Indian Journal of Experimental Biology

Vol.52, March 2014, pp. 261-266

A new spectrophotometric method for quantification of potassium solubilized

by bacterial cultures
Mahendra Vikram Singh Rajawat, Surender Singh & Anil Kumar Saxena*
Division of Microbiology, Indian Agricultural Research Institute, New Delhi 110 012, India
Received 26 March 2013; revised 8 October 2013
A new spectrophotometric method was developed for the quantification of potassium in the culture broth supernatant of
K-solubilizing bacteria. The standard curve of potassium with the new method, which is based on the measurement of
cobalt, showed a regression coefficient (R2) of 0.998. The quantification values of potassium obtained with flame
photometric method and the newly developed method showed a significant correlation (r) of 0.978. The new method
depends on the precipitation of sodium cobaltinitrite with solubilized potassium in liquid medium as potassium sodium
cobaltinitrite, which develops bluish green colour by the addition of conc. HCl. The intensity of developed colour can be
recorded at 623 nm. This method involves less number of steps, is easy and time saving, and can be used for the reliable
estimation of available potassium in culture broth supernatant of K-solubilizing bacteria
Keywords: Cobalt nitrite, Flame photometry, K-solubilizing bacteria, Potassium solubilization

Bacteria are known to solubilize potassium from

minerals like mica, feldspar, aluminosilicate minerals,
phosphorite, K-rich shale, rock powder, microcline,
orthoclase, and muscovite1-6. Plate assay based on
observations of halo zone around the colony in
Aleksandrov medium with potassium aluminosilicates
as an insoluble source of potassium is available for
qualitative estimation of potassium solubilized2,7.
Quantitative estimation of potassium, however,
primarily relies on flame photometry8 or atomic
absorption spectrophotometer9 or inductively coupled
plasma-atomic emission spectrometry10. These
methods are sensitive, but sometimes give erroneous
results when used for estimation of potassium in broth
cultures of bacteria. This may be due to production of
copious amounts of polysaccharide by potassium
solubilizing bacteria2, 4, which reduces the sensitivity
and reliability of the observations. There are several
methods for the quantitative estimation of potassium
from blood serum and serum which rely on the
property of insolubility of potassium cobaltinitrite
compound. The amount of potassium may be
estimated by using volumetric, spectrophotometric
and gasometric estimation of the nitrite. Most of these

* Correspondent author
Telephone: 91-11-25847649
Fax: 91-11-25846420
E-mail: saxena461@yahoo.com

methods employ the ability of potassium to precipitate

sodium cobaltinitrite as sparingly soluble sodium
potassium cobaltinitrite and the subsequent estimation
of one of the constituents of the precipitate. Methods
based on estimation of less stable nitrite radicals11,12
or estimation of stable constituent of precipitate
e.g. cobalt is available. But such methods are tedious,
involving a number of steps13-15.
Therefore, the present study has been undertaken
with an aim to develop an easy, convenient and less
time consuming method for the analysis of potassium
in culture broth supernatant of K-solubilizing bacteria,
based upon the development of a stable and colour
compound of cobalt.
Materials and Methods
Bacterial culturesBacterial isolates from
Jaisalmer, Rajasthan (India), available in the germplasm
collection at the Division of Microbiology, Indian
Agricultural Research Institute (IARI), New Delhi
were screened for potassium solubilisation, using both
qualitative and quantitative methods. On the basis of
quantification of solubilized potassium, six bacterial
strains Bacillus cereus IARI-J-2 (Accession Number:
JN411401.1), Bacillus mycoides IARI-J-4 (Accession
Number: JN411403.1), Bacillus cereus IARI-J-6
(Accession Number: JN411405.1), Bacillus sp. IARIJ-11 (Accession Number: JN411408.1), Bacillus sp.
IARI-J-20 (Accession Number: JN411414.1), and

262

INDIAN J EXP BIOL, MARCH 2014

Bacillus firmus IARI-J-28 (Accession Number:

JN411422.1) were selected.
Growth conditions and potassium solubilization in
brothPotassium solubilization studies were carried
out in Aleksandrov liquid medium, as described by
Hu et al2. The composition of medium (g/L) was as
follows: 5.0 glucose, 0.5 magnesium sulphate, 0.005
ferric chloride, 0.1 calcium carbonate, 2 calcium
phosphate and 2 potassium aluminosilicates minerals.
The pH of the medium was adjusted to 7.2 by adding
1 N NaOH. Potassium aluminosilicates (Hi-Media
Laboratories, India) was used in solubilization
medium as the insoluble source of potassium. Conical
flasks (150 mL) containing 40 mL of liquid medium
were inoculated with each of the bacterial cultures
tested. Autoclaved liquid medium without
inoculation, served as control. The flasks were
incubated for 5 d at 30 C, with continuous shaking at
140 rpm. After incubation, broth was centrifuged at
10,000 rpm for 10 min and the supernatant was
collected for estimation of available potassium using
both flame photometer and the newly developed
spectrophotometric method.
Optimization of cobaltinitrite concentration for
maximum precipitation with potassiumSodium
cobaltinitrite powder (25 g) was dissolved in 100 mL
of distilled water and filtered before use. Potassium
chloride (KCl) was used for the precipitation of
sodium cobaltinitrite solution. KCl solution was
prepared by dissolving 0.149 g KCl in 200 mL
distilled water to achieve a 400 ppm concentration of
K in stock solution. An equal volume of KCl solution
in each test tube was used to optimize the
precipitation of sodium cobaltinitrite in presence of
potassium. Different volumes of 25% sodium
cobaltinitrite solution (0, 1, 2, 3, 4, 5 mL) were added
slowly to each test tube and volume was made up to
10 mL by adding distilled water. The final
concentration of potassium in 10 mL of reaction
mixture was kept as 40 ppm for all the treatments;
while the sodium cobaltinitrite concentration varied
from 0, 2.5, 5, 7.5, 10 and 12.5% in the different
treatments. Incubation was done at 37 C for 45 min
to accomplish the maximum precipitation of
potassium. After incubation, reaction mixture was
centrifuged at 13000 rpm for 5 min to settle down the
precipitate. Supernatant was decanted in fresh test
tube and precipitate was collected. The amount of
potassium retained in reaction mixture was measured
by flame photometer. The concentration of sodium

cobaltinitrite solution used in reaction mixture

separately without potassium served as control.
Quantitative estimation of available K by flame
photometric method in culture broth supernatant
Various concentrations (20, 30, 40 ppm) of KCl
solution were used as standard for the quantification
of potassium through flame photometer. For the
estimation of potassium in culture supernatant, 5 mL
of broth culture was centrifuged at 13,000 rpm for
5 min, the supernatant was collected in fresh tubes
and used for precipitation of cobaltinitrite as
described above. Following precipitation, the
supernatant was used for estimation of potassium by
flame photometer.
Based on the results, cobaltinitrite concentration of
12.5% was selected for development of standard
curve for potassium and for estimation of K in culture
supernatant of selected bacteria. Different concentrations
of KCl solution, ranging from 0-100 ppm, were
used for the preparation of standard curve for the
spectrophotometric estimation of potassium. Sodium
cobaltinitrite solution (5 mL) was added slowly to
each test tube containing varied concentrations of
potassium and volume was made to 10 mL by adding
distilled water. The reaction mixture was incubated at
37 C for 45 min to precipitate the potassium and
centrifuged at 13,000 rpm for 5 min to permit the
precipitate to settle down in the tube. The supernatant
was decanted, precipitate collected and washed twice
with distilled water and once with absolute ethanol.
After careful washing, 10 mL of conc. HCl was added
to precipitate and incubated at 37 C for 15-20 min
to develop the colour and absorbance was measured
at 623 nm. Following the same procedure and
conditions, potassium was estimated in 5 mL of
culture supernatant, with reference to the standard
curve generated.
Statistical analysisThe correlation coefficient
between the values obtained with flame photometric
method and the developed spectrophotometric method
was determined using SPSS 16.0 statistical software.
The mean values and the critical difference between
treatments were calculated at 1% level of significance
and denoted as C.D. (Critical Difference) in the tables.
Results
Optimization of cobaltinitrite concentration for
maximum precipitation with potassiumVariations
were observed in the precipitation of potassium
chloride with different concentrations of sodium
cobaltinitrite solution (Fig. 1). The amount of

RAJAWAT et al.:SPECTROPHOTOMETRIC METHOD FOR QUANTIFICATION OF POTASSIUM

263

potassium in the reaction mixture decreased gradually

with an increase in percent of sodium cobaltinitrite
and at 12.5% cobaltinitrite concentration, no
potassium was detected by flame photometry. Thus,
in further estimations, 12.5% of sodium cobaltinitrite
in final reaction volume was used for precipitation.
Standard curve for the spectrophotometric
estimation of potassiumCobaltinitrite precipitates
obtained for varied concentrations of potassium
chloride solution were used for the preparation of
standard curve for quantification of potassium
spectrophotometrically. On addition of conc. HCl
to the potassium cobaltinitrite precipitate, the solution
became turbid. The details of steps involved are
given in Fig. 2. After incubation, the solution

became transparent and developed a bluish green

colour, which was found to be stable for several
hours.
The standard curve maintained its linearity up to
100 ppm with R2 value as high as 0.998 (Fig. 3),
indicating a strict correlation between the intensity of
bluish green colour and the quantity of potassium
present as cobaltinitrite complex. The standard curve
was used for estimation of potassium in culture
filtrates of different bacterial isolates.
Estimation of potassium in culture supernatant
The analyses of data revealed a significant correlation
(r=0.978) at 0.01 level between the two values

Fig. 1Correlation between concentration of sodium

cobaltinitrite and potassium in solution, following precipitation by
potassium chloride.

Fig. 3Spectrophotometric quantification of potassium in different

standard solutions of potassium chloride by the method developed.
The intensity of bluish green colour was read at 623 nm.

Fig. 2Schematic diagram of steps involved in the newly developed method for the quantitative estimation of potassium in culture
broth supernatant

INDIAN J EXP BIOL, MARCH 2014

264

obtained for quantification of potassium by newly

developed spectrophotometric method and flame
photometric method (Table 1). ANOVA revealed that
the average of the differences in the values generated
by the two methods were not significantly different
(3.77 vis a vis 5.38). This showed that the new
developed method is accurate and satisfactory for the
quantification of potassium from culture supernatant
on the basis of developed colour intensity. In general,
the values obtained by the newly developed method
were lower than that obtained by flame photometer up
to 60 ppm potassium; however, beyond 60 ppm K, the
values obtained by the new method were slightly
higher than that obtained by flame photometer, but
not significantly different. Among the cultures
screened, Bacillus cereus IARI-J-6 was found to be
the most efficient for potassium solubilisation,
exhibiting significantly higher values by both
methods.
The interference of different cations on this method
for K estimation was studied by using varied
concentrations of different cations (Li+, Na+, Ca2+,
Mg2+, Cu2+, Mn2+, Al3+, Fe3+) in reaction mixture. No
interference was observed with Fe3+ and Na+ at
concentration up to 35 ppm in reaction mixture.
However, Mn2+ at 25 ppm, Ca2+ and Al3+ at 20 ppm,
Li+ and Cu2+ at 10 ppm, Mg2+ at 5 ppm interfere with
the second step of the assay that involves dissolution
of K-cobaltinitrite precipitate with conc. HCl.

Discussion
Several methods are available for estimation of
potassium based on the ability of potassium to
precipitate sodium cobaltinitrite from blood serum.
However, the spectrophotometric estimation of
potassium in the culture supernatant of microbes has
not been attempted. One of the main disadvantages
among the available methods is the indirect analysis
of potassium through precipitation in different
complex forms and detection of components, other
than potassium. Another disadvantage is that all the
methods for the estimation of potassium are time
consuming, and not suitable for bacterial cultures,
where
the
bacterial
metabolites,
including
polysaccharides/mucilage
interfere
with
the
spectrophotometric observations.
In the present study, a new spectrophotometric
method was developed to quantify potassium in the
culture
broth
supernatant.
There
are
spectrophotometric methods available for estimation
of cobalt, rather than nitrite13-16. Among these, one
method involves dissolving the precipitate with dilute
HNO3, followed by heating and addition of alcoholic
ammonium thiocyanate solution after cooling for
colour development13. Another method employs the
suspension of the precipitate in distilled water,
followed by immersion in boiling water bath for
complete dissolution. To the cooled solution choline
hydrochloride and sodium ferrocyanide solutions are

Table 1Comparative quantification of available K in the supernatant of bacterial culture broth by flame photometric and developed
spectrophotometric methods
Bacterial cultures

Available potassium (in ppm)

Flame photometric
method

Newly developed
spectrophotometric
method

Differences in the values

obtained by the two
methods

SEM

CD (0.01)

Control

21.6

20.3

1.3

1.396

3.86

Bacillus cereus IARI-J-2

60.8

54.7

6.1

1.695

4.68

Bacillus mycoides IARI-J-4

66.4

67.5

1.1

1.184

3.27

Bacillus cereus IARI-J-6

72.8

77.4

4.6

2.654

7.34

Bacillus sp. IARI-J-11

52

45.5

6.5

2.053

5.67

Bacillus sp. IARI-J-20

57.2

52.8

4.4

1.757

4.86

Bacillus firmus IARI-J-28

44.8

42.4

2.4

2.884

7.97

SEM

2.410

1.177

CD (0.01)

6.66

3.25

3.77

5.38

Average
*Correlation coefficient: 0.978; Correlation was significant at the 0.01 level.

RAJAWAT et al.:SPECTROPHOTOMETRIC METHOD FOR QUANTIFICATION OF POTASSIUM

added respectively to initiate spectrophotometric

reaction14. Sobel and Kramer16 developed a method in
which the sodium cobaltinitrite precipitate with
potassium was washed and degraded by adding 6 N
HCl, and heating in steam bath or flame. The clear
solution obtained was placed in oven at 105-125 C to
dryness and the residue dissolved in potassium
pyrophosphate solution. An olive green colour was
developed by adding of cysteine hydrochloride
solution, followed by the addition of H2O2, leading to
an intense yellow coloured solution. Water is added to
the precipitate of potassium and heated on a boiling
water bath until the precipitate dissolved to give a
transparent solution, followed by the addition of
glycine, sodium carbonate and FolinCiocalteu
phenol reagent for spectrophotometric estimation of
potassium from serum15.
The new method depends upon the precipitation
of cobaltinitrite with potassium, followed by
dissolving the precipitate in conc. HCl, and reading
the colour intensity spectrophotometrically. When
cobalt reacts with small amount of HCl, it gives blue
colour because of the formation of tetrachlorocobalt
(II) complex in presence of excess amount of
chlorine ions in reaction. But with high
concentration of HCl, it develops bluish green
colour. The complex involved in the bluish green
colour development is not well known. Hypothetically,
the spectrophotometric complex formed may be
[CoCl5]3- or [HCoCl5]2-. ANOVA revealed no
significant differences among the values generated
by the two methods, illustrating the promise of this
time saving technique.
The present method developed is time and costeffective, requiring less number of steps and a
reliable technique, aiding in screening large number
of samples/cultures of K-solubilizers. Newly
developed method is a two step assay and requires
the use of relatively inexpensive chemicals, sodium
cobaltinitrite and HCl. In addition, the reaction is
carried out at room temperature and thus saves
energy. Addition of conc. HCl after precipitation
with sodium cobaltinitrite is sufficient to develop the
colour within 15-20 min. The new method is
sensitive, reliable and depends upon estimation of
stable constituent of precipitate that is cobalt. The
earlier reported colorimetric methods require several
steps to develop colour13-20 and involve the use of
expensive chemicals like choline hydrochloride14,
dipicrylamine17, chloroplatinic acid18,19, sodium
tetraphenyl boron20.

265

Acknowledgement
The authors are thankful to Department of
Biotechnology (DBT) for financial assistance.
References
1

Bennet P C, Rogers J R, Choi W J & Hiebert F K, Silicates,

silicate weathering, and microbial ecology, Geomicrobiol J,
18 (2001) 3.

Hu X, Chen J & Guo J, Two phosphate- and

potassium- solubilizing bacteria isolated from Tianmu
Mountain, Zhejiang, China, World J Microbiol Biotech,
22 (2006) 983.

Sugumaran P & Janarthanam B, Solubilization of potassium

containing minerals by bacteria and their effect on plant
growth, World J Agric Sci, 3 (2007) 350.

Girgis M G Z, Khalil H M A & Sharaf M S, In vitro

evaluation of rock phosphate and potassium solubilizing
potential of some Bacillus strains, Aust J Basic Appl Sci,
2 (2008) 68.

Lian B, Wang B, Pan M, Liu C & Teng H H, Microbial

release of potassium from K-bearing minerals by
thermophilic fungus Aspergillus fumigatus, Geochim
Cosmochim Acta, 72 (2008) 87.

Lopes-Assad M L, Avan sini S H, Erler G, Rosa M M, Porto

de Carvalho J R & Ceccatop- Antonini S R, Rock powder
solubilization by Aspergillus niger as a source of potassium
for agroecological systems, in: 19th World Congress of Soil
Science, Soil Solutions for a changing World 1-6 August
2010, Brisbane, Australia, (2010), 219.

Aleksandrov V G, Blagodyr R N & Illev I P, Liberation of

phosphoric acid from apatite by silicate bacteria, Mikrobiol
Zh (Kiev), 29 (1967) 111.

Jackson M L, Soil chemical analysis, (Prentice Hall India,

New Delhi) 1973, 498.

Manib M, Zahra M K, Abdel A L & Heggo A, Role of

silicate bacteria in releasing K and Si from biotite and
orthoclase, in: Soil biology and conservation of the
biosphere, edited by Szegi J. (Akademiai Kiado, Budapest).
1986, 733.

10 Buss H L, Luttge A & Brantley S L, Etch pit formation in

iron silicate surfaces during siderophore-promoted
dissolution, Chem Geol, 240 (2007) 326.
11 Briggs A P, A study of the inorganic elements of blood
plasma, J Biol Chem, 57 (1923) 351.
12 Looney J M & Dyer C G, Photoelectric method for
determination of potassium in blood serum, J Lab Clin Med,
28 (1942) 355.
13 Breh F & Gaebler O H, The determination of potassium in
blood serum, J Biol Chem, 87 (1930) 81.
14 Jacobs H R D & Hoffman W S, A new spectrophotometric
method for the estimation of potassium, J Biol Chem,
93 (1931) 685.
15 Abul-Fadl M A M, Spectrophotometric determination of
potassium by Folin-Ciocalteu Phenol Reagent, J Biol Chem,
44 (1949) 282.

266

INDIAN J EXP BIOL, MARCH 2014

16 Sobel A E & Kramer B, A new colorimetric method for the

quantitative estimation of small amounts of potassium,
J Biol Chem, 100 (1933) 561.
17 Harington C R, The micro-determination of potassium,
J Biol Chem, 35 (1941) 545.
18 Shohl A T & Bennett H B, A micro method for the determination
of potassium as iodoplatinate, J Biol Chem, 78 (1928) 643.

19 Cunningham B, Kirk P L & Brooks S C, Quantitative drop

analysis. (XV) Determination of potassium, J Biol Chem,
139 (1941) 21.
20 Stephen W I, Contribution to spot colorimetry
using the weisz ring Oven, Mikrochim Acta, 44 (1956)
1540.