Soil and plant inoculation with heterotrophic zinc-solubilizing bacteria (ZSB) is considered a promising approach for increasing zinc (Zn) phytoavailability and enhancing crop growth and nutritional quality. Nevertheless, it is necessary to understand the underlying bacterial solubilization processes to predict their repeatability in inoculation strategies. Acidification via gluconic acid production remains the most reported process. In this study, wheat rhizosphere soil serial dilutions were plated on several solid microbiological media supplemented with scarcely soluble Zn oxide (ZnO), and 115 putative Zn-solubilizing isolates were directly detected based on the formation of solubilization halos around the colonies. Eight strains were selected based on their Zn solubilization efficiency and siderophore production capacity. These included one strain of , two of, three strains of , one of, and one strain of In ZnO liquid solubilization assays, the presence of glucose clearly stimulated organic acid production, leading to medium acidification and ZnO solubilization. While solubilization by and was attributed to the accumulated production of six and seven different organic acids, respectively, the other strains solubilized Zn via gluconic, malonic, and oxalic acids exclusively. In contrast, in the absence of glucose, ZnO dissolution resulted from proton extrusion (e.g., via ammonia consumption by strains) and complexation processes (i.e., complexation with glutamic acid in cultures of ). Therefore, while gluconic acid production was described as a major Zn solubilization mechanism in the literature, this study goes beyond and shows that solubilization mechanisms vary among ZSB and are strongly affected by growth conditions. Barriers toward a better understanding of the mechanisms underlying zinc (Zn) solubilization by bacteria include the lack of methodological tools for isolation, discrimination, and identification of such organisms. Our study proposes a direct bacterial isolation procedure, which prevents the need to screen numerous bacterial candidates (for which the ability to solubilize Zn is unknown) for recovering Zn-solubilizing bacteria (ZSB). Moreover, we confirm the potential of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) as a quick and accurate tool for the identification and discrimination of environmental bacterial isolates. This work also describes various Zn solubilization processes used by wheat rhizosphere bacteria, including proton extrusion and the production of different organic acids among bacterial strains. These processes were also clearly affected by growth conditions (i.e., solid versus liquid cultures and the presence and absence of glucose). Although highlighted mechanisms may have significant effects at the soil-plant interface, these should only be transposed cautiously to real ecological situations.

Please cite this article as: Meyer, G., Maurhofer, M., Frossard, E., Gamper, H.A., Mäder, P., Mészáros, É., Schönholzer-Mauclaire, L., Symanczik, S., Oberson, A., Pseudomonas protegens CHA0 does not increase phosphorus uptake from 33 P labeled synthetic hydroxyapatite by wheat grown on calcareous soil, Soil Biology and Biochemistry (2019), doi: https://doi. Abstract 18Soil microorganisms exuding organic acids have the potential to solubilize inorganic 19 phosphorus (P), which could improve the P availability to plants growing on calcareous 20 soil. The gluconic acid exuding bacteria Pseudomonas protegens CHA0 can solubilize 21 P under glucose rich in vitro conditions, but evidence on the effectiveness in soil is 22 from the two bacterial inoculants suggested that overall microbial activity, i.e., via 36 protonation due to respiration, increased inorganic P mobilization. P solubilizing 37 bacteria should be evaluated using a tracer and an appropriate bacterial control in 38 order to reveal the mechanisms involved in increased plant available P in soil 39 inoculated with P solubilizing bacteria. 40

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