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Do you have questions, want to discuss the issues raised in this column, or read the comments of other aquarists and the answers from columnist Tim Hovanec? You can do so by going to:Topical Science Interactive. What Are the Ammonia-Oxidizing Bacteria Really?
For the last few months the topic of discussion in this column has been nitrite-oxidizing bacteria. This month, I will give the first group of bacteria in the nitrification process a little equal time by discussing a recent paper that examined just which ones are responsible for ammonia-oxidation in wastewater treatment plants.For close to 30 years or more the commonly held opinion in hundreds of books and magazines related to fishkeeping was that the bacteria that oxidize ammonia to nitrite belong to the genus Nitrosomonas, and, in fact, that they are actually the species Nitrosomonas europaea. However, there had been little research done to demonstrate whether this was truly the case or not.
A few years ago I published a paper on some experiments I had done using modern molecular methods to identify the ammonia-oxidizing bacteria in aquaria. The results of the tests showed that Nitrosomonas europaea and its close relatives could not be found in the biofilters of any aquaria. Those of you who wish to read the original paper can find it under the following reference: Hovanec, T. A. and E. F. DeLong. 1996. Comparative Analysis of Nitrifying Bacteria Associated with Freshwater and Marine Aquaria. App Env Microbiol 62:2888-2896. I also wrote a “layman’s” version for fish hobbyists not wishing to wade through a scientific paper. It was published as a two-part article in Aquarium Fish Magazine (December 1996 and January 1997).
The point of this research is that I could not find bacteria that are closely related to Nitrosomonas europaea in freshwater aquaria. Nor could I find any other known species of ammonia-oxidizer, even though the water chemistry demonstrated that ammonia — either added to tank in the form of ammonium chloride or excreted by fish — was being rapidly converted to nitrate, the end product of nitrification. I was not able to figure out which bacteria were actually doing the job and am still working on that answer today.
However, there are those in the fish hobby business who have misinterpreted my paper. They maintain that I found that Nitrosomonas europaea does not oxidize ammonia and, in fact, that Nitrobacter winogradskyi does not oxidize nitrite. These statements are wrong. What I actually found was that Nitrosomonas europaea does oxidize ammonia and Nitrobacter winogradskyi does oxidize nitrite — they just don’t seem to be present in freshwater aquaria. Further, there are those who think I was just plain wrong...but that’s another story.
So, why do we assume that the ammonia-oxidizer in our freshwater aquaria is Nitrosomonas europaea? Because, among other things, those are the bacteria that are believed to oxidize ammonia in wastewater treatment plants. But there are problems with this thinking. First, just because a bacterium is found in one environment that contains the substrate it prefers does not mean it would automatically be found in another environment that also has that substrate.
Second, no one has proven that N. europaea is really the major ammonia-oxidizer in wastewater facilities. In fact, recent studies have shown that while N. europaea can be found in the microbial assemblage of wastewater plants, they may not be the dominant ammonia-oxidizer.
This month I will discuss one of the papers that addresses this topic. The formal reference is: Juretschko, S., G. Timmermann, M. Schmid, K.-H. Schleifer, A. Pommerening-Roser, H.-P Koops and M. Wagner. 1998. Combined molecular and conventional analyses of nitrifying bacterium diversity in activated sludge: Nitrosococcus mobilis and Nitrospira-like bacteria as dominant populations. App Env Microbiol 64:3042-3051.
In this study, the authors collected activated sludge samples from a wastewater treatment facility and analyzed them by a variety of methods. The methods included one called FISH! FISH stands for “fluorescent in situ hybridization.” FISH is a way to label an individual cell with a fluorescent probe that can be seen using a special type of microscope. The power of FISH is that the probe can be tailored to hybridize (or match) an individual species of bacterium or a group of related bacteria by using the genetic code that is specific to the species or group. By using different color probes for different species or groups, the researchers are able to count the individual bacteria cells for several species or groups in one sample at the same time.
The reason this is important is that most bacteria look pretty much the same, so how can you distinguish the different species? If your job was to count the number of cocker spaniels, German shepherds and pugs in a room full of dogs, you could figure out a pretty easy way to do this because they all look very different from each other, even though they are all the same species. Bacteria, however, are much more difficult. Additionally, the methods, even those that rely on molecular information, such as DNA, can be hard to get to work. In any case, the researchers were able to develop a suite of FISH probes for various groups of ammonia-oxidizing bacteria so they could see what was in the sludge.
They also examined nucleic acid extracts from the sludge using the PCR process with primers specific for ammonia- and nitrite-oxidizing bacteria. Finally, they used the conventional microbiological method for studying bacteria, which is to attempt to grow them in enrichment cultures. Thus, they examined the sludge for ammonia- and nitrite-oxidizing bacteria by three different methods.
The results of their investigation showed that there were at least three different groups of ammonia-oxidizing bacteria in the sludge. One of the groups consisted of bacteria that were closely related to Nitrosomonas europaea, but this group was not numerically dominant, consisting of only 16 to 20 percent of the cells in the sample. Instead, the most numerous ammonia-oxidizing cells were related to Nitrosococcus mobilis. The third group of ammonia-oxidizing bacteria seemed to be composed of a novel, uncharacterized bacterium (or bacteria) that they were not yet able to culture. So, while Nitrosomonas europaea and its close relatives were present in the samples, they were not the most numerous.
This is the first time Nitrosococcus mobilis-like cells have been reported to be important in wastewater nitrification. In the past, they were isolated from brackish water environments. Another interesting finding was that when the researchers enriched samples to grow the ammonia-oxidizing bacteria they found that the numbers of N. europaea types of bacteria rose to 50 percent, while the number of Nitrosococcus mobilis types declined to 50 percent. This shows that culturing had a selection effect on the species of bacteria. These results have also been demonstrated by other researchers in the past.
What does all this mean? For one thing it means that when you try to isolate and grow ammonia-oxidizers, N. europaea may grow better than other species and may numerically dominate in the culture vessel, even though they were not the dominant ammonia-oxidizing bacteria in the natural setting. And, because most of the conclusions about which species of bacteria are important for various biogeochemical and industrial processes were made from the results of just such culture enrichment studies, one has to wonder how much was missed.
It is important to know what bacteria are really responsible for nitrification because, in order to mimic nature, we have to understand what we are trying to copy. There are many brands of bacterial mixtures for jump starting or accelerating the break-in period of a newly set-up aquariums on the market. On what basis were they formulated? From research that has been done thus far we know that just putting some N. europaea and Nitrobacter winogradskyi in a bottle does not work. Of course, using this type of mixture is appealing to a lot of people because most of us don’t like to wait. However, there is a lot evidence accumulating from various research efforts that shows that the nitrifying bacteria are not dominated by just two species of bacteria — one for ammonia oxidation and the other for nitrite oxidation. Further, the species that we had previously thought to be important may not be the critical ones.
Note: Because this study was really about ammonia oxidizers, the authors did find that there were no Nitrobacter cells at all in the sludge. But they did find that about 9 percent of the cells in the sludge samples were Nitrospira-like. Here is yet another example showing that Nitrobacter was not found where it was assumed to be and that the nitrite-oxidizing organism was actually a Nitrospira-like bacteria.
This finding is repeating itself in many different environments. I think there can be little doubt that the nitrite-oxidizing bacteria in our aquaria are members of the genus Nitrospira. Have you switched the name you’re using for the nitrite-oxidizing bacteria yet?
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