Adaptation of estuarine bacteria to toluene at low concentrations in sea water: cometabolism of toluene

Abstract

The ability of sediment-water interface bacteria to adapt to micromolar concentrations of water-dissolved toluene was determined. Control and experimental chambers of 200 cm2 of undisturbed sediment were fitted for continuous replacement of overlying seawater. The experimental chamber received toluene in sterile seawater at a rate determined to give concentrations of 4 to 17 μM during adaptation. Samples of seawater and sediment surface were removed at intervals and analyzed for bacterial numbers and rates of assimilation of radiolabeled glutamate, phenol, and toluene. Adaptation occurred in 3 to 5 days in the experimental chamber and was characterized by rapid increases in phenol and toluene assimilation. An increase in phenol assimilation by sediment surface bacteria was the first detectable adaptation. Kinetic analyses indicated bacteria in seawater overlying the sediments had a greater affinity for toluene (half-saturation at 50 nM) following adaptation than for phenol (half-saturation at 3.9 μM). The first-order rate constant for toluene assimilation at low concentrations decreased 10-fold on prolonged continuous culture of the seawater community at millimolar concentrations of toluene. Total numbers of bacteria by direct count and plate count, and bacterial activity measured with [14C]glutamate, did not change appreciably in the control and experimental chambers. During the early stages of adaptation the meta-ring-fission product of 3-methylcatechol accumulated in the seawater to a concentration of 4.9 μM. Tests of isolates from plate counts on phenol and toluene vapours revealed 30% formed diffusable yellow metabolites of these substrates in the absence of significant growth. One of these isolates showed partial metabolism of toluene and meta-toluate to the first metabolite following meta-ring fission, 2-hydroxy-6-keto-2,4-heptadienoate, by resting cells and cells growing on alternative carbon sources.