Mineral phosphate solubilization activity of gluconacetobacter diazotrophicus under P-limitation and plant root environment

doi:10.4236/as.2011.21003

Paper Menu >>

Journal Menu >>

Vol.2, No.1, 16-22 (2011) Agricultural Sciences

doi:10.4236/as.2011.2 1003

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

ABSTRACT

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

1. INTRODUCTION

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

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-

ties.

2. MATERIALS AND METHODS

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

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.

3. RESULTS

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-

type.

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

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

-1

)

200

400

600

800

1000

1200

Gluconic acid (g l

-1

)

O.D.

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.

HY non-BN FHY BNFHY BNF (MES )

P Solubilization (%)

100

120

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

non-BNF BNF non-BNF BNF

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

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-

limitation.

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.

4. DISCUSSION

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

(a)

(1) (2) (3)

(b)

(1) (2) (3)

(4)

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

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

shown.

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.

REFERENCES

[1] Goldstein, A.H. (1986) Bacterial mineral phosphate so-

lubilization: Historical perspective and future prospects.

American Journal of Alternative Agricultur e, 1, 57-65.

[2] Sashidhar, B. and Podile, A.R. (2010) Mineral phosphate

solubilization by rhizosphere bacteria and scope for ma-

nipulation of the direct oxidation pathway involving

glucose dehydrogenase. Journal of Applied Microbiology,

109, 1-12.

[3] Rodriguez, H. and Fraga, R. (1999) Phosphate solubiliz-

ing bacteria and their role in plant growth promotion.

Biotechnology Advan ces, 17, 319-339.

doi:10.1016/S0734-9750(99)00014-2

[4] Gyaneshwar, P., Kumar, G.N., Parekh, L.J. and Poole, P.S.

(2002) Role of soil microorganisms in improving P nutri-

tion of plants. Plant and Soil, 245, 83-93.

doi:10.1023/A:1020663916259

[5] Intorne, A.C., de Oliveira, M.V., Lima, M.L., da Silva,

J.F., Olivares, F.L. and de Souza Filho, G.A. (2009) Iden-

tifcation and characterization of Gluconacetobacter di-

azotrophicus mutants defective in the solubilization of

phosphorus and zinc. Archives of Microbiology, 191,

477-483. doi:10.1007/s00203-009-0472-0

J. M. Crespo et al. / Agricultural Sciences 2 (2011) 16-2 2

[6] Pedraza, R.O. (2008) Recent advances in nitrogen-fixing

acetic acid bacteria. International Journal of Food Mi-

crobiology, 125, 25-35.

doi:10.1016/j.ijfoodmicro.2007.11.079

[7] Luna, M.F., Galar, M.L., Aprea, J., Molinari M.L. and

Boiardi, J.L. (2010) Colonization of sorghum and wheat

by seed inoculation with Gluconacetobacter diazotro-

phicus. Biotechnology Letters, 32, 1071-1076.

doi:10.1007/s10529-010-0256-2

[8] Maheshkumar, K.S., Krishnaraj, P.U. and Alagwadi, A.R.

(1999) Mineral solubilising activity of Acetobacter di-

azotrophicus, a bacterium associated with sugarcane.

Current Science, 76, 874-875.

[9] Galar, M.L. and Boiardi, J.L. (1995) Evidence for a

membrane-bound pyrroloquinoline quinone-linked glu-

cose dehydrogenase in Acetobacter diazotrophicus. Ap-

plied Microbiology and Biotechnology, 43, 713-716.

doi:10.1007/BF00164778

[10] Luna, M.F., Bernardelli, C.E., Galar, M.L. and Boiardi,

J.L. (2006) Glucose metabolism in batch and continuous

cultures of Gluconacetobacter diazotrophicus PAL 3.

Current Microbiology, 52, 163-168.

doi:10.1007/s00284-005-4563-0

[11] Luna, M.F., Mignone, C.F. and Boiardi, J.L. (2000) The

carbon source influences the energetic efficiency of the

respiratory chain of N2-fixing Acetobacter diazotrophicus.

Applied Microbiology and Biotechnology, 54, 564-569.

doi:10.1007/s002530000425

[12] Stephan, M.P., Oliveira, M., Teixeira, K.R.S.,

Martínez-Drets, G. and Döbereiner, J. (1991) Physiology

and dinitrogen fixation of Acetobacter diazotrophicus.

FEMS Microbiology Letters, 77, 67-72.

doi:10.1111/j.1574-6968.1991.tb04323.x

[13] Nautiyal, C.S. (1999) An effcient microbiological growth

medium for screening phosphate solubilizing microor-

ganisms,” Microbiology Letters, 170, 265-270.

doi:10.1111/j.1574-6968.1999.tb13383.x

[14] Rodriguez, H., Gonzalez, T. and Selman, G. (2000) Ex-

pression of a mineral phosphate solubilizing gene from

Erwinia herbicola in two rhizobacterial strains. Journal

of Biotechnology, 84, 155-161.

doi:10.1016/S0168-1656(00)00347-3

[15] Clesscerl, L.S., Greenberg, A.E. and Eaton, A.D. (1998)

Standard methods for the examination of water and

wastewater. 20th Edition, APHA-AWWA-WEF, Wash-

ington, DC.

[16] Matsushita, K. and Ameyama, M. (1982) D-Glucose

dehydrogenase from Pseudomonas fluorescens, mem-

brane-bound. Methods in Enzymology, 89, 149-155.

doi:10.1016/S0076-6879(82)89026-5

[17] Matsushita, K., Shinagawa, E. and Ameyama, M. (1982)

D-gluconate dehydrogenase from bacteria, 2-keto-D-

gluconate yielding, membrane bound. Methods in Enzy-

mology, 89, 187-193.

doi:10.1016/S0076-6879(82)89033-2

[18] Fähraeus, G. (1957) The infection of clove root hairs by

nodule bacteria studied by simple glass slide technique.

Journal of General Microbiology, 16, 347-381

[19] Mehta, S. and Nautiyal, C.S. (2001) An efficient method

for qualitative screening of phosphate solubilizing bacte-

ria. Current Microbiology, 43, 51-55.

doi:10.1007/s002840010259

[20] Olsthoorn, A.J. and Duine, J.A. (1998) On the mecha-

nism and specificity of soluble, quinoprotein glucose

dehydrogenase in the oxidation of aldose sugars. Bio-

chemistry, 37, 13854-13861. doi:10.1021/bi9808868

[21] Attwood, M.M., van Dijken, J.P. and Pronk, J.T. (1991)

Glucose metabolism and gluconic acid production by

Acetobacter diazotrophicus. Journal of Fermentation and

Bioengineering, 72, 101-105.

doi:10.1016/0922-338X(91)90317-A

[22] Luna, M.F. and Boiardi, J.L. (2008) Growth yields and

glucose metabolism of N2-fixing Gluconacetobacter di-

azotrophicus at different culture pH values. World Jour-

nal of Microbiology and Biotechnology, 24, 587-590.

doi:10.1007/s11274-007-9507-3

[23] Russell, J.B., and Cook, G.M. (1995) Energetics of bac-

terial growth: balance of anabolic and catabolic reactions.

Microbiology and Molecular Biology Reviews, 59, 48-62.

[24] Luna, M.F., Bernardelli, C.E., Mignone, C.F. and Boiardi,

J.L. (2002) Energy generation by extracellular aldose

oxidation in N2-fixing Gluconacetobacter diazotrophicus.

Applied and Environmental Microbiology, 64, 2054-2056.

doi:10.1128/AEM.68.4.2054-2056.2002

[25] Lugtenberg, B.J.J. Kravchenko, L.V. and Simons, M.

(1999) Tomato seed and root exudate sugars: composi-

tion, utilization by Pseudomonas biocontrol strains and

role in rhizosphere colonization. Environmental Microbi-

ology, 1, 439-446.

doi:10.1046/j.1462-2920.1999.00054.x

[26] Wang, P., Bi, S., Wang, S. and Ding, Q. (2006) Variation

of wheat root exudates under aluminum stress. Journal of

Agricultural and Food Chemistry, 54, 10040-10046.

doi:10.1021/jf061249o