Biotransformation of polychlorinated biphenyls by Pseudomonas strain LB400, influence of growth substrate and surfactant supplementation

Abstract

Studies were undertaken to characterize biotransformation of polychlorinated biphenyls (PCBs) by Pseudomonas LB400, a bacterium that was previously isolated from contaminated soil and exhibits a broad specificity towards a range of PCB congeners.

Resting cells of Pseudomonas LB400 are known to transform PCBs when the cells are previously grown on biphenyl. In this study, PCB transformation was also observed in resting cells grown on other substrates such as glucose and glycerol. The presence of PCB congeners in the growth medium increased the lag phase for growth of cells on a biphenyl substrate, but not on a glycerol substrate. Supplementation of the degradation medium with biphenyl dramatically decreased the rate of PCB congener transformation while the presence of glycerol or glucose had little or no effect on PCB transformation rates. Removal rates of individual congeners with biphenyl-grown cells were found to vary depending on chlorination pattern of the congener. When compared to cells grown on glucose and glycerol, the relative rates of congener disappearance were not constant. The presence of PCBs adversely affected the viability of biphenyl-grown cells over a 48-h incubation period and may explain the decline observed in PCB conversion capacity over the same incubation period. A major objective of this study was to investigate the significance of using biphenyl as carbon source for growth of Pseudomonas LB400 cells capable of PCB transformation. The findings indicate that, whereas higher rates of transformation of PCBs are observed with biphenyl-grown cells, cells grown on other carbon sources retain PCB-transforming enzymes.

When resting cells of Pseudomonas LB400 grown on biphenyl were incubated with different Aroclor mixtures, greater degradation of total PCBs was observed in Aroclors containing lower-chlorinated congeners. Cells grown on glucose or glycerol also transformed Aroclors, to lesser extents. Time courses of transformation of individual congeners in the Aroclors were plotted and used to determine the transformation rate constants (le). By analysis of the rate constants, it was concluded that the order of degradation of the different congeners in an Aroclor were similar regardless of growth substrate. In general, k values for conversion of a particular congener were lower for cells grown on glucose or glycerol compared to cells grown on biphenyl. The data allowed congeners to be grouped according to their relative rates of degradation.

The effectiveness of a variety of commercial surfactants in solubilizing PCBs sorbed to glass was dependent on surfactant concentration and dissolution time. Most surfactants were fully or partially effective as PCB-solubilizing agents at concentrations greater than their critical micelle concentration (CMC) value. Among ethoxylate surfactants tested, those with lower CMCs were more efficient PCB-solubilizing agents. PCBs were not solubilized with a no-surfactant water control, nor with the nonionic block copolymer. At a concentration of 10 g/1, three out of eleven surfactants, a diethanolamide/ethoxylate blend, an alkane sulphonate and a twin alcohol ethoxylate, inhibited bacterial growth. Only two surfactants, Sorbax PMO-20, a fatty acid ethoxylate and Witcomul 3235, an anionic/nonionic blend, supported growth of Pseudomonas LB400 as sole carbon sources. In general, at surfactant concentrations above their CMC, anionic surfactants promoted, whereas nonionic surfactants inhibited PCB transformation compared to a water control. Transformation rate constants of each congener in the presence of Pseudomonas LB400 and selected surfactants were compared. The inhibitory effect of lgepal C0-630, an alkylphenol ethoxylate, on PCB degradation could be eliminated by diluting the surfactant solution to a lower concentration.

These surfactants were then tested for their abilities to wash PCBs from weathered contaminated soil. While none were effective at solubilizing the PCBs at a surfactant concentration of 1 g/1, six surfactants, at a concentration of 10 g/1, removed greater than 75 % of the hexane-extractable PCBs from the soil. The most effective surfactants in soil washing tests were the nonionic alcohol ethoxylate, Bio Soft EN-600, and lgepal C0-630. The PCB congeners in the soil washings were then transformed by resting cells of Pseudomonas LB400, previously grown on biphenyl. Solutions containing the latter efficient soil washing surfactants manifested lower rates of PCB biodegradation, with only 16 or 32% of congeners transformed over a 48-h incubation period. In contrast, two anionic surfactants, Hostapur SAS 60 and Nansa LSS38/ AS, exhibited highest rates and extents of PCB degradation (S2-67% of congeners transformed over a 48-h incubation period), although they were less efficient surfactants in the soil washing process.

Cells of Pseudomonas LB400, grown on biphenyl, glucose or glycerol, transformed PCB congeners into chlorobenzoic acid (CBA) metabolites. Rates and extents of PCB transformation and metabolite formation were highest with biphenyl grown cells. Intermediate rates of metabolite production were observed with glycerol-grown cells and lowest rates of production were found with glucose-grown cells. Regardless of carbon source, the rate of degradation of congeners was faster than the rate of production of CBAs. Relative rates of PCB transformation and metabolite production from different congeners with cells grown on a particular substrate followed the same general order, 2,3- CBA (from 2,3-CBP) > 2-CBA (from 2,2'-CBP) > 4-CBA (from 2,5,4'-CBP) >2,4-CBA (from 2,4,2',4'-CBP). Pseudomonas LB400 appeared unable to grow on any of the chlorobenzoic acids. However, Pseudomonas LB400 cells grown on biphenyl appeared capable of degrading 2-CBA and 2,3-CBA but not 4-CBA nor 2,4-CBA Cells grown on glycerol appeared unable to metabolize any CBAs.