Stephanie N. Merbt

Differential photoinhibition of bacterial and archaeal ammonia oxidation

Inhibition by light potentially influences the distribution of ammonia oxidizers in aquatic environments and is one explanation for nitrite maxima near the base of the euphotic zone of oceanic waters. Previous studies of photoinhibition have been restricted to bacterial ammonia oxidizers, rather than archaeal ammonia oxidizers, which dominate in marine environments. To compare the photoinhibition of bacterial and archaeal ammonia oxidizers, specific growth rates of two ammonia-oxidizing archaea (Nitrosopumilus maritimus and Nitrosotalea devanaterra) and bacteria (Nitrosomonas europaea and Nitrosospira multiformis) were determined at different light intensities under continuous illumination and light/dark cycles. All strains were inhibited by continuous illumination at the highest intensity (500 μE m(-2) s(-1)). At lower light intensities, archaeal growth was much more photosensitive than bacterial growth, with greater inhibition at 60 μE m(-2) s(-1) than at 15 μE m(-2) s(-1), where bacteria were unaffected. Archaeal ammonia oxidizers were also more sensitive to cycles of 8-h light/16-h darkness at two light intensities (60 and 15 μE m(-2) s(-1)) and, unlike bacterial strains, showed no evidence of recovery during dark phases. The findings provide evidence for niche differentiation in aquatic environments and reduce support for photoinhibition as an explanation of nitrite maxima in the ocean.

Wastewater Treatment Plant Effluents Change Abundance and Composition of Ammonia-Oxidizing Microorganisms in Mediterranean Urban Stream Biofilms

Streams affected by wastewater treatment plant (WWTP) effluents are hotspots of nitrification. We analyzed the influence of WWTP inputs on the abundance, distribution, and composition of epilithic ammonia-oxidizing (AO) assemblages in five Mediterranean urban streams by qPCR and amoA gene cloning and sequencing of both archaea (AOA) and bacteria (AOB). The effluents significantly modified stream chemical parameters, and changes in longitudinal profiles of both NH4+ and NO3− indicated stimulated nitrification activity. WWTP effluents were an allocthonous source of both AOA, essentially from the Nitrosotalea cluster, and mostly of AOB, mainly Nitrosomonas oligotropha, Nitrosomonas communis, and Nitrosospira spp. changing the relative abundance and the natural composition of AO assemblages. Under natural conditions, Nitrososphaera and Nitrosopumilus AOA dominated AO assemblages, and AOB were barely detected. After the WWTP perturbation, epilithic AOB increased by orders of magnitude whereas AOA did not show quantitative changes but a shift in population composition to dominance of Nitrosotalea spp. The foraneous AOB successfully settled in downstream biofilms and probably carried out most of the nitrification activity. Nitrosotalea were only observed downstream and only in biofilms exposed to either darkness or low irradiance. In addition to other potential environmental limitations for AOA distribution, this result suggests in situ photosensitivity as previously reported for Nitrosotalea under laboratory conditions.

Small-scale heterogeneity of microbial N uptake in streams and its implications at the ecosystem level.

Large-scale factors associated with the environmental context of streams can explain a notable amount of variability in patterns of stream N cycling at the reach scale. However, when environmental factors fail to accurately predict stream responses at the reach level, focusing on emergent properties from small-scale heterogeneity in N cycling rates may help understand observed patterns in stream N cycling. To address how small-scale heterogeneity may contribute to shape patterns in whole-reach N uptake, we examined the drivers and variation in microbial N uptake at small spatial scales in two stream reaches with different environmental constraints (i.e., riparian canopy). Our experimental design was based on two ¹⁵N additions combined with a hierarchical sampling design from reach to microhabitat scales. Regardless of the degree of canopy cover, small-scale heterogeneity of microbial N uptake ranged by three orders of magnitude, and was characterized by a low abundance of highly active microhabitats (i.e., hot spots). The presence of those hot spots of N uptake resulted in a nonlinear spatial distribution of microbial N uptake rates within the streambed, especially in the case of epilithon assemblages. Small-scale heterogeneity in N uptake and turnover rates at the microhabitat scale was primarily driven by power relationships between N cycling rates and stream water velocity. Overall, fine benthic organic matter (FBOM) assemblages responded clearly to changes in the degree of canopy cover, overwhelming small-scale heterogeneity in its N uptake rates, and suggesting that FBOM contribution to whole-reach N uptake was principally imposed by environmental constraints from larger scales. In contrast, N uptake rates by epilithon showed no significant response to different environmental influences, but identical local drivers and spatial variation in each study reach. Therefore, contribution of epilithon assemblages to whole-reach N uptake was mainly associated with emerging properties from small-scale heterogeneity at lower spatial scales.

Development of a 16S rRNA-targeted fluorescence in situ hybridization probe for quantification of the ammonia-oxidizer Nitrosotalea devanaterra and its relatives

The Thaumarchaeota SAGMCG-1 group and, in particular, members of the genus Nitrosotalea have high occurrence in acidic soils, the rhizosphere, groundwater and oligotrophic lakes, and play a potential role in nitrogen cycling. In this study, the specific oligonucleotide fluorescence in situ hybridization probe SAG357 was designed for this Thaumarchaeota group based on the available 16S rRNA gene sequences in databases, and included the ammonia-oxidizing species Nitrosotalea devanaterra. Cell permeabilization for catalyzed reporter deposition fluorescence in situ detection and the hybridization conditions were optimized on enrichment cultures of the target species N. devanaterra, as well as the non-target ammonia-oxidizing archaeon Nitrosopumilus maritimus. Probe specificity was improved with a competitor oligonucleotide, and fluorescence intensity and cell visualization were enhanced by the design and application of two adjacent helpers. Probe performance was tested in soil samples along a pH gradient, and counting results matched the expected in situ distributions. Probe SAG357 and the CARD-FISH protocol developed in the present study will help to improve the current understanding of the ecology and physiology of N. devanaterra and its relatives in natural environments.

Differences in ammonium oxidizer abundance and N uptake capacity between epilithic and epipsammic biofilms in an urban stream

The capacity of stream biofilms to transform and assimilate N in highly N-loaded streams is essential to guarantee the water quality of freshwater resources in urbanized areas. However, the degree of N saturation experienced by urban streams and their response to acute increases in N concentration are largely unknown. We measured changes in the rates of NH4+ uptake (UNH4) and oxidation (UAO) resulting from experimental increases in NH4+-N concentration in mature biofilms growing downstream of a wastewater treatment plant (WWTP) and, thus, naturally exposed to high N concentration. We investigated the responses of UNH4 and UAO to NH4+-N increases and the abundance of NH4+ oxidizing bacteria and archaea (AOB and AOA) in epilithic and epipsammic biofilms. UNH4 and UAO increased with increasing NH4+-N concentration for the 2 biofilm types, suggesting no N saturation under ambient levels of NH4+-N. Thus, these biofilms can contribute to mitigating N excesses and the variability of NH4+-N concentrations from WWTP effluent inputs. The 2 biofilm types exhibited different Michaelis–Menten kinetics, indicating different capacity to respond to acute increases in NH4+-N concentration. Mean UNH4 and UAO were 5× higher in epilithic than epipsammic biofilms, coinciding with a higher abundance of AOA+AOB in the former than in the later (76 × 104 vs 14 × 104 copies/cm2). AOB derived from active sludge dominated in epilithic biofilms, so our results suggest that WWTP effluents can strongly influence in-stream NH4+ processing rates by increasing N inputs and by supplying AOA+AOB that are able to colonize some stream habitats.

Day–night ammonium oxidation in an urban stream: the influence of irradiance on ammonia oxidizers

Efficient NH4+ oxidation is a critical issue in human-impaired streams receiving high N loads from the effluent of wastewater treatment plants (WWTP). Archaeal (AOA) and bacterial (AOB) ammonia oxidizers are strongly photoinhibited in laboratory cultures, so we expected that light availability would affect the distribution of AOA and AOB and NH4+ oxidation rates at the reach scale. We selected 2 contiguous reaches downstream of a WWTP input in La Tordera river (northeastern Spain) that strongly differed in canopy cover (open and shaded). Against expectations and despite significant differences in light availability, the 2 reaches showed similar abundance of AOA and AOB and similar daily rates of ecosystem respiration, gross primary productivity, and NH4+ oxidation. The abundance of ammonia oxidizers was not correlated with biomass in biofilms protected from light, whereas a positive relationship was found for light-exposed biofilms. This result suggests that biomass accrual could provide light protection to ammonia oxidizers in light-exposed biofilms. The contribution of NH4+ oxidation to whole-reach NH4+ uptake reached up to 89%, indicating a high potential for NH4+ oxidation in the 2 reaches. NH4+ oxidation rates were similar between night and day, but their contribution to whole-reach NH4+ uptake tended to be higher at night than during the day. Altogether, these findings highlight that environmental factors other than irradiance drive reach-scale NH4+ oxidation in this urban stream.