Ecological Engineering 81 (2015) 340–347

Potassium solubilizing rhizobacteria (KSR): Isolation, identification,and K-release dynamics from waste mica

Vijay Singh Meena a,b, Bihari Ram Maurya a, Jai Prakash Verma c, Abhinav Aeron d,e,Ashok Kumar a, Kangmin Kim e,*, Vivek K. Bajpai f,**aDepartment of Soil Science and Agricultural Chemistry, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi 221-005, UP, IndiabCrop Production Division, Vivekananda Institute of Hill Agriculture, (ICAR),Almora 263-601, UTK, Indiac Institute of Environment and Sustainable Development, Banaras Hindu University, Varanasi 221-005, UP, Indiad School of Basic Sciences and Research, Sharda University, Greater Noida 201-306, Uttar Pradesh, IndiaeDivision of Biotechnology, College of Environmental and Bioresource Sciences, Chonbuk National University, 79 Gobong-ro, Iksan-si 570-752, Joellabuk-do(Jeonbuk), South KoreafDepartment of Applied Microbiology and Biotechnology, School of Biotechnology, Yeungnam University, Gyeongsan, Gyeongbuk 712-749, South Korea

A R T I C L E I N F O

Article history:Received 9 December 2014Received in revised form 11 March 2015Accepted 9 April 2015Available online xxx

Keywords:RhizospherePotassium solubilizing rhizobacteria (KSR)MuscoviteBiotite

A B S T R A C T

Injudicious application of chemical fertilizers in India has a considerable negative impact on economyand environmental sustainability. There is a growing need to turn back to nature or sustainable agentsthat promote evergreen agriculture. Among such natural bio-agents, the potassium solubilizingrhizobacteria (KSR), which solubilize fixed forms of potassium (K) to plant available K by variousmechanisms including acidolysis, chelation, exchange reactions, complexolysis, and production oforganic acids are considered one such available viable alternative. KSR represent an enormous potentialto transform the problems associated with the agrarian sector. Twelve KSR were isolated fromrhizosphere of common Kharif crops (maize, banana, sugarcane, potato, pigeon pea, and tobacco) basedon their ability to solubilize waste mica (muscovite and biotite) in plate assay. All these KSR were capableof K-solubilization from waste mica in both solid and liquid medium in-vitro. On the basis of 16Sribosomal DNA (16S rDNA) sequencing, out of 12 KSR, 7 strains belonged to Agrobacterium tumefaciensspecies, 2 strains each representing Rhizobium pusense and Flavobacterium anhuiense clade, while onestrain showed affiliation to Rhizobium rosettiformans. As a result, among the assessed 12 KSR, A.tumefaciens OPVS 11 and R. pusense OPVS6 occurred at the highest K-solubilizing frequency. Studies onmechanism of K-solubilization by these strains demonstrated significant reduction in media pH andincreased K release with incubation period under both waste muscovite and biotite as a sole source ofinsoluble K mineral.

ã 2015 Elsevier B.V. All rights reserved.

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1. Introduction

Plants can uptake potassium (K) through the soil minerals,organic materials, and synthetic fertilizers. Consumption of K wasexceeded 260 lakh tons for two consecutive years (2011 and 2012)in India, and all the K fertilizers were imported across the globe tomeet the demand for agricultural productivity (FAI, 2013),indicating the injudicious application of K fertilizers. K deficiencyin the rhizosphere of economically important crops has become an

* Corresponding author. Tel: +82 53 810 3842; fax: +82 53 813 4620.** Corresponding author. Tel: +82 63 850 0836; fax: +82 63 850 0834.

E-mail addresses: activase@jbnu.ac.kr (K. Kim), vbajpai04@yahoo.com(V.K. Bajpai).

http://dx.doi.org/10.1016/j.ecoleng.2015.04.0650925-8574/ã 2015 Elsevier B.V. All rights reserved.

important limiting factor responsible for sustainable developmentof evergreen agriculture in India (Naidu et al., 2011). Ironically,most of the K fertilizers are imported from other countries in India,which may also result with negative impact on agro-basedeconomy of India in longer term. In addition, extensive use ofchemical fertilizers is proven to destroy soil structures anddecrease the amount of organic matter in the soil (SOM), as wellas aggravate environmental pollution by contaminating under-ground water.

Potassium (K) is one of the major plant macronutrientsinfluencing plant growth, development and grain quality, its playsa key role in the synthesis of cells, enzymes, proteins, starch,cellulose, and vitamins. Moreover, K not only participates innutrient transportation and uptake, but also confers resistance toabiotic and biotic stresses, leading to enhanced production of

V.S. Meena et al. / Ecological Engineering 81 (2015) 340–347 341

quality crops and provides resistance to plant diseases (Epstein andBloom, 2005; Pettigrew, 2008; Maqsood et al., 2013). The K isabsorbed by plants in large amount than any other mineralelement except nitrogen (N) and, in some cases, calcium (Ca).Chemical or synthetic K fertilizers are the largest available sourcesof K rhizosphere, therefore, larger amounts of K fertilizers can beused to promote the availability of K for plant uptake (Li et al.,2007). The concentration of K in straw and grain serves as anindicator whether the K status of crop is deficient or sufficient (Raoet al., 2010). However, K uptake by aboveground parts of plants isassimilated mainly into the straw but not into the grain (Basak andBiswas, 2009).

Although application of low amount of chemical-based Kfertilizers which implore the conversion of K-feldspar to K fertilizerthrough transformation has assisted significantly to improve theplant productivity, use of such chemicals is still difficult andinfeasible. This has emerged in the use of novel types ofrhizospheric microorganisms or K-solubilizing rhizobacteria(KSR) as the best possible alternatives to mitigate the rhizosphericK dilemma. These KSR can help in enhancing the availability ofnutrients playing an essential role in dynamic soil environment bycontributing release of key nutrients from primary minerals andores. These key macronutrients are central for nutrition ofmicrobial population present in the soil and in turn also effectuatethe benefit to plant nutritional status (Sheng and He, 2006;Nishanth and Biswas, 2008; Abou-el-Seoud and Abdel-Megeed,2012; Maurya et al., 2014; Meena et al., 2014a).

Economically important crops need to be supplied with solubleK fertilizers, the demand of which is expected to increasesignificantly, particularly in developing regions of the world. Inthe soil, all kinds of micro- and macro-nutrients have beneficialeffects on efficient plant growth either via nitrogen fixation,phosphate and potassium solubilization or mineralization. Inaddition, release of plant growth regulating substances, productionof antibiotics, biodegradation of organic matter, and nutrientscycling in the soil by KSR can also be benefited for crop productivityand ecological sustainability (Meena et al., 2013, 2014b; Zhanget al., 2013; Maurya et al., 2014; Zorb et al., 2014). Some of theselected rhizobacteria such as Pseudomonas spp., Acidothiobacilli-cus ferrooxidans,Bacillus mucilaginosus, B. edaphicus and B. mega-terium have already been shown to release K from K-bearingminerals by excreting organic acids (Sheng et al., 2008; Meenaet al., 2014b; Zhang and Kong, 2014). Recent investigations haveshown that organic exudates of few selected bacteria also play akey role in releasing otherwise unavailable K from K-bearingminerals (Maurya et al., 2014; Meena et al., 2014b). Mechanism ofK-solubilization could be mainly attributed to excrete organic acidswhich either directly dissolve rock K or chelate silicon ions to bringK into solution (Prajapati et al., 2013).

In light of above facts, it can be observed that application of Kcan contribute to sustainable high yield and high K use efficiency.Therefore, the objective of this research was to isolate, characterizeKSR and further, to evaluate their K-solubilizing capacity withinsoluble waste mica with muscovite (potash mica) and biotite asthe sole sources of highly insoluble K. In this respect, K-solubilizersreferred to be as KSR were isolated from rhizosphere of Kharifcrops such as maize (Zea mays), banana (Musa paradisiacal), potato

Table 1Elemental composition of waste mica (muscovite and biotite).

Minerals Silica Iron Potash Magnesium oxide

Sl2O3 (%) Fe2O3 (%) K (%) MgO (%)

Muscovite 45.10 2.54 9.82 0.61

Biotite 38.42 16.24 9.70 11.94

(Solamum tuberosum), sugarcane (Saccharum officinarum), pigeonpea (Cajanus cajan) and tobacco (Nicotiana tabacum), wereevaluated for their efficiency to release K from waste mica.

2. Materials and methods

2.1. Soil sampling

The soil samples were taken from the root attached soils of plants.Some top-surface soil was removed before the collection of soilsamples. The surface soil was digged to 10 cm soil layer, where rootsof crops were concentrated. From about 0–2.5 mm away from theroot surface, a zone of soil is located that is significantly influenced bylivingroots and is referredto asthe rhizosphere.Rhizosphere soilandroots were separated from the bulk of the soil by hand. The 45 plantsamples belonging to maize, banana, tobacco, sugarcane, pigeonpea,and potato crops intact with roots and soil were taken randomly inevery block of Varanasi district and 20 g of rhizosphere soil wascollected around each crop plants. All soil samples were sealed insterilized zip lock bags, stored in an ice chest, and used within10–14 h of collection. Rhizospheric soil samples from plants broughtin laboratory were collected from different crop fields covering atotal area of 2535 km2 from June to August 2012. All crop fields werelocated at Indo-Gangetic Plains of Ganges river in the holy cityVaranasi (El 80.71 m [264.80 ft]; Lt 25.3333�N; Lg 83.0000�E), whichis also known by the names such as Banaras/Kaashi in the state ofUttar Pradesh, North Central Part of India. Rhizosphere soil sampleswere collected in rainy season (June–August), which is peak growingseason for the crops mentioned above.

2.2. Waste mica

Waste mica, a K-bearing mineral, was obtained from surround-ings of mica mines located at Koderma district of Jharkhand, Indiathat is produced during dressing of raw mica blocks. It was groundin a Wiley mill to 2 mm size. Ground waste mica was analyzed forits chemical characteristics (Table 1 and Fig. 1) following thestandard procedures. The sieved powder was submerged insterilized water for 48 h to eliminate soluble K.

2.3. Isolation of K-solubilizing rhizobacteria

Modified solid Aleksandrov medium (MAMs) supplementedwith waste mica powder as a sole source of K, was used to screenK-solubilizing rhizobacteria. The serially diluted (in sterile normalsaline, 0.87%) soil samples were plate on MAMs containing (perliter) 5 g glucose, 0.005 g MgSO4�7H2O, 0.1 g FeCl3, 2.0 g CaCO3,3.0 waste mica (muscovite and biotite) as a potassium mineral(2.0 g used in original media), 2.0 g calcium phosphate and 20 gagar–agar (Hu et al., 2006). The plates were then incubated at28�2 �C. After 7 days, colonies showing formation of clear zonearound them were considered to be KSR and selected for furtherstudies using Aleksandrov broth medium (Sugumaran andJanartham, 2007). Screened isolates were gram-stained forpresumptive identification and pure colonies were transferredto sterile slants on nutrient agar medium (Hi-Media). Further, inanother set of experiment, zone of solubilization of purified KSR

Sodium oxide Manganese oxide Phosphorus Calcium oxideNa2O (%) MnO (%) P (%) CaO (%)

0.37 Traces 0.022 0.230.24 Traces 0.019 0.14

Fig. 1. Waste mica (muscovite and biotite) used during the investigation, after processing in Wiley mill and sieved through 2 mm sieve. (Material collected from surroundingsof mica mines located at Koderma district of Jharkhand, India).

342 V.S. Meena et al. / Ecological Engineering 81 (2015) 340–347

isolates was measured using a scale after 7 days of plating onMAMs. The diameter of the solubilization zone was measured incentimeter, and the values were reported as mean � standarddeviation (SD) for each sample. Morphological characteristics ofthese KSR were studied using standard phenotypic techniques(Holt et al., 1994).

2.4. Potassium solubilizing ability of KSR

Waste mica powder was added to the modified Aleksandrovbroth (MAB) as the sole source of K to test the K-solubilizationability of the isolates from waste mica K-mineral. Briefly, 50 mL ofMAB was added into 250 mL flask. The flasks with the MAB weresterilized by autoclaving at 0.1 MPa for 20 min. The cooled flaskswere inoculated with 1 mL K-solubilizing rhizobacteria cultureraised to a population of 108 cfu mL�1 by overnight shaking(150 rpm, 28 � 1 �C, OD600 0.5). Uninoculated MAB served as acontrol in the experiment. Three flasks each (for statisticalreplication) were incubated at 150 rpm and 28 � 1 �C for 7, 14,and 21 days. Available K content was periodically measuredquantitatively using flamephotometric method (Sugumaran andJanarthanam, 2007).

0.100.150.20

Fig. 2. Phylogenetic relationships among 16S rDNA sequences of KSR. Bootstrap

2.5. Quantification analysis of K and analysis of spent medium for pH

The incubated broth solutions containing KSR (50 mL) werevortexed for 10 min to eliminate insoluble materials. The brothcultures (50 mL) were then centrifuged at 10,000 rpm for 10 min(REMI) to separate the supernatant (CFCF) from the grown cellsand insoluble waste mica (muscovite and biotite). K content andpH in the CFCF (muscovite and biotite) were determined byflamephotometry and pH meter, respectively, and compared withuninoculated control (Sugumaran and Janarthanam, 2007).Potassium chloride (KCl) solution was used to prepare the standardcurve for the measurement of free K. The experiment was repeatedthree times each having three replicates for statistical comparison.The results were reported as mean � standard deviation for pooledvalues from each sample.

2.6. Amplification of 16 S rDNA genes by polymerase chain reaction(PCR)

The universal primers, forward 27F (50-AGAGTTTGATCCTGGCT-CAG-30) and reverse 1492R (50-TACGGTTAC CTTGTTACGACTT-30)were used for the amplification of 16S rDNA gene for all bacterial

A. tumefaciens OPVS01

A. tumefacie ns OPVS 09

A. tumefaciens OPVS 02

A. tumefaciens OPVS 12

A. tumefaciens OPVS 10

A.tumefaciens OPVS 07

A.tumefaciens OPVS 11

R. pusense OPVS 03

R. pusense OPVS 06

R.rosettiforman s OPVS 04

F. anhuie nse OPVS 05

F.an huie nse OPVS 08

87

86

95

99

0.000.05

values (>50%) are indicated at branch points and scale bar 0.05 variation.

V.S. Meena et al. / Ecological Engineering 81 (2015) 340–347 343

isolates. These primers were custom synthesized by BangaloreGenei Pvt. Ltd., Bangalore, India. The 50 mL of reaction mixtureconsisted of 50 ng of genomic DNA, 2.5 U of Taq polymerase, 5 mL of10� buffer (100 mM Tris–HCl, 500 mM KCl pH 8.3), 200 mM dNTP,1.5 mM MgCl2 and 10 pmol of each primer. Amplification wasperformed under the following PCR (PCR System 2720, AppliedBiosystems, Singapore) conditions: initial denaturation at 94 �C for5 min, followed by 34 cycles of denaturation at 94 �C for 1 min,annealing at 52 �C for 1.5 min, extension at 72 �C for 2 min and afinal extension at 72 �C for 7 min.

Amplified PCR products (5 mL) were resolved on a 1.5% (w/v)agarose gel at 100 V for 45 min in 1X TAE buffer containingethidium bromide (EtBr) along with 500 bp DNA ladder (BangaloreGenei Pvt. Ltd., Bangalore, India). Size of PCR products wasobserved under a UV lightin dark conditions (1500 bp approx.). PCRproducts were purified using PCR purification kit, using the manualas provided by the manufacturer (Bangalore Genei, Bangalore,India and the samples were kept at �20 �C for further analysis of16S rDNA gene sequencing).

2.7. DNA sequencing

Sequencing of 16S rDNA was outsourced and carried out atBangalore Genei Pvt. Ltd., Bangalore, India. The 16S rDNAsequences were analyzed using DNA sequence analysis software(DNASTAR), and the nucleotide database available at the GenBankwas used for BLAST for similarity searching (www.ncbi.nlm.nih.gov) and identification of the species of the KSR isolated. Thesequences were submitted to NCBI GenBank and a phylogenetictree was prepared using Molecular Evolutionary Genetic AnalysisSoftware (version 6.0) to ascertain relationships among closelymatching 16S rDNA sequences (Fig. 2).

2.8. Statistical analysis

All data obtained in the laboratory experiments were subjectedto analysis of variance (ANOVA), assessed by Duncan’s multiplerange tests (Duncan, 1955) with a probability, p = 0.05, by usingSPSS version 10.0.

3. Results and discussion

3.1. Isolation and characterization of KSR

A total of 30 different types colonies were able to grow on MAMs

which were from rhizosphere of different crops from Indo GangeticPlain (IGP) of India. Among these isolated colonies, only 12 werefound to make a zone of clearance indicating K-solubilization onMAMs. Out of these 12 rhizobacterial isolates, 4 belonged to

Table 2Colony characteristics of KSR isolated from different crops on (MAMs).

Isolates Color Margin Colony elevation

Gram reaction Slightly raised

OPVS-1 White Smooth + _

OPVS-2 Whitish Rough + _

OPVS-3 White Smooth + +

OPVS-4 Creamy Rough + _

OPVS-5 White Rough + +

OPVS-6 Creamy Smooth + +

OPVS-7 Creamy Smooth + +

OPVS-8 White Rough – _

OPVS-9 White Smooth + _

OPVS-10 Creamy Smooth + +

OPVS-11 White Rough – –

OPVS-12 Creamy Smooth – –

rhizosphere of maize, 2 each of banana, sugarcane and potato and1 each to pigeon pea and tobacco rhizosphere (Table 3). KSR can beobtained from the crop rhizosphere easily as also done by Altamareet al. (1999) and replicated in our studies. All rhizobacterial strainsisolated in our study produced slime; however, magnitude ofproduction varied from low to high depending upon the capacity ofindividual bacterial strain (Table 2). Colony morphology revealedwhite, creamy and whitish texture with smooth and rough surface.OPVS-2, 9, and 11 were noted higher slime producers, while OPVS-1,6, and 10 had medium capacity to produce slime. Out of12 rhizobacterial strains, 6 strains showed entire smooth margin.OPVS-3, 5, 6, 7, and 10 showed slightly elevated colonies whilecolonies of OPVS-1, 2, 4, 8, 9,11, and 12 were highly raised. On MAMs,colony formed by most of the isolates appeared to be translucentexcept colonies of OPVS-2, 3, and 4 which appeared to be opaque. Allthe isolates were gram +ve, rods as also reported by other workers(Archana et al., 2012; Maurya et al., 2014). A potassium source(waste mica) powder agar plate was used in the quantitativemeasurement of the K-solubilization zone ability of the KSR. All therhizobacteria were found to be capable of K-solubilization, and thesolubilization zone ranged from 1.29 to 2.34 cm in diameter. StrainOPVS 11 showed most pronounced ability to solubilize K (2.34 cm indiameter) followed by OPVS 6 and 10. However, OPVS 1 solubilizedthe least amount of K as observed by weak zone of 1.29 cm ascompared to other strains used in this study (Fig. 3). Data recordedon zone of solubilization revealed that OPVS-11 which was procuredfrom rhizosphere of Z. mays formed highest zone of solubilizationcompared to other rhizobacterial strains. The solubilizing zones ofdifferent strains were found to vary from 1.29 to 2.34 cm on MAMs.Interestingly, strains from cereals (maize) caused much greaterzone of solubilizationwhen compared with the strains isolatedfrompulses (pigeon pea). Several studies have shown that rhizospherecontains a variety of KSR as also found out in our study (Friedrichet al., 2004; Meena et al., 2013). These rhizospheric microorganismsmineralize or decompose silicate minerals such as K-feldspar andwaste mica. They solubilize fixed form of K in the soil into plantavailable K that can be directly absorbed by plants, leading toenhanced plant growth promotion and development, although theymight also secrete other active plant growth promoting substances(Maurya et al., 2014; Meena et al., 2014a). Recently, the use ofK-solubilizers as biological fertilizers has been considered as ahotspot while studying agriculture and environmental conserva-tion (Meena et al., 2014b).

3.2. Impact of KSR on pH dynamics

Initial pH of uninoculated waste mica added broth was 7.6 whichdid not influence much by incubationperiod. However, slight changeinpHvalueswithincubationperiodwasobserved.Thismay be due to

Optical density Slime production

Highly raised Translucent Opaque

+ + _ Medium+ _ + High_ _ + Low+ _ + Low_ + _ High_ + _ Medium_ + _ Low+ + _ Low+ + _ High– + – Medium+ + – High+ – + Low

Table 3Genetic characterization of selected KSR to genus level.

Rhizosphere GeneBank accession number Closest species with accession number Similarity (%) Isolates denoted

Zea mays KJ410659 A. tumefaciens strain D254 (KJ499777) 100 A. tumefaciens strain OPVS01Musa paradisiaca KJ410660 A. tumefaciens, isolate BD18-R03 (HF584882) 100 A. tumefaciens strain OPVS02Solamum tuberosum KJ410661 R. pusense strain BN-23 (AB969785) 100 R. pusense strain OPVS03Cajanus cajan KJ410662 R. rosettiformans strain W3 (NR116445|) 99 R. rosettiformans strain OPVS04Nicotiana tabacum KJ410663 F. ahuensis strain THWCS2 (GQ284450) 91 F. anhuiense strain OPVS05Saccharum officinarum KJ410664 R. pusense strain BN-23 (AB969785) 100 R. pusense strain OPVS06Zea mays KJ410665 A. tumefaciens isolate Y36 (KF730752) 100 A. tumefaciens strain OPVS07Zea mays KJ410666 F. ahuensis strain THWCSN17 (GQ284447) 94 F. anhuiense strain OPVS08Musa paradisiaca KJ410667 A. tumefaciens strain SWFU-W99 (KF773134) 100 A. tumefaciens strain OPVS09Solamum tuberosum KJ410668 A. tumefaciens strain KRM13 (KJ124585) 100 A. tumefaciens strain OPVS10Zea mays KJ410669 A. tumefaciens strain SWFU-W98 (KF773133) 100 A. tumefaciens strain OPVS11Saccharum officinarum KJ410670 A. tumefaciens strain SWFU-W90 (KF773132) 99 A. tumefaciens strain OPVS12

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production of H+ during the hydrolysis of added waste mica. Binbinand Bin (2011) also reported very minute change in pH of mineraladded broth with increase in incubation periods. The pH value ofinoculated broth supplemented with waste mica decreasedsignificantly by all rhizobacterial strains with increase in incubationperiod. In case of waste muscovite at 14 and 21 DAI, all therhizobacterial strains caused significant decrease in pH of broth ascompared to uninoculated waste mica broth (Fig. 4a and b). At all theincubation periods, strain OPVS 11 showed significantly lower pHvalue as compared to all other KSR strains followed by OPVS 3 and 9.However, in case of waste biotite, at 7 DAI, significantly lower pHvalue was recorded with OPVS 8 followed by OPVS 4. Similar patternwas observed in pH values for OPVS 8 and 6 at 14 and 21 DAI (Fig. 4cand d). Obvious difference in loweringof pH was recorded among the

Fig. 3. Solubilizing zone on MAMs at 7 days after incubation, Rhizobium pusense strain OFlavobacterium anhuiense strain OPVS08.

K-solubilizers. Reduction in pH may be due to production ofdifferent kinds of organic and inorganic acids by K-solubilizers(Maurya et al., 2014). This statement is in accordance to thefindings of several workers who have reported that K-solubilizersproduce mono-, di- and tri-organic acids i.e., gluconic, acetic, oxalic,fumaric, tartaric and citric, which resulted in lowering the pH of thespent medium (Han et al., 2006; Girgis et al., 2008; Meena et al.,2014b). The KSR characterized in this study might had producedseveral kinds of organic acids which possibly broken down thewaste mica structure to satisfy their Si+4 and K+ requirementsbringing them into solution, consequently lowering the pHof the inoculated broth. Lowest pH values in our studies wererecorded at 21 DAI in both cases of waste muscovite and biotiteminerals.

PVS06, Agrobacterium tumefaciens strain OPVS11; R. rosettiformans strain OPVS04;

Fig. 4. (a) Effect of KSR onpH reaction ofMAB (wastemuscovite) at different days of inoculation (7,14 and 21 DAI, respectively). (Initial pH of brothwas 7.60). Each bar represents amean (�SD) from three different experiments withthree replicates each; bars followed by a similar letter between treatments within a days of incubation are not significantly different at p<0.05 level of significance according to Duncan’s Multiple Range Test. (b) Potassiumsolubilizing activity (mgmL�1) of KSR inMAB (waste muscovite). Each error bars represent standard errors for three samples from three replications. Bars followed by a similar letter between treatments within a days of incubationare not significantly different at p<0.05 level of significance according to Duncan’sMultiple Range Test. (c) Effect of KSR onpH reaction ofMAB (waste biotite) at different days of inoculation (7,14, and 21 DAI, respectively). Initial pHof broth was 7.60. Each bar represents a mean (�SD) from three different experiments with three replicates each. Bars followed by a similar letter between treatments within a days of incubation are not significantly different atp<0.05 level of significance according to Duncan’sMultiple Range Test. (d) Potassium solubilizing activity (mgmL�1) of KSR inMAB (waste biotite). Each Error bars represent standard errors for three samples from three replications.Bars followed by a similar letter between treatments within a days of incubation are not significantly different at p<0.05 level of significance according to Duncan’s Multiple Range Test.

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3.3. Impact on K-solubilizing dynamics by KSR

Very less content of K in uninoculated waste mica broth wasmight be due the structural disturbance in waste mica caused byhydrolysis which resulted in the release of K in broth (Zhao et al.,2008; Liu et al., 2012). Besides, organic acids produced by the KSRmight have also influenced the frame work destabilizing surfacecomplexes or by complexion metals in solution (Stillings et al.,1996).

In this study, release of K from insoluble waste mica wassignificantly influenced by inoculants, incubation periods and theirinteractions. The ability of all 12 KSR from the rhizosphere tosolubilize potassium was assessed with both waste muscovite andbiotite, and the quantity of K-solubilization tended to increase withincubation period. All of the rhizobacteria examined were able tosolubilize K but not always to the same extent. In case of wastemuscovite, ability of the KSR to solubilize K ranged from 2.86 to12.86, 6.30 to 11.40 and 9.26 to 16.20 mg mL�1at 7, 14, and 21 DAI,respectively (Fig. 4a). However, KSR strains also solubilized wastebiotite minerals in a range from 8.88 to 13.31, 13.25 to 21.60 and30.34 to 49.73 mg mL�1 at 7, 14, and 21 DAI, respectively (Fig. 4c).Although KSR exhibited significantly more K release as comparedto uninoculated broth, they varied in their capacity to release Kfrom the waste mica. Hence, it is hypothesized that the release of Kmay be due to the production of different kind of organic acids bythe strains. OPVS 1 showed highest K-solubilizing capacity(16.20 and 49.73 mg mL�1) with waste muscovite and biotite,respectively, which was significantly superior when comparedwith other KSR strains isolated from different crop rhizosphere. Itindicates that KSR isolates procured from same type of soil variedin their capacity of K-solubilization from waste mica. Furtherexamination of the solubilization activity of the KSR showed thatsolubilization activity varied, the same isolate also showeddifferent levels of K solubilization activity which was consistentwith the findings of previous studies who also reported thatK-solubilizers produce organic acids and cause reduction in pH ofthe resulting solution (Mikhailouskaya and Tcherhysh, 2005;Sugumaran and Janarthanam, 2007; Prajapati et al., 2012; Chishi,2010; Meena et al., 2013; Maurya et al., 2014). Although, no definiterelationship in pH of broth and release of K by the KSR strains wasobserved, the release of K was greatly influenced by the incubationperiod.

3.4. Phylogenic analysis of KSR

In this study, we examined 12 isolates affirming to 4 differentspecies of KSR, based on 16S phylogeny, found in maize, banana,sugarcane, potato, pigeon pea, and tobacco rhizosphere. The 16SrDNA sequences of the 12 KSR strains were compared to those ofknown 16S rDNA sequences using BLAST and the results indicatethat the 12 KSR strains isolated from different rhizosphere canbe basically categorized into four groups (Table 3 and Fig. 2).Most of the KSR obtained so far by other workers have beenisolated from rhizosphere of different plants (Gopal et al., 2005;Zhou et al., 2006). Previously KSR have been successfully isolatedfrom rhizosphere of rice, corn, and coconut plantationscorroborating our findings (Gopal et al., 2005). B. mucilaginosus,Bacillus and Pseudomonas species are the most widely studiedspecies of KSR (Zhou et al., 2006; Sugumaran and Janarthanam,2007). However, in our presented research, we studied differentspecies of KSR with potential for K-solubilization and bioferti-lization. Seven isolates (58.33%) were found to be closelyphylogenetically related to Agrobacterium, showing about 99–100% similarity in their 16S rDNA sequences, making Agro-bacterium the most dominant genus namely Agrobacteriumtumefaciens strain (58.33%/ 7 out of 12), Rhizobium pusense

strain (16.66%/ 2 out of 12), Flavobacterium anhuiense strain(16.66%/ 2 out of 12), and Rhizobium rosettiformans strain (8.33%/1 out of 12). The results indicate that there is a greater diversityof K-solubilizing rhizobacteria waiting to be discovered and thevast benefits they bring with them is the present answer tomany of the synthetic fertilizer conundrum.

4. Conclusion

Application of KSR on soil-plant system under greenhouse/fieldconditions can be a valuable tool for increased crop productivity;therefore, in this study, examination of a total of 12 KSR tosolubilize K from waste mica was carried out under in-vitrocondition. Influence of 12 KSR on the K release and pH dynamicunder waste mica (muscovite and biotite) at different growthintervals revealed that all the strains were able to efficientlysolubilize the insoluble K and, therefore showed a great potentialfor use as novel biofertilizers. A. tumefaciens OPVS 11 followed by R.pusense OPVS 6 significantly reduced pH of broth indicatingacidolysis as one of the chief mechanisms among these KSR. Krelease from waste muscovite and biotite was significantlyinfluenced by A. tumefaciens OPVS 1 and A. tumefaciens OPVS2 pointing out to fact that each species has differential capability tosolubilize K, perhaps, making it necessary to screen out diversity ofrhizobacteria for their K release potential. The plant growthpromoting KSR (biological K-fertilizers) which enhance the Kavailability in agricultural soils with combined use of waste micamay reduce the negative impact on Indian economy whileintegrating with slogan of the sustainable development. Furtherstudies on the mechanism by which KSR solubilized K and theeffectiveness of their use in the field are needed to promoteevergreen agriculture.

Acknowledgements

Sincere thanks to Head, Department of Soil Science andAgricultural Chemistry, Institute of Agricultural Sciences, BanarasHindu University. VSM is thankful to University Grants Commis-sion (UGC, New Delhi), Government of India (GOI) for DoctoralFellowship.

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