The greenhouse gas nitrous oxide (N2O) is produced mainly by the microbial processes of nitrification and denitrification. I hypothesized that microbial community structure (composition and abundance) is linked to differences in soil N2O emissions from these two processes. Microbial community composition (type and number of nitrifier and denitrifier genotypes), abundance and N2O emission activity were determined and compared for soils from two landscapes characteristic of the North American prairie pothole region (cultivated vs. uncultivated wetlands). The landscape difference in composition of individual microbial communities was not predictive of soil N2O emissions, indicating that there is redundancy in each microbial community in relation to N2O emission activities. However, community factors influenced the pattern and distribution of N2O emission from the soils of the study site. For example, nitrification was the dominant N2O emitting process for soils of all landforms. However, neither nitrifier amoA abundance nor community composition had predictive relationships with nitrification associated N2O emissions. This lack of relationship may be a consequence of using amoA as the gene target to characterize nitrifiers. For denitrifying bacteria, there was a temporal relationship between community composition and N2O emissions. However, this may be related to the change in water-filled pore space over time. Alternatively, the presence of fungi can be linked directly to N2O emissions from water accumulating landform elements. Under hypoxic conditions, there may be two fungal pathways contributing to N2O release: fungal denitrification via P450nor and fungal heterotrophic nitrification. Results suggest that the relative importance of these two processes is linked to root exudates such as formate. It is the interaction between the seasonal fluctuations of the microbial and environmental factors that determine the level of N2O emissions from soils.

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Thesis (M.S. in E.A.S.)--School of Earth and Atmospheric Sciences, Georgia Institute of Technology, 2004. Directed by Ellery Ingall. / Includes bibliographical references (leaves 133-140).

Denitrification is the process by which bacteria reduce nitrate to dinitrogen gas. Most denitrifying bacteria perform these reactions under anaerobic conditions only. Thiosphaera pantotropha is one of a number of species capable of aerobic denitrification. During aerobic growth T. pantotropha expresses a periplasmic nitrate reductase but under anaerobic growth conditions nitrate is reduced by a membrane-bound nitrate reductase. The periplasmic nitrate reductase is relatively insensitive to azide and does not reduce chlorate. Aerobic denitrification provides a mechanism to dispose of excess reducing equivalents during growth on reduced carbon sources. Numbers of nitrate reducing bacteria, and nitrate and ammonia concentrations were monitored in a Norfolk broad over a 12 month period. Several novel microorganisms capable of aerobic nitrate respiration were isolated from the sediment of this broad. All were shown to express a periplasmic nitrate reductase activity, and the effects of growth rate and carbon substrate on the activity of this enzyme were studied. Of the nine isolates studied, five were shown to be able to reduce nitrate at oxygen concentrations up to 80% of air saturation. The remaining four were shown to be able to reduce nitrate under anaerobic conditions. Analysis of 16S rDNA sequences was used to identify the isolates, seven were assigned to the Genus Aeromonas, and two to the Genus Pseudomonas. Sediment samples were used to establish a microcosm in which changes in the concentration of nitrate, nitrite and ammoniacal nitrogen were monitored.

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.

Nitrate reductase (Nar) of F. canadensis is membrane-bound. Glucose is the most effective reductant to support nitrate uptake, and methyl and benzyl viologens are good electron donors to Nar both in intact cells and in membrane fractions. Nitrate uptake depends upon nitrate reduction, and requires the presence of active Nar. Nitrate transport depends upon the transmembrane pH gradient. / Oxygen reversibly inhibits nitrate uptake, and the minimal air saturation for this inhibition is about 2-4%. Oxygen inhibits denitrification at the level of nitrate transport rather than its reduction. The reduction of both nitric oxide (NO) and nitrous oxide by F. canadensis is relatively tolerant to oxygen, and its nitrite reductase (Nir) is much more sensitive to oxygen than the other reductases. Neither copper- nor heme-type Nir DNA probes from Pseudomonas species hybridized with the total DNA of F. canadensis, indicating that F. canadensis Nir may possess unique properties. / F. canadensis keeps the NO concentration very low under normal conditions. However, ionophores (carbonyl cyanide m-chlorophenylhydrazone (CCCP), carbonyl cyanide p-trifluoromethoxylphenylhydrazone (FCCP), and nigericin), high concentrations of nitrite, and low pH stimulate net NO production during reduction of nitrite. NO consumption by F. canadensis inhibited by several inhibitors. They are azide, cyanide, CCCP, FCCP, nigericin, sulfide, hydroxylamine, carbon monoxide, diethyldithiocarbamate, and Triton X-100. NO is toxic to Nor (nitric oxide reductase) only at concentrations $>$67 nM. / Studies on chloramphenicol inhibition of denitrification enzyme activity indicate that chloramphenicol inhibits denitrification at the levels of nitrate reduction and NO consumption in F. canadensis.

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Methane can be a potentially inexpensive, widely available electron donor for biological denitrification of wastewater, landfill leachate or drinking water, while no studies have clearly shown nitrate reduction to nitrogen gas. Recently anaerobic methane oxidation (AMO) coupled to partial denitrification (nitrite to nitrogen gas) was found by several studies. A microbial consortium, enriched from anoxic sediments, oxidized methane to carbon dioxide coupled to denitrification in the complete absence of oxygen, though the rates and pathways of AMO coupled to denitrification are still poorly understood. In this study, direct AMO coupled to denitrificaiton of nitrate was proved to be possible and its kinetic parameters were experimentally determined. Using a set of batch experiments designed to provide the best estimates of each parameter, these parameters were obtained: maximum specific growth rate (??max) = 0.121/day, maximum substrate consumption rate (qmax) = 1.63 mg COD/mg cells-day, true yield (Y) = 0.074 mg cells/mg COD, half maximum-rate substrate concentration (Ks) = 85 ??M CH4, and endogenous decay rate (b) = 0.03/day. This study firstly characterized kinetic parameters of anaerobic methanotrophic denitrifiers, which will substantially help understand anaerobic methane oxidation in natural systems and accelerate methane-utilizing denitrification in engineering systems.

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