Background
New, uncycled fish tanks pose a grave danger to their inhabitants: organic material and fish waste turn into ammonia, a toxic chemical that can be deadly to fish at levels above 1.0 ppm (even lower levels are still harmful). In older, well-established tanks, populations of beneficial microorganisms that are able to convert ammonia into nitrite and nitrite into nitrate will have accumulated, thus making the aquariums far safer environments for the aquatic animal(s) inside.
The commonly-held belief within the aquaria community is that bacteria are primarily responsible for the conversion of ammonia to nitrite to nitrate.
For years, people have known about the nitrogen cycle and how the element nitrogen moves across the Earth in various forms. In certain closed ecosystems such as freshwater aquaria, it is quite unlikely that all of the nitrogen cycle's numerous steps can be observed. Thus, when aquarists reference the nitrogen cycle, they are generally referring to a portion of the nitrogen cycle as opposed to the entire cycle. That portion is known as nitrification and is only possible under aerobic conditions. Nitrification can be broken down into a few simple steps:
- The conversion of ammonia (NH3) or ammonium (NH4+) to nitrite (NO2−)
- The conversion of nitrite to nitrate (NO3−)
Currently, the commonly-held belief within the aquaria community is that a number of bacteria—Nitrosomonas, Nitrosospira, Nitrobacter, and Nitrospira—are primarily responsible for the conversion of ammonia to nitrite to nitrate: members of the genera Nitrosomonas and Nitrosospira (the ammonia-oxidizing bacteria, or AOB) gain energy through the oxidation of ammonia, while those classified as Nitrobacter or Nitrospira rely upon the oxidation of nitrite ions.
Similar to the proverb “a chain is only as strong as its weakest link,” any given process can only occur as fast as its slowest step—the rate-limiting step. Nitrification is often limited by the oxidation of ammonia. That given, nitrification can only occur as fast as ammonia is converted into nitrite.
The Discovery
This summer (2017), I have had the opportunity to participate in the “Stanford Earth Young Investigators” program. As an intern, I have been investigating phytoplankton populations in the world’s most southern sea, the Ross Sea. In my time at Stanford, I have had the opportunity to meet numerous people performing cutting-edge research, some of which is quite pertinent to my interests as an aquarist.
While discussing archaea and the nitrogen cycle with Ph.D. student Emily Cardarelli, I learned that archaea are virtually everywhere and that research has been done on their presence in aquaria. Emily has published a paper, “Unraveling the black box of aquaria biofilter function: FISHing for novel ammonia-oxidizing archaea associations”, that detailed “how AOA associate with the ammonia-oxidizing bacteria, and the specific spatial interaction between AOA/AOB and nitrite-oxidizing bacteria.” Intrigued by our conversation (and by Emily's beautiful photos of archaea), I decided to look more into scientific literature involving archaea and aquaria...
In 2005, researchers Martin Könneke, Anne E. Bernhard, José R. de la Torre, Christopher B. Walker, John B. Waterbury, and David A. Stahl published a paper entitled “Isolation of an autotrophic ammonia-oxidizing marine archaeon” in which they detailed their discovery of archaea linked with the nitrogen cycle. The abstract reads:
“Here we report the isolation of a marine crenarchaeote that grows chemolithoautotrophically by aerobically oxidizing ammonia to nitrite--the first observation of nitrification in the archaea [emphasis added].” (Könneke et al. 2005).
While archaea have been known to science since 1977, Könneke was the first to observe ammonia oxidation in archaea. Scientists now have a special name for these organisms: ammonia-oxidizing archaea, or AOA. Following Könneke's 2005 discovery, other groups of scientists came to realize the abundance and importance of AOA.
The majority of ammonia–nitrite conversion in freshwater aquaria is done by archaea, not by bacteria as was previously believed.
Six years later (2011), a group from the University of Waterloo’s Department of Biology (Laura A. Sauder, Katja Engel, Jennifer C. Stearns, Andre P. Masella, Richard Pawliszyn, and Josh D. Neufeld) researched the presence of AOA in small-scale aquaria:
“Twenty-seven freshwater and eight saltwater aquarium filter samples were collected from retail and residential locations in three cities (Table S1). All biofilters sampled in this study were derived from standalone aquaria in homes (or offices), or from display tanks in retail outlets, reflecting conditions common to most residential or retail aquaria, respectively. In addition to aquarium biofilters, we included two aquarium supplements in the analysis. The aquaria selected for sampling ranged in pH from 7.6 to 9.2 and varied in their fish and live plant composition. The aquaria contained a variety of fish including mixed tropical, goldfish, South American cichlids and African cichlids. Three aquaria had received antibiotic treatment in the previous six months (SW4, SW5, FW8) and several were known to have received doses of bacterial filter supplement when first established (e.g. FW12, FW13, FW19, FW25).”
The results published in their paper, “Aquarium Nitrification Revisited: Thaumarchaeota Are the Dominant Ammonia Oxidizers in Freshwater Aquarium Biofilters,” are impressive: “AOA were numerically dominant in 23 of 27 freshwater biofilters, and in 12 of these biofilters AOA contributed all detectable amoA genes. [...] For freshwater aquaria, the proportion of amoA genes from AOA relative to AOB was inversely correlated with ammonium concentration.”
Though the study only analyzed twenty-seven freshwater aquariums, its results can not be ignored—almost half of the samples had zero ammonia monooxygenase (amoA) genes from bacteria. Ammonia monooxygenase is an enzyme involved in the oxidation of ammonia to hydroxylamine (part of the ammonia to nitrite step of the nitrogen cycle). Thus, if archaea were not present in these aquaria, ammonia could not be converted into nitrite. In the majority of the aquariums (23/27), AOB were present, but outnumbered by AOA. The conclusions of this study are groundbreaking—the majority of ammonia–nitrite conversion in freshwater aquaria is done by archaea, not by bacteria as was previously believed.
In all of the eight saltwater aquariums that were investigated, “[b]oth thaumarchaeal and bacterial amoA genes were detected [...], with AOA genes outnumbering AOB genes in five of eight biofilters.” Five out of eight is not a significant enough result to definitively state that AOA outnumber AOB in saltwater aquaria, but such a result does certainly still demonstrate the presence and importance of AOA in saltwater aquaria.
The Potential Impact
Since the discovery of AOA, a handful of aquarium websites including Advanced Aquarist, American Aquarium Products, The Aquarium Wiki, and The Reef Tank have reported their existence. That said, knowledge of AOA in aquaria is still quite uncommon, even amongst seasoned aquarists.
While the existence of a tiny, new microorganism that performs the same processes as AOB may seem inconsequential, the discovery of ammonia-oxidizing archaea should have received far more coverage in the aquaria community than it has so far. A variety of factors including the overall opacity of scientific literature to the layperson and the lack of perceived importance of the discovery have likely contributed to this phenomenon.
An AOA-based nitrifying supplement has the potential to revolutionize the cycling process as we know it.
Currently, there are products on the market such as Tetra's SafeStart Plus that claim to “[accelerate] the establishment of the bio-filter in newly set-up freshwater aquariums.” Their page explains further, “New filters are sterile and take 30 to 40 days for bacteria to naturally form. With SafeStart Plus, all of the beneficial bacteria are seeded from the beginning, for instant, safe results.”
However, Sauder et. al writes of two nitrifying supplements (Big Al’s Multi-Purpose Bio-Support and Hagen/Fluval's Nutrafin Cycle) also tested in their 2011 experiment: “[b]acterial amoA genes were abundant in both supplements, but thaumarchaeal amoA and 16S rRNA genes could not be detected.” Four of the freshwater tanks in their study had “received bacterial aquarium supplements when first established” (product unknown), but, in each of those tanks, “thaumarchaeal amoA genes represented the entire detected amoA gene signal.” There were no more bacteria converting ammonia into hydroxylamine.
Thus, it appears that “bacteria-in-a-bottle” products do not always seed aquaria as is widely claimed. This is not to say that these products are entirely useless, however. Instead of seeding a biofilter with bacteria that will grow to dominate the filter biofilm, these products provide a population of AOB that work to oxidize ammonia until populations of AOA can fully establish themselves.
This raises the possibility of a new product, an AOA-based nitrifying supplement that has the potential to revolutionize the cycling process as we know it. Given the dominance of AOA over AOB in freshwater aquaria (Sauder et al., 2011), a product that primarily populates the biofilter of a freshwater aquarium with AOA instead of AOB may be more effective than the current AOB-only products on the market. In established saltwater tanks, AOA are still present, albeit less dominant than in freshwater ones (Sauder et al., 2011). A product containing a cocktail of AOA and AOB may thus be useful for saltwater aquaria.
While research regarding AOA is still relatively sparse and literature regarding AOA in aquaria is even more scarce, it is certainly in our best interests to learn more about these perplexing microorganisms that play a key role in the nitrogen cycle. Archaea may be tiny organisms, but their importance in aquaria is certainly still a huge discovery.
Final Thoughts
I am so grateful that I am able to be a Stanford Earth Young Investigator this summer. This summer opportunity has so far taught me not only about phytoplankton composition in the Antarctic, but also about the misassumption we have made about bacteria and the nitrogen cycle, the role of AOA in aquariums, and about the potential for AOA-in-a-bottle products.