The metagenome of the marine anammox bacterium 'Candidatus Scalindua profunda' illustrates the versatility of this globally important nitrogen cycle bacterium.

Anaerobic ammonium-oxidizing (anammox) bacteria are responsible for a significant portion of the loss of fixed nitrogen from the oceans, making them important players in the global nitrogen cycle. To date, marine anammox bacteria found in marine water columns and sediments worldwide belong almost exclusively to the 'Candidatus Scalindua' species, but the molecular basis of their metabolism and competitive fitness is presently unknown. We applied community sequencing of a marine anammox enrichment culture dominated by 'Candidatus Scalindua profunda' to construct a genome assembly, which was subsequently used to analyse the most abundant gene transcripts and proteins. In the S. profunda assembly, 4756 genes were annotated, and only about half of them showed the highest identity to the only other anammox bacterium of which a metagenome assembly had been constructed so far, the freshwater 'Candidatus Kuenenia stuttgartiensis'. In total, 2016 genes of S. profunda could not be matched to the K. stuttgartiensis metagenome assembly at all, and a similar number of genes in K.stuttgartiensis could not be found in S. profunda. Most of these genes did not have a known function but 98 expressed genes could be attributed to oligopeptide transport, amino acid metabolism, use of organic acids and electron transport. On the basis of the S. profunda metagenome, and environmental metagenome data, we observed pronounced differences in the gene organization and expression of important anammox enzymes, such as hydrazine synthase (HzsAB), nitrite reductase (NirS) and inorganic nitrogen transport proteins. Adaptations of Scalindua to the substrate limitation of the ocean may include highly expressed ammonium, nitrite and oligopeptide transport systems and pathways for the transport, oxidation, and assimilation of small organic compounds that may allow a more versatile lifestyle contributing to the competitive fitness of Scalindua in the marine realm.

Effects of nitrogen dioxide and anoxia on global gene and protein expression in long-term continuous cultures of Nitrosomonas eutropha C91.

Nitrosomonas eutropha is an ammonia-oxidizing betaproteobacterium found in environments with high ammonium levels, such as wastewater treatment plants. The effects of NO(2) on gene and protein expression under oxic and anoxic conditions were determined by maintaining N. eutropha strain C91 in a chemostat fed with ammonium under oxic, oxic-plus-NO(2), and anoxic-plus-NO(2) culture conditions. Cells remained viable but ceased growing under anoxia; hence, the chemostat was switched from continuous to batch cultivation to retain biomass. After several weeks under each condition, biomass was harvested for total mRNA and protein isolation. Exposure of N. eutropha C91 to NO(2) under either oxic or anoxic conditions led to a decrease in proteins involved in N and C assimilation and storage and an increase in proteins involved in energy conservation, including ammonia monooxygenase (AmoCAB). Exposure to anoxia plus NO(2) resulted in increased representation of proteins and transcripts reflective of an energy-deprived state. Several proteins implicated in N-oxide metabolism were expressed and remained unchanged throughout the experiment, except for NorCB nitric oxide reductase, which was not detected in the proteome. Rather, NorY nitric oxide reductase was expressed under oxic-plus-NO(2) and anoxic-plus-NO(2) conditions. The results indicate that exposure to NO(2) results in an energy-deprived state of N. eutropha C91 and that anaerobic growth could not be supported with NO(2) as an oxidant.

Diversity and enrichment of nitrite-dependent anaerobic methane oxidizing bacteria from wastewater sludge.

Recently discovered microorganisms affiliated to the bacterial phylum NC10, named "Candidatus Methylomirabilis oxyfera", perform nitrite-dependent anaerobic methane oxidation. These microorganisms could be important players in a novel way of anaerobic wastewater treatment where ammonium and residual dissolved methane might be removed at the expense of nitrate or nitrite. To find suitable inocula for reactor startup, ten selected wastewater treatment plants (WWTPs) located in The Netherlands were screened for the endogenous presence of M. oxyfera using molecular diagnostic methods. We could identify NC10 bacteria with 98% similarity to M. oxyfera in nine out of ten WWTPs tested. Sludge from one selected WWTP was used to start a new enrichment culture of NC10 bacteria. This enrichment was monitored using specific pmoA primers and M. oxyfera cells were visualized with fluorescence oligonucleotide probes. After 112 days, the enrichment consumed up to 0.4 mM NO(2)(-) per day. The results of this study show that appropriate sources of biomass, enrichment strategies, and diagnostic tools existed to start and monitor pilot scale tests for the implementation of nitrite-dependent methane oxidation in wastewater treatment at ambient temperature.

Liquid chromatography-mass spectrometry-based proteomics of Nitrosomonas.

During the last century, the research on aerobic ammonium-oxidizing bacteria (AOB) lead to many exciting physiological and biochemical discoveries. Nevertheless the molecular biology of AOB is not well understood. The availability of the genome sequences of several Nitrosomonas species opened up new possiblities to use state of the art transcriptomic and proteomic tools to study AOB. With the currect technology, thousands of proteins can be analyzed in several hours of measurement and translated proteins can be detected at femtomole and attomole concentrations. Moreover, it is possible to use mass spectrometry-based proteomics approach to analyze the expression, subcellular localization, posttranslational modifications, and interactions of translated proteins. In this chapter, we describe our LC-MS/MS methodology and quality control strategy to study the protein complement of Nitrosomonas eutropha C91.

New anaerobic, ammonium-oxidizing community enriched from peat soil.

Anaerobic ammonium-oxidizing (anammox) bacteria have been recognized as an important sink for fixed nitrogen and are detected in many natural environments. However, their presence in terrestrial ecosystems has long been overlooked, and their contribution to the nitrogen cycling in natural and agricultural soils is currently unknown. Here we describe the enrichment and characterization of anammox bacteria from a nitrogen-loaded peat soil. After 8 months of incubation with the natural surface water of the sampling site and increasing ammonium and nitrite concentrations, anammox cells constituted 40 to 50% of the enrichment culture. The two dominant anammox phylotypes were affiliated with "Candidatus Jettenia asiatica" and "Candidatus Brocadia fulgida." The enrichment culture converted NH(4)(+) and NO(2)(-) to N(2) with the previously reported stoichiometry (1:1.27) and had a maximum specific anaerobic ammonium oxidation rate of 0.94 mmol NH(4)(+)·g (dry weight)(-1)·h(-1) at pH 7.1 and 32°C. The diagnostic anammox-specific lipids were detected at a concentration of 650 ng·g (dry weight)(-1), and pentyl-[3]-ladderane was the most abundant ladderane lipid.

Identification and quantification of anammox bacteria in eight nitrogen removal reactors.

Various studies have revealed anaerobic ammonium oxidation (anammox) as a very attractive alternative process suitable for nitrogen removal from wastewater. Here we investigated anammox bacteria in eight different nitrogen removal reactors. The diversity and abundance of anammox bacteria were determined by the 16S rRNA gene analysis, fluorescence in situ hybridization with specific probes and real-time quantitative PCR (qPCR). In these reactors, at least eight unique near full length anammox 16S rRNA gene sequences were detected, which were distributed over two genera; Candidati Brocadia and Kuenenia. FISH results confirmed that only one anammox bacterium dominated the community in each of the eight reactors investigated in this study. qPCR analysis revealed that anammox bacteria were present in seven of the reactors in the order of 10(9) cells/ml and 10(7) cells/ml in reactor A1. The dominant and divergent Brocadia-like anammox phylotype in one reactor represented a novel species for which we propose the name Candidatus Brocadia sinica. Taken together, these results indicated that a single seeding source could be used to seed anammox reactors designed to treat different types of wastewater, which could lead to a faster start-up of bioreactors.

Ammonium concentrations in produced waters from a mesothermic oil field subjected to nitrate injection decrease through formation of denitrifying biomass and anammox activity.

Community analysis of a mesothermic oil field, subjected to continuous field-wide injection of nitrate to remove sulfide, with denaturing gradient gel electrophoresis (DGGE) of PCR-amplified 16S rRNA genes indicated the presence of heterotrophic and sulfide-oxidizing, nitrate-reducing bacteria (hNRB and soNRB). These reduce nitrate by dissimilatory nitrate reduction to ammonium (e.g., Sulfurospirillum and Denitrovibrio) or by denitrification (e.g., Sulfurimonas, Arcobacter, and Thauera). Monitoring of ammonium concentrations in producing wells (PWs) indicated that denitrification was the main pathway for nitrate reduction in the field: breakthrough of nitrate and nitrite in two PWs was not associated with an increase in the ammonium concentration, and no increase in the ammonium concentration was seen in any of 11 producing wells during periods of increased nitrate injection. Instead, ammonium concentrations in produced waters decreased on average from 0.3 to 0.2 mM during 2 years of nitrate injection. Physiological studies with produced water-derived hNRB microcosms indicated increased biomass formation associated with denitrification as a possible cause for decreasing ammonium concentrations. Use of anammox-specific primers and cloning of the resulting PCR product gave clones affiliated with the known anammox genera "Candidatus Brocadia" and "Candidatus Kuenenia," indicating that the anammox reaction may also contribute to declining ammonium concentrations. Overall, the results indicate the following: (i) that nitrate injected into an oil field to oxidize sulfide is primarily reduced by denitrifying bacteria, of which many genera have been identified by DGGE, and (ii) that perhaps counterintuitively, nitrate injection leads to decreasing ammonium concentrations in produced waters.