The effect of environmental conditions, especially ammonium concentration, on community composition and nitrification activity of nitrifying bacterial biofilms in a pilot wastewater treatment plant was examined. A decreasing ammonium gradient was created when four aerated tanks with suspended carrier material were serially fed with wastewater. Community composition was analysed using fluorescence in situ hybridization (FISH) probes as well as partial 16S rRNA and amoA gene analysis using polymerase chain reaction-denaturating gradient gel electrophoresis (PCR-DGGE) and sequencing. Fluorescence in situ hybridization probes identified at least five ammonia-oxidizing bacterial (AOB) and two nitrite-oxidizing bacterial (NOB) populations. A change in nitrifying community was detected in the tanks, indicating that ammonium was an important structuring factor. Further, we found support for different autoecology within the Nitrosomonas oligotropha lineage, as at least one population within this lineage increased in relative abundance with ammonium concentration while another population decreased. Absolute numbers of AOB and NOB growing in biofilms on the carriers were determined and the cell specific nitrification rates calculated seemed strongly correlated to ammonium concentration. Oxygen could also be limiting in the biofilms of the first tank with high ammonium concentrations. The response of the nitrifying community to increased ammonium concentrations differed between the tanks, indicating that activity correlates with community structure.

Recent investigations have highlighted the relative importance of the winter season for emissions of N(2)O from boreal soils. However, our understanding of the processes and environmental controls regulating these emissions is fragmentary. Therefore, we investigated the potential for, and relative importance of, N(2)O formation at temperatures below 0 degrees C in laboratory experiments involving incubations of a Swedish boreal forest soil. Our results show that frozen soils have a high potential for N(2)O formation and subsequent emission. Net N(2)O production rates at -4 degrees C equaled those observed at +10 to +15 degrees C at moisture contents >60% of the soil's water-holding capacity. The source of this N(2)O was found to be denitrification occurring in anoxic microsites in the frozen soil and temperature per se did not control the denitrification rates at temperatures around 0 degrees C. Furthermore, both net nitrogen-mineralisation and nitrification were observed in the frozen soil samples. Based on these findings we propose a conceptual model for the temperature response of N(2)O formation in soils at low temperatures.

cIn biofilms, microbial activities form gradients of substrates and electron acceptors, creating a complex landscape of microhabitats, often resulting in structured localization of the microbial populations present. To understand the dynamic interplay between and within these populations, quantitative measurements and statistical analysis of their localization patterns within the biofilms are necessary, and adequate automated tools for such analyses are needed. We have designed and applied new methods for fluorescence in situ hybridization (FISH) and digital image analysis of directionally dependent (anisotropic) multispecies biofilms. A sequential-FISH approach allowed multiple populations to be detected in a biofilm sample. This was combined with an automated tool for vertical-distribution analysis by generating in silico biofilm slices and the recently developed Inflate algorithm for coaggregation analysis of microbial populations in anisotropic biofilms. As a proof of principle, we show distinct stratification patterns of the ammonia oxidizers Nitrosomonas oligotropha subclusters I and II and the nitrite oxidizer Nitrospira sublineage I in three different types of wastewater biofilms, suggesting niche differentiation between the N. oligotropha subclusters, which could explain their coexistence in the same biofilms. Coaggregation analysis showed that N. oligotropha subcluster II aggregated closer to Nitrospira than did N. oligotropha subcluster I in a pilot plant nitrifying trickling filter (NTF) and a movingbed biofilm reactor (MBBR), but not in a full-scale NTF, indicating important ecophysiological differences between these phylogenetically closely related subclusters. By using high-resolution quantitative methods applicable to any multispecies biofilm in general, the ecological interactions of these complex ecosystems can be understood in more detail.

Cryosectioned biofilm from three depths (0.5, 3.0 and 6.0 m) in a full-scale nitrifying trickling filter (NTF) were studied using fluorescence in situ hybridization (FISH). A large number of sections were used to determine how the biofilm thickness, structure and community composition varied with depth along the ammonium concentration gradient in the NTF, and how the ammonia-oxidizing bacteria (AOB) and nitrite-oxidizing bacteria (NOB) were distributed vertically within the biofilm. Both the biofilm thickness and relative biomass content of the biofilm decreased with depth, along with structural differences such as void size and surface roughness. Four AOB populations were found, with two Nitrosomonas oligotropha populations dominating at all depths. A smaller population of Nitrosomonas europaea was present only at 0.5 m, while a population of Nitrosomonas communis increased with depth. The two N. oligotropha populations showed different vertical distribution patterns within the biofilm, indicating different ecophysiologies even though they belong to the same AOB lineage. All NOB were identified as Nitrospira sp., and were generally more associated with the biofilm base than the surface-associated dominating AOB population. Additionally, a small population of anaerobic ammonia-oxidizers was found at 6.0 m, even though the biofilm was well aerated.

Aims: To study the effects of different solids retention time (SRT) on the nitrification activity and community composition of ammonia-oxidizing bacteria (AOB) in two full-scale activated sludge processes during a 5-month period. Methods and Results: The AOB community composition was analysed using fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE), and the identified populations were enumerated by quantitative FISH. Potential nitrification rates were determined in batch tests and the in situ rates were calculated from mass balances of nitrogen in the plants. Increased SRT reduced the nitrification activity, but neither the number per mixed liquor suspended solids nor community composition of AOB were affected. Two dominant AOB populations related to Nitrosomonas europaea and Nitrosomonas oligotropha were identified by FISH, whereas only the latter could be detected by DGGE. Conclusions: The effect of a longer SRT on the activity was probably because of physiological changes in the AOB community rather than a change in community composition. Significance and Impact of the Study: Physiological alterations of a stable AOB community are possible and may stabilize activated sludge processes. The commonly used FISH probes designed to target all betaproteobacterial AOB does not detect certain Nitrosomonas oligotropha populations, leading to an underestimation of AOB if a wider set of probes is not used.