Course:EOSC 475/ResearchProject/AnammoxBacteria

A diagram illustrating the marine nitrogen cycle. Anammox is being discovered to be a more important process in the marine nitrogen cycle than originally thought

Electron micrograph of an anammox bacterium showing internal compartmentalization

“Anammox” is a widely-used acronym for anaerobic ammonium oxidation. This is the process by which ammonia is converted, using either nitrate or nitrite as the electron acceptor, to N₂. The reaction is as follows:

NH₄⁺ + NO₂^- → N₂ + 2H₂O

The anammox reaction is performed by an elite group of chemoautolithotrophic bacteria, mostly comprised of select species from the genus Planctomycetales, discovered in the early 1990s in waste-water sludge. This group of bacteria grows very slowly, approximately dividing only once every two weeks Unlike other described bacteria, the Planctomycetales exhibit many membrane-bound intracellular compartments, both single- and double-layered. These intracellular compartments, including the double-membrane surrounded “anammoxosome” which is crucial to anammox catabolism, are common among all known anammox bacteria. Upon examining cell surfaces, researchers also discovered circular-electron dense areas on their cell surfaces, known as crateriform structures and lack peptidoglycan in their cell walls. The discovery of anammox bacteria led to the awareness of their overall importance in nitrogen turnover (up to 50%) in the marine environment.

Background

The Nitrogen Cycle

Overview of the nitrogen cycle.

List of compounds involved in the global nitrogen cycle

The global nitrogen cycle includes various nitrogen-containing compounds, both organic and inorganic, and describes the fluxes between various sinks and reservoirs, the largest being the atmosphere which is primarily composed (80%) of dinitrogen gas, N₂. Other major reservoirs include soil, sediments of lakes, rivers and oceans, dissolved N in surface and groundwater and in the biomass of living organisms. Key processes involved in the nitrogen cycle include:

• Nitrogen fixation – bacterial conversion of N₂ to fixed organic nitrogen
• Ammonification – the decomposition of organic nitrogen, forming NH₄⁺ (ammonia)
• Nitrification – the conversion of NH₄⁺ to NO₃^2- by nitrifying bacteria
• Denitrification – in anaerobic conditions, denitrifying bacteria use NO₃^- to metabolize, releasing N₂ as a byproduct.

Nitrogen is crucial to life as it is used in the construction of amino acids (and thus proteins) and nucleic acids and is usually a limiting nutrient to marine autotrophs, among other organisms

Denitrification

It was previously thought that denitrificiation (converting nitrate to N₂) by heterotrophic bacteria was the only major sink for fixed inorganic nitrogen in the ocean. The gradual process of denitrification can be outlined as follows:

NO₃²⁻ → NO₂⁻ → NO + N₂O → N₂ (g)

Denitrification is carried out by many genera and physiological types of bacteria and most of these denitrifying bacteria carry out these processes in anaerobic conditions. However, removal of ammonium from anaerobic environments coupled with nitrite disappearance suggested that another process was taking place in order to regenerate dinitrogen gas, a process that became known as “Anammox”

Rising interest in "Anammox"

Transmission electron micrograph of the known anammox bacterium Candidatus Kuenenia stuttgartiensis, part of the Planctomycetes. The scale bar represents 200 nm. Note the large intracellular compartment known as the anammoxosome.

In their article, Graaf et al. propose that because anaerobic ammonium oxidation is exergonic, in theory, it could produce energy for chemoautolithotrophs however, as of 1994, there was no published evidence proving the anaerobic removal of ammonium coupled with nitrate (or nitrite) reduction in marine ecosystems. Despite not having an actual "poster child" for the anammox reaction, later discovered in the Planctomycetes, Strous, Kuenen and Jetten sought to describe the full physiology of anaerobic ammonium oxidation and compared physiological parameters between anaerobic and aerobic ammonium oxidation (anammox and nitrification, respectively) Finally, around 1999, the "missing lithotroph" that could perform the already well-studied anammox process, was finally discovered to be a member of the Planctomycetes (of which, not all bacteria are capable of the anammox reaction)

Anammox bacteria and the anammoxosome

All known anammox bacteria contain an intracellular compartment known as the “anammoxosome.” The intracellular membrane of the anammoxosome is a dense membrane composed almost entirely of ladderdane lipids, explained later. This compartment contains the hydrazine/hydroxylamine oxidoreductase enzyme which is essential for anammox catabolism. During aerobic oxidation of ammonium, the intermediate produced is hydroxylamine (NH₂OH), but in anaerobic oxidation of ammonium (anammox), bacteria can use this aerobic oxidation intermediate in addition to nitrate/nitrite to produce the intermediate, hydrazine (N₂H₄). This intermediate is eventually converted into N₂, using the hydrazine/hydroxylamine oxidoreductase enzyme present in the anammoxosome.

Anaerobic ammonium oxidation by anammox bacteria in the Black Sea

Marcel M. M. Kuypers, A. Olav Sliekers, Gaute Lavik, Markus Schmid, Bo Barker Jergensen, J. Gijs Kuenen, Jaap S. Sinninghe Damste, Marc Strous and Mike S. M. Jetten

Aim

Kuypers et al. provide evidence for bacteria anaerobically oxidizing ammonium (NH₄⁺) with nitrite (NO₂) to produce N₂ in the world’s largest anoxic aquatic environment, the Black Sea. They show that ammonia diffusing up from the sediments is consumed by anammox bacteria below the oxic layer of the Black Sea, illustrating for the first time that bacteria that can undergo the anammox process are directly linked to the removal of fixed inorganic nitrogen in aquatic environments. Their research suggests that the anammox process may be a major player in the overall global nitrogen cycle.

Results

Morphology and physiology of anammox bacteria and their role in the marine nitrogen cycle

Nutrient Analyses

Methods

Once water samples were obtained, nitrate, nitrite and ammonium concentrations were measured immediately with an autoanalyser.

Analysis

By using both microbiological and biogeochemical methods, Kuypers et al. observed maximum nitrite levels just below the oxic zone in the Black Sea. They presumed that this maximum was due to both the mineralization of organic nitrogen derived by phytoplankton and aerobic nitrification by nitrifying bacteria. They found ammonium concentrations to be high in deep waters, but were very low in the top 100 meters of the water column. Since aerobic nitrification, which can only occur in approximately the top 80 meters of the water column, cannot be the cause of low ammonia levels, and nitrite persists in deeper waters, and nitrite is an intermediate in the denitrification process, they supposed that nitrite could be the oxidizer of ammonium in deeper waters.

By anaerobically incubating water samples and adding distinguishable isotopes of nitrite and ammonium ([¹⁴N] nitrite and [¹⁵N] ammonium), the depth distribution of nitrogen isotopes indicated that the anammox reaction could be occurring in the suboxic zone, while no anammox activity was shown to occur outside this zone.

Lipid Analysis

Structures of various ladderane lipids. Ladderane substructures are bolded and lipid headgroups are blue, red and green.

Ladderane lipids are the primary subunits of the bacterial membrane surrounding the anammoxosome. They were used to trace anammox bacteria in particulate organic matter throughout the suboxic zone. They consist of stringed cyclobutane rings, thus forming a molecular ladder

Methods

Particulate organic matter for lipid analysis was captured using in situ filtration of ~1000L water samples through glass fiber filters with a pore size of 0.7 µm. As filtration through pores this small could cause a loss of anammox bacteria themselves, thereby reducing cell counts, the proposed concentrations of ladderane lipids are minimum values. After an internal standard was added, thus separating fatty acids fractions and neutral lipids, aliquots of extracted particulate organic matter were saponified. The fatty acid fractions were methylated and neutral lipid fractions were silyated so that analysis by gas chromatography – mass spectrometry could be performed and ladderane lipids could be identified and quantified.

Analysis

Three different ladderane lipids were discovered, all exhibiting a depth distribution in the water column comparable to that of anammox activity. This further indicated that anammox bacteria may be responsible for the anaerobic removal of ammonia.

Phylogenetic tree of 16s rRNA gene sequences showing the Black Sea clone, as related to other known Anammox bacteria

Clone Library Produced

Anammox bacteria fluorescently labeled with the oligotrophic FISH probe

A clone library was produced from DNA taken from the Black Sea water samples at the depth where maximum ladderane lipids were found and amplification of the 16s ribosomal RNA gene with primers unique to Planctomycetes bacteria. Phylogenetic analysis of these sequences confirmed that the bacteria present in the Black Sea are closely related to known anammox bacteria. An oligonucleotide probe was created (using FISH, or fluorescence in situ hybridization), specific for the physiological shape of known anammox bacteria and ladderane biomaerkers along with cells attaching to the FISH probe were identified in the suboxic zone. Average number of anammox bacteria was averaged based on counts from 20 different slides.

Calculating Anammox Rates

An anammox rate of ~0.007 µM/day in the suboxic zone was calculated using a reaction diffusion model which compared to aerobic ammonium oxidation rates of 0.005 – 0.05 µM/day typical of the Black Sea. Kuypers et al. deduced that 300 – 3000 anammox cells per mL would be required to see the anammox rate calculated, and these cells were accounted for using the FISH probe, which gave counts of ~1900 +/- 800 cells per mL.

Conclusions

Although they recognize the obvious assumptions made during the above calculations, Kuypers et al. propose that these calculated rates show that reduction of nitrate by denitrifying bacteria along with anammox accounts for a large amount of fixed inorganic nitrogen lost from suboxic waters in the Black Sea. They deduced that anammox may consume up to 40% of ammonia sinking to the deep anoxic waters in the Black Sea. They also prove the high abundance of anammox bacteria in this anoxic environment, and the importance of anammox bacteria in the Black Sea’s nitrogen cycle.

Critical Analysis

(A critical summary of the strengths and weaknesses of this paper.) This should be the most important section of you wiki project

Weaknesses

In their final cell counts of anammox bacteria in Black Sea waters, we can see the major uncertainty involved. Uncertainty in cell counts measured over 40% of actual cell “counts.” This is primarily due to the various assumptions made throughout the calculations in order to get to a final cell count. There was also tremendously large uncertainty used for cells required to exhibit measured anammox levels (300 – 3000 cells/mL). With these large variances, one could question the validity of the final cell counts calculated in the Black Sea samples.

Strengths

Overall, Kuypers et al. were very thorough in their procedures. They used modern technology to their advantage, by using various computer programs to create models which assisted in their calculations. By using a new oligonucleotide FISH probe which binds specifically to Annamox bacteria, they were able to effectively use the fluorescent stain DAPI (4',6-diamidino-2-phenylindole) to perform cell counts. They also clearly outlined the uncertainty involved in their results, indicating that, although we cannot be completely sure, we can assume that Anammox performs a more prominent role in the nitrogen cycle of the Black Sea and subsequently, other anoxic environments.

Personal Opinion

Although they performed cell count calculations, the uncertainty of error was very high, making the average reader somewhat uncertain about the significance of the findings. Also, although the information presented was interesting and unique (as anammox has never been studied in the Black Sea), the broad significance of anammox and nitrogen flux rates in the Black Sea was left unaddressed.

Impact on the scientific community

Further Research Performed

Much further research has been done on anammox and the specific bacteria that can perform the process. Prior to the research done by Kuypers et al., most, if not all, of the research had centered around marine environments and waste water treatment plants however, Schubert et al. discovered anammox occurring in freshwater Lake Tanganyika, the second largest lake by volume in the world, located in Southern Africa. By describing the rate anammox occurs in the freshwater environment of Lake Tanganyika, they showed that anammox bacteria are present and active in freshwater environments, in addition to waste water and marine environments They suggest that further research be done on anammox in freshwater environments to better assess the role of anammox in freshwater ecosystems in the global nitrogen cycle.

Industrial Importance of Anammox

Originally found in ammonia-rich waste water treatment plants, anammox bacteria provide amazing opportunities in removal of excess ammonia, nitrate and nitrite, forming nitrogen gas using biological rather than chemical processes, thus returning N₂ to the atmosphere. A process has been patented, termed the SHARON (single reactor system for high-rate ammonium removal over nitrate), which converts ammonium to nitrite only, rather than nitrate, which is extremly costly to remove. This process was found to produce a 50:50 ratio of ammonium:nitrite, thus facilitating the anammox process almost perfectly.

Future Research Opportunities

Although much has been discovered about the anammox reaction and anammox bacteria in the last two decades, there is still a lot left to learn. Future research opportunities should be geared towards the role of anammox in the global nitrogen cycle, rather than just in specific ecosystems.

References

Annotated Bibliography

1. Thamdrup, B. & Dalsgaard, T. Production of N₂ through anaerobic ammonium oxidation coupled to nitrate reduction in marine sediments. Applied and Environmental Microbiology. 68(3), 1312-1318 (2002).

   This article distinguishes between the process of denitrification and the anaerobic oxidation of ammonium (united with nitrate reduction) and identifies the latter as a significant contributor of N₂ in marine sediments.

2. Strous, M. et al. Missing lithotroph identified as new planctomycete. Nature. 400, 446–449 (1999).

   This article describes a lithotrophic bacterium of the order ‘’Planctomycetales’’ and identifies it to be responsible for converting ammonium in anoxic waters.</blockquote

3. Van de Graaf, A. A., Mulder, A., De Bruijn, P., Jetten, M.S.M., et al. Anaerobic oxidation of ammonium is a biologically mediated process. Applied and Environmental Microbiology. 61(4), 1246-1251 (1995).

   This article proposes that the Annamox process uses equal amounts of ammonium and nitrate in order to produce N₂.

4. Kawagoshi, Y., Nakamura, Y., Kawashima, H., et al. Enrichment culture of marine anaerobic ammonium oxidation (anammox) bacteria from sediment of sea-based waste disposal site. Journal of Bioscience and Bioengineering. 107(1), 61-53 (2009).

   This study aims to create an improved culture of anammox bacteria in marine environments using samples obtained at waste-disposal sites located near marine environments.

5. Schubert, C.J., Durisch-Kaiser, E., Wehrli, B., et al. Anaerobic ammonium oxidation in tropical freshwater system (Lake Tanganyika). Environmental Microbiology. 8(10), 1857-1863 (2006).

   This article offers innovative evidence showing the anammox process in a lacustrine system, showing the importance of anammox bacteria in the production of N₂.

6. Schmid, M. C., Maas, B., Dapena, A., et al. Biomarkers for in situ detection of anaerobic ammonium-oxidizing (anammox) bacteria. Applied and Environmental Microbiology. 71(4), 1677-1684 (2005).

   This article uses rRNA- and non-rRNA-based methods to fully study the actions of anammox bacteria.

7. Op den Camp, H. J. M., Kartal, B., Guven, D., et al. Global impact and application of the anaerobic ammonium-oxidizing (anammox) bacteria. Biochemical Society Transactions. 34(1), 174-178 (2006).

   This review analyzes recent research suggesting that, when facing low levels of ammonium, anammox bacteria may use organic acids to convert nitrate/nitrite into N₂.

8. Tal, Yossi, Watts, Joy E. M., Schreier, Harold J. Anaerobic ammonium-oxidizing (anammox) bacteria and associated activity in fixed-film biofilters of a marine recirculating aquaculture system. Applied and Environmental Microbiology. 72(4), 2896-2904 (2006).

   This article compares the processes of denitrification and anammox and proposes ways that the anammox process may be an alternative to denitrification in some situations.

9. Kartal, Boran, Kuypers, Marcel M. M., Lavik, Gaute, et al. Anammox bacteria disguised as denitrifiers: nitrate reduction to dinitrogen gas via nitrite and ammonium. Environmental Microbiology. 9(3), 635-642 (2007).

   This study investigates the breakdown of nitrate (NO₃^2-) by anammox bacteria to produce ammonium (NH₄⁺) and nitrite (NO₃^-) in order to use the two to make N₂.

10. Jetten, M. S. M., Sliekers, O., Kuypers, M., et al. Anaerobic ammonium oxidation by marine and freshwater planctomycete-like bacteria. Appl Microbiol Biotechnol. 63(2). 107-114 (2003).

    This article discusses the cooperation of two groups of ammonium-oxidizing bacteria (anaerobic anammox bacteria and aerobic ammonium-oxidizing bacteria) in order to create a system to remove ammonia from highly polluted waste water.