Boran Kartal

Deciphering the evolution and metabolism of an anammox bacterium from a community genome

Anaerobic ammonium oxidation (anammox) has become a main focus in oceanography and wastewater treatment. It is also the nitrogen cycles major remaining biochemical enigma. Among its features, the occurrence of hydrazine as a free intermediate of catabolism, the biosynthesis of ladderane lipids and the role of cytoplasm differentiation are unique in biology. Here we use environmental genomics—the reconstruction of genomic data directly from the environment—to assemble the genome of the uncultured anammox bacterium Kuenenia stuttgartiensis from a complex bioreactor community. The genome data illuminate the evolutionary history of the Planctomycetes and allow us to expose the genetic blueprint of the organisms special properties. Most significantly, we identified candidate genes responsible for ladderane biosynthesis and biological hydrazine metabolism, and discovered unexpected metabolic versatility.

Sewage Treatment with Anammox

Wastewater treatment including high rate anammox processes have the potential to become energy-neutral or even energy-producing. Organic matter must be removed from sewage to protect the quality of the water bodies that it is discharged to. Most current sewage treatment plants are aimed at removing organic matter only. They are energy-inefficient, whereas potentially the organic matter could be regarded as a source of energy. However, organic carbon is not the only pollutant in sewage: Fixed nitrogen such as ammonium (NH4+) and nitrate (NO3−) must be removed to avoid toxic algal blooms in the environment. Conventional wastewater treatment systems for nitrogen removal require a lot of energy to create aerobic conditions for bacterial nitrification, and also use organic carbon to help remove nitrate by bacterial denitrification (see the figure). An alternative approach is the use of anoxic ammonium-oxidizing (anammox) bacteria, which require less energy (1) but grow relatively slowly. We explore process innovations that can speed up the anammox process and use all organic matter as much as possible for energy generation.

Molecular mechanism of anaerobic ammonium oxidation.

Two distinct microbial processes, denitrification and anaerobic ammonium oxidation (anammox), are responsible for the release of fixed nitrogen as dinitrogen gas (N2) to the atmosphere. Denitrification has been studied for over 100 years and its intermediates and enzymes are well known. Even though anammox is a key biogeochemical process of equal importance, its molecular mechanism is unknown, but it was proposed to proceed through hydrazine (N2H4). Here we show that N2H4 is produced from the anammox substrates ammonium and nitrite and that nitric oxide (NO) is the direct precursor of N2H4. We resolved the genes and proteins central to anammox metabolism and purified the key enzymes that catalyse N2H4 synthesis and its oxidation to N2. These results present a new biochemical reaction forging an N–N bond and fill a lacuna in our understanding of the biochemical synthesis of the N2 in the atmosphere. Furthermore, they reinforce the role of nitric oxide in the evolution of the nitrogen cycle.

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.

Biochemistry and molecular biology of anammox bacteria

Anaerobic ammonium-oxidizing (anammox) bacteria are one of the latest additions to the biogeochemical nitrogen cycle. These bacteria derive their energy for growth from the conversion of ammonium and nitrite into dinitrogen gas in the complete absence of oxygen. These slowly growing microorganisms belong to the order Brocadiales and are affiliated to the Planctomycetes. Anammox bacteria are characterized by a compartmentalized cell architecture featuring a central cell compartment, the “anammoxosome”. Thus far unique “ladderane” lipid molecules have been identified as part of their membrane systems surrounding the different cellular compartments. Nitrogen formation seems to involve the intermediary formation of hydrazine, a very reactive and toxic compound. The genome of the anammox bacterium Kuenenia stuttgartiensis was assembled from a complex microbial community grown in a sequencing batch reactor (74% enriched in this bacterium) using a metagenomics approach. The assembled genome allowed the in silico reconstruction of the anammox metabolism and identification of genes most likely involved in the process. The present anammox pathway is the only one consistent with the available experimental data, thermodynamically and biochemically feasible, and consistent with Ockham’s razor: it invokes minimum biochemical novelty and requires the fewest number of biochemical reactions. The worldwide presence of anammox bacteria has now been established in many oxygen-limited marine and freshwater systems, including oceans, seas, estuaries, marshes, rivers and large lakes. In the marine environment over 50% of the N2 gas released may be produced by anammox bacteria. Application of the anammox process offers an attractive alternative to current wastewater treatment systems for the removal of ammonia-nitrogen. Currently, at least five full scale reactor systems are operational.

Complete nitrification by a single microorganism

Nitrification is a two-step process where ammonia is first oxidized to nitrite by ammonia-oxidizing bacteria and/or archaea, and subsequently to nitrate by nitrite-oxidizing bacteria. Already described by Winogradsky in 1890, this division of labour between the two functional groups is a generally accepted characteristic of the biogeochemical nitrogen cycle. Complete oxidation of ammonia to nitrate in one organism (complete ammonia oxidation; comammox) is energetically feasible, and it was postulated that this process could occur under conditions selecting for species with lower growth rates but higher growth yields than canonical ammonia-oxidizing microorganisms. Still, organisms catalysing this process have not yet been discovered. Here we report the enrichment and initial characterization of two Nitrospira species that encode all the enzymes necessary for ammonia oxidation via nitrite to nitrate in their genomes, and indeed completely oxidize ammonium to nitrate to conserve energy. Their ammonia monooxygenase (AMO) enzymes are phylogenetically distinct from currently identified AMOs, rendering recent acquisition by horizontal gene transfer from known ammonia-oxidizing microorganisms unlikely. We also found highly similar amoA sequences (encoding the AMO subunit A) in public sequence databases, which were apparently misclassified as methane monooxygenases. This recognition of a novel amoA sequence group will lead to an improved understanding of the environmental abundance and distribution of ammonia-oxidizing microorganisms. Furthermore, the discovery of the long-sought-after comammox process will change our perception of the nitrogen cycle.

Propionate Oxidation by and Methanol Inhibition of Anaerobic Ammonium-Oxidizing Bacteria

ABSTRACT Anaerobic ammonium oxidation (anammox) is a recently discovered microbial pathway and a cost-effective way to remove ammonium from wastewater. Anammox bacteria have been described as obligate chemolithoautotrophs. However, many chemolithoautotrophs (i.e., nitrifiers) can use organic compounds as a supplementary carbon source. In this study, the effect of organic compounds on anammox bacteria was investigated. It was shown that alcohols inhibited anammox bacteria, while organic acids were converted by them. Methanol was the most potent inhibitor, leading to complete and irreversible loss of activity at concentrations as low as 0.5 mM. Of the organic acids acetate and propionate, propionate was consumed at a higher rate (0.8 nmol min−1 mg of protein−1) by Percoll-purified anammox cells. Glucose, formate, and alanine had no effect on the anammox process. It was shown that propionate was oxidized mainly to CO2, with nitrate and/or nitrite as the electron acceptor. The anammox bacteria carried out propionate oxidation simultaneously with anaerobic ammonium oxidation. In an anammox enrichment culture fed with propionate for 150 days, the relative amounts of anammox cells and denitrifiers did not change significantly over time, indicating that anammox bacteria could compete successfully with heterotrophic denitrifiers for propionate. In conclusion, this study shows that anammox bacteria have a more versatile metabolism than previously assumed.

A microdiversity study of anammox bacteria reveals a novel Candidatus Scalindua phylotype in marine oxygen minimum zones

The anaerobic oxidation of ammonium (anammox) contributes significantly to the global loss of fixed nitrogen and is carried out by a deep branching monophyletic group of bacteria within the phylum Planctomycetes. Various studies have implicated anammox to be the most important process responsible for the nitrogen loss in the marine oxygen minimum zones (OMZs) with a low diversity of marine anammox bacteria. This comprehensive study investigated the anammox bacteria in the suboxic zone of the Black Sea and in three major OMZs (off Namibia, Peru and in the Arabian Sea). The diversity and population composition of anammox bacteria were investigated by both, the 16S rRNA gene sequences and the 16S-23S rRNA internal transcribed spacer (ITS). Our results showed that the anammox bacterial sequences of the investigated samples were all closely related to the Candidatus Scalindua genus. However, a greater microdiversity of marine anammox bacteria than previously assumed was observed. Both phylogenetic markers supported the classification of all sequences in two distinct anammox bacterial phylotypes: Candidatus Scalindua clades 1 and 2. Scalindua 1 could be further divided into four distinct clusters, all comprised of sequences from either the Namibian or the Peruvian OMZ. Scalindua 2 consisted of sequences from the Arabian Sea and the Peruvian OMZ and included one previously published 16S rRNA gene sequence from Lake Tanganyika and one from South China Sea sediment (97.9-99.4% sequence identity). This cluster showed only <or= 97% sequence identity to other known Candidatus Scalindua species. Based on 16S rRNA gene and ITS sequences we propose that the anammox bacteria of Scalindua clade 2 represent a novel anammox bacterial species, for which the name Candidatus Scalindua arabica is proposed. As sequences of this new cluster were found in the Arabian Sea, the Peruvian OMZ, in Lake Tanganyika and in South China sediment, we assume a global distribution of Candidatus Scalindua arabica as it is observed for Candidatus Scalindua sorokinii/brodae (or Scalindua clade 1).

Enrichment and characterization of marine anammox bacteria associated with global nitrogen gas production

Microbiological investigation of anaerobic ammonium oxidizing (anammox) bacteria has until now been restricted to wastewater species. The present study describes the enrichment and characterization of two marine Scalindua species, the anammox genus that dominates almost all natural habitats investigated so far. The species were enriched from a marine sediment in the Gullmar Fjord (Sweden) using a medium based on Red Sea salt. Anammox cells comprised about 90% of the enrichment culture after 10 months. The enriched Scalindua bacteria displayed all typical features known for anammox bacteria, including turnover of hydrazine, the presence of ladderane lipids, and a compartmentalized cellular ultrastructure. The Scalindua species also showed a nitrate-dependent use of formate, acetate and propionate, and performed a formate-dependent reduction of nitrate, Fe(III) and Mn(IV). This versatile metabolism may be the basis for the global distribution and substantial contribution of the marine Scalindua anammox bacteria to the nitrogen loss from oxygen-limited marine 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.