A Sphingobium sp. strain isolated from radioactive solid waste management site (RSMS) completely degraded 7.98 g/L of tributyl phosphate (TBP) from TBP containing suspensions in 3 days. It also completely degraded 20 mM dibutyl phosphate (DBP) within 2 days. The strain tolerated high levels of TBP and showed excellent stability with respect to TBP degradation over several repeated subcultures. On solid minimal media or Luria Bertani media supplemented with TBP, the RSMS strain showed a clear zone of TBP degradation around the colony. Gas chromatography and spectrophotometry analyses identified DBP as the intermediate and butanol and phosphate as the products of TBP biodegradation. The RSMS strain utilized both TBP and DBP as the sole source of carbon and phosphorous for its growth. The butanol released was completely utilized by the strain as a carbon source thereby leaving no toxic residue in the medium. Degradation of TBP or DBP was found to be suppressed by high concentration of glucose which also inhibited TBP or DBP dependent growth. The results highlight the potential of Sphingobium sp. strain RSMS for bioremediation of TBP and for further molecular investigation.

A tri- and dibutyl phosphate (TBP/DBP) non-degrading spontaneous mutant, Sphingobium SS22, was derived from the Sphingobium sp. strain RSMS (wild type). Unlike the wild type strain, Sphingobium SS22 could not grow in a minimal medium supplemented with TBP or DBP as the sole source of carbon or phosphorous. Sphingobium SS22 also did not form any of the intermediates or end products of TBP or DBP degradation, namely DBP, butanol or inorganic phosphate. Proteomic analysis revealed the absence of three prominent proteins in Sphingobium SS22 as compared to wild type. These proteins were identified by MALDI mass spectrometry, and they showed similarities to phosphohydrolase- and exopolyphosphatase-like proteins from other bacteria, which belong to the class of phosphoesterases. Cellular proteins of Sphingobium SS22 showed none or negligible phosphodiesterase (PDE) and phosphomonoesterase (PME) activities at pH 7 and displayed approximately five- and approximately twofold less DBP and monobutyl phosphate (MBP) degradation activity, respectively, in comparison to the wild type strain. In-gel zymographic analysis revealed two PDE and PME activity bands in the wild type strain, one of which was absent in the Sphingobium SS22 mutant. The corresponding proteins from the wild type strain could degrade DBP and MBP. The results demonstrate the involvement of phosphoesterase enzymes in the TBP degradation pathway elucidated earlier.

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