The purpose of this research was to examine the impact of cation addition on an industrial activated sludge system and to determine the applicability of the findings of a previous VPI&SU doctoral student (Higgins, 1995) to this industrial sludge. From this research, the implications on the full-scale industrial wastewater treatment system could be postulated. The addition of sodium and potassium to the feed stream of a laboratory-scale activated sludge system improved sludge volume index (SVI), but produced high supernatant solids. Sodium addition resulted in weak flocs and a deterioration in sludge dewatering. Potassium addition also resulted in poor sludge dewatering but did not significantly affect floc stability. The addition of magnesium improved SVI, supernatant solids, and floc stability, but did not significantly affect sludge dewatering. Increasing the monovalent/divalent cation ratio in the reactor feed stream, reduced SVI but produced high supernatant solids. The soluble and bound extracellular protein content was observed to increase with as this ratio increased. No trend was observed with soluble or bound extracellular polysaccharide content. Batch addition of both calcium and magnesium improved sludge settling and dewatering, although this effect was not observed when sludge with low extracellular polymer content was examined. This research confirmed that the effect of cations on activated sludge properties is highly dependent on the feed wastewater composition and starting and operating conditions. This implies the need for laboratory or pilot-scale testing before the effect of cation addition on a given sludge can be determined. For the industrial sludge studied here, the addition of 7 to 12 mM magnesium produced the most desirable sludge characteristics including improved SVI, supernatant solids and floc stability. / Master of Science

Microorganisms have developed ways to preserve cellular functions under low temperature conditions using a variety of biochemical adaptations including the modification of ice formation. In order to conduct a limited survey of microbial ice-associating strategies, a bacterial community associated with frost-exposed leaves was assessed by the construction of a 16S rDNA library, followed by the characterization of some isolates. Fifteen different species were identified based on their 16S rDNA. Among these, Pseudomonas syringae J6 had ice nucleation activity (INA), which promoted ice formation close to 0ºC, whereas Erwinia billingiae, Flavobacterium sp. and Sphingobacterium kitahiroshimense inhibited the recrystallization of small ice crystals at temperatures close to melting. The Erwinia billingiae isolate showed adhesive and swarming behaviour, which can be associated with biofilm formation. Visualization using negative staining, transmission electron microscopy and scanning electron microscopy confirmed the presence of flagella in addition to the presence of slimy biofilm architecture in these Erwina billingiae cultures. Subsequent purification of the extracellular polymeric substance followed by mass spectrometry allowed the identification of a putative outer membrane protein A, which may be involved in the protection of this bacterium to freeze-thaw cycles. To further explore bacterial ice nucleation activity, an ice nucleation protein was cloned from Pseudomonas borealis, a bacterium originating from tundra soil, using degenerative PCR and chromosome walking. The sequence of the putative ice nucleation protein gene (inaPb) was cloned and expressed in Escherichia coli, and its identification was confirmed in the recombinant cells. Although the INPPb was more divergent than other plant-related bacterial INPs, it retained the highly conserved, repetitive core region. The protein may fold so that it has two flat faces, one for protein-protein interactions and the other for ice binding. Expression of the INPPb coding region fused to jelly fish green fluorescent protein showed a temperature-dependent polarized distribution of the recombinant protein in E. coli. In summary, results from this thesis suggests that low temperature survival may be associated with a number of ice-associating adaptations including the presence of biofilm formation in Erwina billingiae amongst other bacteria, INA in P. borealis and INA-expressing recombinant E. coli. / Thesis (Ph.D, Biology) -- Queen's University, 2010-01-27 11:47:02.385

Os exopolissacarídeos (EPS) insolúveis produzidos em processos de tratamento anaeróbios de certos tipos de água residuárias são indesejáveis devido a problemas operacionais que podem vir causar, inviabilizando, muitas vezes, o processo. O reator anaeróbio operado em bateladas seqüenciais contendo biomassa imobilizada (ASBBR) é uma nova configuração de reator que vem sendo estudada no tratamento de águas residuárias cujos resultados são positivos, no entanto em algumas condições observou-se a produção de EPS que inviabilizou o processo. Para evitar estes problemas operacionais em escala real é importante saber os fatores que favorecem a produção de EPS nestes sistemas. Portanto, os objetivos deste trabalho foram verificar a influência de diferentes fontes de carbono e a relação carbono/nitrogênio (C/N), na produção e composição de polímeros extracelulares (EPS) e no desempenho do ASBBR contendo biomassa imobilizada em espuma de poliuretano. As fontes de carbono utilizadas foram glicose, extrato de carne, óleo de soja, as três fontes de carbono juntas, e os ácidos acético, butírico e propiônico juntos. Nos experimentos variando-se a relação C/N utilizou-se glicose como fonte de carbono e uréia como fonte de nitrogênio. As relações C/N foram 13,6 (C/N1), 6,8 (C/N2) e 3,4 (C/N3) gC/gN. Para verificar a influência das condições de microaerofilia na produção do EPS, foi feito também um experimento borbulhando gás nitrogênio, mantendo sob anaerobiose estrita e utilizando glicose como fonte de carbono. Em todos os experimentos o reator foi alimentado com 1000 mgDQO/L. O reator atingiu eficiências médias de remoção da matéria orgânica de 82% em todas as condições, exceto quando o reator foi alimentado com óleo de soja, pois constatou-se o fenômeno de adsorção no material suporte. A produção de EPS por grama de fonte de carbono fornecida foi de 23,6 mg, 13,3 mg, 9,0 mg, 5,9 mg, 5,7 mg, 2,6 mg e 1,4 mg quando o reator foi alimentado com glicose na relação C/N1, óleo de soja, ácidos voláteis, glicose na relação C/N2, glicose/extrato de carne/óleo de soja, glicose na relação C/N3 e extrato de carne, respectivamente. Em condições de anaerobiose estrita não foi observada a produção de EPS. Os resultados sugerem que, a fonte de carbono, condição de microaerofilia e alta relação C/N favorecem a produção de EPS. Por meio de ensaios enzimáticos, com celulase combinada, verificou-se que o principal exopolissacarídeo presente no material polimérico foi celulose. Comparando os espectros de infravermelho das amostras de EPS constatou-se que as diferentes condições nutricionais não influenciaram na composição do polímero. A celulose bacteriana produzida, nesses casos, apresenta organizações alomórficas diferentes quando há glicose no substrato, constatada pelas análises de raios-X. / The insoluble exopolysaccharides (EPS) produced in anaerobic treatment processes from some kinds of wastewater are unwelcome due to operational problems that it may cause, making the process unfeasible most of times. The anaerobic sequential batch biofilm reactor (ASBBR), containing immobilized biomass, is a new configuration of reactor that has been studied for wastewater treatment with positive results. Nevertheless, it was verified the production of EPS in some conditions, which made the process impracticable. To avoid these operational problems in full scale it is important to know the factors that enhance EPS production on those systems. Thus, the objectives of this work were investigating the influence of different carbon sources and the relation carbon/nitrogen (C/N), on the production and composition of extracellular polymers (EPS) and on the performance of the ASBBR with immobilized biomass in polyurethane foam. The carbon sources used were glucose, meat extract, soybean oil, the three carbon sources combined and acetic, butyric and propionic acids combined. On the experiments with variation of C/N relation, glucose was used as carbon source and urea as nitrogen source. The C/N relations were: 13.6 (C/N1), 6.8 (C/N2) and 3.4 (C/N3) gC/gN. Another experiment was accomplished to verify the influence of microerophilic conditions on EPS production, bubbling nitrogen gas, keeping under strict anaerobic conditions and using glucose as carbon source. The reactor was fed with 1000 mgDQO/L in all experiments, achieving 82% of organic substance removal average efficiency, except when fed with soybean oil. At this condition it was verified the adsorption phenomenon in the material support. The yield of EPS was 23.60 mg/g carbon, 13.3 mg/g carbon , 9.0 mg/g carbon, 5.9 mg/g carbon, 5.70 mg/g carbon, 2.6 mg/g carbon and 1.4 mg/g carbon when the reactor was fed with glucose in the relation C/N2, glucose/meat extract/soybean oil, glucose in the relation C/N3 and meat extract, respectively. The production of EPS was not observed at strict anaerobic conditions. Results suggest that the carbon source, microerophilic condition and high C/N relation favor EPS production. Through enzymatic tests with combined cellulase, it was verified that cellulose was the main exopolysaccharide present at the polymeric material. Furthermore, polymers composition is not influenced by different nutritional conditions, comparing infrared spectra of EPS samples. In these cases, the produced bacterial cellulose presents different allomorphic organizations as showed by X-rays analysis.

Os exopolissacarídeos (EPS) insolúveis produzidos em processos de tratamento anaeróbios de certos tipos de água residuárias são indesejáveis devido a problemas operacionais que podem vir causar, inviabilizando, muitas vezes, o processo. O reator anaeróbio operado em bateladas seqüenciais contendo biomassa imobilizada (ASBBR) é uma nova configuração de reator que vem sendo estudada no tratamento de águas residuárias cujos resultados são positivos, no entanto em algumas condições observou-se a produção de EPS que inviabilizou o processo. Para evitar estes problemas operacionais em escala real é importante saber os fatores que favorecem a produção de EPS nestes sistemas. Portanto, os objetivos deste trabalho foram verificar a influência de diferentes fontes de carbono e a relação carbono/nitrogênio (C/N), na produção e composição de polímeros extracelulares (EPS) e no desempenho do ASBBR contendo biomassa imobilizada em espuma de poliuretano. As fontes de carbono utilizadas foram glicose, extrato de carne, óleo de soja, as três fontes de carbono juntas, e os ácidos acético, butírico e propiônico juntos. Nos experimentos variando-se a relação C/N utilizou-se glicose como fonte de carbono e uréia como fonte de nitrogênio. As relações C/N foram 13,6 (C/N1), 6,8 (C/N2) e 3,4 (C/N3) gC/gN. Para verificar a influência das condições de microaerofilia na produção do EPS, foi feito também um experimento borbulhando gás nitrogênio, mantendo sob anaerobiose estrita e utilizando glicose como fonte de carbono. Em todos os experimentos o reator foi alimentado com 1000 mgDQO/L. O reator atingiu eficiências médias de remoção da matéria orgânica de 82% em todas as condições, exceto quando o reator foi alimentado com óleo de soja, pois constatou-se o fenômeno de adsorção no material suporte. A produção de EPS por grama de fonte de carbono fornecida foi de 23,6 mg, 13,3 mg, 9,0 mg, 5,9 mg, 5,7 mg, 2,6 mg e 1,4 mg quando o reator foi alimentado com glicose na relação C/N1, óleo de soja, ácidos voláteis, glicose na relação C/N2, glicose/extrato de carne/óleo de soja, glicose na relação C/N3 e extrato de carne, respectivamente. Em condições de anaerobiose estrita não foi observada a produção de EPS. Os resultados sugerem que, a fonte de carbono, condição de microaerofilia e alta relação C/N favorecem a produção de EPS. Por meio de ensaios enzimáticos, com celulase combinada, verificou-se que o principal exopolissacarídeo presente no material polimérico foi celulose. Comparando os espectros de infravermelho das amostras de EPS constatou-se que as diferentes condições nutricionais não influenciaram na composição do polímero. A celulose bacteriana produzida, nesses casos, apresenta organizações alomórficas diferentes quando há glicose no substrato, constatada pelas análises de raios-X. / The insoluble exopolysaccharides (EPS) produced in anaerobic treatment processes from some kinds of wastewater are unwelcome due to operational problems that it may cause, making the process unfeasible most of times. The anaerobic sequential batch biofilm reactor (ASBBR), containing immobilized biomass, is a new configuration of reactor that has been studied for wastewater treatment with positive results. Nevertheless, it was verified the production of EPS in some conditions, which made the process impracticable. To avoid these operational problems in full scale it is important to know the factors that enhance EPS production on those systems. Thus, the objectives of this work were investigating the influence of different carbon sources and the relation carbon/nitrogen (C/N), on the production and composition of extracellular polymers (EPS) and on the performance of the ASBBR with immobilized biomass in polyurethane foam. The carbon sources used were glucose, meat extract, soybean oil, the three carbon sources combined and acetic, butyric and propionic acids combined. On the experiments with variation of C/N relation, glucose was used as carbon source and urea as nitrogen source. The C/N relations were: 13.6 (C/N1), 6.8 (C/N2) and 3.4 (C/N3) gC/gN. Another experiment was accomplished to verify the influence of microerophilic conditions on EPS production, bubbling nitrogen gas, keeping under strict anaerobic conditions and using glucose as carbon source. The reactor was fed with 1000 mgDQO/L in all experiments, achieving 82% of organic substance removal average efficiency, except when fed with soybean oil. At this condition it was verified the adsorption phenomenon in the material support. The yield of EPS was 23.60 mg/g carbon, 13.3 mg/g carbon , 9.0 mg/g carbon, 5.9 mg/g carbon, 5.70 mg/g carbon, 2.6 mg/g carbon and 1.4 mg/g carbon when the reactor was fed with glucose in the relation C/N2, glucose/meat extract/soybean oil, glucose in the relation C/N3 and meat extract, respectively. The production of EPS was not observed at strict anaerobic conditions. Results suggest that the carbon source, microerophilic condition and high C/N relation favor EPS production. Through enzymatic tests with combined cellulase, it was verified that cellulose was the main exopolysaccharide present at the polymeric material. Furthermore, polymers composition is not influenced by different nutritional conditions, comparing infrared spectra of EPS samples. In these cases, the produced bacterial cellulose presents different allomorphic organizations as showed by X-rays analysis.

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