Anaerobic digestion (AD) is an essential process in wastewater treatment to stabilize waste organic solids and produce biogas. This research is comprised of two projects in the discipline of anaerobic digestion. First, the effect of high sulfate concentration on anaerobic digestion of wastewater sludge was investigated. Secondly, the performance of acetoclastic methanogens Methanosaeta spp. and Methanosarcina spp. were investigated under intensified AD operation conditions (i.e., elevated acetate concentrations, vigorous mixing, etc.). In the sulfate experiments, the cumulative biogas and methane production decreased linearly with increasing initial sulfate doses (0 – 3,300 mg S L-1) and the correlation between the sulfate dose and methane production was verified with theoretical predictions, indicating complete reduction of sulfate to sulfide in AD. The examined sulfate concentrations resulted in no clear negative effects on the COD (chemical oxygen demand) removal or VSS (volatile suspended solids) destruction of the wastewater sludge, indicating that previous findings on sulfide toxicity might have been attributed to potential COD overestimation of digested sludge with high levels of sulfide. To avoid potential misinterpretation of AD performance on sulfide toxicity effects, we proposed a new method for COD correction for digested sludge. In the second project focused on acetoclastic methanogens, vigorous mixing conditions substantially decreased Methanosarcina spp. growth and methane production, and the decreased methanogenesis was more pronounced at higher acetate concentrations. Methanosarcina spp. prefer to grow in clusters and the vigorous mixing can disrupt cluster formation; as a result, reduced chances for cluster formation limited the growth of Methanosarcina spp.. While Methanosarcina spp. growth and methane production increased with the increasing acetate concentration, Methanosaeta spp. growth was unaffected by the examined vigorous mixing and soluble substrate conditions with negligible relative growth. Thus, rapid enrichment of Methanosarcina spp. is critical for successful operation intensified of AD processes under high organic loading conditions. / Thesis / Master of Applied Science (MASc)

The purpose of this thesis was to experimentally investigate the influence of increasing sulfide concentrations on the 34S isotope enrichment in sulfate during dissimilatory sulfate reduction (DSR). Two independent batch culture experiments with different maximum sulfide concentrations of up to 20 mM in the first and up to 40 mM in the second experiment were conducted using the marine sulfate reducer Desulfobacter latus. A comparison of the results from both experiments revealed a distinct offset towards more positive δ34S(SO42-) values in the 'high-sulfide' experiment, compared to the 'low-sulfide' experiment. While a Rayleigh type fractionation model was able to match the slopes - i.e., enrichment factors - of both experiments, it failed to reproduce the proper y-axis intercept in the 'high-sulfide' experiment. I therefore propose a new fractionation model that allows for a backward flow of ambient H2S into the bacterial cell and a subsequent enzymatically mediated oxidation of H2S to sulfate. The new backward flow increases with elevated H2S concentrations and is described as a first order rate constant. Unlike a Rayleigh type fractionation model, my model explains the slope and y-intercept of both experiments with a single parameter set. The new model with H2S-reflux further suggests that it can be used to determine growth kinetic parameters like the half-saturation constant through δ34S measurements. These findings support the hypothesis of microbially mediated, bi-directional S-fluxes between oxidized and reduced sulfur species. Because the S-transport during DSR appears to be bi-directional, great care must be taken when evaluating culture experiments with a Rayleigh type fractionation model, owing to the fact that an evident S-backward flow violates the prerequisites for applying the Rayleigh model. A variable S-backward flow results in variable enrichment factors which increased from -11 (no H2S) to ≈-17 ‰ (40 mM of H2S) in my experiments. I show for the first time the significance of a bi-directional H2S transport across the cell membrane during DSR and its consequences for the 34S-isotope fractionation in sulfate.

The purpose of this thesis was to experimentally investigate the influence of increasing sulfide concentrations on the 34S isotope enrichment in sulfate during dissimilatory sulfate reduction (DSR). Two independent batch culture experiments with different maximum sulfide concentrations of up to 20 mM in the first and up to 40 mM in the second experiment were conducted using the marine sulfate reducer Desulfobacter latus. A comparison of the results from both experiments revealed a distinct offset towards more positive δ34S(SO42-) values in the 'high-sulfide' experiment, compared to the 'low-sulfide' experiment. While a Rayleigh type fractionation model was able to match the slopes - i.e., enrichment factors - of both experiments, it failed to reproduce the proper y-axis intercept in the 'high-sulfide' experiment. I therefore propose a new fractionation model that allows for a backward flow of ambient H2S into the bacterial cell and a subsequent enzymatically mediated oxidation of H2S to sulfate. The new backward flow increases with elevated H2S concentrations and is described as a first order rate constant. Unlike a Rayleigh type fractionation model, my model explains the slope and y-intercept of both experiments with a single parameter set. The new model with H2S-reflux further suggests that it can be used to determine growth kinetic parameters like the half-saturation constant through δ34S measurements. These findings support the hypothesis of microbially mediated, bi-directional S-fluxes between oxidized and reduced sulfur species. Because the S-transport during DSR appears to be bi-directional, great care must be taken when evaluating culture experiments with a Rayleigh type fractionation model, owing to the fact that an evident S-backward flow violates the prerequisites for applying the Rayleigh model. A variable S-backward flow results in variable enrichment factors which increased from -11 (no H2S) to ≈-17 ‰ (40 mM of H2S) in my experiments. I show for the first time the significance of a bi-directional H2S transport across the cell membrane during DSR and its consequences for the 34S-isotope fractionation in sulfate.

A tecnologia anaeróbia tem sido utilizada com sucesso no tratamento de água residuária contendo compostos fenólicos. Recentes pesquisas incluem tais compostos entre aqueles que podem ser degradados através desse processo. O objetivo desse trabalho foi avaliar a degradação do fenol em diferentes condições nutricionais, com ênfase na redução do sulfato. Os experimentos foram realizados com meio de cultura específico para esses microrganismos anaeróbios. Foram realizados ensaios de degradação em reatores em batelada alimentados nas seguintes condições: (1) fenol e sulfato, a diferentes concentrações, com inóculo previamente enriquecido; (2) fenol, sulfato e co-substratos e; (3) fenol, sulfato e extrato de levedura. Todos os ensaios foram realizados em temperatura de 30 graus Celsius, sob agitação de 150 rpm. Foi avaliado o consumo de fenol e sulfato e, produção de metano, em função do tempo, para diferentes concentrações iniciais de fenol e sulfato. Nos ensaios com reatores alimentados com fenol (329,3 mg/l); fenol (307,3 mg/l) e sulfato (160 mg/l); fenol (322.3 mg/l), sulfato (160 mg/l) e lactato (478,16 mg/l); fenol (332,1 mg/l), sulfato (150 mg/l) e etanol (129,76 mg/l), a remoção foi de, respectivamente, 99,8%, 98,2%, 98,8% e 98,8%. Os reatores alimentados com fenol (239,7 mg/l) obtiveram 100% de eficiência na degradação em apenas 11 dias e, os reatores alimentados com fenol (234,3 mg/l) e sulfato (162,5 mg/l) e fenol (256,0 mg/l) e sulfato (500 mg/l) tiveram eficiências de degradação de, respectivamente, 98,8% e 99,3% com 17 dias de operação. Tais eficiências foram obtidas pelo acréscimo de extrato de levedura nos reatores, no início dos ensaios. A caracterização morfológica foi realizada através de microscopia óptica. A diversidade microbiana referente aos Domínios Bacteria e Archaea, além do grupo de bactérias redutoras de sulfato foi avaliada através da técnica de PCR DGGE, onde foram observadas alterações nas populações microbianas, em função das condições nutricionais. Para o Domínio Archaea não foram observadas diferenças nos ensaios realizados. Para o Domínio Bacteria e Grupo das BRS essas diferenças foram, mais facilmente, percebidas com relação ao inóculo e entre os diversos reatores. A alteração na diversidade microbiana pode ter sido decorrente da composição do meio que, nesse caso, foi específico para BRS e a composição do inóculo que continha parte previamente adaptada às BRS. Essas condições adequadas puderam propiciar surgimento e desenvolvimento de populações microbianas capazes de degradar fenol, utilizando sulfato. / The anaerobic technology has been successfully used to treat wastewater containing phenolic compounds. Recent research includes such compounds among those that can be degraded through this process. The goal of this project was to assess phenol degradation in different nutritional conditions, focusing on sulfate reduction. The essays were carried out in a specific culture mean for these kinds of anaerobic microorganisms. Degradation essays were carried out in a batch reactor fed in the following ways: (1) phenol and sulfate, in different concentrations, with previously enriched inoculum; (2) phenol, sulfate and co-substrates and; (3) phenol, sulfate and yeast extract. All the essays were carried out at 30 Celsius degrees, under 150 rpm agitation. The consumption of phenol and sulfate and, methane production, in function of time, for different initial concentrations of phenol and sulfate was assessed. In the essays the reactors were fed with phenol (329.3 mg/l); phenol (307.3 mg/l) and sulfate (160 mg/l); phenol (322.3 mg/l), sulfate (160 mg/l) and lactate (478.16 mg/l); phenol (332.1 mg/l), sulfate (150 mg/l) and ethanol (129.76 mg/l), the removal was of, respectively, 99.8%, 98.2%, 98.8% and 98.8%. The reactors fed with phenol (239.7 mg/l) obtained 100% degradation efficiency in only 11 days. The reactors fed with phenol (234.3 mg/l) and sulfate (162.5 mg/l) and phenol (256.0 mg/l) and sulfate (500 mg/l) obtained degradation efficiency of 98.8% and 99.3%, respectively, in only 17 days of operation. Such efficiencies were obtained by the increase of yeast extract in the reactors, in the beginning of the essays. The morphological characterization was carried out by optical microscopy. The microbial diversity regarding the Bacteria and Archaea Domain, besides the sulfate reducing bacteria group was assessed through the PCR DGGE technique, where alterations were seen in the microbial population, due to nutritional conditions. For the Archaea Domain, no differences were seen in all the essays. For the Bacteria Domain and the SRB Group these differences were easily seen, noticed by the inoculum and the diverse reactors. The alteration in the microbial diversity might have occurred due to culture mean composition that, in this case, was specific for SRB and also due to inoculum composition that contained a part previously adapted to SRB. These adequate conditions could promote appearance and development of microbial population capable of degrading phenol, using sulfate.

This thesis is an investigation into biogenic sulfide oxidation and sulfate reduction associated with hydrothermal systems at oceanic spreading centers. First, the production of sulfur floc and 'snowblower' events due to sulfide oxidizing bacteria is investigated. The effects of sulfur floc on the pososity is shown to be negligible. 'Snowblower' events are shown to be sulfur floc that is stored over long periods of time mixed with a component of sulfur floc being created in a bloom event. Secondly, biogenic sulfate reduction in hydrothermal recharge zones is investigated and the effects on the concentration profiles is considered.

The overall objective of the research presented here was to combine multiple geochemical parameters and molecular characterizations to provide a novel view of active microbial community ecology of sediments in a large-river deltaic estuary. In coastal and estuarine environments, a large portion of benthic respiration has been attributed to sulfate reduction and implicated as an important mechanism in hypoxia formation. The use of high-resolution sampling of individual sediment cores and high throughput nucleic acid extraction techniques combined with 454 FLX sequencing provided a robust understanding of the metabolically active benthic microbial community within coastal sediments. This was used to provide further understanding and show the importance of simultaneously analyzing the connectivity of sulfur and iron cycling to the structure and function of the microbial population. Although aqueous sulfide did not accumulate in the sediments of the northern Gulf of Mexico, active sulfate reduction was observed in all locations sampled. Microbial recycling and sequestration as iron sulfides prevented the release of sulfide from the sediment. Prominent differences were observed between the sample locations and with depth into the sediment column. This study emphasized the importance of combining novel molecular techniques with simultaneous traditional geochemical measurements to show the interdependence of microbiology and geochemistry. In addition, this study highlights the need to consider microbial community biogeography along with small-scale variations in geochemistry and biology that impact the overall cycling of redox elements when constructing biogeochemical models in marine sediments.

The treatment of Mn and SO42- contaminated mine drainage via a sulfate reducing bioreactor is expected to result in near-permanent immobilization of significant amounts of Mn and a portion of the sulfates within the matrix. This study tested several different combinations of organic amendments and inorganic substrates in an attempt to optimize sulfate reducing conditions and Mn removal capacity. Five different organic carbon sources, including corn mash, wood mulch, biosolids, soybean oil, and sorghum syrup in combination with five different inorganic substrates, including creek sediment, marble and limestone chips, polished gravel, and sand were tested in batch experiments. Results indicate a widely Mn variant removal potential among the treatments, ranging from 35% for soybean oil to 97% for the mulch mixture, with respective Eh ranges of +60 mV and -320 mV. Sulfate removal ranged from less than 10% to 85%. The most favorable combinations were tested in small scale bioreactors under dynamic conditions. Greater than 90% of Mn and 70% of sulfate was removed over a 65 day test period. Results indicate Mn removal mechanisms include sulfide, oxide, and carbonate formation and simple sorption and SO42- removal mechanisms of sulfide gas evolution, gypsum and MnS precipitation, and anion sorption/cation bridging.

Abstract Dissimilatory microbial sulfate reduction (MSR) The specific objectives of the study were to provide the first measurements of sulfur isotope fractionation associated with acidophilic sulfate reducing-microorganisms, and to examine whether pH influences sulfur fractionation during MSR. The fractionation associated with the strains investigated was comparable to that of neutrophilic strains with similar metabolisms (4-12‰), but varied with pH. Two fractionation regimes were identified: one regime is consistent with fractionation during exponential growth, while the other – not identified previously - is not linked to active sulfate reduction and may result from internal sulfate accumulation. This would represent the first measurement of sulfur fractionation during sulfate uptake, the first step of MSR. Geological processes at the Cretaceous-Paleogene (KPg) boundary The KPg boundary is associated with one of the largest biological extinctions in the history of our planet. Two major geologic events - the Chicxulub bolide impact with evaporite terrane and the eruption of the Deccan continental flood basalts - coincide with the KPg boundary and have been identified as possible triggers for the extinctions, but their relative timing remains unresolved. The objectives of this study were to identify the contribution of these processes to the sulfur burden in the sedimentary environment of two freshwater KPg sections, and to determine their relative timing. The results demonstrate that the peak of Deccan volcanism post-dates the Chicxulub impact and the associated abrupt KPg mass extinction, thus precluding a direct volcanic causal mechanism, but shedding light on the underlying causes for the delayed recovery of ecosystems in the early Paleogene.

No

A tecnologia anaeróbia tem sido utilizada com sucesso no tratamento de água residuária contendo compostos fenólicos. Recentes pesquisas incluem tais compostos entre aqueles que podem ser degradados através desse processo. O objetivo desse trabalho foi avaliar a degradação do fenol em diferentes condições nutricionais, com ênfase na redução do sulfato. Os experimentos foram realizados com meio de cultura específico para esses microrganismos anaeróbios. Foram realizados ensaios de degradação em reatores em batelada alimentados nas seguintes condições: (1) fenol e sulfato, a diferentes concentrações, com inóculo previamente enriquecido; (2) fenol, sulfato e co-substratos e; (3) fenol, sulfato e extrato de levedura. Todos os ensaios foram realizados em temperatura de 30 graus Celsius, sob agitação de 150 rpm. Foi avaliado o consumo de fenol e sulfato e, produção de metano, em função do tempo, para diferentes concentrações iniciais de fenol e sulfato. Nos ensaios com reatores alimentados com fenol (329,3 mg/l); fenol (307,3 mg/l) e sulfato (160 mg/l); fenol (322.3 mg/l), sulfato (160 mg/l) e lactato (478,16 mg/l); fenol (332,1 mg/l), sulfato (150 mg/l) e etanol (129,76 mg/l), a remoção foi de, respectivamente, 99,8%, 98,2%, 98,8% e 98,8%. Os reatores alimentados com fenol (239,7 mg/l) obtiveram 100% de eficiência na degradação em apenas 11 dias e, os reatores alimentados com fenol (234,3 mg/l) e sulfato (162,5 mg/l) e fenol (256,0 mg/l) e sulfato (500 mg/l) tiveram eficiências de degradação de, respectivamente, 98,8% e 99,3% com 17 dias de operação. Tais eficiências foram obtidas pelo acréscimo de extrato de levedura nos reatores, no início dos ensaios. A caracterização morfológica foi realizada através de microscopia óptica. A diversidade microbiana referente aos Domínios Bacteria e Archaea, além do grupo de bactérias redutoras de sulfato foi avaliada através da técnica de PCR DGGE, onde foram observadas alterações nas populações microbianas, em função das condições nutricionais. Para o Domínio Archaea não foram observadas diferenças nos ensaios realizados. Para o Domínio Bacteria e Grupo das BRS essas diferenças foram, mais facilmente, percebidas com relação ao inóculo e entre os diversos reatores. A alteração na diversidade microbiana pode ter sido decorrente da composição do meio que, nesse caso, foi específico para BRS e a composição do inóculo que continha parte previamente adaptada às BRS. Essas condições adequadas puderam propiciar surgimento e desenvolvimento de populações microbianas capazes de degradar fenol, utilizando sulfato. / The anaerobic technology has been successfully used to treat wastewater containing phenolic compounds. Recent research includes such compounds among those that can be degraded through this process. The goal of this project was to assess phenol degradation in different nutritional conditions, focusing on sulfate reduction. The essays were carried out in a specific culture mean for these kinds of anaerobic microorganisms. Degradation essays were carried out in a batch reactor fed in the following ways: (1) phenol and sulfate, in different concentrations, with previously enriched inoculum; (2) phenol, sulfate and co-substrates and; (3) phenol, sulfate and yeast extract. All the essays were carried out at 30 Celsius degrees, under 150 rpm agitation. The consumption of phenol and sulfate and, methane production, in function of time, for different initial concentrations of phenol and sulfate was assessed. In the essays the reactors were fed with phenol (329.3 mg/l); phenol (307.3 mg/l) and sulfate (160 mg/l); phenol (322.3 mg/l), sulfate (160 mg/l) and lactate (478.16 mg/l); phenol (332.1 mg/l), sulfate (150 mg/l) and ethanol (129.76 mg/l), the removal was of, respectively, 99.8%, 98.2%, 98.8% and 98.8%. The reactors fed with phenol (239.7 mg/l) obtained 100% degradation efficiency in only 11 days. The reactors fed with phenol (234.3 mg/l) and sulfate (162.5 mg/l) and phenol (256.0 mg/l) and sulfate (500 mg/l) obtained degradation efficiency of 98.8% and 99.3%, respectively, in only 17 days of operation. Such efficiencies were obtained by the increase of yeast extract in the reactors, in the beginning of the essays. The morphological characterization was carried out by optical microscopy. The microbial diversity regarding the Bacteria and Archaea Domain, besides the sulfate reducing bacteria group was assessed through the PCR DGGE technique, where alterations were seen in the microbial population, due to nutritional conditions. For the Archaea Domain, no differences were seen in all the essays. For the Bacteria Domain and the SRB Group these differences were easily seen, noticed by the inoculum and the diverse reactors. The alteration in the microbial diversity might have occurred due to culture mean composition that, in this case, was specific for SRB and also due to inoculum composition that contained a part previously adapted to SRB. These adequate conditions could promote appearance and development of microbial population capable of degrading phenol, using sulfate.