The aim of this work was to develop a production process for the enzyme xyloglucan endo-transglycosylase from Populus tremula x tremuloides (PttXET16-34). The natural transglycosylating activity of this enzyme has previously been employed in a XET-Technology. This chemo enzymatic method is useful for biomimetic modification of cellulose surfaces and holds great potential for industrial applications. Thus, it requires that the XET-enzyme can be produced in larger scale. This work also shows how the wildtype PttXET16-34 was modified into a glycosynthase. By mutation of the catalytic nucleophile into an alanine, glycine or serine residue, enzymes capable of synthesising defined xyloglucan fragments were obtained. These defined compounds are very valuable for further detailed studies of xyloglucan active-enzymes, but are also useful in molecular studies of the structurally important xyloglucan-cellulose interaction. A heterologous production system for PttXET16-34 was previously developed in the methylotrophic yeast Pichia pastoris. A methanol-limited fed-batch process was also previously established, but the yield of active XET was low due to proteolysis problems and low productivity. Therefore, two alternative fed-batch techniques were investigated for the production of PttXET16-34: a temperature-limited fed-batch (TLFB) and an oxygen-limited high-pressure fed-batch (OLHPFB). For the initial recovery of XET after the fermentation process, two different downstream processes were investigated: expanded bed adsorption (EBA) and cross-flow filtration (CFF). / <p>QC 20101108</p>

<p>The two techniques aqueoustwo-phase partitioning and expanded bed adsorption that bothare suitable for primary protein recovery were studied. Most ofthe work was focused on partition in aqueous two-phase systemsand in particular on the possibility to effect the partitionbehaviour by fusion of short peptide tags or protein domains tothe target protein.</p><p>The partitioning of fusionproteins between different variants of the domain tag Z and thenaturally occurring protein DNA Klenow polymerase were studiedin Breox/Reppal aqueous two-phase systems. Most studies wereperformed with cell homogenate. The Breox/Reppal system was infocus because if the fusion protein can be partitioned to theBreox-rich top phase the next step can be a thermoseparatingaqueous two-phase system. When the Breox phase is heated to50°C it switches from a one-phase system to a two-phasesystem resulting in an almost pure water rich top phase andhighly concentrated Breox-rich bottom phase. The Breox can thenbe reused and the protein recovered from the water phase. TheZ-domain was genetically modified in different ways to Z_basic1, Z_acid2and Z_trp12and fused to the Klenow protein to try toenhance partitioning to the Breox-rich phase. From theexperiments it was not possible to observe any effects on thepartition behaviour irrespectively of tested properties of thedomain tag. Despite the absence of domain tag effects highK-values, i.e. partition to the Breox-rich top phase, wereobserved in the Breox/Reppal system. However, the proteinK-values seemed to be rather sensitive to the cell homogenateload and showed a tendency to decrease with increased cellhomogenate load. Also increased phosphate concentration reducedthe K-values. The partitioning of cell debris also seemed todependent on the cell homogenate load. At higher homogenateload (<=20g DW/L) clear Breox-rich top phases were observedwith the cell debris collected in Reppal-rich bottomphases.</p><p>Two different tetrapeptides,AlaTrpTrpPro and AlaIleIlePro were inserted near the C-terminusof the protein ZZT0. The Trp-rich peptide unit stronglyincreased both the partitioning of ZZT0 into the poly(ethyleneglycol) (PEG)-rich phase in a PEG/potassium phosphate aqueoustwo-phase system and its retention on PEG and propylhydrophobic interaction chromatographic columns with potassiumphosphate as eluent in isocratic systems. Both the partitioningand the retention increased with increasing number of Trp-richpeptide units inserted into ZZT0. Insertion of Ile-richtetrapeptide units affected the partitioning and retention to amuch lesser extent. Partition and modelling data also indicateda folding of inserted Trp and Ile tetrapeptide units, probablyto minimise their water contact. It was also investigated howto predict the partitioning of proteins in isoelectricPEG/phosphate aqueous two-phase systems.</p><p>The capture ofß-galactosidase from<i>E. coli</i>cell homogentate (50g DW/L) by metal chelatexpanded bed adsorption was studied. These experiments showedthat capture, with a certain degree of selectivity, andclarification of ß-galactosidase could be achieved from acell homogenate. However, a rather low recovery of about 35 %was obtained at a capacity of 0.25mg/mL of gel. Thus, severalparameters remain to be optimised like the load buffercomposition and the cell homogenate load.</p><p><b>Keywords:</b><i>E. coli</i>, aqueous two-phase systems, fusion proteins,hydrophobic interaction chromatography, expanded bedadsorption, ß-galactosidase, Klenow polymerase, Z-domain,peptide tags</p>

O Alquilbenzeno Linear Sulfonado (LAS) é um surfactante aniônico considerado prioridade de risco ambiental entre os contaminantes emergentes atuais. Estudos anteriores reportaram a degradação anaeróbia de LAS por co-digestão da água residuária de lavanderia comercial e co-substratos sintéticos (extrato de levedura e etanol), em reatores em escala de bancada. Todavia, na literatura ainda são limitados os estudos envolvendo co-substratos economicamente viáveis, como o esgoto doméstico. O objetivo deste estudo foi avaliar a eficiência de remoção do LAS, de água residuária de lavanderia comercial em co-digestão com esgoto doméstico. O reator de leito granular expandido (EGSB) escala piloto de 62,0 L foi operado em condição mesofílica, em TDH de 40 h, por 480 dias. O reator foi inoculado com 12,0 L de lodo proveniente de reator UASB usado no tratamento de água residuária de abatedouro de aves. Na primeira etapa, de adaptação, com duração de 60 dias, o reator foi alimentado apenas com esgoto doméstico. Em seguida, o reator foi alimentado com água residuária de lavanderia comercial diluída em esgoto doméstico para obtenção de concentrações de LAS afluente de 5 ± 4; 19 ± 10 e 36 ± 19 mg L-1, nas etapas II, III e IV, respectivamente. Nestas etapas o reator foi monitorado por 60, 270 e 90 dias. Verificou-se diminuição da eficiência de remoção de matéria orgânica de 73 ± 23 % na etapa de adaptação, para 61 ± 25%, 50 ± 15% e 44 ± 15%, respectivamente, para as etapas II, III e IV. Assim como, verificou-se diminuição da eficiência de remoção do LAS de 73 ± 31% para 55 ± 29% e 32 ± 17% nas três últimas etapas. Verificou-se aumento significativo de sulfeto da etapa II para a etapa III e IV, respectivamente de 8 ± 2 mg L-1 para 29 ± 12 mg L-1 e 31 ± 5 mg L-1, respectivamente. Foi realizada a caracterização taxonômica da biomassa do leito do lodo do reator EGSB ao final de cada etapa experimental, sendo identificadas bactérias semelhantes a Bellinea (domínio Bacteria) e Methanosaeta (domínio Archaea), as quais foram as de maior abundância relativa ao longo de toda operação. A tecnologia empregada para remoção do surfactante foi adequada para concentrações afluente de até 20 mgLAS L-1. Aclimatação da biomassa microbiana foi observada, após longo período e exposição aos compostos recalcitrantes da água residuária de lavanderia. / Classified as an emergent contaminant, Alkylbenzene Linear Sulfonate (LAS) is an anionic surfactant of high priority given its environmental risk. Previous studies reported LAS anaerobic degradation from laundry wastewater in co-digestion with synthetic substrates (such as methanol, ethanol and yeast extract). However, studies with more economic viable substrates as domestic sewage still lacking. This study assessed LAS removal from laundry wastewater diluted in domestic sewage, using an Expanded Bed Granular Sludge (EGSB) pilot scale reactor. The reactor was kept in mesophilic temperature, with 62,0 L total volume and it was operated with a hydraulic retention time (HRT) of 40 h. The inoculum was 12,0 L of sludge from an UASB used on a poultry slaughterhouse wastewater treatment plant. On the first stage, of adaptation, the reactor was fed only with domestic sewage for 60 days. Then, commercial laundry wastewater was diluted in domestic sewage to obtain LAS affluent concentrations of 5 ± 4; 19 ± 10 and 36 ± 19 mg L-1, on stages II, III and IV, respectively. These stages lasted for 60, 270 and 90 days. The increase of commercial laundry wastewater proportion on fed resulted in lower organic matter removal efficiencies. These parameter decreased from 73 ± 23% on adaptation, to 61 ± 25%, 50 ± 15% and 39 ± 23% on following stages. As well as LAS removal efficiencies that dropped from 73 ± 31% to 55 ± 29% and 44 ± 15 % on three last stages. Sulphide production enhanced from stage II (8 ± 2 mg L-1) to stage III (29 ± 12 mg L-1) and remained 31 ± 5 mg L-1 on stage IV. Taxonomic characterization of granular sludge biomass in the end of each stage was performed and genders similar to Bellinea (Bacteria domain) and Methanosaeta (Archae domain) were the ones with higher relative abundance in all stages. The technology applied was suitable for anaerobic surfactant removal with LAS affluent concentration of 20 mg L-1. Acclimation of microbial biomass was observed after long exposure of microbial community to recalcitrant compounds from laundry wastewater.

Os reatores anaeróbios de tratamento de esgoto possibilitam elevada remoção de matéria orgânica, com menor custo em comparação com os processos aeróbios. Entretanto, esses reatores geralmente são pouco eficientes na remoção de nutrientes e seu efluente pode apresentar valores relativamente elevados de DQO e de sólidos suspensos. Por isso, investigou-se a aplicação de cloreto férrico e auxiliares de floculação no afluente de um reator anaeróbio de leito expandido em escala real (Volume = 32 m3; TDH = 3,2 h), no intuito de elevar as eficiências de remoção de DQO, turbidez, fósforo e sólidos. Foram realizados testes de Atividade Metanogênica para avaliar a toxicidade do cloreto férrico ao lodo do reator, mas nenhum efeito tóxico foi detectado para concentrações de até 500 mg FeCl3/l. Em seguida, foram realizados testes em reator de bancada (jar test), quando foi investigado o uso de cloreto férrico (dosagens entre 40 e 200 mg/l) em conjunto com um polímero catiônico, um polímero aniônico e amido natural de batata (dosagens entre 0,5 e 2,0 mg/l), sendo obtidos os melhores resultados com cloreto férrico e polímero aniônico. As eficiências médias diárias de remoção no reator de leito expandido foram elevadas de 64% para 71%, para DQO; de 47% para 59%, para turbidez; de 17% para 31%, para fósforo; e de 52% para 77%, para SST, quando aplicadas dosagens de 40 mg/l de cloreto férrico e 1,0 mg/l de polímero aniônico. / Anaerobic reactors for wastewater treatment have high capacity in removing organic matter and lower costs in comparison with aerobic processes. Nevertheless, anaerobic reactors generally are less efficient in nutrient removal and their effluents may present relatively high COD and total suspended solid (TSS) concentrations. Hence, the application of ferric chloride (FeCl3) and flocculant aids to a full-scale (Volume = 32 m3; HRT = 3,2 h) anaerobic expanded bed reactor influent was investigated to improve removal of COD, turbidity, phosphorous and solids. Methanogenic activity tests were conducted to evaluate toxicity of ferric chloride against methanogenic anaerobic reactor sludge. Tests showed that no toxic effect occurred before concentrations of FeCl3 reached 500 mg/l. Conventional bench-scale jar tests were conducted and ferric chloride was tested in combination with a cationic polymer, an anionic polymer and natural potato starch. Best results were obtained when ferric chloride was employed with anionic polymer. Average daily removal rates were increased from 64% to 71% for COD, from 47% to 59% for turbidity, from 17% to 31% for phosphorous and from 52% to 77% for TSS when dosages close to 40 mg/l of ferric chloride and 1,0 mg/l of anionic polymer were applied to anaerobic expanded bed reactor influent.

A presente pesquisa teve como objetivo principal a avaliação do desempenho de um reator de leito expandido (159,0 \'M POT.3\'), em escala plena, operado sob condições anaeróbia e anaeróbia-aeróbia sobrepostas, tratando esgoto sanitário. Como objetivos específicos, foi avaliado o uso de areias comerciais para complementação do leito de partículas, bem como a distribuição do material suporte ao longo da altura do reator. Além disso, houve a simulação da flotação do efluente do sistema a fim de otimizar a eficiência na remoção de sólidos, visto pela ineficiência do sistema na separação dos sólidos biológicos. Dentre os materiais selecionados para compor o leito de partículas, foi escolhida a areia com tamanho médio dos grãos igual a 0,69 mm e massa específica de 2690 kg.\'M POT.-3\'. Esta pesquisa foi dividida em duas etapas experimentais, sendo que a primeira o sistema foi operado somente sob a condição anaeróbia, enquanto na segunda, o sistema foi submetido à condição anaeróbia e aeróbia sobrepostas. Para a primeira etapa, o sistema obteve maior eficiência na remoção de matéria orgânica, com TDH de 14 horas, quando foi removido 0,84 kgDQObruta/\'M POT.3\'.dia, sendo que foi aplicado 1,26 kgDQObruta/\'M POT.3\'.dia. As eficiências médias de remoção de DQObruta, DQOfilt, DBObruta, DBOfilt, SST e SSV foram de 65%; 64%; 80%; 70%; 58% e 69%. O efluente apresentou, em média, 245 mgDQObruta/L, 117 mgDQOfilt/L, 104 mgDBObruta/L, 76 mgDBOfilt/L; 88 mgSST/L e 69 mgSSV/L. O grau de amonificação obtido foi de 90%, evidenciando a potenciabilidade do sistema na conversão de matéria nitrogenado orgânico em amoniacal. Para a segunda etapa, a maior eficiência na remoção de matéria orgânica foi obtida com TDH de 16 horas, quando foi removido 0,29 kgDQObruta/\'M POT.3\'.dia, sendo que foi aplicado 0,84 kgDQObruta/\'M POT.3\'.dia. As eficiências médias de remoção de DQOfilt e DBOfiltr foram de 80% e 95%, respectivamente. O efluente apresentou, em média, 72 mgDQOfiltr/L, e 13 mgDBOfiltr/L. O grau de amonificação obtido foi de 85%, no entanto houve baixa eficiência na oxidação do \'N\'-amoniacal, no máximo 25%, sendo que as concentrações máximas no efluente foram de 4 mg\'N\'-\'NO IND.2\'POT.-\'/L e 2 mg\'NO IND.3\'POT.-\'/L, respectivamente. Em relação à distribuição do material suporte, foi verificada que a proporção de carvão ativado granular e carvão antracitoso foi superior em relação à areia, nos diferentes pontos de coleta, sendo que essa proporção tendeu a se reduzir ao longo das amostragens, além disso, foram verificadas que as características do fluxo não estão adequadas para proporcionar a fluidificação/expansão do leito de partículas. Os melhores resultados obtidos na simulação da flotação do efluente, quando foi aplicada recirculação com concentração de ar de 17,5 mgar/Lam, e velocidade ascensional de 10 cm/min. As eficiências de remoção de SST; SSF; SSV; DQO, cor e turbidez obtidas foram de 64; 82; 62; 62; 53 e 73%, respectivamente. O efluente flotado apresentou 63 mgSST/L; 5 mgSSF/L; 59 mgSSV/L; 127 mgDQObruta/L, 431 uC e 29 uT. / This present research had as main objective the evaluation of the performance of a full scale expanded bed reactor (159.0 \'M POT.3\'), operated under anaerobic and overlapping anaerobic-aerobic, treating sanitary wastewater. As specific objectives, it was evaluated using commercial sand to complement the bed of particles and distribution of material support along the height of reactor. Besides, it was simulated the flotation of the effluent system in order to optimize the efficient removal of solids, due to inefficiency of the system in the separation of biological solids. Among the materials selected to compose the bed of particles was chosen with the average size of sand grains equal to 0.69 mm and density of 2690 kg.\'M POT.-3\'. This research was divided into two experimental stages, the first being that the system was operated only under the anaerobic condition, while the second, the system was submited to anaerobic and aerobic conditions overlaped. For the first step, the system achieved greater efficiency in removing organic matter, with hydraulic detention time of 14 hours, when it was removed from 0.84 kgCODraw/\'M POT.3\'.day, which was applied 1.26 kgCOD/\'M POT.3\'.day. The mean removal efficiencies of DQOraw, DQOfiltered, BODraw, BODfiltered, TSS and VSS were 65%; 64%; 80%; 70%; 58% and 69%. The effluent had an average of 245 mgCODraw/L; 117 mgCODfiltered/L; 104 mgBODraw/L; 76 mgBODfiltered/L; 88 mgTSS/L and 69 mgVSS/L. The ammonification obtained was 90%, demonstrating the potencial of the system in the conversion of organic matter into ammonia nitrogen. For the second stage, the most efficient in the removal of organic matter was obtained with hydraulic detention time of 16 hours when it was removed from 0.29 kgCODraw/\'M POT.\'3.day, which was applied 0.84 kgCODraw/\'M POT.3\'.day. The mean removal efficiencies of CODfiltered and BODfiltered were 80% and 95%, respectively. The effluent had an average of 72 mgCODfiltered/L, and 13 mgBODfiltered/L. The ammonification obtained was 85%, however there was low efficiency in oxidation of ammonia-\'N\', at most 25%, and the maximum concentrations in the effluent were 4 mg\'N\'-\'NO IND.2\'POT.-\'/L and 2 mg\'N\'-\'NO IND.3\'POT.-\'/L, respectively. Regarding the distribution of material support, it was found that the proportion of activated granular carbon and anthracite carbon was higher on sand, in different points of collection, whereas this proportion tended to drop over the sample, moreover, was found that the characteristics of the flow are not adequate to provide fluidization/expansion of the bed of particles. The best results obtained in the simulation of the flotation of the effluent when it was applied with recirculation of air concentration of 17.5 mgair/Lsample, and ascension velocity of 10 cm/min. Removal efficiencies of TSS; FSS; VSS; COD, color and turbidity were obtained from 64; 82; 62; 62; 53 and 73%, respectively. The effluent had 63 mgTSS/L; 5 mgFSS/L; 59 mgVSS/L; 127 mgCODraw/L; 431 uC and 29 uT.

A presente pesquisa teve como objetivo principal a avaliação do desempenho de um reator de leito expandido (159,0 \'M POT.3\'), em escala plena, operado sob condições anaeróbia e anaeróbia-aeróbia sobrepostas, tratando esgoto sanitário. Como objetivos específicos, foi avaliado o uso de areias comerciais para complementação do leito de partículas, bem como a distribuição do material suporte ao longo da altura do reator. Além disso, houve a simulação da flotação do efluente do sistema a fim de otimizar a eficiência na remoção de sólidos, visto pela ineficiência do sistema na separação dos sólidos biológicos. Dentre os materiais selecionados para compor o leito de partículas, foi escolhida a areia com tamanho médio dos grãos igual a 0,69 mm e massa específica de 2690 kg.\'M POT.-3\'. Esta pesquisa foi dividida em duas etapas experimentais, sendo que a primeira o sistema foi operado somente sob a condição anaeróbia, enquanto na segunda, o sistema foi submetido à condição anaeróbia e aeróbia sobrepostas. Para a primeira etapa, o sistema obteve maior eficiência na remoção de matéria orgânica, com TDH de 14 horas, quando foi removido 0,84 kgDQObruta/\'M POT.3\'.dia, sendo que foi aplicado 1,26 kgDQObruta/\'M POT.3\'.dia. As eficiências médias de remoção de DQObruta, DQOfilt, DBObruta, DBOfilt, SST e SSV foram de 65%; 64%; 80%; 70%; 58% e 69%. O efluente apresentou, em média, 245 mgDQObruta/L, 117 mgDQOfilt/L, 104 mgDBObruta/L, 76 mgDBOfilt/L; 88 mgSST/L e 69 mgSSV/L. O grau de amonificação obtido foi de 90%, evidenciando a potenciabilidade do sistema na conversão de matéria nitrogenado orgânico em amoniacal. Para a segunda etapa, a maior eficiência na remoção de matéria orgânica foi obtida com TDH de 16 horas, quando foi removido 0,29 kgDQObruta/\'M POT.3\'.dia, sendo que foi aplicado 0,84 kgDQObruta/\'M POT.3\'.dia. As eficiências médias de remoção de DQOfilt e DBOfiltr foram de 80% e 95%, respectivamente. O efluente apresentou, em média, 72 mgDQOfiltr/L, e 13 mgDBOfiltr/L. O grau de amonificação obtido foi de 85%, no entanto houve baixa eficiência na oxidação do \'N\'-amoniacal, no máximo 25%, sendo que as concentrações máximas no efluente foram de 4 mg\'N\'-\'NO IND.2\'POT.-\'/L e 2 mg\'NO IND.3\'POT.-\'/L, respectivamente. Em relação à distribuição do material suporte, foi verificada que a proporção de carvão ativado granular e carvão antracitoso foi superior em relação à areia, nos diferentes pontos de coleta, sendo que essa proporção tendeu a se reduzir ao longo das amostragens, além disso, foram verificadas que as características do fluxo não estão adequadas para proporcionar a fluidificação/expansão do leito de partículas. Os melhores resultados obtidos na simulação da flotação do efluente, quando foi aplicada recirculação com concentração de ar de 17,5 mgar/Lam, e velocidade ascensional de 10 cm/min. As eficiências de remoção de SST; SSF; SSV; DQO, cor e turbidez obtidas foram de 64; 82; 62; 62; 53 e 73%, respectivamente. O efluente flotado apresentou 63 mgSST/L; 5 mgSSF/L; 59 mgSSV/L; 127 mgDQObruta/L, 431 uC e 29 uT. / This present research had as main objective the evaluation of the performance of a full scale expanded bed reactor (159.0 \'M POT.3\'), operated under anaerobic and overlapping anaerobic-aerobic, treating sanitary wastewater. As specific objectives, it was evaluated using commercial sand to complement the bed of particles and distribution of material support along the height of reactor. Besides, it was simulated the flotation of the effluent system in order to optimize the efficient removal of solids, due to inefficiency of the system in the separation of biological solids. Among the materials selected to compose the bed of particles was chosen with the average size of sand grains equal to 0.69 mm and density of 2690 kg.\'M POT.-3\'. This research was divided into two experimental stages, the first being that the system was operated only under the anaerobic condition, while the second, the system was submited to anaerobic and aerobic conditions overlaped. For the first step, the system achieved greater efficiency in removing organic matter, with hydraulic detention time of 14 hours, when it was removed from 0.84 kgCODraw/\'M POT.3\'.day, which was applied 1.26 kgCOD/\'M POT.3\'.day. The mean removal efficiencies of DQOraw, DQOfiltered, BODraw, BODfiltered, TSS and VSS were 65%; 64%; 80%; 70%; 58% and 69%. The effluent had an average of 245 mgCODraw/L; 117 mgCODfiltered/L; 104 mgBODraw/L; 76 mgBODfiltered/L; 88 mgTSS/L and 69 mgVSS/L. The ammonification obtained was 90%, demonstrating the potencial of the system in the conversion of organic matter into ammonia nitrogen. For the second stage, the most efficient in the removal of organic matter was obtained with hydraulic detention time of 16 hours when it was removed from 0.29 kgCODraw/\'M POT.\'3.day, which was applied 0.84 kgCODraw/\'M POT.3\'.day. The mean removal efficiencies of CODfiltered and BODfiltered were 80% and 95%, respectively. The effluent had an average of 72 mgCODfiltered/L, and 13 mgBODfiltered/L. The ammonification obtained was 85%, however there was low efficiency in oxidation of ammonia-\'N\', at most 25%, and the maximum concentrations in the effluent were 4 mg\'N\'-\'NO IND.2\'POT.-\'/L and 2 mg\'N\'-\'NO IND.3\'POT.-\'/L, respectively. Regarding the distribution of material support, it was found that the proportion of activated granular carbon and anthracite carbon was higher on sand, in different points of collection, whereas this proportion tended to drop over the sample, moreover, was found that the characteristics of the flow are not adequate to provide fluidization/expansion of the bed of particles. The best results obtained in the simulation of the flotation of the effluent when it was applied with recirculation of air concentration of 17.5 mgair/Lsample, and ascension velocity of 10 cm/min. Removal efficiencies of TSS; FSS; VSS; COD, color and turbidity were obtained from 64; 82; 62; 62; 53 and 73%, respectively. The effluent had 63 mgTSS/L; 5 mgFSS/L; 59 mgVSS/L; 127 mgCODraw/L; 431 uC and 29 uT.

Made available in DSpace on 2014-12-17T15:01:49Z (GMT). No. of bitstreams: 1 JorgeSC_TESE.pdf: 1730447 bytes, checksum: 39fc94239eeb6e994140c5c0f9a2cc23 (MD5) Previous issue date: 2010-06-22 / Expanded Bed Adsorption plays an important role in the downstream processing mainly for reducing costs as well as steps besides could handling cells homogenates or fermentation broth. In this work Expanded Bed Adsorption was used to recover and purify whey proteins from coalho cheese manufacture using Streamline DEAE and Streamline SP both ionic resins as well as a hydrophobic resin Streamline Phenyl. A column of 2.6 cm inner diameter with 30 cm in height was coupled to a peristaltic pump. Hydrodynamics study was carried out with the three resins using Tris-HCl buffer in concentration of 30, 50 and 70 mM, with pH ranging from 7.0 to 8.0. In this case, assays of the expansion degree as well as Residence Time Distribution (RTD) were carried out. For the recovery and purification steps, a whey sample of 200 mL, was submitted to a column with 25mL of resin previously equilibrated with Tris/HCl (50 mM, pH 7.0) using a expanded bed. After washing, elution was carried out according the technique used. For ionic adsorption elution was carried out using 100 mL of Tris/HCl (50 mM, pH 7.0 in 1M NaCl). For Hydrophobyc interaction elution was carried out using Tris/HCl (50 mM, pH 7.0). Adsorption runs were carried out using the three resins as well as theirs combination. Results showed that for hydrodynamics studies a linear fit was observed for the three resins with a correlation coefficient (R2) about 0.9. In this case, Streamline Phenyl showed highest expansion degree reaching an expansion degree (H0/H) of 2.2. Bed porosity was of 0.7 when both resins Streamline DEAE and Streamline SP were used with StremLine Phenyl showing the highest bed porosity about 0.75. The number of theorical plates were 109, 41.5 and 17.8 and the axial dipersion coefficient (Daxial) were 0.5, 1.4 and 3.7 x 10-6 m2/s, for Streamline DEAE, Streamline SP and Streamline Phenyl, respectively. Whey proteins were adsorved fastly for the three resins with equilibrium reached in 10 minutes. Breakthrough curves showed that most of proteins stays in flowthrough as well as washing steps with 84, 77 and 96%, for Streamline DEAE, Streamline SP and Streamline Phenyl, respectively. It was observed protein peaks during elution for the three resins used. According to these peaks were identified 6 protein bands that could probably be albumin (69 KDa), lactoferrin (76 KDa), lactoperoxidase (89 KDa), &#946;-lactoglobulin (18,3 KDa) e &#945;-lactoalbumin (14 KDa), as well as the dimer of beta-lactoglobulin. The combined system compound for the elution of Streamline DEAE applied to the Streamline SP showed the best purification of whey proteins, mainly of the &#945;-lactoalbumina / A adsor??o em leito expandido vem se destacando como uma t?cnica promissora dentro do downstream processing por ser de f?cil manuseio, baixo custo, diminuir etapas de processamento e utilizar o material particulado no seu estado natural. Portanto, o presente trabalho teve como objetivo recuperar e purificar prote?nas presentes no soro de queijo tipo coalho, atrav?s da t?cnica de adsor??o em leito expandido, utilizando resinas de troca ani?nica Streamline DEAE e troca cati?nica Streamline SP e intera??o hidrof?bica Streamline Phenyl,. Foi utilizada uma coluna de 2,6 cm de di?metro interno por 30 cm de altura, acoplada a uma bomba perist?ltica. Para o estudo do sistema foram realizados testes de hidrodin?mica e corridas de adsor??o, com as tr?s resinas, na presen?a de tamp?es Tris-HCl nas concentra??es 30, 50 e 70 mM, com pHs ajustados usando HCl para 7,0; 7,5 e 8,0. Para os testes hidrodin?micos foram estudados a expans?o do leito e a Distribui??o do Tempo de Resid?ncia (DTR). Na etapa de recupera??o e purifica??o, uma amostra de solu??o de soro de 200 mL foi aplicada, a temperatura ambiente, a uma coluna contendo resina (25 mL) previamente equilibrada em tamp?o Tris/HCl (50 mM e pH 7,0), ap?s lavagem efetuou-se a elui??o de acordo com o tipo de t?cnica utilizada. Dessa forma, para adsor??o com troca i?nica a elui??o ocorria com adi??o do eluente 100 mL Tris/HCl (50 mM, pH 7,0 em NaCl 1M). No caso de intera??o hidrof?bica, o eluente consistia de Tris/HCl (50 mM e pH 7,0). Os ensaios de adsor??o foram realizados com as resinas Streamline DEAE, Streamline SP e Streamline Phenyl e suas combina??es. Os resultados mostraram que para as condi??es em que foram realizados os ensaios fluidodin?micos e para o tipo de coluna utilizada, houve uma tend?ncia a linearidade, o coeficiente de correla??o (R2) foi da ordem de 0,9 e que a resina Streamline Phenyl obteve um maior grau de expans?o que as outras resinas, chegando a uma rela??o H0/H de 2,2. A porosidade do leito usando as resinas DEAE e SP foi de 0,70 e da resina Phenyl foi um pouco maior, em torno de 0,75. O n?mero de pratos te?ricos foi 109, 41,5 e 17,8 e o coeficiente de dispers?o axial (Daxial) foi de 0,5, 1,4 e 3,7 x 10-6 m2/s, para as resinas Streamline DEAE, Streamline SP e Streamline Phenyl, respectivamente. As prote?nas do soro s?o adsorvidas nas tr?s resinas e a concentra??o de prote?na em solu??o diminui rapidamente nos primeiros instantes do processo de adsor??o, sendo o equil?brio alcan?ado nos primeiros 10 minutos. Ao se aplicar o soro bruto sem tratamento para as tr?s resinas at? a satura??o (ruptura), embora exista adsor??o das prote?nas para essas resinas, perde-se grande parte dessas prote?nas nas etapas de passante e lavagem. Essas perdas somam 84, 77 e 96%, para as resinas Streamline DEAE, Streamline SP e Strealine Phenyl, respectivamente. Entretanto, pode-se recuperar 16, 23 e 4%, respectivamente, para as tr?s resinas. As tr?s resinas estudadas apresentaram picos de prote?nas na elui??o. De acordo com esses picos, foram identificadas 6 bandas de prote?nas. Provavelmente essas prote?nas sejam: albumina (69 KDa), lactoferrina (76 KDa) e lactoperoxidase (89 KDa), &#946;-lactoglobulina (18,3 KDa) e &#945;-lactoalbumina (14 KDa), d?mero da &#946; -lactoglobulina. Portanto, as resinas estudadas s?o compat?veis para serem utilizadas em leito expandido. O sistema formado pela elui??o da Streamline DEAE quando foi aplicada na resina Streamline SP, tende a uma melhor purifica??o das prote?nas do soro, principalmente da &#945;-lactoalbumina

Os reatores anaeróbios de tratamento de esgoto possibilitam elevada remoção de matéria orgânica, com menor custo em comparação com os processos aeróbios. Entretanto, esses reatores geralmente são pouco eficientes na remoção de nutrientes e seu efluente pode apresentar valores relativamente elevados de DQO e de sólidos suspensos. Por isso, investigou-se a aplicação de cloreto férrico e auxiliares de floculação no afluente de um reator anaeróbio de leito expandido em escala real (Volume = 32 m3; TDH = 3,2 h), no intuito de elevar as eficiências de remoção de DQO, turbidez, fósforo e sólidos. Foram realizados testes de Atividade Metanogênica para avaliar a toxicidade do cloreto férrico ao lodo do reator, mas nenhum efeito tóxico foi detectado para concentrações de até 500 mg FeCl3/l. Em seguida, foram realizados testes em reator de bancada (jar test), quando foi investigado o uso de cloreto férrico (dosagens entre 40 e 200 mg/l) em conjunto com um polímero catiônico, um polímero aniônico e amido natural de batata (dosagens entre 0,5 e 2,0 mg/l), sendo obtidos os melhores resultados com cloreto férrico e polímero aniônico. As eficiências médias diárias de remoção no reator de leito expandido foram elevadas de 64% para 71%, para DQO; de 47% para 59%, para turbidez; de 17% para 31%, para fósforo; e de 52% para 77%, para SST, quando aplicadas dosagens de 40 mg/l de cloreto férrico e 1,0 mg/l de polímero aniônico. / Anaerobic reactors for wastewater treatment have high capacity in removing organic matter and lower costs in comparison with aerobic processes. Nevertheless, anaerobic reactors generally are less efficient in nutrient removal and their effluents may present relatively high COD and total suspended solid (TSS) concentrations. Hence, the application of ferric chloride (FeCl3) and flocculant aids to a full-scale (Volume = 32 m3; HRT = 3,2 h) anaerobic expanded bed reactor influent was investigated to improve removal of COD, turbidity, phosphorous and solids. Methanogenic activity tests were conducted to evaluate toxicity of ferric chloride against methanogenic anaerobic reactor sludge. Tests showed that no toxic effect occurred before concentrations of FeCl3 reached 500 mg/l. Conventional bench-scale jar tests were conducted and ferric chloride was tested in combination with a cationic polymer, an anionic polymer and natural potato starch. Best results were obtained when ferric chloride was employed with anionic polymer. Average daily removal rates were increased from 64% to 71% for COD, from 47% to 59% for turbidity, from 17% to 31% for phosphorous and from 52% to 77% for TSS when dosages close to 40 mg/l of ferric chloride and 1,0 mg/l of anionic polymer were applied to anaerobic expanded bed reactor influent.

O Alquilbenzeno Linear Sulfonado (LAS) é um surfactante aniônico considerado prioridade de risco ambiental entre os contaminantes emergentes atuais. Estudos anteriores reportaram a degradação anaeróbia de LAS por co-digestão da água residuária de lavanderia comercial e co-substratos sintéticos (extrato de levedura e etanol), em reatores em escala de bancada. Todavia, na literatura ainda são limitados os estudos envolvendo co-substratos economicamente viáveis, como o esgoto doméstico. O objetivo deste estudo foi avaliar a eficiência de remoção do LAS, de água residuária de lavanderia comercial em co-digestão com esgoto doméstico. O reator de leito granular expandido (EGSB) escala piloto de 62,0 L foi operado em condição mesofílica, em TDH de 40 h, por 480 dias. O reator foi inoculado com 12,0 L de lodo proveniente de reator UASB usado no tratamento de água residuária de abatedouro de aves. Na primeira etapa, de adaptação, com duração de 60 dias, o reator foi alimentado apenas com esgoto doméstico. Em seguida, o reator foi alimentado com água residuária de lavanderia comercial diluída em esgoto doméstico para obtenção de concentrações de LAS afluente de 5 ± 4; 19 ± 10 e 36 ± 19 mg L-1, nas etapas II, III e IV, respectivamente. Nestas etapas o reator foi monitorado por 60, 270 e 90 dias. Verificou-se diminuição da eficiência de remoção de matéria orgânica de 73 ± 23 % na etapa de adaptação, para 61 ± 25%, 50 ± 15% e 44 ± 15%, respectivamente, para as etapas II, III e IV. Assim como, verificou-se diminuição da eficiência de remoção do LAS de 73 ± 31% para 55 ± 29% e 32 ± 17% nas três últimas etapas. Verificou-se aumento significativo de sulfeto da etapa II para a etapa III e IV, respectivamente de 8 ± 2 mg L-1 para 29 ± 12 mg L-1 e 31 ± 5 mg L-1, respectivamente. Foi realizada a caracterização taxonômica da biomassa do leito do lodo do reator EGSB ao final de cada etapa experimental, sendo identificadas bactérias semelhantes a Bellinea (domínio Bacteria) e Methanosaeta (domínio Archaea), as quais foram as de maior abundância relativa ao longo de toda operação. A tecnologia empregada para remoção do surfactante foi adequada para concentrações afluente de até 20 mgLAS L-1. Aclimatação da biomassa microbiana foi observada, após longo período e exposição aos compostos recalcitrantes da água residuária de lavanderia. / Classified as an emergent contaminant, Alkylbenzene Linear Sulfonate (LAS) is an anionic surfactant of high priority given its environmental risk. Previous studies reported LAS anaerobic degradation from laundry wastewater in co-digestion with synthetic substrates (such as methanol, ethanol and yeast extract). However, studies with more economic viable substrates as domestic sewage still lacking. This study assessed LAS removal from laundry wastewater diluted in domestic sewage, using an Expanded Bed Granular Sludge (EGSB) pilot scale reactor. The reactor was kept in mesophilic temperature, with 62,0 L total volume and it was operated with a hydraulic retention time (HRT) of 40 h. The inoculum was 12,0 L of sludge from an UASB used on a poultry slaughterhouse wastewater treatment plant. On the first stage, of adaptation, the reactor was fed only with domestic sewage for 60 days. Then, commercial laundry wastewater was diluted in domestic sewage to obtain LAS affluent concentrations of 5 ± 4; 19 ± 10 and 36 ± 19 mg L-1, on stages II, III and IV, respectively. These stages lasted for 60, 270 and 90 days. The increase of commercial laundry wastewater proportion on fed resulted in lower organic matter removal efficiencies. These parameter decreased from 73 ± 23% on adaptation, to 61 ± 25%, 50 ± 15% and 39 ± 23% on following stages. As well as LAS removal efficiencies that dropped from 73 ± 31% to 55 ± 29% and 44 ± 15 % on three last stages. Sulphide production enhanced from stage II (8 ± 2 mg L-1) to stage III (29 ± 12 mg L-1) and remained 31 ± 5 mg L-1 on stage IV. Taxonomic characterization of granular sludge biomass in the end of each stage was performed and genders similar to Bellinea (Bacteria domain) and Methanosaeta (Archae domain) were the ones with higher relative abundance in all stages. The technology applied was suitable for anaerobic surfactant removal with LAS affluent concentration of 20 mg L-1. Acclimation of microbial biomass was observed after long exposure of microbial community to recalcitrant compounds from laundry wastewater.

The two techniques aqueoustwo-phase partitioning and expanded bed adsorption that bothare suitable for primary protein recovery were studied. Most ofthe work was focused on partition in aqueous two-phase systemsand in particular on the possibility to effect the partitionbehaviour by fusion of short peptide tags or protein domains tothe target protein. The partitioning of fusionproteins between different variants of the domain tag Z and thenaturally occurring protein DNA Klenow polymerase were studiedin Breox/Reppal aqueous two-phase systems. Most studies wereperformed with cell homogenate. The Breox/Reppal system was infocus because if the fusion protein can be partitioned to theBreox-rich top phase the next step can be a thermoseparatingaqueous two-phase system. When the Breox phase is heated to50°C it switches from a one-phase system to a two-phasesystem resulting in an almost pure water rich top phase andhighly concentrated Breox-rich bottom phase. The Breox can thenbe reused and the protein recovered from the water phase. TheZ-domain was genetically modified in different ways to Zbasic1, Zacid2and Ztrp12and fused to the Klenow protein to try toenhance partitioning to the Breox-rich phase. From theexperiments it was not possible to observe any effects on thepartition behaviour irrespectively of tested properties of thedomain tag. Despite the absence of domain tag effects highK-values, i.e. partition to the Breox-rich top phase, wereobserved in the Breox/Reppal system. However, the proteinK-values seemed to be rather sensitive to the cell homogenateload and showed a tendency to decrease with increased cellhomogenate load. Also increased phosphate concentration reducedthe K-values. The partitioning of cell debris also seemed todependent on the cell homogenate load. At higher homogenateload (&lt;=20g DW/L) clear Breox-rich top phases were observedwith the cell debris collected in Reppal-rich bottomphases. Two different tetrapeptides,AlaTrpTrpPro and AlaIleIlePro were inserted near the C-terminusof the protein ZZT0. The Trp-rich peptide unit stronglyincreased both the partitioning of ZZT0 into the poly(ethyleneglycol) (PEG)-rich phase in a PEG/potassium phosphate aqueoustwo-phase system and its retention on PEG and propylhydrophobic interaction chromatographic columns with potassiumphosphate as eluent in isocratic systems. Both the partitioningand the retention increased with increasing number of Trp-richpeptide units inserted into ZZT0. Insertion of Ile-richtetrapeptide units affected the partitioning and retention to amuch lesser extent. Partition and modelling data also indicateda folding of inserted Trp and Ile tetrapeptide units, probablyto minimise their water contact. It was also investigated howto predict the partitioning of proteins in isoelectricPEG/phosphate aqueous two-phase systems. The capture ofß-galactosidase fromE. colicell homogentate (50g DW/L) by metal chelatexpanded bed adsorption was studied. These experiments showedthat capture, with a certain degree of selectivity, andclarification of ß-galactosidase could be achieved from acell homogenate. However, a rather low recovery of about 35 %was obtained at a capacity of 0.25mg/mL of gel. Thus, severalparameters remain to be optimised like the load buffercomposition and the cell homogenate load. <b>Keywords:</b>E. coli, aqueous two-phase systems, fusion proteins,hydrophobic interaction chromatography, expanded bedadsorption, ß-galactosidase, Klenow polymerase, Z-domain,peptide tags / NR 20140805