Activated sludge microorganisms can remove many hazardous substances from wastewater by adsorbing and concentrating them on their surfaces. Biosorption of these substances onto activated sludge surfaces are influenced by the chemical properties of the substance in question as well as the surface properties of the microorganisms. The purpose of this study is to identify the biosorption mechanisms of heavy metals and the effect of extracellular polymer (ECP) composition of activated sludge on the biosorption of Pb(II), Cd(II), Cu(II), Zn(II) and Ni(II). Microorganisms cultured under different growth conditions are expected to have different compositions of ECPs and hence, different biosorption capacities. For this purpose, three sets of reactors with C/N ratios of 9, representing a carbonlimited case / 21, representing conventional municipal wastewater treatment plant activated sludge and 43, representing nitrogen-limited condition, were set up. The semi continuous reactors were fed synthetically and operated at a sludge age of 8 days. Isotherm and kinetic experiments that were held with three different C/N ratios was indicated that the biosorptive capacity of activated sludge was highly dependent on metal species and the C/N ratio. Although, the dependence of biosorptive capacity on C/N ratio was different for each metal, biosorption properties of activated sludge were found to be directly related with ECP composition. Among the heavy metals tested, Pb(II) was the one that was adsorbed at the highest capacity at all C/N ratios. Also, with the purpose of understanding the mechanism of the process, Ca(II) and Mg(II) ions and carbohydrates released into the solution were also monitored and it was indicated that an ion exchange process is involved in the biosorption of heavy metals especially at high metal concentrations but the whole metal removal can not be explained by ion exchange.

Este projeto estudou a influência do substrato na dinâmica do processo de formação do biofilme em espuma de poliuretano, em reatores anaeróbios horizontais de leito fixo diferenciais, alimentados com extrato de carne, glicose, amido, lipídeos e esgoto sanitário sintético. O estudo constitui em acompanhar a colonização das matrizes de espuma, ao longo do tempo, em relação a quantidade de biomassa aculumada, polímero extracelular produzido e características morfológicas das células presentes nos suportes retirados dos reatores diferenciais. O uso dessas técnicas permitiu o melhor entendimento do processo de aderência, além da verificação da composição morfológica e a estrutura do biofilme aderido ao suporte, possibilitando correlacionar a ocorrência de determinadas morfologias com cada etapa da colonização observada. A influência do substrato na dinâmica de aderência foi verificada devido aos diferentes padrões de colonização encontrados. Foi observada uma grande variabilidade morfológica em relação ao substrato utilizado. Todavia, uma grande ocorrência de organismos semelhantes a Methanosaeta sp foi verificada em todos os ciclos, principalmente em relação a Methanosarcina sp. O fenômeno de excreção de polímeros pareceu ser de fundamental importância no processo de colonização de matrizes de poliuretano, estando vinculado provavelmente à fixação das células aos suportes. A produção de polímeros, na etapa de aderência inicial, apresentou comportamento diferenciado para cada substrato utilizado. De acordo com os modelos propostos e as análises de microscopia eletrônica de varredura, verificou-se que os polímeros extracelulares podem estar ligados ao entupimento de reatores de leito fixo, permanecendo no interior dos suportes e nos interstícios do leito, causando problemas operacionais. As rápidas partidas observadas em trabalhos utilizando reatores de leito fixo e espuma de poliuretano como suporte podem estar vinculadas à rápida aderência dos organismos em todos os ciclos estudados. / This work focused on the influence of the type of substrate on the process of biofilm formation onto polyurethane foam matrices, in differential horizontal anaerobic immobilized sludge reactors, fed with meat extract (protein), glucose, starch, lipid and synthetic domestic wastewater. It consisted of accompanying the colonization of foam matrices with time, regarding to biomass amount, extracellular polymers produced and the morphological characteristics of the cells present on the supports packed in the differential reactors. These techniques permitted a better understanding of the adhesion process, besides a verification of the morphological composition and the structure of the biofilm attached to the support, making possible a correlation of the particular morphologies occurrences with each colonization step. The influence of the substrate was verified due to the different colonization patterns found. It was observed considerable morphological variety depending on the substrate utilized. However, the presence of Methanosaeta sp.- like organisms was often verified in all cycles, specially Methanosarcina sp. The excretion of polymers seemed to be crucial in the colonization process of the polyurethane matrices, being probably related to the cell fixation on the support. The polymeric production, in the inital adhesion step, showed a particular behavior for each substrate employed. According to the proposed models and the scanning electronic microscopy analysis, it was verified that the extracellular polymers can be related to the clogging of the fixed-bed reactors, as it keeps into the supports and the bed interstices, causing operational problems. The fast start-ups observed in works using fixed-bed reactors and polyurethane foam as support can be related to the fast cell adhesion during all cycles studied.

Bioflocculation, which can be defined as aggregation of bacterial flocs, has important implications on the physical characteristics of sludge. It is especially critical to settling and dewatering systems which impacts the overall economy of the process greatly. One of the most common problems in activated sludge systems to negatively influence the settleability is sludge bulking which can be defined as non-settling situation of microbial mass. The first objective of this research is to investigate the effect of calcium ion on sludge bulking in a phosphorus deficient medium and the second objective is to improve the settling, dewatering, and pumping of activated sludge by adjusting the calcium (Ca) ion concentration of the feed. For this purpose, 7 semi-continuous laboratory scale activated sludge reactors were operated with a mixed aerobic culture. The reactors had 8 days of sludge residence time and aerated with air pumps to provide a dissolved oxygen concentration of at least 3 mg/L. All the analyses were conducted after the reactors reached steady state condition. In the first part of the research, the effect of strictly phosphorus-limited medium on bulking of activated sludge was studied at different calcium ion concentration. Three reactors were set up having 5, 10 and 20 meq/L calcium concentrations. From the results it was observed that, phosphorus deficiency caused viscous bulking independent from the calcium ion concentration. It was found out that bulking of activated sludges due to phosphorus deficiency could be cured by the addition of phosphorus. Furhermore, microorganisms starved for phosphorus, seemed to accumulate polyphosphate granules when they were exposed to a phosphorus source. In the second part of the study, the effect of calcium ion on physical, chemical and surface chemical properties of activated sludge was investigated at 4 different concentrations (0.27, 5, 10 and 20 meq/L) under sufficient phosphorus concentration. Calcium addition resulted in significant changes in the quantity and quality of extracellular polymeric substances (EPS). Total EPS increased depending on the calcium concentration. Carbohydrate content of EPS dominated over the protein content for calcium concentration of 5 meq/L and above. The amount of calcium ions incorporated into the sludge floc matrix also increased with the dose of calcium added. Settleability and dewaterability of sludge improved significantly at 5 meq/L dose of calcium. However, settleability did not change any further with increasing calcium dose, whereas dewaterability increased for all increasing calcium concentrations. Sludge viscosity also decreased considerably at 5 meq/L concentration indicating better pumpability but it did not change further above 10 meq/L calcium addition.

Thesis (MSc Food Sc)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: The start-up period of Upflow Anaerobic Sludge Bed (UASB) reactors can significantly be reduced by enhancing the time-consuming granulation process through the batch cultivation of anaerobic granular sludge and thus seeding the reactor with this cultivated granular sludge, instead of raw anaerobic sludge. The precise mechanism for granule formation is not well known, but it is believed that extracellular polymers (ECP) play a critical role in the granulation process. Information on the precise role of ECP is also limited and no universal standardised method for ECP extraction is used at present. Therefore, comparison of results from different researchers has to be made with great caution. The objectives of this study were to evaluate an ECP extraction methód so as to optimise the extraction time, and then to correlate ECP composition of UASB granules with granule metabolic activity. The impact of changes in the environmental conditions, such as sludge sources (Paarl and Kraaifontein-sludge), carbon growth substrates (yeast extract lactate, glucose medium and fruit cocktail effluent) and batch cultivation techniques (roller-table and shake-waterbath), on batch cultivation studies was also evaluated in terms of granule activity, ECP composition and granule formation. A physical extraction method was used to quantify the ECP content of UASB granules from six different sources. The optimal extraction time was taken as the time needed before cell lysis took place, and before intracellular material started contributing to the ECP content of the granules. It was concluded that the ECP composition was affected by the wastewater composition fed to the original UASB reactors, It was also found that the activity test results could be used to indirectly predict the activity of the different trophic groups present in the UASB granules. A correlation was found between the activity test results and the total ECP content, and this showed that the granules with the higher ECP yields exhibited greater biogas (SB) and methanogenic (SM) activities. However, based on the activity data and total ECP content, it appeared that a protein:carbohydrate ratio < 1 affected the activity of the granules, The sludge source used as inoculum for batch cultivation of anaerobic granular sludge had a significant effect on granule formation. The use of a pre- granulated raw anaerobic sludge, such as the Paarl-sludge, resulted in a greater increase in granule numbers at the end of the cultivation period. The acetic acid activity profiles showed that the acetoclastic methanogens that are involved in initiation of granulation by nucleus formation, were inactive or absent in the different batch systems, with the exception of the roller-table glucose cultivated Kraaifontein-sludge (RKG) batch system. The addition of glucose as carbon growth substrate for batch cultivation not only enhanced the activity of the acidogenic population, but also led to the establishment to a greater variety of granule trophic groups within all the glucose cultivated batch systems. The addition of fruit cocktail effluent as carbon substrate enhanced ECP production in the Paarl-sludge cultivated batch systems. However, the addition of carbon substrates showed no discernible trend on granule formation itself. The roller-table cultivation technique resulted in the higher increase in granule numbers, and it was speculated that the more vigorous shake-waterbath technique probably shortened the contact time between biomass and substrate. Large variations in the ECP composition of the different batch systems were found, and these were ascribed to the composition heterogeneity of different sludges. For future studies, it is advisable to characterise sludge, both chemically and microbiologically before using as inoculum. The selection of an appropriate sludge inoculum should then lead to optimisation of the granulation process. / AFRIKAANSE OPSOMMING: Die aanvangsperiode van "Upflow Anaerobic Sludge Bed" (UASB) bioreaktors kan noemenswaardig verminder word deur die tydsame granulasie proses te versnel deur die vooraf lot-kweking van anaërobe granulêre slyk waarmee 'n reaktor dus geïnokuleer kan word in plaas van rou anaërobe slyk. Die presiese meganisme van granulevorming is nog nie welbekend nie, maar daar word beweer dat ekstrasellulêre polimere (ECP) wel 'n kritiese rol speel in die granulasie proses. Inligting; oor die presiese rol van ECP is ook nog beperk, en tans word daar nog , geen universele standaard metode vir ECP-ekstraksie gebruik nie. Gevolglik moet resultate vanaf verskeie navorsers met groot omsigtigheid vergelyk word. Die doelwitte van hierdie studie was om 'n ECP ekstraksiemetode te evalueer deur die ekstraksietyd te optimiseer, en dan te korreleer met die ECP samestelling en metaboliese aktiwiteit van die UASB granules. Die inwerking van veranderinge in omgewingskondisies, soos slykbronne (Paarl- en Kraaifontein-slyk), koolstofbronne (gisekstrak-Iaktaat-, glukose-medium en vrugtekelkie-uitvloeisel) en lot-kwekingstegnieke (rol-tafel en skud-waterbad) op lot-kweking studies in terme van granule aktiwiteit, ECP-samestelling en granulevorming is ook ondersoek. 'n Fisiese ekstraksie metode is gebruik om die ECP-inhoud van UASB granules vanaf ses verskillende bronne te bepaal. Die optimale ekstraksietyd is geneem as die tyd benodig voordat sellise sal plaasvind en die intrasellulêre materiaal 'n bydrae sal lewer tot die ECP-inhoud van granules. Dit is afgelei dat die ECP-samestelling beïnvloed word deur die samestelling van die afvalwater wat vir die oorspronklike UASB bioreaktors gevoer is. Voorts is gevind dat die aktiwiteitstoets resultate indirek gebruik kan word vir die voorspelling van aktiwiteit van die verskillende trofiese groepe wat in die UASB granules teenwoordig is. 'n Korrelasie is gevind tussen die aktiwiteitstoets resultate en die totale ECP-inhoud wat aangedui het dat granules met hoër ECP opbrengste, beter biogas (SB) en metanogeniese (SM) aktiwiteit getoon het. Volgens die aktiwiteitsdata en totale ECP-inhoud het dit egter geblyk dat 'n proteïen:koolhidraat verhouding < 1 die aktiwiteit van granules beïnvloed het. Die slykbron wat as inokulum gebruik is vir lot-kweking van' anaërobiese granulêre slyk het 'n noemenswaardige invloed gehad op granulevorming. Die gebruik van 'n rou anaërobe slyk wat reeds 'n mate van granulasie getoon het, soos die Paarl-slyk, het 'n hoër toename in granule getalle aan die einde van die kwekingsperiode teweeg gebring. Die asynsuur aktiwiteitsprofiele het aangedui dat die asetoklastiese metanogene, wat hoofsaaklik betrokke is by inisiëring van granulasie deur kernvorming, onaktief of afwesig was in al die verskillende lotsisteme, met die uitsondering van die roltafel glukose-gekultiveerde Kraaifonteinslyk (RKG) lot-sisteem. Die toevoeging van glukose as koolstofbron vir lot-kweking het nie alleenlik die aktiwiteit van die asidogene populasie verhoog nie, maar het ook bygedra tot die vestiging van 'n groter verskeidenheid van granule trofiese groepe. Die toevoeging van vrugtekelkie-uitvloeisel as koolstofbron het die produksie van ECP verhoog in die Paarl-slyk gekweekte lot-sisteme. Die toevoeging van koolstofsubstrate het egter geen merkbare verandering getoon in granulasie opsigself nie. Die rol-tafel kwekingstegniek het 'n hoër toename in granule getalle tot gevolg gehad, en dit is gespekuleer dat die meer kragtige skud-waterbad tegniek waarskynlik die kontaktyd tussen die substraat en biomassa verkort het. Groot variasies is gevind in die ECP-samestelling van die verskillende lotsisteme, en dit is toegeskryf aan die heterogene samestelling van die verskillende slyke. Vir toekomstige navorsing is dit raadsaam om slyk voor gebruik as inokulum beide chemies en mikrobiologies te karakteriseer. Die keuse van 'n geskikte slyk-inokulum sal bydra tot die optimisering van die granulasie proses.

Este projeto estudou a influência do substrato na dinâmica do processo de formação do biofilme em espuma de poliuretano, em reatores anaeróbios horizontais de leito fixo diferenciais, alimentados com extrato de carne, glicose, amido, lipídeos e esgoto sanitário sintético. O estudo constitui em acompanhar a colonização das matrizes de espuma, ao longo do tempo, em relação a quantidade de biomassa aculumada, polímero extracelular produzido e características morfológicas das células presentes nos suportes retirados dos reatores diferenciais. O uso dessas técnicas permitiu o melhor entendimento do processo de aderência, além da verificação da composição morfológica e a estrutura do biofilme aderido ao suporte, possibilitando correlacionar a ocorrência de determinadas morfologias com cada etapa da colonização observada. A influência do substrato na dinâmica de aderência foi verificada devido aos diferentes padrões de colonização encontrados. Foi observada uma grande variabilidade morfológica em relação ao substrato utilizado. Todavia, uma grande ocorrência de organismos semelhantes a Methanosaeta sp foi verificada em todos os ciclos, principalmente em relação a Methanosarcina sp. O fenômeno de excreção de polímeros pareceu ser de fundamental importância no processo de colonização de matrizes de poliuretano, estando vinculado provavelmente à fixação das células aos suportes. A produção de polímeros, na etapa de aderência inicial, apresentou comportamento diferenciado para cada substrato utilizado. De acordo com os modelos propostos e as análises de microscopia eletrônica de varredura, verificou-se que os polímeros extracelulares podem estar ligados ao entupimento de reatores de leito fixo, permanecendo no interior dos suportes e nos interstícios do leito, causando problemas operacionais. As rápidas partidas observadas em trabalhos utilizando reatores de leito fixo e espuma de poliuretano como suporte podem estar vinculadas à rápida aderência dos organismos em todos os ciclos estudados. / This work focused on the influence of the type of substrate on the process of biofilm formation onto polyurethane foam matrices, in differential horizontal anaerobic immobilized sludge reactors, fed with meat extract (protein), glucose, starch, lipid and synthetic domestic wastewater. It consisted of accompanying the colonization of foam matrices with time, regarding to biomass amount, extracellular polymers produced and the morphological characteristics of the cells present on the supports packed in the differential reactors. These techniques permitted a better understanding of the adhesion process, besides a verification of the morphological composition and the structure of the biofilm attached to the support, making possible a correlation of the particular morphologies occurrences with each colonization step. The influence of the substrate was verified due to the different colonization patterns found. It was observed considerable morphological variety depending on the substrate utilized. However, the presence of Methanosaeta sp.- like organisms was often verified in all cycles, specially Methanosarcina sp. The excretion of polymers seemed to be crucial in the colonization process of the polyurethane matrices, being probably related to the cell fixation on the support. The polymeric production, in the inital adhesion step, showed a particular behavior for each substrate employed. According to the proposed models and the scanning electronic microscopy analysis, it was verified that the extracellular polymers can be related to the clogging of the fixed-bed reactors, as it keeps into the supports and the bed interstices, causing operational problems. The fast start-ups observed in works using fixed-bed reactors and polyurethane foam as support can be related to the fast cell adhesion during all cycles studied.

Os objetivos deste trabalho foram avaliar o desempenho do reator anaeróbio operado em batelada seqüencial quando submetido ao aumento progressivo da concentração de glicose e estimar os parâmetros cinéticos da degradação da glicose. Inicialmente o reator foi operado com ciclos de 8 horas, tratando glicose nas concentrações, aproximadas, de 500, 1000 e 2000 mg/L. Não foi detectada glicose no efluente nas três condições. O reator operou de maneira estável, tratando aproximadamente 500 mg/L de glicose, com eficiência na remoção da DQO filtrada entre 93% e 97%. Na operação com concentrações de glicose no afluente próximas de 1000 mg/L e 2000 mg/L, observou-se instabilidade operacional, principalmente devido à produção de polímeros extracelulares (EPS) que comprometeram a hidrodinâmica e a transferência de massa no sistema. Os valores médios da concentrações de ácidos voláteis no efluente foram de 159 ± 72 mg/L e 374 ± 92 mg/L, respectivamente. Aos perfis de concentração de glicose foi ajustado modelo de primeira ordem, enquanto que um modelo modificado, contemplando concentração residual de matéria orgânica, foi ajustado aos perfis temporais de DQO. Para verificar a formação do EPS, operou-se o reator com 3 horas de ciclo nas concentrações, aproximadas, de 500 e 1000 mg/L. Esta fase teve como objetivo verificar a hipótese, segundo a qual, a produção de EPS seria resultado da exposição da biomassa a baixas concentrações de matéria orgânica por longo período. Dessa forma, reduzindo o tempo de ciclo, a exposição a baixas concentrações também seria reduzida. No entanto, embora o reator tenha operado com relativa estabilidade, verificou-se formação de grande quantidade de EPS logo na primeira condição operacional, com aproximadamente, 500 mg/L de glicose no afluente, indicando que a hipótese não estava correta. / The main objectives of this study were to evaluate the performance of the anaerobic sequencing batch reactor when subjected to a progressive increasing of the influent glucose concentration and estimate the kinetic parameters of glucoses degradation. Initially the reactor was operated with 8-hour cycles, treating glucose at concentrations of 500, 1000 and 2000 mg/L. Glucose was not detected in the effluent in all these three conditions. The reactor showed operating stability treating glucose concentration of approximately 500mg/L, with efficiencies between 93% to 97% in the filtrated COD removal. In the operation with glucose concentrations of 1000 mg/L and 2000 mg/L, approximately, it could be noticed an operational instability, caused mainly by a production of extracellular polymers (EPS) leading to hydrodynamic and mass transfer problems in the reactor. The mean values of volatile acids concentration in the effluent were about 159 ± 72mg/L and 374 ± 92mg/L, respectively. A first order model was adjusted to glucose concentration profiles, and a modified model, including a residual concentration of substrate, was adjusted to COD temporal profiles. To verify the EPS formation, the reactor was operated with 3-hour cycle in the concentrations of 500 and 1000 mg/L This stage had the objective of verifying if the EPS production would result from the exposure of the biomass to low concentration of substrate for a long period of time. Thus, reducing the time cycle, the exposure to low concentrations would also be reduced. Nevertheless, even with the reactor operating with relative stability, the hypotheses could not be verified due the formation of a large amounts of EPS right in the first operational condition with approximately to 500 mg/L of glucose in the influent, showing that the hypothesis was not right.

Os objetivos deste trabalho foram avaliar o desempenho do reator anaeróbio operado em batelada seqüencial quando submetido ao aumento progressivo da concentração de glicose e estimar os parâmetros cinéticos da degradação da glicose. Inicialmente o reator foi operado com ciclos de 8 horas, tratando glicose nas concentrações, aproximadas, de 500, 1000 e 2000 mg/L. Não foi detectada glicose no efluente nas três condições. O reator operou de maneira estável, tratando aproximadamente 500 mg/L de glicose, com eficiência na remoção da DQO filtrada entre 93% e 97%. Na operação com concentrações de glicose no afluente próximas de 1000 mg/L e 2000 mg/L, observou-se instabilidade operacional, principalmente devido à produção de polímeros extracelulares (EPS) que comprometeram a hidrodinâmica e a transferência de massa no sistema. Os valores médios da concentrações de ácidos voláteis no efluente foram de 159 ± 72 mg/L e 374 ± 92 mg/L, respectivamente. Aos perfis de concentração de glicose foi ajustado modelo de primeira ordem, enquanto que um modelo modificado, contemplando concentração residual de matéria orgânica, foi ajustado aos perfis temporais de DQO. Para verificar a formação do EPS, operou-se o reator com 3 horas de ciclo nas concentrações, aproximadas, de 500 e 1000 mg/L. Esta fase teve como objetivo verificar a hipótese, segundo a qual, a produção de EPS seria resultado da exposição da biomassa a baixas concentrações de matéria orgânica por longo período. Dessa forma, reduzindo o tempo de ciclo, a exposição a baixas concentrações também seria reduzida. No entanto, embora o reator tenha operado com relativa estabilidade, verificou-se formação de grande quantidade de EPS logo na primeira condição operacional, com aproximadamente, 500 mg/L de glicose no afluente, indicando que a hipótese não estava correta. / The main objectives of this study were to evaluate the performance of the anaerobic sequencing batch reactor when subjected to a progressive increasing of the influent glucose concentration and estimate the kinetic parameters of glucoses degradation. Initially the reactor was operated with 8-hour cycles, treating glucose at concentrations of 500, 1000 and 2000 mg/L. Glucose was not detected in the effluent in all these three conditions. The reactor showed operating stability treating glucose concentration of approximately 500mg/L, with efficiencies between 93% to 97% in the filtrated COD removal. In the operation with glucose concentrations of 1000 mg/L and 2000 mg/L, approximately, it could be noticed an operational instability, caused mainly by a production of extracellular polymers (EPS) leading to hydrodynamic and mass transfer problems in the reactor. The mean values of volatile acids concentration in the effluent were about 159 ± 72mg/L and 374 ± 92mg/L, respectively. A first order model was adjusted to glucose concentration profiles, and a modified model, including a residual concentration of substrate, was adjusted to COD temporal profiles. To verify the EPS formation, the reactor was operated with 3-hour cycle in the concentrations of 500 and 1000 mg/L This stage had the objective of verifying if the EPS production would result from the exposure of the biomass to low concentration of substrate for a long period of time. Thus, reducing the time cycle, the exposure to low concentrations would also be reduced. Nevertheless, even with the reactor operating with relative stability, the hypotheses could not be verified due the formation of a large amounts of EPS right in the first operational condition with approximately to 500 mg/L of glucose in the influent, showing that the hypothesis was not right.