The Capacity of Soil Bacteria, Bacillus sprif and Pseudomonas sprif, in solubilizing Soil Phosphate and Potassium

Increasing availability of phosphate (P) and potassium (K) in soil can be driven by microbial activities, which are specifically able to dissolve P and K, known as nutrients hard to dissolve. The objectives of this research were to study the solubilising activity toward soil P and K by P- and K- solubilizing bacteria in sterilized and non-sterilized condition of Oxisol and Inceptisol. Marking procedure was performed on P- and K- solubilizing bacteria to scrutinize their activities in the soil. Marking process was conducted by utilizing resistance toward rifampicin antibiotica concentration of 50 µg.ml−1. The results of this study revealed that the increasing availability of P was evident in both soil conditions (sterilized and non-sterilized). In Oxisol, P availability increased by 48.86%, while in Inceptisol it reached an increase by 187.77%, compared to the initial concentration. Likewise, K availability in Oxisol increased by 4.53 times, and it rose by 5.26 times in Inceptisol. The activities of P solubilising bacteria, in addition to being able to increase soil P availability, were also able to enhance soil’s K content. Similarly, the K solubilizing bacteria were also capable of increasing P availabilityy in both soils.


Introduction
Soil is a dynamic system with high extent of microbial heterogeneity. Microorganism activity occuring in the soil exerts bearing impact on soil nutrient cycle. The availability of phosphorus and potassium nutrients is influenced by soil microbial activity in accelerating weathering and solubilisation processes. Potassium is present in four forms in soil, which are K ions (K + ) in soil solution, as an exchangeable cation, tightly held on the surfaces of clay minerals and organic matter. Moreover, it is tightly held or fixed by weathered micaceous minerals and present in the lattice of certain Kcontaining primary minerals. Soil characteristics affecting the availability of K pertain to the number and type of clay minerals, cation exchange capacity, potassium buffer capacity, moisture, temperature, aeration, and soil pH. The inoculation involving bacteria, which can improve P and K availability in soils by producing organic acids and other chemicals, stimulates the growth and mineral uptake of plants [1].
A research result [2] corroborates that yeast Torulaspora globosa dissolves 38% of total alkaline ultramafic rock powder for 15 days of incubation, while Aspergillus niger dissolves 62% -70% of total ultramafic alkaline mineral, subsequent to 35 days of incubation [3]. The double inoculation of PSB (phosphate-solubilising bacteria), Bacillus megaterium, and KSB (potassium-solubilising bacteria) Bacillus mucilaginosus combined with P and K bearing mineral, significantly increases the availability

Materials and Research Method 2.1 Isolate Marker
Testing the viability and activity of isolates Bacillus sp as phosphate-solubilising bacteria and Pseudomonas sp as potassium-solubilising bacteria under non-sterile conditions was marked by using antibiotic resistance Kirby-Bauer method. The administration of markers with the resistance test against Rifampicin antibiotica up to a concentration level of 50 μg.mL -1 was carried out gradually. Both isolates that have been characterized are identified as Bacillus sp rif and Pseudomonas sp rif

Solubility Test in Solid Selective Media by Marked Isolate
The test in this regard scrutinized the solubility of Bacillus sp rif and Pseudomonas sp rif , It was carried out in Alexsandrov and Pikovskaya solid medium by calculating the solubility index (SI) using the following formula on seventh days: Clear zone diameter SI = Colony diameter

The Examination on Soil's phosphate and potassium Solubility
The solubilisation activity test was performed on 2 soils with different characteristics (Table 1) and within two different conditions (sterilized and non-sterilized). Completely randomized design was made operative, which involved soil factors including sterilized Oxisol (O S ), non-sterilized Oxisol (O N ), sterilized Inceptisol (I S ) and non-sterilized inceptisol (I N ). The second factor was concerned with the isolates: control group (B0); PSB (Bacillus sp rif )and KSB (Pseudomonas sp rif ). The study operationalized 12 treatment combinations with three replications. 200 g soil was inoculated with each bacteria at cell density of 10 7 per gram soil. The soil condition was maintained to resemble field capacity condition and was incubated for a month. Soil sterilization is done 2 times with autoclave at 121 0 C for 15-20 minutes at 1 atmosphere pressure. On regular basis, particularly on the tenth day, twentieth day, and thirtieth day, soil pH, phosphate concentration (Bray extract), and concentration of potassium (Citric acid extract) were analyzed.

Antibiotica Resistance
The examination isolate resistance against Rifampicin antibiotica proved that the both bacteria under research were quite resistant at concentration of 50 μg.mL -1 . The use of rifampicin antibiotica was quite effective as a marker in bacteria tested in heterogeneous soil conditions. A previous research [9] corroborated that, for Pseudomonas ecology test in field, Rifampicin antibiotica resistance marker was applied to Pseudomonas putida strain WCS358 to a concentration of 250 μg.ml -1 and it concluded that rifampicin was stable to be applied as a marker for Pseudomonas putida strain WCS358 in potato rhizosphere. Other studies [10; 8] also have concluded that the use of rifampicin in Pseudomonas fluorescens and Pseudomonas putida is useful material for ecological testing of mutants resulting therefrom. Continuous exposure to rifampicin antibiotica resulted in resistance arising from the mutations of β-RNA polymerase sub-unit gene and RNA polymerase, which changed due to normally functional mutation resistant to rifampicin-triggered inhibition.

Solubility Index
The activity of the two mutant bacteria in the solubilisation of P and K in vitro setting within both Pikovskaya and Alexandrov's media was still detected. Fine solubilizing capability was shown by two mutant bacteria namely Bacillus sp rif and Pseudomonas sp rif . Solubilisation by PSB rif-b (Bacillus sp) has a solubility index (SI) of 2.5 with the widest clear zone diameter of 0.75 cm on Pikovskaya media, while KSB rif-p (Pseudomonas sp) displayed an SI value of 3.3 with maximum clear zone diameter of 1 cm on Alexandrov's media. The solubility index evinced the bacteria's ability to dissolve the sources of P and K present in the media. Based on the criteria applied by the researchers on the results of previous studies [6], both bacteria possessed intermediate-degree solubilising capabilities (SI ≤ 2.00 ≤ 4.00), but their rates of solubilisation were dissimilar in that PSB (Bacillus sp rif ) was found at fast solubilising category (<3 days) whereas KSB (Pseudomonas sp rif ) was proven to be at slow solubilising category (5 days).

The Test on Soil's P Solubility
Oxisol and Inceptisol tested have low P and K concentrations, acid conditions with low organic content ( Table 1). The soil conditions affect the activity of the isolates, because for activities it takes The presence of microbial activity of both PSB (Bacillus sp rif ) and KSB (Pseudomonas sp rif ) in Oxisol soil caused P concentration to be lower than that of the control treatment with no microbial activity, although at the end of incubation the P concentration was still found higher than was the initial concentration ( Figure 1). The high ratio of organic carbon to the availability of P in the soil led to the immobilization of P. Under the condition characterized by C : P ratio < 200, the mineralization of P started to occur [11], while the initial data showed C: P ratio available > 200. This condition resulted in a larger immobilization net and showed P in the soil solution used by the existing microbes, thereby declining the P concentration in the soil solution. Oxisol constitutes highly weathered soil, so the phosphate is bound to Al, Fe or occluded-Fe, making it unavailable to plant.
The pattern of P solubilisation between Oxisol and Inceptisol was different, although the concentration at the end of incubation in both soils increased, compared to the initial soil concentrations (Figures 1a and 1b). In Inceptisol, substantial increase was evident after the 20 th day. The dissolved phosphate in Inceptisol soils was greater than that of Oxisol. Besides, P immobilization through the use of phosphate by microbes still maintained the concentration of P available in the soil solution. In Inceptisol, C: P ratio was lower than that in Oxisol. The lower the C: P ratio was, the more likely mineralization of P was to occur and the lower net immobilization would become.
The sterilized and non sterilized soil conditions did not make a significant difference on the solubilisation of P due to the microbial activity applied. PSB (Bacillus sp rif ) and KSB (Pseudomonas sp rif ) were able to perform well in any soil conditions. As a corollary, the ability to compete against indigenous microbes was quite sound. Based on table 2, KSB (Pseudomonas sp rif ) also possessed the ability to solubilise P from both Oxisol and Inceptisol.
In their activity, bacteria secreted several metabolites, including organic acids. The results of a previous study [6] point out that KSB produces organic acids such as Citric, Ferulic and Coumaric. In the same vein, another study [5] corroborates that KSB produces Citric, Oxalic, Malic, Succinic and Tartaric acid. Furthermore, PSB also produces organic acids in its activities, encompassing Citric, Oxalic, Malic, Succinic [12], Citrate, Oxalate, Succinate, Fumarate, Acetate, Propionate, and Butyrate [13]. One of the mechanisms of solubilising P, especially resulting from the presence of organic anions, is done through several mechanisms, inter alia (1) organic anions competing against orthophosphates ion on the surface of the positively-chargeed colloidal site; (2) the release of orthophosphate ion from metal-P bond through the formation of organic metal complexs [14]; and (3) the modification of surface charge of the site by organic ligands [15]. 6  According to [14] the presence of certain organic anions derived from the decomposition of organic matter, microbial activity, or root secretion may affect the absortion of P through the competition of surface site or lower the adhesion site through solubilisation. In addition, organic acids with low molecular weight lowered P by Al oxide, Fe or allofan clay minerals due to their high affinity [16]. The addition of organic acids with low molecular weight also effectively triggered Al and Fe from Fe-P and Al-P, allowing the solubilisation of P to occur [17]. Organic ligands such as tartaric, oxalate, malate, and citrate containing carboxyl (COOH), aliphatic-OH, phenolic-hydroxyl groups were proven highly effective in mineral solubilisation and chelate formation enriched with such elements as Al, Fe, Ca, and other elements, and declining pH level of media [18 ].
The reaction of phosphate solubilisation by organic acids is formulated as following.  (table 3). Oxisol was highly weathered soil, making possible P to be precipitated with Al or Fe. Besides, P also bound with Fe and Al oxides, as well as in the form of occluded P. In Inceptisol, some adsorption complexes were predominantly Ca and Fe ions, so P bound to Ca and/or Fe. Beside P-organic form, P-inorganic form in soil consists of  6 several fraction. The proportion or percentage of each fraction is influenced by several factors, including the type of soil. The distribution of inorganic P fraction (%) in some soil types in Malaysia and Indonesia [19] shows the fractionation of P in Oxisol and Inceptisol soils as follows:  [20] show that the OP is not dissolved with the addition of soil amendment so the concentration is still high, while the other P form increases its solubility. The fractionation of P in Oxisol of West Java was obtained by the sequence of the greatest as follows: reductan Fe-P> Fe-P> Occluded-P> Ca-P> Al-P> soluble-P [13], therefore release of P on Oxisol is lower than Inceptisol.
The soil acidity indicated an increase at all treatments until the end of incubation in both Oxisol and Inceptisol (see Figure. 2). Organic acid was generally produced by phosphate-solubilising bacteria and potassium-solubilising bacteria. However, the presence of organic acid did not diminish the pH level of both soil types. The carboxyl groups from organic acid developed negative charge as the positively charged H was removed. When the pH of a soil was increased, the release of H from carboxyl groups aided in sustaining the increase of pH and at the same time created the CEC (negative charge). The presence of negative charge on the surface of the clay mineral would attract Ca, Fe or Al cations, consequently increasing phosphate availability.

Potassium Solubilisation
The concentration of K-soluble citric acid in two soil types increased along with increasing period of incubation in the same pattern ( Figure 3). The sterilized and non-sterilized soil conditions also did not affect the activity of the two solubilising bacteria in both soil types, resulting in increased concentration of K. PSB (Bacillus sp rif ) and KSB (Pseudomonas sp rif ), both of which are mutant bacteria against rifampicin resistance. The enhanced concentration, however, did not affect the capacity of bacteria as K-solubilising agent. Findings of a research [21] using antibiotica resistant against Mutant potassium-releasing bacterial strain of Bacillus edaphicus NBT with rifampicin antibiotica (150 mg.l -1 ) also reveal fine activity in increasing P and K concentrations in soil, so that P and K uptake by cotton plants and Rape is greater than those without inoculation. Re-isolation at the end of incubation performed on non-sterilized soil conditions showed the presence of both mutant bacteria. In the 5 th week, mutant potassium-releasing bacterial strain of Bacillus edaphicus NBT was still established in cotton and rape rhizosphere. One of the mechanisms for solubilising the soil potassium by the microorganisms activity resulted from excreted organic acids, therefore triggering the organic cation to be stimulated by Si ions and releasing K in the solutions. The concentration of potassium in Oxisol increased by 3.78 to 4.53 times greater than its initial concentration, whereas Inceptisol rose by 4.32 to 5.26. The increase in Inceptisol was more intense than that in Oxisol, because Inceptisol generally was dominated by smectite clay minerals and some kaolinite and it was derived from clay sediment main material [22]. The concentration of soil K was dependent on the amount of smectite clay minerals in soil. Clay mineral Smectite could fixate K at that mineral layer interspace where the fixated K was reserved exch-K for plants through release and desorption processes, resulting in greater release of K in Inceptisol. The availability of soil K relied on the process and dynamic of K in soil, especially sorption and desorption process. Sorption and desorption of K in soil were determined especially by the type and amount of clay minerals. Clay mineral type 2:1 adsorbed K and released K more intensely than did other clay minerals, such as type 2: 1: 1, 1: 1, oxyde, and alophane [22] In general, mineral composition and chemical properties of red soil include the presence of Oxisol particularly in humid tropical area, characterized by sand mineral dominated by quartz and opaque, while clay mineral is dominated by kaolinite with additional mineral gibbsit, goethite, and hematite [23]. By contrast, according to another research [24], the clay fraction of Oxisols is dominated by (a) 1: 1 phyllosilicates; (b) oxides of Fe and/or Al, or the mixtures of (a) and (b). Clay minerals in soils of tropical climates such as Oxisols and Ultisols are dominated by kaolinite and halloysite, in addition to gibbsite and sesquioxides. The soil with dominance of kaolinite clay and halloysite is characterized by the presence or position of K ion which is not on the interspace layer because there is only one octahedral layer and one tetrahedral layer. Consequently, K fixated as a K-reserve which can be released in solution is also low (Figure 4).  Figure 4. The Illustration of clay Mineral Structure (type 1:1), Illite (type 2:1 non exchangeable), and montmorillonit (type 2:1 exchangeable) Both PSB (Bacillus sp rif ) and KSB (Pseudomonas sp rif ) were able to solubilise phosphate and potassium in both soils although each had distinctive solubility capability. This is line with the study [25] which proves the ability of Bacillus mucilaginosus in increasing P and K availability. The same finding is obtained in another research [4] using phosphate solubilizing bacteria, (PSB) Bacillus megaterium var. Phosphaticum and potassium solubilizing bacteria (KSB) Bacillus mucilaginosus. The mechanism of solubilising phosphate and soil potassium was relatively similar in that it was carried out through acidification, chelation, and exchange reactions [26], as well as through the production and excretion of organic acids.

Conclusion
Both isolates of PSB (Bacillus sp rif ) and KSB (Pseudomonas sp rif ) have been found effective to solubilise the phosphate and potassium of Oxisol and Inceptisol soils in both sterilized and nonsterilized conditions. Phosphate or potassium solubilizing in Inceptisol soil has been proven greater than that in Oxisol. The sterilized and non-sterilized soil conditions have revealed no bearing impact on the solubilizing activity in both isolates.