Isolation and characterization of potassium solubilizing bacteria from ceramic industry soil

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CIBTech Journal of Microbiology ISSN: 2319-3867 (Online)
An Online International Journal Available at http://www.cibtech.org/cjm.htm
2012 Vol. 1 (2-3) Jul.-Sept. & Oct.-Dec., pp.8-14/Prajapati and Modi
Research Article
8
ISOLATION AND CHARACTERIZATION OF POTASSIUM
SOLUBILIZING BACTERIA FROM CERAMIC INDUSTRY SOIL
*K.B. Prajapati1 and H.A. Modi2
1Department of Biotechnology, Kadi Sarva Vishwavidyalaya,Gandhinagar
2Life Science Department, School of Sciences, Gujarat University,Ahmedabad
*Author for Correspondence
ABSTRACT
Potassium is vital component of plant nutrition package limiting crop yield and Quality that performs a
multitude of important biological functions to maintain plant growth, Isolation of potassium solubillizers
was carried out using feldspar (insoluble potassium) from the soil samples of ceramic industries, on
Aleksandrow’s agar medium. From the 14 isolated bacterial isolates 5 bacterial strains were selected
which exhibiting highest potassium solubilization on solid medium and characterized on the basis of
cultural, morphological and biochemical characteristics. Solubilization of potassium from the potassium
aluminium silicate minerals by the selected bacterial strains resulted to the action of different organic
acids like Citric, Oxalic, Malic, succinic and Tartric acid.
Key Words: Nutrition, Multitude, Solubilization, Feldspar,
INTRODUCTION
Among the Nitrogen (N), Phosphorus (P) and Potassium (K), Potassium is the third important plant
nutrient. Potassium is essential macronutrient for plant growth and plays significant roles in activation of
several metabolic processes including Protein synthesis, Photosynthesis, Enzymes, as well as in
Resistance to diseases and insects etc (Rehm and Schmitt, 2002). Potassium though present in as
abundant element in soil or is applied to fields as natural or synthetic fertilizers, only one to two percent
of this is available to plants, the rest being bound with other minerals and therefore unavailable to plants.
The most common soil components of potassium, 90 to 98%, are feldspar and mica (McAfee, 2008). Soil
microorganisms influence the availability of soil minerals, playing a central role in ion cycling and soil
fertility (Bin Lian et al., 2010). Very little of this potassium source is available for plant use. Silicate
bacteria were found to resolve potassium, silicon and aluminum from insoluble minerals (Alexander,
1985).Their uses as biofertilizers or biontrol agents for agriculture improvement and environmental
protection have been a focus of recent research.
Certain bacteria are capable of decomposing alumino silicate minerals and releasing a portion of the
potassium contained therein (Biswas and Basak, 2009). A detailed understanding of how bacteria affect
mineral dissolution rates is essential to quantify mineral weathering on global element cycling (Xiufang,
Hu Jishuang and Jiangfeng, 2006).
MATERIALS AND METHODS
Sample Collection
Majorly the ceramic industries are using insoluble source of potassium i.e. feldspar as their raw material
so Samples were collected from the various areas of insoluble potassium mineral containing soils from
the ceramic industries nearby Kadi, Kalol and Himmatnagar region.
Adaptation and Enrichment
Collected soil samples were mixed with insoluble potassium (Feldspar) and incubated for 1 week at room
temperature. After adaptation 1 gm of soil was inoculated in 100 ml liquid medium containing 1%
glucose, 0.05%yeast extract and 0.5% feldspar and incubated at 370 C on 120 rpm for 1 week.
Isolation and Screening of Potassium Solubilizing Microbes

CIBTech Journal of Microbiology ISSN: 2319-3867 (Online)
An Online International Journal Available at http://www.cibtech.org/cjm.htm
2012 Vol. 1 (2-3) Jul.-Sept. & Oct.-Dec., pp.8-14/Prajapati and Modi
Research Article
9
Enriched samples were inoculated after serial dilution up to 10-6 on Aleksandrov agar medium constituted
1% glucose, 0.05% MgSo4.7H2O, 0.0005% FeCl3, 0.01% CaCO3, 0.2% CaPo4 and 0.5% potassium
aluminium silicate, agar 3 % pH-6.5(Sugumaran and Janartham, 2007) and incubated at 37 0 C for 1 week
and incubated at 370c for 1 week. Colonies exhibiting clear zone of potassium solubilization were selected
as potassium solubilizers from the 10-4 , 10-5and 10-6 dilutions containing paltes. Secondary Screening was
carried out on the basis of study of zone activity of the different isolates by using Khandeparkar’s
selection ratio. Ratio = D/d = Diameter of zone of clearance / Diameter of growth
Characterization of Potassium Solubilizing Bacterial Isolates
Bacterial isolates were characterized using different cultural, microscopical and biochemical
characteristics. In addition to other studies including various enzymes production, Antibiogram pattern,
pH optima, temperature optima, growth at different NaCl concentration, different organic acids
productions (Osman, 2009).
Cultural, Morphological and Biochemical Characteristics of Bacterial Strains
The isolates were cultured on Aleksandrov’S agar medium. Colony Characteristics such as size, shape,
texture, consistency and transparency were examined. Gram staining,endospore staining and capsule
staining were carried out for the isolates. Voges-proskauer, Catalase test,Ornithine utilization, Lysine
utilization, H2S production ,Nitrate reduction, Phenylalanine deamination test were performed
(Cappuccino, 1998).Production of acids from various carbohydrates and enzymatic activities were
determined as per procedure outlined by cappuccino and Sherman. Various carbohydrate utilization were
also studied (John, Noel, Peter, James and Stanley, 1997).
Antibiotic sensitivity test.
Antibiotic sensitivity tests were performed according to the Kirby – Bauer method (Prescott, Harley and
Kelin, 2002).
Effect of pH on Growth of K solubilizers
The effects of pH, was studied by using Nutrient broth medium and pH adjusted to the required level by
0.1 M HCl and NaOH. Medium was inoculated with 1 loopful of previously activated bacterial culture in
10 ml N.broth medium and incubated at 370 C for 48 hours. Then the growth was checked visually.
Effect of Temperatures on Growth of K solubilizers
The effects of temperatures was studied by using Nutrient broth medium. Medium was inoculated with 1
loopful of previously activated bacterial culture in 10 ml N.broth medium and incubated at different
Temperatures for 48 hours. Then the growth was checked visually.
Effect of NaCl concentration on Growth of K solubilizers
The effects of NaCl concentration was studied by using N.broth medium with different NaCl
concentration. Medium was inoculated with 1 loopful of previously activated bacterial culture in 10 ml
N.broth medium and incubated at 370 C for 48 hours. Then the growth was checked visually.
Detection of Organic acids produced by bacterial isolates
Organic acids identification was performed by using solvent system n- butanol: Acetic acid: Water (4:1:5)
and Developing Reagent 0.04% Bromocresol Green in Alcohol from the 1/10 th volume concentrated
supernatant of Aleksandrov broth culture incubated at 30 0 C for 10 days (Isherwood and Hanes, 1953).
Potassium solubilisation and identification of best solubilizer
The all five isolates were grown on K enriched 100 ml broth containing 1% Glucose, 0.05% yeast extract,
0.5% feldspar, pH-6.5 and incubated on shaking condition at 300 C on 120 rpm for 7 days by using two
insoluble source of potassium feldspar. After each 24 hours the potassium released were determined by
using Sodium cobaltinitrite and Folin-Ciocalteu Phenol reagent (Abul Fadl, 1948). The higher potassium
solubilizing strain was identified using standard cultural, morphological and biochemical methodology,
but its identity was re-evaluated by 16S rRNA gene sequence analysis.

CIBTech Journal of Microbiology ISSN: 2319-3867 (Online)
An Online International Journal Available at http://www.cibtech.org/cjm.htm
2012 Vol. 1 (2-3) Jul.-Sept. & Oct.-Dec., pp.8-14/Prajapati and Modi
Research Article
10
RESULTS AND DISCUSSION:
Isolation and screening
Colonies exhibiting zone of clearance indicating Potassium solubilization were selected.
The colonies were selected which were morphologically distinct. Total 14 bacterial isolates were isolated
as potassium solubilizers and named as KSB1 to KSB14.(Table-1).
Table-1: Potassium solubilization values of bacterial isolates by Khandeparkar’s selection ratio.
Isolates
Diameter of zone of clearance
(D) mm
Diameter of growth
(d) mm
D/d (ratio)
KSB1
7
6
1.66
KSB2
8.5
7
1.21
KSB3
10
7.5
1.33
KSB4
7.5
6
1.25
KSB5
6
5
1.20
KSB6
12
10
1.20
KSB7
13
9
1.44
KSB8
11
7
1.57
KSB9
8.5
7
1.21
KSB10
6.5
5
1.3
KSB11
9
7
1.28
KSB12
10.5
9
1.16
KSB13
7.2
6.9
1.04
KSB14
6.5
6.1
1.06
From that isolates five bacterial KSB 1, KSB 3 KSB 7 , KSB 8, and KSB 11 isolates exhibiting higher
ratio of clear zone of potassium solubilization by Khandeparkar’s selection ratio were selected.
Cultural, Microscopical and Biochemical Characteristics
The morphological and the Biochemical characteristics of the 5 good Potassium solubilizers are presented
in Tables 2. Most of these isolates are aerobic, capsulated, motile and 2 of them are Gram positive while
3 are gram negative. The biochemical characters are studied for the five isolates of potassium solubilizing
bacteria presented in (Table-2).
Table-2: Colonical, Morphological And biochemical characteristics of best potassium solubilizing
bacterial isolates.
Isolates
Colony characters
Morphological characters
Gram
reaction
& cell
shape
Spore
Formation
Capsule
Formation
Motility
Biochemical
tests
1
2
3
4
5
KSB 1
Small Creamy, transparent,
smooth, Raised,
-ve,
Small rod
Non
sporulating
Non
capsulated
Motile
-
+
-
+
+
KSB 3
Large,White,opaque, smooth
circular, Slimy,
+ve,
Thick rod
Sporulating
Capsulated
Motile
+
-
-
-
-
KSB 7
Large,White,opaque,smooth
circular, Slimy.
+ve,
Thick rod
Sporulating
Capsulated
Motile
-
-
-
-
-
KSB 8
Medium,creamy,opaque,
smooth, spreading, gummy.
-ve,
Small rod
Non
sporulating
Capsulated
Motile
+
+
-
+
+
KSB11
Medium,creamy,opaque,
smooth, spreading, gummy
-ve,
Small rod
Non
sporulating
Capsulated
Motile
+
+
-
+
+
1-V-P test, 2- Ornithine utilization, 3- H2S Production, 4- Lysine utilization,5- Citrate utilization.

CIBTech Journal of Microbiology ISSN: 2319-3867 (Online)
An Online International Journal Available at http://www.cibtech.org/cjm.htm
2012 Vol. 1 (2-3) Jul.-Sept. & Oct.-Dec., pp.8-14/Prajapati and Modi
Research Article
11
Out of five selected bacterial strains three are gram negative while two are gram positive bacteria. All are
motile and four of them are capsule forming organisms.
KSB 8 and KSB 11 shows V-P, ornithine utilization,lysine utilization and Citrate utilization tests positive
while KSB 7 gives all the five test negative. KSB 1 utilize ornithine, lysine and Citrate. All the five
bacterial strains do not produce H2S.
Total nine different enzymes activity and eleven various carbohydrate sources were checked for all the
five bacterial strains. Among them KSB 3 and KSB 7 shows a wide range of carbohydrate utilization
while KSB 1 only able to utilize Glucose as a carbon source.
Table-3: Enzymatic Activity and various carbohydrates utilization by potassium solubilizers.
Isolates
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
KSB 1
-
+
+
-
+
-
-
-
-
+
-
-
-
-
-
-
-
-
-
-
KSB 3
+
+
-
-
-
+
+
-
-
+
-
+
+
-
+
+
+
+
+
+
KSB 7
+
+
-
-
-
+
+
-
-
+
-
+
+
-
+
+
+
+
+
-
KSB 8
-
-
-
+
+
-
+
-
+
+
-
-
+
+
+
+
+
-
-
-
KSB11
-
-
-
+
+
-
+
-
+
+
-
-
+
+
+
+
+
-
-
-
1-Amylase, 2- Protease, 3- Lipase, 4- Urease, 5- Catalase, 6- Alkaline phosphatise, 7- β –galactosidase,
8- Phenylalanine deaminase, 9- Nitrate reducase, 10- Glucose, 11- Adonitol, 12- Lactose, 13- Arabinose,
14- Sorbitol, 15- Sucrose, 16- Mannitol, 17- Trehalose, 18- Maltose,19- Raffinose.
Table-4: Antibiogram pattern of potassium solubilizing bacteria.
Antibiotics
Concentration
µg/ml
Strain
KSB1
Strain
KSB3
Strain
KSB7
Strain
KSB8
Strain
KSB11
Ampicillin
10
18 (R)
-
-
-
-
Ciprofloxacin
10
26 (S)
18 (S)
22 (S)
24 (S)
24 (S)
Colistin
10
16 (I)
16 (I)
12 (R)
10 (R)
10 (R)
Co-Trimoxazol
25
-
16 (S)
16 (S)
22 (S)
22 (S)
Gentamicin
10
24 (S)
18 (S)
22 (S)
18 (S)
16 (S)
Nitrofurantoin
300
-
17 (S)
24 (S)
-
-
Streptomycin
10
14 (I)
-
-
16 (I)
16 (I)
Tetracyclin
30
12 (R)
28 (S)
24 (S)
-
-
Amikacin
10
-
18 (S)
17 (S)
20 (S)
14 (R)
Amoxycillin
10
-
20 (S)
20 (S)
-
12 (R)
Bacitracin
10 U
-
16 (I)
-
-
-
Cephalothin
30
-
14 (R)
16 (I)
-
-
Erythromycin
15
-
24 (S)
20 (S)
-
-
Novoblocin
30
-
24 (S)
22 (S)
8 (R)
-
Oxytetracyclin
30
18 (I)
24 (S)
-
-
-
Vancomycin
30
-
20 (S)
22 (S)
-
-
(S) Susceptible, (R) Resistant, (I) Intermediate

CIBTech Journal of Microbiology ISSN: 2319-3867 (Online)
An Online International Journal Available at http://www.cibtech.org/cjm.htm
2012 Vol. 1 (2-3) Jul.-Sept. & Oct.-Dec., pp.8-14/Prajapati and Modi
Research Article
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Various 16 antibiotics effect were checked for all the bacterial strain all the isolates having various effects
against the different antibiotics which shown in (Table-4).
Table-5: Growth of isolates on different PH levels.
Sr. No.
pH
Strain
KSB1
Strain
KSB3
Strain
KSB7
Strain
KSB8
Strain
KSB11
1
2
-
-
-
-
-
2
4
-
-
-
-
-
3
5
+
++
++
++
++
4
6
+
++
++
++
++
5
7
++
++
++
+++
+++
6
8
+
++
-
++
++
7
10
-
+
-
-
-
8
12
-
-
-
-
-
- No Growth, + Slight growth, ++ Moderate growth, +++ Vigorous growth
Table- 6: Growth of l isolates on different temperatures.
Sr No.
Temp
Strain
KSB1
Strain
KSB3
Strain
KSB7
Strain
KSB8
Strain
KSB11
1
50C
-
-
-
-
-
2
100C
-
+
-
-
-
3
250C
+++
++
++
+++
+++
4
300C
+++
+++
+++
+++
+++
5
350C
+++
++
++
+++
+++
6
400C
+
+
+
-
-
- No Growth, + Slight growth, ++ Moderate growth, +++ Vigorous growth
Table-7: Growth of isolates on different NaCl concentrations.
Sr. No.
NaCl Concentration
Strain
KSB1
Strain
KSB3
Strain
KSB7
Strain
KSB8
Strain
KSB11
1
1 %
+++
+++
+++
+++
+++
2
2 %
++
++
+
++
++
3
4 %
+
+
+
+
+
4
5 %
+
+
+
+
+
5
7 %
-
+
-
-
-
6
10 %
-
-
-
-
-
-No Growth, + Slight growth, ++ Moderate growth, +++ Vigorous growth

CIBTech Journal of Microbiology ISSN: 2319-3867 (Online)
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2012 Vol. 1 (2-3) Jul.-Sept. & Oct.-Dec., pp.8-14/Prajapati and Modi
Research Article
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All the five selected isolates were checked at various pH, temperatures and NaCl concentrations, among
them KSB 3 having a wide range of pH and NaCl concentrations as compare to other isolates. While KSB
1, KSB 3 and KSB 7 able to grow up to 400 C and KSB 3 can grow at 10 0 C also. KSB 3 can grow at
wide range of temperature, pH as well as in higher saline environment as compare to the other isolates
(Table-5, 6, 7).
Organic acids productions
All the five bacterial isolates were solubilize the potassium from the insoluble source of potassium i.e.
feldspar through the acid production and oxalic and citric acid is likely involved in potassium
solubilization (Table-8).
Table-8: Differnt organic acids produced by isolates.
Sr. No.
Isolates
Oxalic Acid
Citric Acid
Malic Acid
Succinic Acid
Tarteric Acid
1
KSB 1
+
+
-
+
-
2
KSB 3
+
+
+
-
-
3
KSB 7
+
+
-
+
-
4
KSB 8
+
+
-
-
-
5
KSB 11
+
-
-
-
-
Potassium solubilization and identification of best solubilizer
Among the all bacterial strains KSB 8 shows highest potassium solubilization when tested on liquid
media and release the higher amount of potassium from the insoluble source of feldspar when determined
by colorimetric method. From the cultural morphological and finally by 16S rRNA gene sequencing the
KSB 8 is identified as Enterobacter hormaechei.
Conclusion
After secondary screening five bacterial isolates were selecte out of 14 isolates. Among them KSB1,
KSB8 and KSB11 are gram negative, short rods while KSB3 and KSB7 are gram positive bacillus central
spore formers. Except KSB 1 all the bacterial isolates are higher exopolysaccharide producers Among 5
bacterial strains KSB3 and KSB7 which are Gram Positive bacilli are utilizing wide range of the
carbohydrate sources while KSB1 only capable of utilizing Glucose as a sole source of carbon. All the
bacterial strains having various enzymatic activity as well as different Biochemical characterization.KSB3
having a high capacity to grow in saline condition and pH range. Oxalic acid and citric acids were likely
to be involved in the solubilization of feldspar among the all 5 acids. So these all characteristics make
these bacterial strains to solubilize the potassium. The best potassium solubilizing bacterial strain is
identified as Enterobacter hormaechei from cultural, morphological and 16S rRNA gene sequencing.
ACKNOWLEDGEMENT
The authors are thankful to the Department of Biotechnology and Management of Kadi Sarva
Vishwavidyalaya (KSV),Gandhinagar, for providing the facilities for this research.
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CIBTech Journal of Microbiology ISSN: 2319-3867 (Online)
An Online International Journal Available at http://www.cibtech.org/cjm.htm
2012 Vol. 1 (2-3) Jul.-Sept. & Oct.-Dec., pp.8-14/Prajapati and Modi
Research Article
14
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