Marinelandâ„¢ ...for the life of your fish
Newsletter Warranty Registration Dealer Area Store Locator View Cart Login
bio-wheel story bio-spira research dr. tim's library visit our laboratory
bio-spira research
bio-spira timeline
bio-spira testimonials
the bio-spira product
order bio-spira

Who's the Ammonia-Oxidizing Bacteria?

by Timothy A. Hovanec, Ph. D.

For the last few months the topic of discussion has been nitrite-oxidizing bacteria. This month, the first group of bacteria in the nitrification process are given a little equal time as I discuss a recent paper which examined who is responsible for ammonia-oxidation in wastewater treatment plants.

For probably close to 30 years or more it has been written in hundreds of books and magazines, related to fishkeeping, that the bacteria which oxidize ammonia to nitrite belong to the genus Nitrosomonas or are flatly the species Nitrosomonas europaea. But there really had not been much research demonstrating whether this was truly the case or not. However, a few years ago I published a paper on some experiments which 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 aquaria. For those who wish to read the original paper the reference is Hovanec, T. A. and E. F. DeLong. 1996. Comparative Analysis of Nitrifying Bacteria Associated with Freshwater and Marine Aquaria. Applied and Environmental Microbiology. Vol. 62:2888-2896. For those not wishing to wade through a scientific paper, I wrote a layman’s version for fish hobbyists which was published as a two-part article in the Dec 1996 and Jan 1997 issues of Aquarium Fish Magazine.

The take home story from that paper was that in freshwater aquaria bacteria which were closely related to Nitrosomonas europaea could not be found. 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 who was actually doing the job and am still working on that answer today. However, some people in the fish hobby business have misinterpreted my paper and are saying that I found that Nitrosomonas europaea does not oxidize ammonia and, by the way, that Nitrobacter winogradskyi does not nitrite. These statements are wrong and are not what I found. Nitrosomonas europaea does oxidize ammonia and Nitrobacter winogradskyi does oxidize nitrite but they don’t seem to be present in freshwater aquaria. Further, some people think that I was just plain wrong but that’s another story.

So why is it assumed that the ammonia-oxidizer is Nitrosomonas europaea? Because, among other things, that’s the bacteria which is believed to oxidize ammonia in wastewater treatment plants. The problems with this thinking it that just because a bacterium in found in one environment which contains the substrate it prefers does not mean it would be automatically found in another environment which also has that substrate. Secondly, no one has proven that N. europaea is really the major ammonia-oxidizer in wastewater facilities. In fact, recent studies has 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 one of those papers is examined. 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. Applied and Environmental Microbiology. Vol. 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 which 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 which 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 spanials, german shepards and pugs in a room full of dogs you could figure out a pretty easy way as they look very different from each other even though they are all the same species.

Bacteria, however, are much more difficult. Further, the methods, even ones which rely of 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 who 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 which were closely related to Nitrosomonas europaea. But this group was not numerically dominant, consisting of only 16 to 20% 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) which they were not yet able to culture. So while Nitrosomonas europaea and close relatives were present in the samples, they were not the most numerous.

This is the first time that 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 bacteria rose to 50% while the number of Nitrosococcus mobilis types declined to 50%. This shows that culturing had a selection effect on the species of bacteria. This results have also been demonstrated before by other researchers. What it means is that when you try to isolate and grow ammonia-oxidizers, N. europaea can growth 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. Since most of the conclusions about which species of bacteria are important for various biogeochemical and industrial processes were made from the results of culture enrichment studies, one has to wonder how much was missed.

The importance of knowing who is really responsible for nitrification is that is 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 aquarium. But what is the basis for how they were formulated? Just putting some N. europaea and Nitrobacter winogradskyi in a bottle does not work. Of course, the use of mixtures is appealing because no one like to wait, but the facts are that there is a lot evidence accumulating from various research efforts which shows that the nitrifying bacteria are not dominated by just two species of bacteria, one for ammonia oxidation the other for nitrite oxidation. Further, the species thought to be important may not be the critical ones.

As aside, since the discussion is really about ammonia-oxidizers, the authors did not find Nitrobacter cells in the sludge. But they did find that about 9% of the cells in the sludge samples were Nitrospira-like. This is another example where Nitrobacter was not found where it was assumed to be, and the nitrite-oxidizing organism was determined to be Nitrospira-like bacteria. This finding is repeating itself in many different environments. I think that there can be little doubt that the nitrite-oxidizing bacteria in aquaria are members of the genus Nitrospira - so have you switched the name you’re using for the nitrite-oxidizing bacteria?

©1998, Timothy A. Hovanec

Originally published in Aquarium Frontiers, Aug. 1998

<< Back


Site Map Contact Security