Thorough investigation of the glycine conjugation pathway has been neglected over the last 30 years. Environmental factors, nutrition, and the chronic use of medications are increasing the exposure of humans to benzoate and drugs that are metabolized to acyl-CoA intermediates. Glycine conjugation of mitochondrial acyl-CoAs, catalysed by glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13), is an important metabolic pathway responsible for maintaining adequate levels of free coenzyme A (CoASH). However, because of the small number of pharmaceutical drugs that are conjugated to glycine, the pathway has not yet been characterised in detail. Therefore, one of the objectives of this thesis was to develop a better understanding of glycine conjugation and its role in metabolism. In humans and animals a number of endogenous and xenobiotic organic acids are conjugated to glycine. Glycine conjugation has generally been assumed to be a detoxification mechanism, increasing the water solubility of organic acids in order to facilitate urinary excretion. However, recently it was proposed that the role of the amino acid conjugations, including glycine conjugation, is to regulate systemic levels of amino acids that are also utilised as neurotransmitters in the central nervous systems of animals. The glycine deportation hypothesis was based on the observation that, compared to glucuronidation, glycine conjugation does not significantly increase the water solubility of aromatic acids. A thorough review of the literature for this thesis showed that the major role of glycine conjugation, however, is to dispose of the end products of phenylpropionate metabolism. The review also introduced the new perspective that mitochondrial glycine conjugation prevents the accumulation of benzoate in the mitochondrial matrix by forming hippuric acid a less lipophilic conjugate that can be more readily transported out of the mitochondria. Although organic anion transporters can export benzoate from the matrix, this process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not the transport out of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine conjugates to re-enter the matrix that is decreased. Lastly, glycine conjugation of benzoate also exacerbates the dietary deficiency of glycine in humans. Because the resulting shortage of glycine can negatively influence brain neurochemistry and the synthesis of collagen, nucleic acids, porphyrins, and other important metabolites, the risks of using benzoate as a preservative should not be underestimated. To date, no defect of the glycine conjugation pathway has been reported and this, together with the fact that GLYAT plays an important role in hepatic metabolism, suggests that this pathway is essential for survival. GLYAT activity affects mitochondrial ATP production, glycine availability, CoASH availability and the toxicity of various organic acids. Therefore, variation in the glycine conjugation pathway could influence liver cancer, musculoskeletal development and mitochondrial energy metabolism. Significant interindividual variation exists in glycine conjugation capacity. The molecular basis for this variability is not known. The main aim of this thesis was to investigate and characterise the genetic variation in the coding region of the GLYAT gene. This was accomplished by firstly, investigating the influence of non-synonymous single nucleotide polymorphisms (SNPs) on the enzyme activity of a recombinant human GLYAT and secondly, by analysing the level of genetic variation in the coding region of the GLYAT gene using existing worldwide population data. To investigate the influence of non-synonymous SNPs in the GLYAT gene on the enzyme activity, a recombinant human GLYAT was prepared, and characterised. Site-directed mutagenesis was used to generate six variants of the enzyme (K16N; S17T; R131H; N156S; F168L; R199C). The variants were expressed, purified, and enzymatically characterised. The enzyme activities of the K16N, S17T and R131H variants were similar to that of the wild-type, whereas the N156S variant was more active, the F168L variant less active, and the R199C variant was inactive. The results showed that SNP variations in the human GLYAT gene can influence the kinetic properties of the enzyme. The genetic variation data of the human GLYAT open reading frame (ORF) available on public databases was investigated by formulating the hypothesis that due to the essential nature of the glycine conjugation pathway, the genetic variation in the ORF of the GLYAT gene should be low and that deleterious alleles will be found at low frequencies. Data from the i) 1000 Genome Project, ii) the HapMap Project, and iii) the Khoi-San/Bantu Sequencing Project was downloaded from available databases. Sequence data of the coding region of a small cohort of South African Afrikaner Caucasian individuals was also generated and included in the analyses. In the GLYAT ORF of the 1537 individuals analysed, only two haplotypes (S156 and T17S156) out of 14 haplotypes were identified in all populations as having the highest haplotype frequencies (70% and 20% respectively). The S156C199 and S156H131 haplotypes, which have a deleterious effect on the enzyme activity of a recombinant human GLYAT, were detected at very low frequencies. The results of this study indicated that the GLYAT ORF is remarkably conserved, which supports the hypothesis that the glycine conjugation pathway is an essential detoxification pathway. The findings presented in this thesis highlight the importance that future investigations should determine the in vivo capacity of the glycine conjugation pathway for the detoxification of benzoate and other xenobiotics. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2015

Thorough investigation of the glycine conjugation pathway has been neglected over the last 30 years. Environmental factors, nutrition, and the chronic use of medications are increasing the exposure of humans to benzoate and drugs that are metabolized to acyl-CoA intermediates. Glycine conjugation of mitochondrial acyl-CoAs, catalysed by glycine N-acyltransferase (GLYAT, E.C. 2.3.1.13), is an important metabolic pathway responsible for maintaining adequate levels of free coenzyme A (CoASH). However, because of the small number of pharmaceutical drugs that are conjugated to glycine, the pathway has not yet been characterised in detail. Therefore, one of the objectives of this thesis was to develop a better understanding of glycine conjugation and its role in metabolism. In humans and animals a number of endogenous and xenobiotic organic acids are conjugated to glycine. Glycine conjugation has generally been assumed to be a detoxification mechanism, increasing the water solubility of organic acids in order to facilitate urinary excretion. However, recently it was proposed that the role of the amino acid conjugations, including glycine conjugation, is to regulate systemic levels of amino acids that are also utilised as neurotransmitters in the central nervous systems of animals. The glycine deportation hypothesis was based on the observation that, compared to glucuronidation, glycine conjugation does not significantly increase the water solubility of aromatic acids. A thorough review of the literature for this thesis showed that the major role of glycine conjugation, however, is to dispose of the end products of phenylpropionate metabolism. The review also introduced the new perspective that mitochondrial glycine conjugation prevents the accumulation of benzoate in the mitochondrial matrix by forming hippuric acid a less lipophilic conjugate that can be more readily transported out of the mitochondria. Although organic anion transporters can export benzoate from the matrix, this process would likely be futile because benzoic acid can simply diffuse back into the matrix. Hippurate, however, is significantly less lipophilic and therefore less capable of diffusing into the matrix. It is therefore not the transport out of the mitochondrial matrix that is facilitated by glycine conjugation, but rather the ability of the glycine conjugates to re-enter the matrix that is decreased. Lastly, glycine conjugation of benzoate also exacerbates the dietary deficiency of glycine in humans. Because the resulting shortage of glycine can negatively influence brain neurochemistry and the synthesis of collagen, nucleic acids, porphyrins, and other important metabolites, the risks of using benzoate as a preservative should not be underestimated. To date, no defect of the glycine conjugation pathway has been reported and this, together with the fact that GLYAT plays an important role in hepatic metabolism, suggests that this pathway is essential for survival. GLYAT activity affects mitochondrial ATP production, glycine availability, CoASH availability and the toxicity of various organic acids. Therefore, variation in the glycine conjugation pathway could influence liver cancer, musculoskeletal development and mitochondrial energy metabolism. Significant interindividual variation exists in glycine conjugation capacity. The molecular basis for this variability is not known. The main aim of this thesis was to investigate and characterise the genetic variation in the coding region of the GLYAT gene. This was accomplished by firstly, investigating the influence of non-synonymous single nucleotide polymorphisms (SNPs) on the enzyme activity of a recombinant human GLYAT and secondly, by analysing the level of genetic variation in the coding region of the GLYAT gene using existing worldwide population data. To investigate the influence of non-synonymous SNPs in the GLYAT gene on the enzyme activity, a recombinant human GLYAT was prepared, and characterised. Site-directed mutagenesis was used to generate six variants of the enzyme (K16N; S17T; R131H; N156S; F168L; R199C). The variants were expressed, purified, and enzymatically characterised. The enzyme activities of the K16N, S17T and R131H variants were similar to that of the wild-type, whereas the N156S variant was more active, the F168L variant less active, and the R199C variant was inactive. The results showed that SNP variations in the human GLYAT gene can influence the kinetic properties of the enzyme. The genetic variation data of the human GLYAT open reading frame (ORF) available on public databases was investigated by formulating the hypothesis that due to the essential nature of the glycine conjugation pathway, the genetic variation in the ORF of the GLYAT gene should be low and that deleterious alleles will be found at low frequencies. Data from the i) 1000 Genome Project, ii) the HapMap Project, and iii) the Khoi-San/Bantu Sequencing Project was downloaded from available databases. Sequence data of the coding region of a small cohort of South African Afrikaner Caucasian individuals was also generated and included in the analyses. In the GLYAT ORF of the 1537 individuals analysed, only two haplotypes (S156 and T17S156) out of 14 haplotypes were identified in all populations as having the highest haplotype frequencies (70% and 20% respectively). The S156C199 and S156H131 haplotypes, which have a deleterious effect on the enzyme activity of a recombinant human GLYAT, were detected at very low frequencies. The results of this study indicated that the GLYAT ORF is remarkably conserved, which supports the hypothesis that the glycine conjugation pathway is an essential detoxification pathway. The findings presented in this thesis highlight the importance that future investigations should determine the in vivo capacity of the glycine conjugation pathway for the detoxification of benzoate and other xenobiotics. / PhD (Biochemistry), North-West University, Potchefstroom Campus, 2015

Humans are continuously challenged by harmful endogenous and xenobiotic substances. Detoxification is the ability to neutralise and remove these substances from the body. Glycine N-acyltransferase, EC 2.3.1.13 (GLYAT) is a key enzyme in detoxification. GLYAT catalyses an amino acid (glycine) conjugation reaction in phase II of detoxification. It is expected that, similar to what has been observed in the Cytochrome P450 enzymes, variations within the GLYAT gene may lead to altered enzyme activity that may affect the efficacy of detoxification. The aim of this study was to identify genetic variations within the GLYAT gene of a cohort of individuals whose GLYAT activity has been biochemically characterized. Biochemical profiles of phase I and II detoxification of a number of individuals was screened to select those with possible aberrant GLYAT activity. Eighteen selected individuals agreed to participate in the study. The 23.21 kb GLYAT gene of the participants was amplified in four fragments and sent for pyrosequencing (Roche GS FLX titanium) at Inqaba Biotec. The results were analysed with the Lasergene software package from DNAStar (Madison, Wisconsin, USA). A total of 94 variations were identified from the Next Generation Sequencing data. Of these three found in the exons were known variations and four variations located in the exons were novel. A total of 62 known and 25 novel variations were identified in the introns of the GLYAT gene. Sanger sequencing verified 70.29% (68 in total) of the variation, which included 12 novel variations, of which one is located in exon six. Real-time quantitative PCR (qPCR) experiments were conducted and the data analysed using CopyCaller software to identify copy number variations within the cohort. It was found that participant 17 may have multiple copies of parts of the 3-terminal end of the gene (exons five and six), which might have an effect on GLYAT activity. Variations could possibly affect GLYAT activity, but the data was inconclusive and must be confirmed. Some of the variations could possibly affect GLYAT activity, but no correlation could be made between the variations identified during this study and the cohort’s detoxification ability. Further studies needs to be conducted to establish the effect of the variations in combination with one another on GLYAT activity. If some of these variations affect GLYAT activity such data might shed some light on variations observed between the glycine conjugation ability of individuals. Such information could eventually be of value in treatment of inborn errors of metabolism. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

Unstable oil markets with rising environmental concerns have revived widespread interest in production of fuel ethanol from renewable materials. Cellulosic materials are abundant and prominent feedstocks for cheap ethanol production. However, due to recalcitrant structure of these materials, pretreatment is a prerequisite. Depending on the biomass, pretreatment and hydrolysis conditions, a number of degradation products and/or toxic components may be released that show strong inhibitory effects on the fermenting microorganisms. This thesis deals with application of encapsulation technology to ferment the highly toxic hydrolyzates without further pretreatment. Free cells could not tolerate presence of 5 g/l furfural in defined medium, and inhibitors in wood and peel hydrolyzates in batch mode of operation and fermentation failed. Continuous cultivation of wood hydrolyzate was only successful at 0.1 h−1 and the majority of cells lost their viability after 5 retention times. Encapsulated cell system could successfully ferment the synthetic medium containing 5 g/l furfural during sequential batch cultivations with ethanol yield of 0.41-0.42 g/g. Cultivation of undetoxified hydrolyzates was also carried out, where glucose and mannose were converted within 10 h without significant lag phase. However, a gradual decrease in cell activity was observed in sequential batches. Continuous cultivation was more successful, and wood hydrolyzate was fermented to ethanol by encapsulated S. cerevisiae at dilution rates up to 0.5 h−1. More than 75% of the encapsulated cells were viable in the worst conditions. Ethanol was produced with yield 0.44 g/g and specific productivity 0.14–0.17 g/g•h at all dilution rates. Contrary to wood hydrolyzate, where there is no preference for permeation of sugars or inhibitors through the capsules’ membrane, encapsulation technology was applied to eliminate inhibition of limonene in fermentation of orange wastes to ethanol. The capsules’ membrane, of hydrophilic nature, is practically impermeable to hydrophobic compounds such as limonene while allowing penetration of nutrients and products. While presence of 0.1% v/v limonene in the medium results in strong inhibition or even failure of cultivation with free cells, using this technique allowed fermentation of a medium containing 1.5% v/v limonene. The impact of encapsulation on the anaerobic growth pattern, morphological and physiological changes of S. cerevisiae over long-term application was investigated. The growth rate, total RNA and protein content of the encapsulated cells decreased gradually over repeated batch cultivations, while stored carbohydrates content increased. Within 20 batch cultivations, total RNA and protein content of encapsulated cells decreased by 39% and 24%, whereas glycogen and trehalose content increased by factors of 4.5 and 4, respectively. / <p>Akademisk avhandling som för avläggande av teknologie doktorsexamen vid Chalmers tekniska högskola försvaras vid offentlig disputation den 18 april 2008.</p>

The main goal of this essay is to show that lifestyle has an influence on alcohol addiction and its process, but most importantly on maintaining abstinence after a treatment. From various research, which where made, is clear, that most of relapses are directly related to lifestyle of addicts (or at least to its parts such as health, social situation, economic status, hobbies etc.). That is why it is important to work on a lifestyle change during a treatment so that the addicts are in balance during the abstinence and are not exposed to risky situations which they do not know how to react to and deal with them. There is Relapse Prevention to learn these pocedures and it should be applied also during the treatment and after. Part of my essay is qualitative research, which should reflect previously mentioned phenomenons among ten respondents of detoxification centre, who experienced relapses before. I tried to prove that lifestyle and its disorders influence and increase the potential of relapse after completed treatment and it is necessary to change it during abstinence.

L'ozone troposphérique est un polluant atmosphérique majeur qui agit comme une phytotoxine. Il pénètre dans les feuilles par les stomates avant d'être dissout dans l'apoplaste en générant des radicaux libres oxygénés (ROS) provoquant ainsi un stress oxydatif. Deux barrières existent pour restreindre les effets de l'ozone : (i) les stomates qui peuvent limiter les flux entrants par contrôle de la conductance stomatique et (ii) le système de détoxication des ROS issus de la dégradation de l'ozone. Nous avons étudié les effets de l'ozone (120 ppb) sur ces deux moyens de défense chez trois génotypes de peuplier euraméricain (Populus deltoides x Populus nigra) placés en conditions contrôlées dans des chambres phytotroniques. Un effet direct de l'ozone sur la photosynthèse et sur les mouvements stomatiques en réponse à des variations de facteurs environnementaux (ralentissement des phénomènes d'ouverture et de fermeture) a été mis en évidence. Les modèles de calcul de la conductance stomatique, sur lesquels se basent les indicateurs de seuil de risque à l'ozone pour la végétation, doivent donc les prendre en compte. De plus, ces travaux ont mis en évidence le rôle prépondérant des concentrations constitutives en antioxidants dans la tolérance à l'ozone ainsi que la complexité de ces mécanismes de détoxication. La notion de flux effectif d'ozone doit prendre en considération ces deux aspects afin de caractériser au mieux les différences de sensibilité à l'ozone intra et inter spécifique / Tropospheric ozone is a major air pollutant that acts as a phytotoxin. It enters the leaf through the stomata before being dissolved in the apoplast by generating reactive oxygen species (ROS) causing oxidative stress. Two defenses exist to restrict the effects of ozone: (i) the stomata which can limit ozone uptake by regulating stomatal conductance and (ii) the detoxification processes of ROS generated by ozone.We studied the effects of ozone (120 ppb) on these two mechanisms of defense in three euramerican poplar genotypes (Populus deltoides x Populus nigra) under controlled conditions in phytotronic chambers. A direct effect of ozone on photosynthesis and stomatal movements in response to changes in environmental factors (by slowing the stomatal opening and closure) has been highlighted. Models of stomatal conductance, on which indicators of critical level of ozone for vegetation are based, must take them into account. In addition, these studies have highlighted the role of constitutive concentrations of antioxidants in tolerance to ozone as well as the complexity of these detoxification mechanisms. The notion of effective ozone flux must consider these two aspects to better characterize the intra-and inter-specific differences in sensitivity to ozone

O cluster Drosophila buzzatii (grupo D. repleta) é constituído por sete espécies crípticas, com desenvolvimento larval ocorrendo exclusivamente em tecidos de cactos em decomposição. Os cactos são constituídos por diversos compostos químicos, que incluem substâncias tóxicas (alcaloides, esteroides e fenóis) para insetos. Nas espécies cactofílicas Drosophila mojavensis e D. buzzatii, o gene GSTD1, da família Glutationa S- transferases, é superexpresso em larvas submetidas a substratos tóxicos. GSTD1 é indicado com função de desintoxicação, sendo importante alvo de seleção e necessário no processo de adaptação em espécies de Drosophila. No presente trabalho, foram isoladas regiões codificadoras completas do gene GSTD1 das sete espécies do cluster D. buzzatii com o objetivo de entender a evolução nas sequências de DNA e nos fenótipos (proteínas), com análise da estrutura e função da proteína GSTD1, no processo de desintoxicação, entre as espécies do cluster. Foram sequenciados 630 pb do gene GSTD1 para cada espécie e a estrutura primária da proteína, composta por 209 resíduos de aminoácidos, foi inferida \"In silico\". Nas inferências filogenéticas Bayesiana com o gene GSTD1, é indicado monofilietismo do cluster, com formação do primeiro aglomerado para D. buzzatii e D. koepferae, o segundo composto por D. antonietae e D. serido e, por último, um agrupamento entre D. gouveai, D. borborema e D. seriema. Sinais de seleção positiva nas sequências de DNA resultam em alterações nos resíduos de aminoácidos nas posições 39, 133, 136 e 209, com mudanças na estrutura química da proteína GSTD1, entre as espécies do cluster. Foram obtidos sete modelos da proteína GSTD1, realizada com modelagem por homologia, uma para cada espécie do cluster D. buzzatii. No docking molecular, entre as proteínas GSTD1 com o ligante mescalina, foi observado que as proteínas de D. seriema, D. gouveai e D. koepferae apresentaram melhores interações químicas com o ligante e menor gasto energético. O ligante mescalina foi posicionado entre duas regiões GSH (glutationa redutase) em D. seriema, D. gouveai e D. koepferae. A evolução do gene GSTD1 entre as espécies do cluster D. buzzatii envolve sinais de seleção positiva em sequências de DNA com mudanças químicas em resíduos de aminoácidos no sítios-G e na estrutura interna da proteína GSTD1, bem como a capacidade do sítio de ligação GSH reconhecer o composto alcaloide. / The cluster Drosophila buzzatii (group D. repleta) is constituted by seven cryptic species, with larval development occurring exclusively in tissues of decomposing cacti. Cacti are composed of several chemical compounds, which include toxic substances (alkaloids, steroids and phenols) for insects. In the cactophilic species Drosophila mojavensis and D. buzzatii, the GSTD1 gene, from the Glutathione S-transferases family, is overexpressed in larvae submitted to toxic substrates. GSTD1 is indicated with detoxification function, being important target of selection and necessary in the adaptation process in Drosophila species. In the present work, complete coding regions of the GSTD1 gene were isolated from the seven species of the D. buzzatii cluster with the objective of understanding the evolution in DNA sequences and phenotypes (proteins), with analysis of the structure and function of the GSTD1 protein in the process detoxification, among the species of the cluster. 630 bp of the GSTD1 gene was sequenced for each species and the primary structure of the protein, composed of 209 amino acids, was inferred \"In silico\". In the Bayesian phylogenetic inferences with the GSTD1 gene, monophyly of the cluster is indicated, with the formation of the first grouping between D. buzzatii and D. koepferae, the second composed by D. antonietae and D. serido and finally, a grouping between D. gouveai, D. borborema and D. seriema. Signs of positive selection on DNA sequences result in changes in amino acid residues at positions 39, 133, 136 and 209, with changes in the chemical structure of the GSTD1 protein among the species in the cluster. We obtained seven models of the GSTD1 protein, performed with homology modeling, one for each species of the D. buzzatii cluster. In the molecular docking between the GSTD1 proteins with the mescaline ligand, it was observed that the proteins of D. seriema, D. gouveai and D. koepferae presented better chemical interactions with the ligand and lower energy expenditure. Interestingly, the mescaline linker was positioned between two GSH (glutathione reductase) regions, in D. seriema, D. gouveai and D. koepferae. The evolution of the GSTD1 gene among D. buzzatii cluster species involves positive selection signals in DNA sequences with chemical changes in amino acid residues at the G-sites and in the internal structure of the GSTD1 protein, as well as the ability of the GSH binding site to recognize the alkaloid compound.

Durante a etapa de pre-tratamento acido, necessaria para o rompimento da matriz lignocelulosica e liberacao de acucares, diversos compostos toxicos sao formados e liberados no hidrolisado hemicelulosico. Estes compostos afetam negativamente o processo de fermentacao, sendo necessaria uma etapa de destoxificacao do hidrolisado hemicelulosico. Este trabalho teve como objetivo contribuir para o desenvolvimento de um metodo de destoxificacao biologica do hidrolisado hemicelulosico. Com este proposito foram selecionadas leveduras, em meio de cultura sintetico, capazes de utilizar furfural, 5- hidroximetilfurfural (HMF), acido acetico, acido ferulico e siringaldeido como fontes de carbono e energia. As leveduras Issatchenkia occidentalis M1 e Issatchenkia occidentalis Y1\'a foram as que demonstraram os melhores resultados de remocao destes compostos em meio de cultura sintetico e consequentemente foram as selecionadas para os experimentos que foram realizados em hidrolisado hemicelulosico de bagaco de cana-de-acucar concentrado. Em um segundo momento, foi verificado a eficiencia na destoxificacao biologica do hidrolisado hemicelulosico para futuras aplicacoes em processos fermentativos de producao de etanol. Para cada levedura selecionada foi realizado um planejamento fatorial completo 22, com tres repeticoes no ponto central. As variaveis, pH (4,0 ou 5,0) e agitacao do sistema (100 rpm ou 300 rpm), foram avaliados e como variavel resposta foi considerada a porcentagem da remocao de HMF, furfural, acido acetico e fenois do hidrolisado hemicelulosico apos 96 horas de tratamento biologico. A partir dos resultados obtidos constatou-se que para a estirpe de Issatchenkia occidentalis M1 o experimento 4 (pH 5,0 e 300 rpm) foi o que possibilitou uma remocao mais eficiente de cerca de 42,89% dos compostos toxicos totais, sendo 94,77% de HMF 99,19% de furfural, 29,17% de fenois e 100% de acido acetico. Para a estirpe de Issatchenkia occidentalis Y1\'a o experimento 3 (pH 4,0 e 300rpm) foi o que permitiu uma remocao mais significatica de cerca de 46,04% dos compostos toxicos totais, sendo 97,68% de HMF, 96,06% de furfural, 27,15% de fenois e 100% de acido acetico. Os resultados obtidos neste trabalho demonstram o potencial das leveduras Issatchenkia occidentalis M1 e Issatchenkia occidentalis Y1\'a no tratamento biologico do hidrolisado hemicelulosico para a remocao de compostos toxicos liberados pela etapa de pre-tratamento acido. / During the acid pretreatment step, required for the disruption of the lignocelluloses matrix and sugars release, several toxic compounds are produced and released in the hemicellulosic hydrolyzates. These compounds negatively affects the fermentation process, being necessary an additional step for detoxification of the hemicellulosic hydrolysates. The present work aimed to contribute for the development of a biological detoxification method of the hemicellulosic hydrolysates. For this purpose yeasts were selected, in synthetic culture medium, capable to metabolize furfural, 5- hydroxymethylfurfural (HMF), acetic acid, ferulic acid and syringaldehyde as carbon and energy sources. The yeasts Issatchenkia occidentalis M1 and Issatchenkia occidentalis Y1\'a demonstrated the best results to remove these compounds present in medium and they were selected to the experiments that had been carried through in concentrated sugarcane bagasse hemicellulosic hydrolyzates. In a second moment, the efficiency of the biological detoxification of the hemicellulosic hydrolyzates was verified for future applications in fermentative processes for ethanol production. For each selected yeast, a 22 full factorial design was carried out. The variables, pH (4.0 or 5.0) and agitation rate (100 rpm or 300 rpm), had been evaluated and as response variable was considered the percentage of HMF, furfural, acetic acid and phenols removed from hemicellulosic hydrolyzates after 96 hours of biological treatment. According with the results, was evidenced that Issatchenkia occidentalis M1 strain, in the experiment conditions 4 (pH 5.0 and 300 rpm), was more efficient removals (about 42.89% of total toxic composites), being 94.77% of HMF, 99.19% of furfural, 29.17% of phenols and 100% of acetic acid. For the Issatchenkia occidentalis Y1\'a strain, in the experiment conditions 3 (pH 4.0 and 300 rpm) was observed significant removal (about 46.04% of total toxic composites), being 97.68% of HMF, 96.06% of furfural, 27.15% of phenols and 100% of acetic acid. The results demonstrate the potential of the yeasts Issatchenkia occidentalis M1 and Issatchenkia occidentalis Y1\'a in the biological detoxification of the sugarcane bagasse hemicellulosic hydrolyzates for removing the toxic compounds released during the acid pretreatment step.

O presente trabalho teve como principal objetivo avaliar o potencial de linhagens termotolerantes da espécie Kluyveromyces marxianus para produção de xilitol a partir do hidrolisado hemicelulósico de palha de arroz. Foi também objetivo deste trabalho estabelecer condições de tratamento alcalino diluído (desacetilação) previamente ao processo de hidrólise ácida da palha de arroz com vistas à obtenção de um hidrolisado com menor toxicidade. Numa primeira etapa do trabalho, foram avaliadas 4 cepas de K.marxianus (Y-6860, Y-6373, Y- 265 e Y-8287) quanto a produção de xilitol em meio semidefinido variando a temperatura de 30 a 40 °C. Com base nos resultados obtidos, foi selecionada a linhagem Y-6373, que apresentou os maiores valores de conversão de xilose em xilitol (YP/S = 0,70 g/g) e produtividade volumétrica (QP = 0,60 g/L.h) na temperatura de 40 °C. Esta linhagem foi então utilizada nos ensaios fermentativos a partir dos hidrolisados obtidos. Para obtenção do hidrolisado hemicelulósico, a palha de arroz foi primeiramente submetida a um tratamento alcalino, em escala de frascos Erlenmeyer, onde se avaliou o efeito da temperatura (50 - 70 °C) e da carga de NaOH (20 - 80 mg/g de biomassa) sobre a remoção de grupos acetil. De acordo com o modelo obtido, a máxima remoção de acetil (97,2 ± 3,4%) pôde ser alcançada empregando 70 °C; 80 mg de NaOH /g de palha por 45 min. Nestas condições do processo, o modelo previu remoções de lignina e de cinzas de 41,3 ± 4,3 e 62,3 ± 5,3%, respectivamente, sem perda significativa das frações açucaradas. Em maior escala (reator de 50L), o tratamento alcalino mostrou resultados de remoção de acetil próximos aos previstos pelo modelo. Com a palha desacetilada, foram então realizados ensaios de hidrólise ácida variando a concentração de H2SO4 (0,5 - 1,5% m/v) e tempo de residência (30 - 90 min) visando estabelecer as condições que maximizem a eficiência de hidrólise da hemicelulose (EHH). Nas condições otimizadas, (H2SO4 1,0% m/v e 85 minutos) foi alcançada uma EHH de 75% em frascos e de 76% em reator. Os resultados referentes à fermentabilidade do hidrolisado empregando a K. marxinuas Y-6373 demonstraram que independentemente da suplementação nutricional, a conversão de xilose em xilitol foi elevada ~ 0,87 g/g e superior a obtida em meio semidefinido contendo apenas xilose como fonte de carbono, porém os valores de produtividade volumétrica foram cerca de 6,5 vezes inferiores ~ 0,13 g/L.h, devido a baixa eficiência de utilização da xilose (XC) ~ 20%, após 72 h de cultivo. A fermentabilidade dos hidrolisados foi melhorada após o procedimento de destoxificação e os resultados variaram com o método empregado. Os melhores resultados foram obtidos em hidrolisado tratado com CaO (YP/S= 0,74 g/g; QP = 0,32 g/L.h e XC =92%). Neste trabalho, foi também demonstrado o efeito repressivo da glicose sobre a assimilação e conversão da xilose em xilitol por K. marxianus, uma vez que em meio semidefinido simulando as concentrações de xilose e glicose presentes no hidrolisado, os valores de XC e YP/S foram reduzidos em torno de 40%. De uma forma geral, conclui-se que a linhagem de K. marxianus selecionada neste estudo, apresenta grande potencial para a produção de xilitol em elevadas temperaturas, porém são ainda necessários estudos para otimizar as condições de produção de xilitol a partir de hidrolisados hemicelulósicos. / In this work the potential of thermotolerant strains of Kluyveromyces marxianus to produce xylitol from rice straw hemicellulosic hydrolyzed was evaluated. In addition, were also established conditions of diluted alkaline treatment (deacetylation) previously to the process of rice straw acid hydrolysis, aiming to decrease the toxicity of the hydrolysate . Initially, four yeast strains of K.marxianus (Y-6860, Y-6373, Y-2265 e Y-8287) were evaluated regarding to the xylitol production in a semi-defined medium, varying the temperature from 30 to 40 ºC. From these results, the strain Y-6373 was selected, due to high values of xylose conversion into xylitol (YP/S = 0.70 g/g) and volumetric productivity (QP = 0.60 g/L.h) at 40ºC. This strain was thus used in the fermentative tests employing the hemicellulosic hydrolysate obtained. The hemicellulosic hydrolysate of rice straw was prepared in two steps; firstly the rice straw was submitted to the alkaline treatment, which was carried out in Erlenmeyer flasks scale. In these experiments, the effects of temperature from 50 to 70 °C and NaOH load from 20 to 80 mg/g biomass on the removal of acetyl groups were evaluated. According to the model obtained, the maximum acetyl removal (97.2 ±3.4%) could be achieved by employing 70 ºC; 80 mg of NaOH/g of straw during 45 minutes. Under these conditions, the model predicted lignin and ashes removals of 41.3±4.3 and 62.3±5.3%, respectively, without significant loss of sugars. On a large scale (reactor of 50L), the alkaline treatment showed outcomes of acetyl removal near the ones predicted by the model. After, the deacetylating straw was submitted to acid hydrolysis process, varying the H2SO4 concentration from 0.5 to 1.5% w/v, and the residence time from 30 to 90 minutes, aiming to stablish the conditions that maximize the efficiency of hemicellulose hydrolysis (EHH). Under optimized conditions, (H2SO4 1.0% w/v and 85 minutes) 75% EHH was achieved in Erlenmeyers flasks and 76% in reactor. As regarding the hydrolysate fermentability employing K. marxinuas Y-6373, the results showed that, regardless of the nutritional supplementation, the conversion of xylose into xylitol was high ~ 0.87 g/g and superior in relation to the one obtained at a semidefined medium containing only xylose as a carbon source. Nevertheless, the values ofvolumetric productivity were approximately 6.5 times inferior ~ 0.13 g/L.h, due to the lowefficiency of xylose consumption (XC) ~ 20%, after 72h of cultivation. The hydrolysate fermentability was improved after detoxification and the results varied with the method employed. The best results were obtained on hydrolysate treated with CaO (YP/S= 0.74 g/g; QP = 0.32 g/L.h and XC = 92%). The glucose effect was further examined in semi-defined medium. The results showed that xylose assimilation and xylitol production by K. marxianus was repressed by glucose, since in a semi-defined media simulating the concentrations of xylose/glucose found in the hydrolyzed, the values of XC e YP/S were reduced in approximately 40%. Based on the results, was concluded that the strain of K. marxianus selected on this study, presents a great potential to produce xylitol at elevated temperatures. However, studies are still necessary to optimize the conditions of this bioconversion from the hemicellulosic hydrolysate.

Este estudo teve como objetivo avaliar a interação entre substâncias húmicas e xenobióticos através de estudos ecotoxicológicos. O principal foco foi avaliar a resposta entre diferentes níveis tróficos em organismos aquáticos (fitoplanctônicos, zooplanctônicos, peixes e macroinvertebrados bentônicos) e terrestres (vegetais superiores, insetos e anelídeos) avaliando os efeitos diretos e indiretos das SH e de sua mistura com xenobióticos sobre os organismos. O reconhecido efeito das SH aumentarem ou reduzirem o efeito tóxico de algumas substâncias foi estudado. Os principais pontos para discussão são: a) SH podem reduzir ou estimular o crescimento algal (P. subcapitata); b) A presença de SH podem proteger os organismos contra efeitos tóxicos de metais, no entanto, a presença de Cd/Cu afeta negativamente o crescimento de C. xanthus. d) Em uma análise integrada de processos de remediação solo/água, a presença de SH afetou negativamente ou positivamente os efeitos tóxicos da atrazina em alguns organismos. A significância das SH como tecnologia é discutida. / This study focuses the interaction of humic substances and xenonbiotics, throw ecotixicological studies. The main point was quantify and qualify the ecotoxicological responses of several throphic levels of freshwater (algae, zooplankton, fishes and benthic organisms) and soil organisms (higth plants, insects and annelids) analyzing the direct and indirect effects of humic substances (HS) and their mixture on organisms. The recognized ability of HS on improve or reduce the toxic effect of same substances has been studied. The main points of discussion are: a) humic can be both reduce or stimulate the algal growth (P. subcapitata); b) The presence of HS can be protect aquatic organisms to negative effects of metals. However, the presence of mixture of Cd/Cu affect negatively the growth of C. xanthus; d) In a integrated analysis of remediation process in soil/water microcosm, the presence of HS displayed negative or positive effects on atrazine toxicity for some organisms. The value of humic technology was discussed.