Vol.2, No.1, 16-22 (2011) Agricultural Sciences
doi:10.4236/as.2011.2 1003
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
Mineral phosphate solubilization activity of
gluconacetobacter diazotrophicus under P-limitation
and plant root environment
J. M. Crespo1, J. L. Boiardi1, M. F. Luna1,2*
1CINDEFI (UNLP-CONICET, CCT La Plata), Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La Plata, Argentina;
2CIC PBA, Comision de Investigaciones Cientificas, Provincia de Buenos Aires, Argentina;
*Corresponding Author: mafla@quimica.unlp.edu.ar
Received 19 October 2010; revised 17 November 2010; accepted 24 November 2010
The ability to solubilize insoluble inorganic pho-
sphate compounds by Gluconacetobacter di-
azotrophicus was studied using different cul-
ture approaches. Qualitative plate assays using
tricalcium phosphate as the sole P-source
showed that G. diazotrophicus produced solu-
bilization only when aldoses were used as the
C-source. Extracellular aldose oxidation via a
pyrroloquinoline quinone-linked glucose dehy-
drogenase (PQQ-GDH) is the main pathway for
glucose metabolism in G. diazotrophicus. In
batch cultures with 5 g l-1 of hydroxyapatite as
the P-source and glucose as the C-source, more
than 98% of insoluble P was solubilized. No
solubilization was observed neither using glyc-
erol nor culturing a PQQ-GDH mutant of G. di-
azotrophicus. Solubilizaton was not affected by
adding 100 mmol l-1 of MES buffer. Continuous
cultures of G. diazotrophicus sho wed significant
activities of PQQ-GDH either under C or P limi-
tation. An intense acidification in the root envi-
ronment of tomato and wheat seedlings inocu-
lated with a G. diazotrophicus PAL5 was ob-
served. Seedlings inoculated with a PQQ-GDH
mutant strain of G. diazotrophicus showed no
acidification. Our results suggest that G. di-
azotrophicus is an excellent candidate to be
used as biofertilizer because in addition to the
already described plant grow th-promoting abili-
ties of this organism, it shows a significant
mineral phosphate solubilization capacity.
Keyw ords: Gluconacetobacter Diazotrophicus;
Phosphate Solubilization; Glucose Dehydrogenase;
Pqq; Biofertilizer
Phosphorus (P) is after Nitrogen, the most important
nutrient limiting agricultural production. Soils are often
abundant in insoluble P, either in organic or inorganic
forms, but deficient in soluble phosphates essential for
growth of most plants and microorganisms. Soluble
forms of phosphate fertilizers are widely applied to ag-
ricultural soils in order to circumvent P-deficiency but
75 to 90% of added P is rapidly precipitated as insoluble
forms and becomes unavailable to plants [1]. Converting
soil insoluble phosphates (both organic and inorganic) to
a form available for plants is a necessary goal to achieve
sustainable agricultural production. Several reports show
the ability of different bacteria to solubilize inorganic
phosphate compounds such as tricalcium phosphate,
dicalcium phosphate, hydroxyapatite and rock phosphate
[2]. Among the bacterial genera reported to express a
mineral phosphate solubilization (MPS) phenotype are
Pseudomonas, Bacillus, Rhizobium, Burkholderia, Ach-
romobacter, Agrobacterium, Microccocus, Aereobacter,
Flavobacterium and Erwinia [3]. A significant body of
evidence has been developed to show that in gram-
negative bacteria the expression of a direct extracellular
oxidative pathway allows superior MPS capabilities [2].
Through this pathway (also called nonphosphorylating
oxidation) glucose is oxidized to gluconic acid and
2-ketogluconic acid directly in the periplasmic space.
These strong organic acids can dissolve poorly soluble
calcium phosphates present in soils. On the other hand it
has been reported that the buffering capacity of soils
could limit P solubilization by microorganisms [4,5].
Gluconacetobacter diazotrophicus is a nitrogen-fixing
endophytic bacterium able to colonize several plant spe-
cies [6,7]. G. diazotrophicus promotes, besides N2-fixa-
tion, other beneficial effects to plants such as phythor-
mones production and biocontrol towards plant patho-
gens [6]. Moreover, in some G. diazotrophicus strains the
ability to promote P solubilization has been demon-
J. M. Crespo et al. / Agricultural Sciences 2 (2011) 16-2 2
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
strated in vitro [8,5]. Considering all these characteris-
tics G. diazotrophicus has been described as a plant
growth-promoting bacterium.
G. diazotrophicus possess a pyrroloquinoline quinone-
linked glucose dehydrogenase (PQQ-GDH) responsible
for the periplasmic conversion of aldoses into the corre-
sponding aldonic acid [9]. This is considered the princi-
pal pathway for glucose metabolism in this bacterium
[10]. It has been reported that in the presence of aldoses
and under N2-fixing conditions, G. diazotrophicus is able
to express an enhanced production of energy linked to a
fully active PQQ-GDH [11]. However, there is no in-
formation about PQQ-GDH expression under P-limi-
tation, a condition likely found in soils and it is not
known whether buffering could affect MPS by G. di-
azotrophicus. Moreover, it is not known whether this
organism expresses an active PQQ-GDH in the ri-
zosphere of plants, where the MPS activity needs to be
expressed in order to provide soluble phosphate to plants.
The objective of this study was to address the above
mentioned issues and assess the potential of G. di-
azotrophicus as a biofertilizer due to its MPS activity
coupled to its well-known plant growth promoting abili-
2.1. Organism and Maintenance
G. diazotrophicus strain PAL 5 (ATCC 49037), kindly
provided by Dr. Caballero-Mellado, was maintained on
agar slants on a potato medium [12]. Strain MF105, an
already described PQQ-GDH negative mutant strain of G.
diazotrophicus [10], was maintained on the same me-
dium supplemented with streptomycin (400 µg ml-1).
2.2. Cultures and Growth Conditions
Plate assays were carried out using the National Bo-
tanical Research Institute's phosphate medium (NBRIP)
[13] containing l-1: Ca3(PO4)2 (TCP), 5 g; MgCl2.6H2O,
5 g; MgSO4.7H2O, 0.25 g; KCl, 0.2 g; (NH4)2SO4, 0.1 g
and 10 g of different carbon sources (Figure 1). Bacterial
cultures of both strains of G. diazotrophicus with a con-
centration of around 1.109 CFU ml-1 were centrifuged
and resuspended in the same volume of saline solution
pH 6.0. A volume of 50 µl of these cell suspensions was
plated onto NBRIP medium. The plates were observed
two days after incubation at 30 °C.
Batch cultures were performed employing the NBRIP
liquid medium with 10 g l-1 of glucose or glycerol as car-
bon source, 5 g l-1 of hydroxyapatite (HY, Ca10(OH)2
(PO4)6) as the sole P source and 2.5 g l-1 of (NH4)2SO 4
(under non-BNF conditions). HY was replaced by
K2HPO4 (2.0 g l-1) in experiments with soluble P. When
the organism was grown under BNF conditions, (NH4)2-
SO4 concentration was decreased to 0.132 g l-1 [12]. To
evaluate the effect of buffering on MPS activity, NBRIP
medium was strongly buffered with morpholineethane-
sulfonic acid (MES) buffer 100 mmol l-1. Initial pH was
adjusted at 6.0 by adding KOH 0.1 mol l-1 or HCl 0.1
mol l-1. Bacteria (both, wild type and mutant) were
grown at 30 °C in 250 ml liquid NBRIP medium (two
flasks per treatment) on a rotary shaker stirred at 200 or
100 rpm for non-BNF or BNF, respectively. Negative
controls (no bacteria) were carried out to quantify the
solublized P in the culture conditions regardless of mi-
crobial activity.
Chemostat cultures were carried out using the modi-
fied LGIM medium described by Luna et al. (2000) [11]
with glucose 10.0 g l-1 or 20.0 g l-1 and NaH2PO4.H2O 10
mmol l-1 or 0.5 mmol l-1 for C- or P-limitation respec-
tively. (NH4)2SO4 (2.50 g l-1) was added to cultures
grown under non-BNF conditions. Cultures under BNF
were carried out without (NH4)2SO4 in the culture me-
dium. The strategies to attain BNF conditions were de-
scribed by Luna et al. (2000) [11]. G. diazotrophicus Pal
5 was grown at 30 °C in a 2-l LH (Incelltech 210) fer-
mentation unit with a working volume of 1.0 l. The
growth rate (dilution rate) was adjusted at 0.05 ± 0.001
h-1. The pH was automatically maintained at 5.5 ± 0.1 by
addition of either 0.5 mol l-1 NaOH or 0.25 mol l-1
H2SO4. Foam formation was prevented by automatic
addition of an antifoam agent. Cultures were flushed
with air (20 to 25 l h-1). The dissolved oxygen concen-
tration was continuously measured using an Ingold
(Wilmington, MA) polarographic probe and maintained
at the desired level of air saturation by varying the agita-
tion speed of the impeller. Cultures were considered to
be under steady-state conditions when biomass concen-
tration and specific rate of oxygen consumption of cul-
tures remained almost constant (varied less than 5 %), as
previously described [11]. After modification in growth
conditions, 5 to 10 volume changes were usually re-
quired to re-obtain steady state.
2.3. Analyses
Samples of batch (at 8-12 h intervals during 5 days) or
continuous cultures (daily during 5-7 days) were taken
for pH, absorbance, biomass dry weight, glucose and
products quantification. Growth was estimated by meas-
urement of the absorbance at 560 nm and biomass dry
weight determined as previously reported [11]. Samples
of batch cultures grown in medium with HY were di-
luted 1:1 (v:v) using 0.1 mol l-1 HCl to dissolve the re-
sidual insoluble phosphate and measured against a blank
identically treated [14]. Samples were centrifuged 10
min at 10,000 g and the resulting supernatant was em-
ployed to assay P, glucose, gluconic acid, and extracel-
J. M. Crespo et al. / Agricultural Sciences 2 (2011) 16-2 2
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
lular polysaccharides (EPS). Glucose concentrations in
media and supernatants were determined with a glucose
oxidase enzymatic kit (Wiener, Argentina). Gluconate
concentrations were assayed using a test-kit (Boehringer,
Mannheim, Germany). EPS dry weight was determined
by adding two volumes of ethanol to culture super-
natants; after storing overnight at 4 °C precipitated ma-
terial was collected by centrifugation (20 min at 7,000 g).
The pellets were resuspended in distilled water and dried
at 60 °C. Soluble P concentration was measured by the
method described by Clesscerl et al. (1998) [15]. Oxy-
gen and carbon dioxide concentrations in the emitted
gases from the fermentor were determined using a para-
magnetic oxygen analyzer (Servomex 1100A, Norwood,
MA) and an infrared carbon dioxide analyzer (Horiba
PIR 2000, Japan). Gas flow rates were measured with a
bubble flow meter. Biomass yields, rates of oxygen
consumption, and carbon dioxide production were cal-
culated as previously described [11].
2.4. Enzyme Assays
PQQ-GDH and gluconate dehydrogenase (GaDH) ac-
tivities were measured spectrophotometrically using
2,6-dichlorophenol-indophenol (DCIP) as the electron
acceptor and glucose or gluconate respectively [16,17].
Samples (20 ml) from batch cultures were taken at ex-
ponential phase, while glucose was still detectable, and
centrifuged for 10 minutes at 12,000 g at 4 ºC. In che-
mostat, once steady-state conditions were attained, an
appropriate volume of culture (70 ml) was withdrawn
and centrifuged as described above. Cells were washed
twice in phosphate buffer 10 mmol l-1 (pH 6.0) contain-
ing 5 mmol l-1 MgCl2 to a final concentration of 4.50 mg
ml-1. This washed cells suspension (WCS) was em-
ployed to determine the PQQ-GDH activity in whole
cells. The final concentration of cells in the reaction
mixture was 0.10 mg ml-1 dry weight.
2.5. Acid Production from Root Exudates
Tomato (Lycopersicum esculentum cv. “platense itali-
ano”) and wheat (Triticum aestevium cv. “baguette”)
seeds were surface sterilized with 2 % sodium hypochlo-
rite for 5 min followed by three washes with sterile water.
Seeds were germinated onto water-agar plates (0.5 %
agar) at 30 °C during 72 h. The seedlings were inocu-
lated by immersion for 15 min in a G. diazotrophicus
suspension (either PAL5 or MF105 centrifugated and
resuspended as described for plates assays) and placed
into tubes containing semi solid agarized Fähraeus me-
dium [18] with 0.1 % methyl red as acid-base indicator.
Uninoculated seedlings and a G. diazotrophicus suspen-
sion were used as negative controls.
Plate assays. Qualitative estimation of P solubilization
was made in agar plates supplemented with TCP. The
MPS phenotype was identified by the production of
clearing zones of solubilization around the colony (Fig-
ure 1). As it can be seen in Figure 1 appearance of
clearing halos was dependent on the nature of the carbon
source. Glucose, arabinose, galactose and xylose (all
substrates of PQQ-GDH) showed an MPS (+) phenotype.
With other carbon sources G. diazotrophicus showed
growth but was not capable of solubilizing phosphates.
When plates were inoculated with strain MF105
(PQQ-GDH (-) mutant) neither the PQQ-GDH substrates
nor the other carbon sources showed a MPS (+) pheno-
Batch cultures. Quantitative estimation of P solubili-
zation was carried out in liquid medium cultures, since
this approach is considered more accurate than plate
assays [19]. Batch cultures performed with glucose and
replacing the soluble P-source by HY, showed a very
similar growth behavior to the one reported using solu-
ble P [10]. Soluble P concentration in the media in-
creased together with the gluconic acid. The culture pH
dropped during the same period from 6.0 to 2.5 with a
concomitant P solubilization that reached around 1,000
(a) (b)
Bacterial strain Carbon
source PAL5 MF105
Glucose + -
Arabinose + -
Galactose + -
Xylose + -
Lactose - -
Maltose - -
Gluconate - -
Glycerol - -
Fructose - -
Figure 1. In vitro P solubilization by G. di-
azotrophicus (a) PAL5 and (b) MF105. Clear-
ing halo formation (+) and no halo formation (–)
by G. diazotrophicus growing on NBRIP me-
dium with TCP and different C-sources.
J. M. Crespo et al. / Agricultural Sciences 2 (2011) 16-2 2
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
ppm (Figure 2). This amount represents more than 98%
of the insoluble P contained in the HY added to the cul-
ture medium (Figure 3). Once glucose was entirely oxi-
dized (around 80 h of growth) soluble P decreased in the
culture supernatants together with gluconate consump-
tion. There was no difference between P solubilization
levels under both BNF and non-BNF conditions (Figure
3). P concentration of negative controls (non inoculated)
remained almost constant, between 2 and 8 mg l-1, along
the experiment.
It was observed that G. diazotrophicus exhibited a
similar P-dissolving capability either in the absence or in
the presence of buffer, even at a concentration of 100
mmol l-1 of MES (Figure 3).
As already described for G. diazotrophicus growing
with soluble P [10], PQQ-GDH was actively synthesized
in glucose-containing batch cultures using HY as the
time (h)
020406080100 120 140 160
P (mg l
Gluconic acid (g l
Figure 2. Soluble P, gluconic acid, pH and O.D. measurements
of G. diazotrophicus PAL5 cultures growing with glucose 20 g
l-1, HY 5 g l-1 and BNF conditions. () soluble P; () gluconic
acid; () O.D. and () pH.
P Solubilization (%)
Figure 3. P solubilization by G. diazotrophicus PAL5 in cul-
tures with glucose 20 g l-1, HY 5 g l-1 under both BNF and
non-BNF condition and with MES buffer 100 mmol l-1. P solu-
bilization percentage was determined dividing the maximum
soluble P value obtained by the initial P amount in HY.
sole P source, either under BNF or non-BNF conditions
(Table 1).
Cultures of G. diazotrophicus PAL5 using glycerol as
the sole carbon source and cultures of strain MF105 with
glucose, both with HY as the sole P-source, showed pH
values over 6.0 all along cultures, indicating no acid
production and levels of soluble P below 10 ppm (data
not shown).
Continuous cultures. G. diazotrophicus was grown
under C- or P-limiting conditions in a chemostat using
glucose as C-source. In order to check that growth was
indeed C- (or P-limited), additions of the corresponding
limiting substrate were made to the culture vessel. An
immediate increase in the steady state biomass concen-
tration was observed after the addition of either limiting
substrate. Moreover, the residual concentration of the
limiting substrate in the supernatants of steady state cul-
tures was below the detection limits of the assays em-
ployed (data not shown).
In cultures grown under P-limitation, either with NH4
or N2 as the N-source, biomass yields were lower than
those observed in glucose-limited cultures and showed a
significant increase of O2 consumption. Growth yields of
C- or P-limited continuous cultures were not signifi-
cantly affected by the nature of the N-source (NH4
+ or N2),
as already observed in a previous work [11] (Table 2).
Table 1 In vitro PQQ-GDH activities of G. diazotrophicus
PAL5 in batch cultures.
Enzymatic activity
HY 5 g l-1
non-BNF HY 5 g l-1 BNF
HY 5 g l-1 BNF
(MES 100
mmol l-1)
PQQ-GDH476* ± 31.87 486* ± 30.70 503* ± 13.17
* Enzymatic activities are expressed as nmoles DCIP reduced min-1 mg
protein-1 (assuming 60% protein content in the biomass). Data are mean of
at least three repetitions.
Table 2 Continuous cultures of G. diazotrophicus PAL5.
C-limitation P-limitation
Yx/s 29.2 34.7 13.9 14.9
QO2 8.44 7.69 11.03 10.37
EPS nd nd 3.6 1.41
Gluconic acidnd nd Nd 9.12
C-recovery 98.6 94.5 74.6 102.3
PQQ-GDH 360 406 343 361
GaDH 100.5 95.2 58.0 52.7
Yx/s is expressed as g biomass mol substrate-1; QO2 as mmol O2 g biomass-1
h-1; EPS as g l-1; gluconic acid as g l-1; C-recovery in %. PQQ-GDH and
GaDH activities are expressed as nmol DCIP reduced min-1 mg protein-1
(assuming 60% protein content in the biomass). Data are mean of at least
three repetitions. SD was never >10%.
EPS was detected in supernatants of both P-limited
J. M. Crespo et al. / Agricultural Sciences 2 (2011) 16-2 2
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
cultures but only in those growing under BNF conditions
gluconic acid could also be detected (Table 2). In cul-
tures grown P-limited and with NH4
+ as the N-source,
C-recovery (taking into account biomass, CO2, EPS and
gluconic acid) was not enough to match the C-input
(glucose feeded to the culture). On the other hand the
supernatant fluids of C-limited cultures contained neither
gluconic acid nor any other detectable extracellular
product (Table 2).
Significant activities of PQQ-GDH were detected in-
dependently of the limitation (C or P) or the N-source
employed. Similarly GaDH activities were detected un-
der all culture conditions tested but, in this case, with a
higher expression under C-limitation in relation to P-
Acid production from root exudates. Figure 4 shows
red zones of acidification in the root environment of
seedlings from both plant species inoculated with a G.
diazotrophicus PAL5 suspension. On the other hand, no
acidification was observed in seedlings inoculated with
the mutant strain MF105. Similarly, no acid production
was observed in control tubes either inoculated with G.
diazotrophicus PAL5 without plants or in tubes where
non-inoculated seeds were placed.
PQQ-GDH of G. diazotrophicus presented the com-
mon behaviour of a broad-substrate aldose dehydro-
genase as reported for others PQQ-GDH [20]. This
(1) (2) (3)
(1) (2) (3)
Figure 4. Acidification in the root environment of (a) wheat
and (b) tomato seedlings. 1- non-inoculated seeds; 2- MF105
inoculated seeds; 3- PAL5 inoculated seeds; 4- inoculated with
PAL5 without seeds.
means the enzyme can oxidize sugars other than glucose
but with an aldose function (Ta b l e 1 ). Only PQQ-GDH
substrates were able to lower the pH of the medium with
the consequent solubilization of poorly soluble calcium
phosphates. The MF105 mutant, deficient in PQQ-GDH
activity, failed to release phosphate from insoluble
compounds for every carbon source assayed. These re-
sults suggest that, in accordance with Intorne et al. (2009)
[5], the periplasmic PQQ-GDH of G. diazotrophicus
PAL 5, which converts aldoses into the corresponding
organic acid, was the responsible for the MPS (+) phe-
notype. Intorne et al. (2009) [5] have shown that muta-
tions affecting the production of gluconic acid by G. di-
azotrophicus dramatically alter the MPS phenotype.
It was reported that the exponential growth phase of
batch cultures of G. diazotrophicus grown in glucose,
fixed nitrogen, and soluble P begins when almost 60% of
the glucose is converted into gluconic acid [21,9]. Dur-
ing this process an intense aerobic metabolism with a
concomitant pH reduction takes place. In our cultures,
soluble P increased together with the gluconic acid con-
centration in the culture medium (Figure 2), showing
that P solubilization was directly related to acid produc-
tion. In Azospirillum brasilense, reduction in soluble
phosphate concentration after a 48 h incubation period
can be explained as an auto-consumption of soluble
phosphates by the growing bacterial population [14].
However, G. diazotrophicus batch cultures performed
with soluble phosphates showed that only 10-15 mg l-1
of P were used for bacterial growth (data not shown).
The reduction of soluble P concentration observed in our
cultures could be ascribed to gluconic acid consumption
followed by an increase in culture pH, instead of soluble
P metabolism by bacterial cells.
PQQ-GDH activity was very similar for all culture
conditions tested indicating a constitutive expression of
the protein as previously reported [22]. No P solubiliza-
tion was observed using glycerol as the sole C-source.
The same applied to strain MF105 cultures. These re-
sults indicate that no P solubilization could take place in
the absence of organic acids derived from PQQ-GDH
activiy. There was no difference between P solubilization
levels under both FBN and non-FBN conditions indicat-
ing that P solubilization by G. diazotrophicus is not af-
fected by the nature of the N-source. GaDH activity was
also observed for all culture conditions indicating the
possible presence of 2-ketogluconic acid in supernatants.
Nevertheless, if this acid was present, its P solubilization
effect would not be as important as the one achieved
with gluconic acid because P solubilization patterns were
directly related to gluconic acid concentration in the
culture supernatants.
It has been reported that the soil buffer capacity af-
J. M. Crespo et al. / Agricultural Sciences 2 (2011) 16-2 2
Copyright © 2011 SciRes. Openly accessible at http://www.scirp.org/journal/AS/
fects MPS by microorganisms [4]. However, in our ex-
periments G. diazotrophicus was able to solubilize P in
the presence of relatively high MES concentration indi-
cating that buffering would not significantly affect the
release of soluble P from HY.
The lower growth yield values observed in chemostat
under P-limitation, compared to those under C-limitation,
were predictable because in C-limited grown microor-
ganisms, catabolism is tightly coupled to anabolism and
high biomass yields are achieved. On the other hand,
cultures grown under C-excess conditions exhibit high
rates of carbon consumption and low biomass yields and
thus, have low energetic growth efficiency as indicated
by an increased specific oxygen consumption rate. This
behavior is generally coupled to the production of ex-
tracellular products (overflow metabolism) [23]. In our
case, significant concentrations of EPS (and gluconic
acid under BNF) could be detected in culture super-
natants when cells were grown under P-limitation and
therefore, C-excess. Moreover, in P-limited cultures
growing with N-fixed, another unknown extracellular
product would have been released since C-recovery
could account only for 75% of the consumed glucose.
In spite of the extra energy expenditure for N2 fixation
the biomass yields of cultures grown under BNF condi-
tions were higher (in the case of C-limitation) or, at least,
comparable (in the case of P-limitation) to those grown
using N-fixed (Table 2). It has been reported that cul-
tures of G. diazotrophicus grown in glucose (or mixtures
of gluconic acid and xylose) under N2-fixing conditions
express an improved growth energetic efficiency be-
cause of a higher coupling of the respiratory chain. It
was demonstrated that this was linked to the expression
of an active aldose oxidation via PQQ-GDH [24]. Once
again, it seems that the expression of an active PQQ-
GDH and N2-fixation were the conditions required by G.
diazotrophicus cultures to direct the electron flow through
a more efficient branch of the respiratory chain. In this
case, the effect was observed under P-limitation.
Table 2 indicates that PQQ-GDH is not induced by
phosphate starvation. Nevertheless, the enzyme was ful-
ly active in all cultures. Moreover, under P-limitation
and N2-fixation significant concentrations of gluconic
acid could be detected in the culture supernatants.
Tubes containing seedlings developed from seeds that
had been inoculated with G. diazotr ophicus PAL5 showed a
significant area of acidification. This acidification can be
ascribed to the production of organic acid/s by PQQ-
GDH expression. This assumption is made on the basis
that no acidification was observed around seedlings from
non-inoculated seeds and from those inoculated with the
mutant strain MF105, impaired in PQQ-GDH expression.
The same was observed in tubes inoculated with G. d i -
azotrophicus but without plants. Therefore the simulta-
neous presence of G. diazotrophicus able to express
PQQ-GDH and growing seedlings were necessary for
acidification. Since root exudates of many plants, in-
cluding the two used in this study, contain significant
amounts of PQQ-GDH substrates [25,26], it is likely that
the acidification areas observed in Figure 4 were caused
by some aldonic acid produced by the inoculated G. di-
azotrophicus cells. This result indicates that the presence
of G. diazotrophicus in the root environment of plants
allows the active expression of PQQ-GDH with the
concomitant production of organic acids that are able to
solubilize P from poorly soluble calcium phosphates, as
Taken together, the results show that G. diazotrophicus
is able to actively express PQQ-GDH with the concomi-
tant production of organic acids and consequent MPS
activity. This activity was not affected by the buffering
capacity of the environment. PQQ-GDH activity was
expressed under conditions of P-limitation (either with
N2 or NH4
+ as N-source) and in the root environment of
different plant species producing acids from the root
exudates. Therefore, in addition to other plant growth
promoting activities already described for G. diazotro-
phicus [6], this organism expresses a MPS (+) phenotype
which allows its consideration as a promising species for
being used as a biofertilizer.
5. Acknowledgements
We are thankful to Dr. Jesús Caballero-Mellado (Centro de Ciencias
Genómicas, Universidad Nacional Autónoma de México) for providing
G. diazotrophicus strain PAL5.
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