Ammonia Excretion by Azotobacter chroococcum

The increasing cost of petroleum products required in nitrogen fertilizer production has
focused attention on the development of biological systems for nitrogen fixation. One approach used is the enhancement of N2 fixation in free living microorganisms. The isolation
of nifderepressed auxotrophic mutants of Klebsiella pneumoniae' and the derepressed control mutants of Azotobacter vinelandii* suggested the possibility of using such mutants for
the fermentative production of ammonia. The recent report of Wallace and Stokes3 on these
mutants, however, suggests that a process for ammonia production using these strains
may not be economically viable. This is because of the dependence of the K . pneumoniae
mutants on glutamine or glutamate for growth and ammonia excretion and the low quantities
of ammonia excreted by the mutants of A . vinelandii.
There are a few reports available in literature on the usefulness of azotobacters in crop
improvement under tropical and subtropical conditions and there is indirect evidence to
suggest that these bacteria may be excreting free ammonia in addition to a variety of growthpromoting factors in the presence of carbon-rich root
As a part of our investigations on ammonia-excreting azotobacters and the development of an economical fermentation process for ammonia production, we have examined a number of strains of Azotobacter for their ability to excrete ammonia while growing in a synthetic medium. We report
in this communication the identification of two strains of Azotobacter chroococcum which
can excrete as much as 45 pg ammonidml of the culture broth in a sucrose supplemented
synthetic medium.
Twenty three Azotobacrer strains which included six from different laboratories in India
and 17 isolated from local soils by standard methods6 were used in this study. Isolations
from the soil were made using modified Jensen agar medium' and the strains were identified
as detailed in Bergey's Manual ofDeterminarive Bacteriology (8th ed. 1974). Of the 17
cultures, 15 were A.chroococcum, one was A.vinelandii, and one was A . paspalum.
Ammonia excretion was determined by incubating 30 ml of Jensen medium containing
2.5% sucrose in 125-ml conical flasks inoculated with Azotobacter cells from 48-hr-old slant
cultures, in an incubator at 27 ? 2C under stationary conditions. Ammonia (NH4+) content
in culture broths or supernatants obtained after centrifuging the broths at 1.2 x lo4 g for
10 min was determined colorimetrically using Chaykin's reagent* after microdiffusion as
described by Burris.' Growth was determined by centrifuging 5 ml of the culture broth at
5000 rpm for 10 min and drying the pellet at 80C. To test whether NH4+ excretion was due
to cell lysis, flasks containing 3 pCi of I4C lysine and 300 pg of cold lysine were inoculated
with Azotobacter cells, 1.5 ml of culture broths withdrawn on 4,8, 12, 16, and 20 days, and
the radioactivity of the cell-free supernatants was determined using a Beckman LS 100
scintillation counter." The protein content of supernatants was determined according to
Lowry et al." and for free nucleic acid the OD 260 of the cell-free supernatants was determined.
In a preliminary study, cultures were incubated in multiples for a total period of 18 days
during which flasks were withdrawn at intervals of 3-4 days and the ammonia level of culture
broths estimated. Table I shows the level of ammonia at the end of 18 days of incubation.
It was found that most strains excrete ammonia but the amount varied from 0.2 to 46 pg!
ml(O.01 to 2.61mM). Among these cultures two (Nit and IA) excreted the highest amounts.
Strain SA2, excreted 41 pg of ammonia. All these were A.chroococcum and, interestingly,
two cultures of A.vinelandii tested did not excrete any ammonia.
To determine the time course of ammonia excretion by these two cultures, these were
grown as described above and flasks were withdrawn at various intervals and analyzed for
Biotechnology and Bioengineering, Vol. XXIII, Pp. 467-470 (1981)
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BIOTECHNOLOGY AND BIOENGINEERING VOL. XXIII (1981)

TABLE I
Ammonia Excretion by Various Azotobacter
Strains"
Strains
1.
2.
3.
4.
5.
6.
1.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.

A . chroococcum
A . chroococcum
A . chroococcum
A . chroococcum
A . chroococcum
A . chroococcum
A . chroococcum
A . chroococcum
A . chroococcum
A . chroococcum
A . chroococcum
A . chroococcum
A . chroococcum
A . chroococcum
A . chroococcum
A . beijerinckii
A . vinelandii
A . chroococcum
A . chroococcum
A . chroococcum
A . chroococcum
A . chroococcum
A . vinelandii

B 5-1
B 5-2
H 29
H 15-2
H 116
B 10-1
B 10-2
B 10-3
B 10-5
B 53
H 73-1
H 56
H 56-1
SA 2
M2
1 B 53
B l
AC L
AC 6
AC 16
Nit
1A

11.0
Traces
0.5
0.4
I .3
1.8
0.6
1.3
23.0
5.9
16.0
I .30
14.0
41 .O
21 .o
5.9
1.5
1.5
1.4
18.0
46.0
46.0
Traces

a Strains 1-17 were local soil isolates; 18-20

were from Punjab Agricultural University, Ludhiana; 21 was from Nitragen Co., Lucknow; 22
was from I.A.R.I., New Delhi; 23 was from University of Agricultural Sciences Bangalore.

cell mass and ammonia level (Fig. 1). Growth, as determined by dry mass increase, increased
steadily and reached a stationary phase by about 12 days while ammonia excretion commenced at this stage and reached a maximum around the 17th day, after which it remained
constant. The results show that ammonia release occurs after the cells have reached the
stationary phase which is similar to the results of Shanmugam et a].'* who found that in
Klebsiella pneumoniae ammonia excretion occurs only under nongrowing conditions. Experiments using I4C lysine have shown that nearly 99% of the radioactivity remains in the
cells when ammonia release occurs. Also, neither protein content nor the OD 260 of the
supernatant showed any significant increase at the time of ammonia release. The increase
in the free ammonia content of the broth could be due to release offree ammonia accumulated
inside the cells. The quantities found in the culture broths are at least 1.5 times more than
reported by Wallace and Stokes3 with K . pneumoniae.
Azotobacters are aerobic organisms and need oxygen for growth and nitrogm fixation.
To test whether ammonia excretion can be enhanced by aeration, flasks were incubated on
a rotary shaker (230 rpm) at 30"C, for a period of 20 days. Flasks were withdrawn at intervals

COMMUNICATIONS T O T H E EDITOR

469

"1

il0

INCUBATON TIME(doys)

Fig. 1. Growth and ammonia excretion in A.chroococcum. Growth: ( x - x )

SA 2;
(wg of NH4'/ml) excreted: (o--o)
SA 2; (-)
1A. Each point
represents an average of three readings.
(H)
IA. Ammonia

and the culture broth was assayed for ammonia. Contrary to expectations, no ammonia
excretion occurred during this period although the cultures grew normally, while under
stationary conditions ammonia excretion began around the 12th day. Therefore, it appears
that for ammonia excretion by these strains, vigorous aeration is not essential, a feature that
has much importance in fermentative production of ammonia.
The results reported herein suggest that there are wild-type strains of A.chroococcum in
nature which have the ability to excrete ammonia. However, all the strains do not release
ammonia. The detection of cultures such as those reported herein which have the ability
to excrete as much as 2.6mM of ammonia is promising for further exploration and development of processes for the fermentative production of ammonia. Since these cultures excrete ammonia in synthetic media and under low aerated conditions, fermentative processes
based on these cultures may be economically viable. The mechanism by which these cultures
excrete ammonia is being investigated with a view to improve on the production levels.

References
I . K. T. Shanmugam and R. C. Valentine, Proc. Natl. Acad. Sci. USA, 72, 136 (1975).
2. J. K. Gordon and W. J. Brill, Proc. Natl. Acad. Sci. USA, 69, 3501 (1972).
3 . C. J. Wallace and B. 0. Stokes, Biotechnol. Bioeng. Symp., 8, 153 (1978).
4. J. Dobereiner, in The Biology of Nitrogen Fixation, A. Quispel, Ed. (North-Holland,
Amsterdam, 1974), p. 86.
5 . R. Knowles, in A Treatise On Dinitrogen Fixation, R. W . F. Hardy, and A. H. Gibson
Eds. (Wiley, New York, 1977), Sec. IV, p. 33.
6. M. E. Brown, S. K. Burlingham, R. M. Jackson, Plant and Soil, 17, 309 (1962).
7. H . L. Jensen, Proc. SOC. Appl. Bacteriol., 14, 89 (1951).
8. S. Chaykin, Anal. Biochem., 31, 375 (1969).
9. R. H. Burris, in Methods in Enzymology, S . P. Colowick and N. 0. Kaplan, Eds.
(Academic, New York, 1972) Vol. 24, p. 415.
10. G. A. Bray, Anal. Biochem., 1279, (1960).
1 1 . 0. H. Lowry, N. J. Rosebrough, A. L. Farr, and R. J. Randall, J . Biol. Chem., 193 265
(1951).
12. K. T. Shanmugam, F. O'gara, K. Anderson, C. Morandi, and R. C. Valentine, in Ni-

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BIOTECHNOLOGY AND BIOENGINEERING VOL. XXIII (1981)

trogen in the Environment Vo/. 2 , D. R. Nielsen and J. G. Mac Donald, Eds. (Academic,
New York, 1978), p. 393.

N. NARULA
K. LAKSHMINARAYANA
P. TAURO
Department of Microbiology,
Haryana Agricultural University,
Hissar, India, 125004
Accepted for Publication August 20, 1980.