Protoplasma (1994) 183: 62- 66
PROTOPLASMA
Comparative study of the response of Azotobacter vinelandii
and Acetobacter diaeotrophicus to changes in pH
R. H. Burris*
Department of Biochemistry, University of Wisconsin-Madison, Madison, Wisconsin
Received March 3, 1994
Accepted July 29, 1994
Dedicated to the memoryof ProfessorJohn G. Torrey
Summary.Curves were established for the p H response of respiration
on eleven snbstrates by Azotobacter vinelandii and Acetobacter dia-
zotrophicus. With every substrate the optimal pH for A. diazotro-
phicus was lower than for A. vinelandii.The optimal hydrogenion
concentration for A. diazotrophicus was 5 fold to 365 fold greater
than for A. vinelandiidependingupon the substrate. In general, A.
diazotrophicus supports respiration overa widerpH range than does
A. vinelandii.
Keywords:Aeetobacter; Azotobacter; pH; Respiration; Substrates.
Introduction
In general, biological N 2 fixation occurs best near neu-
trality and is minimal below pH 5. An examination of
the literature indicates that media suggested for free-
living Nz-fixers usually are adjusted to a pH near 7.
For example: Azotobacter spp. pH 7.3 (Newton et al.,
1953); Frankia spp. pH6.4 (Callaham etal. 1978),
methanogenic bacteria pH 7.6 (Belay et al. 1984) pH 6.7
(Murray and Zinder 1984); Desulfovibrio spp. pH7.6
(Postgate and Kent 1985); family Rhodospirillaceae
pH 6.8 (Madigan et al. 1984); Klebsiella pneumoniae
pH 7.0 (Bachhuber et al. 1976); Rhizobium legumino-
sarum pH 7.0 (DeSmedt and DeLey 1977); Azospirillum
halopraeferens pHS.5 (Reinhold etal. 1987); Azospi-
rillum amazonense pH6.8 (Falk etal. 1985); Bacillus
polymyxa pH 7.7 (Hino and Wilson 1958). In contrast,
Cojho et al. (1993) suggest a pH of 5.5 for growth of
Acetobacter diazotrophicus.
D6bereiner et al. (1972) observed that the nitrogenase
* Correspondence and reprints: Department of Biochemistry,Uni-
versityof Wisconsin-Madison,420 HenryMall,Madison,W153706-
1569, U.S.A.
9 Springer-Verlag 1994
Printed in Austria
activity in the rhizosphere of sugarcane was consid-
erably greater than around the roots of a number of
other plants. Cavalcante and D6bereiner (1988) re-
ported the isolation of "A new acid-tolerant nitrogen-
fixing bacterium associated with sugarcane." They
found that the organism was predominantly in the su-
garcane tissue rather than in the rhizosphere. It grew
in 30% sucrose, established a pH below 3, and growth
and fixation of N 2 occurred at this acidity. They pro-
posed the name Saccharobacter nitrocaptans, and in a
footnote suggested a change to Acetobacter nitrocap-
tans; the accepted name now is Acetobacter diazotro-
phicus (Gillis eta1. 1989). Growth and N2 fixation at
this very low pH is in marked contrast to other N2-
fixing microorganisms.
We have compared how the oxidation of various sub-
strates by Azotobacter vineIandii (a free-living bacter-
ium that grows rapidly and fixes N 2 vigorously) and
A. diazotrophicus varies with the pH. The optimal hy-
drogen ion concentration for 02 uptake on various
substrates by A. diazotrophicus ranges from 6 to 365
times that for A. vinelandii. The peak pH region and
overall pH region of activity for A. diazotrophicus tend
to be broader than for A. vinelandii.
Materials and methods
Cultures of A. vinelandii and A. diazotrophicus were aerated in a
500 x 60 m m test tube at 30 ~ Air was admitted through a sintered
glass sparger. A. vinelandii was grown on Burk's medium (Newton
et al. 1953; p H 7.3) and A. diazotrophicus was grown on A D medium
which consisted of the salts of Burk's medium with 3 mg Fe and
1 m g Mo per liter plus 20 g sucrose. To this was added per liter 14 mg
N as (NH4)2SO4, 2 g MES [2-(N-morpholino) ethanesulfonic acid],
R. H. Burris: Response of Azotobacter vinelandii and Acetobacter diazotrophicus to changes in pH 63

1 g malic acid, 0.1 g Difco yeast extract; pH was adjusted to 6.5 with 100f / ~ ]
NaOH solution. Cultures of the bacteria were harvested at approx- ,oo V
imately the end of log phase by centrifugation, and then they were 4_ 80
resuspended in water, resedimented and resuspended in water for ~ 6 0 [ e/ ~ // 1 60 60
use. They were stored a t 5 ~ and were aerated before use.
Uptake of 02 was measured with a Gilson differential respirometer
~ 40I / 1 // J ~ 40
e
2O
at 30 ~ (Umbreit etal. 1964). The respirometer vessels contained rv 2 0 ~
0
0.5mi cell suspension, 0.5ml 0.3M phosphate-buffer of the appro- 02 4 6 8 10 2 4 6 8 10 2 4 6 8 10

o: ~176176
priate pH and 0.5 ml of 0.02 M substrate. The center well had 0. l ml pH pH pH
20% KOH and a filter paper wick to increase surface for absorbing Sucrose Glucose Fructose
COz. Phosphate buffers were used throughout to avoid introducing
effects that would result from changing the buffer ions. When the 100 ~- ~'_'N~
m
reaction components were mixed, the pH was altered from the buffer ~ 80
pH. The linearity of the curves for 02 uptake indicated that the pH o 60
._>
changed little during the course of the measurements. Immediately ~ 40
after the measurements were completed (measurements usually were
taken for 40 to 150 rain to establish suitable curves), the contents of ~" 20
the respirometer vessels were removed and the pH was measured 0 -
2 4 6 8 10 2 4 6 8 10 2 4 6 8 10
with a glass electrode; this terminal pH was plotted against relative pH pH pH
rate of 02 uptake to yield the curves shown. The most rapid uptake Ethanol Propanot Glycerol
was given the value of 100 and the other rates were plotted relative
Fig.2. Respiration of Azotobacter vinelandii ( 9 and Acetobacter
to it.
diazotrophieus ( 0 ) on various sugars and alcohols in response to
various pHs. Data are expressed relative to 100 for the highest activity
Results
Figure 1, showing a representative run, indicated the
linearity of 02 uptake by A. diazotrophicus with sucrose as the substrate. The buffer pHs and final pHs are
listed, and the relative rates are plotted against the final
(6.00) pHs in the inset curve. The responses of respiratory
6.02 activity of A. vinelandii and A. diazotrophicus with
oo k o~o~..,, 17.o8)
| / (5.00) 0'7.08 changing pH are shown in Fig. 2 and 3, and the ap-
80 L / 5.16 ~_ (7.60) 9
120 60 w 7.40 6.02 proximate pH optima on the various substrates are
indicated in Table 1.

lO0 20~ /~2.68) (8.30_)\ / / Sugars

m 2.60 8133 * y Each sugar was oxidized most effectively at a higher
" 2 ?.4o
80 ~ / pH by A. vinelandii than by A. diazotrophicus. The
oxidation is measurable over a wider pH range (usually
~L about 2 pH units wider) with A. diazotrophicus than
e, 60
with A. vinelandii. The curves above the optimal pH
are quite similar, but respiration of sucrose is supported
40 at a distinctly lower pH by A. diazotrophicus than by
A. vinelandii. The curves for glucose are not greatly
different from those for sucrose except that the optimal
20
range for A. diazotrophicus is broad and extends over
8.33 greater than 2 pH units. Fructose again is similar with
0 rO ~ ~ 2.50
2T0 40 60 80 100
F ~
an optimal pH for A. vinelandii at pH 6 to 6.8 and for
Minutes A. diazotrophicus near 5.0. At pH 3.0 the rate of res-
piration by A. diazotrophicus still is about 30% of its
Fig. 1. Effect of pH on the respiration of sucrose by Acetobaeter
diazotrophicus. Inset Rate plots derived from the slopes of the curves; maximum rate, but respiration is very limited for A.
indicated are the pH of the buffer used (in parentheses) and the pH vinelandii at pH 4.6.
of the reaction mixture at the end of the run as measured with a
glass electrode and plotted on the figure. When the cells, buffer and Alcohols
substrate are mixed the pH changes, but there is little subsequent
change in pH during the run as indicated by the linearity of the Ethanol supports rapid respiration. For A. vinelandii
uptake of 02 pH 6.9 appears optimal, whereas for A. diazotrophicus
64 R . H . Burris: Response of Azotobacter vinelandii and Acetobacter diazotrophicus to changes in pH

Table 1. Optimal pHs for respiration and rates of respiration on various substrates by Azotobacter vinelandii
and Aeetobaeter diazotrophieus, and differences in hydrogen ion concentrations at these optimal pHs

Substrate A. diazotrophicus A. vinelandii Fold

difference [H + ]
pH Q02 (N) = pH Q02 (N)

Sucrose 6.0 577 7.0 1114 10

Glucose 5.3 1311 7.0 364 80
Fructose 5.0 197 6.4 1242 13
Ethanol 4.4 393 6.9 886 365
Propanol 4.1 315 6.7 945 175
Glycerol 5.0 170 6.9 102 49
Formate 6.1 66 7.6 38 15
Acetate 5.7 79 6.3 2076 6
Pyruvate 5.2 92 6.8 1924 23
Lactate 5.2 144 6.2 2227 10
Succinate 3.8 131 5.4 87 49

= Qo2 (N), gl 02 uptake/(h x mg N) at optimal pH

the optimal pH is near 4.4. Thus the optima are at 100 100p ~ 7 100F

I
drastically different hydrogen ion concentrations, the z 8o
hydrogen ion concentration optimal for A. diazotro- >0 60
phicus being about 365 times that for A. vinelandii. With ~ 40 40 I 40=
propanol the optimal pHs are somewhat lower than
a: 20
for ethanol, but the differences in the optimal hydrogen 0
ion concentrations between organisms again are dras- 8 10 2 4 6 8 10 4 6 8 10
~H pH pH
tically different (about 175 fold).

8oL
I
Formate Acetate Succinate
Glycerol is not metabolized very rapidly by either or-
ganism, but there is a difference in optimal pHs of 100 100[- /~'b~
about 1.9 units, or a difference of about 49 fold in ~ 8o
hydrogen ion concentration. Glycerol was metabolized ~ 6o fi0
.>
at a pH as low as 2.5 by A. diazotrophicus, but no "~ ~0 40
D
activity by A. vinelandii was observed at pH 4.7. rv 20 2
0 v

Organic acids
Figure 3 indicates the pH responses on organic acids.
The optimal pH for oxidation of formate by A. dia-
zotrophicus was near 6.1, and good activity remained
at pH 5.1. In contrast to the sharp optimum for A.
diazotrophicus on formate, A. vinelandii exhibited a
rather broad optimum between pH 6.6 and 8.6.
The difference between organisms on acetate was less
marked than that on most substrates; the optimal value
was near pH 6.3 for A. vinelandii and 5.7 for A. dia-
zotrophicus, i.e., a difference of about 6 fold in hydro-
gen ion concentration. The upper limits for oxidation
were similar for the two organisms.
A. vinelandiiexhibited an unusual response to succinate
oxidation with varying pH. Oxidation reached a min-
imum near pH 6.3 and then rose as the pH was in-
4 6 8 10 2 4 10
pH ~H
Pyruvate Lactate
Fig. 3. Respiration of Azotobacter vinelandii (O) and Acetobacter
diazotrophicus (Q) on various organic acids in response to various
pHs. Data are expressed relative to 100 for the highest activity
creased to pH 8. The optimal value was near 5.4. A.
diazotrophicus had optimal oxidation near pH 3.8 and
did not show the rise exhibited by A. vinelandii at high
pH. The spread was 1.6 pH units between optimal aci-
tivities for the two organisms.
The spread between optima of 1.6 units on pyruvate
was similar to the spread on succinate, but the curves
were quite different. Among the substrates tested, only
succinate showed a double peak in response to pH
R. H. Burris: Response of Azotobacter vinelandiialld Acetobacter diazotrophicusto changes in pH
changes. Again on pyruvate A. diazotrophicus sup-
ported active respiration over a greater range of p H
than did A. vinelandii. The shapes of the curves from
their optimal p H to high pHs were quite different.
Lactate with a spread of the optimum from 5.0-5.4 for
A. diazotrophicus and with a peak at p H 6.2 for A.
vinelandii exhibited one of the narrowest differences
among the substrates tested. The shapes of the curves
on lactate were similar, and the overall range of activ-
ities on lactate did not differ as greatly between or-
ganisms as with most of the substrates.
We also compared activities on malate, citrate and
propionate, but they were metabolized so poorly by
one or both organisms that the curves for these sub-
strates were not definitive. The use of dissolved oxygen
by the organisms on several of the substrates was cross-
checked with a Clark electrode. The data are not shown,
but they verified the results obtained with the Gilson
respirometer.
Discussion
Acetobacter diazotrophicus shows a remarkable ability
to grow and fix N 2 at a very low p H and at very high
osmotic pressure. Cavalcante and D6bereiner (1988)
have found that it grows and fixes N2 in 30% sucrose.
Thus, it is adapted to growth inside the sugarcane plant.
As the growth of sugarcane extends over a long period
and cane produces a massive amount of plant material,
the contribution of A. diazotrophicus can be of great
importance in support of growth. The studies of Bod-
dey et al. (1990) in Brazil have indicated that A. dia-
zotrophicus fixes up to 150 kg N/hectare in association
with sugar cane during a growing season. This is com-
parable to fixation in highly effective legume symbioses.
The production of alcohol for fuel in Brazil is de-
pendent upon an abundant supply of low-cost sugar
from cane, and cane commonly is grown on low-ni-
trogen soils in Brazil.
In m a n y field experiments the benefit derived by the
plants from inoculation of the soil with Azospirillum
spp. has been marginal. Smith etal. (1976) reported a
benefit to crops on Florida soils from associative fix-
ation by Azospirillum lipoferum. However, the field re-
sults apparently were not repeatable in subsequent
years. O k o n etal. (1988) found benefit to associated
plants when inoculated with A. lipoferum on alkaline,
sandy soils, but it was not clear whether the response
was to N2 fixation or generation of plant growth sub-
stances by the bacteria. We ran experiments with maize
on good soils in Wisconsin. Growth was excellent and
65
statistical variation was small, but there was no de-
monstrable benefit from inoculation with A. lipoferum
(unpubl. data). The responses are reminiscent of those
observed from inoculation with a free-living organism
such as A. vinelandii. The organism fixes N2 vigorously
when supplied a suitable source of energy in the lab-
oratory, but in the field available sources of energy are
limiting. The situation in surgarcane perhaps is unique
in that energy from the sucrose within the plant is
abundant, and A. diazotrophicus is well adapted to
tolerate the high osmotic pressure and low p H that it
encounters. Given these conditions, fixation may be
150 kg N/hectare rather than the usual 1-2 kg/hectare
contributed by free-living diazotrophs in the soil.
Comparison of p H curves for A. vinelandii and A. dia-
zotrophicus indicates that A. diazotrophicus usually can
metabolize a variety of substrates at about as high a
limiting p H as A. vinelandii. However, it can oxidize
the substrates over a wider p H range, and differences
are dramatic at low pHs. When we examined the p H
optimal for each organism, the optimal p H value was
lower for A. diazotrophicus than for A. vinelandii on
every substrate tested. In some cases the optimal hy-
drogen ion concentration was as much as 365 times as
great for A. diazotrophicus as for A. vinelandii.
When tests have been made for the comparative rates
of oxidation of various substrates by an organism, it
has been customary to compare them at a single p H
rather than at the p H optimal for each substrate. Our
tests show differences in rates of respiration relative to
glucose and other substrates between the two means
of comparison. However, the results do not alter our
general conclusions regarding comparisons of A. dia-
zotrophicus and A. vinelandii, and the data are not
shown.
Acknowledgements
This work was supported by the College of Agricultural and Life
Sciences, University of Wisconsin-Madison and Department of En-
ergy grant DE-FG02-87ER13707.
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