Influence of inceptisol and alfisol’s Potassium Solubilizing Bacteria (KSB) isolates on release of K from waste micaVetos 27-1-181-187 (2014)

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Influence of inceptisol and alfisol’s Potassium Solubilizing Bacteria
(KSB) isolates on release of K from waste mica
B. R. Maurya, Vijay Singh Meena* and O. P. Meena
Received: 22 June 2013 / Revised: 12 Sept 2013 / Accepted: 18 Jan 2014 / Published online: 30 April 2014
This article is published with open access at www.vegetosindia.org
Abstract Major part of potassium in soil exists
in form of insoluble K-minerals. Fraction of K-
solubilized from such minerals through potas-
sium solubilizing bacteria (KSB) will reduce
cost on import of K- fertilizers. Nine and four
isolates of K-solubilizing bacteria from In-
ceptisol (KI) and Alfisol (KA), respectively were
evaluated for their ability to release of K from
waste mica at 7, 14 and 21 days of incubation
in modified Aleksandrov medium containing
powdered waste mica as sole source of po-
tassium. Morphological characteristics, zone of
solubilization at week end and acidity of broth
at different incubation periods were also stud-
ied. Majority of the isolates were entire
smooth margin, raised, translucent, gram +ve
rods and whitish to creamy in appearance. Iso-
lates from cereals caused more zone of solu-
bilization than pulse isolates. Isolates either
from Alfisol or from Inceptisol efficiently de-
creased pH of the broth with increase in incu-
bation periods. Isolates of same soil type dif-
fered in their K-release capacity. KSB isolates
from Inceptisol showed higher K solubilization
potential than isolates from Alfisol. Isolate KI1
& KA19 caused maximum acidity but lowest
release of K from mica indicated that decrease
in pH of the medium is not the only mecha-
nism of K release from native K mineral of soil.
Isolate KI16 and KA59 were high slime producer
and showed highest 23.88 and 13.71 µg mL-1
K
solubilization capacity, respectively and
emerged out as potential isolates of K-
solubilizers as a K- biofertilizers.
Keywords: K-solubilizing bacteria (KSB), In-
ceptisol, Alfisol, waste mica, slime production,
K-solubilization
Introduction
Potassium is third of the primary plant
nutrient elements which involved in many
physiological processes including protein syn-
thesis, enzymes activation, photosynthesis,
development of resistance to diseases and
insects etc. (Anna and Francisco 2012). In soil,
K exists mainly in four different pools: mineral,
not exchangeable, exchangeable and labile K
(Romheld and Kirkby 2010). The concentration
of soluble K in soils is meagre and its major
portion (98%) exists as insoluble minerals
(Goldstein 1994). For optimum crop produc-
tion, soil solution and exchangeable K need to
be replenished continually with non-
exchangeable K through weathering of K
bearing minerals such as waste micas (Liu et
al. 2012, Sparks and Huang 1985) or by addi-
tion of K fertilizers.
In India the state of Jharkhand has the
richest mica deposit in the world. During the
dressing of mica sheet large amounts of mica
waste are generated which are dumped near
the mica mines and as such is not used in ag-
riculture. These are exposed to natural weath-
ering. They contain an appreciable amount of
potassium (8-10% K2O) and may be used as a
source of K-fertilizers, if modified by suitable
means. Management of waste mica through
composting along with crop residues inoculat-
ed with Bacillus mucilaginosus and Bacillus
edaphicus is an alternative viable technology
where signiﬁcant amount of insoluble K pre-
sent in waste mica can be mobilized into plant
available form of K and used as a source of
potassium in crop production, which could
help to reduce the reliance on costly chemical
fertilizers (Nishanth and Biswas 2008).
In contrast to K, the ratio of fertilizer
used to that removed by harvest for N and P is
usually much higher. Imbalance between K
and N is often exacerbated by the sole appli-
cation of N fertilizers. Presumably alluvial soils
are fairly rich in K but intensive cropping and
Department of Soil Science and Agricultural Chemistry, Institute of Agricultural Sciences, Banaras Hindu University,
Varanasi-221005 India
*Corresponding author E-mail vijayssac.bhu@gmail.com
Vol. 27 (1) : 181-187 (2014)VEGETOS

182
less application of potassic fertilizers have ini-
tiated deficiency of K in soil and necessitate
immediate K fertilization to check the aggra-
vation of K imbalances in soil. (Hasan 2002).
Certain rhizospheric bacteria have potentiality
to release the K from soil K-minerals and they
play a pivotal role in natural K cycle. Therefore,
inclusion of K solubilizing bacteria (KSB)
would be a promising approach for increasing
K availability to cope the K-demand of crops
(Vandevivere et al. 1994, Barker et al. 1998,
Archana et al. 2013). Present study deals with
isolation, morphological characterization and
mica K-release capacity of KSB isolates pro-
cured from rhizospheric soils of cereals and
legumes in Inceptisol and Alfisols to screen
out the most efficient KSB isolates for their
use as KSB inoculants.
Materials and Methods
Waste mica, a K-bearing mineral, was
obtained from surroundings of mica mines
located at Koderma district of Jharkhand, India
that generates during dressing of raw mica
blocks .It was ground in a Wiley mill to 2 mm
size. Ground mica was analyzed for its chemi-
cal characteristics (Table 1 and Figure 1) fol-
lowing the standard procedures.
The rhizospheric soils samples were
collected from Inceptisol of Agricultural Re-
search Farm, BHU (250
18’ N latitude, 830
03’ E
longitude and 128.93 m above MSL) 26 05’ N)
and Alfisol of Barkachha, Mirzapur (250
10 N
Fig 1. Waste mica used during the investigation (A) Before processing (B) Ground waste mica in Wiley mill
(Material collected from surroundings of mica mines located at Koderma district of Jharkhand, India).
A B
Fig 2. Effect of bacterial stains isolated from Inceptisol and Alfisol on zone of solubilization on Aleksandrov
media
B. R. Maurya et al.

183
latitude, 820
37’ E longitude and 427 m above
MSL). The soil samples were brought to labor-
atory in polythene bags. Each of the soil sam-
ples were divided in two parts. One part was
kept in refrigerator at 4o
C for microbiological
study and second part was processed for
chemical analysis. Soil samples were air-dried,
processed and sieved through 2-mm sieve.
Soil pH and electrical conductivity were meas-
ured in soil: water ratio of 1:2.5 (Jackson 1973).
Org. C, Available N, P and K were determined
by following the standard procedures as de-
scribed by Walkley and Black (1934), Subbiah
and Asija (1956), Watanabe and Olsen (1965)
and Hanway and Heidel (1952) , respectively.
pH , E C and Org. C of Inceptisol and Alfisol
were 7.94 & 6.49; 0.16 & 0.18 dSm-1
and 4.1 &
4.2 mg kg-1
soil, respectively. Available N, P &
K of these soils were being 125 &100, 16 &12,
156 &113 kg ha-1
, respectively.
The serially diluted soil samples were
plated on Aleksandrov medium containing
(per L) 5 g glucose, 0.005 g MgSO4.7H2O, 0.1 g
Fe Cl3, 2.0g Ca CO3, 3.0 Mica as a potassium
mineral (2.0 g in original media), 2.0 g calci-
um phosphate and 20 g agar- agar Aleksan-
drov broth (Sugumaran and Janartham 2007)
to isolate the potassium solubilizing bacteria.
The plates were incubated at 28±2 ºC. After 3
days, colonies showing clear zone of for-
mation around their colonies were considered
to be the potassium solubilizing bacteria and
selected for further studies. Zone of solubiliza-
tion of purified K- isolates was measured us-
ing scale at 7 days of plating on Aleksandrov
medium. Bacterial isolates of K- solubilizers
were studied for their morphological charac-
teristics following standard techniques (Holt et
al. 1994).
A total of 13 isolates showing zone of
solubilization on Aleksandrov agar were iso-
lated and their ability to release of K from
waste mica in broth culture were studied. Two
mL of overnight broth culture of each isolate
was poured to 50 mL of Aleksandrov broth
containing 0.3 g powdered waste mica in
three replicates. All the inoculated flasks were
incubated for 7, 14 and 21 days in BOD at
28±20
C temperature. The broth cultures were
centrifuged at 10,000 rpm for 10 minutes in
Figs 3-5. Effect of K-solubilizers isolated from Inceptisol on pH reaction of Aleksandrov broth and release of
potassium from waste mica at different days of inoculation (7, 14 and 21 DAI, respectively) initial pH of
broth was 7.60.
Figs 6-8 Effect of K-solubilizers isolated from Alfisol on pH reaction of Aleksandrov broth and release of
potassium from waste mica at different days of inoculation (7, 14 and 21 DAI, respectively) initial pH of
broth was 7.60.
3 4 5
6 7 8
Influence of Inceptisol and Alfisol’s Potassium Solubilizing Bacteria (KSB)

184
the REMI-centrifuge machine to separate the
supernatant from the cell growth and insolu-
ble waste mica. Potassium content in the su-
pernatant solution of culture was determined
by flame photometry and compared with un-
inoculated control (Sugumaran and Janar-
thanam 2007). pH of the broth culture was
measured using the pH meter. After determi-
nation of K, electrical conductivity of centri-
fuged microbial culture suspension was esti-
mated by using the digital EC meter. Influence
of purified KSB isolates on pH, EC and K- solu-
bilizing capacity was studied at 7, 14 and 21
days after incubation. Data generated from
the laboratory experiments were subjected
to the statistical analyses of variance ap-
propriate to the experimental design. Sta-
tistical analysis of the data was carried out us-
ing Analysis of Variance (ANOVA) technique.
Results and Discussion
All the isolates produced slime but
magnitude of its production varied from low
to high depending upon the capacity of the
isolates (Table 2). This observation is in con-
formity to the findings of Sugumaran and Ja-
narthanam (2007) who have also reported the
production of slime by KSB isolates. KI11, KI16,
KI55, KA32 and KA59 were noted as high slime
producers. Out of 13 isolates, 8 isolates
showed entire smooth margin, 4 isolates were
rough and only one isolate had undulated
margin. KI12, KI16, KI30, KI38, KA19, KA32 and KA51
showed slightly elevated colonies while colo-
nies of KI1, KI11, KI13, KI41, KI55 and KA59 were
highly raised. On Aleksandrov agar medium,
colony formed by most of the isolates ap-
peared to be translucent except colony of KI11,
KI12, KI13, KA19 and KA59 which appeared to be
opaque. All the isolates were gram +ve rods.
Colonies of most of the isolates were whitish
to creamy in appearance except the colonies
of KI1 which was grayish and whitish in color,
respectively (Archana et al. 2012). The diame-
ter of zone of solubilization caused by the iso-
lates at seven days of growth ranged from 0.7
to 1.4 cm (Fig 2). Data recorded on zone of
solubilization revealed that KI11 and KA51 which
were procured from rhizosphere of Triticum
aestivum and Zea mays formed highest zone
of solubilization compared to other isolates.
Interestingly isolates from cereals caused
much greater zone of solubilization in com-
parison to isolates of arhar.
Initial pH of uninoculated mica added
broth was 7.6 which did not influence much by
incubation period. However, slight change in
pH values with incubation period was ob-
served. This may be due to production of H+
during the hydrolysis of added waste mica.
Binbin and Bin (2011) have also reported very
minute change in pH of mineral added broth
with increase in incubation periods. pH value
of inoculated broth supplemented with waste
Figs 9-10. Effect of K-solubilizers isolated from Alfisol and Inceptisol on electrical conductivity at different
days of inoculation, respectively.
Table 1. Elemental composition of waste mica
Silica Iron Potash Magnesium Sodium Oxide
Manganese
oxide
Phosphorus
Sl2O3 (%) Fe2O3 (%) K (%) MgO (%) Na2O (%) MnO (%) P (%)
45.10 2.54 9.82 0.61 0.37 Traces 0.022
9 10
B. R. Maurya et al.

185
mica decreased significantly by all stains with
increase in incubation period (Figs 3-8). At 14
and 21 DAI, all the isolates caused significant
decrease in pH of broth as compared to unin-
oculated mica broth. Obvious difference in
lowering of pH was recorded among the K-
solubilizers .Reduction in pH may be due to
production of different kind of organic acids
by K-solubilizers. This statement is in accord-
ance to the findings of (Girgis et al. 2008) who
have reported that K and P-solubilizers pro-
duce mono, di and tri organic acids i.e., glu-
conic, acetic, oxalic, fumaric, tartaric and citric,
which result in lowering the pH. Lowest pH
value 4.46 was recorded at 21 DAI with isolate
KI1 & KA19 which were isolated from Inceptisol
and Alfisol, respectively (Figs 3-8).
Data on EC of broth supplemented
with mined mica significantly influenced by
inoculants, incubation period and their inter-
action (Figs 9-10). All the isolates, except KI1
and KI16 caused significant increase in EC of
broth in comparison to un-inoculated control.
The KSB isolates might had produced several
kind of organic acids which broken down the
mica structure to satisfy their Si+4
and K+
re-
quirements and brought them in to solution
consequently EC of the inoculated broth was
increased (Han et al. 2006; Parmar and Sindhu
2013). It is worth to note that isolate KI1
caused highest acidity to the medium but ex-
hibited lowest EC. This indicated that besides
lowering of pH, other mechanism is also oper-
ative to solubilise the mineral K. Insignificant
change in EC in control at 21 days of incuba-
tion was in conformity to the finding of Gold-
stein (1986), Sheng and He (2006). At 21 DAI,
highest value of EC 2.44 and 2.14 dSm-1
with
KI38 and KA51 under Inceptisol and Alfisol, re-
spectively recorded (Figs 9-10).
Very less content of K in uninoculated
mica broth was might be due the structural
disturbance in mica caused by hydrolysis
which resulted to release of K in broth (Zhao
et al. 2008, Liu et al. 2012). Release of potassi-
um from insoluble waste mica was significant-
ly influenced by inoculants, incubation periods
and their interactions. Quantity of K solubiliza-
tion tended to increase with incubation period
(Figs 3-8). All the isolates of Inceptisol exhibit-
ed significantly more K release in comparison
to un-inoculated broth but they varied in their
capacity to release K from the mica. Release of
K may be due to the production of different
kind of organic acids by the isolates. This fact
is in agreement with findings of Prajapati and
Modi (2012), Chishi (2010) who have also re-
ported that K solubilizers produce organic ac-
ids and cause reduction in pH. No any definite
relation in pH of broth and release of K by the
isolates was observed. Release of K was great-
ly influenced by the incubation period than
isolates. Besides this, organic acids produced
by the KSB isolates also influence forming
Table 2. Colony characteristics of K-solubilizing bacterial isolates on Aleksandrov medium
Isolates Crop Color Margin
Colony elevation Optical Density
Slime produc-
tionSlightly
raised
Highly
raised
Trans-
lucent
Opaq
ue
Inceptisol Isolates
KI1 Maize Grayish Smooth _ + + _ Medium
KI11 Wheat White Rough _ + _ + High
KI12 Sorghum Creamy Rough + _ _ + Low
KI13 Sorghum Whitish Rough _ + _ + Low
KI16 Wheat White Smooth + _ + _ High
KI30 Maize Creamy Smooth + _ + _ Medium
KI38 Wheat White Smooth + _ + _ Low
KI41 Arhar Creamy Smooth _ + + _ Low
KI55 Arhar White Smooth _ + + _ High
Alfisols isolates
KA19 Maize Creamy Undulated + _ _ + Medium
KA32 Maize White Smooth + _ + _ High
KA51 Maize White Smooth + _ + _ Medium
KA59 Maize Creamy Rough _ + _ + High
(K= K-solubilizing bacteria, I= Inceptisol, A= Alfisol)

186
frame work-destabilizing surface complexes or
by complexing metals in solution (Stillings et
al., 1996). Maximum K solubilization capacity
23.88 and 13.71 µg K mL-1
was recorded with
KI16 and KA59 which were significantly superior
to other isolates of their particular soil type
from where they were isolated.
In vitro K solubilization of K ranged
from 2.58 to 33.97µg mL-1
and 2.58 to 17.89
µg mL-1
due to isolates of Inceptisol and Al-
fisols, respectively. Isolate, KA19 showed signif-
icantly more mica K solubilization capacity as
compared to KA32 (Figs 2-7). The pH of mica
broth with KI1 and KI16 though did not differ
much but greatly varied in their K- release ca-
pacity. KI1 and KI16 showed minimum and
maximum release of K from waste mica, re-
spectively. Similar results also reported by
Prajapati et al. (2012); Sheng et al. (2008). This
indicated that isolates procured from same
type of soil vary in their capacity of K-
solubilization from mica. Similar observations
were also recorded with Alfisol isolates KA19
and KA59. It is worth to note that KI16 and KA59
are high slime producers.
Conclusion
Nine potassium solubilizing bacterial
isolates from Inceptisol (KI) and four isolates
from Alfisol (KA) exhibited production of
slime.All the isolates were gram +ve
rods .Isolates from cereals caused more zone
of solubilization than pulse isolates. Isolates
from Inceptisol had greater potential of K sol-
ubilization than isolates of Alfisol. Highest
acidity but lowest solubilization of K of mica
with KI1 and KA19 indicated that decrease in
pH of the medium is not only the mechanism
of K release from native K mineral of soil.
Slime production by the K solubilizers may
also play a role in mineral K solubilization.
However, the actual mechanism of K release
by KSB is not clear and needs further investi-
gation. Isolate KI16 and KA59 were high slime
producers which emerged out as potential
isolate of K- solubilizers.
Acknowledgements
The authors are thankful to the Head, Depart-
ment of Soil Science and Agricultural Chemis-
try, Institute of Agricultural Sciences, BHU, Va-
ranasi, for providing the necessary facilities to
conduct this research work.
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