Methane (CH4) and Nitrous Oxide (N2O) are both potent greenhouse gases which contribute to global warming by an estimated value of 20% and 6% respectively in addition to which their relative concentrations within the atmosphere are also on the increase at a rate of 0.8% and 0.3% yr-1 respectively over the past few decades (IPCC, 2007; Verma et al., 2006; Moiser et al., 1998). With the combined contribution of both CH4 and N2O constitute in relation to global warming, highlighting the importance of research into their reduction. Through the investigation of the process of methane oxidation and denitrification, both of which are bacterial processes which can lead to a reduction in the relative concentrations of both CH4 and N2O respectively within the atmosphere. Highlighting the importance of the research carried out within this thesis, in relation to the investigation of the potential coupling between methanotrophic and denitrifying bacteria, known as methanotrophic dependent denitrification (MDD) to occur within the environment. The initial experimental setup was designed to test whether soil derived and model denitrifying bacteria; Pseudomonas nitroreducens, Pseudomonas citronellolis and Paracoccus denitrificans, were able denitrify to N2 on the presumptive methanotrophic carbon exudates sodium acetate and sodium formate as their sole carbon source (Costa et a; 2000; Hanson & Hanson 1996; Rhee & Fuhs 1978). All of which was carried out in pure cultures, under ≤ 0.4 % O2 v/v headspace. 15N-labelling was carried out in order to obtain a more complete picture in relation to the production of N2, and the creation of a N2O:N2 product ratio for and whether there was any difference in the utilisation between the two carbon sources. The presented data demonstrated that Pseudomonas nitroreducens and Pseudomonas citronellolis were able to produce N2 on sodium acetate but not on sodium formate. This was followed by assessing how different oxygen headspace conditions 10, 3, 2, 1 and 0.4 % O2 v/v would affect the exudation of acetate by Type II soil derived methanotrophic bacteria Methylocystis parvus, Methylocystis rosea and Methylocystis trichosporium. The results demonstrated that the methanotrophic bacteria (i) exudate acetate (ii) the presence of the acetate exudate within the media was only detectable under microaerobic conditions of ≤ 2 % available O2 v/v within the headspace (iii) the concentration of acetate exudate present within the media increased as the available O2 v/v decreased from 2 % to ≤ 0.4 % O2 v/v available within the headspace. This was then followed by a series of experiments designed to assess the ability of Pseudomonas nitroreducens and Pseudomonas citronellolis denitrify to N2 on 0.22 μm filter sterilized acetate exudate bacteria from M. parvus, M. iv rosea and M. trichosporium grown under a ≤ 0.4 % O2 v/v headspace as their sole carbon source when also grown under the same ≤ 0.4 % O2 v/v headspace conditions. The results demonstrated that Pseudomonas nitroreducens and Pseudomonas citronellolis were able to denitrify on filter sterilized acetate exudate from the type II soil derived methanotrophic bacteria, under which the greatest concentration of acetate production was under a ≤ 0.4 % O2 headspace, demonstrating that the amount of acetate which is exuded by the Type II methanotrophic bacteria is great enough to support denitrification to N2. This was followed by a co-culture experimental setup in which both the methanotrophic and denitrifying bacteria were grown simultaneously in the same closed system, in which no bacterial mixing occurred.

Aerobic methanotrophs (MOB) are a functional group of proteobacteria that use methane as their only energy and carbon source. Phylogenetically, such methanotrophs are affiliated with present alpha and gamma proteobacteria. Methanotrophic microorganisms play an essential role in the methane cycle, since they to a great extent reduce potential methane emissions to the atmosphere. In stratified lakes the aerobic methanotrophs are commonly present at the oxic-anoxic interfaces. This study aims to identify the environmental factors that could regulate the distribution of aerobic methanotrophs in the water column of freshwater lakes as well to assess whether a vertical community structure exists there. Lakes Erken and Tämnaren located in central Swedenwere studied using molecular analysis and environmental data. Aerobic methanotrophs were detected throughout the water column of Lake Erken. Two types of methanotroph communities were identified, with distribution patterns that appeared to be correlated to levels of methane in the water. Furthermore one of these communities feature changes in the relative abundance of its member populations along a depth gradient suggesting further differentiation along chemical gradients in the hypolimnion. Sequencing of pmoA amplicons revealed only members of the gamma proteobacteria methanotrophs. Also two novel operational taxonomic units were identified. Methanotrophs communities between Tämnaren and Erken also differ. Together these results indicate the existence of distribution patterns in methanotroph freshwater communities.

The availability of substrates is one of the most important environmental constraints on the diversity and functioning of microorganisms. Substrate quantity and quality as well as the metabolic features of heterotrophic microorganisms determine the efficiency, speed and type of transformation that can occur in nature. As such their interplay with the environment regulates how much carbon and energy is incorporated by bacteria and subsequently reaches higher trophic levels. In lakes the bulk substrate that is available for bacteria is composed of a complex mixture of compounds, varying in lability and distribution in the environment. This thesis addresses the coupling of organic substrates, their metabolic use and the composition and ecology of the microbial community. Controlled laboratory experiments with mixed bacterial communities in either batch cultures or chemostats were designed to shed further light on bacterial use of labile and quantitatively significant carbon compounds. I show that different amino acid substrates only exert a minor influence on bacterioplankton community composition and growth. Hence the ability to use a wide range of such abundantly produced protein monomers seems to be widespread among freshwater bacteria. In contrast, when acetate was provided as the only carbon substrate, in either pulsed or continuous amendments, this very different substrate input mode had a strong effect on bacterial community composition. Biomass yield, for example, was twice as high when acetate was given in the form of pulses rather than provided continuously. In another set of experiments, I show that the oxidation of the globally significant greenhouse gas methane is a process that can potentially take place at the water-ice interface of seasonally ice-covered lakes and was not constrained by temperature as suggested in previous studies. This work also suggests that methane oxidation in ice-covered lakes can be constrained by competition for nutrients between specialized methanotrophs and heterotrophic bacteria. Combined these studies suggest that some labile substrates cause minor selection on bacterial community structure and functioning. This probably reflects the competitive advantage of using a broad range of low molecular weight substrates. However, as in the case of methanotrophs there is specialization for a specific low molecular weight substrate such as methane. In which case, competition with other community members i.e. for nutrients can constrain methane oxidation. In both cases it might however not depend just on the availability of substrate, but also on how substrates are distributed in time and space.

No description available.

Student Number : 9902262G - MSc Dissertation - School of Molecular and Cell Biology - Faculty of Science / Anaerobic fermentative bacteria degrade waste components in landfills where methane (CH4) and carbon dioxide (CO2) are the primary biogases emitted and methanotrophic bacteria in the cover soil oxidise the emitted CH4. Three bi-phasic bench-scale landfill bioreactors were commissioned to evaluate soil nutrient addition effects on CH4 formation and oxidation and to isolate inherent soil methanotrophs using Nitrate Mineral Salts (NMS) medium. Set A soil contained no nutrient additions, Set B soil contained 50 μM nitrate and 150 μM phosphate and Set C soil contained dried sewage cake. Bioreactors were run for a 4 week period and pH, anaerobic gas emissions, volatile fatty acids (VFA), bacterial counts and scanning electron microscopy (SEM) analyses were performed. A pilot study revealed that pH dictated the stability of methanogenesis, where increased VFA levels inhibited methanogenesis. Furthermore, it was revealed that modifications of the NMS medium were needed to enrich for methanotrophs. An in depth study showed that the Set C anaerobic reactor produced the most methane with Set B the least. The hypothesis that methane oxidation in the soil could regulate methane formation in the waste could not be conclusively observed, as a lack of aeration in the soil reactors is believed to have prevented the proliferation of methanotrophs here. No methanotrophs were successfully isolated from soil, but rather major heterotrophic bacterial interference was observed. SEM revealed the presence of rod and cocci forms of bacteria in both leachate and soil, consistent with literature reports, which indicated that the bench-scale landfill bioreactors were capable of promoting bacterial growth.

No description available.

Thesis (Ph. D.)--Auburn University, 2008. / Abstract. Vita. Includes bibliographical references.

Polyhydroxyalkanoates (PHAs) are a type of biologically-produced plastic known for their biocompatibility and biodegradability. They have the potential to replace petroleum-based plastics as an environmentally-friendly alternative. This is beneficial because the release of plastics into environments such as the ocean and the buildup of plastics in landfills are major concerns facing society today. Currently, however, PHAs are significantly more expensive than their petroleum-based counterparts. This is largely due to the cost of carbon sources and of extracting the bioplastics from bacteria. The goal of these studies was to examine replacing traditional carbon sources used in PHA production like sugar and oils with sustainable carbon sources and to improve extraction procedures by inducing secretion of PHAs in bacteria. A few sustainable carbon sources were examined for use in PHA production. First, studies focused on the conversion of food waste into PHAs were reviewed. It was shown that utilizing food wastes as carbon sources may be a viable approach to producing PHAs. A second carbon source examined was methanol. A novel isolate of Methylobacterium that demonstrated the ability to produce PHAs from methanol was identified. A system of secreting PHAs that was constructed using synthetic biological engineering approach was introduced to this isolate. This secretion system was not shown to improve extraction of PHAs in Methylobacterium in its current form.

L'oxydation anaérobie du méthane (AOM) couplé à la réduction du sulfate (AOM-SR) est un processus biologique médié par méthanotrophes anaérobie (ANME) et de bactéries sulfato-réductrices. La communauté scientifique s'inquiète de AOM, en raison de sa pertinence dans la régulation du cycle global du carbone et de la potentielle application biotechnologique pour le traitement de sulfate riches eaux usées.Pour améliorer les connaissances récentes sur les conditions de distribution et d'enrichissement ANME, cette recherche a étudié AOM-SR avec les objectifs suivants: (i) caractériser les communautés microbiennes responsables de AOM dans les sédiments marins, (ii) de les enrichir dans les bioréacteurs avec différentes configurations, à savoir bioréacteur à membrane (MBR), filtre biotrickling (BTF) et bioréacteur à haute pression (HPB), et (iii) d'évaluer l'activité de l'ANME et le processus AOM dans différentes conditions de pression et de température.Les microbes habitant peu profonde dans les sédiments de Marine lac Grevelingen (Pays-Bas) ont été caractérisés et leur capacité de faire AOM-SR a été évaluée. Un test d'activité a été réalisée en discontinu pour 250 jours, AOM-SR est mise en évidence par la production de sulfure et de la prise concomitante de sulfate et de méthane dans des rapports équimolaires et il a été atteint 5 µmoles par gramme de poids par jour de taux de réduction du sulfate. L'analyse des séquences de gènes 16SrRNA a montré la présence de méthanotrophes anaérobie ANME-3 dans les sédiments marins du lac Grevelingen.Deux configurations de bioréacteurs, à savoir MBR et BTF ont été opérés dans des conditions ambiantes pendant 726 jours et 380 jours, respectivement, pour enrichir les micro-organismes de Ginsburg Mud Volcano performantes AOM. Les réacteurs sont exploités en mode fed-batch pour la phase liquide avec un apport continu de méthane. Dans le MBR, une membrane d'ultrafiltration externe a été utilisée pour retenir la biomasse, alors que, dans la BTF, la rétention de biomasse a été accomplie par la fixation de la biomasse sur le matériau d'emballage. AOM-SR a été enregistrée seulement après ~ 200 jours dans les deux configurations de bioréacteurs. L'opération du BTF a montré l'enrichissement de l'ANME dans le biofilm par la méthode Illumina Miseq, en particulier ANME-1 (40%) et ANME-2 (10%). Dans le MBR, les agrégats d'ANME-2 et Desulfosarcina ont été visualisées par CARD-FISH. La production d'acétate a été observée dans le MBR, ce qui indique que l'acétate était un possible intermédiaire d'AOM. Bien que les deux configurations de bioréacteurs ont montré de bonnes performances, le taux de réduction du sulfate était légèrement plus élevée et plus rapide dans la BTF (1,3 mM par jour âpres 280 jours) que le MBR (0,5 mM par jour jour âpres 380 jours).Afin de simuler les conditions de suintement froid et de différencier l'impact des conditions environnementales sur AOM, les sédiments fortement enrichi avec le clade ANME-2a ont été incubées dans HPB à différentes températures (4, 15 et 25 °C à 100 bars) et pressions (20, 100, 200 et 300 bar à 15 °C). L'incubation à une pression de 100 bar et 15 ° C a été observé comme la condition la plus appropriée pour la phylotype ANME-2a, qui est similaire aux conditions in situ (Capitaine Aryutinov Mud Volcano, Golfe de Cadix). L'incubation de ce sédiment aux conditions in situ pourrait être une option privilégiée pour obtenir une activité AOM-SR plus élevée.Dans cette thèse, il a été démontré expérimentalement que la rétention de la biomasse et l'approvisionnement continu de méthane peuvent favoriser la croissance de la lente communauté microbienne qui oxyde le méthane en anaérobiose dans des bioréacteurs, même dans des conditions ambiantes. Par conséquent, la localisation des habitats de ANME dans des environnements peu profonds et l'enrichissant dans des conditions ambiantes peut être avantageuse pour les futures applications de la biotechnologie environnementale / Anaerobic oxidation of methane (AOM) coupled to sulfate reduction (AOM-SR) is a biological process mediated by anaerobic methanotrophs (ANME) and sulfate reducing bacteria. Due to its relevance in regulating the global carbon cycle and potential biotechnological application for treating sulfate-rich wastewater, AOM-SR has drawn attention from the scientific community. However, the detailed knowledge on ANME community, its physiology and metabolic pathway are scarcely available, presumably due to the lack of either pure cultures or the difficulty to enrich the biomass. To enhance the recent knowledge on ANME distribution and enrichment conditions, this research investigated AOM-SR with the following objectives: (i) characterize the microbial communities responsible for AOM in marine sediment, (ii) enrich ANME in different bioreactor configurations, i.e. membrane bioreactor (MBR), biotrickling filter (BTF) and high pressure bioreactor (HPB), and (iii) assess the AOM-SR activity under different pressure and temperature conditions.The microbes inhabiting coastal sediments from Marine Lake Grevelingen (the Netherlands) was characterized and the ability of the microorganisms to carry out AOM-SR was assessed. By performing batch activity tests for over 250 days, AOM-SR was evidenced by sulfide production and the concomitant consumption of sulfate and methane at approximately equimolar ratios and a sulfate reduction rate of 5 µmol sulfate per gram dry weight per day was attained. Sequence analysis of 16S rRNA genes showed the presence of ANME-3 in the Marine Lake Grevelingen sediment.Two bioreactor configurations, i.e. MBR and BTF were operated under ambient conditions for 726 days and 380 days, respectively, to enrich the microorganisms from Ginsburg Mud Volcano performing AOM. The reactors were operated in fed-batch mode for the liquid phase with a continuous supply of gaseous methane. In the MBR, an external ultra-filtration membrane was used to retain the biomass, whereas, in the BTF, biomass retention was achieved via biomass attachment to the packing material. AOM-SR was recorded only after ~ 200 days in both bioreactor configurations. The BTF operation showed the enrichment of ANME in the biofilm by Illumina Miseq method, especially ANME-1 (40%) and ANME-2 (10%). Interestingly, in the MBR, aggregates of ANME-2 and Desulfosarcina were visualized by CARD-FISH. Acetate production was observed in the MBR, indicating that acetate was a possible intermediate of AOM. Although both bioreactor configurations showed good performance and resilience capacities for AOM enrichment, the sulfate reduction rate was slightly higher and faster in the BTF (1.3 mM day-1 at day 280) than the MBR (0.5 mM day-1 at day 380).In order to simulate cold seep conditions and differentiate the impact of environmental conditions on AOM activities, sediment highly enriched with the ANME-2a clade was incubated in HPB at different temperature (4, 15 and 25 oC at 100 bar) and pressure (20, 100, 200 and 300 bar at 15 oC) conditions. The incubation at 100 bar pressure and 15 oC was observed to be the most suitable condition for the ANME-2a phylotype, which is similar to in-situ conditions where the biomass was sampled, i.e. Captain Aryutinov Mud Volcano, Gulf of Cadiz. The incubations at 200 and 300 bar pressures showed the depletion in activities after 30 days of incubation. Incubation of AOM hosting sediment at in-situ condition could be a preferred option for achieving high AOM activities and sulfate reduction rates.In this thesis, it has been experimentally demonstrated that biomass retention and the continuous supply of methane can favor the growth of the slow growing anaerobic methane oxidizing community in bioreactors even under ambient conditions. Therefore, locating ANME habitats in shallow environments and enriching them at ambient conditions can be advantageous for future environmental biotechnology applications

Os microrganismos são responsáveis por diversos processos biológicos essenciais ao ambiente, sendo estes intimamente relacionados com as taxas de decomposição da matéria orgânica e com a persistência da fertilidade nos solos. Apesar da importância e grande diversidade, a identificação de táxons envolvidos em processos específicos está geralmente restrita a uma pequena fração da microbiota que pode ser isolada e cultivada. Sendo assim, pouco se sabe sobre os microrganismos que atuam no ciclo do carbono no solo, como, aqueles que participam da oxidação do metano (CH4), por exemplo. Estes, chamados metanotróficos exercem papel importante no controle da emissão desse gás de efeito estufa para a atmosfera podendo servir como um filtro de metano e mitigar suas emissões. A Terra Preta Antropogênica (TPA) é um importante ecossistema na região amazônica e contém fragmentos cerâmicos e frações orgânicas, como o carvão (biocarvão), que foram incorporados em períodos pré-colombianos. Isso resultou em solos sustentáveis com elevada fertilidade, apresentando cerca de três vezes mais matéria orgânica, setenta vezes mais biocarvão e diversidade microbiana maior quando comparados com os solos adjacentes. Com o presente trabalho, objetivou-se avaliar o efeito do enriquecimento atmosférico com metano sobre a abundância da comunidade bacteriana total e de metanotróficas nos solos de Terra Preta da Amazônia sob floresta e cultivo (TPA Floresta e TPA Cultivada) e seus respectivos solos adjacentes (ADJ Floresta e ADJ Cultivado), coletados Estação Experimental do Caldeirão (Iranduba, AM). Para tanto, foi realizado um experimento de microcosmo no qual os solos foram incubados com atmosfera contendo 10% de metano e meio de cultura NMS (do inglês, Nitrate mineral salts), utilizado para crescimento de metanotróficas, a fim de avaliar a resposta dessas comunidades ao longo de 21 dias. A variação da concentração de metano na atmosfera dos frascos foi monitorada através de cromatografia gasosa e o DNA do solo recuperado nos tempos de coleta durante o experimento foi extraído para utilização na técnica de PCR quantitativo (qPCR), a qual possibilitou a quantificar o número de cópias dos genes 16S rRNA Bacteria e pmoA nas amostras. O solo de Terra Preta da Amazônia se mostrou um potencial dreno de CH4 atmosférico Comparando as respostas dos solos com floresta (TPA Floresta e ADJ Floresta) e cultivados (TPA Cultivada e ADJ Cultivado), notou-se uma menor variação da abundância da comunidade metanotrófica presente nestes últimos, o que indica que alteração do uso do solo afeta a capacidade do mesmo em retirar metano da atmosfera. Os solos Adjacentes apresentaram resposta diferente dos solos de TPA, indicando que a história de formação, ocupação e uso do solo também influenciam na capacidade do solo em drenar o metano da atmosfera. / Microorganisms are responsible for several biological processes essential to the environment, which are closely related to the rates of decomposition of organic matter and with the persistence of fertility in soils. Despite the importance and high diversity, identification of taxa involved in specific processes is usually restricted to a small fraction of the microbiota that can be isolated and cultivated. Thus, little is known about the microorganisms that act on the carbon cycle in the soil, such as those participating in the oxidation of methane (CH4), for example. These, known as methanotrophs play an important role in controlling the emission of greenhouse gas into the atmosphere and may serve as a methane filter and mitigate their emissions. Amazonian Dark Earth (ADE) is an important ecosystem in the Amazonian region and contains ceramic fragments and organic amendments, such as charcoal (biochar), which were incorporated in Pre-Columbian periods. This resulted in sustainable soils with high fertility, presenting about three times more organic matter, seventy times more biochar and higher microbial diversity when compared to adjacent soils. The present work aimed to evaluate the effect of atmospheric methane enrichment on the abundance of the bacterial and methanotrophic community in ADE soils under forest and cultivation (ADE Forest and ADE Cultivated) and their respective adjacent soils (ADJ Forest and ADJ Cultivated), sampled at Caldeirão Experimental Station (Iranduba, AM). For this purpose, a microcosm experiment was performed in which the soils were incubated under an atmosphere containing 10% of methane and NMS (Nitrate mineral salts) culture medium used for methanotrophic growth in order to evaluate the response of these communities over 21 days. The variation of methane concentrations in the atmosphere of the vials was measured by gas chromatography and the soil DNA recovered in the collection time during the experiment was extracted for use in the technique of quantitative PCR (qPCR), which made it possible to quantify the number of copies of 16S rRNA Bacteria and pmoA on samples. The Amazonian Dark Earth soil showed a potential sink for atmospheric CH4. Comparing atmospheric responses of forest soils (ADE Forest and ADJ Forest) and cultivated soils (ADE Cultivated and ADJ Cultivated), noted a minor variation in the abundance of methatroph community in these last, indicating that land use change affects the ability of it to sink the methane atmosphere. Adjacent soils had different responses of ADE soils, indicating that the history formation, occupation and land use also influence the capacity of the soil to drain methane from the atmosphere.