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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. Nitrospira, not Nitrobacter...Again!
In a recent edition of this column I discussed the results of some of my published research, which showed that the nitrite-oxidizing bacteria in aquaria are members of the Nitrospira group of bacteria rather than species of Nitrobacter or its close relatives, as has been written for decades. But, how widespread is this finding? Could Nitrobacter spp. still be the dominant nitrite-oxidizing bacteria found in sewage treatment facilities? How about in lake sediments? The ocean water column?This month I will detail work that was done during the same time I was working on Nitrospira by a group who did not know about my results because they had not yet been published. As often happens in science, two or more groups of researchers can be working independently on the same problem and not even be aware of each other, but they will still come up with the same answer(s). This paper is also about Nitrospira and how it was found in sludge from a wastewater treatment system instead of the familiar nitrite-oxidizing bacterium — Nitrobacter spp. The formal reference for the paper is Burrell, P. C., J. Keller and L.L. Blackall. 1998. Microbiology of a Nitrite-Oxidizing Bioreactor. Applied Env Microbiol 64:1878-1883.
The goal of this study was to determine the species of bacteria that oxidize nitrite in the wastewaters of a domestic sewage treatment plant by inoculating a controlled culturing environment with some mixed liquor from the sewage plant and to analyze the activated sludge of the treatment plant using molecular methods. Mixed liquor is the name given to the mixture of liquid, suspended particles and the active culture of microorganisms that are formed in activated sludge facilities, such as the one sampled.
Nitrification using activated sludge differs from nitrification using trickle filters or rotating biological contactors. In the case of trickle filters and rotating biological contactors, the bacteria grow on solid, fixed surfaces (specially shaped plastic spheres, rocks or disks that turn through the water), while in the activated sludge the bacteria grow on the suspended particles (the sludge) in the treatment channel. Both methods work and the reasons for using one or another are not important for the present discussion.
The researchers add some of the mixed liquor to an inorganic culture medium that was maintained in a special culturing device called a chemostat. The chemostat is a controlled environment — the researchers can manually (or automatically with dosing pumps and probes) adjust pH, dissolved oxygen, nitrite and many other characteristics. In this research the chemostat was operated in a manner that mimics the pathway the activated sludge follows in the sewage plant. This pathway includes a mixing phase, a settling phase and a decanting phase. These are important because in an activated sludge plant the operator cannot let the sludge escape the plant or he’ll lose the nitrifying bacteria because they are part of the sludge.
So, the mixture of sludge and treated water is allowed to settle in a part of the plant. Then, the clean water (on top) is decanted and allowed to leave the plant, while the sludge-containing water is recycled back to the plant to restart the process. The design and operating of the chemostat was such that a cycle from the feeding of fresh media into the unit to decanting the liquid after a settling phase was six hours.
The researchers operated the chemostat for six months and then sampled the sludge. The sample was subjected to microscopic analysis, to plating on agar (for the culturing of bacterial colonies) and nucleic acid analysis, including the polymerase chain reaction (PCR), cloning and DNA sequencing. They found a wide range of bacteria growing on the agar, but none was related to known nitrite-oxidizing bacteria.
This is not really surprising because many recent studies have shown that the best scientists can do is to culture only 1 to 3 percent (or less) of the bacteria present in a sample. Thus, for example, if one goes out to the ocean and grabs a water sample and looks at 1 milliliter of the water with a powerful, special microscope, anywhere from 1000 to 1,000,000 bacterial cells will be seen in that sample. However, if you put that same milliliter of water on agar or in media, only 10 to 3000 bacterial cells will grow. Clearly, we’re not very good at providing the requirements all the bacteria need for growth.
Even on the agar, which contained an inorganic media with nitrite, the researchers found heterotrophic bacteria, not nitrite-oxidizing bacteria. This indicates that there are some fast-growing bacteria that are not too strict in their requirements for growth.
Next, the researchers extracted DNA from the sludge and produced what is called a “clone library.” This is when you take DNA that is actually a mixture of DNA from many organisms and use a cloning process to separate the DNA of the different species. When you clone in this fashion, only the DNA from one species will be inserted into each cloning vector (this is usually a specially grown cell of the Escherichia coli bacterium). Normally, you will get hundreds of individual bacteria cells, each with one segment of foreign DNA, and you can’t tell by just looking at the cells whether the DNA segments are the same or different.
There are different ways to try and group the clones that are similar by using restriction enzymes. This is important because sequencing is expensive and you don’t want to continually sequence the same DNA segment. The details are not important here, just the fact that the researchers were able to group the clones. They examined a large number of clones that came from DNA that was extracted directly from the sludge from the sewage plant (77 clones) and of sludge that grew in the chemostat (102 clones). In the chemostat sludge sample, they found that 90 of those 102 clones were closely related to Nitrospira spp. — the rest of the clones examined were different from each other and the Nitrospira spp. The data from the sludge directly from the sewage plant showed a wider range of microbial diversity compared to the chemostat. This is expected because the chemostat was fed a solution of nitrite and other inorganic salts that would tend to select for those organisms that can grow in that environment.
In the sewage plant sludge sample they did find that there were possibly two clones that may be related to Nitrobacter spp. To be sure of the identity they need to get more sequence data. However, they did find clones that were a match to the Nitrospira group.
The significance of this research is that it provides additional evidence that Nitrospira-like bacteria are probably widespread in aquatic environments in which nitrite is available for oxidation. My work showed that Nitrospira-like bacteria are active nitrite oxidizers in aquaria, and the work noted here extends the presence of Nitrospira spp. to wastewater treatment facilities. It also shows that Nitrobacter spp. were not present in significant numbers in wastewater facilities, as is commonly assumed, just as I did not find Nitrobacter spp. in aquaria. However, these results do not mean that Nitrobacter spp. don’t exist or that Nitrobacter spp. do not oxidize nitrite. Nitrobacter is a bonafide bacterial species that does oxidize nitrite — it just does this in soils and does not seem to play an important role in aquatic environments.
It’s time for hobbyists, technical people and writers of articles in the fish hobby press to start using the correct name for the nitrite-oxidizing bacteria in aquaria — Nitrospira.Why is it important to know the bacteria? A compelling reason is that there are fundamental physiological differences between Nitrobacter and Nitrospira spp., the most important of which may be the fact that the Nitrobacter spp. is not really an obligate aerobe (it would need to be in an environment that contains oxygen) and it can be grown, albeit slowly, heterotrophically (getting the carbon it needs from organic chemicals instead of just from carbon dioxide, which is called autotrophic growth).
On the other hand, so far Nitrospira spp. can only be grown autotrophically and aerobically. This could be an important bit of information when trying to provide an optimal environment. Further, by knowing which bacteria play an important role in the cycling of chemicals, such as nitrite, researchers like me can study and (I’m hopeful) find out why sometimes things go wrong. For instance, a relatively common problem in saltwater aquaria is that it can take a long time for the nitrite to be completely oxidized to nitrate. I have had people tell me that the nitrite concentrations in their newly set-up aquarium have been between 1 and 5 milligrams per liter for 10 to 14 weeks or longer. Why? By being able to target and count the bacteria responsible for nitrite oxidation we can now see whether there are substances that inhibit their growth by actually counting the number of bacterial cells over the course of time during the establishment of nitrification.
Further, experiments can be designed and conducted to examine, among other things, how different chemicals effect the bacteria growth, how well the bacteria grow on different media and how the bacteria associate with other microorganisms in the aquarium. As has been mentioned in this column before, little is actually known about the microbiology of aquarium systems. Most of what is written is a best guess, and with the application of modern molecular methods I think you will find that much will have to be revised as study continues.
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