ISOLATION OF NITROSOMONAS IN PURE CULTURE'
R. F. LEWIS AND D. PRAMER
Department of Agricultural Microbiology, Rutgers, The State University of New Jersey, New Brunswick,
Received for publication May 30, 1958
Although it has been 68 years since Winograd-
sky (1890) accomplished the first isolation of
nitrifying bacteria, the methods employed have
not been improved significantly, and pure cultures
remain incredibly difficult to obtain. The litera-
Nitrosomonas in pure culture. Nevertheless, the
organism has seldom been isolated by dilution
techniques. In the relatively few cases where suc-
cess was claimed, the procedures were tedious
and difficult, and the results often uncertain
1929; Engle and Skallau, 1937; Bomeke, 1939).
The majority of successful isolations have resulted
from the plating of enrichment cultures. However,
the colonies formed by Nitrosomonas on washed
agar or silica gel are not more than 100 ,u in
diameter and micromanipulation is required to
pick colonies or single cells (Kingma Boltjes,
1935; Meiklejohn, 1950).
The most frequently cited explanations for the
difficulties encountered in obtaining Nitrosomo-
nas in pure culture by dilution techniques are:
(a) the autotrophic nitrifiers develop very slowly;
(b) the heterotrophic contaminants in enrichment
cultures develop at a rate equal to or greater than
that of the nitrifying bacteria; and (c) the solution
media employed by Winogradsky, and subse-
quently used by others for the cultivation of
Nitrosomonas, contain insoluble carbonate which
adsorbs the cells and prevents dispersed growth.
A recent report by Goldberg and Gainey (1955)
resolved conflicting views regarding the role of
surface phenomena in nitrification by demon-
strating that appreciable quantities of particulate
matter were not essential for rapid nitrification
in solution medium. This suggested that the
isolation of Nitrosomonas by dilution techniques
should be reconsidered using media as free from
1 Paper of the Journal Series, New Jersey Agri-
cultural Experiment Station, Rutgers, The State
University, Department of Agricultural Micro-
biology, New Brunswick, N. J.
particulate material as possible. The present
report describes such media in which the develop-
ment of Nitrosomonas in enrichment cultures
was greater than that of associated heterotrophic
bacteria. The magnitude of the ratio of the num-
ber of cells of Nitrosomonas to that of hetero-
trophic bacteria was adequate to permit isolation
of Nitrosomonas in pure culture by dilution
EXPERIMENTAL METHODS AND RESULTS
Although the medium employed by Goldberg
and Gainey (1955) was free of particulate material
and permitted rapid nitrification it contained soil
extract and was not defined chemically. There-
studies were made of the
development of a nitrifying enrichment culture
in various chemically defined solution media
formulated to be relatively free of particulate
matter. A simple salt solution of the following
Na2HP04, 3.1 g; KH2P04, 1.1 g; MgSO4 7H20,
0.1 g; (NH4)2S04, 0.5 g; FeCl3.6H02, 14.4 mg;
CaCl2 -2H20, 18.4 mg; distilled water, 1000 ml.
The (NH4)2S04 was sterilized separately and
added aseptically. The reaction of the medium
was pH 7.2. Attempts to eliminate the trace of
insoluble precipitate in this medium by use of
N-hvdroxyethylethylenediaminetriacetic acid) or
methane buffer for the phosphate buffer were not
nitrification was reduced.
The influence of concentration of (NH4)2S04 and
pH on the numbers of Nitrosomonas and hetero-
trophic bacteria that developed in the medium
Studies were made using 100 ml quantities
of medium in 250 ml Erlenmeyer flasks. All
flasks were incubated at 28 C on a rotary shaker.
The inoculum was a nitrifying enrichment cul-
ture obtained from soil and transferred serially in
a conventional CaCO3-containing solution me-
ISOLATION OF NITROSOMONAS IN PURE CULTURE
dium2 at intervals of 1 month for a period of 2
years. Three treatments were employed. In the
first, flasks received only an initial increment of
(NH4)2SO4 (10.6 mg nitrogen); in the second
treatment, flasks received a second increment of
(NH4)2SO4 after 7 days; and in the third treat-
ment, the flasks received 3 increments of (NH4)2-
SO4, the initial amount and additional quantities
after 7 and 14 days of incubation. The reaction
of the solutions of this last treatment was re-
adjusted to the original level (pH 7.2) with
sterile NaOH at weekly intervals. At the time
of inoculation and at 7 day intervals for 3 weeks
thereafter each flask was tested for numbers of
heterotrophic bacteria, numbers of Nitrosomonas,
concentration of nitrite, and pH. The numbers of
heterotrophic bacteria were determined by plate
counts using nutrient agar. The numbers of
Nitrosomonas were determined by dilution in
solution medium. Five tubes were used at each
dilution and the most probable numbers of
Nitrosomonas were obtained by reference to the
tables of Halvorson and Ziegler (1933). The
tubes were incubated for 30 days and then tested
for the presence of nitrite. Nitrite was determined
using the Griess-Ilsovay reagent and a Klett-
electrometrically. All treatments were tested in
triplicate. Representative results are presented
in table 1.
In treatment 1 the concentration of (NH4)2SO4
limited the development of Nitrosomonas. In
treatment 2, where a second addition of (NH4)2-
SO4 was made after incubation for 7 days, pH
Nitrosomonas. The greatest numbers of Nitro-
somonas were obtained in those flasks that re-
ceived 3 increments of (NH4)2SO4 and had the
reaction of the medium adjusted to pH 7.2
at weekly intervals (treatment 3). In each case
the numbers of heterotrophic bacteria increased
greatly during the
decreased the second week, and remained rela-
tively constant thereafter. In treatments 2 and 3
the ratio of the number of cells of Nitrosomonas
to those of heterotrophic bacteria exceeded 100,
indicating that the isolation of Nitrosomonas in
pure culture by dilution teehniques was possible.
0.1 N HCI, 15 ml; distilled water, 1000ml; pH7.2.
first week of incubation,
3.0 g; K2HPO4,
10 mg; Fe2(S04)3-5H5O,
Influence of concentration ofenergysou-ce and of
pH onthe development of Nitrosomonas and
No. of Het-
*For 1, 10.6 mg NH4+-N was added initially;
for 2, 10.6 mg NH4+-N was added both initiallv
and after 1 week incubation; for 3, flasks received
additions of 10.6 mg NH4+-N initially and after
incubation for 1 and 2 weeks, and at the latter 2
periods the reaction was readjusted to pH 7.2
with sterile NaOH.
To test this possibility, a nitrifying enrichment
culture was transferred to the phosphate buffered
medium, additional increments of (NH4)2SO4
were provided to each flask, and the pH of the
solutions was readjusted at 7 day intervals as in
treatment 3 of the previous experiment. After
flask and diluted
medium in test tubes. The tubes were incubated
for 30 days and then tested for nitrite and hetero-
trophic bacteria. The following 4 procedures were
used to detect heterotrophic bacteria: (a)
aliquots were incorporated into pour plates using
nutrient agar; (b) 0.1 ml aliquots were used to
inoculate the surface of nutrient agar plates; (c)
1 ml and 10 ml aliquots were added to flasks of
nutrient broth; and, (d) double strength nutrient
broth was added directly to the tubes. Of 69
tubes inoculated with 10-8 to 10-10 dilutions of the
nitrifying culture, 24 (35 per cent) were found to
be free of heterotrophic contaminants and to
contain Nitrosomonas in pure culture.
yielded pure cultures of Nitrosomonas without
1 ml aliquots were taken from each
LEWIS AND PRAMER
great difficulty, the necessity of adding (NH4)2-
S04 in increments and of adjusting the pH at
weekly intervals was laborious and greatly in-
creased the chances of contamination. A study of
the influence of medium constituents including
buffer concentration and pH on thedevelopment
of Nitrosomonas resulted in the preparation of a
more satisfactory medium. The composition of
this medium was Na2HP04, 13.5 g; KH2P04, 0.7
g; MgSO4.7H20, 0.1 g; NaHC03, 0.5 g; (NH4)2-
804, 2.5 g; FeCl3 6H20, 14.4 mg;CaCl2*2H20,
18.4 mg; distilled water, 1000 ml; final pH, 8.0.
This differed from the medium used in previous
experiments in that the buffer concentration was
increased from 0.03 to 0.1 M and sufficient(NH4)2-
heterotrophic bacteria in two media
pH No.of Ni
S04 (53 mg nitrogen) wassupplied initially to
permit relatively profuse development of Nitro-
somonas. Inaddition,the reaction of the solution
was increased frompH 7.2 to 8.0. This medium
nitrifying enrichment cultures as well as for the
isolation and maintenance ofpure cultures of
Thesuitabilityof the medium for the isolation
of Nitrosomonas inpure culture was determined
by inoculation with
culture that haddeveloped in the conventional
incubation the culture was examined by the
procedures employedinprevious experiments. A
culture that had been maintained in the conven-
Table 2 shows that thedevelopmentof Nitro-
somonas in the mediumcontainingan insoluble
carbonate buffer was limitedby pHand that the
ratio of the numbers of Nitrosomonas to hetero-
trophic bacteria was 2. There was more nitrifica-
tion in thephosphatebuffered medium.Although
theexperiment was terminated before eitherpH
or the concentration of(NH4)2S04 hadbecome
limiting, the ratio of the number of cells of
Nitrosomonas toheterotrophic bacteria exceeded
250.Aliquotstaken from thephosphate buffered
medium and diluted serially in test tubes of
medium were incubated for 30days and tested
a nitrifying enrichment
After 8 days
Figure 1. Electron micrographs of Nitrosomonas europaea. The specimen was air dried and shadowed
with germanium. A, X16,000; B, X20,000.
ISOLATION OF NITROSOMONAS IN PURE CULTURE
for nitrite and heterotrophic bacteria. Of 25 tubes
inoculated with 10-7 to 109 dilutions of the
nitrifying enrichment culture, 22 (88 per cent)
were free of heterotrophic contaminants and were
presumably pure cultures of Nitrosomonas.
The isolates obtained by the foregoing pro-
cedures appear to be Nitrosomonas europaea.
The cells are oval, measuring approximately 1.5
,u in length and 1 ,u in width (figure 1). They are
nonmotile and gram-negative. Pure cultures of
Nitrosomonas have been maintained
phosphate buffered solution medium for more
than 12 months without loss of vitality and
without recontamination. The culture was de-
posited with the American Type Culture Collec-
The recent statement of Meiklejohn (1950)
that "it is impossible to obtain pure cultures
[of Nitrosomonas] by simple serial transfers in
liquid medium" was based on reports such as
Gibbs (1919), and Kingma Boltjes (1935) who
contaminants remained in nitrifying enrichment
cultures. Gibbs (1919) found that after 50 trans-
fers heterotrophic bacteria persisted in equal or
greater numbers than the autotrophic nitrifying
bacteria. The cultures of Nelson (1931) con-
tained 1000 heterotrophic bacteria for each cell of
Nitrosomonas, and Hanks and Weintraub (1936)
similarly observed greater numbers of heterotro-
phic contaminants than
investigations support these early studies, in that
the ratio of the number of cells of Nitrosomonas
to the number of heterotrophic bacteria obtained
when nitrifying enrichment cultures developed in
a solution medium containing an insoluble al-
kaline carbonate as buffer, was approximately 2
and of insufficient magnitude for the isolation of
Nitrosomonas by dilution techniques.
It is apparent that the inability of previous
investigators to isolate Nitrosomonas in pure
culture by dilution techniques resulted from the
presence of an insoluble carbonate in the media
employed. By use of media formulated to be
relatively free of particulate matter it has been
possible to obtain dispersed growth of nitrifying
enrichment cultures and a ratio of
Nitrosomonas to those of heterotrophic bacteria
that exceeded 250, making isolation of Nitro-
somonas in pure culture by dilution techniques a
relatively simple matter.
The report of Goldberg and Gainey (1955),
which demonstrated that particulate matter was
not essential for rapid development of nitrifying
bacteria in solution media, and served as the
basis for the present investigations, was con-
firmed in a recent note by Engel and Alexander
(1958). The medium employed by these investi-
gators was rendered free of insoluble consti-
tuents by the use of a chelate (ethylenediamine
undoubtedly suffice for the isolation of Nitro-
somonas in pure culture by the foregoing dilution
The authors wish to express their gratitude to
Miss Pauline E. Holbert for the electron micro-
graphs, and to Mrs. M. Madama for technical
The development of a nitrifying enrichment
in solution media formulated
relatively free of particulate matter was investi-
gated. Rapid and dispersed growth of Nitro-
somonas was obtained in a medium buffered by
phosphate and containing (NH4)2SO4 as a source
of energy. Under suitable conditions the ratio
of the number of cells of Nitrosomonas to that of
heterotrophic bacteria present in the enrichment
culture exceeded 250, and isolation of Nitroso-
monas in pure culture by dilution techniques
was accomplished with comparative ease.
ENGEL, H. AND SKALLAU, W.
Bakteriol. Parasitenk. Abt. II, 97, 305-311.
ENGEL, M. S. AND ALEXANDER, M.
of Nitrosomonas europaea in media free of
FRANKLAND, G. C. AND FRANKLAND, P. F.
The nitrifying process and its specific ferment.
Trans. Roy. Soc. London, Ser. B, 181, 107-128.
GIBBS, W. M.
The isolation and study of
Nature, 181, 136.
LEWIS AND PRAMER
GOLDBERG, S. S. AND GAINEY, P. L.
of surface phenomena in nitrification.
Sci. 80, 43-53.
HANKS, J. H. AND WEINTRAUB, R. L.
pure culture isolation of ammonia-oxidizing
J. Bacteriol., 32, 653-670.
Untersuchungen uber Nitrit-
Planta, 8, 398-422.
.KINGMA BOLTJES, T. Y.
0. AND ZIEGLER, N. R.
uber die nitrifizierenden Bakterien.
Mikrobiol., 6, 79-138.
somonas europaea in pure culture.
Microbiol., 4, 185-191.
NELSON, D. H.
Zentr. Bakteriol. Parasitenk. Abt.
Pasteur, 4, 213-231, 257-275, 760-771.
The isolation of Nitro-
Isolation and characteri-