Diazotrophic Azotobacter salinestris YRNF3: a probable calcite-solubilizing bio-agent for improving the calcareous soil properties
Authors: Rashad Younes M , Hafez Mohamed , Rashad Mohamed 
Published: November 2023

Calcareous soils are characterized by a high calcium carbonate content (calcite), which plays a crucial role in the soil structure, plant growth, and nutrient availability. The high content of CaCO3 leads to the increment of the soil alkalinity, which results in a lowering of the nutrient availability causing a challenge for the agriculture in these soils. In this study, the calcite-solubilizing potential of the diazotrophic Azotobacter salinestris YRNF3 was investigated in vitro as a probable bio-agent for enhancing the calcareous soils properties such as soil pH and nutrient availability. Twelve diazotrophic bacterial strains were isolated from wheat rhizosphere collected from different wheat-cultivated fields in five Egyptian governorates. Using Nessler’s reagent, all isolated bacterial strains were found to have the ability to produce ammonia. By amplification of nifH gene, a PCR product of 450 bp was obtained for all isolated bacterial strains. For each isolate, three biological and three technical replicates were applied. All isolated diazotrophic bacteria were qualitatively screened for their calcite-solubilizing ability. To quantitatively investigate the calcite-solubilizing potential of A. salinestris YRNF3 in vitro, changes in the contents of soluble calcium (Ca2+), bicarbonate (HCO3−), total nitrogen (TN), total protein (TP), and pH were daily measured in its culture filtrate along 10 days of incubation. The results showed that the pH values in the culture filtrate ranged from 5.73 to 7.32. Concentration of Ca2+ and HCO3− in the culture filtrate significantly decreased with the increment in the incubation time, while concentration of TN increased along the time. The highest TN concentration (0.0807 gL−1) was observed on days 4 and 5, compared to that of the day 0 (0.0014 gL−1). Content of TP in the culture filtrate also significantly increased along the incubation period. The highest TP content was recorded in day 4 (0.0505%), while no TP content was recorded on day 0. Furthermore, data obtained revealed that A. salinestris YRNF3 produced acid phosphatase at low activity (5.4 U mL−1). HPLC analysis of the culture filtrate indicated production of different organic acids, namely lactic acid (82.57 mg mL−1), formic acid (46.8 mg mL−1), while acetic acid was detected in a low quantity (3.11 mg mL−1). For each analysis, three replicates of each treatment were analyzed. Means of the tested treatments were compared using Tukey's HSD test at p ≤ 0.05. In conclusion, findings of this work suggested that A. salinestris YRNF3 has the potential to be a probable bioagent to be used for the reclamation of the calcareous soils by solubilizing CaCO3, improving soil fertility, and promoting plant growth. However, further studies are needed to investigate its field application and their long-term effects on the soil properties and plant productivity. To the best of the author's knowledge, this is the first study reporting the calcite-solubilizing ability of a nitrogen-fixing bacteria. Having these two abilities by one microorganism is a unique feature, which qualifies it as a promising bioagent for reclamation of the calcareous soils.