Phosphate solubilization and acid phosphatase activity of Serratia sp. isolated from mangrove soil of Mahanadi river delta, Odisha, India

Abstract

Phosphorus is an essential element for all life forms. Phosphate solubilizing bacteria are capable of converting phosphate into a bioavailable form through solubilization and mineralization processes. Hence in the present study a phosphate solubilizing bacterium, PSB-37, was isolated from mangrove soil of the Mahanadi river delta using NBRIP-agar and NBRIP-BPB broth containing tricalcium phosphate as the phosphate source. Based on phenotypic and molecular characterization, the strain was identified as Serratia sp. The maximum phosphate solubilizing activity of the strain was determined to be 44.84 μg/ml, accompanied by a decrease in pH of the growth medium from 7.0 to 3.15. During phosphate solubilization, various organic acids, such as malic acid (237 mg/l), lactic acid (599.5 mg/l) and acetic acid (5.0 mg/l) were also detected in the broth culture through HPLC analysis. Acid phosphatase activity was determined by performing p-nitrophenyl phosphate assay (pNPP) of the bacterial broth culture. Optimum acid phosphatase activity was observed at 48 h of incubation (76.808 U/ml), temperature of 45 °C (77.87 U/ml), an agitation rate of 100 rpm (80.40 U/ml), pH 5.0 (80.66 U/ml) and with glucose as a original carbon source (80.6 U/ml) and ammonium sulphate as a original nitrogen source (80.92 U/ml). Characterization of the partially purified acid phosphatase showed maximum activity at pH 5.0 (85.6 U/ml), temperature of 45 °C (97.87 U/ml) and substrate concentration of 2.5 mg/ml (92.7 U/ml). Hence the present phosphate solubilizing and acid phosphatase production activity of the bacterium may have probable use for future industrial, agricultural and biotechnological application.

Keywords: Bacteria; Enzyme; Mangrove ecosystem; Organic acid; Phosphatase.

Figure 1

Phylogenetic tree based on 16S rRNA gene sequences by Neighbor Joining method (using MEGA 6.0), showing the relationship between strain PSB-26 and other members of the Serratia sp. The Genbank nucleotide accession numbers are listed next to the strain names. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) is shown next to the branches. The scale bars represent 0.005 substitution/site.

Figure 2

Time dependent tri-calcium phosphate solubilisation and drop of pH of the NBRIP broth medium by Serratia PSB-37 isolate.

Figure 3

HPLC analysis of organic acids in the culture supernatant of isolates PSB-37 at 144 h of incubation in NBRIP broth. The corresponding peaks detected in culture medium were of lactic acid, malic acid and acetic acids including eight unknown peaks (3.8, 7.0, 7.6, 8.2, 13.0, 13.7, 17.2 and 19.6).

Figure 4

Effect of different growth parameters (a) Temperature, (b) Shaking velocity, (c) pH, (d) Carbon sources and (e) Nitrogen sources on crude acid phosphatase production by the isolate, PSB-37 (*** denotes p-value <0.0001).

Figure 5

Effect of (a) different pH (b) different temperature and (c) different substrate concentration on partially purified acid phosphatase activity (*** denotes p-value <0.0001).