Assessing the life-cycle sustainability of algae and bacteria-based wastewater treatment systems: High-rate algae pond and sequencing batch reactor.

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.

Denitrification as an N2O sink.

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 (VmaxN2O→N2) exceeded the capacity to produce N2O (VmaxNO3→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 = VmaxN2O→N2/VmaxNO3→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.

O2 versus N2O respiration in a continuous microbial enrichment.

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 (KS) 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.

Growth yield and selection of nosZ clade II types in a continuous enrichment culture of N2 O respiring bacteria.

Nitrous oxide (N2 O) reducing microorganisms may be key in the mitigation of N2 O emissions from managed ecosystems. However, there is still no clear understanding of the physiological and bioenergetic implications of microorganisms possessing either of the two N2 O 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 (Ks ) for N2 O. We compared N2 O reducing communities with different nosZI/nosZII ratios selected in chemostat enrichment cultures, inoculated with activated sludge, fed with N2 O 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 N2 O respiration pathway in nosZ clade II systems, as reflected in the growth yield per mole of substrate, or (ii) a higher affinity for N2 O, defined by µmax /Ks , in organisms with nosZ clade II.

Life on N2O: deciphering the ecophysiology of N2O respiring bacterial communities in a continuous culture.

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 µmax/Ks. Thus, the specific growth rate is likely a key factor determining the composition of communities living on N2O respiration under growth-limited conditions.

Exploring microbial N2 O reduction: a continuous enrichment in nitrogen free medium.

N2 O is a potent greenhouse gas, but also a potent electron acceptor. In search of thermodynamically favourable - yet undescribed - metabolic pathways involving N2 O reduction, we set up a continuous microbial enrichment, inoculated with activated sludge, fed with N2 O as the sole electron acceptor and acetate as an electron donor. A nitrogen-free mineral medium was used with the intention of creating a selective pressure towards organisms that would use N2 O directly as source of nitrogen for cell synthesis. Instead, we obtained a culture dominated by microorganisms of the Rhodocyclaceae family growing by N2 O reduction to N2 coupled to N2 fixation. Biomass yields of this culture were 40% lower than those of a previously reported culture grown under comparable conditions but with an NH4+-amended medium, as expected from the extra energy expense of N2 fixation. Interestingly, we found no significant difference in yields whether N2 O or acetate was the growth-limiting substrate in the chemostat in contrast to the study with NH4+-amended medium, in which biomass yields were roughly 30% lower during acetate limiting conditions.

Dark fermentation: isolation and characterization of hydrogen-producing strains from sludges.

To improve bacterial hydrogen production, ten hydrogen-producing strains belonging to Clostridium spp. were isolated from various sludges under low vacuum. Hydrogenogenesis by dark fermentation in batch cultures of these strains was optimal at about 35 degrees C and an initial pH of 6.5, which for all strains gradually dropped to ca. pH 4 during the fermentation. Clostridium roseum H5 and C. diolis RT2 had the highest hydrogen yields per total substrate (120 ml H2/g initial COD). Substrate consumption alone by C. beijerinckii UAM and C. diolis RT2 reached 573 and 475 ml H2/g consumed COD, respectively. Butyric acid fermentation was predominant, with butyrate and acetate as the major by-products and propionate, ethanol, and lactate as secondary metabolites. The acetate:butyrate ratios and fermentation pathways varied depending on the strains and environmental conditions. Hydrogenogenesis was studied in greater detail in C. saccharobutylicum H1. In butyric acid fermentation by this representative strain, acetoacetate was detected as an intermediate metabolite. Hydrogenogenesis was also analyzed in an enrichment culture, which behaved similarly to the axenic cultures.

Dark fermentation: isolation and characterization of hydrogen-producing strains from sludges

To improve bacterial hydrogen production, ten hydrogen-producing strains belonging to Clostridium spp. were isolated from various sludges under low vacuum. Hydrogenogenesis by dark fermentation in batch cultures of these strains was optimal at about 35 ºC and an initial pH of 6.5, which for all strains gradually dropped to ca. pH 4 during the fermentation. Clostridium roseum H5 and C. diolis RT2 had the highest hydrogen yields per total substrate (120 ml H2/g initial COD). Substrate consumption alone by C. beijerinckii UAM and C. diolis RT2 reached 573 and 475 ml H2/g consumed COD, respectively. Butyric acid fermentation was predominant, with butyrate and acetate as the major by-products and propionate, ethanol, and lactate as secondary metabolites. The acetate: butyrate ratios and fermentation pathways varied depending on the strains and environmental conditions. Hydrogenogenesis was studied in greater detail in C. saccharobutylicum H1. In butyric acid fermentation by this representative strain, acetoacetate was detected as an intermediate metabolite. Hydrogenogenesis was also analyzed in an enrichment culture, which behaved similarly to the axenic cultures (AU)

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