RESSALVA Atendendo solicitação do autor, o texto completo desta dissertação será disponibilizado somente a partir de 21/02/2024. Mohammed Anas Zaiter Assessment of tolerance to inhibitors derived from plant biomass hydrolysis and xylose consumption by yeasts isolated from the environment São José do Rio Preto 2022 Câmpus de São José do Rio Preto Mohammed Anas Zaiter Assessment of tolerance to inhibitors derived from plant biomass hydrolysis and xylose consumption by yeasts isolated from the environment Dissertação apresentada como parte dos requisitos para obtenção do título de Mestre em Microbiologia, junto ao Programa de Pós-Graduação em Microbiologia, do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de São José do Rio Preto/SP. Financiadora: CAPES Orientador: Dr. Ronivaldo Rodrigues da Silva São José do Rio Preto 2022 Z21a Zaiter, Mohammed Anas Assessment of tolerance to inhibitors derived from plant biomass hydrolysis and xylose consumption by yeasts isolated from the environment / Mohammed Anas Zaiter. -- São José do Rio Preto, 2024 99 f. Dissertação (mestrado) - Universidade Estadual Paulista (Unesp), Instituto de Biociências Letras e Ciências Exatas, São José do Rio Preto Orientador: Ronivaldo Rodrigues da Silva 1. Microbiologia industrial. 2. Fermentação. 3. Leveduras. 4. Xilose. 5. Bioetanol. I. Título. Sistema de geração automática de fichas catalográficas da Unesp. Biblioteca do Instituto de Biociências Letras e Ciências Exatas, São José do Rio Preto. Dados fornecidos pelo autor(a). Essa ficha não pode ser modificada. Mohammed Anas Zaiter Assessment of tolerance to inhibitors derived from plant biomass hydrolysis and xylose consumption by yeasts isolated from the environment Dissertação apresentada como parte dos requisitos para obtenção do título de Mestre em Microbiologia, junto ao Programa de Pós-Graduação em Microbiologia, do Instituto de Biociências, Letras e Ciências Exatas da Universidade Estadual Paulista “Júlio de Mesquita Filho”, Câmpus de São José do Rio Preto/SP. Financiadora: CAPES Comissão Examinadora Dr. Ronivaldo Rodrigues da Silva UNESP – Câmpus de São José do Rio Preto/SP Orientador Profª. Drª. Tatiane Beltramini Souto Instituto Federal de Minas Gerais, Passos/MG Profª. Drª. Gabriela Salvador de Amo Instituto Federal de São Paulo, Catanduva/SP São José do Rio Preto 21 de fevereiro de 2022 DEDICATION To my mother, Safaa, for her never-ending desire to provide a better future for her children, To my father, Tayser, for being the voice of reason and supporting me on my path, To my wife, Alaa, for standing by my side on the journey of life, To my daughters, Safa and Mariam, for giving me a reason to keep going, To my keenly supportive brothers and sister, for their love and affection, Without you all, I wouldn’t be the person I am today. Thank you. Àqueles que apesar de todas as adversidades sempre acreditaram que eu seria capaz de atingir meus objetivos. ACKNOWLEDGEMENTS I am grateful for the opportunity to be a part of the Postgraduate Program in Microbiology at the Institute of Biosciences, Letters, and Exact Sciences, São Paulo State University. It has, frankly, been the right place for me, both professionally and personally. I have appreciated the collaborative and supportive environment. I am grateful to Profa. Dra. Eleni Gomes, Prof. Dr. Roberto da Silva, and Prof. Dr. Mauricio Boscolo for providing materials, instruments, infrastructure, and the environment conducive to scientific research. I am most grateful to my advisor, supervisor, and mentor, Dr. Ronivaldo Rodrigues da Silva, who taught me the basics of microbiological laboratory and scientific research and showed me the way to become an independent thinker. He has been both patient and lenient when things have been arduous, but has always encouraged me to complete the work to the fullest. For this work to see the light, I thank my committee members, Profª. Drª. Tatiane Beltramini Souto and Profª. Drª. Gabriela Salvador de Amo, for their intellectual guidance, efforts, and support throughout this process. I must thank all the many members of the laboratory of biochemistry and applied microbiology, and Bioenergy laboratory, past and present, for always being so willing to help and solve problems. They were invaluable in assisting me in getting started in the lab and have been wonderful colleagues and friends. I am also very grateful to my friend Ahmed, who encouraged me to go on this path when I was hesitant and uncertain. The present work was carried out with the support of the Coordination of Improvement of Higher Education Personnel - Brazil (CAPES) - Funding Code 001, to which I am grateful. RESUMO A tendência por esgotamento das reservas de petróleo e os problemas ambientais decorrentes do uso de combustíveis de origem fóssil têm evidenciado a necessidade por fontes alternativas de energia sustentáveis e baratas. Isso impulsionou o uso de etanol como combustível líquido, especialmente em países como EUA e Brasil. Contudo, para suprir esta crescente demanda por combustíveis, no Brasil extensas áreas de terras são ocupadas por cana-de-açúcar, e têm competido com outras culturas vegetais, especialmente para suprimento da cadeia de alimentos. Como alternativa, nos últimos anos têm se intensificado estudos para produção de etanol a partir da biomassa vegetal residual, como bagaço de cana-de-açúcar e outros subprodutos da agroindústria, uma vez que este material é subaproveitado e rico em hexoses (C6) e pentoses (C5). Para que a hidrólise enzimática da biomassa vegetal resulte em açúcares fermentescíveis, são necessárias etapas de pré-tratamento deste material, os quais tendem a gerar compostos inibidores de crescimento microbiano. Desta forma, leveduras resistentes a estes inibidores e capazes de co-fermentar açúcares C5 e C6 são amplamente investigadas. Diferentemente da levedura Saccharomyces cerevisae, algumas espécies não-Saccharomyces têm se mostrado capazes de assimilar pentoses, mas ainda são muito pouco exploradas, frente a diversidade de espécies na natureza. Investigar leveduras fermentadoras de xilose, assim como suas tolerâncias a inibidores do crescimento microbiano, pode suportar futuros estudos de engenharia metabólica para capacitação da linhagem industrial S. cerevisiae. Portanto, este trabalho propôs-se a avaliar a capacidade das leveduras Pichia ofunaensis e Trichosporon multisporum para assimilar xilose, e suas tolerâncias a alguns compostos inibitórios, bem como suas capacidades para produção de etanol a partir de xilose. As leveduras foram cultivadas em meio YEPX, pH 4,5 contendo, separadamente, os inibidores hidroximetilfurfural (HMF), furfural, ácido ferúlico, ácido acético, ácido fórmico e vanilina, cultivadas a 28 ºC e 150 rpm por 96 h. A levedura P. ofunaensis apresentou crescimento em todas as concentrações destes inibidores e foi capaz de consumir completamente a xilose presente nos meios de cultivo, exceto em 20 mM de furfural, onde houve consumo de 40% da xilose, e entre 80 a 90% em maiores concentrações de ácido ferúlico (3 mM), ácido fórmico (40 mM) e vanilina (10 mM). Em presença de ácido acético 80 mM, a levedura não cresceu, e nas concentrações de 60 mM e 40 mM consumiu 20% e 90% da xilose presente nos meios, respectivamente. A levedura T. multisporum, em presença de furfural, exibiu crescimento até 15 mM e consumiu 30% da xilose. Em meio com ácido ferúlico, HMF e ácido fórmico, houve crescimento em todas as concentrações e consumo de até 40% (ácido ferúlico e HMF) e 30% (ácido fórmico) da xilose. Com vanilina, observamos crescimento até 5 mM deste inibidor e máximo consumo de 30% da xilose, e em meio contendo ácido acético a levedura não apresentou crescimento a 20 mM, exibindo consumo de até 40% de xilose em concentrações inferiores. A levedura P. ofunaensis se mostrou mais tolerante aos compostos considerados potencialmente tóxicos, exibindo melhor crescimento e consumo de xilose em comparação a levedura T. multisporum. Furfural e ácido acético se mostraram os compostos mais prejudiciais ao crescimento das leveduras. Por fim, na fermentação alcoólica, detectamos a produção de etanol apenas no cultivo da levedura P. ofunaensis, com máxima produção de 0,51 g/L após 96 h de cultivo em meio salino contendo 50 g/L de xilose. Palavras-chave: Biomassa Lignocelulósica, Bioetanol, Ácidos Orgânicos, Fermentação, Leveduras, Pentose, Xilose. ABSTRACT Oil reserves are being depleted at an alarming rate, while the environmental problems arising from fossil fuels usage have highlighted the need for sustainable and cheap alternative energy sources, which has boosted the use of ethanol as a liquid fuel, especially in countries like the USA and Brazil. However, to meet this growing demand for combustibles, extensive land areas in Brazil have been occupied by sugarcane and have competed with other vegetable crops, especially for supplying the food chain. Alternatively, in recent years, studies have been intensified for ethanol production from residual plant biomass, such as sugarcane bagasse and other agro-industrial residues, since this material is underutilized and rich in hexoses (C6) and pentoses (C5). For enzymatic action in the degradation of plant biomass, resulting in fermentable sugars, pre-treatment steps of this material are necessary, which tend to generate compounds that inhibit microbial growth. Thus, yeast resistance to these inhibitors and the capability of co-fermenting C5 and C6 sugars are widely investigated. Unlike the yeast Saccharomyces cerevisiae, some non-Saccharomyces species can assimilate pentoses, but they are still very little explored, given the diversity of species in nature. Investigating xylose- fermenting yeasts, as well as their tolerances to microbial growth inhibitors, may support future studies of metabolic engineering to enable the industrial strain S. cerevisiae. Therefore, this work aimed to evaluate the capacity of Pichia ofunaensis and Trichosporon multisporum yeasts to assimilate xylose and their tolerances to some inhibitory compounds, as well as their capability to produce ethanol from xylose. Yeasts were cultivated in YEPX medium, pH 4.5, containing, separately, the inhibitors hydroxymethylfurfural (HMF), furfural, ferulic acid, acetic acid, formic acid, and vanillin, cultivated at 28 ºC and 150 rpm for 96 h. The yeast P. ofunaensis grew at all concentrations of these inhibitors and was able to completely consume the xylose present in culture media, except in 20 mM furfural, in which 40% of xylose was consumed, and between 80 and 90 % in higher concentrations of ferulic acid (3 mM), formic acid (40 mmM), and vanillin (10 mM). In the presence of 80 mM acetic acid, the yeast did not grow, and at the concentrations of 60 mM and 40 mM, it consumed 20% and 90% of the xylose present in the media, respectively. The yeast T. multisporum, in the presence of furfural, exhibited growth up to 15 mM and consumed 30% of the xylose. In media with ferulic acid, HMF, and formic acid, there was growth at all concentrations and consumption of up to 40% (ferulic acid and HMF) and 30% (formic acid) of xylose. With vanillin, we observed growth up to 5 mM of this inhibitor and maximum consumption of 30% of xylose, and in a medium containing acetic acid the yeast did not grow at 20 mM, exhibiting consumption of up to 40% of xylose at lower concentrations. The yeast P. ofunaensis was more tolerant to compounds considered potentially toxic and showed better growth rates and xylose consumption compared to the yeast T. multisporum. Furfural and acetic acid proved to be the most harmful compounds for yeasts growth. Finally, in the alcoholic fermentation, we detected ethanol production only in the yeast P. ofunaensis cultures, with a maximum output of 0.51 g/L after 96 h of cultivation in a saline medium containing 50 g/L of xylose. Keywords: Bioethanol, Fermentation, Lignocellulosic Biomass, Organic Acids, Pentose, Yeasts, Xylose. ILLUSTRATIONS LIST Figure 1 - Representation of lignocellulose structure showing cellulose, hemicellulose and lignin fractions………………………………………………………………............ 21 Figure 2 - Different regions of cellulose…………………...…………………………… 22 Figure 3 - Degradation products from lignocellulosic biomass as a result of acidic pretreatment……………………………………………………………………………... 27 Figure 4 - Metabolic pathways for xylose catabolism………………………...………... 30 Figure 5 - Yeast with ideal properties for fermenting plant biomass pentose and hexose sugars……………………………………………………………………………. 32 Figure 6 - Some Organic acids produced from lignocellulosic biomass hydrolysates and their main applications……………………………………………………………… 34 Figure 7 - P. ofunaensis (right) and T. multisporon (left) yeasts in a Petri dish………... 36 Figure 8 - P. ofunaensis (right) and T. multisporon (left) yeasts under microscope…... 36 Figure 9 - Cellular Growth in culture media containing furfural inhibitor (2-20 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)……………. 43 Figure 10 - Xylose assimilation in culture media containing furfural inhibitor (2-20 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)……… 44 Figure 11 - pH variation in culture media containing furfural inhibitor (2-20 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)………………… 45 Figure 12 - Cellular Growth in culture media containing HMF inhibitor (2-20 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)………………… 48 Figure 13 - Xylose assimilation in culture media containing HMF inhibitor (2-20 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)……………. 49 Figure 14 - pH variation in culture media containing HMF inhibitor (2-20 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)……………………. 50 Figure 15 - Cellular Growth in culture media containing ferulic acid inhibitor (0.5-3 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)…...…. 56 Figure 16 - Xylose assimilation in culture media containing ferulic acid inhibitor (0.5- 3 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)……. 57 Figure 17 - pH variation in culture media containing ferulic acid inhibitor (0.5-3 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)……………... 58 Figure 18 - Cellular Growth in culture media containing vanillin inhibitor (1-10 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)……………. 61 Figure 19 - Xylose assimilation in culture media containing vanillin inhibitor (1-10 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)………. 62 Figure 20 - pH variation in culture media containing vanillin inhibitor (1-10 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)………………… 63 Figure 21 - Cellular Growth in culture media containing acetic acid inhibitor (20-80 mM) for 96 h at 28 °C and 150 rpm, ofunaensis (A) and T. multisporum (B)…………. 69 Figure 22 - Xylose assimilation in culture media containing acetic acid inhibitor (20- 80 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)…… 70 Figure 23 - pH variation in culture media containing acetic acid inhibitor (20-80 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)……………. 71 Figure 24 - Cellular Growth in culture media containing formic acid inhibitor (10-40 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)………. 74 Figure 25 - Xylose assimilation in culture media containing formic acid inhibitor (10- 40 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)…… 75 Figure 26 - pH variation in culture media containing formic acid inhibitor (10-40 mM) for 96 h at 28 °C and 150 rpm, P. ofunaensis (A) and T. multisporum (B)……………. 76 Figure 27 - Anaerobic cultivation of the yeasts P. ofunaensis and T. multisporum in saline culture media for 96 h at 28 °C and 150 rpm ……………………….…………… 80 Figure 28 - Xylose consumption (symbols) and ethanol production (bars) during anaerobic cultivation of the yeasts P. ofunaensis (circle) and T. multisporum (triangle) in saline culture media for 96 h at 28 °C and 150 rpm………………….………………. 82 Figure 29 - pH variation of the saline media during anaerobic cultivations of the yeasts P. ofunaensis and T. multisporum for 96 h at 28 °C and 150 rpm ………………. 82 TABLES LIST Table 1 - Partial chemical composition of some Brazilian common agro-industrial wastes……………………………………………………………………………………. 24 Table 2 - Analyzed inhibitor compounds and tested concentration range……………... 39 Table 3 - Optical density and pH of YEPX culture media containing furfural after 96 hours of incubation and sugar consumed during fermentation by P. ofunaensis and T. multisporum……………………………………………………………………………... 41 Table 4 - Optical density and pH of YEPX culture media containing HMF after 96 hours of incubation and sugar consumed during fermentation by P. ofunaensis and T. multisporum……………………………………………………………………………... 46 Table 5 - Optical density and pH of YEPX culture media containing ferulic acid after 96 hours of incubation and sugar consumed during fermentation by P. ofunaensis and T. multisporum……………………………………………………………………………... 54 Table 6 - Optical density and pH of YEPX culture media containing vanillin after 96 hours of incubation and sugar consumed during fermentation by P. ofunaensis and T. multisporum……………………………………………………………………………... 59 Table 7 - Optical density and pH of YEPX culture media containing acetic acid after 96 hours of incubation and sugar consumed during fermentation by P. ofunaensis and T. multisporum……………………………………………………………………………... 67 Table 8 - Optical density and pH of YEPX culture media containing formic acid after 96 hours of incubation and sugar consumed during fermentation by P. ofunaensis and T. multisporum……………………………………………………………………………... 72 LIST OF ABBREVIATIONS, ACRONYMS AND SYMBOLS 2,3-BD 2,3-Butanediol ATP Adenosine triphosphate DNS 3, 5 - Dinitrosalicylic Acid h Hours HMF Hydroxymethylfurfural M Molar mL Milliliter mm Millimeter mM Millimolar (10-3 mol L-1) nm Nanometer O.D. Optical density pH Potential of hydrogen rpm Rotations per minute STEX Steam explosion xg Centrifugal force YEP Yest Extract & Peptone YEPD Yest Extract, Peptone & Dextrose YEPX Yest Extract, Peptone & Xylose CONTENTS 1 Introduction ......................................................................................................................... 14 2 GENERAL BIBLIOGRAPHIC REVIEW ........................................................................ 15 2.1 Global population growth and the increasing demand for food and energy .......... 15 2.2 Energy: importance, types and sources ...................................................................... 16 2.3 Different renewable energy types; advantages and disadvantages. ......................... 17 2.3.1 Solar Energy ............................................................................................................. 17 2.3.2 Wind Energy ............................................................................................................ 18 2.3.3 Hydropower Energy ................................................................................................. 18 2.3.4 Geothermal Energy .................................................................................................. 19 2.3.5 Biomass Energy........................................................................................................ 19 2.4 Biofuel ............................................................................................................................ 20 2.5 Lignocellulosic biomass ................................................................................................ 20 2.6 Agro-industrial wastes .................................................................................................. 23 2.7 Inhibitors derived from hydrolysis of lignocellulosic biomass ................................. 24 2.7.1 Furan aldehydes inhibitors ....................................................................................... 25 2.7.2 Aliphatic acids inhibitors ......................................................................................... 25 2.7.3 Phenolic compounds inhibitors ................................................................................ 26 2.8 Xylose ............................................................................................................................. 27 2.8.1 Xylose assimilation and metabolism in yeasts ..................................................... 28 2.8.2 Xylose transport in yeasts ...................................................................................... 30 2.9 Biotechnological applications ....................................................................................... 33 3 OBJECTIVES ...................................................................................................................... 35 3.1 General Objective ......................................................................................................... 35 3.2 Specific Objectives ........................................................................................................ 35 4 MATERIALS AND METHODS ........................................................................................ 35 4.1 Microorganisms ............................................................................................................ 35 4.2 Strains cultivation and quantification of growth in xylose ....................................... 37 4.2.1 The pre-inoculum preparation .................................................................................. 37 4.2.2 Cultivation of microorganisms in media containing xylose and glucose................. 37 4.2.3 Growth quantification, pH measurement, and sugar assimilation of culture media containing xylose............................................................................................................... 38 4.2.4 Standardization of the inoculum .............................................................................. 38 4.2.5 Yeast tolerance test to different inhibitor compounds ............................................. 39 4.2.6 Yeast tolerance test to combined inhibitor compounds ........................................... 39 4.3 Ethanol production assay ............................................................................................. 40 4.4Ethanol identification and quantification .................................................................... 40 5 RESULTS & DISCUSSIONS ............................................................................................. 41 5.1 Furan aldehydes inhibitors .......................................................................................... 41 5.1.1 Furfural effect on cellular growth and xylose consumption .................................... 41 5.1.2 HMF effect on cellular growth and xylose consumption ......................................... 46 5.1.3 Discussion ................................................................................................................ 51 5.2 Phenolic compounds inhibitors .................................................................................... 54 5.2.1 Ferulic acid effect on cellular growth and xylose consumption............................... 54 5.2.2 Vanillin effect on cellular growth and xylose consumption .................................... 59 5.2.3 Discussion ................................................................................................................ 64 5.3 Aliphatic acids inhibitors ............................................................................................. 67 5.3.1 Acetic acid effect on cellular growth and xylose consumption ............................... 67 5.3.2 Formic acid effect on cellular growth and xylose consumption .............................. 72 5.3.3 Discussion ................................................................................................................ 77 5.4 The combined impact of the tested inhibitors on the two yeasts .............................. 79 5.5 Ethanol production, identification and quantification .............................................. 79 6 CONCLUSIONS .................................................................................................................. 83 REFERENCES ....................................................................................................................... 84 14 1 Introduction According to United Nations, the world population has exceeded 7 billion people. Overpopulation, along with the rapid development of civilized lifestyles, has contributed to the rise in energy demand, as global consumption has grown by around 50% in the last twenty years, and carbon-based fuels such as coal, oil, and gas have been the principal sources. This puts us in front of three crucial problems: high energy demand; significant depletion of non-renewable energy resources; and environmental concerns such as global warming, and pollution, which show the urgent need to search for alternative renewable, environmentally friendly, and cheaper sources of energy. One of these sources is lignocellulosic biomass. It is an abundant bio-renewable resource, is inexpensive, broadly available, and has been identified as a sustainable carbon source. The use of this biomass points to the opportunity to add value to agro-industrial residues and does not compete with the food supply at the same time. This energy has much lower gas emissions compared to fossil fuels and lower costs compared to other renewable energy types. This lignocellulosic biomass is composed of three main parts: cellulose, which is the dominant part of the lignocellulosic biomass, is a linear homopolymer that has D-glucose units; hemicellulose, representing 20 to 35% of the lignocellulosic biomass, is a highly diverse heteropolysaccharide, composed of several types of hexoses and pentose, and xylan is the most predominant type of hemicellulose, which has a main chain formed by xylose monomers; and lignin is an amorphous three-dimensional polymer that gives rigidity and resistance to the cell wall. Xylose is an important sugar derived from the hydrolysis of plant biomass and can constitute up to 25% of the total dry weight of some forest and agricultural residues, which denotes the importance of its use in fermentation processes. However, the yeast S. cerevisiae is unable to efficiently co-assimilate glucose and xylose. Thus, prospecting for non- Saccharomyces yeasts capable of assimilating xylose may provide a better knowledge of the xylose transport mechanisms in the cell and offer support for the metabolic engineering of S. cerevisiae to assimilate it. In the laboratory of biochemistry and applied microbiology - Unesp, São José do Rio Preto, SP, Brazil, a research group under the supervision of Profa. Dra. Eleni Gomes, keeps prospecting for microorganisms capable of fermenting xylose and producing ethanol and other valuable products. This work is a continuation of what a previous master's student (VAZ, 2020) had done to study the two yeasts, P. ofunaensis and T. multisporum. 83 6 CONCLUSIONS The yeast P. ofunaensis exhibited more tolerant abilities toward potentially harmful chemicals than T. multisporum. Acetic acid had the most potent inhibitory effect, while HMF had the weakest inhibitory effect on yeast growth. The yeast T. multisporum had a poorer tolerance for the various inhibitors investigated, and its performance in terms of cellular growth and sugar consumption is considerably lower than that of P. ofunaensis. Despite low ethanol production and low tolerance to the inhibitors tested, the yeast P. ofunaensis was able to consume all of the xylose in the YPX medium at pH 4.5, implying the possibility of using xylose/H+ symporters, which have been described as transporters with higher affinity for D-xylose. These findings support the continuation of research on xylose transporters. 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Zaiter ficha Dissertation-M. A. Zaiter 1 Introduction 2 GENERAL BIBLIOGRAPHIC REVIEW 2.1 Global population growth and the increasing demand for food and energy 2.2 Energy: importance, types and sources 2.3 Different renewable energy types; advantages and disadvantages. 2.3.1 Solar Energy 2.3.2 Wind Energy 2.3.3 Hydropower Energy 2.3.4 Geothermal Energy 2.3.5 Biomass Energy 2.4 Biofuel 2.5 Lignocellulosic biomass 2.6 Agro-industrial wastes 2.7 Inhibitors derived from hydrolysis of lignocellulosic biomass 2.7.1 Furan aldehydes inhibitors 2.7.2 Aliphatic acids inhibitors 2.7.3 Phenolic compounds inhibitors 2.8 Xylose 2.8.1 Xylose assimilation and metabolism in yeasts 2.8.2 Xylose transport in yeasts 2.9 Biotechnological applications 3 OBJECTIVES 3.1 General Objective 3.2 Specific Objectives 4 MATERIALS AND METHODS 4.1 Microorganisms 4.2 Strains cultivation and quantification of growth in xylose 4.2.1 The pre-inoculum preparation 4.2.2 Cultivation of microorganisms in media containing xylose and glucose 4.2.3 Growth quantification, pH measurement, and sugar assimilation of culture media containing xylose 4.2.4 Standardization of the inoculum 4.2.5 Yeast tolerance test to different inhibitor compounds 4.2.6 Yeast tolerance test to combined inhibitor compounds 4.3 Ethanol production assay 4.4Ethanol identification and quantification 5 RESULTS & DISCUSSIONS 5.1 Furan aldehydes inhibitors 5.1.1 Furfural effect on cellular growth and xylose consumption 5.1.2 HMF effect on cellular growth and xylose consumption 5.1.3 Discussion 5.2 Phenolic compounds inhibitors 5.2.1 Ferulic acid effect on cellular growth and xylose consumption 5.2.2 Vanillin effect on cellular growth and xylose consumption 5.2.3 Discussion 5.3 Aliphatic acids inhibitors 5.3.1 Acetic acid effect on cellular growth and xylose consumption 5.3.2 Formic acid effect on cellular growth and xylose consumption 5.3.3 Discussion 5.4 The combined impact of the tested inhibitors on the two yeasts 5.5 Ethanol production, identification and quantification 6 CONCLUSIONS 7 REFERENCES << /ASCII85EncodePages false /AllowTransparency false /AutoPositionEPSFiles true /AutoRotatePages /All /Binding /Left /CalGrayProfile (Gray Gamma 2.2) /CalRGBProfile (sRGB IEC61966-2.1) /CalCMYKProfile (U.S. Web Coated \050SWOP\051 v2) /sRGBProfile (sRGB IEC61966-2.1) /CannotEmbedFontPolicy /Warning /CompatibilityLevel 1.7 /CompressObjects /Off /CompressPages true /ConvertImagesToIndexed true /PassThroughJPEGImages true /CreateJobTicket false /DefaultRenderingIntent /Default /DetectBlends false /DetectCurves 0.0000 /ColorConversionStrategy /LeaveColorUnchanged /DoThumbnails false /EmbedAllFonts true /EmbedOpenType false /ParseICCProfilesInComments true /EmbedJobOptions true /DSCReportingLevel 0 /EmitDSCWarnings false /EndPage -1 /ImageMemory 1048576 /LockDistillerParams true /MaxSubsetPct 100 /Optimize true /OPM 1 /ParseDSCComments true /ParseDSCCommentsForDocInfo true /PreserveCopyPage true /PreserveDICMYKValues true /PreserveEPSInfo true 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