High Rate Algae Ponds (HRAPs) are a promising technology for the treatment of municipal wastewater in locations with sufficient space and solar radiation. Algae-based processes do not require aeration, and thus have the potential to be less energy-intensive than activated sludge processes. We used a combination of LCA and LCCA analysis to evaluate the sustainability of HRAP systems, using data from the construction and operation of two demonstration-scale systems in Almería and Cádiz, Spain. As a reference for comparison, we used data from an activated sludge-based Sequencing Batch Reactor (SBR) treatment system in operation in Leppersdorf, Germany, which has comparable removal rates for a similar inflow. We focused solely on the actual wastewater treatment aspect of these technologies, excluding sludge treatment from this analysis. Based on our analysis, the current HRAP technology is more energy-efficient than activated sludge-based SBRs and requires only 22% of its electricity consumption. In addition, HRAP is more advantageous both economically (0.18 €/m³ versus 0.26 €/m³) and environmentally, with both lower global warming and eutrophication potentials (146.27 vs. 458.27 × 10⁻³ kg CO₂ equiv./m³; 126.14 vs. 158.01 × 10⁻⁶ kg PO₄ equiv./m³). However, the Net Environmental Benefit of SBR was slightly more favorable than of HRAP because of the higher removal rate for nutrients of SBR.

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The strong greenhouse gas nitrous oxide (N₂O) can be emitted from wastewater treatment systems as a byproduct of ammonium oxidation and as the last intermediate in the stepwise reduction of nitrate to N₂ by denitrifying organisms. A potential strategy to reduce N₂O emissions would be to enhance the activity of N₂O reductase (NOS) in the denitrifying microbial community. A survey of existing literature on denitrification in wastewater treatment systems showed that the N₂O reducing capacity (V_maxN2O→N2) exceeded the capacity to produce N₂O (V_maxNO3→N2O) by a factor of 2–10. This suggests that denitrification can be an effective sink for N₂O, potentially scavenging a fraction of the N₂O produced by ammonium oxidation or abiotic reactions. We conducted a series of incubation experiments with freshly sampled activated sludge from a wastewater treatment system in Oslo and found that the ratio α = V_maxN2O→N2/V_maxNO3→N2O fluctuated between 2 and 5 in samples taken at intervals over a period of 5 weeks. Adding a cocktail of carbon substrates resulted in increasing rates, but had no significant effect on α. Based on these results – complemented with qPCR and metaproteomic data – we discuss whether the overcapacity to reduce N₂O can be ascribed to gene/protein abundance ratios (nosZ/nir), or whether in-cell competition between the reductases for electrons could be of greater importance.

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Reduction of the greenhouse gas N₂O to N₂ is a trait among denitrifying and non-denitrifying microorganisms having an N₂O reductase, encoded by nosZ. The nosZ phylogeny has two major clades, I and II, and physiological differences among organisms within the clades may affect N₂O emissions from ecosystems. To increase our understanding of the ecophysiology of N₂O reducers, we determined the thermodynamic growth efficiency of N₂O reduction and the selection of N₂O reducers under N₂O- or acetate-limiting conditions in a continuous culture enriched from a natural community with N₂O as electron acceptor and acetate as electron donor. The biomass yields were higher during N₂O limitation, irrespective of dilution rate and community composition. The former was corroborated in a continuous culture of Pseudomonas stutzeri and was potentially due to cytotoxic effects of surplus N₂O. Denitrifiers were favored over non-denitrifying N₂O reducers under all conditions and Proteobacteria harboring clade I nosZ dominated. The abundance of nosZ clade II increased when allowing for lower growth rates, but bacteria with nosZ clade I had a higher affinity for N₂O, as defined by μ_max/K_s. Thus, the specific growth rate is likely a key factor determining the composition of communities living on N₂O respiration under growth-limited conditions.

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With its rapidly rising concentration in the atmosphere and its high global warming potential, N2O is arguably the greenhouse gas of the 21st century. The research carried out within the Nitrous Oxide Research Alliance (NORA) – of which this thesis forms part of – focused on the microbial conversions of N2O within the nitrogen cycle, the ultimate aim being to develop N2O mitigation strategies for natural and managed ecosystems such as agricultural soils and wastewater treatment plants. A variety of pathways in the nitrogen cycle produce N2O, but respiratory N2O reduction to N2 by microorganisms harboring an N2O reductase enzyme (encoded by the gene nosZ), is the only known microbial conversion that consumes N2O. N2O-respiring microorganisms may thus be key in this endeavour. Studies in literature reporting the cultivation of denitrifying bacteria with N2O as a sole electron acceptor date back to the 1950s and in recent years, there have been important discoveries of novel groups of denitrifying and non-denitrifying N2O reducing bacteria and archaea, and their importance for N2O reduction in the environment. Nevertheless, essential aspects of N2O reduction remain unclear and the aim of this thesis was to fill in some of the existing knowledge gaps regarding N2O-reducer ecophysiology, using wastewater treatment as a frame of reference. Our main approach was to study simplified, naturally selected, N2O reducing bacterial communities in chemostat enrichment cultures fed with N2O as the sole electron acceptor and acetate as electron donor. Continuous cultivation, which selects for a fairly simple community, is ideal for ecophysiology studies as it bridges the gap between ecosystem studies and pure culture work. Furthermore, it allows for cultivation under constant and limiting conditions...@en

Nitrous oxide (N₂O) reducing microorganisms may be key in the mitigation of N₂O emissions from managed ecosystems. However, there is still no clear understanding of the physiological and bioenergetic implications of microorganisms possessing either of the two N₂O reductase genes (nosZ), clade I and the more recently described clade II type nosZ. It has been suggested that organisms with nosZ clade II have higher growth yields and a lower affinity constant (K_s) for N₂O. We compared N₂O reducing communities with different nosZI/nosZII ratios selected in chemostat enrichment cultures, inoculated with activated sludge, fed with N₂O as a sole electron acceptor and growth limiting factor and acetate as electron donor. From the sequencing of the 16S rRNA gene, FISH and quantitative PCR of nosZ and nir genes, we concluded that betaproteobacterial denitrifying organisms dominated the enrichments with members within the family Rhodocyclaceae being highly abundant. When comparing cultures with different nosZI/nosZII ratios, we did not find support for (i) a more energy conserving N₂O respiration pathway in nosZ clade II systems, as reflected in the growth yield per mole of substrate, or (ii) a higher affinity for N₂O, defined by μ_max/K_s, in organisms with nosZ clade II.

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Despite its ecological importance, essential aspects of microbial N₂O reduction—such as the effect of O₂ availability on the N₂O sink capacity of a community—remain unclear. We studied N₂O vs. aerobic respiration in a chemostat culture to explore (i) the extent to which simultaneous respiration of N₂O and O₂ can occur, (ii) the mechanism governing the competition for N₂O and O₂, and (iii) how the N₂O-reducing capacity of a community is affected by dynamic oxic/anoxic shifts such as those that may occur during nitrogen removal in wastewater treatment systems. Despite its prolonged growth and enrichment with N₂O as the sole electron acceptor, the culture readily switched to aerobic respiration upon exposure to O₂. When supplied simultaneously, N₂O reduction to N₂ was only detected when the O₂ concentration was limiting the respiration rate. The biomass yields per electron accepted during growth on N₂O are in agreement with our current knowledge of electron transport chain biochemistry in model denitrifiers like Paracoccus denitrificans. The culture’s affinity constant (K_S) for O₂ was found to be two orders of magnitude lower than the value for N₂O, explaining the preferential use of O₂ over N₂O under most environmentally relevant conditions.

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