Who's the Ammonia-Oxidizing Bacteria?
by Timothy A. Hovanec,
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 laymans version
for fish hobbyists which was published as a two-part
article in the Dec 1996 and Jan 1997 issues of Aquarium
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 dont seem to be present
in freshwater aquaria. Further, some people think that
I was just plain wrong but thats another story.
So why is it assumed that the ammonia-oxidizer is Nitrosomonas
europaea? Because, among other things, thats 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
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.
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 youre using for
the nitrite-oxidizing bacteria?
©1998, Timothy A. Hovanec
Originally published in Aquarium
Frontiers, Aug. 1998