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Sludge management is the highest operating cost in municipal wastewater treatment. Anaerobic digestion (AD) is used to stabilize the sludge and reduce biosolids generation. Hydrolysis kinetics limit the rate of anaerobic digestion and must be improved to increase the overall process rate. In this study a new sludge characterization analysis was used to evaluate hydrolysis in a lab-scale pretreatment process operated at 55℃, 65℃, and 75℃. The experimental results were used to develop a new AD mathematical model, the hydrolysis digestion model (HDM). The model developed is easier to use, as the number of processes and variables were reduced by half, in comparison to existing models. The model variables can be measured using standard sludge characterization analysis, and the hydrolysis reactions included the fermenting microorganism to more accurately model the two-phase hydrolysis model. Model simulations were found to be a good fit of the experimental results, accurately predicting the rate and extent of hydrolysis in the pretreatment digester. / Thesis / Master of Applied Science (MASc)

Studies were conducted to determine the role of bioflocculation in the activated sludge unit processes. Laboratory and full-scale studies revealed that bioflocculation is important in determining settling, dewatering, effluent and digested sludge properties (activated sludge properties) and may be vital to the function of all processes related to the above properties. In these studies, it was shown that divalent cations such as calcium and magnesium improved activated sludge properties, whereas monovalent cations such as sodium, potassium and ammonium ions were detrimental to these properties. The divalent cations promoted bioflocculation through charge bridging mechanisms with negatively charged biopolymers (mainly protein and polysaccharide). It was found that oxidized iron plays a major role in bioflocculation and determination of activated sludge properties through surface interactions between iron and biopolymers. Oxidized iron was effective in removing colloidal biopolymers from solution in coagulation and conditioning studies. The research included experiments evaluating effects of potassium and ammonium ions on settling and dewatering properties; effects of magnesium on settling properties; effects of sodium, potassium, calcium and magnesium on effluent quality; effect of solids retention time on effluent quality; and evaluation of floc properties during aerobic and thermophilic digestion. A floc model is proposed in which calcium, magnesium and iron are important to bioflocculation and the functionality of aeration tanks, settling tanks, dewatering equipment and aerobic or anaerobic digesters. It is shown that activated sludge floc properties affect wastewater treatment efficiency. / Ph. D.

Autothermal thermophilic aerobic digestion (ATAD) of sludge has been used to produce class A biosolids. With stringent EPA guidelines, more and more municipalities are looking to use this process for digestion of sludge. However the large polymer costs associated with dewatering these sludges has made the use of this technology unfavorable. Several studies have been conducted in the past which have looked into the mechanism leading to such a poor dewatering of sludge. Some of these studies have attributed the release of protein and polysaccharide during the high temperature digestion to be responsible for the poor dewatering. However the exact mechanism leading to the poor dewatering is still not totally clear. Laboratory scale studies were conducted to evaluate the mechanism leading to the poor dewatering of these sludges and also to be able to economically condition these sludges. ATAD sludge samples were collected from ATAD processing facilities in Ephrata, PA, Cranberry, PA, Titusville, FL and College Station, TX. The research included experiments evaluating the protein and polysaccharide concentrations in solution, cations and anions, iron and aluminum, zeta potential and capillary suction time. It was found that during digestion large amounts of protein and polysaccharide were released which were in the colloidal range, and the dewatering of each of these sludges became poorer as the amount of protein and polysaccharide in the solution increased. The release of protein and polysaccharide was related to the monovalent to divalent cation ratio and the iron and aluminum concentration in the sludge. Also during the digestion process, the pH of the sludge increased appreciably and the divalent cations precipitated out. The zeta potential of the ATAD digested sludge was also found to be positive. Different chemical coagulants were used to condition the sludge, but even with high polymer doses the dewatering of the sludge was not satisfactory. A combination of iron (or cationic polymer) followed by anionic polymer was found to improve the dewatering to a desired level. The use of this combination of sludge conditioning also provides an economical solution to the problem of dewatering. The role of iron in improving the dewatering of the sludges was found to be important, with the sludge dewatering being better for sludges with a high iron content. The combination of high pH, divalent cation precipitation, iron deficiency and biopolymer release all contribute to the poor dewatering of ATAD sludge. / Master of Science

Thermal stability of theG37 tRNA methyltransferase proteins from Thermotoga maritima and Aquifex aeolicus have been compared using Differential Scanning Calorimetry. It was shown that the Thermotoga protein is remarkably stable and is denatured at temperatures in excess of 100 degrees Centigrade. The Aquifex aeolicus protein was less stable, denaturing broadly at temperatures between 55oC and 100oC. In contrast, the mesophilic E. coli protein was completely denatured at 55oC. Enzymatic activity of the proteins was measured at various temperatures. Both the Thermotoga and Aquifex enzymes are active at ambient temperatures, and display a significant decrease in activity when the temperature is raised above 50oC. This may relate to subtle changes in protein structure causing an effect on the tRNA based assay. Both enzymes contain inter subunit disulfide bonds which might contribute to thermal stability. Assays of the enzymes in the presence of high concentrations of Dithiothreitol (DTT) did not significantly reduce activity at higher temperatures, but did stimulate activity at lower temperatures. Site directed mutagenesis of non -conserved protein sequences within Thermotoga maritima were initiated in order to determine what structures might confer heat stability on the protein. Alanine mutagenesis of lysine residues 103,104 led to reduced catalytic activity, but did increased activity at higher temperatures. Aspartate is the most common residue at the relative position 166 in the variable loop of most TrmD genes. It has been shown that in E. coli this is essential for catalytic activity and possibly the residue which carries out N1 deprotonation on residue G37 in tRNA. In Thermotoga glutamate is present at this position. Alanine mutagenesis of this residue did not eliminate activity suggesting another nearby residue may function in this capacity in the Thermotoga TrmD protein.

O estudo das temperaturas preferenciais e comportamento termofílico é essencial na compreensão da termobiologia das serpentes, todavia, a maioria dos trabalhos tem focado somente os animais diurnos e de clima temperado e pouco se sabe a respeito das serpentes noturnas tropicais e sub-tropicais. O presente estudo utilizou uma arena com gradiente térmico monitorada por vídeo para mensurar o intervalo térmico preferencial procurado durante os períodos de atividade e inatividade (Tset) e a resposta termofílica pós-prandial frente a diferentes quantidades de ingesta em 29 serpentes Bothrops moojeni . Os resultados obtidos mostraram que o intervalo térmico preferencial de B.moojeni diferiu conforme o fotoperíodo (20,93°C a 22,20°C durante o dia e 22,81°C a 24,42°C durante a noite) e foi similar a outros intervalos encontrados para outras serpentes crotalinae. Os dados mostraram que há uma correlação inversa entre intensidade da resposta termofílica pós-prandial e quantidade de alimento ingerido, uma vez que os animais alimentados com uma porção correspondente a 10% de seu peso corporal apresentaram maior resposta termofílica pós-prandial quando comparados àqueles que foram alimentados com porções maiores (20 e 40% do peso corporal). Esta diferença parece estar relacionada com a diminuição de mobilidade e a maior SDA (Specific Dynamic Action) nos animais que ingeriram a maior quantidade de alimento. As serpentes crotalinae são tidas como animais preponderantemente noturnos sendo que a maioria dos dados a respeito dessas atividades foram obtidos em pesquisas de campo. A detecção do período de atividade, assim como o conhecimento de como o estado nutricional influencia a taxa de trânsito, são extremamente importantes na compreensão da ecofisiologia destes animais. No estudo foram utilizadas 29 exemplares de Bothrops moojeni, monitoradas quanto ao horário e o tempo de deslocamento durante o jejum e nos 5 dias subseqüentes a alimentação. A alimentação das serpentes variou entre 10, 20 e 40% do peso da própria serpente. Os resultados confirmaram Bothrops moojeni como espécie essencialmente noturna, sendo que a atividade foi aumentada em mais de 45 vezes durante o período. O pico de atividade ocorreu entre as 19:00 e 20:00 horas, não havendo diferença estatística entre machos e fêmeas. Com relação à atividade pós-prandial foi notada diferença estatística nas taxas de deslocamento dos animais que comeram as maiores proporções (20-40%). Os resultados obtidos corroboram com os dados prévios obtidos em estudos de campo que pontuam Bothrops moojeni como um animal noturno e de baixo grau de mobilidade. A interação entre a quantidade de alimento ingerido pela serpente, o balanço energético e o grau de mobilidade dos animais é bastante complexa e discutível. As serpentes foram consideradas por muito tempo como incapazes de gerar calor através de seu metabolismo aeróbico, à exceção das pythons no choco. Entretanto, estudos recentes mostraram que algumas espécies são capazes de aumentar a temperatura corporal sem recorrer a fontes externas de calor ou contrações musculares. Esta termogênese ocorre devido aos enormes níveis metabólicos atingidos durante a digestão (Specific Dynamic Action - SDA). Em geral as serpentes têm um baixo custo energético de manutenção e a maioria das espécies é capaz de permanecer longo período em jejum e depois se alimentar de uma grande ingesta. A energia produzida durante a digestão destas grandes refeições pode gerar um significativo aumento na temperatura corpórea dos animais. O presente estudo investigou a termogênese pós-prandial em Bothrops moojeni, através de imagens térmicas tomadas em um ambiente termoestável a 30°C. As 12 serpentes foram divididas em 2 grupos e acompanhadas pelas 72 horas após ingerirem refeições equivalentes a 10-60 20% e 30-40% de seu próprio peso, respectivamente. Os resultados mostraram que a resposta termogênica pós-prandial levou a um aumento de 1,6°C da temperatura da pele das serpentes. O pico térmico ocorreu entre 33 e 36 horas após a alimentação em ambos os grupos e a duração da termogênese variou conforme o tamanho da ingesta. Os resultados mostraram uma alta correlação entre a termogênese pós-prandial e a SDA das serpentes. O aumento significativo da temperatura corporal e a sua manutenção por certo tempo sugerem que estes animais são capazes de manter altas taxas metabólica durante a digestão independente da temperatura do ambiente. / The study of the preferred temperatures and thermophilic behavior is essential to understand the snakes thermal biology. Although some studies have been reported, most of them are focused in temperate and diurnal species. Thus, little is known about the tropical and sub-tropical nocturnal species. In the present study , a video-monitored arena with a thermal gradient was used to investigate the preferred thermal range during activity and inactivity (Tset) and the post-prandial thermophilic response after different levels of food intake in 29 Brazilian lanceheads (Bothrops moojeni). The results showed that the preferred thermal range of B.moojeni changes depending on the photoperiod (20.93°C to 22.20°C in daytime and 22.81°C to 24.42°C in night time), being similar to other crotalinae snakes. Our data suggests that there is an inverse correlation between the post-prandial thermophilic response intensity and food intake, as animals fed with a portion corresponding to 10% of its bodyweight presented a higher thermophilic response in comparison to those who were fed with larger portions (20 and 40% of bodyweight). This difference may be related to the decreased mobility and increased SDA (Specific Dynamic Action) in animals that consumed the largest amount of food. Crotalinae are considered to be mainly nocturnal and most of the gathered data on these snakes came from field work. The knowledge of activity pattern and how nutritional status affect wandering rate are determinant to understand the ecophysiology of the snakes. In this study the daily activity pattern was followed in 29 subjects of the lancehead Bothrops moojeni in order to record displacement rate in fasting and feed snakes for five days after feeding meal of 10, 20 and 40% of their own body weight. The results show that B.moojeni is prevalently nocturnal, and activity increased 45 times at night, peaking between 19 to 20 h, in both males and females. Activity level changed significantly after feeding in snakes which ate a larger meal (20-40%). The results confirm previous field data for B.moojeni as a nocturnal species with low mobility. The complex interactions between the amount of food taken by the snake, energetic balance and mobility are discussed. Snakes were for long considered incapable to generate heat fueled by aerobic metabolism, except for brooding pythons. However, recent studies proved that some species are able to elevate their body temperature without muscle contraction or external heat source. Such thermogenic capacity is supported by high metabolic rate that follows digestion and assimilation of food (Specific Dynamic Action - SDA). Snakes have a low maintenance cost and most species can stand for long fasting time, particularly after ingesting a great meal. The energy produced during digestion of such meal can generate significant increase in body temperature. The present study investigated thermogenic response after feeding in the Brazilian lancehead, Bothrops moojeni, using thermal images taken in temperature controlled environment at 30°C. The 12 snakes were divided into two groups and followed for 72 hours after fed a meal representing 10-20% and 30-40% of their body weight, respectively. The results showed thermogenic digestive response with an increase up to 1.6 °C of skin temperature. Thermal heat production peak occurred between 33 to 36 hours after feeding in both groups, and the duration of thermogeny varied with the meal size. The result showed high correlation between thermogenic data and SDA in snakes. The significant increase of body temperature after feeding and its maintenance for extended time suggest a physiological advantage to keep high metabolic rate despite of environmental temperature.

A viabilidade técnica de um sistema termofílico combinado de reatores anaeróbio-aeróbio foi avaliada para tratamento de efluente sintético da indústria de pasta de celulose não branqueada. Estas indústrias apresentam efluente com temperatura superior a 40ºC e o tratamento termofílico pode eliminar a necessidade do resfriamento, necessário para a faixa mesofílica de tratamento, reduzindo custos. Os reatores foram inoculados com lodo mesofílico com gradual adaptação para a condição termofílica. Na faixa mesofílica (37,5 a 42,5ºC) a eficiência do sistema foi de (69±3)%, com maior eficiência do reator anaeróbio (56±7)%. Depois que os reatores apresentaram equilíbrio dinâmico aparente, a temperatura foi gradualmente aumentada de 45,0 para 52ºC. As eficiências de remoção de DQO do sistema variaram de 57% a 88%. A porcentagem de metano no biogás foi decrescendo com o aumento de temperatura, de 77% para valores abaixo do limite de detecção do método cromatográfico utilizado. A eficiência do reator anaeróbio foi baixa e houve acúmulo de ácidos, principalmente acético. O reator aeróbio apresentou maior eficiência de remoção de DQO porém a concentração no licor misto foi baixa. Após a alteração no tempo de detenção hidráulica dos reatores UASB e de lodos ativados para 24 horas e 8 horas, respectivamente, os reatores operaram em faixa de temperatura de 52 a 57ºC por 85 dias. As eficiências de remoção de DQO do sistema variaram de 54% a 70%. A eficiência do reator UASB continuou baixa enquanto o reator aeróbio apresentou bom desempenho, com eficiências variando de 3% a 35% e de 43% a 75%, respectivamente. As concentrações de SSV no licor misto aumentaram, variando de 122 mg/L a 2940 mg/L. Em faixa de temperatura termofílica houve predomínio de Methanosaetas sp e bacilos no reator anaeróbio, não havendo diferenciação significativa de morfologias comparando com a fase mesofílica. No reator aeróbio houve diferenciação a partir da temperatura de 42,5ºC e predominaram bacilos, cocos e filamentos em temperatura termofílica. Os resultados demonstram a viabilidade do tratamento termofílico, mas outros experimentos são necessários antes da aplicação industrial do processo. / The technical feasibility of a combined thermophilic anaerobic-aerobic biological reactors configuration was evaluated in this work using a synthetic unbleached pulp mill effluent. These industries introduce effluent with temperature greater 40ºC and the treatment would eliminate the need of cooling that is required for the mesophilic range, reducing the costs. The reactors were inoculated with mesophilic sludge with gradual adaptation to the termophilic condition. In the mesophilic range (37.5 to 42.5ºC) the efficiency of the combined system was (69±3)%, with the higher efficiency in the anaerobic reactor (56±7)%. After the reactors reached apparent steady state regime the temperature was gradually increased from 42.5 to 52ºC. The system efficiency ranged from 57% to 88%. The percentage of methane in the biogas decreased with the increase in temperature, from 77% to values below the detection limit of the chromatographic measurement used in this work. The anaerobic efficiency was low and the reactor accumulated fatty acid, mostly acetic. The aerobic reactor presented higher efficiency removal of COD but the mixed liquor concentration was lower. After adjustment in the hydraulic retention time of both reactors, with the UASB operating with 24 hours HRT and the aerobic with 8 hours HRT, the reactors operated in the temperature range of 52 to 57ºC during 85 days. The overall removal efficiency ranged from 54% to 70%. The efficiency of the UASB reactor remained lower while the aerobic reactor presented good performance, with COD removal efficiency in the range of 3% to 35%, respectively. The mixed liquor VSS concentration increased with the increase in temperature from 122 mg/L to 2940 mg/L. In thermophilic temperature there was predominance of Methanosaetas sp and baccilus in the anaerobic reactor, without significant differentiation of morphology in comparison with the mesophilic temperature. In the aerobic reactor there was differentiation in temperature of 42.5ºC and baccilus, cocus and filamentous predominated in the thermophilic temperature. These results indicated the feasibility of the thermophilic treatment but other experiments are needed before industrial application of this process.

Made available in DSpace on 2017-07-21T18:53:03Z (GMT). No. of bitstreams: 1 Priscila Judacewski.pdf: 1953850 bytes, checksum: 9d42323aa08ff06768c755a6db587472 (MD5) Previous issue date: 2015-02-12 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The Camembert is considered one of the finest cheeses. Presents as main characteristic the surface covered with a white mycelium of the fungus Penicillium candidum, which provides peculiar sensory characteristics greatly appreciated. In Brazil, little has been studied about this cheese, in this way, in order to generate scientific technical knowledge about factors that may influence the standard of quality cheeses such as Camembert, this work was divided into two parts. In the first (Chapter II), the objective was to evaluate the application of different primary cultures, and different concentrations of salt in the curing process. In the second (Chapter III), the objective was to develop and evaluate a protocol for obtaining fresh biomass of Penicillium candidum for use as a catch crop. Were used as primary cultures mesophilic homofermentative, heterofermentative mesophilic and thermophilic. The concentration of the brine used was 15, 20, 25 and 29º Brix. The biomass of P. candidum obtained in synthetic medium was recovered, and micro fragmented sprayed onto cheeses, with score of 2.106 spores / cheese. Of physical and chemical composition analyzes were performed, maturation index, colorimetric analysis and texture profile in the samples during maturation. The mycelia growth was established by the whiteness index (WI), which proved effective for this purpose, it is fast and inexpensive. Although the texture profile parameters were similar during maturation with different primary cultures, resilience showed lower value for processed cheeses with thermophilic culture, and was the only parameter that showed significant differences between cultures. Another positive factor for cheeses with this culture was superficial development of white mold mycelium with one-day gain, compared to mesophilic. Samples with different salt concentrations, showed significant differences (p <0.05) between the moisture, protein, dry matter, ash, sodium and sodium chloride, and did not affect the growth of the fungus P. candidum. The physical and chemical composition of cheeses matured with biomass was similar to cheeses matured with spores, but proteolysis of cheeses matured with biomass was more intense, with the end of 15 days of aging, free amino acid content of 14% more, compared with samples containing only spores. Among the samples with the application of the inoculums, the tyrosine levels and fat, as well as the cohesiveness of the texture profile parameter showed significant difference. The mycelium overlaid cheese with a day in advance when compared only with the inoculums of spores and showed greater height. This work demonstrates ways to monitor and speed up the mycelium closing within two days, using colorimetric analysis, thermophilic culture and micro fragmented biomass. These results can be readily used by companies to establish quality standards for cheese like Camembert. / O Camembert é considerado um dos principais queijos finos. Apresenta como principal característica a superfície recoberta por um micélio branco do fungo Penicillium candidum, que proporciona características sensoriais peculiares muito apreciadas. No Brasil pouco foi estudado sobre este queijo, desta forma com o propósito de gerar conhecimento técnico científico sobre fatores que podem influenciar o padrão de qualidade de queijos tipo Camembert, esta dissertação foi dividida em duas partes. Na primeira (Capítulo II) o objetivo foi avaliar a aplicação de diferentes culturas primárias, e diferentes concentrações de sal no processo de salga. Na segunda (Capítulo III) o objetivo foi desenvolver e avaliar um protocolo de obtenção de biomassa fresca de Penicillium candidum para utilização como cultura secundária. As culturas primárias utilizadas foram mesofílica homofermentativa, mesofílica heterofermentativa e termofílica. As concentrações das salmouras foram de 15, 20, 25 e 29º Brix. A biomassa de P. candidum obtida em meio sintético foi recuperada, microfragmentada e aspergida sobre os queijos, com contagem de 2.106 de esporos/queijo. Foram realizadas análises de composição físico-química, índice de maturação, análise colorimétrica e perfil de textura nas amostras durante a maturação. O crescimento do micélio foi estabelecido pelo índice de brancura (WI) o qual se mostrou eficiente para este propósito, sendo rápido e de baixo custo. Embora os parâmetros do perfil de textura tenham sido semelhantes durante a maturação com diferentes culturas primárias, a resiliência apresentou menor valor para os queijos processados com cultura termofílica, e foi o único parâmetro que apresentou diferença significativa entre as culturas. Outro fator positivo para os queijos com esta cultura, foi o desenvolvimento superficial do micélio do mofo branco com ganho de um dia, em comparação às mesofílicas. As amostras com diferentes concentrações de sal, apresentaram diferença significativa (p< 0,05) entre os teores de umidade, proteína, extrato seco, cinzas, cloreto de sódio e sódio, e não interferiram no crescimento do fungo P. candidum. A composição físico-química dos queijos maturados com biomassa foi semelhante aos queijos maturados com esporos, porém a proteólise dos queijos maturados com biomassa foi mais intensa, apresentando ao final de 15 dias de maturação, teor de aminoácidos livres de 14% a mais, comparado com amostras contendo apenas esporos. Entre as amostras com as aplicações dos inóculos, os teores de tirosina e gordura, assim como o parâmetro coesividade do perfil de textura, apresentaram diferença significativa. O micélio recobriu o queijo com um dia de antecedência quando comparado apenas com o inóculo de esporos e apresentou maior altura. Este trabalho demonstra formas de monitorar e acelerar o fechamento do micélio em até dois dias, utilizando análise colorimétrica, cultura termofílica e biomassa microfragmentada. Estes resultados podem ser prontamente utilizados por empresas a fim de estabelecer padrões de qualidade para queijos tipo Camembert.

DNA is constantly subjected to chemical and mechanical damage. The ability to repair the lesions sustained is essential for all life. Double stranded DNA (dsDNA) breaks are especially toxic as both antiparallel strands of DNA are severed. The most high fidelity mechanism available to repair this damage is homologous recombination, a mechanism that uses homology from the sister chromatid to replace any lost information. Key proteins involved in maintaining genomic stability this way are conserved in all domains of life. One such component is the Mre11/Rad50 complex that is involved in the initial recognition of damage and recruitment of subsequent repair factors. Understanding the function of this DNA repair complex and any associated proteins has implications for human cancers and aging. The proteins of thermophilic archaea present an excellent opportunity to study these systems in a robust, tractable and eukaryote-like system. Archaea are in many ways biochemically unique, for example they are the only domain capable of methanogenesis. However archaea share a high level of homology with eukaryotes in many essential cellular processes such as DNA replication, homologous recombination and protein degradation. In thermophilic archaea the mre11/rad50 genes are clustered in an operon with the herA/nurA genes that form a helicase/nuclease complex. This has lead to speculation that the four proteins work together during homologous recombination to produce the 3' overhangs required by RadA to identify homology. As part of this investigation I have performed extensive bioinformatic searches of a variety of archaeal/bacterial systems. These analyses have revealed operonic linkages to other known recombinational helicase/nucleases, such as AddAB and RecBCD. These genomic linkages are especially prevalent in thermophilic organisms suggesting their functional relevance is particularly acute in organisms exposed to a high amount of genomic stress. Comparison of the evolutionary trees, constructed for each protein, makes a single genomic linkage event the most likely scenario, but cannot definitively exclude other possibilities. Exhaustive attempts were made to demonstrate an interaction between Mre11/Rad50 and HerA/NurA. Despite analysis by nickel/cobalt pulldown, immunoprecipitation, analytical gel filtration, ITC and OCTET an interaction could not be confirmed or definitively dismissed. However in the process an interesting Rad50 tetrameric assembly was identified and attempts were made to crystalize it. Hexameric helicases and translocases are key to the replication and DNA packaging of all cellular life and multiple viruses. The hexameric translocase HerA is a robust model for investigating the common features of multimeric ATPases as it is extremely stable and experimentally tractable. Here it is revealed that HerA exists in a dynamic equilibrium fluctuating between hexameric and heptameric forms with rapidly interchanging subunits. This equilibrium can be shifted to heptamer by buffering conditions or towards the hexamer by the physical interaction with the partnering nuclease NurA, raising the possibility that these alternate states may play a role in translocase assembly or function. A novel C-terminal brace, (revealed by a collaborative crystallographic structure) is investigated; as well as stabilizing the assembly, this brace reaches over the ATPase active site of its neighbouring subunit. It is seemingly involved in the conversion of energy generated by ATP hydrolysis into physical movement in the central channel of the hexamer. The regulation of homologous recombination is extremely important to prevent aberrant activity, resulting in mutations and genome reorganization. In eukaryotic organisms, it is well established that post-translational modifications and protein turnover at the proteosome play important roles in this control. In particular, there is significant interest currently in the ubiquination-proteasome destruction pathway as a mechanism for extracting DNA repair components from chromatin at the termination of the DNA repair process. To date no Ubiquitin proteins have been identified in the Archaea, however related proteins URMs/SAMPs (Ubiquitin Related Modifier/Small Archaeal Modifier Protein) have previously been identified. URMs are thought to have evolved from a common antecedent to eukaryotic ubiquitin and likely represent an evolutionary 'missing link' in the adaption of sulphur transfer proteins for covalent modifications. There has been speculation that Urm1 may play a similar role to ubiquitin in the proteasome degradation pathway and we have recently provided evidence to corroborate this. Here the potential for modification of Mre11/Rad50/HerA/NurA by Urm1 was investigated. Indeed Rad50 shows evidence of clear urmylation both in vivo and in vitro. Western blotting and mass spec analysis confirmed the covalent attachment of Urm1 to Rad50. Furthermore I present preliminary evidence that this urmylation can lead to the destruction of Rad50 via a direct physical interaction with the proteasome. This is the first evidence of such a regulatory system for Rad50. Investigating the urmylation and destruction of Rad50 was closely linked to investigating the archaeal proteasome, a close homologue of the eukaryotic proteasome. To date the majority of archaeal core proteasomes examined were believed to consist of only two subunits; alpha and beta. The subunits are arranged into heptameric rings, which then form an alpha/beta/beta/alpha stack with a single channel running through the centre of all four rings. Here we reveal that in Sulfolobus species the inner catalytic chambers are made up of mixed beta rings composed of two subunits. The first plays a crucial structural role but appears catalytically inert, while the second conveys catalytic activity. Here we investigate an inactive complex, containing only the structural beta subunit, and an active complex, containing both beta subunits. First, electron microscopy was performed on both complexes revealing the expected four-layered toroidal stack. Both complexes were subsequently investigated crystallographically. A 3.8 Å structure was determined for the inactive complex. As well as being one of the few archaeal core proteasome structures, this is also an important first step towards structurally investigating the novel three-subunit proteasome. The discovery of active and inactive beta subunits in the archaea brings them even closer to eukaryotic proteasomal systems, making the archaea even more valuable as model systems.

A viabilidade técnica de um sistema termofílico combinado de reatores anaeróbio-aeróbio foi avaliada para tratamento de efluente sintético da indústria de pasta de celulose não branqueada. Estas indústrias apresentam efluente com temperatura superior a 40ºC e o tratamento termofílico pode eliminar a necessidade do resfriamento, necessário para a faixa mesofílica de tratamento, reduzindo custos. Os reatores foram inoculados com lodo mesofílico com gradual adaptação para a condição termofílica. Na faixa mesofílica (37,5 a 42,5ºC) a eficiência do sistema foi de (69±3)%, com maior eficiência do reator anaeróbio (56±7)%. Depois que os reatores apresentaram equilíbrio dinâmico aparente, a temperatura foi gradualmente aumentada de 45,0 para 52ºC. As eficiências de remoção de DQO do sistema variaram de 57% a 88%. A porcentagem de metano no biogás foi decrescendo com o aumento de temperatura, de 77% para valores abaixo do limite de detecção do método cromatográfico utilizado. A eficiência do reator anaeróbio foi baixa e houve acúmulo de ácidos, principalmente acético. O reator aeróbio apresentou maior eficiência de remoção de DQO porém a concentração no licor misto foi baixa. Após a alteração no tempo de detenção hidráulica dos reatores UASB e de lodos ativados para 24 horas e 8 horas, respectivamente, os reatores operaram em faixa de temperatura de 52 a 57ºC por 85 dias. As eficiências de remoção de DQO do sistema variaram de 54% a 70%. A eficiência do reator UASB continuou baixa enquanto o reator aeróbio apresentou bom desempenho, com eficiências variando de 3% a 35% e de 43% a 75%, respectivamente. As concentrações de SSV no licor misto aumentaram, variando de 122 mg/L a 2940 mg/L. Em faixa de temperatura termofílica houve predomínio de Methanosaetas sp e bacilos no reator anaeróbio, não havendo diferenciação significativa de morfologias comparando com a fase mesofílica. No reator aeróbio houve diferenciação a partir da temperatura de 42,5ºC e predominaram bacilos, cocos e filamentos em temperatura termofílica. Os resultados demonstram a viabilidade do tratamento termofílico, mas outros experimentos são necessários antes da aplicação industrial do processo. / The technical feasibility of a combined thermophilic anaerobic-aerobic biological reactors configuration was evaluated in this work using a synthetic unbleached pulp mill effluent. These industries introduce effluent with temperature greater 40ºC and the treatment would eliminate the need of cooling that is required for the mesophilic range, reducing the costs. The reactors were inoculated with mesophilic sludge with gradual adaptation to the termophilic condition. In the mesophilic range (37.5 to 42.5ºC) the efficiency of the combined system was (69±3)%, with the higher efficiency in the anaerobic reactor (56±7)%. After the reactors reached apparent steady state regime the temperature was gradually increased from 42.5 to 52ºC. The system efficiency ranged from 57% to 88%. The percentage of methane in the biogas decreased with the increase in temperature, from 77% to values below the detection limit of the chromatographic measurement used in this work. The anaerobic efficiency was low and the reactor accumulated fatty acid, mostly acetic. The aerobic reactor presented higher efficiency removal of COD but the mixed liquor concentration was lower. After adjustment in the hydraulic retention time of both reactors, with the UASB operating with 24 hours HRT and the aerobic with 8 hours HRT, the reactors operated in the temperature range of 52 to 57ºC during 85 days. The overall removal efficiency ranged from 54% to 70%. The efficiency of the UASB reactor remained lower while the aerobic reactor presented good performance, with COD removal efficiency in the range of 3% to 35%, respectively. The mixed liquor VSS concentration increased with the increase in temperature from 122 mg/L to 2940 mg/L. In thermophilic temperature there was predominance of Methanosaetas sp and baccilus in the anaerobic reactor, without significant differentiation of morphology in comparison with the mesophilic temperature. In the aerobic reactor there was differentiation in temperature of 42.5ºC and baccilus, cocus and filamentous predominated in the thermophilic temperature. These results indicated the feasibility of the thermophilic treatment but other experiments are needed before industrial application of this process.