Candidatus Brocadia anammoxidans

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A Microbial Biorealm page on the genus Candidatus Brocadia anammoxidans

Classification

Higher order taxa

Domain: Bacteria

Phylum: Planctomycetes

Class: Planctomycetacia

Order: Planctomycetales

Family: unclassified Planctomycetales

Genus: Candidatus Brocadia

Species

Candidatus Brocadia anammoxidans

NCBI: Taxonomy

Description and significance

Candidatus Brocadia anammoxidans (Ca. B. anammoxidans) are aquatic autotrophs best known for their unique ability to anarobically oxidize ammonia to dinitrogen gas, a reaction that has been patented and is otherwise known as the “anammox” reaction (5). The bacteria were first discovered in a wastewater treatment plant in the Netherlands where it was observed that ammonia concentrations dropped while dinitrogen gas concentrations rose in airtight effluent reactors (5). Ca. B. anammoxidans were subsequently held responsible for this process and were deemed to be the first bacteria to demonstrate anaerobic ammonia oxidation abilities (5). The bacteria were isolated from enrichment cultures by density centrifugation and have received ample attention from ecologists who suspect the bacteria’s participation in consuming substantial amounts of nitrogen in the ocean, and from researchers who see the bacteria’s metabolism as a potential in revolutionizing wastewater treatment (3, 8). These microbes are no bigger than one micron in diameter and grow optimally in a pH range of 6.4-8.3 and in a temperature range of 20-43 oC (6, 9). The bacteria is named as such, as Candidatus indicates an uncultureable yet well-characterized organism, Brocadia refers to the Gist-Brocades, the place of its discovery, and anammoxidans describes the process of anaerobic ammonium oxidation (3, 10).

Genome structure

The genome of Ca. B. anammoxidans has yet to be sequenced. The unculturable nature of the bacteria (and of its closest relatives) has made it difficult to ascertain its genomic particulars. Researchers have found through PCR, however, the complete sequences of the 16S ribosomal RNA gene, tRNA-Ile gene, and tRNA-Ala genes, and a partial sequence of the 23S RNA gene. The known sequences of these genes total 4032 contiguous base pairs. The size of the genome, the shape and number of chromosomes, and the presence or absence of plasmids are not yet known (7).

Cell structure and metabolism

Ca. B. anammoxidans is a spherical bacterium that lacks peptidoglycan, a common compound found in most microbial cell walls, and displays small cavities known as ‘crateriform structures’ on its surface. The organism also exhibits a compartmentalized cytoplasm -- a rare find in bacteria. The anammoxosome, one of the cellular compartments, comprises 30-60% of the cell volume and is arguably the most integral structure Ca. B. anammoxidans possesses, as it plays the chief role in the bacteria’s unique metabolic process (3).

Nitrogen tracer studies have shown that Ca. B. anammoxidans obtain their energy by anaerobically combining ammonia and nitrite to produce dinitrogen gas (3, 6):

NH4+ + NO2- → N2 + 2H2O

The reaction, which takes place inside the ammoxosome, yields and requires two toxic intermediates, hydroxylamine (NH2OH) and hydrazine (N2H4, otherwise known as rocket-fuel). These intermediates serve as electron generators for the initial step of the anammox reaction, the reduction of nitrite (2, 3). The safe containment of the noxious intermediates would be impossible were it not for the unparalleled molecular structure of the anammoxosome membrane. Lipids composed of five linearly cis-linked cyclobutane rings make the membrane unusually dense, limiting the diffusion of hydroxylamine and hydrazine. The diffusion limiting aspect of the membrane not only protects the rest of the cell from the toxic intermediates, but also prevents a substantial decrease in the bacteria’s metabolism. From a bioenergetics perspective, if one molecule of hydrazine diffuses through the anammoxosome, a 50% decrease in the catabolic activity will ensue. The bacteria have low growth rates to begin with; in an optimal environment, they double once every eleven days at best (9).

Ecology

Ca. B. anammoxidans plays a substantial role perpetuating the nitrogen cycle in the ocean. Nitrogen tracer studies and calculations have demonstrated that Ca. B. anammoxidans consume 20-40% of inorganic nitrogen that drops into the suboxic zones of the ocean (4). The bacterium use nitrite as the electron acceptor to anaerobically oxidize ammonia to dinitrogen gas, which promotes the growth and productivity of aquatic organisms by limiting the amount of inorganic nitrogen found in the ocean. It is impossible, however, for Ca. B. anammoxidans to carry this reaction out in oxic ocean areas. The anammox bacteria are extremely sensitive to concentrations of oxygen (as low as 2 μM) and will terminate metabolic processes upon oxygen sensation (4). Albeit this limitation, Ca. B. anammoxidans can cooperate with aerobic ammonium oxidizing bacteria to carry out the anammox reaction if it finds itself in slightly oxygenated marine areas. For example, at the oxic/anoxic ocean interface, Ca. B. anammoxidans cooperates with members of the genus Nitrosomonas. The Nitrosomonas bacteria aerobically oxidize ammonia to nitrite and suppress oxygen concentrations, while Ca. B. anammoxidans accepts the nitrite and combines it with ammonia to produce dinitrogen gas anaerobically (3, 4).

Pathology

Ca. B. anammoxidans is not known to cause any disease.

Application to Biotechnology

The performance of the anammox reaction by Ca. B. anammoxidans has revolutionized wastewater treatment. Before the discovery of Ca. B. anammoxidans, effluent treatments were carried out by aerobic bacteria that were obligated to perform nitrification in conjunction with denitrification in order to free the wastewater of ammonia (5). Furthermore, the long aerobic process necessitated an expensive supply of methanol (5). The use of the anammox reaction of Ca. B. anammoxidans in wastewater treatment eliminates these substantial inconveniences. The anaerobic removal of ammonia from wastewater by Ca. B. anammoxidans leads to a faster treatment and a 90% reduction in operational costs, as the anammox process bypasses the denitrification step of the nitrogen cycle completely and does not require expensive methanol as fuel (3). Effluent cure is arguably the most pragmatic application of Ca. B. anammoxidans, and is the frequent study of modern anammox research (11, 12).

Current Research

1) A recent phylogenic study at the University of Queensland was conducted to investigate the similarities and differences of the primary and secondary genetic sequences of Candidatus Brocadia anammoxidans and Candidatus Kuenenia stuttgartiensis. The two planctomycetes’ ribonuclease P RNA gene sequences and secondary structures were compared and found to be identical in helix number. In phylogenetic studies, the two bacteria were repeatedly found to be closely related to Gemmata obscuriglobus, a sister planctomycete. It was verified that P13, a unique bacterial helix insert was found in only these three planctomycetes. This study has been deemed significant as it shows that since the helix insert is exclusively shared between the three planctomycetes, it can be deduced that there is a shared common ancestor for ribonuclease P RNA molecules among these three species (1).

2) Recent and significant improvements on the quality of wastewater treatment with Ca. B. anammoxidans have been made through new anammox reactor developments. The newly developed anammox reactors yield a nitrogen removal rate of 25 kg-N m-3 per day, a number that triples the current nitrogen removal rate, 8.7 kg-N m-3 per day. The bacteria were also able to double in an unprecedented 3.6-5.4 days -- a period that is less than half the doubling time of the bacteria mentioned in current reports. According to the study, the increased rate of nitrogen removal and bacterial doubling is attributed to special inter-reactor fabric sheets and a high total nitrogen loading rate. These two improvements allow for a higher density of anammox bacteria (70% of total bacteria) to be used in the new effluent reactors. The newly developed non-woven fabric sheets are used as biofilm carriers and effectively keep the bacteria active inside the reactor, and the high total nitrogen loading rate encourages internal substrate transport, which plays a part in encouraging anammox bacteria division. These improvements made in effluent reactors promise a more effective means of anaerobic wastewater treatment (12).

3) A study in Hokkaido University, Japan, is innovating current quantification methods of Ca. B. anammoxidans by quantifying the bacteria using a real-time polymerase chain reaction (PCR). The quantification of the bacteria has traditionally been carried out using fluorescence in situ hybridization (FISH) but the method has proved to be impractical. Anammox bacteria have low counts of rRNA molecules per cell and tend to form heavy clusters. These obstacles make FISH difficult to perform. The use of real-time PCR for quantification is shown in the study to be a more convenient and advantageous method for anammox bacteria enumeration. The real-time method is better adapted to quantify dense microbial clusters and is sensitive enough to quantify the slow-growing Ca. B. anammoxidans in an uncultured environment, as it is based on continual fluorescent monitoring. Real-time PCR was used to quantify the 16S rRNA gene of anammox bacteria subsequent to the development of specific PCR primers for an enrichment culture of anammox bacteria from a rotating disk reactor biofilm (11). The autotrophic and obligate anaerobic nature of Ca. B. anammoxidans makes culturing the bacteria very difficult, but the quantification of the bacteria’s 16S rRNA gene using real-time PCR sheds light on the intricate details of the bacteria’s physiology and kinetics, which is an important step in isolating Ca. B. anammoxidans in pure culture (11).

References

1. Butler M.K., Op den Camp H.J.M., Harhangi H.R., Lafi F.F., Strous M., Fuerst J.A. “Close relationship of RNase P RNA in Gemmata and anammox planctomycete bacteria”. Federation of European Microbiological Societies. 2007. Volume 268. p. 244-253.

2. Francis C.A., Beman J.M., Kuypers M.M.M. “New processes and players in the nitrogen cycle: the microbial ecology of anaerobic and archaeal ammonia oxidation”. The ISME Journal. 2007. Volume 1. p. 19-27.

3. Jetten M.S.M., Wagner M., Fuerst J., vanLoosdrecht M., Kuenen G., Strous M. “Microbiology and application of the anaerobic ammonium oxidation (‘anammox’) process”. Current Opinion in Biotechnology. 2001. Volume 12. p. 283-288.

4. Kuypers M.M.M., Sliekers A.O., Lavik G., Schmid M., Jorgensen B.B., Kuenen J. G., Damste J.S.S., Strous M., Jetten M.S.M. “Anaerobic ammonium oxidation by anammox bacteria in the Black Sea”. Nature. 2003. Volume 422 p. 608-611.

5. Mulder A., van de Graaf A.A., Robertson L.A., Kuenen J.G. “Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor”. FEMS Microbiology Ecology. 1995. Volume 16. p. 177-184.

6. Schmidt I., Sliekers O., Schmid M., Cirpus I., Strous M., Bock E., Kuenen J.G., Jetten M.S.M. “Aerobic and anaerobic ammonia oxidizing bacteria – competitors or natural partners?”. FEMS Microbiology Ecology. 2002. Volume 39. p. 175-181.

7. Schmid M., Schmitz-Esser S., Jetten M., Wagner M. “16S-23S rDNA intergenic spacer and 23S rDNA of anaerobic ammonium-oxidizing bacteria: implication for phylogeny and in situ detection”. Environmental Microbiology. 2001. Volume 3. p. 450-459.

8. Schmid M.C., Mass B., Dapena A., van de Pas-Schoonen K., van de Vossenberg J., Kartal B., van Niftrik L., Schmidt I., Cirpus I., Kuenen J.G., Wagner M., Damste J.S.S., Kuypers M., Revsbech N.P., Mendez R., Jetten M.S.M., Strous M. “Biomarkers for In Situ Detection of Anaerobic Ammonium-Oxidizing (Anammox) Bacteria”. Applied and Environmental Microbiology. 2005. Volume 71. p. 1677-1684.

9. Sinninghe-Damste J.S., Strous M., Rijpstra W.I., Hopmans E.C., Geenevasen J.A.A., Van Duin A.C.T., Van Niftrik L.A., Jetten M.S.M. “Linearly concatenated cyclobutate lipids form a dense bacterial membrane”. Nature. 2002. Volume 419. p. 708-712.

10. Stackebrandt E., Frederiksen W., Garrity G.M., Grimont P.A.D., Kampfer P., Maiden M.C.J., Nesme X., Rossello-Mora R., Swings J. Truper H.G., Vauterin L., Ward A.C., Whitman W.B. “Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology”. Internation Journal of Systematic and Evolutionary Microbiology. 2002. Volume 52. p. 1043-1047.

11. Tsushima I., Kindaichi T., Okabe S. “Quantification of anaerobic ammonium-oxidizing bacteria in enrichment cultures by real-time PCR”. Water Research. 2007. Volume 41 p. 785-794.

12. Tsushima I., Ogasawara Y., Kindaichi T., Satoh H., Okabe S. “Development of high-rate anaerobic ammonium-oxidizing (anammox) biofilm reactors”. Water Research. 2007. Volume. 41. p. 1623-1634.

Edited by Daniel Calaguas, student of Rachel Larsen

Edited by KLB