Chemosphere 66 (2007) 17941798

www.elsevier.com/locate/chemosphere

Technical Note

Solubilization of zinc compounds by the diazotrophic, plant growth

promoting bacterium Gluconacetobacter diazotrophicus
a,b,*
V.S. Saravanan , M. Madhaiyan b, M. Thangaraju c

a
School of Bioengineering and Biosciences, Vellore Institute of Technology (VIT), Vellore 632 014, Tamil Nadu, India
b
Department of Agricultural Chemistry, Chungbuk National University, Cheongju, Chungbuk 361-763, Republic of Korea
c
Department of Agricultural Microbiology, Tamil Nadu Agricultural University, Coimbatore 641003, Tamil Nadu, India

Received 31 December 2005; received in revised form 21 July 2006; accepted 23 July 2006
Available online 7 September 2006

Abstract

Gluconacetobacter diazotrophicus an endophytic diazotroph also encountered as rhizosphere bacterium is reported to possess dierent
plant growth promoting characteristics. In this study, we assessed the zinc solubilizing potential of G. diazotrophicus under in vitro con-
ditions with dierent Zn compounds using glucose or sucrose as carbon sources. G. diazotrophicus showed variations in their solubili-
zation potential with the strains used and the Zn compounds tested. G. diazotrophicus PAl5 eciently solubilized the Zn compounds
tested and ZnO was eectively solubilized than ZnCO3 or Zn3(PO4)2. The soluble Zn concentration was determined in the culture super-
natant through Atomic Absorption Spectrophotometer. Gas chromatography coupled Mass Spectrometry analysis revealed 5-ketoglu-
conic acid, a derivative of gluconic acid as the major organic acid produced by G. diazotrophicus PAl5 cultured with glucose as carbon
source. This organic anion may be an important agent that helped in the solubilization of insoluble Zn compounds.
! 2006 Elsevier Ltd. All rights reserved.

Keywords: Gluconacetobacter diazotrophicus; Zinc solubilization; Zinc oxide; Atomic absorption spectrophotometer; 5-Ketogluconic acid; Phytoreme-
diation

1. Introduction Zn solubilization by microorganisms in this context proves

Zinc is an essential micronutrient for plant growth pro-
motion. It is a vital constituent of various metabolic
enzymes and its poor mobility in plants suggests the need
for a constant supply of available zinc for optimum
growth. Zn supplied to plants in the form of fertilizers like
zinc sulfate which inturn transformed into dierent insolu-
ble forms depending upon the soil types, soil chemical reac-
tions and totally unavailable in the environment within
seven days of application (Rattan and Shukla, 1991). Apart
from phosphorus, micronutrients like Zn, Fe and Mn were
found to be deficient in most of the soils with Zn as a fore-
most nutrient throughout the world (Alloway, 2004). So
*
Corresponding author. Address: Department of Agricultural Chemis-
try, Chungbuk National University, Cheongju, Chungbuk 361-763,
Republic of Korea. Tel.: +82 43 2612561; fax: +82 43 2715921.
E-mail address: saraphd2003@yahoo.com (V.S. Saravanan).
to be beneficial and economical. Heterotrophic Zn solubi-
lization has been widely studied in fungi (White et al.,
1997) and few studies have also documented solubilization
of insoluble Zn compounds by bacteria (Di Simine et al.,
1998; Fasim et al., 2002).
Gluconacetobacter diazotrophicus originally isolated from
sugarcane (Cavalcante and Dobereiner, 1988) was consid-
ered to be an endophytic diazotroph. Since then reports on
G. diazotrophicus from the rhizosphere soils of sugarcane,
coee, ragi and rice reveals it as a prominent rhizosphere
dwelling organism (Li and Mac Rae, 1991; Jimenez-Salgado
et al., 1997; Loganathan et al., 1999; Muthukumarasamy
et al., 2005). Moreover, the detection of Gluconacetobacter
azotocaptans, a close relative of G. diazotrophicus only in
the rhizosphere soil of corn strongly suggests its occurrence
and survival in soil (Mehnaz et al., in press). Production of
plant growth hormones indole-3-acetic acid (IAA) and
gibberellins (Bastian et al., 1998), in vitro phosphate

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doi:10.1016/j.chemosphere.2006.07.067
 V.S. Saravanan et al. / Chemosphere 66 (2007) 17941798
solubilization (Madhaiyan et al., 2004; Madhaiyan et al.,
2006), and biocontrol of Xanthomonas albilineans a sugar-
cane pathogen (Pinon et al., 2002) by G. diazotrophicus have
also been reported. In this study we report in vitro solubiliza-
tion of Zn compounds by G. diazotrophicus and the produc-
tion of 5-ketogluconic acid a major gluconic acid derivative
product that aid in the solubilization process.
2. Materials and methods
2.1. Bacterial strains and culture conditions
G. diazotrophicus PAl5 was kindly provided by Dobere-
iner (EMBRAPA, Brazil) and the strains R10 and L3 were
from Culture Collection of the Centre of Advanced Stud-
ies in Agricultural Microbiology, Tamil Nadu Agricultural
University, Coimbatore, India. The G. diazotrophicus
strains were cultured in LGI medium (g l!1 composition:
sucrose 100, K2HPO4 0.2, KH2PO4 0.6, MgSO4. 7H2O
0.2, CaCl2. 2H2O 0.02, Na2MoO4. 2H2O 0.002, FeCl3.
6H2O 0.018, bromothymol blue, 0.5% in 0.2 M KOH
5 ml l!1, pH 5.5) routinely (Cavalcante and Dobereiner,
1988). Glucose and sucrose at 10% concentration were used
as carbon sources to access the solubilization potential of
the zinc compounds. Zinc oxide at 0.12%, 0.24%, zinc car-
bonate or zinc phosphate (Loba Chemie, Mumbai, India)
at 0.21%, 0.42% were added to the media to get a final con-
centration of zinc at 0.1% and 0.2%.
2.2. In vitro zinc solubilization assay
G. diazotrophicus strains grown in LGI broth for 48 h
(6 108 cfu ml!1) were spotted at 10 ll volume on to plates
containing LGI medium supplemented with insoluble Zn
compounds. The plates were incubated at 28 1 "C for
3 d and the solubilization halo was observed against an
opaque background. The soluble Zn present in the culture
broth was determined using Atomic Absorption Spectro-
photometer (AAS-Model Varian C) at 24 and 48 h of incu-
bation. For the broth assay, G. diazotrophicus PAl5 was
grown in 100 ml LGI broth with insoluble zinc sources at
0.1% concentration at 28 "C in a controlled environment
incubator shaker (model: G27, New Brunswick, USA) at
120 rpm. G. diazotrophicus PAl5 originally cultured in
LGI broth for 24 h was used as mother inoculum. One
ml sample diluted to 50 ml with sterile distilled water was
filtered through 0.4 lm filter and directly fed into AAS
and the available zinc cation was recorded. The pH of
the broth was also determined simultaneously. Three repli-
cations were maintained for each treatment and uninocu-
lated media with insoluble Zn sources served as control.
2.3. Organic acid analysis by GC-MS
The presence of dominant organic acids in the sample
was identified by the Gas Chromatography coupled Mass
Spectrometry (GC-MS) analysis equipped with Cbp 5 col-
1795
umn (25 m, 0.5 mm ID) with helium as carrier gas (Model
QP 7000, Shimadzu, Japan) following the procedure by
Fasim et al. (2002). First the sample was freeze dried in
lyophilizer (Christ LOC-1M, Germany) to concentrate
acids and dissolved in 500 ll methanol. To produce trimeth-
ylsilyl derivatives, methanol extracts of lyophilized samples
flushed with nitrogen and 100 ll of N-Methyl-N-(trimethyl-
silyl) trifluoroacetamide (Sigma Chemical Co., St. Louis,
Missouri, USA) and 100 ll of pyridine were added and
again nitrogen was flushed. The reactions were carried out
in sealed septum vials, heated gently in a water bath at
60 "C for 30 min and left overnight for stabilization. The
samples were analyzed in GC-MS with a source tempera-
ture of 200 "C and ionizing voltage of 70 eV and operated
in a scan mode (50700 m/z) using a temperature gradient
of 70260 "C and compared with known standards.
2.4. Statistical analysis
The data were subjected to statistical analysis and signif-
icant dierences were calculated at P 6 0.05 using SAS
package, Version 9.1 (SAS, 2004).
3. Results and discussion
Preliminary plate assays were conducted to assess the Zn
solubilization potential of G. diazotrophicus strains supple-
menting with various Zn compounds at 0.1 and 0.2% Zn
concentrations in LGI medium containing either glucose
or sucrose as carbon sources. The diameter of solubiliza-
tion halo produced by G. diazotrophicus strains with glu-
cose as carbon source at 0.1% insoluble Zn is presented
in Fig. 1. G. diazotrophicus strains PAl5 and L3 showed
greater diameter of solubilization with the Zn compounds
tested under both carbon sources at 0.1 and 0.2% Zn con-
centrations. Strain R10 showed no solubilization zones
when sucrose was used and comparatively less solubili-
zation with glucose than the other two strains. Also no
solubilization zone was observed with R10 when Zn was
supplemented in the medium at 0.2% concentration
(Table 1). Our results revealed that the eciency to solubi-
lize insoluble Zn compounds varied between the strains
and the carbon source provided. Similar results were
observed with Pseudomonas aeruginosa that showed higher
Fig. 1. Solubilization halos produced by G. diazotrophicus strains (R10,
PA15 and L3) on LGI medium containing 10 g l!1 glucose supplemented
with 0.1% (w/v) zinc sources (A) ZnO, (B) ZnCO3, and (C) Zn3(PO4)2.
1796 V.S. Saravanan et al. / Chemosphere 66 (2007) 17941798

Table 1
Solubilization of insoluble zinc compounds (per cent) in solid medium by G. diazotrophicus with dierent carbon sources
Treatment* Solubilization zone of dierent zinc compounds (mm)
ZnO ZnCO3 Zn3(PO4)2
0.10 0.20 0.10 0.20 0.10 0.20
L3 + sucrose 28 1.73 c 24 1.73 a 38 1.73 a 20 1.15 c 21 0.58 c 14 1.15 c
R10 + sucrose
PAl5 + sucrose 30 1.15 cb 23 1.73 a 40 1.15 a 28 1.15 a 23 1.15 c 12 0.58 d
L3 + glucose 32 1.73 b 14 1.73 b 20 0.58 c 23 1.15 b 30 2.31 b 27 1.15 a
R10 + glucose 0.5 0.06 d 0.3 0.06 d 0.3 0.06 d
PAl5 + glucose 36 2.31 a 15 0.58 b 25 0.58 b 23 1.73 b 35 2.31 a 25 1.15 b
LSD (P 6 0.05) 3.00 2.74 2.09 2.23 3.30 1.79
*
The G. diazotrophicus strains PAl5, L3 and R10 were grown in LGI agar medium containing 10% sucrose or 10% glucose as the carbon source. Each
value represents a mean SE of three replicates per treatment. In the same column, significant dierences according to (least significant dierence) LSD at
P 6 0.05 levels are indicated by dierent letters no solubilization zone observed.

Table 2
Total soluble zinc estimated in LGI broth on G. diazotrophicus strain PAl5 inoculation
Zinc source at 0.1 per cent Zinc solubilized (mg l!1)
Uninoculated Inoculated
24 h 48 h 24 h 48 h
ZnO 10.0 0.94 a 10.0 1.41 a 402.0 5.66 a 420.0 7.20 a
ZnCO3 10.0 0.47 a 10.0 0.47 a 151.0 5.19 b 157.0 5.66 b
Zn3(PO4)2 5.0 0.47 b 5.0 0.94 b 152.0 6.13 b 300.0 8.49 b
LSD (P 6 0.05) 3.46 2.27 32.09 9.44
Each value represents mean SE of three replicates per treatment. In the same column, significant dierences according to LSD at P 6 0.05 levels are
indicated by dierent letters.

Fig. 2. GC spectrum of culture supernatants (a) G. diazotrophicus unamended (b) G. diazotrophicus amended with ZnO. Peaks identified as 5-ketogluconic
acid and pentanoic acids (ribitol pentanoic and erythro pentanoic acid) are indicated. 5-ketogluconic acids represented 32 and 34% (a) and 24 and 31% (b)
of total peak area of the analysed components; (c and d) comparative ion fragmentation pattern of 17.2 and 17.5 min peak of 5-ketogluconic acid from a
sample and (e) standard peak. 5-KGA 5-ketogluconic acid.

solubilization of ZnO than Zn3(PO4)2 (Fasim et al., 2002). for Zn solubilization by plate assays has revealed strain
Also previous study with G. diazotrophicus strains tested level dierences (Madhaiyan et al., 2004).
 V.S. Saravanan et al. / Chemosphere 66 (2007) 17941798
Screened by the plate assay, G. diazotrophicus PAl5 with
glucose as carbon source supplemented with Zn com-
pounds at 0.1% Zn concentration was selected for broth
assay to measure the soluble Zn concentrations at dierent
growth periods. The amount of total soluble Zn present in
the culture supernatants as measured by AAS at 24 and
48 h of growth is presented in Table 2. G. diazotrophicus
PAl5 was able to solubilize the insoluble Zn compounds
added to the media and produced 420, 300, 157 mg l!1
Zn from ZnO, Zn3(PO4)2 and ZnCO3 at 48 h respectively
with 42, 30 and 16% solubilization. According to European
Commission norms, the critical limit for toxicity is
300 mg kg!1 of soil, whereas deficiency occurs when the
soil available zinc concentration is below 2.0 mg kg!1 of
soil (Alloway, 2004). The huge quantity of solubilized Zn
(420 mg l!1) observed with ZnO treatment might be attrib-
uted to the use of 10% glucose as carbon source. However,
it may not superimpose under field conditions, since solubi-
lization in rhizosphere directly depends on the carbon exu-
dation from the crop plants and microbial activity
(Grayston et al., 1997). The pH in the zinc phosphate treat-
ment decreased from 5.8 to 4.5. No decrease in pH was
observed in both ZnO and ZnCO3 amended medium dur-
ing the study period. This might be due to the intrinsic buf-
fering potential of the Zn compounds as demonstrated by
Franz et al. (1991) with Penicillium simplicissimum. Also,
ZnO may act as an excellent buer consuming two mol
of protons per mol for solubilization.
The presence of 5-ketogluconic acid was detected by
GC-MS analysis in the Zn amended culture broth as well
as in the control. The peaks 2 and 3 were prominently iden-
tified as 5-ketogluconic acid based on the retention time
(17 min) and by referring to the standard in the GC-MS
software library data (Fig. 2). 5-ketogluconic acid was
found to be dominant acid with higher concentrations
(based on the peak area) in the control compared to zinc
amended sample. The peaks 4 and 5 were identified as ribi-
tol pentanoic acid and erythro pentanoic acid (Fig. 2a and
b). Production of 5-ketogluconic acid by G. diazotrophicus
was noticed both under Zn unamended and amended con-
ditions, it may have eected the solubilization process.
Ketogluconates mediated Zn solubilization was already
reported in Pseudomonas fluorescens (Di Simine et al.,
1998) and P. aeruginosa (Fasim et al., 2002).
The ability of G. diazotrophicus to solubilize Zn has the
ecological advantage of making the cation available for the
plant in zinc deficient soils as well as in microbe mediated
phytoextraction or phytoremediation of metal contami-
nated soils. However, further pot culture and field studies
are necessary to confirm the Zn solubilization potential
to assess its practical feasibility.
4. Summary
The experiments performed in vitro showed that G. dia-
zotrophicus an agriculturally important bioinoculant solu-
bilized the insoluble Zn compounds [ZnO, ZnCO3 and
1797
Zn3(PO4)2] with strain level variations. Glucose was the
preferred carbon source over sucrose and higher solubiliza-
tion resulted at 0.1% insoluble Zn compound amendment.
The organic anion 5-ketogluconic acid identified may be
one of the agents that aided solubilization.
Acknowledgements
The authors are grateful to Jesus Munoz-Rojas, Uni-
versidad Nacional Autonoma de Mexico, Mexico and S.
Poonguzhali, Chungbuk National University, Republic of
Korea for their valuable suggestions in manuscript prepa-
ration. University Grants Commission, India is gratefully
acknowledged for their financial support.
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