A new pioneer colorimetric micro-plate method for the estimation of ammonia production by plant growth promoting rhizobacteria (PGPR)

Abstract

The ability of Plant Growth Promoting Rhizobacteria (PGPR) to produce ammonia (NH3) is one of the key mechanisms for the growth and development of plants. Ammonia spectrophotometric quantification assay using Nessler’s reagent remains laborious and seems to be not suitable for large-scale PGP screening assay. Here, we performed a new accurate, easy, and cost-effective micro-plate protocol for large-scale ammonia quantification in bacterial supernatants as an alternative to the spectrophotometric method. In order to validate the accuracy of our innovative microplate assay, 9 bacterial strains were explored for their ability to produce ammonia using both the conventionally described assay and the newly performed micro-plate one. The ammonia 96-well microplate assay was successfully performed by scaling down the spectrophotometric methods to reduce the volume of bacterial supernatant as well as Nessler’s reagent. The reduction was estimated to 90%of the total used volume in comparison to the conventional test. The micro-test is 10-fold cheaper and 26 times faster than the conventional method. All bacterial isolates were positive for ammonia production. Bacillus inaquorsum and Bacillus mojavensis produced the highest ammonia concentration of about 371 and 370μM respectively. Furthermore, the application of the ammonia micro-plate assay reduces drastically the reagent waste and toxicity hazard of K2HgI4 (Nessler’s reagent) in the environment, thus, we can classify it as eco-friendly respecting the Green Chemistry concept according to Environmental Protection Agency (EPA). The statistical data obtained from both assays are significantly correlated (r = 0.985, R squared = 0.9329, and p < 0.001) proving the accuracy of the micro-plate assay. The proposed NH3 micro-assay is a reliable, rapid, eco-friendly, and cost-effective method to screen plant growth-promoting potential of more than 25 bacterial strains in one micro-plate. It could be an alternative for the conventional NH3 assay as a routine research tool.