J. Gijs Kuenen

Missing lithotroph identified as new planctomycete

With the increased use of chemical fertilizers in agriculture, many densely populated countries face environmental problems associated with high ammonia emissions. The process of anaerobic ammonia oxidation (‘anammox’) is one of the most innovative technological advances in the removal of ammonia nitrogen from waste water,. This new process combines ammonia and nitrite directly into dinitrogen gas. Until now, bacteria capable of anaerobically oxidizing ammonia had never been found and were known as “lithotrophs missing from nature”. Here we report the discovery of this missing lithotroph and its identification as a new, autotrophic member of the order Planctomycetales, one of the major distinct divisions of the Bacteria. The new planctomycete grows extremely slowly, dividing only once every two weeks. At present, it cannot be cultivated by conventional microbiological techniques. The identification of this bacterium as the one responsible for anaerobic oxidation of ammonia makes an important contribution to the problem of unculturability.

Anaerobic ammonium oxidation by anammox bacteria in the Black Sea

The availability of fixed inorganic nitrogen (nitrate, nitrite and ammonium) limits primary productivity in many oceanic regions. The conversion of nitrate to N2 by heterotrophic bacteria (denitrification) is believed to be the only important sink for fixed inorganic nitrogen in the ocean. Here we provide evidence for bacteria that anaerobically oxidize ammonium with nitrite to N2 in the worlds largest anoxic basin, the Black Sea. Phylogenetic analysis of 16S ribosomal RNA gene sequences shows that these bacteria are related to members of the order Planctomycetales performing the anammox (anaerobic ammonium oxidation) process in ammonium-removing bioreactors. Nutrient profiles, fluorescently labelled RNA probes, 15N tracer experiments and the distribution of specific ‘ladderane’ membrane lipids indicate that ammonium diffusing upwards from the anoxic deep water is consumed by anammox bacteria below the oxic zone. This is the first time that anammox bacteria have been identified and directly linked to the removal of fixed inorganic nitrogen in the environment. The widespread occurrence of ammonium consumption in suboxic marine settings indicates that anammox might be important in the oceanic nitrogen cycle.

Anammox bacteria: from discovery to application

Anaerobic ammonium oxidation (anammox) bacteria, which were discovered in waste-water sludge in the early 1990s, have the unique metabolic ability to combine ammonium and nitrite or nitrate to form nitrogen gas. This discovery led to the realization that a substantial part of the enormous nitrogen losses that are observed in the marine environment — up to 50% of the total nitrogen turnover — were due to the activity of these bacteria. In this Timeline, Gijs Kuenen recalls the discovery of these unique microorganisms and describes the continuing elucidation of their roles in environmental and industrial microbiology.

New concepts of microbial treatment processes for the nitrogen removal in wastewater

Many countries strive to reduce the emissions of nitrogen compounds (ammonia, nitrate, NOx) to the surface waters and the atmosphere. Since mainstream domestic wastewater treatment systems are usually already overloaded with ammonia, a dedicated nitrogen removal from concentrated secondary or industrial wastewaters is often more cost-effective than the disposal of such wastes to domestic wastewater treatment. The cost-effectiveness of separate treatment has increased dramatically in the past few years, since several processes for the biological removal of ammonia from concentrated waste streams have become available. Here, we review those processes that make use of new concepts in microbiology: partial nitrification, nitrifier denitrification and anaerobic ammonia oxidation (the anammox process). These processes target the removal of ammonia from gases, and ammonium-bicarbonate from concentrated wastewaters (i.e. sludge liquor and landfill leachate). The review addresses the microbiology, its consequences for their application, the current status regarding application, and the future developments.

Biomarkers for In Situ Detection of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria

The existence of anaerobic ammonium oxidation (anammox) was hypothesized based on nutrient profiles and thermodynamic calculations (5, 31, 44). It was first discovered about 1 decade ago (25) in a pilot plant treating wastewater from a yeast-producing company in Delft, The Netherlands. The anammox reaction is the oxidation of ammonium under anoxic conditions with nitrite as the electron acceptor and dinitrogen gas as the product. Hydroxylamine and hydrazine were identified as important intermediates (51). Due to their very low growth rates (doubling time in enrichments is at best 11 days) the cultivation of the anammox bacteria proved to be tedious and required very efficient biomass retention (41, 43). A physical purification of anammox organisms from enrichment cultures was achieved with percoll density centrifugation (42). The purified cells performed the anammox reaction after activation by hydrazine. Based on phylogenetic analysis, the discovered anammox organism branched deep in the Planctomycetes phylum (Fig. 1A and B, [42]) and was named “Candidatus Brocadia anammoxidans” (19). FIG. 1. (A) 16S rRNA gene-based phylogenetic tree reflecting the relationship of “Ca. Scalindua,” “Ca. Brocadia,” and “Ca. Kuenenia” to other Planctomycetes and other reference organisms. Tree reconstruction was ... After the first discovery, nitrogen losses, which could only be explained by the anammox reaction, were reported in other wastewater treatment facilities including landfill leachate treatment plants in Germany, Switzerland, and England (11, 14, 15, 36), as well as in semitechnical wastewater treatment plants in Germany (34), Belgium (30), Japan (12), Australia (48), and the United States (10, 45). Molecular techniques showed the presence of organisms affiliated with the anammox branch within the Planctomycetes in all these wastewater treatment plants. Nutrient profiles and 15N tracer studies in suboxic marine and estuarine environments indicated that anammox is also a key player in the marine nitrogen cycle (8, 46, 49). In addition, 16S rRNA gene analysis, fluorescence in situ hybridization (FISH), the distribution of specific anammox membrane lipids, nutrient profiles, and tracer experiments with [15N]ammonia showed the link between the anammox reaction and the occurrence of the anammox bacterium “Candidatus Scalindua sorokinii” in the suboxic zone of the Black Sea (20). The anammox reaction has also been tested for implementation for full-scale removal of ammonia in wastewater treatment (13, 52, 53). The detection and identification of active anammox organisms in environmental samples combined with information on environmental conditions can facilitate the search for possible biomass sources to be used as an inoculum for laboratory, semitechnical, or full-scale anammox reactors. Additionally, such information could provide insights into the niche differentiation of anammox organisms. This review summarizes the recent advances made in the 16S rRNA gene-based techniques for the detection of anammox bacteria. A convenient PCR detection method for anammox organisms is presented in which anammox-specific FISH probes were used as primers. Furthermore, methods which link activity and the detection of anammox bacteria, such as the combination of FISH and microautoradiography (FISH-MAR) (22) as well as FISH targeting the intergenic spacer region (ISR) between the 16S and 23S rRNA are discussed and compared to conventional methods to detect anammox activity. Each of these approaches by itself only addresses limited aspects, such as abundance, activity, or physiology. Thus, a combination of rRNA-based and non-rRNA-based methods is necessary to allow a comprehensive study of anammox bacteria in their ecosystems.

1994–2004: 10 years of research on the anaerobic oxidation of ammonium

The obligately anaerobic ammonium oxidation (anammox) reaction with nitrite as primary electron acceptor is catalysed by the planctomycete-like bacteria Brocadia anammoxidans, Kuenenia stuttgartiensis and Scalindua sorokinii. The anammox bacteria use a complex reaction mechanism involving hydrazine as an intermediate. They have a unique prokaryotic organelle, the anammoxosome, surrounded by ladderane lipids, which exclusively contains the hydrazine oxidoreductase as the major protein to combine nitrite and ammonia in a one-to-one fashion. In addition to the peculiar microbiology, anammox was shown to be very important in the oceanic nitrogen cycle, and proved to be a very good alternative for treatment of high-strength nitrogenous waste streams. With the assembly of the K. stuttgartiensis genome at Genoscope, Evry, France, the anammox reaction has entered the genomic and proteomic era, enabling the elucidation of many intriguing aspects of this fascinating microbial process.

Analysis of Diversity and Activity of Sulfate-Reducing Bacterial Communities in Sulfidogenic Bioreactors Using 16S rRNA and dsrB Genes as Molecular Markers

ABSTRACT Here we describe the diversity and activity of sulfate-reducing bacteria (SRB) in sulfidogenic bioreactors by using the simultaneous analysis of PCR products obtained from DNA and RNA of the 16S rRNA and dissimilatory sulfite reductase (dsrAB) genes. We subsequently analyzed the amplified gene fragments by using denaturing gradient gel electrophoresis (DGGE). We observed fewer bands in the RNA-based DGGE profiles than in the DNA-based profiles, indicating marked differences in the populations present and in those that were metabolically active at the time of sampling. Comparative sequence analyses of the bands obtained from rRNA and dsrB DGGE profiles were congruent, revealing the same SRB populations. Bioreactors that received either ethanol or isopropanol as an energy source showed the presence of SRB affiliated with Desulfobulbus rhabdoformis and/or Desulfovibrio sulfodismutans, as well as SRB related to the acetate-oxidizing Desulfobacca acetoxidans. The reactor that received wastewater containing a diverse mixture of organic compounds showed the presence of nutritionally versatile SRB affiliated with Desulfosarcina variabilis and another acetate-oxidizing SRB, affiliated with Desulfoarculus baarsii. In addition to DGGE analysis, we performed whole-cell hybridization with fluorescently labeled oligonucleotide probes to estimate the relative abundances of the dominant sulfate-reducing bacterial populations. Desulfobacca acetoxidans-like populations were most dominant (50 to 60%) relative to the total SRB communities, followed by Desulfovibrio-like populations (30 to 40%), and Desulfobulbus-like populations (15 to 20%). This study is the first to identify metabolically active SRB in sulfidogenic bioreactors by using the functional gene dsrAB as a molecular marker. The same approach can also be used to infer the ecological role of coexisting SRB in other habitats.

Mariprofundus ferrooxydans PV-1 the First Genome of a Marine Fe(II) Oxidizing Zetaproteobacterium

Mariprofundus ferrooxydans PV-1 has provided the first genome of the recently discovered Zetaproteobacteria subdivision. Genome analysis reveals a complete TCA cycle, the ability to fix CO2, carbon-storage proteins and a sugar phosphotransferase system (PTS). The latter could facilitate the transport of carbohydrates across the cell membrane and possibly aid in stalk formation, a matrix composed of exopolymers and/or exopolysaccharides, which is used to store oxidized iron minerals outside the cell. Two-component signal transduction system genes, including histidine kinases, GGDEF domain genes, and response regulators containing CheY-like receivers, are abundant and widely distributed across the genome. Most of these are located in close proximity to genes required for cell division, phosphate uptake and transport, exopolymer and heavy metal secretion, flagellar biosynthesis and pilus assembly suggesting that these functions are highly regulated. Similar to many other motile, microaerophilic bacteria, genes encoding aerotaxis as well as antioxidant functionality (e.g., superoxide dismutases and peroxidases) are predicted to sense and respond to oxygen gradients, as would be required to maintain cellular redox balance in the specialized habitat where M. ferrooxydans resides. Comparative genomics with other Fe(II) oxidizing bacteria residing in freshwater and marine environments revealed similar content, synteny, and amino acid similarity of coding sequences potentially involved in Fe(II) oxidation, signal transduction and response regulation, oxygen sensation and detoxification, and heavy metal resistance. This study has provided novel insights into the molecular nature of Zetaproteobacteria.

Microbial Thiocyanate Utilization under Highly Alkaline Conditions

ABSTRACT Three kinds of alkaliphilic bacteria able to utilize thiocyanate (CNS−) at pH 10 were found in highly alkaline soda lake sediments and soda soils. The first group included obligate heterotrophs that utilized thiocyanate as a nitrogen source while growing at pH 10 with acetate as carbon and energy sources. Most of the heterotrophic strains were able to oxidize sulfide and thiosulfate to tetrathionate. The second group included obligately autotrophic sulfur-oxidizing alkaliphiles which utilized thiocyanate nitrogen during growth with thiosulfate as the energy source. Genetic analysis demonstrated that both the heterotrophic and autotrophic alkaliphiles that utilized thiocyanate as a nitrogen source were related to the previously described sulfur-oxidizing alkaliphiles belonging to the gamma subdivision of the division Proteobacteria (theHalomonas group for the heterotrophs and the genusThioalkalivibrio for autotrophs). The third group included obligately autotrophic sulfur-oxidizing alkaliphilic bacteria able to utilize thiocyanate as a sole source of energy. These bacteria could be enriched on mineral medium with thiocyanate at pH 10. Growth with thiocyanate was usually much slower than growth with thiosulfate, although the biomass yield on thiocyanate was higher. Of the four strains isolated, the three vibrio-shaped strains were genetically closely related to the previously described sulfur-oxidizing alkaliphiles belonging to the genus Thioalkalivibrio. The rod-shaped isolate differed from the other isolates by its ability to accumulate large amounts of elemental sulfur inside its cells and by its ability to oxidize carbon disulfide. Despite its low DNA homology with and substantial phenotypic differences from the vibrio-shaped strains, this isolate also belonged to the genusThioalkalivibrio according to a phylogenetic analysis. The heterotrophic and autotrophic alkaliphiles that grew with thiocyanate as an N source possessed a relatively high level of cyanase activity which converted cyanate (CNO−) to ammonia and CO2. On the other hand, cyanase activity either was absent or was present at very low levels in the autotrophic strains grown on thiocyanate as the sole energy and N source. As a result, large amounts of cyanate were found to accumulate in the media during utilization of thiocyanate at pH 10 in batch and thiocyanate-limited continuous cultures. This is a first direct proof of a “cyanate pathway” in pure cultures of thiocyanate-degrading bacteria. Since it is relatively stable under alkaline conditions, cyanate is likely to play a role as an N buffer that keeps the alkaliphilic bacteria safe from inhibition by free ammonia, which otherwise would reach toxic levels during dissimilatory degradation of thiocyanate.

Improved nitrogen removal by application of new nitrogen-cycle bacteria

In order to meet increasingly stringentEuropean discharge standards, new applicationsand control strategies for the sustainableremoval of ammonia from wastewater have to beimplemented. In this paper we discuss anitrogen removal system based on the processesof partial nitrification and anoxic ammoniaoxidation (anammox). The anammox process offersgreat opportunities to remove ammonia in fullyautotrophic systems with biomass retention. Noorganic carbon is needed in such nitrogenremoval system, since ammonia is used aselectron donor for nitrite reduction. Thenitrite can be produced from ammonia inoxygen-limited biofilm systems or in continuousprocesses without biomass retention. Forsuccessful implementation of the combinedprocesses, accurate biosensors for measuringammonia and nitrite concentrations, insight inthe complex microbial communities involved, andnew control strategies have to be developed andevaluated.