A refined set of rRNA-targeted oligonucleotide probes for in situ detection and quantification of ammonia-oxidizing bacteria

Ammonia-oxidizing bacteria (AOB) of the betaproteobacterial genera Nitrosomonas and Nitrosospira are key nitrifying microorganisms in many natural and engineered ecosystems. Since many AOB remain un-cultured, fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes has been one of the most widely used approaches to study the community composition, abundance, and other features of AOB directly in environmental samples. However, the established and widely used AOB-specific 16S rRNA-targeted FISH probes were designed up to two decades ago, based on much smaller rRNA gene sequence datasets than available today. Several of these probes cover their target AOB lineages incompletely and suffer from a weak target specificity, which causes cross-hybridization of probes that should detect different AOB lineages. Here, a set of new highly specific 16S rRNA-targeted oligonucleotide probes was developed and experimentally evaluated that complements the existing probes and enables the specific detection and differentiation of the known, major phylogenetic clusters of betaproteobacterial AOB. The new probes were successfully applied to visualize and quantify AOB in activated sludge and biofilm samples from seven pilotand full-scale wastewater treatment systems. Based on its improved target group coverage and specificity, the refined probe set will facilitate future in situ analyses of AOB. (c) 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/

Report and preliminary results of R/V POSEIDON cruise POS539, Varna (Bulgaria) - Varna (Bulgaria) November 6 - November 21, 2019

The R/V POSEIDON cruise POS539 took place in the northwestern basin of the Black Sea (42°30’N to 44°N and 29°E to 31°E). The overarching aim of the campaign was to obtain sediment and water samples, including suspended particle material, from the various redox zones of the Black Sea. The campaign lasted between November 6th and November 21st 2019 and the collected samples were taken in order to investigate the activity and physiology of microorganisms involved in the conversion of nitrogen compounds and degradation of organic carbon under various oxygen conditions. The main topics of the cruise were: (a) to quantify the contribution of archaeal nitrifiers to the nitrogen and carbon cycles, b) to measure the production and consumption of the powerful greenhouse gases CH4 and N2O, c) to record palaeoenvironmental changes in high resolution, and d) to describe the complexity and identity of biopolymers. For this, water and sediment samples were retrieved from 10 discrete shelf and slope stations. First, ‘deep water’ transect was conducted, which included three stations with water depths over 2000 m. The second perpendicular transect encompassed stations that gradually transitioned from the deep parts of the slope towards the shelf (ca. 80 m depth). Additionally, two stations were setup north and south of the shelf transect, respectively, for paleoceanographic studies. Throughout the cruise the weather conditions were overwhelmingly good, only towards the end of the campaign gusty winds of 7 Bft were recorded. The recorded oceanographic conditions were in agreement with the expected water properties at all stations. Station activities were completed on November 20th at 14:00 local board time. On November 21st at 10:30 local time, R/V POSEIDON reached the port of Varna, Bulgaria, thus concluding the POS539 expedition. Analyses and results from the samples and experiments will provide a basis for our understanding of the microbial control on the carbon and nitrogen cycle of the Black Sea.13032

Expanding the knowledge on nitrifying bacteria and their ecological niches

Im globalen Stickstoffkreislauf ist chemolithotrophe Nitrifikation ein Schlüsselprozess, der ausschließlich von spezialisierten Mikroorganismen – nämlich von Ammoniak-oxidierenden Bakterien und Archeen sowie Nitrit-oxidierenden Bakterien – ausgeführt wird. Die Kultivierung und Kulturerhaltung von Ammoniak- und Nitritoxidierern sind äußerst schwierig, weshalb nur beschränktes Wissen über ihre Physiologie vorhanden ist. In dieser Studie wurden sowohl Anreicherungs- und Kultivierungstechniken als auch kultivierungsabhängige Methoden angewandt, um mehr Einblick in die Physiologie, Evolution und Nischendifferenzierung dieser wichtigen funktionellen Gruppe zu erhalten. Ein besonderes Augenmerk lag dabei auf Nitritoxidierern. Zuerst wurde das Denitrifikationspotential einer Reinkultur des Nitritoxidierers Nitrospira moscoviensis weiter erforscht. N. moscoviensis-Biomasse wurde mit Nitrat (NO3 ) und verschiedenen Intermediaten und Vorstufen des Zitronensäure-Zyklus sowie mit verzweigten Aminosäuren, Ethanol oder Formiat inkubiert. Von den getesteten Substraten konnte N. moscoviensis lediglich Formiat und NO3 zur Energiegewinnung nutzen. Die Verwendung dieser Substrate könnte N. moscoviensis eine alternative Energiegewinnungsmöglichkeit in Abwesenheit von Sauerstoff als terminalem Elektronenakzeptor eröffnen. Zusätzlich könnte dieser Stoffwechselweg auch eine Symbiose zwischen aerob und anaerob lebenden N. moscoviensis-Populationen in der Umwelt ermöglichen. Im zweiten Teil der Studie wurde eine Methode zur Untersuchung von möglichen Unterschieden auf Genom-Ebene und der sich daraus ergebenden funktionellen Differenzierung zwischen mikrodiversen koexistierenden Nitrifikanten in Belebtschlamm weiterentwickelt und in einer Vorstudie bewertet. Belebtschlamm wurde hierfür unter nitrifizierenden Bedingungen mit schweren Isotopen (13C und D2O) inkubiert, um aktive Mikroorganismen zu markieren. Danach wurden die Belebtschlammflocken durch Sonifizieren in Mikroflocken zerkleinert, wobei gezeigt werden konnte, dass diese Methode geeignet ist, einzelne Nitrifikanten-Kolonien in Mikroflocken zu erhalten. Danach wurden Raman-Spektren von Mikroflocken aufgenommen. Raman-Spektroskopie ermöglicht die Identifizierung aktiver Nitrifikanten, weil diese charakteristische Zytochrom-Signaturen aufweisen und der Einbau schwerer Isotope in Biomasse in den Spektren sichtbar ist. Es konnte gezeigt werden, dass D2O als Aktivitätsmarker gut geeignet ist, nicht jedoch 13C, da manche Nitrifikanten in ihren Raman-Spektren Peaks in der Region des 13C-Aktivitätsmarkers aufweisen. Ausgewählte Mikroflocken wurden mittels optischer Pinzette sortiert. Die so vereinzelten Mikroflocken wurden lysiert, ihre DNA mittels multiple displacement amplification (MDA) amplifiziert und MDA-Produkte durch PCR auf Anwesenheit von bakterieller und Nitrifikanten-DNA überprüft. Dadurch konnte gezeigt werden, dass gezieltes Sortieren von Nitrifikanten-Mikroflocken aufgrund ihres Raman-Spektrums im Vergleich zu zufälligem Sortieren nicht zu einer Anreicherung von Nitrifikanten-DNA in den erhaltenen MDA-Produkten führte. Nichtsdestotrotz enthielten MDA-Produkte von Mikroflocken mit Zytochrom-Signaturen signifikant häufiger Nitrifikanten-DNA als MDA-Produkte von Mikroflocken ohne Zytochrom-Signaturen. Diese Ergebnisse deuten darauf hin, dass die eigentliche Selektion auf Nitrifikanten bereits durch das Zerkleinern von Belebtschlammflocken erfolgte, da Nitrifikanten als sehr robuste Mikrokolonien wachsen. Des Weiteren bedeutet dies, dass der Hauptvorteil der Raman-Spektroskopie darin liegt, aktive Mikroorganismen zu identifizieren. Aufgrund der PCR-Screening-Ergebnisse wurden 24 MDA-Produkte ausgewählt, um die Metagenome der sortierten Mikroflocken zu sequenzieren. Obwohl noch einige Schritte im Workflow optimiert werden könnten, ist die hier angewandte Methode für die Analyse von Genomen mikrodiverser Nitrifikanten aus Belebtschlamm sehr gut geeignet. Die in dieser Studie gewonnenen Erkenntnisse werden in Zukunft das Erstellen und Überprüfen von Hypothesen zur Nischendifferenzierung, Symbiose und Evolution von Koexistenz mikrodiverser Nitrifikanten in Belebtschlamm ermöglichen. Im letzten Teil dieser Studie wurde eine neue Nitrotoga-Spezies aus Belebtschlamm angereichert, charakterisiert und höchstwahrscheinlich in Reinkultur erhalten. Diese neue Spezies wurde provisorisch Candidatus Nitrotoga fabula (fabula, lat. „kleine Bohne“, nach der charakteristischen Morphologie dieser Spezies) genannt. Diese neue Art gehört einer Untergruppe von Nitrotoga an, in der bisher keine beschriebenen Anreicherungen zu finden sind. Außerdem unterscheidet sie sich physiologisch deutlich von den zwei beschriebenen Anreicherungen, die beide bei niedrigen Temperaturen und Substratkonzentrationen angereichert wurden. Ca. N. fabula ist mit einem Temperaturoptimum von 28 °C und einer Substrattoleranz von etwa 15 mM NO2- die erste beschriebene mesophile Spezies in der Gattung Nitrotoga. Dies impliziert, dass die ökologische Nische der Gattung Nitrotoga viel breiter ist als zuvor angenommen. Zusätzlich wurde eine neue Art der Festmedium-Zubereitung angewandt, welche die Kultivierung von Ca. N. fabula auf Agarose-Platten ermöglichte. Diese Kultivierungsmethode wird in Zukunft die Forschung an diesen evolutiv höchst interessanten und kaum erforschten Nitritoxidierern erleichtern. Zusammengefasst erweitern die Erkenntnisse, die in dieser Studie gewonnen wurden, das Wissen über mögliche Substrate von Nitrospira moscoviensis abseits von Nitritoxidation und die physiologischen Anpassungen der bisher kaum erforschten Gattung Nitrotoga. Außerdem wurde eine Methode zur Untersuchung von mikrodiversen koexistierenden Nitrifikanten in Belebtschlamm weiterentwickelt und beurteilt, die in Zukunft Erkenntnisse von unschätzbarem Wert bezüglich Nischendifferenzierung, Symbiose und Evolution dieser wichtigen Organismen ermöglichen wird.Chemolithotrophic nitrification is a key process in the global nitrogen cycle, and is exclusively carried out by specialized microorganisms – namely ammonia oxidizing bacteria and archaea, and nitrite oxidizing bacteria. As these organisms are difficult to obtain and keep in axenic culture, only limited knowledge is available on their physiology. In the present study, both cultivation dependent and cultivation independent approaches were taken to gain further insight into physiology, evolution, and niche differentiation of these organisms, with the main focus on nitrite oxidizing bacteria. First, the denitrification potential of an axenic culture of Nitrospira moscoviensis, a nitrite oxidizing bacterium, was assessed. Biomass was incubated under anoxic conditions in the presence of nitrate (NO3 ) and either tricarboxylic acid cycle precursors or intermediates, branched amino acids, ethanol or formate. It was shown that of the tested compounds, N. moscoviensis can only utilize formate and NO3 for energy generation under anoxic conditions. This may allow N. moscoviensis to gain energy during periods of anoxia, or even enable a syntrophy between aerobically and anaerobically growing N. moscoviensis populations in the environment. Second, a method to obtain genomes of microdiverse coexisting nitrifiers from activated sludge was further developed and evaluated in a pre-study to gain insights into mechanisms allowing for coexistence and niche differentiation. Activated sludge was incubated under nitrifying conditions in the presence of heavy isotopes (13C or D2O) to allow for detection of active microorganisms. Incubated sludge was dissected by sonication, and it was shown by FISH that this method is suitable for obtaining single nitrifier microcolonies within microflocs. Subsequently, Raman spectra were acquired for the obtained microflocs. Raman spectra allowed for detecting active nitrifiers based on the presence of cytochrome signatures, which are indicative of nitrifiers, and presence of activity marker signatures. It was shown that incubation is feasible with D2O, but not with 13C as activity marker, as some nitrifiers possessed inherent peaks in the 13C marker region. Obtained microflocs were sorted by optical tweezing. Cells of sorted microflocs were lysed, their DNA amplified by multiple displacement amplification (MDA), and MDA products were screened by PCR for bacterial and nitrifier DNA. It was shown that targeted sorting of active nitrifiers did not significantly enrich for nitrifiers in comparison to random sorting. Nonetheless, microflocs whose Raman spectra contained cytochrome signatures significantly more often contained nitrifier DNA than those without. These observations imply that the main enrichment for nitrifiers already occurred during sonication, as nitrifiers grow in very rigid microcolonies, which are resistant to dissection. This also implies that the main advantage of using Raman spectroscopy is to identify active microorganisms. Lastly, 24 MDA products were selected for microfloc metagenome sequencing. While some steps in the workflow can still be optimized, overall the employed strategy seems well suited to obtain (at least partial) genomes from nitrifiers in activated sludge, which will allow for the generation of hypotheses regarding niche differentiation, symbiosis and evolution of coexistence among microdiverse nitrifiers. Finally, a novel species of the genus Nitrotoga, tentatively named Candidatus Nitrotoga fabula (fabula, lat. “small bean”, named after the characteristic morphology of this species), was enriched from activated sludge from WWTP Klosterneuburg, characterized, and likely obtained in axenic culture. This novel species belongs to a sublineage of Nitrotoga without described relatives and shows drastically different physiology when compared to the two previously described enrichments, which were obtained at low substrate concentrations and temperatures. Ca. N. fabula is the first described mesophilic species of the genus, with a NO2- tolerance of approximately 15 mM and a temperature optimum of 28 °C, indicating that the environmental niche of Nitrotoga is likely much wider than previously assumed. Additionally, a novel solid medium preparation was employed, which allowed cultivation of Ca. N. fabula on plates and will simplify further research on this evolutionarily highly interesting and hardly investigated nitrite oxidizers. Taken together, the insights gained in this study have expanded the knowledge on nitrifying bacteria regarding substrate utilization of N. moscoviensis and physiological adaptations within the hardly investigated genus Nitrotoga. Additionally, the further development and evaluation of the workflow for studying microdiverse coexisting nitrifiers is expected to yield invaluable information on niche differentiation, symbiosis and evolution of these important organisms

Metabolic versatility of nitrifiers playing a key role in global nitrogen cycling

Nitrifikation, die Oxidation von Ammoniak zu Nitrat über Nitrit, ist ein Schlüsselprozess im biogeochemischen Stickstoffkreislauf und wird von spezialisierten Mikroorganismen ausgeführt, den Nitrifikanten. Trotz der Bedeutung dieser Organismen für künstliche und natürliche Systeme sind viele Aspekte ihrer Physiologie unerforscht. Hier wurden kultivierungsabhängige und kultivierungsunabhängige Isotopen-basierte Ansätze kombiniert, um die metabolische Vielseitigkeit von Nitrifikanten und ihre Relevanz in der Umwelt zu untersuchen. Die Isolierung, physiologische und genomische Beschreibung des neuen Nitrit Oxidierer Candidatus Nitrotoga fabula aus einer Kläranlage eröffnete neue Einblicke in die Evolutionsgeschichte der Nitrit Oxidation und zeigte, dass Nitrotoga das Potential hat, Wasserstoff und Sulfit als alternative Elektronendonoren zu nutzen. Metabolische Vielseitigkeit kennzeichnete auch Nitrifikanten in der Umwelt. Ammoniak-oxidierende Archaeen (AOA), die bis zu vierzig Prozent der marinen Mikroorganismen ausmachen, verwendeten im Golf von Mexiko (GoM) die organischen Stickstoffverbindungen Harnstoff und Cyanat als zusätzliche Energie- und Stickstoffquellen, obwohl ihnen bekannte Enzyme für den Cyanat-Abbau fehlen. Im Vergleich zu den AOA sind Nitrospinae, die wichtigsten marinen Nitrit Oxidierer, sehr selten. Da Nitrit im Ozean jedoch nicht angereichert ist, müssen Nitrospinae hochaktiv sein. Dies wurde anhand von Einzelzellmessungen im GoM nachgewiesen. Nitrospinae hatten außerdem eine deutlich höhere Energie-Effizienz und Wachstumsraten als AOA. Zusätzlich deckten Nitrospinae den Großteil ihres Stickstoffbedarfs durch organische Stickstoffverbindungen. Die Verwendung von organischen Stickstoffverbindungen durch Nitrifikanten ist vermutlich ein Grund für ihren Erfolg in der Umwelt. Außerdem wurde eine neue Methode für Inkubationen mit stabilen Isotopen entwickelt, die cross-feeding in mikrobiellen Gemeinschaften unterbindet und die direkte Identifikation von Mikroorganismen ermöglicht, die für Substratumwandlungen in der Umwelt relevant sind.Nitrification, the oxidation of ammonia to nitrate via nitrite, is a key process of biogeochemical nitrogen cycling, carried out by specialized microorganisms, the nitrifiers. Despite their importance in man-made and natural systems, many aspects of their physiology are unknown. Here, cultivation-dependent and -independent stable-isotope-based approaches were used to study nitrifier metabolic versatility and their environmental relevance. The isolation, physiological and genomic characterization of the novel nitrite oxidizer Candidatus Nitrotoga fabula from wastewater provided new insights into the evolution of nitrite oxidation and showed that Nitrotoga has the potential to utilize hydrogen and sulfite as alternative electron donors. Metabolic versatility is also important for nitrifiers in the environment. Ammonia oxidizing archaea (AOA), which can constitute up to forty percent of the marine microbial community, utilized the organic nitrogen compounds urea and cyanate as additional energy and nitrogen-sources in the Gulf of Mexico (GoM), despite lacking known enzymes for cyanate breakdown. Compared to AOA, Nitrospinae, the main marine nitrite oxidizers, are rare. Nitrite does not accumulate in the ocean, therefore, Nitrospinae must be highly active. This was shown using single cell measurements in the GoM. Nitrospinae additionally had much higher energy efficiency and growth rates than AOA. Additionally, Nitrospinae met most of their nitrogen demand by assimilating organic nitrogen. The utilization of organic nitrogen compounds is likely a key factor explaining the environmental success of AOA and Nitrospinae. Additionally, a new method for stable isotope incubations was developed that eliminates cross-feeding and thereby allows to directly link specific microorganisms to environmental substrate-turnover

Taurine as a key intermediate for host-symbiont interaction in the tropical sponge Ianthella basta

Marine sponges are critical components of marine benthic fauna assemblages, where their filter-feeding and reef-building capabilities provide bentho-pelagic coupling and crucial habitat. As potentially the oldest representation of a metazoan-microbe symbiosis, they also harbor dense, diverse, and species-specific communities of microbes, which are increasingly recognized for their contributions to dissolved organic matter (DOM) processing. Recent omics-based studies of marine sponge microbiomes have proposed numerous pathways of dissolved metabolite exchange between the host and symbionts within the context of the surrounding environment, but few studies have sought to experimentally interrogate these pathways. By using a combination of metaproteogenomics and laboratory incubations coupled with isotope-based functional assays, we showed that the dominant gammaproteobacterial symbiont, ‘Candidatus Taurinisymbion ianthellae’, residing in the marine sponge, Ianthella basta, expresses a pathway for the import and dissimilation of taurine, a ubiquitously occurring sulfonate metabolite in marine sponges. ‘Candidatus Taurinisymbion ianthellae’ incorporates taurine-derived carbon and nitrogen while, at the same time, oxidizing the dissimilated sulfite into sulfate for export. Furthermore, we found that taurine-derived ammonia is exported by the symbiont for immediate oxidation by the dominant ammonia-oxidizing thaumarchaeal symbiont, ‘Candidatus Nitrosospongia ianthellae’. Metaproteogenomic analyses also suggest that ‘Candidatus Taurinisymbion ianthellae’ imports DMSP and possesses both pathways for DMSP demethylation and cleavage, enabling it to use this compound as a carbon and sulfur source for biomass, as well as for energy conservation. These results highlight the important role of biogenic sulfur compounds in the interplay between Ianthella basta and its microbial symbionts.</p

Quantification of archaea-driven freshwater nitrification from single cell to ecosystem levels

Deep oligotrophic lakes sustain large populations of the class Nitrososphaeria (Thaumarchaeota) in their hypolimnion. They are thought to be the key ammonia oxidizers in this habitat, but their impact on N-cycling in lakes has rarely been quantified. We followed this archaeal population in one of Europe’s largest lakes, Lake Constance, for two consecutive years using metagenomics and metatranscriptomics combined with stable isotope-based activity measurements. An abundant (8–39% of picoplankton) and transcriptionally active archaeal ecotype dominated the nitrifying community. It represented a freshwater-specific species present in major inland water bodies, for which we propose the name “Candidatus Nitrosopumilus limneticus”. Its biomass corresponded to 12% of carbon stored in phytoplankton over the year´s cycle. Ca. N. limneticus populations incorporated significantly more ammonium than most other microorganisms in the hypolimnion and were driving potential ammonia oxidation rates of 6.0 ± 0.9 nmol l‒1 d‒1, corresponding to potential cell-specific rates of 0.21 ± 0.11 fmol cell–1 d–1. At the ecosystem level, this translates to a maximum capacity of archaea-driven nitrification of 1.76 × 109 g N-ammonia per year or 11% of N-biomass produced annually by phytoplankton. We show that ammonia-oxidizing archaea play an equally important role in the nitrogen cycle of deep oligotrophic lakes as their counterparts in marine ecosystems.publishe

Exploring the upper pH limits of nitrite oxidation: diversity, ecophysiology, and adaptive traits of haloalkalitolerant Nitrospira

Contains fulltext : 225858.pdf (publisher's version ) (Open Access)13 p