Microbial community composition and function in wastewater treatment plants

Biological wastewater treatment has been applied for more than a century to ameliorate anthropogenic damage to the environment. But only during the last decade the use of molecular tools allowed to accurately determine the composition, and dynamics of activated sludge and biofilm microbial communities. Novel, in many cases yet not cultured bacteria were identified to be responsible for filamentous bulking and foaming as well as phosphorus and nitrogen removal in these systems. Now, methods are developed to infer the in situ physiology of these bacteria. Here we provide an overview of what is currently known about the identity and physiology of some of themicrobial key players in activated sludge and biofilm systems.Deutsche Forschungsgemeinschaft - project WA1558/1

Nitrifying and heterotrophic population dynamics in biofilm reactors: effects of hydraulic retention time and the presence of organic carbon

Two biofilmreactors operated with hydraulic retention times of 0.8 and 5.0 h were used to study the links between population dynamics and reactor operation performance during a shift in process operation from pure nitrification to combined nitrification and organic carbon removal. The ammonium and the organic carbon loads were identical for both reactors. The composition and dynamics of the microbial consortia were quantified by fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes combined with confocal laser scanning microscopy, and digital image analysis. In contrast to past research, after addition of acetate as organic carbon nitrification performance decreased more drastically in the reactor with longer hydraulic retention time. FISH analysis showed that this effect was caused by the unexpected formation of a heterotrophic microorganism layer on top of the nitrifying biofilm that limited nitrifiers oxygen supply. Our results demonstrate that extension of the hydraulic retention time might be insufficient to improve combined nitrification and organic carbon removal in biofilm reactors.Ministério da Ciência, Tecnologia e Ensino Superior. Fundação para a Ciência e a Tecnologia (FCT) - PRAXIS XXI BD/15943/98). Deutscher Akademischer Austauschdienst (A/99/06961). European Comission - T.M.R. BioToBio project. Deutsche Forschungsgemeinschaft

Phylogeny and abundance of ammonia oxidizing bacteria

Ammoniak oxidierend Bakterien (AOB) spielen im globalen Stickstoffkreislauf, in der Landwirtschaft, sowie bei der Abwasserreinigung eine essentielle Rolle. Da AOB nur sehr schwer und unter großem Zeitaufwand kultivierbar sind, erfolgt ihr Nachweis und ihre Identifizierung heute überwiegend molekular, mit Hilfe der vergleichenden Sequenzanalyse ihrer 16S rRNS- sowie amoA-Gene. Im Zuge dieser Arbeit wurden 16S rRNS- und amoA-Datenbanken erstellt, die alle veröffentlichten Sequenzen von AOB beinhalten. Zudem wurden die beiden Markermolekülsequenzen für weitere 44 AOB-Isolate ermittelt. Auf Basis der aktualisierten und erweiterten Datensätze wurden umfassende phylogenetische Analysen durchgeführt und ein "Genospezies-Konzept" für den Nachweis bisher unbekannter AOB-Arten in Umweltproben entworfen. Darüber hinaus wurde die Spezifität aller veröffentlichten Sonden und Primer für den Nachweis von AOB überprüft sowie Verbreitungsmuster der einzelnen AOB-Gruppen in der Umwelt aufgedeckt.Ammonia oxidizing bacteria (AOB) play an important role in the global nitrogen cycle, in agriculture, and in wastewater treatment. Cultivation of AOB is difficult and very time consuming. Therefore, today these bacteria are often detected and identified using molecular methods, especially comparative sequence analysis of their 16S rRNA- and amoA- genes. In this thesis 16S rRNA and amoA databases containing all published AOB sequences have been established. Moreover, the sequences of both marker molecules of 44 additional AOB isolates have been determined. Based on the updated and extended databases thorough phylogenetic analyses have been performed and a "genospecies-concept" for the detection of to-date unknown AOB species has been proposed. In addition, the specificity of all published primers and probes for the detection of AOB has been revaluated and natural distribution patterns of distinct AOB-groups have been revealed

Study of nitrifying bacteria diversity on biofilms using a molecular approach

The purpose of this work was to investigate the microbial community structure of nitrifying biofilms. Biofilm samples were collected from two reactors operated with distinct retention times. The composition and spatial distribution of nitrifying consortia in biofilms was quantified by fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes combined with confocal laser scanning microscopy (CLSM), and digital image analysis. High-resolution analyses os ammonia-oxidizer diversity in both reactors were performed using the gene that encodes the catalytic subunit of the ammonia-monooxygenase enzyme (amoA) as a marker. At least two populations of bete-subclass ammonia-oxidizing bacteria (AOB) were detected in both reactors. As demonstrated by oligonucleotide probing and comparative amoA sequence analysis, one of these populations was closely related to the model organism Nitrosomonas europaea, while the other polpulation surprisingly showed no close relationship with recognized ammonia-oxidizers. Nitrospira-like bacteria was the dominant nitrite-oxidizing bacteria (NOB) in the biofilm reactors studied. According to our results biofilms formed in the two studied reactors with distinct retention time were similar in their microbial diversity and spatial distribution of AOB and NOB populations. Differences occurred, however, in the relative abundance of AOB, which was higher in the reactor operated with shorter retention time. Despite of the fact that the environmental conditions within the reactors represented a common situation, the bacterial diversity was surprisingly diverse

Community Structure and Activity Dynamics of Nitrifying Bacteria in a Phosphate-Removing Biofilm

The microbial community structure and activity dynamics of a phosphate-removing biofilm from a sequencing batch biofilm reactor were investigated with special focus on the nitrifying community. O(2), NO(2)(−), and NO(3)(−) profiles in the biofilm were measured with microsensors at various times during the nonaerated-aerated reactor cycle. In the aeration period, nitrification was oxygen limited and restricted to the first 200 μm at the biofilm surface. Additionally, a delayed onset of nitrification after the start of the aeration was observed. Nitrate accumulating in the biofilm in this period was denitrified during the nonaeration period of the next reactor cycle. Fluorescence in situ hybridization (FISH) revealed three distinct ammonia-oxidizing populations, related to the Nitrosomonas europaea, Nitrosomonas oligotropha, and Nitrosomonas communis lineages. This was confirmed by analysis of the genes coding for 16S rRNA and for ammonia monooxygenase (amoA). Based upon these results, a new 16S rRNA-targeted oligonucleotide probe specific for the Nitrosomonas oligotropha lineage was designed. FISH analysis revealed that the first 100 μm at the biofilm surface was dominated by members of the N. europaea and the N. oligotropha lineages, with a minor fraction related to N. communis. In deeper biofilm layers, exclusively members of the N. oligotropha lineage were found. This separation in space and a potential separation of activities in time are suggested as mechanisms that allow coexistence of the different ammonia-oxidizing populations. Nitrite-oxidizing bacteria belonged exclusively to the genus Nitrospira and could be assigned to a 16S rRNA sequence cluster also found in other sequencing batch systems

Microbial population dynamics versus nitrification performance in biofilm reactors operated with different hydraulic retention times during a shift from pure ammonia oxidation to combined organic carbon and ammonia oxidation

ln order to improve biological reactors operation and design it is important to study the effects of changes in process parameters with regard to the microbial community structure and, vice versa, the effects of community structure and dynamics on the reactors performance. Two biofilm reactors operated with hydraulic retention times of 1 h and 6 h were used to study the links between population dynamics and reactor operation performance during a shift in process operation from pure ammonia oxidation to combined organic carbon and ammonia oxidation, under oxygen limiting conditions. During the entire experimental period both reactors received identical ammonium and organic carbon loads. The composition of lhe microbial consortia in both reaclors was quantified with rRNA-targeted oligonucleotide probes combined with fluorescence in situ hybridization, confocal laser scanning microscopy, and digital image analysis. Furthermore, finescale diversity analyses or ammonia-oxidizers in both reactors were performed using the gene (amoA) encoding the catalytic subunit or the ammonia-monooxygenase enzyme as a marker. The observed population dynamics (microscale phenomena) correlated well with the nitrification perforrnance of the reactors and biofilm parameters like thickness and mass 77 concentration (macroscale phenomena). The decrease in nitrification, efficiency after acetate addition was more drastic in the reactor operated with 6 h retention time due to the unexpected formation of a thicker heterotrophic layer on top of the nitrifying biofilm that increased the resistance to oxygen mass transfer and the nitrifiers became oxygen Iimited. This fact can probably be explained by the decrease in the shear forces acting in the biofilm caused by the increase in the liquid phase viscosity due to the higher growth of suspended heterotrophic bacteria observed in that reactor