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Degradation of chrysene, a four ringed highly carcinogenic polycyclic aromatic hydrocarbon (PAH) has been demonstrated by bacterial mixed culture Biorem-CGBD comprising Achromobacter xylosoxidans, Pseudomonas sp. and Sphingomonas sp., isolated from crude oil polluted saline sites at Bhavnagar coast, Gujarat, India. A full factorial Central Composite Design (CCD) using Response Surface Methodology (RSM) was applied to construct response surfaces, predicting 41.93% of maximum chrysene degradation with an experimental validation of 66.45% chrysene degradation on 15th day, using a combination of 0.175, 0.175 and 0.385 mL of OD600 = 1 inoculum of A. xylosoxidans, Pseudomonas sp. and Sphingomonas sp., respectively and a regression coefficient (R2) of 0.9485 indicating reproducibility of the experiment. It was observed that chrysene degradation can be successfully enhanced using RSM, making mixed culture Biorem-CGBD a potential bioremediation target for PAH contaminated saline sites.

The proposed method of kinetic analysis of aqueous-phase biodegradation of polycyclic aromatic hydrocarbons (PAH) mixture presupposes representation of kinetic curves for each pair of mixture components, S(x) and S(y), in double-logarithmic coordinates (ln S(x); ln S(y)). If PAH mixture conversion corresponds to the multisubstrate model with a common active site, then the graphs in double-logarithmic coordinates are straight lines with the angular coefficients equal to the ratio of respective first-order rate constants k(y)(x)= V(y)K(x)/K(y)V(x), where K(x) and K(y) are half-saturation constants, V(x) and V( y ) are the maximum conversion rates for substrates S(x) and S(y); the graph slope does not depend on any concentrations and remains constant during the change of reaction rates as a result of inhibition, induction/inactivation of enzymes or biomass growth. The formulated method has been used to analyze PAH mixture conversion by the culture of Sphingomonas sp. VKM B-2434. It has been shown that this process does not satisfy the multisubstrate model with a single active site. The results suggest that the strain VKM B-2434 contains at least two dioxygenases of different substrate specificity: one enzyme converts phenanthrene and fluoranthene and the other converts acenaphthene and acenaphthylene. The ratios of first-order rate constants have been obtained for these pairs of substrates.

The nature of bioavailability of DNA-intercalated PAHs in aqueous solution was investigated. The degradability of different PAHs including anthracene, phenanthrene and pyrene by Sphingomonas sp. was not inhibited even at a high DNA concentration of 2%. The DNA was stable against the PAH-degrader as indicated by the unchanged electrophoresis gel chromatograms after treatment. This shows that a structural change in the polymer is not necessary for the release of PAHs. Partitioning experiments using phenanthrene as a model PAH illustrated the presence of an initial passive uptake by autoclaved cells. Subsequent intracellular degradation became apparent from parallel data with live cells. Phenanthrene transfer from the DNA was diffusion-controlled and the exit of this molecule from their intercalation sites is favored in lieu of the presence of stronger hydrophobic binding sites in the cell membrane.

Quantitative structure-activity relationships (QSARs) were developed for three Monod-type parameters--qmax, Ks, and qmax/Ks--that express the kinetics of polycyclic aromatic hydrocarbon (PAH) biotransformation by Sphingomonas paucimobilis strain EPA505. The training sets contained high-quality experimental values of the kinetic parameters for 20 unsubstituted and methylated PAHs as well as values of 41 meaningful molecular descriptors. A genetic function approximation algorithm was used to develop the QSARs. Statistical evaluation of the developed QSARs showed that the relationships are statistically significant and satisfy the assumptions of linear-regression analysis. The Organization for Economic Co-operation and Development principles for (Q)SAR validation were followed to evaluate the developed QSARs, which showed that the QSARs are valid. The QSARs contain spatial, spatial and electronic, topological, and thermodynamic molecular descriptors. Whereas spatial descriptors were essential in explaining biotransformation kinetics, electronic descriptors were not. Mechanistic interpretation of the QSARs resulted in evidence that is consistent with the hypothesis of membrane transport as being the rate-limiting process in PAH biotransformation by strain EPA505. The present study demonstrates the value of QSAR not only as a predictive tool but also as a framework for understanding the mechanisms governing biodegradation at the molecular level.

There has been growing interest in employing electro-bioremediation, a hybrid technology of bioremediation and electrokinetics for the treatment of contaminated soil. Knowledge however on the effect of weak electrokinetic conditions on the activity and viability of pollutant-degrading microorganisms is scarce. Here we present data about the influence of direct current (DC) on the membrane integrity, adenosine triphosphate (ATP) pools, physico-chemical cell surface properties, degradation kinetics and culturability of fluorene-degrading Sphingomonas sp. LB126. Flow cytometry was applied to quantify the uptake of propidium iodide (PI) and the membrane potential-related fluorescence intensities (MPRFI) of individual cells within a population. Adenosine tri-phosphate contents and fluorene biodegradation rates of bulk cultures were determined and expressed on a per cell basis. The cells' surface hydrophobicity and electric charge were assessed by contact angle and zeta potential measurements respectively. Relative to the control, DC-exposed cells exhibited up to 60% elevated intracellular ATP levels and yet remained unaffected on all other levels of cellular integrity and functionality tested. Our data suggest that direct current (X=1 V cm(-1); J=10.2 mA cm(-2)) as typically used for electrobioremediation measures has no negative effect on the activity of the polycyclic aromatic hydrocarbon (PAH)-degrading soil microorganism, thereby filling a serious gap of the current knowledge of the electrobioremediation methodology.

Sphingomonas yanoikuyae B1 possesses several different multicomponent oxygenases involved in metabolizing aromatic compounds. Six different pairs of genes encoding large and small subunits of oxygenase iron-sulfur protein components have previously been identified in a gene cluster involved in the degradation of both monocyclic and polycyclic aromatic hydrocarbons. Insertional inactivation of one of the oxygenase large subunit genes, bphA1c, results in a mutant strain unable to grow on naphthalene, phenanthrene, or salicylate. The knockout mutant accumulates salicylate from naphthalene and 1-hydroxy-2-naphthoic acid from phenanthrene indicating the loss of salicylate oxygenase activity. Complementation experiments verify that the salicylate oxygenase in S. yanoikuyae B1 is a three-component enzyme consisting of an oxygenase encoded by bphA2cA1c, a ferredoxin encoded by the adjacent bphA3, and a ferredoxin reductase encoded by bphA4 located over 25kb away. Expression of bphA3-bphA2c-bphA1c genes in Escherichia coli demonstrated the ability of salicylate oxygenase to convert salicylate to catechol and 3-, 4-, and 5-methylsalicylate to methylcatechols.

An investigation of the mobility, viability, and activity of polycyclic aromatic hydrocarbon (PAH) degrading bacteria in an electric field is presented. Bench-scale model aquifers were used to test electrophoresis and electroosmosis as potential mechanisms for bacterial dispersion in contaminated sites. Glass beads, alluvial sand from Lake Geneva, and historically polluted clayey soil were used as packing materials. The green-fluorescent protein labeled PAH-degrading bacteria Sphingomonas sp. L138 and Mycobacterium frederiksbergense LB501TG were used as test organisms because of the known differing physicochemical surface and adhesion properties of the corresponding wild-type strains. No adverse effects of the electric current on bacterial viability and PAH-degradation were observed in the system chosen. Up to 90% of the weakly negatively charged and moderately adhesive cells of strain L138 were transported by electroosmosis, whereas 0-20% were transported by electrophoresis. By contrast, poor electrokinetic transport of strongly charged and highly adhesive cells of M. frederiksbergense LB501TG occurred in the different model aquifers. Treatment of bacteria with the nonionic surfactant Brij35 resulted in up to 80% enhanced electrokinetic dispersion of both strains. Our findings demonstrate that electroosmosis may be a valuable mechanism to transport bacteria in the subsurface with transport efficiencies heavily depending on the retention of the bacteria by the solid phase.