Isolation and Characterization of Phosphorus Solubilizing Bacteria With Multiple Phosphorus Sources Utilizing Capability and Their Potential for Lead Immobilization in Soil

Abstract

Phosphorus solubilizing bacteria (PSB) can promote the level of plant-absorbable phosphorus (P) in agro-ecosystems. However, little attention has been paid to PSB harboring abilities in utilizing multiple phosphorus sources and their potentials for heavy metal immobilization. In this study, we applied the strategy of stepwise acclimation by using Ca₃(PO₄)₂, phytate, FePO₄, and AlPO₄ as sole P source. We gained 18 PSB possessing abilities of multiple P sources utilization, and these bacteria belonged to eight genera (Acinetobacter, Pseudomonas, Massilia, Bacillus, Arthrobacter, Stenotrophomonas, Ochrobactrum, and Cupriavidus), and clustered to two apparent parts: Gram-positive bacteria and Gram-negative bacteria. The isolate of Acinetobacter pittii gp-1 presented good performance for utilizing Ca₃(PO₄)₂, FePO₄, AlPO₄, and phytate, with corresponding P solubilizing levels were 250.77, 46.10, 81.99, and 7.91 mg/L PO₄ ^3--P, respectively. The PSB A. pittii gp-1 exhibited good performance for solubilizing tricalcium phosphate in soil incubation experiments, with the highest values of water soluble P and available P were 0.80 and 1.64 mg/L, respectively. Additionally, the addition of A. pittii gp-1 could promote the immobilization of lead (Pb), and the highest Pb immobilization efficiency reached 23%. Simultaneously, we found the increases in abundances of both alkaline phosphatase gene (phoD) and β-propeller phytase gene (bpp) in strain gp-1 added soils. Besides, we observed the expression up-regulation of both pyrroloquinoline quinone gene (pqq) and polyphosphate kinases gene (ppk), with the highest relative expression levels of 18.18 and 5.23, respectively. We also found the polyphosphate particles using granule staining. To our knowledge, our findings first suggest that the solubilizing of tricalcium phosphate by phosphorus solubilizing bacterium belonging to Acinetobacter is coupled with the synthesis of polyphosphate. Taken together, A. pittii gp-1 could be a good candidate in improving soil fertility and quality.

Keywords: Acinetobacter pittii gp-1; P-cycling-related gene; Pb immobilization; multiple phosphorus source utilizing capacity; phosphorus solubilizing bacteria; ppk and pqq genes.

FIGURE 1

Shifts in bacterial community composition during four acclimation stages. (A) Relative abundance of 16S rRNA gene sequences classified to phylum level. (B) Relative abundance of 16S rRNA gene sequences classified to genus level.

FIGURE 2

Neighbor-joining phylogenetic tree of 18 phosphorus solubilizing bacteria based on 16S rRNA gene sequences. The numbers at the nodes indicate the levels of bootstrap support based on data for 1000 replicates. The scale bar represents 0.05 sequence divergence.

FIGURE 3

The P solubilizing levels of 18 PSB in liquid NBRIP medium containing Ca₃(PO₄)₂ (A), sodium phytate (B), FePO₄ (C), and AlPO₄ (D), and in solid NBRIP medium containing Ca₃(PO₄)₂ (E), sodium phytate (F), FePO₄ (G), and AlPO₄ (H). The results are the mean value of three replicates, error bars represent standard error. The lowercase letters above the columns denote significant level (P < 0.05).

FIGURE 4

The Pb immobilization efficiency and phosphorus content in soils with different addition rate of tricalcium phosphate. The results are the mean value of three replicates, error bars represent standard error. The lowercase letters above the columns denote significant level (P < 0.05).

FIGURE 5

The abundances of 16S rRNA gene (A), gcd gene (B), bpp gene (C), and phoD gene (D) in soils with different addition rate of tricalcium phosphate. The results are the mean value of three replicates, error bars represent standard error. The lowercase letters above the columns denote significant level (P < 0.05).

FIGURE 6

Neighbor-joining phylogenetic tree of ppk gene (A) and pqq gene (B) based on sequence similarity. The numbers at the nodes indicate the levels of bootstrap support based on data for 1000 replicates. The scale bar represents 2% sequence divergence.

FIGURE 7

Gene abundance and phosphorus determination during 24 h. (A) shows the shift in P-cycling-related gene abundance. (B) reflects the concentration of extracellular phosphorus. (C) shows the granular staining. The results are the mean value of three replicates, error bars represent standard error. The lowercase letters above the columns denote significant level (P < 0.05).