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:
[email protected]
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 cultures—Bacterial 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
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Bacillus firmus IARI-J-28 (Accession Number: JN411422.1) were selected. Growth conditions and potassium solubilization in broth—Potassium 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 potassium—Sodium 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 analysis—The 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 potassium—Variations were observed in the precipitation of potassium chloride with different concentrations of sodium cobaltinitrite solution (Fig. 1). The amount of
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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 potassium—Cobaltinitrite 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. 1—Correlation between concentration of sodium cobaltinitrite and potassium in solution, following precipitation by potassium chloride.
Fig. 3—Spectrophotometric 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. 2—Schematic diagram of steps involved in the newly developed method for the quantitative estimation of potassium in culture broth supernatant
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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 1—Comparative 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.
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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 Folin–Ciocalteu 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.
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