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Plant and Soil 90, 193-202 (1986).
9 1986 Martinus Ni/hoffPublishers, Dordrecht. Printed in the Netherlands.
Aspects o f n i t r o g e n f i x a t i o n and d e n i t r i f i c a t i o n b y
Ms. NF 16
Azospirillum
G. DANNEBERG, A. KRONENBERG, G. NEUER and H. BOTHE
Botanisches Institut, Universiti2t Koln, Gyrhofstrasse 15, D-5000 Koln 41, FR G
Key words Azospirillum
Nitrogen fixation
Denitrifieation Nitrate respiration Nitrite reductase
Summary Model experiments were performed to investigate the nitrogen fixation (C2H 2
reduction) and denitrification (N~O formation) capabilities of Azospirillum spp. in association
with wheat. Plants and bacteria were grown together for a week and then assayed for activities.
This association performed C2H2 reduction or N20 formation, depending on the concentrations
of nitrate and oxygen in the vessels. Both activities depended on the Azospirillum strains used.
The newly isolated Azospirillum amazonense strains Y1 and Y6 showed significant C2H2
reduction and low N20 formation in association with wheat under the conditions employed
and axe possibly useful in practice. A cell-free preparation from Azospirillum brasilense Sp 7
possessed a cytochrome cd type dissimilatory nitrite reductase.
Introduction
Since its discovery some ten years ago 7, the association between
cereals and bacteria of the genus Azospirillum has attracted special
attention. It is the general hope that nitrogen-fixing Azospirillum spp.
living on the surface of roots or inside them provide fixed nitrogen
to crop plants. This could reduce the demand for nitrogen fertilizer,
or could increase crop productivity, or both. Studies under diverse
environmental and soil conditions in general gave positive and encouraging increases (5-30%) over controls in yields of grasses and
cereals6"13. However, recently published drawbacks 16 have also to be
rflentioned in this context.
Although Azospirillum has been reported to perform N 2 fixation
in association with cereals even under field conditions, the mode of
interaction between plants and bacteria is not understood currently. It
is not clear whether the bacteria provide an excess of fixed nitrogen
which is supplied to the plants. Azospirillum may also be of shorter life
than the grasses and may be decomposed, thereby helping plant growth.
Azospirillum also produces plant hormones (auxin, gibberellin and/or
cytokinin) which may stimulate growth of the roots and possibly
enhance the uptake of minerals by them (see6'13). The interrelationship between bacterial phytohormone production and plant growth
in the association has not yet, however, been investigated.
Azospirillum is also distinguished by its versatility with respect to
nitrogen metabolism 3 . The bacterium can perform all the reactions
193
194
DANNEBERG, KRONENBERG, NEUER and BOTHE
o f the nitrogen cycle except nitrification. The ability to perform
denitrification is of particular interest both in pure and applied research. Under anaerobic conditions, Azospirillum utilizes nitrate as
a respiratory electron acceptor and reduces it to molecular nitrogen
via nitrite and nitrous oxide. The biochemistry of denitrification
has not been studied in AzospMllum. No information is available
on whether denitrification by Azospirillum affects the growth of the
plants in the association.
In an attempt to gain information on this latter aspect, we have
recently studied nitrogen fixation (C2 H2 reduction) and denitrification
( N 2 0 formation) b y a wheat-Azospirillum association 4 . For this,
germinated wheat seedlings and Azospirillum were grown in semisolid agar medium for a week in air and then assayed under microaerobic conditions These conditions were chosen in order to simplify
the experiments and to obtain reproducible results. The association
performed either N 2 0 formation or C2H2 reduction, depending on the
amount of nitrate and oxygen in the medium. This investigation has
now been extended to experiments with different Azospirillum strains
and the results are presented here. In addition, experiments to characterize dissimilatory nitrite reductase o f Azospirillum brasilense Sp 7
biochemically are described.
Materials and methods
Experiments with the wheat-Azospidllum association
The experiments were performed in exactly the same way as described previously 4. The
bacteria were grown in malate medium 4 for 24 h at 30~ Before germination, wheat seeds were
surface sterilized by treatment with 70% ehtanol for 10 min, followed by treatment with 0.1%
HgC12 dissolved in 0.05 N HC1 for 10 min and by washing 5 times with sterile distilled H20. The
seeds were placed on sterile wet filter papers in Petri dishes and germinated for 2 - 3 days in a
growth chamber with light/dark cycles (12.5 h in the light at 33~ 11.5 h in the dark at 23~
Wheat and bacteria were then incubated together in I1 flasks each containing 100ml of the
sterilized medium described previously and 0.8% agar (Merck). Twenty five germinated seeds
and 1 ml of the different bacterial suspensions each containing ca t.3 • 109 cells were placed
aseptically on the surface of the soft agar. The flasks were covered with sterile cotton wool and
incubated for a week in a growth chamber (12.5 h in the light at 33~ and 11.5 h in the dark at
23~
The cotton wool was then replaced by sterilized rubber stoppers and the flasks were
repeatedly evacuated and filled with argon. The O 7 content in the gas phase was ca 0.2% at the
start of the experiment. Five ml of C2H 2 was injected into the flasks which were then incubated
for 2 4 h in the growth chamber under the conditions just described. C2H 2 reduction and
N20 formation were determined by gas chromatography as described previously4. The Azo.
spirillum brasilense strains Sp 7, Cd 442 and 122, and A. amazonense Y1 and Y6 were kindly
supplied by Dr. J. Dobereiner, Serop6dica, Brazil, the mutant FP 10 of A. brasilense Sp 7 by
Dr. F. Pedrosa, Brighton, UK and the A. lipoferum strain by Dr. G. Jagnow, Braunschweig,
FRG.
Preparation o f dissimilatory nitrite reductase and assay conditions for the enzyme
Azospirillum brasilense Sp 7 was grown in continuous culture with nit~ite as the respiratory
N 2-FIXATION AND DENITRIFICATION BY AZOSPIRILLUM
195
electron acceptor (see2~ D,L-malate instead of L-malate served as the carbon source for
growth in these experiments. The cells were harvested by centrifugation (10min, 16,000g)
and stored at -- 18~ When used, they were resuspended in 10 mmol/1 K-phosphate buffer, pH
7.5, washed twice in the same buffer and broken twice in a French Press at 20,000 psi (= 140,000
hPa). The extract was centrifuged (30min, 1 2 , 0 0 0 g ) a n d the supernatant was assayed for
nitrite reductase activity (formation of N~O from NO~). The experiments were performed in
7 ml Fernbach flasks each containing in a final volume of 3 ml: enzyme extract, 0.84 mg protein, and the following in ~tmol: K-phosphate buffer, pH 7.5, 500; NaNO2, 20, phenazinemethosulphate, 0.8 Na-ascorbate, 50. The assays were performed at 30~ for 45 min under
argon. N20 was determined by gas chromatography and nitrite colorimetrically by the naphthylamine/sulphanilic acid reagent 4. For recording the absorption spectra, the extract was
further centrifuged (2 h, 100,000g). Approximately 30% of the N20-formation activity was
present in the pellet and 70% in the supernatant. The supernatant was subjected to (NH4)2SO 4precipitation. The brown-greenish fraction precipitating between 55-90% (NH4)~SO 4 was
centrifuged, suspended in 10 mmol/1 K-phosphate buffer, dialyzed overnight and taken for recording the spectra on a Perkin-Elmer 555 spectrophotometer. This preparation had a N 2 O formation activity of 53 mmol/min X mg protein. For the reduction of the enzyme, the gas phase in the
cuvettes was replaced by argon. Anaerobic solutions of phenazinemethosulphate (final concentration in the cuvette 0.025 mmol/l) and NADH (0.5 mmol/1) were added by injection. An
anaerobic solution of NaNO 2 (final concentration 12.5 mmol/1) was added for the reoxidation
of nitrite reductase.
Results and discussion
Experiments with the wheat-Azospirillum association
The data obtained previously from the experiments with the wheat-
Azospirillum brasilense Sp 7 association 4 can be summarized as follows:
(1) Both C2H2 reduction and N 2 0 formation were strictly dependent
on the presence o f plants and Azospirillum.
(2) Both activities were detectable after 3 - 5 h incubation under
microaerobic conditions and proceeded linearly for at least 24 h.
(3) The addition o f carbohydrates did not enhance either activity.
The bacteria multiplied at least tenfold during the week and must have
utilized the carbon c o m p o u n d s supplied by the plants.
(4) Nitrate in the medium suppressed CzH 2 reduction and was
required for N 2 0 formation.
(5) N 2 0 formation was observed only in the presence o f C2H2.
Without C2H2 in the vessels, Azospirillum reduced nitrate to Nz. CzH2
is also k n o w n to block specifically nitrous oxide reductase also in other
organisms 1, 19
(6) NzO formation required the exclusion o f 02 from the vessels.
It is k n o w n that 02 is the preferred respiratory electron acceptor
in denitrifying bacteria s . Thus, Azospirillum is no exception to this
general rule.
(7) C2H2 reduction was optimal at 0 . 5 - 2 % 02 in the gas phase.
This is not surprising, because Azospirillum can perform N2 fixation
only under reduced oxygen tensions also in liquid cultures 6 .
196
DANNEBERG, KRONENBERG, NEUER AND BOTHE
(8) Both C2H2 reduction and N 2 0 formation were high when the
association was incubated at higher temperatures (12.5 h at 33 ~ in the
light and 11.5 at 23 ~ in the dark ('tropical' conditions), and marginal
at conditions o f the 'temperate' zone (12.5 h at 23 ~ in the light and
11.5 h at 16 ~ in the dark). This observation may explain the failure
of some investigators to detect N 2 fixation activities by the association
both under laboratory conditions or in field studies. Azospirillum 2
strains occur in soils of the temperate zone but are particularly abundant
in the tropics where they may have impact on the nitrogen supply to
crops either by nitrogen fixation or denitrification.
These experiments have now been extended to different Azospirillum strains (Table 1). The data indicate that the actiyity o f the wheatAzospirillum association is dependent on the Azospirillum strain
used. Some strains did not show any significant C2H2 reduction or
N 2 0 formation in the wheat-Azospirillum association.
In particular, the data in Table 1 show that C2H2 reduction and N 2 0
formation by the association were comparable when either the type
strain, A. brasilense Sp 7 or the red pigmented strain Cd was used. The
latter strain is often claimed to be particularly suitable for practical
application because it possesses carotenoids which were suggested to
function in the protection o f nitrogenase from damage by oxygen 12.
However, we could not find any difference in the sensitivity o f N2
fixation to 02 in recent experiments 2 with liquid suspensions o f both
strains. We have also expressed difficulties in imagining a mechanism by
which carotenoids could protect against damage b y 0 2 . The two wheat
strains A. brasilense 122 and 422 were not particularly active in the
association (Table 1). This was also true for an Azospirillum strain
which was isolated near Braunschweig, F R G 8. This strain showed some
activity when the association was grown for 12.5 h at 23~ in the light
and 11.5 h at 16~ in the dark (data in Table 1). The association had
virtually no activity when grown for 12.5 h at 33~ in the light and
11.5 h at 23~ in the dark (data not presented) which indicates its
true nature as a strain of Central Europe. In contrast, strain FP 10,
a nitrogenase negative mutant of Azospirillum brasilense Sp 714 ,
showed little C2H4 formation, but strong denitrification activity in
the association. It is n o t e w o r t h y that the newly isolated A. amazonense
strains Y 1 and Y6 showed significant C 2 H 2 reduction and low N 2 0 formation activities under the conditions employed. They may be particularly suitable for use in Azospirillum/plant associations, especially
in acid tropical soils.
F o r the experiments with the wheat-Azospirillum association, the
wheat seeds were surface sterilized (see Materials and Methods). C2H2
N~-FIXATION AND DENITRIFtCATION BY AZOSPIRILL UM
197
Table 1, Nitrogen fixation (C2H 2 reduction) and denitrification (N20 f o r m a t i o n ) b y wheatAzospirillum associations
Azospirillum
strain
C2H2 reduction
0 mmol/l KNO 3
3.5 mmol/1 KNO~
N20 formation
3.5 mmol/1 KNO3
A. brasilense Sp 7
A. brasilense Sp 7
mutant FP 10
A. brasilense Cd
A. brasilense 442
A. brasilense 122
A. lipoferum 'Jagnow' x)
A. amazonense Y1
A. amazonense Y6
14.8 • 3.2 (13)
0.5 • 0.2 (12)
10.4 • 3.0 (16)
1.9
16.9
8.1
6,7
6.9
4.6
4.4
• 0.7
• 3.5
• 1.8
• 1.8
-+ 1.3
• 0.8
• 0.9
(4)
(7)
(7)
(6)
(4)
(8)
(6)
0.13
1.3
0.5
0.5
1.1
0.2
0.2
•
•
•
•
•
•
•
0.07 (5)
0.4 (8)
0.3 (7)
0.2 (5)
0.2 (3)
0.1 (6)
0.1 (5)
36,8
19.4
8.4
5.3
0.0
4.1
0.0
• 1.6
• 2.8
• 1.3
_+2.8
• 0.0
• 0.8
• 0.0
(4)
(12)
(7)
(7)
(4)
(6)
(6)
Rates are given in t~mol C2H , or N20 formed/day X flask. Numbers in brackets indicate the
numbers of repetitions performed.
x) Association grown 12.5 h in the light at 23~ and 11.5 h in the dark in the case of this strain.
Table 2, Dissimiiatory nitrite-reduction by crude extracts from A. brasilense Sp 7
Assay condition~
nmol N20 formed/min X mg protein
Complete
Complete + C2H 2 (2%)
NaNO 2
phenazinemethosulf~ite
Na-ascorbate
extract
Extract boiled for 20 rain
Complete, but assay performed in air
43.1
41.3
0.0
~ 0.5
~< 0.5
0,0
<~ 0.5
2.9
-
-
-
-
For experimental conditions see Materials and methods.
reduction and N 2 0 formation activities were not due to bacteria
living inside the seeds which had n o t been affected b y the sterilisation.
This critical statement can be made on the basis of three lines o f
evidence listed below.
(1) Controls with the Azospirillurn medium but w i t h o u t Azospirillum did n o t show significant activities.
(2) When surface-sterilized wheat seeds were incubated in the medium supplemented with Difco nutrient broth for 2 - 5 days, bacteria
often developed. When these suspensions (taking the same cell numbers
as with the Azospirillum suspensions) were used as the inoculum,
denitrification and nitrogen fixation activities were marginal (maximally 10% o f those with Azospirillum).
(3) The data of Table 1 indicate that the activity of the wheatAzospirillum association is a matter o f the Azospirillum strain used.
198
DANNEBERG, KRONENBERG, NEUER AND BOTHE
r./
2
c~30_
E
9
9
9 PMS
./
......................
/
O
Na - a s c o r b a t e
o . o ~176176
i..d"
x
q ,i 9
.g
-o 20-
/f
_*_
,
* NAN02
-J(
_~
tO
0
0
10
6
b
o12
1~0
20
30
,
2'0
,
06
3~0
" NaNO2
. PMS
" Na - a s c o r b a t e
lmM]
Fig. 1. N20 formation by crude extracts from Azospirillum brasilense Sp 7. 9 ..... e, dependence
of N20 formation on the a m o u n t of PMS in the vessel; o . . . . o, dependence of N20 formation
on Na ascorbate; *
9 dependence of N20 formation on NaNO:. For the experimental conditions see Materials and Methods.
As noted above, there were strains which did not show significant
C2H2 reduction or N 2 0 formation in the wheat-Azospirillum association, and the data were statistically sound.
Properties of the dissimilatory nitrite reductase from
Azospirillum brasilense Sp 7
Two different laboratories have recently performed experiments on
the physiology of denitrification by Azospirillum 3,a~176
Azospirillum brasilense can grow in continuous culture with nitrate 3,~~ ,
nitrite 2~ and in batch culture with nitrous oxide ls,ls as respiratory
electron acceptor. The biochemical properties of the enzymes of
dissimilatory nitrate reduction of Azospirillum have not yet been
elucidated. We have now prepared a cell-free extract from A. brasilense Sp 7 to investigate some o f the basic properties o f dissimilatory
nitrite reductase. The crude extract containing soluble and membrane
proteins catalyzed a formation of N 2 0 which was strictly dependent
on nitrite, phenazinemethosulphate (PMS) and Na-ascorbate (Table
2, Fig. 1). The activity was not enhanced by the addition o f C2H2,
indicating that N 2 0 was not further reduced to N2 under the conditions employed. Nitrous oxide formation was suppressed when the
assays were performed in air. Oxygen is apparently the preferred
N2-FIXATION AND D E N I T R I F I C A T I O N BY AZOSPIRILLUM
199
~15
1.0-
-0.3
It
~io!!
,
~ao
i
lil
iii
g
o.5-
li
',
..... reduced
\\
\t
........ r e d u c e d
. NoNO2
\i'
-0.2
ii
,
;,
iX
u
c
0
Jo
L_
ii
ii
519
i\
ii
/,"~ !/'i
',Y i\(l
\'/
~.
-0.1
i
~6o
soo
,.
",,
.-
66o
650
740
i,
.l [ n m ]
Fig. 2. Absorption spectra o f t h e dissimilatory nitrite reductase preparation. The preparation
used was obtained after centrifugation at high speed and after (NH4)2SO4-precipitation. - - ,
si~ectrum of t h e oxidized e n z y m e (as isolated).. - ..... , spectrum after reduction by NADH and
PMS; . . . . . , spectrum after reoxidation o f t h e reduced e n z y m e by NaNO 2 .
respiratory electron acceptor also in the experiments with crude extracts.
Nitrous oxide formatio under anaerobic conditions proceeded linearly
up to 45 min and 1 mg protein/vessel. The reaction showed a broad
o p t i m u m around pH 7.3. The apparent Michaelis constants in these
experiments with crude extracts were 0.9 mmol/1 for NaNO2 and 0.07
mmol/1 for PMS.
Nitrous oxide formation was also accompanied b y a disappearance
o f nitrite. The rate o f nitrate reduction was 5 - 8 times higher than
that of N 2 0 formation. This is partly explained by the fact that the
crude extract also contained assimilatory nitrite reductase catalyzing
the reduction o f nitrite to ammonia without any formation o f N20.
Two distinct types of dissimilatory nitrite reductases have been
reported in the literature s . One enzyme contains copper and is able
to reduce NH2OH. The other does not contain copper and cannot
catalyze the reduction o f NH2OH. The latter enzyme is a two-haem
200
DANNEBERG, KRONENBERG, NEUER AND BOTHE
548
- -
0.08"
0)
0
cO
..o
t._
o
r e d u c e d minus o x i d i z e d
....... r e d u c e d minus o x i d i z e d
+NaNO 2
0.04-
..Q
<
658
"
&0
560
606
660
"",.
760
/[nm]
Fig. 3. Difference spectra of the dissimilatozy nitrite reductase preparation. - - , difference
spectrum: enzyme reduced by NADH and PMS minus oxidized enzyme (in the reference
cuvette); . . . . , difference spectrum: enzyme reduced by PMS and reoxidized by NaNO 2 minus
oxidized enzyme (in the reference cuvette).
protein containing the so-called c y t o c h r o m e cd. Previous experiments
with A. brasilense Sp 72~ have shown that intact cells do not reduce
NH2OH which, however, acts as an inhibitor o f dissimilatory nitrite
reduction in Azospirillum. This suggests that Azospirillum possesses a
c y t o c h r o m e cd type dissimilatory nitrite reductase. This can be shown
directly in a preparation from A. brasilense Sp 7. To do this, the crude
extract used above was centrifuged (2 h, 100,000 g) and the supernatant
was fractionated by (NH4)2SO 4 precipitation. The fraction precipitating
between 55 and 90% (NH4)2SO4 was used to record the absorption
spectra at room temperature. When directly measured, the preparation
showed absorption maxima at 408, 522 and around 638 nm (Fig. 2).
The spectrum did not change significantly upon the addition o f K3Fe(CN) 6 indicating that nitrite reductase was in the oxidized form when
isolated. Maxima at 415, 460, 519, 548 and around 6 5 0 n m became
visible upon reduction with NADH and PMS (Fig. 2). The difference
spectrum (Fig. 3) indicates that the latter broad maximum around 650
nm can be resolved into two peaks at wavelengths of 606 and 650 nm.
Similar spectra were published for the dissimilatory nitrite reductases
from other organisms 9'1.'=. The bands at 415, 519 and 548 nm might
be associated with cytochrome c, and those at 460, 606 and 650 nm
with c y t o c h r o m e d (= c y t o c h r o m e a2). Remarkably, c y t o c h r o m e cd in
nitrite reductase o f Azospirillum was reoxidized to ca 60% by an anaerobic solution o f NaNO2. We are currently attempting to characterize
the c y t o c h r o m e cd containing nitrite reductase in more detail, parti-
N~-FIXATION AND DENITRIFICATION BY AZOSPIRILLUM
201
cularly its association w i t h m e m b r a n e s and its e l e c t r o n d o n o r specificity.
E x p e r i m e n t s are also in progress o n the n i t r o u s o x i d e r e d u c t a s e f r o m
Azospirillum w h i c h m a y 21 or m a y n o t ~7 be a c o p p e r e n z y m e in den i t r i f y i n g bacteria.
Acknowledgement This work was kindly supported by grants from the Bundesministerium
for Forschung und Technologic (PTB Jiilich).
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Na-FIXATION AND DENITRIFICATION BY AZOSPIRILLUM
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