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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.

The newly developed droplet digital PCR (DD-PCR) has shown promise as a DNA quantification technology in medical diagnostic fields. This study evaluated the applicability of DD-PCR as a quantitative tool for soil DNA using quantitative real-time PCR (qRT-PCR) as a reference technology. Cupriavidus sp. MBT14 and Sphingopyxis sp. MD2 were used, and a primer/TaqMan probe set was designed for each (CupMBT and SphMD2, respectively). Standard curve analyses on tenfold dilution series showed that both qRT-PCR and DD-PCR exhibited excellent linearity (R (2) = 1.00) and PCR efficiency (≥92 %) across their detectable ranges. However, DD-PCR showed a tenfold greater sensitivity than qRT-PCR. MBT14 and MD2 were added to non-sterile soil at 0 ~ 5 × 10(8) and 0 ~ 5 × 10(7) cells per gram of soil, respectively (n = 5). This bacterial load test indicated that DD-PCR was more sensitive and discriminating than qRT-PCR. For instance, DD-PCR showed a gradual DNA increase from 14 to 141,160 MBT14 rDNA copies μL DNA extract(-1) as the bacterial load increased, while qRT-PCR could quantify the DNA (6,432 copies μL DNA(-1)) at ≥5 × 10(5) MBT14 per gram of soil. When temporal DNA changes were monitored for 3 weeks in the amended soils, the two technologies exhibited nearly identical changes over time. Linearity tests (y = a · x) revealed excellent quantitative agreement between the two technologies (a = 0.98, R (2) = 0.97 in the CupMBT set and a = 0.90, R (2) = 0.94 in the SphMD2 set). These results suggest that DD-PCR is a promising tool to examine temporal dynamics of microorganisms in complex environments.

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.

Polycyclic aromatic hydrocarbon (PAHs)-degrading bacteria may enhance the bioavailability of PAHs by excreting biosurfactants, by production of extracellular polymeric substances, or by forming biofilms. We tested these hypotheses in pure cultures of PAHs-degrading bacterial strains. Most of the strains did not substantially reduce the surface tension when grown on PAHs in liquid shaken cultures. Thus, pseudo-solubilization of PAHs in biosurfactant micelles seems not to be a general strategy for these isolates to enhance PAHs-bioavailability. Three semi-colloid Sphingomonas polysaccharides all increased the solubility of PAHs (Gellan 1.3- to 5.4-fold, Welan 1.8- to 6.0-fold and Rhamsan 2.4- to 9.0-fold). The increases were most pronounced for the more hydrophobic PAHs. The polysaccharide-sorbed PAHs were bioavailable. Mineralization rates of 9-[14C]-phenanthrene and 3-[14C]-fluoranthene by Sphingobium EPA505, were similar with and without sphingans, indicating that mass-transfer rates from PAHs crystals to the bulk liquid were unaffected by the polysaccharides. Biofilm formation on PAHs crystals may favor the diffusive mass transfer of PAHs from crystals to the bacterial cells. A majority of the PAHs-degraders tested formed biofilms in microtiter wells coated with PAHs crystals. For strains capable of growing on different PAHs; the more soluble the PAHs, the lower the percentage of cells attached. Biofilm formation on PAHs-sources was the predominant mechanism among the tested bacteria to overcome mass transfer limitations when growing on poorly soluble PAHs.