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Publications in peer reviewed journals

10 Publications found
  • Genomic and kinetic analysis of novel Nitrospinae enriched by cell sorting

    Mueller AJ, Jung MY, Strachan CR, Herbold CW, Kirkegaard RH, Wagner M, Daims H
    2020 - ISME J., in press


    Chemolithoautotrophic nitrite-oxidizing bacteria (NOB) are key players in global nitrogen and carbon cycling. Members of the phylum Nitrospinae are the most abundant, known NOB in the oceans. To date, only two closely affiliated Nitrospinae species have been isolated, which are only distantly related to the environmentally abundant uncultured Nitrospinae clades. Here, we applied live cell sorting, activity screening, and subcultivation on marine nitrite-oxidizing enrichments to obtain novel marine Nitrospinae. Two binary cultures were obtained, each containing one Nitrospinae strain and one alphaproteobacterial heterotroph. The Nitrospinae strains represent two new genera, and one strain is more closely related to environmentally abundant Nitrospinae than previously cultured NOB. With an apparent half-saturation constant of 8.7±2.5 µM, this strain has the highest affinity for nitrite among characterized marine NOB, while the other strain (16.2±1.6 µM) and Nitrospina gracilis (20.1±2.1 µM) displayed slightly lower nitrite affinities. The new strains and N. gracilis share core metabolic pathways for nitrite oxidation and CO2 fixation but differ remarkably in their genomic repertoires of terminal oxidases, use of organic N sources, alternative energy metabolisms, osmotic stress and phage defense. The new strains, tentatively named “Candidatus Nitrohelix vancouverensis” and “Candidatus Nitronauta litoralis”, shed light on the niche differentiation and potential ecological roles of Nitrospinae.

  • Anaerobic bacterial degradation of protein and lipid macromolecules in subarctic marine sediment

    Pelikan C, Wasmund K, Glombitza C, Hausmann H, Herbold CW, Flieder M, Loy A
    2020 - ISME J, In press


    Microorganisms in marine sediments play major roles in marine biogeochemical cycles by mineralizing substantial quantities of organic matter from decaying cells. Proteins and lipids are abundant components of necromass, yet the taxonomic identities of microorganisms that actively degrade them remain poorly resolved. Here, we revealed identities, trophic interactions and genomic features of bacteria that degraded 13C-labelled proteins and lipids in cold anoxic microcosms containing sulfidic subarctic marine sediment. Supplemented proteins and lipids were rapidly fermented to various volatile fatty acids within five days. DNA-stable isotope probing (SIP) suggested Psychrilyobacter atlanticus was an important primary degrader of proteins, and Psychromonas members were important primary degraders of both proteins and lipids. Closely related Psychromonas populations, as represented by distinct 16S rRNA gene variants, differentially utilized either proteins or lipids. DNA-SIP also showed 13C-labeling of various Deltaproteobacteria within ten days, indicating trophic transfer of carbon to putative sulfate-reducers. Metagenome-assembled genomes revealed the primary hydrolyzers encoded secreted peptidases or lipases, and enzymes for catabolism of protein or lipid degradation products. Psychromonas species are prevalent in diverse marine sediments, suggesting they are important players in organic carbon processing in situ. Together, this study provides new insights into the identities, functions and genomes of bacteria that actively degrade abundant necromass macromolecules in the seafloor.

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

    Lukumbuzya M, Kristensen JM, Kitzinger K, Pommerening-Roser A, Nielsen PH, Wagner M, Daims H, Pjevac P
    2020 - Water Res., 186: 116372
    ammonia oxidizing bacteria FISH picture


    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 uncultured, 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 pilot- and 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.

  • Flow-through stable isotope probing (Flow-SIP) minimizes cross-feeding in complex microbial communities.

    Mooshammer M, Kitzinger K, Schintlmeister A, Ahmerkamp S, Nielsen JL, Nielsen PH, Wagner M
    2020 - ISME J, in press


    Stable isotope probing (SIP) is a key tool for identifying the microorganisms catalyzing the turnover of specific substrates in the environment and to quantify their relative contributions to biogeochemical processes. However, SIP-based studies are subject to the uncertainties posed by cross-feeding, where microorganisms release isotopically labeled products, which are then used by other microorganisms, instead of incorporating the added tracer directly. Here, we introduce a SIP approach that has the potential to strongly reduce cross-feeding in complex microbial communities. In this approach, the microbial cells are exposed on a membrane filter to a continuous flow of medium containing isotopically labeled substrate. Thereby, metabolites and degradation products are constantly removed, preventing consumption of these secondary substrates. A nanoSIMS-based proof-of-concept experiment using nitrifiers in activated sludge and C-bicarbonate as an activity tracer showed that Flow-SIP significantly reduces cross-feeding and thus allows distinguishing primary consumers from other members of microbial food webs.

  • Composition and activity of nitrifier communities in soil are unresponsive to elevated temperature and CO, but strongly affected by drought.

    Séneca J, Pjevac P, Canarini A, Herbold CW, Zioutis C, Dietrich M, Simon E, Prommer J, Bahn M, Pötsch EM, Wagner M, Wanek W, Richter A
    2020 - ISME J, in press
    soil nitrifier response to climate change


    Nitrification is a fundamental process in terrestrial nitrogen cycling. However, detailed information on how climate change affects the structure of nitrifier communities is lacking, specifically from experiments in which multiple climate change factors are manipulated simultaneously. Consequently, our ability to predict how soil nitrogen (N) cycling will change in a future climate is limited. We conducted a field experiment in a managed grassland and simultaneously tested the effects of elevated atmospheric CO, temperature, and drought on the abundance of active ammonia-oxidizing bacteria (AOB) and archaea (AOA), comammox (CMX) Nitrospira, and nitrite-oxidizing bacteria (NOB), and on gross mineralization and nitrification rates. We found that N transformation processes, as well as gene and transcript abundances, and nitrifier community composition were remarkably resistant to individual and interactive effects of elevated CO and temperature. During drought however, process rates were increased or at least maintained. At the same time, the abundance of active AOB increased probably due to higher NH availability. Both, AOA and comammox Nitrospira decreased in response to drought and the active community composition of AOA and NOB was also significantly affected. In summary, our findings suggest that warming and elevated CO have only minor effects on nitrifier communities and soil biogeochemical variables in managed grasslands, whereas drought favors AOB and increases nitrification rates. This highlights the overriding importance of drought as a global change driver impacting on soil microbial community structure and its consequences for N cycling.

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

    Daebeler A, Kitzinger K, Koch H, Herbold CW, Steinberger M, Schwarz J, Zechmeister T, Karst S, Albertsen M, Nielsen PH, Wagner M, Daims H
    2020 - ISME J, in press
    Ca. N. alkalitolerans


    Nitrite-oxidizing bacteria of the genus Nitrospira are key players of the biogeochemical nitrogen cycle. However, little is known about their occurrence and survival strategies in extreme pH environments. Here, we report on the discovery of physiologically versatile, haloalkalitolerant Nitrospira that drive nitrite oxidation at exceptionally high pH. Nitrospiradistribution, diversity, and ecophysiology were studied in hypo- and subsaline (1.3-12.8 g salt/l), highly alkaline (pH 8.9-10.3) lakes by amplicon sequencing, metagenomics, and cultivation-based approaches. Surprisingly, not only were Nitrospira populations detected, but they were also considerably diverse with presence of members of Nitrospira lineages I, II and IV. Furthermore, the ability of Nitrospira enrichment cultures to oxidize nitrite at neutral to highly alkaline pH of 10.5 was demonstrated. Metagenomic analysis of a newly enriched Nitrospira lineage IV species, “Candidatus Nitrospira alkalitolerans”, revealed numerous adaptive features of this organism to its extreme environment. Among them were a sodium-dependent N-type ATPase and NADH:quinone oxidoreductase next to the proton-driven forms usually found in Nitrospira. Other functions aid in pH and cation homeostasis and osmotic stress defense. “Ca. Nitrospira alkalitolerans” also possesses group 2a and 3b [NiFe] hydrogenases, suggesting it can use hydrogen as alternative energy source. These results reveal how Nitrospira cope with strongly fluctuating pH and salinity conditions and expand our knowledge of nitrogen cycling in extreme habitats.

  • Activity and metabolic versatility of complete ammonia oxidizers in full-scale wastewater treatment systems.

    Yang Y, Daims H, Liu Y, Herbold CW, Pjevac P, Lin JG, Li M, Gu JD
    2020 - mBio, 11: e03175-19


    The recent discovery of complete ammonia oxidizers (comammox) contradicts the paradigm that chemolithoautotrophic nitrification is always catalyzed by two different microorganisms. However, our knowledge of the survival strategies of comammox in complex ecosystems, such as full-scale wastewater treatment plants (WWTPs), remains limited. Analyses of genomes and transcriptomes of four comammox organisms from two full-scale WWTPs revealed that comammox were active and showed a surprisingly high metabolic versatility. A gene cluster for the utilization of urea and a gene encoding cyanase suggest that comammox may use diverse organic nitrogen compounds in addition to free ammonia as the substrates. The comammox organisms also encoded the genomic potential for multiple alternative energy metabolisms, including respiration with hydrogen, formate, and sulfite as electron donors. Pathways for the biosynthesis and degradation of polyphosphate, glycogen, and polyhydroxyalkanoates as intracellular storage compounds likely help comammox survive unfavorable conditions and facilitate switches between lifestyles in fluctuating environments. One of the comammox strains acquired from the anaerobic tank encoded and transcribed genes involved in homoacetate fermentation or in the utilization of exogenous acetate, both pathways being unexpected in a nitrifying bacterium. Surprisingly, this strain also encoded a respiratory nitrate reductase which has not yet been found in any other genome and might confer a selective advantage to this strain over other strains in anoxic conditions. The discovery of comammox in the genus changes our perception of nitrification. However, genomes of comammox organisms have not been acquired from full-scale WWTPs, and very little is known about their survival strategies and potential metabolisms in complex wastewater treatment systems. Here, four comammox metagenome-assembled genomes and metatranscriptomic data sets were retrieved from two full-scale WWTPs. Their impressive and-among nitrifiers-unsurpassed ecophysiological versatility could make comammox an interesting target for optimizing nitrification in current and future bioreactor configurations.

  • Single cell analyses reveal contrasting life strategies of the two main nitrifiers in the ocean.

    Kitzinger K, Marchant HK, Bristow LA, Herbold CW, Padilla CC, Kidane AT, Littmann S, Daims H, Pjevac P, Stewart FJ, Wagner M, Kuypers MMM
    2020 - Nat Commun, 1: 767
    Nitrospina AOA in situ growth rates


    Nitrification, the oxidation of ammonia via nitrite to nitrate, is a key process in marine nitrogen (N) cycling. Although oceanic ammonia and nitrite oxidation are balanced, ammonia-oxidizing archaea (AOA) vastly outnumber the main nitrite oxidizers, the bacterial Nitrospinae. The ecophysiological reasons for this discrepancy in abundance are unclear. Here, we compare substrate utilization and growth of Nitrospinae to AOA in the Gulf of Mexico. Based on our results, more than half of the Nitrospinae cellular N-demand is met by the organic-N compounds urea and cyanate, while AOA mainly assimilate ammonium. Nitrospinae have, under in situ conditions, around four-times higher biomass yield and five-times higher growth rates than AOA, despite their ten-fold lower abundance. Our combined results indicate that differences in mortality between Nitrospinae and AOA, rather than thermodynamics, biomass yield and cell size, determine the abundances of these main marine nitrifiers. Furthermore, there is no need to invoke yet undiscovered, abundant nitrite oxidizers to explain nitrification rates in the ocean.

  • Transcriptomic Response of Nitrosomonas europaea Transitioned from Ammonia- to Oxygen-Limited Steady-State Growth.

    Sedlacek CJ, Giguere AT, Dobie MD, Mellbye BL, Ferrell RV, Woebken D, Sayavedra-Soto LA, Bottomley PJ, Daims H, Wagner M, Pjevac P
    2020 - mSystems, 1: e00562-19
    N. europaea electron flow


    Ammonia-oxidizing microorganisms perform the first step of nitrification, the oxidation of ammonia to nitrite. The bacterium is the best-characterized ammonia oxidizer to date. Exposure to hypoxic conditions has a profound effect on the physiology of , e.g., by inducing nitrifier denitrification, resulting in increased nitric and nitrous oxide production. This metabolic shift is of major significance in agricultural soils, as it contributes to fertilizer loss and global climate change. Previous studies investigating the effect of oxygen limitation on have focused on the transcriptional regulation of genes involved in nitrification and nitrifier denitrification. Here, we combine steady-state cultivation with whole-genome transcriptomics to investigate the overall effect of oxygen limitation on Under oxygen-limited conditions, growth yield was reduced and ammonia-to-nitrite conversion was not stoichiometric, suggesting the production of nitrogenous gases. However, the transcription of the principal nitric oxide reductase (cNOR) did not change significantly during oxygen-limited growth, while the transcription of the nitrite reductase-encoding gene () was significantly lower. In contrast, both heme-copper-containing cytochrome oxidases encoded by were upregulated during oxygen-limited growth. Particularly striking was the significant increase in transcription of the B-type heme-copper oxidase, proposed to function as a nitric oxide reductase (sNOR) in ammonia-oxidizing bacteria. In the context of previous physiological studies, as well as the evolutionary placement of sNOR with regard to other heme-copper oxidases, these results suggest sNOR may function as a high-affinity terminal oxidase in and other ammonia-oxidizing bacteria. Nitrification is a ubiquitous microbially mediated process in the environment and an essential process in engineered systems such as wastewater and drinking water treatment plants. However, nitrification also contributes to fertilizer loss from agricultural environments, increasing the eutrophication of downstream aquatic ecosystems, and produces the greenhouse gas nitrous oxide. As ammonia-oxidizing bacteria are the most dominant ammonia-oxidizing microbes in fertilized agricultural soils, understanding their responses to a variety of environmental conditions is essential for curbing the negative environmental effects of nitrification. Notably, oxygen limitation has been reported to significantly increase nitric oxide and nitrous oxide production during nitrification. Here, we investigate the physiology of the best-characterized ammonia-oxidizing bacterium, , growing under oxygen-limited conditions.

  • Archaeal nitrification is constrained by copper complexation with organic matter in municipal wastewater treatment plants.

    Gwak JH, Jung MY, Hong H, Kim JG, Quan ZX, Reinfelder JR, Spasov E, Neufeld JD, Wagner M, Rhee SK
    2020 - ISME J, 2: 335-346


    Consistent with the observation that ammonia-oxidizing bacteria (AOB) outnumber ammonia-oxidizing archaea (AOA) in many eutrophic ecosystems globally, AOB typically dominate activated sludge aeration basins from municipal wastewater treatment plants (WWTPs). In this study, we demonstrate that the growth of AOA strains inoculated into sterile-filtered wastewater was inhibited significantly, in contrast to uninhibited growth of a reference AOB strain. In order to identify possible mechanisms underlying AOA-specific inhibition, we show that complex mixtures of organic compounds, such as yeast extract, were highly inhibitory to all AOA strains but not to the AOB strain. By testing individual organic compounds, we reveal strong inhibitory effects of organic compounds with high metal complexation potentials implying that the inhibitory mechanism for AOA can be explained by the reduced bioavailability of an essential metal. Our results further demonstrate that the inhibitory effect on AOA can be alleviated by copper supplementation, which we observed for pure AOA cultures in a defined medium and for AOA inoculated into nitrifying sludge. Our study offers a novel mechanistic explanation for the relatively low abundance of AOA in most WWTPs and provides a basis for modulating the composition of nitrifying communities in both engineered systems and naturally occurring environments.

Book chapters and other publications

1 Publication found
  • Is too much fertilizer a problem?

    Sedlacek CJ, Giguere AT, Pjevac P
    2020 - Frontiers for Young Minds, 8: 63


    Fertilizers are added to crops in order to produce enough food to feed the human population. Fertilizers provide crops with nutrients like potassium, phosphorus, and nitrogen, which allow crops to grow bigger, faster, and to produce more food. Nitrogen in particular is an essential nutrient for the growth of every organismon Earth.Nitrogen is all around us and makes up about 78% of the air you breathe. However, plants and animals cannot use the nitrogen gas in the air. To grow, plants require nitrogen compounds fromthe soil,which can be produced naturally or be provided by fertilizers. However, applying excessive amounts of fertilizer leads to the release of harmful greenhouse gases into the atmosphere and the eutrophication of our waterways. Scientists are currently trying to find solutions to reduce the environmentally harmful effects of fertilizers, without reducing the amount of food we can produce when using them.

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