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La biohydrogénation (BH) ruminale des acides gras polyinsaturés (AGPI) est à l'origine de la production d'intermédiaires trans retrouvés dans les productions de ruminants (essentiellement le lait). Il existe deux voies de BH produisant des acides gras (AG) trans qui auraient des propriétés différentes : les isomères t11 auraient des effets bénéfiques pour la santé des consommateurs et les isomères t10 seraient responsables d'une forte diminution du taux butyreux du lait, représentant une contrainte majeure pour les éleveurs. Dans des conditions physiologiques normales, la voie t11 est fortement majoritaire, par contre avec des rations à base d'ensilage de maïs, riches en concentrés et surtout si elles comprennent des suppléments lipidiques riches en AGPI, une déviation de la voie t11 à la voie t10 peut se produire avec une augmentation significative des isomères t10 au détriment des isomères t11. L'objectif de cette thèse est d'expliquer les modalités de cette déviation, afin de mieux la maîtriser en élevage. Nos travaux permettent de conclure que les facteurs alimentaires de maîtrise de la déviation de la voie t11 vers la voie t10, sont la teneur en amidon rapidement fermentescible et la teneur en c9,c12-C18:2. Lorsque la quantité de c9,c12-C18:2 présente dans le rumen est faible, même avec une ration riche en amidon et un pH bas dans le rumen, la déviation n'a pas lieu, la voie t11 suffisant à assurer l'hydrogénation des AGPI puisque dans ces conditions, la ∆9 isomérisation est elle aussi peu efficace à pH bas. En revanche, lorsqu'en plus de l'amidon, du c9,c12-C18:2 est ajouté dans la ration, la voie t11 devient insuffisante et c'est la voie t10 qui prend le relais. Le pyroséquençage 454 couplé à une régression multiple SPLS nous ont permis d'établir des corrélations entre les taxons identifiés et la proportion d'AG (t10 ou t11) dans le rumen. Il s'avère que les genres bactériens corrélés fortement et positivement aux AG t10 sont plus ou moins impliqués dans le métabolisme ruminal du lactate ainsi qu'au faible pH ruminal. Cependant, l'identification des taxons les plus corrélés aux AG t11 était moins précise, elle s'arrête à l'ordre des Clostridiales. Enfin, dans des conditions de déviation de la voie t11 à la voie t10, l'addition de vitamine E dans la ration des vaches n'a pas permis de restaurer un ratio déjà élevé. Ces résultats ont abouti à une meilleure compréhension de cette déviation et orientent vers une meilleure maîtrise en élevage. / Rumen biohydrogenation (BH) of polyunsaturated fatty acids (PUFA) is responsible of the production of trans intermediates found in ruminant products (mainly milk). There are two BH pathways leading to trans fatty acids (FA) with different biological properties: t11 isomers have beneficial effects for consumer's health and t10 isomers result in low milk fat content, representing a major constraint for farmers. In most conditions, t11 FA are the major trans FA, but in some conditions, especially with diets based on corn silage and including lipid supplements rich in PUFA, a shift from t11 to t10 pathway can occur with a significant increase of t10 isomers at the expense t11 isomers. The objective of this work was to explain modalities of this shift to better control it in animal production. Our results demonstrated that dietary factors responsible of the shift from t11 to t10 pathway are starch rapidly fermentable and c9, c12-C18:2 contents. When the amount of c9, c12-C18:2 present in the rumen is low, even though the diet is rich in starch and the pH is low in the rumen, the shift does not occur, t11 pathway being sufficient to ensure the hydrogenation of PUFA since in these conditions, the 9 isomerization is also poorly effective at low pH. However, when c9, c12-C18:2 is supplemented to the diet in addition to starch, t11 pathway becomes insufficient for FA BH, and t10 pathway becomes dominant. 454 pyrosequencing coupled to a multiple sPLS regression allowed us to establish correlations between some identified taxa and FA proportions (t10 or t11) in the rumen. It appears that bacterial genera that are strongly and positively correlated with t10 FA are more or less involved in the metabolism of ruminal lactate and also positively correlated with a low ruminal pH. However, identification of taxa correlated with t11 FA was less accurate, stopping at Clostridiales order. Finally, once the shift occurred, the subsequent addition of vitamin E was not able to counteract this process. These results lead to a better understanding of this shift to better control it in animal livestock.

Le veau nouveau-né possède un rumen peu développé et non fonctionnel. Au cours des premiers mois, les fonctions digestives s’établissent, avec l’implantation du microbiote composé majoritairement de bactéries, archées et protozoaires. Les objectifs de ce travail étaient doubles : i) caractériser et comprendre la séquence d’implantation taxonomique des microorganismes du rumen chez le veau par des techniques de biologie moléculaire et de dénombrement, ainsi que la séquence de mise en place des paramètres fermentaires (AGV et ammoniac) et des activités principales enzymatiques chez le veau en périodes pré- et post-sevrage, ii) étudier l’effet de l’addition de levures vivantes sur la mise en place de cet écosystème ruminal en périodes pré- et post-sevrage. D’une part, nos travaux ont permis de confirmer qu’à la naissance, le rumen chez le veau, est dépourvu de micro-organismes, d’AGV, d’activité xylanasique et amylasique, avec un pH proche de la neutralité et un Eh fortement positif. La colonisation du rumen se fait dès la naissance, pendant les 15 premiers jours de la vie de l’animal par un microbiote complexe prédominé par les bactéries (phyla Proteobacteria et Bacteroidetes) et comprenant aussi des archées (majoritairement Methanobrevibacter). En même temps, le Eh devient fortement négatif. Ces communautés entraînent la production de produits fermentaires grâce à leurs activités enzymatiques. Entre 15 jours et le sevrage, avec l’ingestion d’aliments solides, la composition du microbiote du rumen évolue pour se rapprocher de celle de ruminants adultes, sans atteindre pour autant la maturité en termes de densités et abondances relatives. A cette période, le phylum Bacteroidetes est majoritaire, avec le genre Prevotella. Après le sevrage de légers changements apparaissent sur certains paramètres fermentaires comme les AGV sans doute en raison d’une évolution du microbiote qui devient moins diversifié et plus adapté à la dégradation d’aliments solides. L’apparition, à partir de 90 jours, des protozoaires ciliés dans le rumen semble conditionnée par la présence d’animaux adultes à proximité. A 4 mois d’âge, l’écosystème ruminal tend à devenir proche de celui observé chez les animaux adultes en matière de paramètres fermentaires, activités enzymatiques et composition taxonomique du microbiote. D’autre part, nos travaux ont permis de conclure que, avant sevrage, une supplémentation en levures vivantes (Saccharomyces cerevisiae) diminue l’ingestion de concentrés, conduit à une apparition plus précoce de la communauté des protozoaires et une plus grande densité d’archées, mais a peu d'effets sur la densité et la diversité de la communauté bactérienne, à l'exception de variations d’abondances de quelques taxa mineurs. L’apport de levures entraîne une diminution de la protéolyse, une augmentation de la proportion d'acétate ruminal et une diminution de la proportion de propionate. Au cours de la période post-sevrage, les veaux supplémentés en levures consomment plus de foin et la densité en archées est plus importante alors qu’une réduction de la diversité et de la densité de la communauté bactérienne est observée, mais accompagnée d’une augmentation de l’abondance relative des bactéries dégradant l’amidon, les pectines, les protéines et majoritairement les parois cellulaires en fonction des substrats présents dans le rumen. Ces changements sont probablement à relier aux augmentations de l'activité xylanasique et de la proportion d'acétate. L’ensemble des résultats acquis dans ce travail de thèse a permis d’apporter un certain nombre de connaissances et une meilleure compréhension de la mise en place de l’écosystème ruminal chez le veau de race laitière en périodes pré- et post-sevrage, ainsi que quelques pistes pour orienter ou améliorer cette implantation pour une meilleure maîtrise de l’élevage des veaux d’élevage. / The newborn calf has a little and non-fonctional rumen. During the first months of life, digestive functions establish, in relationship with the colonization by a microbiota mainly composed of bacteria, archaea and protozoa. This study had two objectves: i) characterize and understand the sequence of establishment of ruminal microbiota in calves by molecular biology and counting techniques abd describe the appearance of fermentation parameters (VFA and ammonia) and enzyme activities during the pre- and post-weaning periods, ii) define the effect of yeast supplementation on the establishment of the ruminal ecosystem in pre-and post-weaning periods. On the one hand, our work confirmed that at birth, calf rumen is devoid of micro-organisms, AGV, xylanase and amylase activities, with a pH close to neutrality and a strongly positive Eh. From 2 to 15 days of age, the rumen is colonized by a complex microbiota dominated by bacteria (Proteobacteria and Bacteroidetes phyla) and also containing archaea (with mainly the Methanobrevibacter genus), and Eh becomes strongly negative. These communities result in the production of fermentation products due to their enzymatic activities. Between 15 days and weaning, with the ingestion of solid food, the composition of rumen microbiota changes to become closer to that of adult ruminants without reaching maturity in term of densities and relative abundances. At this time, the phylum Bacteroidetes is predominant with the Prevotella genus. After weaning, slight differences occurs on some fermentative parameters such as VFA, probably related to a change in the microbiota that becomes less diversified and more adapted to the degradation of solid food. From 90 days, the establishment of ciliated protozoa in the calf rumen seems conditioned by the proximity with adult animals. From 4 months of age, considering fermentation, enzymatic and taxonomic composition of the microbiota, the ruminal ecosystem tends to be similar to that observed in adult animals. On the other hand, our work showed that during the pre-weaning period, the supplementation with live yeast (Saccharomyces cerevisiae) results in a lower concentrate intake, an earlier establishment of ciliated protozoa and a higher archaeal community density, but poorly affects the density and diversity of the bacterial community, with the exception of changes in abundances of some minors taxa. Yeast supplementation reduces proteolysis, increases the proportion of acetate and decreases that of propionate. During the post-weaning period, yeast supplemented calves consumed more hay, had a higher archaeal density, but a lower diversity and density of the bacterial community with an increased relative abundance of fiber, protein, starch, pectine degrading bacteria compared to control according to the substrates present in the rumen. These changes are probably related to increases of the xylanolytic activity and the proportion of acetate. Taken together, results obtained in his thesis have improved knowledge and understanding of the establishment of the ruminal ecosystem in the dairy calf in pre- and post-weaning periods, and carried some possibilities to orientate or improve the ruminal establishment for better control of calves rearing.

A aplicação da digestão anaeróbia aparece como opção para processamento da vinhaça, visto que por meio deste processo é possível aliar a recuperação de energia (hidrogênio e metano) ao enquadramento ambiental deste resíduo sem interferir em suas qualidades como biofertilizante. Nesse sentido, este trabalho avaliou a aplicação da digestão anaeróbia da vinhaça em sistema combinado acidogênico e metanogênico. Inicialmente, avaliou-se a influência de materiais suportes (argila expandida, carvão vegetal, cerâmica porosa e polietileno de baixa densidade) na produção de hidrogênio, em reatores de leito empacotado (APBR) operados em condição mesofílica (25°C) (Etapa 1). De uma forma geral, apenas traços de hidrogênio foram observados nos reatores preenchidos com partículas de carvão vegetal e cerâmica porosa (2 - 7,9 mL-H2.d-1.L-1 reator). Por outro lado, os valores para a produção volumétrica de hidrogênio (PVH) nos reatores com argila expandida e polietileno de baixa densidade foram dez vezes superior (74,3 - 84,2 mL-H2.d-1.L-1 reator), todavia, estatisticamente iguais. O critério de seleção do material suporte ocorreu com base em relatos na literatura que indicaram rebaixamento do leito e entupimento da saída de reatores APBR preenchidos com argila expandida. Portanto, o polietileno de baixa densidade foi escolhido como melhor opção dentre os suportes avaliados. Não obstante, a baixa relação C/N da vinhaça associada à microaeração do sistema (presença de microrganismos anóxicos) afetou severamente os reatores APBR com diferentes materiais suportes, levando-os à falência. Na segunda etapa, adotou-se a operação dos APBR preenchidos com polietileno de baixa densidade, em condição termofílica (55°C) a fim de diminuir o rendimento da biomassa acidogênica e a solubilidade do oxigênio. Adicionalmente, foi avaliada influência da Carga Orgânica Volumétrica aplicada (COVa - 36,4 - 108,6 kg- DQO.m-3.d-1), por meio da variação do Tempo de Detenção Hidráulica (TDH - 8 - 24 h). Produção contínua de hidrogênio foi observada em todos os reatores operados em condição termofílica (Etapa 2). Nessa etapa, estabeleceu-se a condição ótima de operação com COVa de 84,2 kg-DQO.m-3.d-1 e TDH de 10 h, resultando em PVH de valor 575,3 mL-H2.d-1.L-1 reator e rendimento de hidrogênio (Y1H2) de 1,4 mol-H2.mol-1 carboidratos totais. Na Etapa III, essas condições foram impostas na operação do APBR, resultando em aumento de 18,2% e 14,2% nos valores de PVH e Y1H2, respectivamente, em relação aos dados obtidos na Etapa II. Em paralelo, foram operados dois reatores metanogênicos do tipo manta de lodo (UASB), compondo um sistema único (UASB) e um sistema combinado (APBR/UASB). A produção de energia no sistema combinado foi 25,7% superior ao observado no sistema único. A eficiência de remoção da matéria orgânica total e solúvel aumentou de 60,7 ± 0,3% e 72,6% ± 1,2% no sistema único e 74,6 ± 0,3% e 96,1 ± 1,7% no sistema combinado, respectivamente, sob COVa de 25 kg-DQO.m-3.d-1 (calculada para os sistemas metanogênicos). / Anaerobic digestion application appears as an option for sugar cane vinasse processing, since via this process it is possible to combine the energy recovery (hydrogen and methane) to the environmental framework of this residue without interfering in their qualities as biofertilizer. In this sense, this study evaluated the application of anaerobic digestion of vinasse in two-stage system (acidogenic and methanogenic). Initially, it was evaluated the influence of support material (expanded clay, charcoal, porous ceramics, and low-density polyethylene) on hydrogen production in acidogenic packed bed reactors (APBR) operated under mesophilic condition (25°C) (Phase 1). In general, only traces of hydrogen were observed in APBR filled with charcoal and porous ceramics particles (2 - 7.9 mL-H2.d-1.L-1 reactor). On the other hand, in APBR with expanded clay and low-density polyethylene as support, the values for volumetric hydrogen production (VHP) were ten times higher (74.3 - 84.2 mL-H2.d-1.L-1 reactor), however, statistically equal. The selection criteria of support material was based on reports in the literature that indicated bed lowering and clogging in APBR outlet with expanded clay as support. Therefore, the low-density polyethylene was chosen as the best support among those evaluated. Nevertheless, the low C/N ratio of vinasse associated to microaeration condition in the systems (presence of microorganisms anoxic) severely affected the APBR with different support materials, leading them to bankruptcy. In the second phase, it was adopted the operation of APBR filled with low density polyethylene in thermophilic conditions (55°C) in order to reduce the acidogenic biomass yield and oxygen solubility. Moreover, it was evaluated the influence of organic loading rate (OLR - 36.4 to 108.6 kg-COD.m-3.d-1) by hydraulic retention time varying (HRT - 8 - 24 h). Continuous hydrogen production was observed in all reactors operated at 55°C (Phase 2). In this phase, it was established the optimum condition of operation, OLR of 84.2 kg-COD.m-3.d-1 and HRT of 10 h, resulting in values for PVH and hydrogen yield (Y1H2) of 575.3 mL-H2.d-1.L-1 reactor and 1.4 mol-H2.mol-1 total carbohydrates, respectively. In Phase III, these conditions were imposed on the operation of the APBR, resulting in an increase of 18.2% and 14.2% in the values of PVH and Y1H2, respectively, compared to the data obtained in Phase II. In parallel, two methanogenic reactors were operated (Up-flow Anaerobic Sludge Blanket UASB type), which composed a single stage system (UASB) and a two-stage system (APBR/UASB). The energy production in the two-stage system was 25.7% higher compared to the single stage system. The values for total and soluble organic matter removal were 60.7 ± 0.3% and 72.6 ± 1.2% to single stage system and 74.6 ± 0.3% and 96.1 ± 1.7% to two-stage system, respectively, at OLR of 25 kg-COD.m-3.d-1 (calculated for the methanogenic reactors).

A aplicação da digestão anaeróbia aparece como opção para processamento da vinhaça, visto que por meio deste processo é possível aliar a recuperação de energia (hidrogênio e metano) ao enquadramento ambiental deste resíduo sem interferir em suas qualidades como biofertilizante. Nesse sentido, este trabalho avaliou a aplicação da digestão anaeróbia da vinhaça em sistema combinado acidogênico e metanogênico. Inicialmente, avaliou-se a influência de materiais suportes (argila expandida, carvão vegetal, cerâmica porosa e polietileno de baixa densidade) na produção de hidrogênio, em reatores de leito empacotado (APBR) operados em condição mesofílica (25°C) (Etapa 1). De uma forma geral, apenas traços de hidrogênio foram observados nos reatores preenchidos com partículas de carvão vegetal e cerâmica porosa (2 - 7,9 mL-H2.d-1.L-1 reator). Por outro lado, os valores para a produção volumétrica de hidrogênio (PVH) nos reatores com argila expandida e polietileno de baixa densidade foram dez vezes superior (74,3 - 84,2 mL-H2.d-1.L-1 reator), todavia, estatisticamente iguais. O critério de seleção do material suporte ocorreu com base em relatos na literatura que indicaram rebaixamento do leito e entupimento da saída de reatores APBR preenchidos com argila expandida. Portanto, o polietileno de baixa densidade foi escolhido como melhor opção dentre os suportes avaliados. Não obstante, a baixa relação C/N da vinhaça associada à microaeração do sistema (presença de microrganismos anóxicos) afetou severamente os reatores APBR com diferentes materiais suportes, levando-os à falência. Na segunda etapa, adotou-se a operação dos APBR preenchidos com polietileno de baixa densidade, em condição termofílica (55°C) a fim de diminuir o rendimento da biomassa acidogênica e a solubilidade do oxigênio. Adicionalmente, foi avaliada influência da Carga Orgânica Volumétrica aplicada (COVa - 36,4 - 108,6 kg- DQO.m-3.d-1), por meio da variação do Tempo de Detenção Hidráulica (TDH - 8 - 24 h). Produção contínua de hidrogênio foi observada em todos os reatores operados em condição termofílica (Etapa 2). Nessa etapa, estabeleceu-se a condição ótima de operação com COVa de 84,2 kg-DQO.m-3.d-1 e TDH de 10 h, resultando em PVH de valor 575,3 mL-H2.d-1.L-1 reator e rendimento de hidrogênio (Y1H2) de 1,4 mol-H2.mol-1 carboidratos totais. Na Etapa III, essas condições foram impostas na operação do APBR, resultando em aumento de 18,2% e 14,2% nos valores de PVH e Y1H2, respectivamente, em relação aos dados obtidos na Etapa II. Em paralelo, foram operados dois reatores metanogênicos do tipo manta de lodo (UASB), compondo um sistema único (UASB) e um sistema combinado (APBR/UASB). A produção de energia no sistema combinado foi 25,7% superior ao observado no sistema único. A eficiência de remoção da matéria orgânica total e solúvel aumentou de 60,7 ± 0,3% e 72,6% ± 1,2% no sistema único e 74,6 ± 0,3% e 96,1 ± 1,7% no sistema combinado, respectivamente, sob COVa de 25 kg-DQO.m-3.d-1 (calculada para os sistemas metanogênicos). / Anaerobic digestion application appears as an option for sugar cane vinasse processing, since via this process it is possible to combine the energy recovery (hydrogen and methane) to the environmental framework of this residue without interfering in their qualities as biofertilizer. In this sense, this study evaluated the application of anaerobic digestion of vinasse in two-stage system (acidogenic and methanogenic). Initially, it was evaluated the influence of support material (expanded clay, charcoal, porous ceramics, and low-density polyethylene) on hydrogen production in acidogenic packed bed reactors (APBR) operated under mesophilic condition (25°C) (Phase 1). In general, only traces of hydrogen were observed in APBR filled with charcoal and porous ceramics particles (2 - 7.9 mL-H2.d-1.L-1 reactor). On the other hand, in APBR with expanded clay and low-density polyethylene as support, the values for volumetric hydrogen production (VHP) were ten times higher (74.3 - 84.2 mL-H2.d-1.L-1 reactor), however, statistically equal. The selection criteria of support material was based on reports in the literature that indicated bed lowering and clogging in APBR outlet with expanded clay as support. Therefore, the low-density polyethylene was chosen as the best support among those evaluated. Nevertheless, the low C/N ratio of vinasse associated to microaeration condition in the systems (presence of microorganisms anoxic) severely affected the APBR with different support materials, leading them to bankruptcy. In the second phase, it was adopted the operation of APBR filled with low density polyethylene in thermophilic conditions (55°C) in order to reduce the acidogenic biomass yield and oxygen solubility. Moreover, it was evaluated the influence of organic loading rate (OLR - 36.4 to 108.6 kg-COD.m-3.d-1) by hydraulic retention time varying (HRT - 8 - 24 h). Continuous hydrogen production was observed in all reactors operated at 55°C (Phase 2). In this phase, it was established the optimum condition of operation, OLR of 84.2 kg-COD.m-3.d-1 and HRT of 10 h, resulting in values for PVH and hydrogen yield (Y1H2) of 575.3 mL-H2.d-1.L-1 reactor and 1.4 mol-H2.mol-1 total carbohydrates, respectively. In Phase III, these conditions were imposed on the operation of the APBR, resulting in an increase of 18.2% and 14.2% in the values of PVH and Y1H2, respectively, compared to the data obtained in Phase II. In parallel, two methanogenic reactors were operated (Up-flow Anaerobic Sludge Blanket UASB type), which composed a single stage system (UASB) and a two-stage system (APBR/UASB). The energy production in the two-stage system was 25.7% higher compared to the single stage system. The values for total and soluble organic matter removal were 60.7 ± 0.3% and 72.6 ± 1.2% to single stage system and 74.6 ± 0.3% and 96.1 ± 1.7% to two-stage system, respectively, at OLR of 25 kg-COD.m-3.d-1 (calculated for the methanogenic reactors).

Supplementation of trace metals such as Co and Ni may improve anaerobic digestion of organic material for biogas formation. Which trace metals that are needed and the quantity to apply are, at least partly, related to metal speciation and bioavailability. According to the common perception, metals have to be dissolved to be available for microbial uptake. However, the impact of trace metal speciation on bioavailability is still unclear. The purpose of the present study was to investigate the effect of Fe-, Co- and Ni-addition on the biogas process performance of stillage-fed lab-scale biogas tank reactors. Metal speciation was determined by sequential extraction (SE), extraction of acid volatile sulfides (AVS) and continuously extracted metals (AVS-Me). Sulfur forms, which may be associated to metal speciation, were studied with S XANES (sulfur X-ray absorption near edge structure). The effect of different Co- and Ni-concentrations on process microflora composition was examined with quantitative PCR (qPCR) and 454-pyrosequencing. The results showed that Co- and Ni-supplementation stimulated and stabilized the biogas process performance by increasing methane production and substrate utilization and by establishing low concentrations of volatile fatty acids. 10-20% of the total Co-amount was found in the dissolved phase, which shows that Co was relatively available for microbial uptake. Nickel was entirely associated to organic matter/sulfides and AVS, and was therefore considered to be non-bioavailable. Nevertheless, Ni-supplementation had stimulatory effects on the biogas process performance. This implies that Ni was available for microbial uptake despite its extensive association to sulfides and that other mechanisms than solubility govern the availability of this trace metal. The microbial analyzes revealed that it was primarily the methane producers which were affected by the concentration of Co and Ni. At stimulatory Co- and Ni-concentrations, the archaeal methanogenic community was dominated by aceticlastic Methanosarcinales. At lower Co- or Ni-levels, when biogas process performance was poor, an increase in hydrogenotrophic Methanomicrobiales was observed. This indicates a shift in the methanogenic flora, from being dominated by acetate utilizers to increased importance of hydrogen utilizers, and that the former was more dependent on Co and Ni. / Tillsats av spårmetaller kan förbättra rötning av organiskt material till biogas. Typ och mängd av respektive spårmetall som behöver tillsättas för att uppnå stimulerande effekter, varierar mellan processer. Detta är delvis kopplat till specieringen och biotillgängligheten av metallerna. Endast fria metalljoner och vissa metallkomplex antas vara tillgängliga för mikrobiellt upptag. Det är dock i många fall oklart hur metallernas speciering påverkar biotillgängligheten. Syftet med föreliggande studie var därför att undersöka effekten av tillsats av Fe, Co och Ni för biogasproduktion från drank, en restprodukt i produktion av bioetanol från spannmål, samt att undersöka hur dessa metallers speciering påverkar deras biotillgänglighet. Effekten av tillsatserna av Fe, Co och Ni undersöktes på biogasreaktorer i lab-skala. Metallernas speciering bestämdes genom sekventiell extraktion (SE), extraktion av AVS (acid volatile sulfide) och kontinuerligt extraherade metaller (AVS-Me). Svavelformer med betydelse för metallspeciering studerades med S XANES (sulfur X-ray absorption near edge structure). Effekten av olika Co- och Ni-koncentrationer på processens mikroflora undersöktes molekylärbiologiskt med kvantitativ PCR (qPCR) och 454-pyrosekvensering. Resultaten visade att Co och Ni stimulerade och stabiliserade biogasprocessen genom ökad metanproduktion, ökad utrötningsgrad samt låga halter av flyktiga fettsyror i det studerade systemet. 10-20% av totala mängden Co återfanns i löst fas, vilket visar att Co var relativt lättillgängligt för mikroorganismerna. Nickel var däremot enbart bundet till organiskt material/sulfider och AVS och kunde alltså betraktas som otillgängligt. Trots detta hade även tillsatsen av Ni stimulerande effekter på biogasprocessen. Det innebär att mikroorganismerna har förmåga att komma åt Ni bundet i svårlösliga sulfidföreningar och att andra mekanismer än löslighet reglerar tillgängligheten av denna spårmetall. De molekylärbiologiska analyserna visade att framför allt de metanbildande mikroorganismerna påverkades av halten av Co och Ni. De halter, som gav välfungerande processer, dominerades helt av acetiklastiska Methanosarcinales. Vid lägre halter av Co eller Ni, då processerna gick sämre, tillkom vätgasutnyttjande metanogener. Det tyder på ett skift i bildningen av metan från att ha dominerats av acetatklyvning till att vätgasutnyttjarna fått större betydelse och att de förra är mer beroende av Co och Ni.

The meals from many oilseed crops contain biocidal chemicals that are known to inhibit the growth and activity of several soil pathogens, though little is known concerning impacts on whole soil microbial communities. We investigated the effect of oilseed meals (SMs) from both brassicaceous plants, including mustard and camelina, as well as non-brassicaceous plants, including jatropha and flax, on P. omnivora (the casual agent of cotton root rot) in Branyon clay soil (at 1 and 5% application rates). We also investigated the effect of SMs from camelina, jatropha, flax, and wheat straw on microbial communities in Weswood loam soil. We also used four types of isothiocyanates (ITCs) including allyl, butyl, phenyl, and benzyl ITC to test their effects on P. omnivora growth on potato dextrose agar (PDA), as well as on soil microbial communities in a microcosm study. Community qPCR assays were used to evaluate relative abundances of soil microbial populations. Soil microbial community composition was determined through tag-pyrosequencing using 454 GS FLX titanium technology, targeting ITS and 16S rRNA gene regions for fungal and bacterial communities, respectively. The results showed that all tested brassicaceous and jatropha SMs were able to inhibit P. omnivora sclerotial germination and hyphal growth, with mustard SM being the most effective. Flax didn't show any inhibitory effects on sclerotial germination. All tested ITCs inhibited P. omnivora OKAlf8 hyphal growth, and the level of inhibition varied with concentration and ITC type. Total soil fungal populations were reduced by ITC addition, and microbial community compositions were changed following SM and ITC application. These changes varied according to the type of SM or ITC added. Our results indicated that SMs of several brassicaceous species as well as jatropha may have potential for reducing cotton root rot as well as some other pathogens. Different SMs releasing varied ITCs may result in differential impacts on soil microorganisms including some pathogens.

In aquatic environments the availability of nitrogen (N) generally limits primary production. N2-fixing prokaryotes (diazotrophs) can convert N2 gas into ammonium and provide significant input of N into the oceans. Cyanobacteria are thought to be the main N2-fixers but diazotrophs also include a wide range of heterotrophic bacteria. However, their activity and regulation in the water column is largely unknown. In this thesis the distribution, diversity, abundance, and activity of marine and estuarine heterotrophic diazotrophs was investigated. With molecular methods targeting the nifH gene, encoding the nitrogenase enzyme for N2 fixation, it was shown that diverse nifH genes affiliating with heterotrophic bacteria were ubiquitous in surface waters from ten marine locations world-wide and the estuarine Baltic Sea. Through enrichment cultures of Baltic Sea surface water in anaerobic N-free medium, heterotrophic N2 fixation was induced showing that there was a functional N2-fixing community present and isolates of heterotrophic diazotrophs were obtained. In Sargasso Sea surface waters, transcripts of nifH related to heterotrophic bacteria were detected indicating heterotrophic N2-fixing activity. Nitrogenase expression is thought to be highly regulated by the availability of inorganic N and the presence of oxygen. Low oxygen zones within the water column can be found in association with plankton. The presence of diazotrophs as symbionts of heterotrophic dinoflagellates was investigated and nifH genes related to heterotrophic diazotrophs rather than the cyanobacterial symbionts were found, suggesting that a symbiotic co-existence prevailed. Oxic-anoxic interfaces could also be potential sites for heterotrophic N2 fixation. The Baltic Sea contains large areas of anoxic bottom water. At the chemocline and in anoxic deep water heterotrophic diazotrophs were diverse, abundant and active. These findings extend the currently known regime of N2 fixation to also include ammonium-rich anaerobic waters. The results of this thesis suggest that heterotrophic diazotrophs are diverse and widely distributed in marine and estuarine waters and that they can also be active. However, limits in the knowledge on their physiology and factors which regulate their N2 fixation activity currently prevent an evaluation of their importance in the global marine N budget.

Reverse genetics systems that are based on either viral transcripts or cDNA genome segments cloned in plasmids have recently been reported for some of the dsRNA viruses of the Reoviridae family, namely African horsesickness virus, bluetongue virus and orthoreovirus. For rotaviruses, three reverse genetics systems which only allow the manipulation of a single genome segment have been described. These rotavirus single genome segment reverse genetics systems are not true stand-alone systems because they require a helper virus and a recombinant virus selection step. A true selection-free, plasmid- only or transcript-based reverse genetics system for rotaviruses is lacking. This study sought to identify and characterise the factors that need to be understood and overcome for the development of a rotavirus reverse genetics system using mRNA derived from the in vitro transcription of a consensus nucleotide sequence as well as from double-layered particles. The consensus whole genome sequence of the prototype rotavirus DS-1 and SA11 strains was determined using sequenceindependent whole genome amplification and 454® pyrosequencing. For the rotavirus DS-1 strain, a novel isoleucine in a minor population variant was found at position 397 in a hydrophobic region of VP4. NSP1 contained seven additional amino acids MKSLVEA at the N-terminal end due to an insertion in the consensus nucleotide sequence of genome segment 5. The first 34 nucleotides at the 5'- terminus and last 30 nucleotides at the 3'-terminal end of genome segment 10 (NSP4) of the DS-1 strain were determined in this study. The consensus genome segment 11 (NSP5/6) sequence was 821 bp in length, 148 bp longer than previously reported. The 454® pyrosequence data for a rotavirus SA11 sample with no known passage history revealed a mixed infection with two SA11 strains. One of the strains was a reassortant which contained genome segment 8 (NSP2) from the bovine rotavirus O agent. The other ten consensus genome segments of the two strains could not be differentiated. Novel minor population variants of genome segments 4 (VP4), 9 (VP7) and 10 (NSP4) were identified. Molecular clock phylogenetic analyses of the rotavirus SA11 genomes showed that the two SA11 strains were closely related to the original SA11-H96 strain isolated in 1958. Plasmids containing inserts of the consensus cDNA of the rotavirus DS-1 strain were purchased and used to generate exact capped transcripts by in vitro transcription with a T7 polymerase. Wild-type transcripts of rotavirus SA11 were obtained from in vitro transcription using purified rotavirus SA11 double-layered particles. The purified rotavirus DS-1 and SA11 transcripts were transfected into BSR, COS-7 and MA104 cells. Work on MA104 cells was discontinued due their very low transfection efficacy. In BSR and COS-7 cells, rotavirus DS-1 and SA11 transcripts induced cell death. However, no viable rotavirus was recovered following attempts to infect MA104 cells with the BSR and COS-7 transfected cell lysates. The cell death was determined to be due to apoptotic cell death mechanisms. Immunostaining showed that the DS-1 genome segment 6 (VP6) and SA11 transcripts were translated in transfected BSR and COS-7 cells. Based on visual inspection, the translation seemed to be higher in the retinoic acid-inducible gene-I (RIG-I) deficient BSR cells than in COS-7 cells. This suggested that the transfection of rotavirus transcripts induced an innate immune response which could lead to the development of an antiviral state. Therefore, the innate immune response to rotavirus transcripts was investigated in HEK 293H cells using qRT-PCR and western blot analyses. Results of this investigation showed that RIG-I, but not MDA5 sensed rotavirus transcripts in transfected HEK 293H cells. Furthermore, rotavirus transcripts induced high levels of cellular mRNA encoding the cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α. Other cytokines namely, IFN-α, IL-10, IL-12 p40 and the kinase RIP1 were not significantly induced. Inhibiting the RNA-dependent protein kinase R (PKR) reduced the induction of cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α, but the expression levels were not abrogated. The importance of a consensus sequence and the insights gained in the current study regarding the role of the innate immune response after transfection of rotavirus transcripts into cells in culture, should aid the development of a true rotavirus reverse genetics system. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2013

Reverse genetics systems that are based on either viral transcripts or cDNA genome segments cloned in plasmids have recently been reported for some of the dsRNA viruses of the Reoviridae family, namely African horsesickness virus, bluetongue virus and orthoreovirus. For rotaviruses, three reverse genetics systems which only allow the manipulation of a single genome segment have been described. These rotavirus single genome segment reverse genetics systems are not true stand-alone systems because they require a helper virus and a recombinant virus selection step. A true selection-free, plasmid- only or transcript-based reverse genetics system for rotaviruses is lacking. This study sought to identify and characterise the factors that need to be understood and overcome for the development of a rotavirus reverse genetics system using mRNA derived from the in vitro transcription of a consensus nucleotide sequence as well as from double-layered particles. The consensus whole genome sequence of the prototype rotavirus DS-1 and SA11 strains was determined using sequenceindependent whole genome amplification and 454® pyrosequencing. For the rotavirus DS-1 strain, a novel isoleucine in a minor population variant was found at position 397 in a hydrophobic region of VP4. NSP1 contained seven additional amino acids MKSLVEA at the N-terminal end due to an insertion in the consensus nucleotide sequence of genome segment 5. The first 34 nucleotides at the 5'- terminus and last 30 nucleotides at the 3'-terminal end of genome segment 10 (NSP4) of the DS-1 strain were determined in this study. The consensus genome segment 11 (NSP5/6) sequence was 821 bp in length, 148 bp longer than previously reported. The 454® pyrosequence data for a rotavirus SA11 sample with no known passage history revealed a mixed infection with two SA11 strains. One of the strains was a reassortant which contained genome segment 8 (NSP2) from the bovine rotavirus O agent. The other ten consensus genome segments of the two strains could not be differentiated. Novel minor population variants of genome segments 4 (VP4), 9 (VP7) and 10 (NSP4) were identified. Molecular clock phylogenetic analyses of the rotavirus SA11 genomes showed that the two SA11 strains were closely related to the original SA11-H96 strain isolated in 1958. Plasmids containing inserts of the consensus cDNA of the rotavirus DS-1 strain were purchased and used to generate exact capped transcripts by in vitro transcription with a T7 polymerase. Wild-type transcripts of rotavirus SA11 were obtained from in vitro transcription using purified rotavirus SA11 double-layered particles. The purified rotavirus DS-1 and SA11 transcripts were transfected into BSR, COS-7 and MA104 cells. Work on MA104 cells was discontinued due their very low transfection efficacy. In BSR and COS-7 cells, rotavirus DS-1 and SA11 transcripts induced cell death. However, no viable rotavirus was recovered following attempts to infect MA104 cells with the BSR and COS-7 transfected cell lysates. The cell death was determined to be due to apoptotic cell death mechanisms. Immunostaining showed that the DS-1 genome segment 6 (VP6) and SA11 transcripts were translated in transfected BSR and COS-7 cells. Based on visual inspection, the translation seemed to be higher in the retinoic acid-inducible gene-I (RIG-I) deficient BSR cells than in COS-7 cells. This suggested that the transfection of rotavirus transcripts induced an innate immune response which could lead to the development of an antiviral state. Therefore, the innate immune response to rotavirus transcripts was investigated in HEK 293H cells using qRT-PCR and western blot analyses. Results of this investigation showed that RIG-I, but not MDA5 sensed rotavirus transcripts in transfected HEK 293H cells. Furthermore, rotavirus transcripts induced high levels of cellular mRNA encoding the cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α. Other cytokines namely, IFN-α, IL-10, IL-12 p40 and the kinase RIP1 were not significantly induced. Inhibiting the RNA-dependent protein kinase R (PKR) reduced the induction of cytokines IFN-1β, IFN-λ1, CXCL10 and TNF-α, but the expression levels were not abrogated. The importance of a consensus sequence and the insights gained in the current study regarding the role of the innate immune response after transfection of rotavirus transcripts into cells in culture, should aid the development of a true rotavirus reverse genetics system. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2013