Solubilization of hydroxyapatite by Enterobacter agglomerans and cloned Escherichia coli in culture medium

Phosphate-solubilizing bacteria (PSB) possessing the ability to solubilize insoluble inorganic phosphate were isolated from the rhizosphere soil of wheat. A laboratory study was conducted to investigate the solubilization of phosphate by a known PSB, Enterobacter agglomerans, and by a genetically manipulated bacterium, Escherichia coli. A second laboratory study investigated the release of P from E. agglomerans compared with known acids. For the first laboratory study, a cosmid (pHC79) library of phosphate-solubilizing gene(s) from E. agglomerans chromosome DNA was constructed in E. coli JM109. The clone JM109 (pKKY) showing phosphate solubilization properties was screened on standard medium containing hydroxyapatite (HY). The P concentration significantly increased at 5 and 10 days for JM109 (pKKY) compared with JM109 (pHC79), the control. Although the P concentration increased, there was no significant change in their pHs. Furthermore, an increase in colony-forming units (CFUs) was seen at 5 and 10 days for JM109 (pKKY) but not for JM109 (pHC79). Artificial acidification of the culture medium with HCl, citric acid, oxalic acid, and lactic acid was achieved by shaking for 48 h. Acidification with these selected acids solubilized more HY than E. agglomerans growing for 42 h at similar pHs. However, a high P concentration was measured in culture medium with E. agglomerans growing for 84 h despite similar pHs. Our results suggest that acid production may play an important role in HY solubilization, but is not the sole reason for the increase in P concentration in culture medium.