P28

TOLUENE DEGRADATION ACTIVITY AND LONGEVITY OF RESTING PpF1 CELLS GROWN UNDER AEROBIC AND HYPOXIC CONDITIONS

Toluene dioxygenase (TDO) in Pseudomonas putida Fl (PpFl) is an ideal enzyme to use as a model for bioremediation studies. It is common in nature and has a broad substrate specificity, enabling it to catalyze the insertion of two molecules of molecular oxygen into the aromatic nucleus of many aromatic contaminants such as benzene, toluene, and xylenes (BTX). PpFl was used as a model bacterium to investigate how low dissolved oxygen (DO) levels affect catabolic enzyme expression and biodegradation activity, and to determine how long the enzyme stays active after the inducing substrate (i.e., toluene) has been removed.

Studies of TDO expression were conducted using resting PpFl cells grown under different dissolved oxygen concentrations. Cells were grown in a continuous culture fermenter (BioStat E) equipped with a dissolved oxygen monitor and air flow regulators which permitted the control of dissolved oxygen concentration in the growth medium. Toluene vapors were supplied as the sole carbon and energy source using a separate air flow line. Induced cells were harvested and subsequently washed and resuspended in air-saturated sterile mineral medium prior to conducting biodegradation assays.

Activity of TDO was assessed over time by measuring the rate of toluene disappearance (corrected for abiotic controls) in the presence of chloramphenicol (350 mg/L). Chloramphenicol is a bacteriostatic antibiotic that inhibits de novo enzyme synthesis. This ensures a constant level of enzyme activity during the biodegradation assay. Toluene degradation activity assays were thus conducted on previously induced cells grown under different DO concentrations and allowed to rest for different durations. Dissolved oxygen concentration was only a factor during growth, as all in vivo enzyme activity assays were conducted under fully aerobic conditions.

Preliminary results show that activity of the TDO enzyme present in resting cells decreased rapidly with time, but toluene degradation activity persisted at low levels for several weeks. Experiments conducted with cells grown at various dissolved oxygen concentrations (0.1 to 8 mg/L) yielded enzyme expression over the range of DO concentrations used, even at DO levels below which denitrification commonly occurs (i.e. <0.3 mg/L). This suggests the possibility of aerobic transformations under denitrifying conditions. Nevertheless, cells growing under hypoxic conditions exhibited lower toluene degradation activities than cells grown under fully aerobic conditions. Whether this effect was due to changes in availability of required NADH, TDO enzyme conformation, or another mechanism, such as reduced ability to assimilate Fe(II) and incorporate it in the active center of TDO, remains to be determined.

Key words: bioremediation, BTX, toluene dioxygenase activity