Nitrospira: The Real Nitrite-Oxidizing Bacteria in Aquaria

[Mr.Reefer]

Nitrospira: The Real Nitrite-Oxidizing Bacteria in Aquaria

by Timothy A. Hovanec, Ph.D.
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 biological oxidation of ammonia to nitrite, and the nitrite to nitrate, is necessary to keep these substances from reaching toxic concentrations and killing the inhabitants of your aquarium.
It has been a paradigm in biology, and the aquarium hobby, that there are two bacteria responsible for nitrification. One, named 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. But 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 efficacy 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 enumerating them. However, modern molecular biology techniques are becoming available which allow one to quantify the nitrifying bacteria, in some cases by species, so that one cannot only measure their increase over time but also determine where in the filter they prefer to live.
This month the paper I will review is one I wrote which was published in January 1998. The citation is Hovanec, T. A., L. T. Taylor, A. Blakis, and E. F. DeLong. 1998. Nitrospira-like bacteria associated with nitrite oxidation in freshwater aquaria. Applied and Environmental Microbiology 64:258-264. The results in this paper continue, in part, the earlier work which I published on investigating the actual bacteria responsible for nitrification in aquaria (Hovanec, T. A. and E. F. DeLong. 1996. Comparative analysis of nitrifying bacteria associated with freshwater and marine aquaria. Applied and Environmental Microbiology 62:2888-2896.)
The first part of this study was to find out who the actual nitrite-oxidizing bacteria were in aquaria. 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 aquaria. 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. In a very simplified form what you are doing in this procedure is taking a piece of filter covered with bacteria, extracting and purifying the DNA. Now you have the DNA from all the bacteria which were on the filter but you have to separate them. This is done by cloning as the DNA from only one bacteria will be inserted into one clone cell. After this is done, you can retrieve the insert and sequence it.
I won't go further into the many details of doing this except to say that it takes weeks of lab work to perform the many tasks associated with finally getting sequence data to analyze. This 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 which were related to Nitrobacter winogradskyi and its close relatives. However, there was a sequence which 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 had found was closely related to both of these.
So now I, at least, had a target in that there was a bacterium in the filters which was related to known nitrite-oxidizing bacteria. So I developed two molecular probes for the bacteria and its closest relatives which would allow to me quantify the amount of this bacterium in nucleic acid samples extracted from aquarium filters. I then did an experiment were I took nucleic acids from many aquarium set-ups, including freshwater, saltwater, with fish in the tanks, tanks which were dosed with ammonium chloride, heavily and lightly stocked tanks, and probed them with the molecular probes for both Nitrobacter and Nitrospira. The results were that 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 aquaria 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 the filters. So next I want to see if I could tell when the Nitrospira-like bacteria appear in a newly set-up filter and 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 the 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 then 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 pertaining to a particular species of bacteria) are separated in the gel so you can see a band in the gel which 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 represents. When you combine the data from the clone libraries with the DGGE data you have strong evidence of as to the make-up of the bacterial assemblage in your system.
With DGGE you can run many samples side by side and by running controls, and from sequence knowledge, I knew which band in the gel lane would correspond to the Nitrospira-like bacteria. So I did two experiments: in one a set-up aquaria and ran them for nearly 140 days and took bacterial samples every 7 days. In the other, I set the aquaria up and ran them for 35 days, taking bacterial samples every day. In both experiments, I collected water samples (3 times a week for the first test, daily for the second test) 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 a nitrate was increasing rapidly.
In this fashion, I was able to show a correlation between the appear in the Nitrospira-like bacteria in the samples (and so on the filter) and the prevailing water chemistry in the aquarium.
For the final test which I reported in this paper I looked at the effects of adding a bacterial additive to aquaria during the start-up phase. Replicate aquaria were set-up and dosed with ammonium chloride. To one set, a commercially available bacterial additive was added on a weekly basis as per the manufacturers instructions (Kathleen, the additive was Cycle by Hagen - you can name it or not, that's up to you). The other set did not receive an 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.
The results were that 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 they fail to become established. The possible positive part of 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 which received the additive. While there are more statistics to do, it seems that the additive did have a kind of fertilization effect. What I surmise is that there are nutrients in the additive which the Nitrospira-like bacteria can use so their numbers increase faster than in the tanks without the additive. Wether this is a significant increase or not I cannot answer at this time, further the nitrite concentration in the additive tanks still rose to toxic levels so the additive did not short-circuit the break in period.
In conclusion, the results of the many tests which I report in this paper demonstrate that Nitrobacter winogradskyi and its close relatives are not the nitrite-oxidizing bacteria in aquaria. Rather that task falls to Nitrospira-like bacteria. So another paradigm falls which is the natural course of science as data is collected which allows one 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.