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A new gene cluster, designated sepABC and a divergently transcribed sepR, was found downstream of the two-component todST phosphorelay system that regulates toluene degradation (the tod pathway) in Pseudomonas putida F1 (PpF1). The deduced amino acid sequences encoded by sepABC show a high homology to bacterial proteins known to be involved in solvent efflux or multidrug pumps. SepA, SepB and SepC are referred to be periplasmic, inner membrane and outer membrane efflux proteins respectively. Effects on growth of various PpF1 mutants compared to that of the wild type in the presence of toluene indicated a possible protective role of the solvent efflux system in a solvent-stressed environment. Growth tests with the complemented mutants confirmed the involvement of the Sep proteins in conferring solvent tolerance. The sepR gene encodes a 260-residue polypeptide that is a member of the E. coli IclR repressor protein family. The repressor role of SepR was established by conducting tests with a sep-lacZ transcriptional fusion in Escherichia coli and PpF1, expression of SepR as a maltose-binding fusion protein in a DNA binding assay, and mRNA analysis. Southern hybridization experiments and analysis of the P. putida KT2440 genome sequence indicated that sepR is a relatively rare commodity compared to homologues of the sepABC genes. We developed a whole-cell bioluminescent biosensor, PpF1G4, which contains a chromosomally based sep-lux transcriptional fusion. The biosensor showed significant induction of the sepABC genes by a wide variety of aromatic molecules, including benzene, toluene, ethylbenzene, and all three isomers of xylene (BTEX), naphthalene, and complex mixtures of aliphatic and aromatic hydrocarbons. PpF1G4 represents a second-generation biosensor that is not based on a catabolic promoter but is nonetheless inducible by aromatic pollutants and moreover functional under nutrient-rich conditions.

ABSTRACT:The whole-cell bioluminescent biosensor Pseudomonas putida F1G4 (PpF1G4), which contains a chromosomally-based sep-lux transcriptional fusion, was used as a tool for direct measurement of the bioavailability of hydrophobic organic compounds (HOCs) partitioned into surfactant micelles. The increased bioluminescent response of PpF1G4 in micellar solutions (up to 10 times the critical micellar concentration) of Triton X-100 and Brij 35 indicated higher intracellular concentrations of the test compounds, toluene, naphthalene, and phenanthrene, compared to control systems with no surfactants present. In contrast, Brij 30 caused a decrease in the bioluminescent response to the test compounds in single-solute systems, without adversely affecting cell growth. The decrease in bioluminescent response in the presence of Brij 30 did not occur in the presence of multiple HOCs extracted into the surfactant solutions from crude oil and creosote. The effect of the micellar solutions on the toluene biodegradation rate was consistent with the bioluminescent response in single-solute systems. None of the surfactants were toxic to PpF1G4 at the doses employed in this study, and PpF1G4 did not produce a bioluminescent response to the surfactants nor utilize them as growth substrates. TEM images suggest that the surfactants did not rupture the cell membranes. The results demonstrate that for Pseudomonas putida F1, nonionic surfactants such as Triton X-100 and Brij 35, at doses between 2 and 10 CMC, may increase the bioavailability and direct uptake of micellar phase HOCs that are common pollutants at contaminated sites. Biotechnol. Bioeng. 2008;99: 86-98.

Lignin, a major polymeric constituent of woody plant tissue, is an abundant source of natural organic matter available as a waste product from the pulp and paper and the fuel ethanol industries. In this study, the sorptive capacity of acid hydrolysis lignin for naphthalene, a representative nonpolar hydrophobic organic compound (HOC), was investigated. When powdered lignin is mixed with distilled water, dissolved and/or colloidal organic matter leaches into the aqueous phase imparting a cloudy yellowish colour. A washing and filtering protocol was developed for pretreating the lignin employed in the sorption studies. Results from batch sorption experiments showed that acid hydrolysis lignin has a strong affinity for naphthalene. The Freundlich isotherm coefficients obtained indicate that the sorption isotherm for naphthalene on hydrolysis lignin is nearly linear. A modified Freundlich equation was employed in order to compare sorption data for HOCs on lignin and activated carbon through the use of unit equivalent coefficients. The results presented in this research and in the literature suggest that the two sorbents are comparable in terms of sorption coefficients. It was determined that acid hydrolysis lignin is unsuitable for use in a packed bed since pumping a naphthalene solution through a column packed with lignin caused the wet lignin to become significantly hardened over time, resulting in a large pressure drop across the system.

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