influence of inceptisol and alfisol’s potassium solubilizing bacteria (ksb) isolates on release of...

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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 [email protected]

Vol. 27 (1) : 181-187 (2014) VEGETOS

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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

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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 4oC 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±20C 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)

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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

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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-tion Slightly

raised Highly raised

Trans-lucent

Opaque

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)


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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

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