Comparative in silico analysis of PCR primers suited for diagnostics and cloning of ammonia monooxygenase genes from ammonia-oxidizing bacteria

Over recent years, several PCR primers have been described to amplify genes encoding the structural subunits of ammonia monooxygenase (AMO) from ammonia-oxidizing bacteria (AOB). Most of them target amoA, while amoB and amoC have been neglected so far. This study compared the nucleotide sequence of 33 primers that have been used to amplify different regions of the amoCAB operon with alignments of all available sequences in public databases. The advantages and disadvantages of these primers are discussed based on the original description and the spectrum of matching sequences obtained. Additionally, new primers to amplify the almost complete amoCAB operon of AOB belonging to Betaproteobacteria (betaproteobacterial AOB), a primer pair for DGGE analysis of amoA and specific primers for gammaproteobacterial AOB, are also described. The specificity of these new primers was also evaluated using the databases of the sequences created during this study

Stream drying drives microbial ammonia oxidation and first-flush nitrate export

Acknowledgments We thank Roser Ventosa for technical assistance at the Nutrient Analytical Service of the CEAB-CSIC, Unai Perez de Arenaza Basauri for field assistance and Iñaki Odriozola and Aitor Larrañaga for statistical advice. We also acknowledge two anonymous reviewers for valuable feedback and constructive comments on the manuscript. S. N. Merbt was supported by a JAE predoctoral fellowship from the Spanish National Research Council (CSIC). This research was granted by the projects DARKNESS (CGL2012-32747, MINECO) to E. O. Casamayor and MED_FORESTREAM (CGL2011-30590-CO2-02, MINECO) and REFRESH (244121 FP7 EU Commission) to E. Martí.Peer reviewedPostprin

Comparative analysis of ammonia monooxygenase (amoA) genes in the water column and sediment-water interface of two lakes and the Baltic Sea

The functional gene amoA was used to compare the diversity of ammonia-oxidizing bacteria (AOB) in the water column and sediment-water interface of the two freshwater lakes Plusssee and Schöhsee and the Baltic Sea. Nested amplifications were used to increase the sensitivity of amoA detection, and to amplify a 789-bp fragment from which clone libraries were prepared. The larger part of the sequences was only distantly related to any of the cultured AOB and is considered to represent new clusters of AOB within the Nitrosomonas/Nitrosospira group. Almost all sequences from the water column of the Baltic Sea and from 1-m depth of Schöhsee were related to different Nitrosospira clusters 0 and 2, respectively. The majority of sequences from Plusssee and Schöhsee were associated with sequences from Chesapeake Bay, from a previous study of Plusssee and from rice roots in Nitrosospira-like cluster A, which lacks sequences from Baltic Sea. Two groups of sequences from Baltic Sea sediment were related to clonal sequences from other brackish/marine habitats in the purely environmental Nitrosospira-like cluster B and the Nitrosomonas-like cluster. This confirms previous results from 16S rRNA gene libraries that indicated the existence of hitherto uncultivated AOB in lake and Baltic Sea samples, and showed a differential distribution of AOB along the water column and sediment of these environment

Die Sauerstoffbestimmung in See- und Süßwasser mit Hilfe der Quecksilbertropfelektrode und ihre Anwendung bei physiologischen Untersuchungen

Nach einer Übersicht über die Grundlagen der polarographischen Sauerstoffbestimmung werden der Aufbau und die Handhabung einer Analyseneinrichtung beschrieben, die für Sauerstoff-Serienbestimmungen in See- und Süßwasser geeignet ist. Die Genauigkeit bei der Analyse luftgesättigter Lösungen beträgt etwa 0,5%. Die Abhängigkeit der polarographischen Methode von verschiedenen Faktoren wird untersucht. Maxima erster Art entstehen vornehmlich bei niedrigen, Maxima zweiter Art vorwiegend bei höheren Elektrolytkonzentrationen. Es wird eine auch für physiologische Untersuchungen unbedenkliche Vorbehandlung des Versuchswassers angegeben, die das Auftreten von Maxima verhindert. Das elektrokapillare Nullpotential liegt in den untersuchten Elektrolytkonzentrationen einheitlich bei -0,55 Volt. Die geringe Abhängigkeit der Stufenhöhe von der Elektrolytkonzentration beruht auf der Veränderung des Sauerstoff-Diffusionskoeffizienten mit der Elektrolytkonzentration. Der Diffusionskoeffizient beträgt bei 20°C und Luftsättigung in Leitungswasser 2,92, in 16 ‰ Seewasser 2,80 und in 35 ‰ Seewasser 2,72 cm2 mal 10-5cm2/sec. Der Temperaturkoffizient der Stufenhöhe wird zu 1,5% (bezogen auf den Diffusionsstrom bei 20°C) bestimmt. Es wird ein Verfahren beschrieben, das die direkte Ablesung der absoluten Sauerstoffkonzentration an der Galvanometerskala gestattet. Es werden Versuchsanordnungen angegeben, um den Sauerstoffverbrauch wasserlebender Tiere in einer abgeschlossenen Wassermenge und in fließendem Wasser zu bestimmen. Ferner wird eine Methode beschrieben, die zur Bestimmung des Sauerstoffverbrauches isolierten Gewebes geeignet ist

Biological sources and sinks of nitrous oxide and strategies to mitigate emissions

Nitrous oxide (N 2 O) is a powerful atmospheric greenhouse gas and cause of ozone layer depletion. Global emissions continue to rise. More than two-thirds of these emissions arise from bacterial and fungal denitrification and nitrification processes in soils, largely as a result of the application of nitrogenous fertilizers. This article summarizes the outcomes of an interdisciplinary meeting, ‘Nitrous oxide (N 2 O) the forgotten greenhouse gas’, held at the Kavli Royal Society International Centre, from 23 to 24 May 2011. It provides an introduction and background to the nature of the problem, and summarizes the conclusions reached regarding the biological sources and sinks of N 2 O in oceans, soils and wastewaters, and discusses the genetic regulation and molecular details of the enzymes responsible. Techniques for providing global and local N 2 O budgets are discussed. The findings of the meeting are drawn together in a review of strategies for mitigating N 2 O emissions, under three headings, namely: (i) managing soil chemistry and microbiology, (ii) engineering crop plants to fix nitrogen, and (iii) sustainable agricultural intensification. </jats:p

Ammonia oxidation: Ecology, physiology, biochemistry and why they must all come together

Ammonia oxidation is a fundamental core process in the global biogeochemical nitrogen cycle. Oxidation of ammonia (NH3) to nitrite (NO2 −) is the first and rate-limiting step in nitrification and is carried out by distinct groups of microorganisms. Ammonia oxidation is essential for nutrient turnover in most terrestrial, aquatic and engineered ecosystems and plays a major role, both directly and indirectly, in greenhouse gas production and environmental damage. Although ammonia oxidation has been studied for over a century, this research field has been galvanised in the past decade by the surprising discoveries of novel ammonia oxidising microorganisms. This review reflects on the ammonia oxidation research to date and discusses the major gaps remaining in our knowledge of the biology of ammonia oxidation

Ammonia‐oxidizing archaea and nitrite‐oxidizing nitrospiras in the biofilter of a shrimp recirculating aquaculture system

This study analysed the nitrifier community in the biofilter of a zero discharge, recirculating aquaculture system ( RAS ) for the production of marine shrimp in a low density (low ammonium production) system. The ammonia‐oxidizing populations were examined by targeting 16 S rRNA and amoA genes of ammonia‐oxidizing bacteria ( AOB ) and archaea ( AOA ). The nitrite‐oxidizing bacteria ( NOB ) were investigated by targeting the 16 S rRNA gene. Archaeal amoA genes were more abundant in all compartments of the RAS than bacterial amoA genes. Analysis of bacterial and archaeal amoA gene sequences revealed that most ammonia oxidizers were related to N itrosomonas marina and N itrosopumilus maritimus . The NOB detected were related to N itrospira marina and N itrospira moscoviensis, and Nitrospira  marina ‐type NOB were more abundant than N . moscoviensis ‐type NOB . Water quality and biofilm attachment media played a role in the competitiveness of AOA over AOB and Nitrospira  marina‐ over N . moscoviensis‐ type NOB .Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/95109/1/fem1448.pd

Ammonia-oxidizing archaea and ammonia-oxidizing bacteria in six full-scale wastewater treatment bioreactors

In this study, dideoxy sequencing and 454 high-throughput sequencing were used to analyze diversities of the ammonia monooxygenase (amoA) genes and the 16S rRNA genes of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in six municipal wastewater treatment plants. The results showed that AOB amoA genes were quite diverse in different wastewater treatment plants while the 16S rRNA genes were relatively conserved. Based on the observed complexity of amoA and 16S rRNA genes, most of the AOB can be assigned to the Nitrosomonas genus, with Nitrosomonas ureae, Nitrosomonas oligotropha, Nitrosomonas marina, and Nitrosomonas aestuarii being the four most dominant species. From the sequences of the AOA amoA genes, most AOA observed in this study belong to the CGI.1b group, i.e., the soil lineage. The AOB amoA and 16S rRNA genes were quantified by quantitative PCR and 454 high-throughput pyrosequencing, respectively. Although the results from the two approaches show some disconcordance, they both indicated that the abundance of AOB in activated sludge was very low

Diversity and quantity of ammonia-oxidizing Archaea and Bacteria in sediment of the Pearl River Estuary, China

The diversity and abundance of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) in the sediment of the Pearl River Estuary were investigated by cloning and quantitative real-time polymerase chain reaction (qPCR). From one sediment sample S16, 36 AOA OTUs (3% cutoff) were obtained from three clone libraries constructed using three primer sets for amoA gene. Among the 36 OTUs, six were shared by all three clone libraries, two appeared in two clone libraries, and the other 28 were only recovered in one of the libraries. For AOB, only seven OTUs (based on 16S rRNA gene) and eight OTUs (based on amoA gene) were obtained, showing lower diversity than AOA. The qPCR results revealed that AOA amoA gene copy numbers ranged from 9.6 × 106 to 5.1 × 107 copies per gram of sediment and AOB amoA gene ranged from 9.5 × 104 to 6.2 × 105 copies per gram of sediment, indicating that the dominant ammonia-oxidizing microorganisms in the sediment of the Pearl River Estuary were AOA. The terminal restriction fragment length polymorphism results showed that the relative abundance of AOB species in the sediment samples of different salinity were significantly different, indicating that salinity might be a key factor shaping the AOB community composition