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Bacteria Revealed

After all these years, it turns out that the nitrifying bacteria are not what we thought.

Biological filtration is the critical filtration component in every aquarium. Whether the biological filter is live rock, trickle media, a sponge or any other substrate, the oxidation of ammonia to nitrite and then to nitrate is necessary to keep these substances from reaching toxic concentrations in our aquariums. It has been a basic tenent in biology and the aquarium hobby that there are two bacteria responsible for nitrification. The first, called Nitrosomonas europaea, oxidizes ammonia to nitrite, while the second, Nitrobacter winogradskyi, oxidizes nitrite to nitrate.

These organisms are called nitrifiers and are classified as belonging to the same family of bacteria. However, recent work on the phylogenetics of these organisms and their close relatives has shown that this classification is wrong and needs to be revised.

The efficiency of a biological filter is usually evaluated from the standpoint of ammonia and nitrite removal. The bacteria responsible for actually doing the work are not directly quantified because of the inherent difficulties with identifying and counting them. However, modern molecular biology techniques that allow one to quantify the nitrifying bacteria, in some cases by species, are becoming available, so one can measure not only their increase over time, but can also where in the filter they prefer to live.

The paper reviewed here is one I wrote that was published in January 1998 (Hovanec, T. A., L. T. Taylor, A. Blakis and E.F. DeLong. 1998. Nitrospira-like bacteria associated with nitrite oxidation in freshwater aquaria. Appl Environ Microbiol 64:258-264). The results in this paper are a continuation, in part, of the earlier work I published on investigating the actual bacteria responsible for nitrification in aquariums (Hovanec, T. A. and E. F. DeLong. 1996. Comparative analysis of nitrifying bacteria associated with freshwater and marine aquaria. Appl Environ Microbiol 62:2888-2896).

The purpose of the first part of this study was to identify the actual nitrite-oxidizing bacteria in aquariums. My earlier paper had shown that Nitrobacter winogradskyi and its close relatives in the alpha subdivision of the Proteobacteria are not present in measurable quantities in freshwater or saltwater tanks. So, I first had to develop what is called a "clone library" and sequence a fragment of DNA from the many clone colonies that were produced. To put it simply, in this procedure a piece of filter covered with bacteria is processed, leaving the DNA from all the bacteria that were on the filter. Then they have to be separated. This is done by cloning, in which the DNA from only one bacterium will be inserted into one clone cell. After this is done, the insert can be retrieved and sequenced. Without going into the many details of doing this, it takes weeks of lab work to perform the many tasks associated with finally getting sequence data to analyze. The data is in the form of the 16S ribosome DNA gene, which is a standard used to compare and relate species of organisms.

From the DNA data, I found that there were no sequences in my clone libraries that were related to Nitrobacter winogradskyi and its close relatives. However, there was a sequence that was related to known nitrite-oxidizing bacteria of the species Nitrospira. There are, so far, two known species of Nitrospira (Nitrospira marina and Nitrospira moscoviensis). The organism I found was closely related to both of these. Now, I at least had a target in that there was a bacterium in the filters that was related to known nitrite-oxidizing bacteria.

So, I developed two molecular probes for the bacteria and its closest relatives that would allow me to quantify the amount of this bacterium in nucleic acid samples extracted from aquarium filters. I then did an experiment. I took nucleic acids from many aquarium setups (freshwater and saltwater, tanks with fish, tanks dosed with ammonium chloride, heavily andlightly stocked tanks) and probed them with the molecular probes for both Nitrobacter and Nitrospira. In no case did I find Nitrobacter (same results as in my earlier paper), but in every freshwater aquarium I found Nitrospira, and in each saltwater aquarium I found evidence of Nitrospira. Thus, I am able to conclude that Nitrobacter and its close relatives are not the nitrite-oxidizing bacteria in aquarium filters, and that Nitrospira-like bacteria are present in filters.

The next thing I wanted to ascertain was whether I could tell when the Nitrospira-like bacteria appear in a newly set-up filter and then relate their appearance to the water chemistry. Meaning, could I see a correlation between the appearance of the Nitrospira-like bacteria and the oxidation of nitrite to nitrate.

For this, I used a process called the polymerase chain reaction (PCR) on aquarium samples. This procedure allows you to amplify (increase) the amount of target DNA in your sample when you mix it with special reagents and place it in a machine called a thermal cycler. I took this PCR product and ran it in a special electrophoresis machine called a denaturing gradient gel electrophoresis (DGGE). When you run DNA in the DGGE, the individual segments of DNA (each segment perhaps pertaining to a particular species of bacteria) are separated in the gel so you can see a band in the gel that corresponds to the bacteria you are interested in, as well as other bacteria in the sample. As an aside, the bands can also be cut out of the gel, purified and then sequenced just like in the clone library. This is one way for you to identify what species of bacteria the band may represent.

When you combine the data from the clone libraries with the DGGE data, you have strong evidence as to the makeup of the bacterial assemblage in your system. Using DGGE you can run many samples side by side, and by running controls (and from sequence knowledge) I would know which band in the gel lane would correspond to the Nitrospira-like bacteria.

So, I performed two experiments. In the first, I set up aquariums and ran them for nearly 140 days, taking bacterial samples every seven days. In the other, I ran the aquariums for 35 days, taking bacterial samples every day. I collected water samples (three times a week for the first test, daily for the second test) from each group and analyzed them for ammonia, nitrite and nitrate.

Using the DGGE technique, I ran the PCR-amplified samples to see what banding pattern was present. In the first test, the Nitrospira-like bacteria did not appear in the pattern with a strong signal until about day 22. After that they remained in the samples and were present in large numbers, as evidenced by the intensity of the band.

In the second test, the Nitrospira-like bacteria appeared starting on day 12, which was also the day an increase in nitrate was measurable in the samples. By day 18, the signal was quite strong and the water chemistry data showed that nitrate was increasing rapidly. In this manner I was able to show a correlation between the appearance of the Nitrospira-like bacteria in the samples (and thus on the filter) and the prevailing water chemistry in the aquarium.

For the final test, I looked at the effects of adding a bacterial additive to aquariums during the start-up phase. Duplicate aquariums were set up and dosed with ammonium chloride. A commercially available bacterial additive was added to one set on a weekly basis as per the manufacturer's instructions. The other set did not receive any additive. I measured water chemistry three times a week and took filter samples for bacterial analysis. I used the molecular probes for Nitrobacter and Nitrospira-like bacteria on these samples.

I did not detect Nitrobacter in either situation, but I did detect Nitrospira-like bacteria in both cases. Thus, even when adding Nitrobacter to the system, these bacteria fail to become established. The only possible benefit to adding the additive was that a greater percentage of the total bacteria DNA in the samples was from the Nitrospira-like bacteria in the tanks that received the additive.

While there are more tests to be performed, it seems that the additive did have a kind of "fertilization" effect. What I surmise is that there are nutrients in the additive that the Nitrospira-like bacteria can use to increase their numbers faster than in tanks without the additive. Whether this is a significant increase or not I cannot answer at this time. In addition, the nitrite concentration in the additive tanks still rose to toxic levels, indicating that the additive did nothing to "shorten" the break in period for the tank.

Finally, the results of the many tests I report in this paper demonstrate that Nitrobacter winogradskyi and its close relatives are not the nitrite-oxidizing bacteria in aquariums. Rather, this task falls to the Nitrospira-like bacteria. So, another myth is toppled. This is the natural course of science -- to test and re-test what is known and not known about the world we live in, or, in our case, a small environment called a fish aquarium.

Timothy Hovanec is the Director of Aquatic Research for Marineland Aquarium Products and has extensive professional experience in aquatic systems design, water chemistry and aquaculture. He holds a Ph.D. candidate in aquatic ecology from the University of California.

 © Timothy Hovanec. May not be reproduced without permission.

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