Optimization of Potassium Dissolution by Pseudomonas fluorescens Using Placket-Burman Design and Response Surface Methodology

Among the elements, potassium (K) is the third important macronutrient for plant nutrition that plays a significant role in plant growth and development. The development of intensively managed agriculture has led to the consumption of increasing amounts of K, low K supply has therefore become an important yield-limiting factor in agriculture. However, more than 98% of potassium in the soil exists in the form of silicate minerals such as illite and lattice K in K-feldspars which K cannot be directly absorbed by plants. Potassium and other minerals can be released when these minerals are weathered. Some microorganisms can play a role in releasing K from minerals. They solubilize K-bearing minerals through different mechanisms including chelation, acidolysis, pH reduction, exchange reaction, complexation, biofilm formation and secretion of organic acid and polysaccharides. Since the use of potassium solubilizing microorganisms (KSMs) as K-biofertilizers reduces the agrochemicals application and supports eco-friendly agriculture, so it is imperative to isolate the KSMs and optimize various growth parameters so as to improve their activity.

The present study was an attempt to model and evaluate the effects of pH, incubation time and different amounts of carbon source on K release by Pseudomonas fluorescens using Placket-Burman design and response surface methodology with a central composite design. At the first step, 12 experiments based on Placket-Burman design were carried out to screen and identify the effective carbon source in potassium release. According to the results of the first step, response surface methodology with the central composite design was employed to evaluate and model the effects of the coded independent variables including pH (3-10), incubation time (1-18 days) and carbon source (0.6-12 g L-1) on K release from feldspar and phlogopite. After the completion of each period, samples were centrifuged at 3000 rpm for 10 minutes and filtered using Whatman paper (No. 41). Potassium concentration of samples was measured by flame photometer. Used minerals in the experiment including feldspar and phlogopite were grounded and filtered through a 230 mesh sieve. In order to remove exchangeable K, the samples were saturated by calcium chloride solution (with a ratio of 2:1), after washing with HCl, samples were then dried at 105oC for 48 hours.

Results showed that there was no difference between carbon sources, applied at the first step of the experiment, so each can be employed as alternatives to each other in the culture medium. The central composite design showed R2 of 0.944 and 0.918 with RMSE of 0.82 and 1.47 for predicting K release of feldspar and phlogopite, respectively, indicating high efficiency. Sensitivity analysis of the central composite design revealed that the pH is the most important factor in K release. The highest concentration of the K was observed at the highest levels of pH. Incubation time also had an impact on potassium release. In the early stages of the incubation time, the trend of potassium release was increasing, in middle stages, K amount decreased but it was accelerated over long times of incubation. The maximum potassium release in presence of phlogopite and feldspar was 121.16 and 96/82 mg L-1, respectively, which was observed at pH= 10.36, sucrose amount= 6.5 g L-1 during 10 days. Potassium amount in this treatment hence increased by 31.52% as compared to feldspar. According to central composite design, maximum potassium release of feldspar and phlogopite was obtained at pH= 10.36 and 10.34, sucrose concentrations of 2.26 and 6.92 g L1 at 18 and 2 days, respectively.

Our results showed that pH had a significant impact on K release by Pseudomonas fluorescens using response surface methodology. Overall, increasing incubation time along with high pH leads to the high amounts of K release from minerals. Different minerals released different content of potassium. Application of soil K-bearing minerals in combination with efficient potassium solubilizing bacterial strains as biofertilizers is required to replace chemical fertilizers and reduce the crop cultivation cost. Many bacterial strains have been found to solubilize minerals and improve plant growth under laboratory and greenhouse conditions, but their ability under field conditions remains unexplored. The capability of these bacteria, considering the soil and plant type, and environmental factors, should be thus evaluated under field conditions.