A New Spectrophotometric Method for Quantification of Potassium Solubilized by Bacterial Cultures

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 methods employ the ability of potassium to precipitate minerals like mica, feldspar, aluminosilicate minerals, sodium cobaltinitrite as sparingly soluble sodium phosphorite, K-rich shale, rock powder, microcline, potassium cobaltinitrite and the subsequent estimation orthoclase, and muscovite1-6. Plate assay based on of one of the constituents of the precipitate. Methods observations of halo zone around the colony in based on estimation of less stable nitrite radicals11,12 Aleksandrov medium with potassium aluminosilicates or estimation of stable constituent of precipitate as an insoluble source of potassium is available for e.g. cobalt is available. But such methods are tedious, qualitative estimation of potassium solubilized2,7. involving a number of steps13-15. Quantitative estimation of potassium, however, Therefore, the present study has been undertaken primarily relies on flame photometry8 or atomic with an aim to develop an easy, convenient and less absorption spectrophotometer9 or inductively coupled time consuming method for the analysis of potassium plasma-atomic emission spectrometry10. These in culture broth supernatant of K-solubilizing bacteria, methods are sensitive, but sometimes give erroneous based upon the development of a stable and colour results when used for estimation of potassium in broth compound of cobalt. cultures of bacteria. This may be due to production of copious amounts of polysaccharide by potassium Materials and Methods solubilizing bacteria2, 4, which reduces the sensitivity Bacterial cultures—Bacterial isolates from and reliability of the observations. There are several Jaisalmer, Rajasthan (India), available in the germplasm methods for the quantitative estimation of potassium collection at the Division of Microbiology, Indian from blood serum and serum which rely on the Agricultural Research Institute (IARI), New Delhi property of insolubility of potassium cobaltinitrite were screened for potassium solubilisation, using both compound. The amount of potassium may be qualitative and quantitative methods. On the basis of estimated by using volumetric, spectrophotometric quantification of solubilized potassium, six bacterial and gasometric estimation of the nitrite. Most of these strains Bacillus cereus IARI-J-2 (Accession Number: JN411401.1), Bacillus mycoides IARI-J-4 (Accession —————— Number: JN411403.1), Bacillus cereus IARI-J-6 * Correspondent author (Accession Number: JN411405.1), Bacillus sp. IARI- Telephone: 91-11-25847649 Fax: 91-11-25846420 J-11 (Accession Number: JN411408.1), Bacillus sp. E-mail: [email protected] IARI-J-20 (Accession Number: JN411414.1), and 262 INDIAN J EXP BIOL, MARCH 2014 Bacillus firmus IARI-J-28 (Accession Number: cobaltinitrite solution used in reaction mixture JN411422.1) were selected. separately without potassium served as control. Growth conditions and potassium solubilization in Quantitative estimation of available K by flame broth—Potassium solubilization studies were carried photometric method in culture broth supernatant— out in Aleksandrov liquid medium, as described by Various concentrations (20, 30, 40 ppm) of KCl Hu et al2. The composition of medium (g/L) was as solution were used as standard for the quantification follows: 5.0 glucose, 0.5 magnesium sulphate, 0.005 of potassium through flame photometer. For the ferric chloride, 0.1 calcium carbonate, 2 calcium estimation of potassium in culture supernatant, 5 mL phosphate and 2 potassium aluminosilicates minerals. of broth culture was centrifuged at 13,000 rpm for The pH of the medium was adjusted to 7.2 by adding 5 min, the supernatant was collected in fresh tubes 1 N NaOH. Potassium aluminosilicates (Hi-Media and used for precipitation of cobaltinitrite as Laboratories, India) was used in solubilization described above. Following precipitation, the medium as the insoluble source of potassium. Conical supernatant was used for estimation of potassium by flasks (150 mL) containing 40 mL of liquid medium flame photometer. were inoculated with each of the bacterial cultures Based on the results, cobaltinitrite concentration of tested. Autoclaved liquid medium without 12.5% was selected for development of standard inoculation, served as control. The flasks were curve for potassium and for estimation of K in culture incubated for 5 d at 30 °C, with continuous shaking at supernatant of selected bacteria. Different concentrations 140 rpm. After incubation, broth was centrifuged at of KCl solution, ranging from 0-100 ppm, were 10,000 rpm for 10 min and the supernatant was used for the preparation of standard curve for the collected for estimation of available potassium using spectrophotometric estimation of potassium. Sodium both flame photometer and the newly developed cobaltinitrite solution (5 mL) was added slowly to spectrophotometric method. each test tube containing varied concentrations of potassium and volume was made to 10 mL by adding Optimization of cobaltinitrite concentration for distilled water. The reaction mixture was incubated at maximum precipitation with potassium—Sodium 37 °C for 45 min to precipitate the potassium and cobaltinitrite powder (25 g) was dissolved in 100 mL centrifuged at 13,000 rpm for 5 min to permit the of distilled water and filtered before use. Potassium precipitate to settle down in the tube. The supernatant chloride (KCl) was used for the precipitation of was decanted, precipitate collected and washed twice sodium cobaltinitrite solution. KCl solution was with distilled water and once with absolute ethanol. prepared by dissolving 0.149 g KCl in 200 mL After careful washing, 10 mL of conc. HCl was added distilled water to achieve a 400 ppm concentration of to precipitate and incubated at 37 °C for 15-20 min K in stock solution. An equal volume of KCl solution to develop the colour and absorbance was measured in each test tube was used to optimize the at 623 nm. Following the same procedure and precipitation of sodium cobaltinitrite in presence of conditions, potassium was estimated in 5 mL of potassium. Different volumes of 25% sodium culture supernatant, with reference to the standard cobaltinitrite solution (0, 1, 2, 3, 4, 5 mL) were added curve generated. slowly to each test tube and volume was made up to Statistical analysis—The correlation coefficient 10 mL by adding distilled water. The final between the values obtained with flame photometric concentration of potassium in 10 mL of reaction method and the developed spectrophotometric method mixture was kept as 40 ppm for all the treatments; was determined using SPSS 16.0 statistical software. while the sodium cobaltinitrite concentration varied The mean values and the critical difference between from 0, 2.5, 5, 7.5, 10 and 12.5% in the different treatments were calculated at 1% level of significance treatments. Incubation was done at 37 °C for 45 min and denoted as C.D. (Critical Difference) in the tables. to accomplish the maximum precipitation of potassium. After incubation, reaction mixture was Results centrifuged at 13000 rpm for 5 min to settle down the Optimization of cobaltinitrite concentration for precipitate. Supernatant was decanted in fresh test maximum precipitation with potassium—Variations tube and precipitate was collected. The amount of were observed in the precipitation of potassium potassium retained in reaction mixture was measured chloride with different concentrations of sodium by flame photometer. The concentration 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 became transparent and developed a bluish green with an increase in percent of sodium cobaltinitrite colour, which was found to be stable for several and at 12.5% cobaltinitrite concentration, no hours. potassium was detected by flame photometry. Thus, The standard curve maintained its linearity up to in further estimations, 12.5% of sodium cobaltinitrite 100 ppm with R2 value as high as 0.998 (Fig. 3), in final reaction volume was used for precipitation. indicating a strict correlation

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