar wäre, erreichte eine energetische Amortisationszeit von 9 Monaten, eine Vermeidung in Höhe der während der Herstellung emittierten Treibhausgase innerhalb von 3 Monaten und eine ökonomische Amortisationszeit von 3 Jahren 3 Monaten, wohingegen Rindergülle keine positiven Effekte zeigte.
Hydrolysis and digestibility of cattle waste as feedstock for anaerobic digestion were improved by thermobarical treatment in lab-scale experiments. The effects of this improvement on greenhouse gas emissions, energy balance and economic benefit was assessed in a full-scale model application. Thermobarical treatment temperatures in lab-scale experiments were 140 to 220°C in 20 K steps for a 5-minute duration. Methane yields could be increased by up to 58 % at a treatment temperature of 180°C. At 220°C, the abundance of inhibitors and other non-digestible substances led to lower methane yields than those obtained from untreated material. In an extended analysis, it could be demonstrated that there is a functional correlation between the methane yields after 30 days and the formation rate and methane yield in the acceleration phase. It could be proved in a regression of these correlation values that the optimum treatment temperature is 164°C and that the minimum treatment temperature should be above 115°C. The theoretical application of a full-scale model was used for assessing energy balance and greenhouse gas emissions following an LCA approach according to ISO 14044 (2006) as well as economy. A model device for thermobarical treatment has been suggested for and theoretically integrated in a biogas plant. The assessment considered the replacement of maize silage as feedstock with liquid and / or solid cattle waste. The integration of thermobarical pretreatment is beneficial for raw material with high organic dry matter content that needs pretreatment to be suitable for anaerobic digestion: Solid cattle waste revealed very short payback times, e.g. 9 months for energy, 3 months for greenhouse gases, and 3 years 3 months for economic amortization, whereas, in contrast, liquid cattle waste did not perform positive replacement effects in this analysis.

La biomasse lignocellulosique en Malaisie peut être considérée comme l'une des sources d'énergie renouvelable prometteuse. Elle est principalement composée de cellulose, d'hémicellulose et de lignine et est adaptée pour des applications dans les domaines de l'énergie et de la chimie en raison de sa disponibilité suffisante, de son faible coût et de son caractère renouvelable. La production de biomasse lignocellulosique en Malaisie est considérée comme élevée et est issue en grande partie de l'industrie de l'huile de palme (environ 60 millions de tonnes de déchets d'huile de palme sont générés en un an). Les déchets de l’industrie de l'huile de palme pourraient être utilisés comme ressources alternatives pour la production de papier et de carton. Cependant, dans ce contexte, d'énormes quantités de lignine seraient rejetées (par l'industrie des pâtes et papier) en raison du manque de prise de conscience de son potentiel. Avec une teneur élevée en groupes fonctionnels divers (-OH phénoliques et aliphatiques, les carbonyles, les carboxyles, etc.), la lignine pourrait être utilisée en substitution de produits actuels dans des applications industrielles telles que l'inhibition de la corrosion des métaux et alliages. Les frondes de palmier à huile (OPF) étant l'un des plus gros contributeurs de déchets de biomasse en Malaisie, elles ont donc été utilisées comme matière première dans cette étude. Afin d'améliorer l'extractabilité de la lignine et ses propriétés, l'extraction a été effectuée de différentes façons (par délignification directe et / ou des méthodes de pré-traitement combiné). Cependant, la forte hydrophobicité de la lignine limite sa capacité à agir comme inhibiteur de corrosion efficace. Par conséquent, des modifications de la structure de la lignine OPF ont été effectuées de deux manières ; (1) en incorporant des piégeurs de recondensation de la lignine (2-naphtol et 1,8-dihydroxyanthraquinone) pendant le prétraitement par autohydrolyse avant le traitement organosolv (pourcentage de rendement de la lignine: AHN EOL = 13,42 ± 0,71% et AHD EOL = 9,64 ± 0,84%) et (2) le fractionnement de la lignine à partir de procédés de délignification directs (Kraft, à la soude et organosolv) par l'intermédiaire d'une technique d'ultrafiltration à membrane (rendement en pourcentage de fractions de lignine perméat: Kraft = 5,41 ± 2,04%; soude = 12,29 ± 0,54% et organosolv = 1,48 ± 0,15%). Les propriétés physiques et chimiques des lignines modifiées ont été évaluées en utilisant l'infrarouge à transformée de Fourier (FTIR), la résonance magnétique nucléaire (RMN), chromatographie par perméation de gel (GPC), l'analyse thermique et la Chromatographie liquide à haute performance (HPLC). Des fractions de lignine modifiée présentant des teneurs en OH phénoliques élevées, des poids moléculaires, polydispersité et contenus en OH aliphatiques faibles ont abouti à des valeurs plus élevées de l'activité antioxydante. L'activité antioxydante semble être dépendante de la teneur en OH phénolique et en ortho-méthoxyle, grâce à la stabilité du radical formé et la capacité de réduire les ions Fe3+ en Fe2+ ions. En effet, les propriétés physico-chimiques améliorées et une activité anti-oxydante de lignine modifiée a donné une corrélation positive avec l'inhibition de la corrosion de l'acier doux dans l'action solution de HCl 0,5 M qui a été évaluée par spectroscopie d'impédance électrochimique (SIE), de polarisation et de la perte de poids mesure potentiodynamique. La meilleure efficacité de pourcentage d'inhibition (ex: 81 à 90%) a été obtenu à la concentration de 500 ppm pour les inhibiteurs de la lignine, mais a diminué avec l'augmentation de la température (303 à 333 K). Les données thermodynamiques indiquent que l'adsorption de la lignine modifiée sur l'acier doux a été spontanée et que les inhibiteurs ont été principalement adsorbés physiquement (physisorption), ce résultat étant confirmé par l'énergie d'activation de l'adsorption, Ea. [...]
Lignocellulosic biomass in Malaysia can be considered as one of the promising sources of renewable energy. It is mainly composed of cellulose, hemicellulose, and lignin and best-suited for energy and chemical applications due to its sufficient availability, inexpensive and is sustainable. In general, the production of lignocellulosic biomass in Malaysia was considered high and mainly derived from the palm oil industries (approximately 60 million tonnes of oil palm waste were generated in a year). The oil palm biomass waste could possibly be used as alternative resources for the production of paper and cardboard. However, massive amounts of lignin by-product could also be discarded in huge quantities (by the pulp and paper industry) due to lack of awareness on its potential. Having high content of diverse functional groups (phenolic and aliphatic –OH, carbonyls, carboxyls, etc.) and phenylpropanoid structure, lignin can lead to substitutes in industrial applications such as in corrosion inhibition of metals and alloys. Since the oil palm fronds (OPF) are one of the largest biomass waste contributors in Malaysia, it was therefore used as raw material in this study. In order to improve the lignin extractability and properties, the extraction was conducted in different ways (via direct delignification and/or combined pretreatment methods). Due to the high hydrophobicity of lignin, it limits the capability to act as efficient corrosion inhibitors. Hence, modifications of the OPF lignin structure were conducted in two ways; (1) by incorporating organic scavengers (2-naphthol and 1,8-dihydroxyanthraquinone) during autohydrolysis pretreatment before organosolv treatment (percentage yield of lignin: AHN EOL = 13.42±0.71 % and AHD EOL = 9.64±0.84 %) and (2) fractionation of lignin from direct delignification processes (Kraft, soda and organosolv) via ultrafiltration membrane technique (percentage yield of permeate lignin fractions: Kraft = 5.41±2.04 %; soda = 12.29±0.54 % and organosolv = 1.48±0.15 %). The physical and chemical properties of the modified lignins were evaluated by using Fourier Transform Infrared (FTIR) spectroscopy, nuclear magnetic resonance (NMR) spectroscopy, gel permeation chromatography (GPC), thermal analysis and high performance liquid chromatography (HPLC). Modified lignin fractions with higher phenolic –OH content but lower molecular weight, polydispersity as well as aliphatic –OH content resulted in higher values of antioxidant activities. The antioxidant activity seems be dependent on the increase of their free phenolic –OH and ortho-methoxyl content, through the stability of the radical formed and the ability to reduce Fe3+ ions to Fe2+ ions. Indeed, the improved physicochemical properties and antioxidant activity of modified lignin gave positive correlation with the mild steel corrosion inhibition action in 0.5 M HCl solution that were evaluated by electrochemical impedance spectroscopy (EIS), potentiodynamic polarization and weight loss measurements. The best percentage of inhibition efficiencies (IE: 81 – 90 %) were attained at the concentration of 500 ppm for all lignin inhibitors but decreased with the increase in temperature (303 – 333 K). Thermodynamic data indicated that the adsorption of the modified lignin onto the mild steel was spontaneous and the inhibitors were mainly physically adsorbed (physiosorption), supported by the activation energy of adsorption, Ea. The enhanced protective properties of the modified lignin will pave way for an alternative approach for the utilization of these natural waste materials

Dissertação de mestrado em Bioengenharia
The progressive depletion of fossil fuels reserves in the last years led to the necessity for biotechnological manufacturing based on lignocellulosic feedstocks. Lignocellulosic biomass, such as straw, is an abundant low-cost source for production of biofuels, such as bioethanol, that does not compete for food needs. However, lignocellulose-to-ethanol process involves pre-treatment of biomass to obtain readily fermentable sugars, which leads to the accumulation of inhibitory by-products (e.g. furan derivatives, phenolic compounds, organic acids). Significant progress has been made in the understanding of the determinants of yeast tolerance to lignocellulose biomass-derived inhibitors, as well as to high ethanol concentrations. Nevertheless, further knowledge at the genetic level is of essential importance for the improvement of second generation bioethanol conversion technology. In a previous work, 5 genes, ERG2, PRS3, RAV1, RPB4 and VMA8, were found to contribute to the maintenance of cell viability and/or for maximal fermentation rate in wheat straw hydrolysate. Taking into account the negative effects reported from single overexpression of ERG2, RAV1 and VMA8 under non-stressful conditions, these genes were not considered as good targets for genetic engineering in the present work. Furthermore, ZWF1, a gene essential for yeast response to the presence of acetic acid, was added to the set of genes considered in the present study. To attempt to overcome the fermentation hurdles resultant from the inhibitory load mentioned above, molecular biology tools were used to: (1) unravel HAA1, PRS3 and RPB4 role in adaptation to toxic biomass hydrolysates, evaluating their expression levels, by qRT-PCR, in the outstanding-fermenting Saccharomyces cerevisiae PE-2 when exposed to acetic acid, HMF and furfural, and (2) improve yeast tolerance and adaptation by overexpressing these genes in the auxotrophic S. cerevisiae BY4741, using multi-copy vectors, and assessing the effects in Eucalyptus globulus wood hydrolysate. Increased HAA1, PRS3 and RPB4 expression levels were observed at the late lag and/or initial stationary phases of the fermentation in the presence of inhibitors. However, the overexpression of these genes under the control of the strong constitutive ScPGK1 promoter has not resulted in improved growth and fermentation profiles. On the other hand, the overexpression of HAA1 and PRS3 genes under the regulation of their native promoters resulted in fermentations profiles with a reduced lag-phase. These results indicate that PRS3 and principally HAA1 overexpression play an important role in the adaptation to lignocellulosic-based stress, and are good candidates for yeast engineering to improve bioethanol production.
A diminuição progressiva das reservas de combustíveis fósseis nestes últimos anos levou à necessidade de uma indústria biotecnológica baseada em matérias-primas lenhocelulósicas. A biomassa lenhocelulósica, tal como a palha, é uma fonte abundante de baixo preço para a produção de biocombustíveis, como o bioetanol, que não compete com as necessidades alimentares. Contudo, o processo de conversão de biomassa lenhocelulósica a etanol envolve um pré-tratamento da biomassa para obtenção imediata de açúcares fermentescíveis, levando à acumulação de produtos inibitórios (ex. derivados de furano, compostos fenólicos, ácidos orgânicos). Avanços significativos têm sido efectuados no que concerne à compreensão de determinantes da tolerância de leveduras a inibidores derivados da biomassa lenhocelulósica, tal como a concentrações elevadas de etanol. No entanto, um maior conhecimento a nível genético é essencial para o melhoramento de tecnologias para a conversão de bioetanol de segunda geração. Num trabalho anterior, 5 genes, ERG2, PRS3, RAV1, RPB4 e VMA8 foram identificados como importantes para a manutenção da viabilidade celular e/ou para maximizar a taxa de fermentação em hidrolisados de palha de trigo. Considerando os efeitos negativos reportados da sobre-expressão singular dos genes ERG2, RAV1 e VMA8 na ausência de stress, estes genes foram considerados, neste trabalho, como não sendo bons alvos para engenharia genética. Adicionalmente, o gene HAA1, essencial na resposta à presença de ácido acético em leveduras, foi acrescentado ao conjunto de genes considerado neste estudo. Na tentativa de ultrapassar os problemas fermentativos acima referidos, ferramentas de biologia molecular foram usadas para: (1) desvendar o papel dos genes HAA1, PRS3 e RPB4, na adaptação a hidrolisados de biomassa tóxicos, avaliando os seus níveis de expressão por qRT-PCR, no excepcional organismo fermentativo Saccharomyces cerevisiae PE-2 quando exposto a ácido acético, HMF e furfural, e (2) melhorar a tolerância e adaptação da levedura através da sobre-expressão destes genes na estirpe auxotrófica S. cerevisiae BY4741, usando vectores multi-cópia, e avaliar os efeitos em hidrolisado de madeira de Eucalyptus globulus. Níveis de expressão aumentados dos genes HAA1, PRS3 e RPB4 foram observados no final da fase de adaptação e/ou no inicio da fase estacionária da fermentação na presença de inibidores. Todavia, a sobre-expressão destes genes sob o controlo do promotor constitutivo e forte ScPGK1 não demonstrou um melhoramento dos perfis de crescimento e fermentativos. Em contrapartida a sobre-expressão dos genes HAA1 e PRS3 sob a regulação dos seus promotores nativos resultaram em perfis de fermentação com reduzida fase de adaptação. Estes resultados indicam que a sobre-expressão do PRS3 e principalmente do HAA1 tem um papel importante na adaptação ao stress derivado de biomassa lenhocelulósica, sendo bons candidatos para a engenharia genética de leveduras, de modo a obter melhorias na produção de bioetanol.
Fundação para a Ciência e a Tecnologia (FCT) - Project GlycoCBMs FCT PTDC/AGR-FOR/3090/2012 – FCOMP-01-0124-FEDER-027948

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

archives@tulane.edu
As fossil fuel resources decrease while pollution and greenhouse gas emissions from gasoline increase, the need for an alternative transportation fuel is pressing (Cassia et al. 2018). Current substitutes include ethanol-mixed gasolines, which have lower efficiency than gasoline and still rely on fossil fuels. Butanol, another possible gasoline substitution, is promising. It is a much more efficient fuel than ethanol and can be directly substituted for gasoline (Seggiani et al. 2012). Current techniques to mass-produce butanol depend on fermentation of sugars for butanol production. While sugar can be found in most natural matter, such as lumber or agriculture, it is an expensive material to process. In order to decrease the expense, agricultural waste, known as lignocellulosic biomass, is fermented instead (Baral et al. 2016). Another substrate that undergoes fermentation is crude glycerol, the waste product of producing biodiesel. Both crude glycerol and lignocellulosic biomass contain sugars that are fermentable. Certain strains of a bacterial genus Clostridium can ferment biomass and create acetone, butanol, and ethanol in a process known as ABE fermentation. A specific strain, Clostridium pasteurianum, is used in this study, as it has the ability to ferment sugars found in lignocellulosic biomass and crude glycerol (Venkataramanan et al. 2012). Unfortunately, both crude glycerol and lignocellulosic biomass also contain many toxic compounds, which kill or damage the bacteria and decrease butanol production. To ensure efficiency, it is important to determine which acids in these two substrates are causing inhibitory effects on Clostridium pasteurianum, specifically. This specific study investigates six different acids and their inhibitory effects on this strain of bacteria: D-glucuronic acid, furfural, hydroxymethylfurfural, levulinic acid, linoleic acid, and oleic acid. Based on these findings, furfural, linoleic, and oleic acid are the most toxic to Clostridium pasteurianum, and a tolerance to these specific acids should be engineered in order to create a more efficient butanol production system.
1
Caroline Duncan

碩士
國立雲林科技大學
環境與安全衛生工程系
105
Lignocellulose is the most abundant biomass on Earth, and its cellulosic polysaccharide is suitable used as raw material for bioenergy production. To release polysaccharide from lignocellulose and further hydrolyze polysaccharide into simple sugar, pretreatment is necessary and beneficial for subsequent fermentation process. Thermal acid hydrolysis is extensively applied and econamic pretreatment method to deal with lignocellulosic biomasses, but inhibitors are inevitably formed during this process. 5-Hydroxymethylfurfural (5-HMF) is the main inhibitor compound produced, and other inhibitors, such as organic acids, furfural and phenols are also commonly generated during thermal acid hydrolysis. These inhibitors seriously influence downstream bioenergy production. Therefore, inhibitors removal is an important issue. In 2004, the US Department of Energy announced 12 top biomass platform molecules for the sustainable future. 2,5-Furan-dicarboxylic acid (FDCA) is present in this list. FDCA can be obtained by 5-HMF biotransformation, and it can replace terephthalic acid (TA) to produce synthetic green plastic material, polyethylene-2,5- furandicarboxylate (PEF). Compared with suspended cells, immobilized cells have several advantages. The adventages include easy solid-liquid separation, low separation cost, high cell density maintenance and toxic compounds resistance. Based on the connection between 5-HMF detoxification in lignocellulosic hydrolysates and FDCA production, our previous isolate strain Burkholderia cepacia H-2 capable of biotransforming 5-HMF into FDCA was used in this study. In order to evaluate the feasibility of the immobilized Burkholderia cepacia H-2 for 5-HMF biotransformation. First, 5-HMF biotransformation using suspended cells and immobilized cells was compared. Then, the optimal inoculum size and stability of immobilized cells were studied. Finally, the effects of various inhibitors/salinity and thier concentrations on 5-HMF biotransformation were investigated. The results showed that the immobilized cells had better 5-HMF biotransformation and FDCA production efficiencies, and stable 5-HMF conversion at low pH. 1851 mg/L 5-HMF could be completely converted into FDCA within 32 hours. As increasing bacterial concentrations in the immobilized cells, 5-HMF biotransformation time was shortened. The optimal inoculum size in the immobilized cells was 175 mg/L (equal to O.D. 0.3). 1894 mg/L 5-HMF could be entirely biotransformed within 24 hours, and 1917 mg/L FDCA was received at the end of experiment. Immobilized cells with and without dead bacterial cells could adsorb 5-HMF and FDCA. As reuse cycles numbers of the immobilized cells increased, 5-HMF conversion efficiency decreased. 5-HMF conversion efficiency was higher than 75% after 16 reuse cycles. The higher the formic acid concentration, the lower the 5-HMF conversion rate. 5-HMF conversion efficiency was not significantly affected as formic acid concentration was 3000 mg/L, but enhanced as formic acid concentration was lower than 1000 mg/L. When increasing formic acid concentration to 5000 mg/L, FDCA production and 5-HMF conversion efficiencies obviously declined. In addition, strain Burkholderia cepacia H-2 was capable of degrading formic acid. Acetic acid concentration lower than 4390 mg/L did not affect 5-HMF biotransformation and FDCA formation, and 5-HMF conversion efficiency was between 87~90%. High furfural concentration had a negative effect on 5-HMF conversion and FDCA generation rates. When furfural concentration was higher than 1000 mg/L, 2000 mg/L 5-HMF could not be completely converted. Besides, furfural was utilized by immobilized strain Burkholderia cepacia H-2. There was no significant effect of levulinic acid on 5-HMF biotransformation. Furthermore, strain Burkholderia cepacia H-2 could use levulinic acid as carbon source to support biomass growth. The higher the phenol concentration, the lower the 5-HMF conversion rate. However, phenol had no significant effect on 5-HMF conversion efficiency. With the increase of NaCl concentration, 5-HMF conversion and FDCA production rates both obviously decreased. When NaCl concentration was higher than 6%, 5-HMF could not be completely biotransformed, but 5-HMF conversion efficiency was not influenced.

The use of lignocellulosic materials to replace fossil resources for the industrial production of fuels, chemicals, and materials is increasing. The carbohydrate composition of lignocellulose (i.e. cellulose and hemicellulose) is an abundant source of sugars. However, due to the feedstock recalcitrance, rigid and compact structure of plant cell walls, access to polysaccharides is hindered and release of fermentable sugars has become a bottle-neck. Thus, to overcome the recalcitrant barriers, thermochemical pretreatment with an acid catalyst is usually employed for the physical or chemical disruption of plant cell wall. After pretreatment, enzymatic hydrolysis is the preferred option to produce sugars that can be further converted into liquid fuels (e.g. ethanol) via fermentation by microbial biocatalysts. However, during acid pretreatment, several inhibitory compounds namely furfural, 5-hydroxymethyl furfural, phenols, and aliphatic acids are released from the lignocellulose components. The presence of these compounds can greatly effect both enzymatic hydrolysis and microbial fermentation. For instance, when Avicel cellulose and acid treated spruce wood hydrolysate were mixed, 63% decrease in the enzymatic hydrolysis efficiency was observed compared to when Avicel was hydrolyzed in aqueous citrate buffer. In addition, the acid hydrolysates were essentially non-fermentable. Therefore, the associated problems of lignocellulose conversion can be addressed either by using feedstocks that are less recalcitrant or by developing efficient pretreatment techniques that do not cause formation of inhibitory byproducts and simultaneously give high sugar yields. A variety of lignocellulose materials including woody substrates (spruce, pine, and birch), agricultural residues (sugarcane bagasse and reed canary grass), bark (pine bark), and transgenic aspens were evaluated for their saccharification potential. Apparently, woody substrates were more recalcitrant than the rest of the species and bark was essentially amorphous. However, the saccharification efficiency of these substrates varied based on the pretreatment method used. For instance, untreated reed canary grass was more recalcitrant than woody materials whereas the acid treated reed canary grass gave a higher sugar yield (64%) than the woody substrates (max 34%). Genetic modification of plants was beneficial, since under similar pretreatment and enzymatic hydrolysis conditions, up to 28% higher sugar production was achieved from the transgenic plants compare to the wild type. As an alternative to the commonly used acid catalysed pretreatments (prior to enzymatic hydrolysis) lignocellulose materials were treated with four ionic liquid solvents (ILs): two switchable ILs (SILs) -SO2DBUMEASIL and CO2DBUMEASIL, and two other ILs [Amim][HCO2] and [AMMorp][OAc]. viii After enzymatic hydrolysis of IL treated substrates, a maximum amount of glucan to glucose conversion of between 75% and 97% and a maximum total sugar yields of between 71% and 94% were obtained. When using acid pretreatment these values varied between 13-77% for glucan to glucose conversion and 26-83% for total sugar yield. For woody substrates, the hemicellulose recovery (max 92%) was higher for the IL treated substrates than compared to acid treated samples. However, in case of reed canary grass and pine bark the hemicellulose recovery (90% and 88%, respectively) was significantly higher for the acid treated substrates than the IL treated samples. To overcome the inhibitory problems associated with the lignocellulose hydrolysates, three chemical conditioning methods were used 1. detoxification with ferrous sulfate (FeSO4) and hydrogen peroxide (H2O2) 2. application of reducing agents (sulfite, dithionite, or dithiothreitol) and 3. treatment with alkali: Ca(OH)2, NaOH, and NH4OH. The concentrations of inhibitory compounds were significantly lower after treatments with FeSO4 and H2O2 or alkali. Using reducing agents did not cause any decrease in the concentration of inhibitors, but detoxification of spruce acid hydrolysates resulted in up to 54% improvement of the hydrolysis efficiency (in terms of sugar release) compared to untreated samples. On the other hand, application of detoxification procedures to the aqueous buffer resulted in up to 39% decrease in hydrolysis efficiency, thus confirming that the positive effect of detoxification was due to the chemical alteration of inhibitory compounds. In addition, the fermentability of detoxified hydrolysates were investigated using the yeast Saccharomyces cerevisiae. The detoxified hydrolysates were readily fermented to ethanol yielding a maximum ethanol concentration of 8.3 g/l while the undetoxified hydrolysates were basically non-fermentable.